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Infections in Endocrinology: Tuberculosis

ABSTRACT

 

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), is responsible for the largest number of deaths worldwide caused by a single organism. Over 25% of the world population is infected with M. tuberculosis, though active infections account only for a small percentage. Though some degree of endocrine dysfunction is invariable in all patients with TB, clinically significant endocrinopathy other than glucose intolerance is rare. This chapter reviews endocrine dysfunction and endocrinopathies associated with TB infection related to the adrenal, thyroid and pituitary glands. Additionally, functional derangement of sodium and calcium homeostasis is also covered. Adrenal involvement can be found in up to 6% of patients with active TB, however isolated adrenal involvement is seen only in a fourth of these. The most common clinical manifestation is Addison’s disease (AD). Clinical manifestations of AD appear only after 90% of the adrenal cortices have been compromised. Thyroid tuberculosis (TTB) is very rare, even in countries with a high prevalence of TB. TB has been seen to involve the thyroid in 0.1 to 1% of patients. Primary pituitary TB (in the absence of systemic involvement and/or constitutional symptoms) is extremely rare, and secondary pituitary TB is more commonly encountered in clinical practice. Pituitary TB should be considered in the differential of a suprasellar mass especially in developing countries, as the condition is potentially curable with treatment. Hyponatremia has been commonly seen in patients admitted to the hospital with TB. The commonest cause of hyponatremia is the syndrome of inappropriate antidiuresis (SIAD). Other causes include untreated primary or secondary adrenal insufficiency, volume depletion, hyponatremia associated with volume excess and hypoalbuminemia and rare cases of cerebral salt wasting seen with tuberculous meningitis. The prevalence of hypercalcemia in patients with TB has ranged from 2-51% in various studies. The primary determinant in the development of hypercalcemia among patients with TB appears to be their Vitamin D status and nutritional calcium intake.

 

INTRODUCTION

 

Mycobacterium tuberculosis the etiological agent of tuberculosis (TB) was directly responsible for 1.3 million deaths in 2019. A majority of these deaths happen in patients without human immunodeficiency virus (HIV) co-infection making M. tuberculosis the pathogen responsible for the largest number of deaths in the world by a single organism. Additionally, TB is among the top ten causes of death worldwide (1).

 

Most cases of primary TB infections are clinically, bacteriologically, and radiologically inapparent. This primary infection in 5-10% patients leads to active disease after a period of latency within 2 years of contracting the infection. In another 5% the disease becomes active much later in life after a decline in general immunity. It is thought that over 25% of the world’s current population is infected with M. tuberculosis though active infections account only for a small percentage. In the year 2019 over 10 million patients were newly diagnosed with clinical TB. South East Asia accounted for over 44% of these along with Africa (25%), Western Pacific (18%), Eastern Mediterranean (8.2%), Americas (2.9%) and Europe (2.5%). The eight countries of India (26%), Indonesia (8.5%), China (8.4%), Philippines (6.0%), Pakistan (5.7%), Nigeria (4.4%), Bangladesh (3.6%) and South Africa (3.6%) account for two thirds of the world’s newly diagnosed cases last year (1).

 

As previously noted most active TB infections are reactivation of latent primary TB though a small but significant percentage of patients have active TB related to new exogenous re-infection. The most common primary site of adult active TB are the highly aerated upper lobes of the lungs. The defining pathology includes the presence of granulomas containing epithelioid cells, Langhan’s giant cells surrounded by lymphocytes with a center of caseous necrosis and varying degrees of fibrosis. This chapter focuses on the endocrinology of tuberculous infection (2, 3).

 

ALTERED IMMUNE-NEUROENDOCRINE COMMUNICATION IN TUBERCULOSIS        

 

The two-way communication between the immune system and the neuroendocrine system is well known and documented. An activated immune cascade can affect all the endocrine systems of the body. Adrenal steroids are the primary hormones that modify immune responses. The up-regulation of the hypothalamic-pituitary adrenal (HPA) axis by inflammation related to infections is primarily mediated by the action of inflammatory cytokines on the hypothalamic releasing factors. Cytokines like Interleukin-6 (IL-6), Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) stimulate the secretion of corticotrophin releasing hormone (CRH) from the hypothalamus leading to corticotrophin (ACTH) secretion from the pituitary. The action of ACTH on the adrenal cortex leads to secretion of both cortisol and dehydroepiandrosterone (DHEA). Cortisol inhibits the T- lymphocyte mediated Th1 response while DHEA antagonizes the cortisol action on Th1 response. This intense immune-endocrine response to acute infection leads to early mobilization of the immune cells and a robust immune response by the host against the offending pathogen (4, 5).

 

However, in TB the chronic persistent activation of the immune-endocrine axis leads to misuse of the immune-endocrine axis and can exacerbate damage to the host. Primarily the prolonged activation of the HPA and resultant increase in glucocorticoid (GC) secretion leads to a change in the T-lymphocyte response from Th1 to Th2 response (6). Beyond this GCs can interfere with gene expression of certain transcription factors like nuclear factor kappa-β(NF-κβ) (7), inhibit the proliferation of effector T cells and cause an increased rate of apoptosis of the regulatory T cells (8). Clinical studies in patients with TB have shown increased circulating levels of cytokines like IL-6, IL-10, interferon-α (IFN-α) and cortisol. However, DHEA levels have been consistently shown to be well below normal levels. In summary, GCs appear to have an adverse effect on the anti-TB immune response while DHEA appears to have a favorable effect. This balance is adversely impacted with the chronic inflammation seen in TB.

 

Some of these changes in immune-endocrinology have also been implicated in the morbidity associated with TB. In vitro studies suggest that negative immune response to mycobacterial antigens was associated with increased IL-6 production which in turn was associated with lower body weights among patients with TB. Higher circulating IL-6 was also associated with loss of appetite (9). The increased circulating GCs additionally mobilize peripheral lipid stores and inhibit protein synthesis and favor loss of lean body mass. Hypothalamic CRH secretion also appear to have direct catabolic effects on the body other than its effect mediated through increased GC secretion (10). Among the adipocyte hormones there is a decrease in leptin and increased secretion of adipocytokines in TB. In an acute infection the above adaptive response appears to be useful by directing limited energy stores to the immune response away from the body’s physiological needs. However, in chronic infections like TB these changes lead to a chronic metabolic deficit leading to cachexia which in turn then affects the further ongoing immune response and disease outcome (11, 12).  

 

Some of these alterations in the immune-endocrine axis in M. tuberculosis infection are summarized in Figure 1.

Figure 1. Immune-Endocrine changes in male patients with tuberculosis (TB). Cytokine release by the T Lymphocytes stimulate the production of releasing factors (RFs) particularly Corticotrophin releasing factor (CRF) by the hypothalamus. Increased corticotrophin release from the pituitary is followed by the increased production of cortisol and dehydroepiandrosterone (DHEA). Transforming growth factor beta (TGF-β) which is increased in TB, in turn, inhibits DHEA production by adrenal cells despite corticotrophin related stimulation to produce increased DHEA. Overall, in patients with TB there is a decrease in the adrenal DHEA production in contrast to patients with acute infections. This unbalanced cortisol/DHEA production ratios from the adrenal cortex along with a reduction in testosterone from the testes favor a Th1→Th2 T Lymphocyte immune shift. The action of cytokines and cortisol on the adipose tissue leads to reduced amounts of leptin production. Leptin is also an immune-stimulant. TB patients also display an increased production of growth hormone (GH) and prolactin probably related to the protracted inflammation, in addition to augmented levels of thyroid hormones via an increase in the pituitary production of thyroid stimulating hormone (TSH). However, despite an increase in TSH there is no change in Free T4 and a decline in Free T3 hormones because of the inhibitory effect of TGF- β This overall pattern is responsible for anorexia, low food intake, lipid mobilization, decrease in protein synthesis which all contribute to the state of cachexia seen in patients with advanced TB (adapted from D’Atillio et al) (12).

ENDOCRINOPATHIES IN PATIENTS WITH TUBERCULOSIS        

 

Though some degree of endocrine dysfunction is invariable in all patients with TB, clinically significant endocrinopathies other than glucose intolerance is rare. In a small study of 50 patients hospitalized with sputum-positive pulmonary TB in South Africa the commonest endocrine dysfunction noted was a low free T3 state as part of sick euthyroid syndrome in over 90% of patients. The other common endocrine dysfunction noted in the study was a 72% prevalence of hypogonadotropic hypogonadism among male patients and a 64% prevalence of hyponatremia of whom almost half of them (17/50) had documented syndrome of inappropriate diuresis (SIAD). No patients in this study had clinically significant adrenal insufficiency and one patient had hypercalcemia (13).

 

In disseminated TB, seeding of the various endocrine glands with mycobacteria and formation of tubercules is common. In an autopsy study performed in over 100 patients who succumbed to disseminated TB done in the eighties, 53% had involvement of the adrenals, 14% had seeding into the thyroid gland, 5% had direct involvement of the testes, and 4% had seeding into the pituitary gland. Among these 100 patients only one had antemortem clinical adrenal insufficiency (14).

 

The full spectrum of possible endocrine abnormalities seen with tuberculosis is summarized in Table 1.

 

Table 1. Endocrine Abnormalities Seen with Mycobacterium Tuberculosis Infection and with Anti-Tubercular Therapy

Hypothalamus

Diabetes Insipidus

Pituitary

1.     Sellar mass lesion

2.     Tuberculous abscess

3.     Sellar Tuberculoma

4.     Thickened stalk with pituitary interruption syndrome

5.     Isolated hyperprolactinemia

6.     Incidental partial or complete hypopituitarism 

7.     Isolated hypogonadotropic hypogonadism

8.     Pituitary dysfunction seen with Tuberculous meningitis

Thyroid

1.     Tubercular thyroiditis

2.     Cold abscess of the thyroid

3.     Chronic fibrosing thyroiditis

4.     Sick euthyroid syndrome

5.     Para-amino-salicylic acid (PAS) related goiter

6.     Ethionamide and rifampicin related thyroid dysfunction

Parathyroid

Inflammation

Pancreas

1.     Stress hyperglycemia

2.     Frank diabetes

3.     Pancreatic abscess 

Testes

1.     Isolated TB orchitis

2.     TB epididymitis

3.     Epididymo-orchitis 

4.     Primary gonadal failure

Ovaries

1.     Tubo-ovarian abscess

2.     Tubal blockage

3.     Unexplained infertility

Water Metabolism

1.     Hyponatremia

2.     Syndrome of inappropriate anti-diuresis (SIAD)

3.     Cerebral salt wasting (CSW)

Vitamin D-Calcium Metabolism

1.     Parathyroid hormone independent hypercalcemia

2.     Vitamin D deficiency/hypocalcemia related to isoniazid and rifampicin

Adrenals

1.     Tubercular adrenalitis

2.     Addison’s disease

3.     Reversible adrenal insufficiency

4.     Isolated DHEA deficiency 

 

In this chapter we will review endocrine dysfunction and endocrinopathies associated with TB infection related to the adrenal, thyroid and pituitary glands. Additionally, functional derangement of sodium, and calcium homeostasis will be covered. Glucose intolerance, diabetes and tuberculosis is a large area of public health and will not covered in this chapter.

 

ADRENALS AND TUBERCULOSIS

 

TB can involve both adrenal glands primarily or the involvement may be part of disseminated TB. Both conditions may present with primary adrenal insufficiency (Addison’s Disease). Anti-tuberculous therapy (ATT)-related enzyme induction abnormalities can also lead to adrenal dysfunction and in some cases unmask subclinical adrenal insufficiency. Chronic steroid therapy used in the treatment of some types of tuberculous infection can lead to suppression of HPA axis and secondary adrenal insufficiency. Finally, it is important to remember that pituitary involvement in central nervous system (CNS) TB can sometimes lead to isolated corticotropin deficiency with adrenal insufficiency or it can be part of generalized hypopituitarism 

 

Tuberculosis and Addison’s Disease

 

Thomas Addison in 1855 first described chronic adrenal failure, or Addison’s disease (AD), due to Mycobacterium tuberculosis infection involving both the adrenal glands. In his paper describing AD, 6/11 patients had tuberculous involvement of the adrenal glands. In 1930, Guttman reported a large series of 566 cases with AD, of which 70% was due to tuberculous adrenalitis (15). In 1956 only 25% of AD was related to TB infection (16). The decreasing incidence of tubercular adrenal failure in Western literature was highlighted in a recent large study of 615 cases of AD from Italy in 2011; in this series only 9% of cases were due to TB (17).

 

This decline in the number of patients with AD related to TB has not been seen in countries endemic for TB like India and South Africa. In India, tuberculous etiology was found in 47% of patients with AD, and of them 85% had enlargement of one or both adrenal glands on imaging (18). The differences between AD due to TB and those with idiopathic AD is summarized in Table 2. In South Africa, 32% of patients with AD had tubercular etiology (19). The most common cause of AD worldwide, however, is autoimmune adrenalitis.

 

Table 2. Differences in Clinical Presentation of Tubercular Addison’s Disease (AD) versus Idiopathic AD (18)

Clinical Features

Tubercular

Idiopathic

p-value

Mean age (in years)

42

35

NS

Durations of symptoms before diagnosis (in months)

14

21

NS

Sex Ratio (M: F)

10:1

14:8

< 0.05

Presentation as crisis (%)

40%

23%

NS

Evidence of other autoimmune disease (%)

10%

27%

< 0.05

Evidence of extra-adrenal TB (%)

55%

9%

< 0.05

Adrenal Cytoplasmic Antibodies (%)

17%

50%

< 0.05

 

PATHOPHYSIOLOGY OF TUBERCULAR ADRENALITIS            

 

Adrenal TB develops from hematogenous or lymphatic spread, hence is often associated with extra-adrenal infection. The rich vascularity of the adrenal gland and high levels of local corticosteroids that suppress cell mediated immunity create an ideal microenvironment for the growth of Mycobacterium tuberculosis (20). Adrenal involvement can be found in up to 6% of patients with active TB, however isolated adrenal involvement is seen only in a fourth of these (1.5-3% of cases with tubercular infection) (21, 22). Clinical manifestations of AD appear only after 90% of the adrenal cortices have been compromised (23).

 

The patterns of adrenal gland involvement in TB are summarized below and in Figure 2 (24):

  1. Chronic infection of the adrenal gland, with clinical manifestations of primary adrenal insufficiency appearing years after initial infection. Pathologically these patients have small atrophic fibrous glands with or without calcification.
  2. Isolated adrenal gland involvement early in the course of disease usually within 2 years of the primary infection. Pathologically these patients most commonly present with bilateral adrenal enlargement because of mass lesions secondary to production of cold abscesses within the adrenal glands. Milder enlargement can be seen in patients with extensive granulomas within the adrenal gland. Lastly, patients with isolated adrenal tuberculosis may also present with normal sized glands with granulomatous inflammation seen microscopically. Calcifications maybe seen in these cases as well.
  3. Secondary adrenal insufficiency due to prolonged steroid therapy in disseminated TB or tubercular involvement of the pituitary or hypothalamus.
  4. Subclinical steroid deficiency unmasked by ATT-related enzyme induction.

Figure 2. Mechanisms of adrenal insufficiency with tuberculosis. Both primary adrenal failure and secondary adrenal insufficiency are possible. The presentation of primary adrenal failure can be both acute and chronic. In patients with acute presentation usually within 2 years of tuberculous infection, the pathological presentations could be one of the three noted. Chronic primary adrenal failure is pathologically defined by atrophic and fibrosed glands. [ATT-Anti-tuberculous therapy

CLINICAL FEATURES

 

Adrenal TB can be found in any age, however is more commonly seen in adults. Rare cases have also been described in the pediatric age group (25, 26). Thomas Addison’s first description of AD showed a constellation of symptoms like “general languor and debility, remarkable feebleness of heart’s action, and a peculiar change in the color of the skin.” Classic manifestations of AD in the form of malaise or fatigue, anorexia, weight loss, nausea, vomiting, muscle and joint pain, orthostatic hypotension, skin hyperpigmentation and salt craving are often present. Mineralocorticoid deficiency leads to postural hypotension, while hyperpigmentation occurs due to activation of the melanocortin 1 receptors (MC1R) in turn because of high ACTH levels (27). In some patients, however, hyperpigmentation can be absent due to reduced stimulation of MC1R from adrenocorticotropin hormone (ACTH), resulting in an alabaster-like appearance (27). A prior history of TB may also be provided in some patients.

 

RADIOLOGIC FINDINGS

 

Computed tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET) are useful non-invasive tools in the diagnosis of adrenal TB. CT has been regarded as the modality of choice for diagnosing adrenal TB, and should include both non-contrast and contrast-enhanced techniques. Adrenal involvement is usually bilateral (28), and findings vary according to course of disease.

 

  • Early stage: In the first two years, CT shows noncalcified enlarged adrenals with areas of lucency reflecting caseous necrosis, and a peripheral rim of contrast-enhanced parenchyma. Contours of the adrenal glands are generally preserved.
  • Late stage: As disease progresses, the adrenals normalize in size, then shrink, and have calcific foci with irregular margins. Calcifications are best visualized on non-contrast CT scans. These may be either diffuse, focal or punctate in nature (22). These findings correlate with long-standing fibrosis and dystrophic calcification seen with tuberculous granulomas.

 

LABORATORY FINDINGS

 

Common findings in patients with adrenal TB include hyponatremia, hyperkalemia and normochromic anemia (27). Hyponatremia can occur due to decreased inhibitory control of vasopressin secretion, resulting in mild SIAD (29). The Mantoux (tuberculin) test usually is strongly positive and erythrocyte sedimentation rate (ESR) is elevated.

 

  • In primary AD, baseline serum ACTH levels are higher than 100 pg/ml, plasma renin levels are elevated and serum aldosterone levels are low. In secondary AD, ACTH levels will be low or inappropriately normal, while mineralocorticoid secretion will be normal (27).
  • Serum DHEAS will also be low in patients with both primary and secondary AD (27).
  • Adrenal insufficiency can be demonstrated by low morning plasma cortisol with a reduced response to synthetic ACTH (27). Impaired ACTH-stimulated cortisol responses have also been observed with lower baseline cortisol levels, and both higher and lower cortisol responses to ACTH stimulation in patients with isolated pulmonary TB without adrenal involvement (24). Injectable tetracosactide hexa-acetate, ACTH 1-24 (Synacthen®) (SST), is not marketed or easily available in many developing countries in the world including India. An alternative ACTH which is injectable long-acting porcine sequence, ACTH 1-39 (Acton Prolongatum®) (APST), is easily available and much cheaper. In a study done recently in India by Nair et al in 20 patients with established adrenal insufficiency and 27 controls, the area under the curve of APST (at 120 min) was 0.986 when compared to the standard SST, thus proving its high accuracy. A serum cortisol cut off value of 19.5 µg/dL at 120-min following APST showed a sensitivity of 100% and specificity of 88% (30).

 

PATHOLOGY OF ADRENAL TUBERCULOSIS

 

Macroscopic involvement of the adrenal gland can be seen in up to 46% of patients with adrenal TB. Bilateral involvement is seen in nearly 70% of patients, however they may not be equally affected. Mean combined weight of adrenal gland ranges from 10-37 gm (mean 17 gm) (31). Caseous necrosis can be seen grossly within a large cavity or within multiple scattered tubercles.

 

Histopathologically, destruction of both the cortex and the medulla is seen with the following patterns of adrenal gland involvement (24):

  1. Presence of granulomas, with or without necrosis. The granulomas show epithelioid cell collections with typical Langhan’s giant cells and an admixture of lymphocytes and plasma cells. Ziehl-Neelsen stain is very useful for detecting acid fast bacilli (AFB) within the necrotic areas, as well as within the granulomas.
  2. Glandular enlargement with destruction of parenchyma by necrotizing granulomas.
  3. Mass lesion secondary to formation of cold abscesses. In these cases, CT-guided fine needle aspiration cytology (FNAC) is helpful for demonstration of AFB, polymerase chain reaction (PCR), and culture for Mycobacterium tuberculosis In most cases, a combination of histopathology, PCR and culture may be required to confirm the diagnosis.
  4. Adrenal atrophy secondary to fibrosis resulting from long-standing tuberculous infection (32).

 

DIFFERENTIAL DIAGNOSIS

 

The differential diagnosis for adrenal enlargement includes primary or metastatic tumors, lymphomas, fungal infections like cryptococcus and histoplasma, amyloidosis, sarcoidosis, hemangiomas and adrenal cortical hyperplasia (24, 28). Tissue sampling for microbiological (PCR and culture) and pathological analysis should adequately distinguish between them.

 

TREATMENT

 

Treatment for active adrenal TB is similar to the regimen followed for extrapulmonary TB with use of multidrug ATT. Rifampicin induces hepatic enzymes that increase the metabolism of glucocorticoids; hence higher doses of replacement glucocorticoids may be required. Rarely, Rifampicin may trigger an adrenal crisis.

 

In cases of chronic disease, adrenal gland function is unlikely to recover due to massive destruction of the gland (20, 28). However, a few authors report improvement in adrenal function when patients are given ATT early in the course of disease (33-35). This may be in part due to the remarkable regenerative capacity of the adrenal cortex to undergo hyperplasia and hypertrophy during active infection (20).

 

Hormone replacement for primary and secondary adrenal insufficiency related to TB follow the same principles as autoimmune or idiopathic primary AD or secondary adrenal insufficiency. In addition to appropriate glucocorticoid replacement mineralocorticoid replacement may be required. Care must be taken to educate patients about stress dosing and the need for parenteral steroids when the patient may not be able to take or absorb oral glucocorticoids. 

 

THYROID AND TUBERCULOSIS

 

The thyroid gland is an uncommon site for infection by M. tuberculosis. Thyroid TB (TTB) is therefore very rare, even in places with a high prevalence of TB. The primary presentation of TTB is as a mass or a goiter. Overt hormonal dysfunction is very uncommon in TTB. However, in patients with tuberculosis affecting any organ clinically insignificant abnormalities in thyroid function tests are very common. ATT also causes both structural and functional thyroid dysfunction. Pre-operative diagnosis of TTB can be made only with a high index of suspicion while evaluating thyroid nodules especially in communities with a high prevalence of TB (36).

 

Epidemiology

 

  1. tuberculosis has been documented to be involved in the thyroid gland of 0.1 to 1% of patients who underwent thyroid tissue sampling for any indication (37-39). In an autopsy series of patients with advanced disseminated TB occurring in the pre- and post-antibiotic era, 14% had evidence of thyroid gland involvement (14). In a large cohort of 2,426 patients from Morocco, only eight had evidence of TB (0.32%). These were in the form of goiter or as a solitary thyroid nodule. In a study from India, thyroid involvement has been seen in 0.43% of specimens obtained from FNAC (40), while among Turkish patients undergoing thyroidectomy, 0.25 - 0.6% showed thyroid involvement by M. tuberculosis (41, 42).

 

Pathogenesis

 

Thyroid involvement in TB is very uncommon. A few postulated intrinsic properties of the thyroid which are proposed not to allow Mycobacterium tuberculosis bacilli to survive include (24, 36):

  • Presence of iodine-containing colloid possessing bacteriostatic activity.
  • High blood flow within the thyroid gland with the presence of intracellular iodine.
  • Increased phagocytosis within the gland, seen in hyperthyroidism.
  • Rich lymphatic supply to the thyroid.
  • Thyroid hormones themselves exercise anti-TB roles.

 

TTB can be primary or secondary

  1. Primary TTB is involvement of the thyroid gland alone, with no evidence of TB elsewhere in the body.
  2. Secondary TTB is usually the result of hematogenous, lymphatic and/or direct spread from an active tubercular focus involving the cervical lymph nodes or larynx. Secondary TTB is much more commonly encountered than primary TTB, and TTB may go undiagnosed in many cases especially where clinical signs are non-specific (24).

 

Clinical Features

 

TTB occurs slightly more commonly in women as compared to men (M: F = 1:1.4) and occurs over a wide age range of 14 to 83 years, median age of 40 ± 16 years for men and 43 ± 17 years for women (36).

 

TTB can manifest as a localized swelling with cold abscess mimicking carcinoma, as multinodular goiter, as a solitary thyroid nodule without cystic component, or very rarely as an acute abscess. The various presentations are summarized in Figure 3. Rarely TTB may present as a goiter or a chronic fibrosing thyroiditis. Presence of cervical lymphadenopathy may raise suspicion of malignancy (36). Clinical presentation is often subacute, but may be acute in cases of abscess (43). Pain associated with swelling, thyroid tenderness, fever and localized extra-thyroidal findings such as dysphagia, dysphonia or recurrent laryngeal nerve palsy are less common in TTB as compared to patients with acute bacterial thyroiditis (24). However, some patients with TTB may present with pyrexia of unknown origin. Table 3 documents with differences in clinical presentation between TTB and bacterial thyroiditis.

Figure 3. Presentations of thyroid tuberculosis (TTB). Relatively common presentations in green and rarer ones in yellow and orange colors.

 

Table 3. Clinical Features that Help Differentiate Between Tuberculous Thyroiditis and Bacterial Thyroiditis

Clinical Features

Tuberculous Thyroiditis

Bacterial Thyroiditis

Pain

-

+++

Pyrexia

+1

+++

Duration of illness (mean duration) (Ref;24)

105 days

18 days

Dysphagia

++

+++

Dysphonia

++

+++

Recurrent Laryngeal Nerve Palsy (Hoarseness)

++

+++

History of previous thyroid illness

-

+

Tenderness over the gland

-

+++

Leukocytosis

-

++

Elevated Erythrocyte Sedimentation Rate (ESR)

+++

+

1Rare reports of presentation as pyrexia of unknown origin

 

Most patients with TTB are euthyroid and do not have pre-existing thyroid disease. Very rarely TTB can be associated with hypothyroidism, with a period of subclinical hyperthyroidism preceding the hypothyroidism (44). Myxedema can occur in cases with extensive destruction of the thyroid gland by disseminated TB, which can also be fatal (45). Past history of TB may be elicited in some cases, and patients may have history of cervical lymphadenopathy (43).

 

Radiological and Laboratory Findings

 

Chest X-ray, ESR, and tuberculin skin test should be performed in all cases of suspected TTB. The diagnosis is made only after FNAC or histopathological examination of the surgical specimen when FNAC is negative (43). Sputum AFB may rarely assist in diagnosis in cases with associated pulmonary TB.

 

Ultrasonography usually shows a heterogenous, hypoechoic mass similar to a neoplastic nodule. Anechoic areas with internal echoes may be seen in abscesses. Contrast-enhanced CT scan can determine the location of the necrotic lesions (46).

 

On MRI the normal thyroid is homogenously hyperintense relative to the neck muscles on both T1 and T2-weighted images. TTB may show intermediate signal intensity due to granulomatous inflammation, however, this appearance is also seen in thyroid carcinoma. Abscesses appear hypointense on T1 and hyperintense on T2-weighted images, and may show peripheral rim of contrast enhancement (47).

 

Thyroid function tests (TFT) are usually normal in patients with TTB. Thyrotoxicosis in the initial stage of rapid release of thyroid hormone, and myxedema in the later stage of thyroid gland destruction have also been noted, and patients may have abnormal TFT accordingly (24). Only 5.2% of patients with TTB have abnormal TFT (36).

 

Pathology of Thyroid TB

 

In most cases, TTB can be diagnosed on FNAC which typically shows epithelioid cell granulomas with Langhan’s giant cells, peripheral lymphocytic infiltration and purulent caseous necrosis. The yield of AFB by the Zeihl Neelsen stain is more with FNAC samples than in biopsies. The aspirates can be sent for TB culture or PCR. TB-PCR is much more sensitive in detecting M. tuberculosis deoxyribonucleic acid (DNA) from FNA samples, and is an alternative to rapid diagnosis of TB in AFB-negative cases (40). The diagnosis is substantiated by histopathology which typically shows granulomas, Langhan’s giant cells and necrosis (Figure 4). Few cases show dense lymphocytic infiltrate with prominent germinal centers, resembling lymphocytic or Hashimoto thyroiditis (Figure 5).

Five pathological varieties of TTB have been described (36):

  1. Multiple miliary lesions throughout the thyroid gland
  2. Goiter with caseation necrosis
  3. Cold abscess
  4. Chronic fibrosing tuberculosis
  5. Acute abscess

Fig 4. Case of TTB showing granulomas within the thyroid parenchyma comprised of epithelioid cells with Langhan’s giant cells (yellow arrows) and foci of necrosis (black arrows). Hematoxylin and eosin, 100x.

Figure 5. Case of TTB showing dense lymphocytic infiltrate with prominent germinal centers in the thyroid parenchyma (arrow). Hematoxylin and eosin, 100x.

 

Differential Diagnosis

 

TTB, although rare, should be considered in the list of differentials for solitary or multinodular thyroid nodules, and abscesses (36). Reidel’s thyroiditis may mimic chronic fibrosing tuberculosis clinically, however histopathology clinches the diagnosis (42).

 

Treatment

 

ATT remains the cornerstone of treatment. Surgery has a limited role with drainage of abscess, avoiding total destruction of gland and subsequent hypothyroidism. However inadvertent total thyroidectomies are performed as the pre-operative diagnosis is commonly a malignancy. In cases were TB was diagnosed prior to surgery, ATT is well tolerated with resolution of symptoms, reduction in thyroid mass symptoms, and with favorable reversal of thyroid hormonal dysfunction. Standard ATT schedules are followed. Thyroid hormone levels should be monitored before, during, and after treatment. Despite strict ATT, recurrence and failure rate is 1% due to resistance to ATT drugs (48).

 

Functional and Structural Alterations of Thyroid Functions with Active Tuberculosis and with Anti-tubercular Therapy

 

Among hospitalized patients with TB without any evidence of involvement of the thyroid gland sick euthyroid syndrome with low free T3 is common. The estimates vary between 63-92% and probably is the commonest endocrinopathy seen in patients with TB (13,49). As with other unwell patients the degree of reduction in Free T3 serves both as a marker for severity of the disease and mortality. In the study by Chow et al, all patients who survived the hospitalization had normal TFT within one month of initiation of ATT. In community dwelling patients with TB, the prevalence of thyroid dysfunction is unclear.

 

Thyroid hormones are metabolized in the liver and the kidneys. In the liver, the enzyme CYP3A4 belonging to the hepatic cytochrome P450 family is responsible for the metabolism. Rifampicin is a potent activator of the P450 system and this leads to an increase in T4 turnover. In most adults with normal a hypothalmo-pituitary-thyroid axis this increase in turnover is compensated by an increase in the production of thyroid hormones and a slight increase in thyroid volume. This may be noted biochemically as a slight increase in free T3 and total T3 levels after rifampicin administration. There are no changes in free T4 and TSH concentrations (50). Among patients with pre-existing thyroid disease with a limited capacity to increase production of thyroid hormones, the rifampicin-mediated increase in free T4 turnover might lead to the need for an increase in thyroid hormone replacement therapy. In a retrospective cohort of patients on levothyroxine replacement therapy, the addition of rifampicin as part of ATT led to a need for a 26% increase in dose in patients on thyroid hormone replacement therapy and a 50% increase in patients on suppressive therapy post thyroidectomy for differentiated thyroid cancer (51).

 

Older anti-tubercular agents have more profound effects on thyroid physiology. Studies by Munkner et al demonstrated an association between the use of p-amino salicylic acid (PAS) and the development of goiter (52). PAS and ethionamide were also associated with significant risk of developing hypothyroidism (53, 54). However, these agents are currently not used as first line agents. It is prudent to monitor TFTs 6-8 weeks after initiation of any of these three agents in patients who have pre-existing thyroid dysfunction.

 

PITUITARY AND TUBERCULOSIS

 

Direct involvement of the pituitary gland by Mycobacterium Tuberculosis is very rare. Some of the earliest published reports of pituitary TB include von Rokitansky who noted tubercles in the hypophysis as early as 1844, Letchworth in 1924 who reported a case of primary pituitary tuberculoma on autopsy examination, and Coleman and Meredith documented a case of pituitary TB in 1940 (55, 56).

 

The spectrum of involvement (Figure 6) of the pituitary gland with TB includes sellar, parasellar, and stalk tuberculomas and sellar tubercular abscesses. Patients with tuberculous meningitis exhibit a range of functional pituitary dysfunction even in the absence of any evidence of direct invasion/extension of the disease into the sella. Among survivors of tubercular meningitis hypopituitarism was noted 10 years after the primary disease. Hypothalamic pituitary dysfunction such as isolated hypogonadotropic hypogonadism may accompany cachexia and weight loss that can complicate more extensive disease. Infiltrative tubercular disease of the stalk can produce pituitary interruption syndrome including isolated diabetes insipidus and hyperprolactinemia.

 

In most cases the diagnosis of tuberculosis of the pituitary is established on histopathology, often in the absence of confirmatory culture studies or positive acid-fast stains (57). Although the diagnosis is difficult on clinical and radiological examination, pituitary TB should be considered in the differential of a suprasellar mass especially in developing countries, as the condition is potentially curable with ATT (58, 59).

 

Figure 6. Spectrum of structural and functional disease of the pituitary seen with tuberculosis

Sellar Tuberculoma/Abscess

 

EPIDEMIOLOGY  

 

The incidence of pituitary TB is very low. In an autopsy series of 3,533 cases, only 2 of 89 intracranial tuberculomas involved the sella turcica, while in another autopsy series of 14,160 cases, only 2 cases of TB were encountered involving the anterior pituitary lobe (50).  In patients with late generalized TB, the incidence of pituitary involvement is 4% (14). Nearly 70% of pituitary TB reported worldwide has been reported from the Indian subcontinent, probably attributable to the higher prevalence of TB in this location (60). In the largest series from India, 18 cases of sellar TB were diagnosed based on histopathology from 1148 pituitary surgeries (60).

 

PATHOGENESIS

 

Pituitary TB can arise either from hematogenous seeding, in the presence or absence of miliary disease, or from direct extension from the brain, meninges or sinuses. TB can either involve the pituitary gland alone, or involve the adjacent and/or distant organs as well (60). Both the adenohypophysis and neurohypophysis may be involved by TB. Supra-sellar extension is common in pituitary TB with only rare cases confined to the sella (57). 

 

CLINICAL FEATURES

 

Pituitary TB occurs at a mean age of 34.1 ± 13.6 years (age range 6 to 68 years), and is more common in women (F:M = 2.7:1). Young children are at high risk of progression of TB including CNS disease. Clinical presentation is often indolent. Duration of symptoms average 4 months (60, 61).

 

Pituitary involvement, either as a sellar abscess or tuberculoma presents primarily with symptoms of a sellar mass. The common presentations clinically are gradual onset of headache (85.2%), visual loss (48.1%) (Figure 7), seizures and cranial nerve palsies. Patients with infiltration of the stalk by tuberculomas may present with central diabetes insipidus with polyuria (8.6%) or menstrual abnormalities related to hyperprolactinemia like amenorrhea in women (37.3%) and galactorrhea (23.7%) (60, 61). Growth retardation and hypogonadism are rare findings in children with pituitary TB (61). Hyperphagia resulting in obesity or weight gain has also rarely been documented which may occur due to the loss of sensitivity of the appetite-regulating network in the hypothalamus to afferent peripheral humoral signals (62). Apoplexy, characterized by acute infarction and/or hemorrhage in the pituitary gland, is an uncommon presentation of pituitary TB (63). Systemic and constitutional symptoms may or may not be present; low grade fever may be seen in 14.8% of patients. Other organs may show evidence of TB in 26.9% (60, 64). Tuberculous meningitis may be associated in a few cases.

Figure 7. Bitemporal hemianopsia demonstrated on perimetry.

RADIOLOGICAL FINDINGS

 

The diagnosis of primary pituitary TB is challenging and often difficult. Radiologically pituitary TB can mimic pituitary adenoma, arachnoid cyst, pyogenic abscess, metastasis, or craniopharyngioma. MRI typically shows a sellar mass which may extend into the suprasellar region, involving the optic nerves and inter-carotid space (Figure 8). T1-weighted MR images appear isointense. T2-weighted images show central hyperintensity corresponding to caseous necrosis, and gadolinium contrast imaging may show thick ring enhancement in the periphery with central hypointense areas. Meningeal enhancement with enhancement of the thickened pituitary stalk may favor non-adenoma etiology. Additional findings like sellar/suprasellar calcification and sellar floor erosion have also been described (57, 63-65).

Figure 8. Magnetic resonance imaging of a patient with pituitary tuberculosis shows a sellar mass lesion measuring 2.1 cm x 1.9 cm x 1.4 cm with suprasellar extension A) heterogenous predominantly increased signal intensity on T2 weighted imaging and B) hypointense on T1 weighted imaging. C and D) Significant homogenous post contrast enhancement of the mass lesion on axial (C) and sagittal (D) views, respectively. Involvement of the pituitary stalk and superior displacement of the optic chiasma is also seen. Bright signal of posterior pituitary is maintained.

MR spectroscopy can detect elevated lipid peaks in a tuberculoma at 0.9, 1.3, 2.0 and 2.8 ppm, and a phosphoserine peak at 3.7 ppm. Lipid resonance at 0.9 and 1.3 ppm occur due to methylene and terminal methyl groups on fatty acids found in caseous necrosis (66)

 

LABORATORY FINDINGS

 

Panhypopituitarism may be encountered on evaluation of anterior pituitary hormones like thyroid stimulating hormone (TSH), early morning cortisol, growth hormone, prolactin, luteinizing hormone (LH) and follicle-stimulating hormone (FSH).

 

Testing for HIV and immunocompromised states should be considered in the appropriate clinical setting. Positive tuberculin test and elevated ESR may be seen in patients with systemic involvement.

 

PATHOLOGY OF PITUITARY TUBERCULOSIS

 

The most common pattern of tuberculous involvement of the pituitary on histopathological examination is granulomas without caseation necrosis (59.6%) as show in Figure 9A. Granulomas may be miliary or may coalesce together to form a conglomerate mass. Reticulin stain helps demonstrate loss of normal reticulin pattern of the pituitary (Figure 9B). The granulomas are composed of epithelioid cells, Langhan’s giant cells, and lymphocytes (Figure 9C-D). Immunohistochemistry (IHC) with CD68 can help confirm the presence of epithelioid histiocytes in cases of unequivocal morphology, while CD3, CD20 and CD138 can highlight a mixture of T-lymphocytes, B-lymphocytes and plasma cells, respectively (Figure 10A-D). Pus and caseation necrosis are seen less commonly, and in these cases the yield of AFB is greater than with cases without necrosis. As with other sites, demonstration of AFB on biopsy material is very low. In such cases growth of M. Tuberculosis organisms on culture and TB PCR aid in diagnosis.

Figure 9. Pituitary tuberculosis. Biopsy of the pituitary showing nests of pituicytes (black arrows) destroyed and separated by confluent granulomas (red arrow). A) low power view, Hematoxylin and Eosin (H&E), 100x; B) corresponding area showing reticulin-free zones (asterisks) occupied by granulomas, Gordon and Sweet’s silver reticulin stain, 100x; C & D) higher power views showing non-caseating granulomas comprised of epithelioid cells with occasional Langhan’s giant cells (top left) and lymphocytes, H&E, 400x.

Figure 10. Immunohistochemistry in pituitary tuberculous granulomas shows mixed inflammatory infiltrate made up of epithelioid histiocytes (CD68), T-lymphocytes (CD3), B-lymphocytes (CD20) and occasional plasma cells (CD138) (A-D, respectively). Diamino benzidine chromogen, 100x.

DIFFERENTIAL DIAGNOSIS

 

Sarcoidosis must be considered in the differential of non-caseating granulomatous hypophysitis, and shows naked granulomas without infiltrating lymphocytes. IgG4-related disease typically shows increase in plasma cells of the IgG4 subtype with storiform fibrosis. Histiocytic lesions like Langerhans cell histiocytosis (LCH) usually involve the infundibulum and typically show presence of Langerhans’s histiocytes with eosinophils. LCH is more common in children and young adults. Other inflammatory lesions involving the pituitary stalk are lymphocytic infundibuloneurohypophysitis (LINH), Wegener’s granulomatosis, and pituitary stalk parasitosis (67). Fungal granulomas involving the hypophyseal region can be ruled out by performing fungal stains on tissue sections (60).

 

TREATMENT

 

Transsphenoidal approach is preferred for surgery and is used for diagnosis and decompression of adjacent structures. Typical intra-operative findings are firm to hard, non-suckable greyish tissue with thickening of the dura. Pituitary TB can be managed conservatively if the diagnosis is confirmed with cerebrospinal fluid TB PCR and other tests.

ATT may be given for up to 18 months and patients should be on periodic follow up with assessment of hormonal profile. Lifelong replacement of hormones may be required in some patients (68). Recurrence of TB in lymph nodes despite completion of 18 months of ATT has been reported to occur due to resistance of M.tuberculosis bacilli to Rifampicin (69).

 

Pituitary Dysfunction in Patients with Tuberculous Meningitis (TBM)

 

In an Indian study of 75 patients with tuberculous meningitis, common pituitary functional abnormalities included hyperprolactinemia (49%), cortisol insufficiency (43%), central hypothyroidism (31%) and multiple hormone deficiencies (29%) (70). Prevalence of functional pituitary abnormalities seen in TBM in multiple studies from India is summarized in Table 4 (71, 72).  In addition, there may be hyponatremia.

 

Table 4. Pituitary Involvement in Patients with Tuberculous Meningitis

 

Delhi (70)

Chandigarh (71)

Lucknow (72)

Number of patients

75

63

115

Any Involvement of Pituitary

 

84.2%

53.9%

Single Axis Involvement

 

39.8%

30.4%

More than one axis (Panhypopituitarism)

29.3%

44.4%

23.5%

Hypogonadotropic Hypogonadism

NR

38.1%

33.9%

Hyperprolactinemia

49.3%

49.2%

22.6%

Secondary Adrenal insufficiency

42.7%

42.9%

13%

Central hypothyroidism

30.7%

9.5%

17.4%

Isolated Growth hormone deficiency

NR

NR

7.8%

Syndrome of Inappropriate anti-diuresis 

NR

NR

9.%

Diabetes Insipidus

Nil

Nil

Nil

NR- Not reported

 

Even among patients who survive tuberculous meningitis, pituitary dysfunction may persist. A study done by Lam in Hong Kong showed growth hormone deficiency to be the most common finding in patients younger than 21 years of age with tuberculous meningitis after 10 years of surviving tuberculous meningitis (73). 

 

WATER IMBALANCE AND TUBERCULOSIS

 

Hyponatremia has been commonly seen in patients admitted to the hospital with TB. Though data about the prevalence of hyponatremia among community treated patients with uncomplicated pulmonary TB is sparse, among inpatients admitted with TB, hyponatremia has been seen in 10-76% of patients (74-78). The commonest cause of hyponatremia is the SIAD. Other causes include untreated primary or secondary adrenal insufficiency, volume depletion, hyponatremia associated with volume excess, and hypoalbuminemia and rare cases of cerebral salt wasting seen with tuberculous meningitis (79) (Figure 11). Hypernatremia is rarely encountered and usually signifies involvement of the hypothalamus or the pituitary stalk leading to diabetes insipidus.

Figure 11. Causes of hyponatremia in patients with Tuberculosis. [SIAD-Syndrome of inappropriate anti-diuresis]. Green boxes are common causes, yellow is less common and the red boxes are rare.

Syndrome of Inappropriate Antidiuresis (SIAD)

 

In the absence of adrenal deficiency, patients with non-CNS TB who are adequately hydrated (euvolemic) the hyponatremia is almost always a consequence of retention of free water despite low serum osmolality (inappropriate antidiuresis). Wiess and Katz first noted the association between active untreated TB and syndrome of inappropriate antidiuresis (SAID). In four patients with active TB and hyponatremia they noted excessive urinary sodium excretion. When these four patients were put on fluid restriction there was an improvement in the serum sodium levels. All patients who survived also had gradual normalization of serum sodium levels and SAID with treatment of TB (80).

 

Three different mechanisms have been proposed for the development of SIAD in patients with tuberculosis without evidence of adrenal involvement.

  1. The first proposed mechanism in common with other pulmonary diseases is the stimulation of baroreceptors by chronic hypoxemia that can accompany extensive pulmonary TB. There is release of anti-diuretic hormone (ADH) in response to baroreceptor stimulation which leads on to SIAD (81).
  2. The second possible mechanism proposed is a shift of the “osmostat” towards the left as seen in patients with decreased effective circulating volume leading to ADH release at lower serum osmolality. Investigators have noted higher circulating ADH levels in the serum despite hyponatremia which subsequently declined when free water was administered. The intact response to hypoosmolality suggested that the osmoregulation set up in the hypothalamus was functioning normally but at a lower osmolar threshold for ADH release (82).
  3. The third mechanism proposed is the ectopic secretion of ADH by the tubercular granuloma. This mechanism was proposed by the authors of a case where a patient with well-established diabetes insipidus developed SIAD and hyponatremia after contracting pulmonary tuberculosis (83).

 

Patients with CNS TB have a higher prevalence of hyponatremia compared to those with pulmonary infections. In adult patients with TBM the prevalence of a low sodium state has varied from 45-65% in different studies (84-86). In children with TBM the prevalence varied from 38-71% in different studies (87-89). In children with TBM and hyponatremia there appears to be an association with mortality and increased intracranial pressures (87, 90). A recent review from India compiled data from over 11 studies comprising a total of 642 patients with TBM and found the prevalence of hyponatremia to be 44%. Unlike non-CNS TB the commonest etiology of hyponatremia among patients with CNS TB is cerebral salt wasting (CSW) rather than SIAD (36% vs 26%) (86). The other less common causes of hyponatremia encountered in TBM include the following

  1. Dehydration and hypovolemic hyponatremia due to anorexia, vomiting, nausea and diarrhea
  2. Drug induced including use of diuretics, osmotic agents like mannitol and anti-seizure medications like carbamazepine and phenytoin.
  3. Secondary adrenal insufficiency and rarely primary adrenal insufficiency

 

CLINICAL PRESENTATION AND TREATMENT OF SIAD ASSOCIATED WITH TUBERCULOSIS        

 

Most patients with SIAD and TB are asymptomatic and do not require any treatment. The hyponatremia accompanying SIAD self corrects itself when ATT is started (82). Fluid restriction is only required in symptomatic patients or in patients with severe hyponatremia. Prior to restricting fluids in patients with non-CNS TB it is important to rule out dehydration either by assessing volume status clinically, assessing volume status with urine spot sodium levels, or measuring central venous pressures in unwell patients. In patients with CNS TB, it is important to rule out CSW prior to initiating fluid restriction. Hypertonic saline infusions are limited to patients with life threatening symptoms like seizures and deep coma attributable to hyponatremia (86). Care should be taken to correct hyponatremia at a rate not faster than 8-10 mEq/L in 24 hours to avoid central pontine myelinolysis.  

 

Cerebral Salt Wasting (CSW)

 

CSW refers to changes in renal salt handling that accompanies CNS disorders which leads to natriuresis and hypovolemia. The accompanying dehydration and decrease in effective circulating volume triggers ADH release via baroreceptors. The action of ADH on collecting tubules then leads to selective water resorption and relative water excess and hyponatremia despite overall hypovolemia. The putative renal natriuretic triggers include atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and c-type natriuretic peptide. In TBM the most likely natriuretic trigger is BNP (91). What induces BNP release in patients with TBM is less well characterized. The putative mechanisms that trigger BNP release include sympathoadrenal activation, increase in intracranial pressure, and vasospasm in cerebral arteries (92). In patients with CNS TB statistically CSW is more likely to be the cause of hyponatremia and should always be ruled out prior to labelling them as SIAD and starting fluid restriction. A simple diagnostic criterion proposed by Kalita et al (86) includes meeting all the 3 essential criteria and meeting at least 3 out of 5 additional supportive criteria to label the patient has having CSW. Table 5 lists the clinical and biochemical differences between SIAD and CSW.

 

Essential Criteria (meets all three)

  1. Polyuria (>3 liters of urine in 24 hours over 2 days)
  2. Documented hyponatremia
  3. Exclusion of other cause of natriuresis like adrenal insufficiency, salt losing nephropathies, use of diuretics, hepatic and cardiac failure.

 

Additional criteria (meet 3 out of 5)

  1. Clinical evidence of hypovolemia and dehydration
  2. Documented negative fluid balance either by careful weight monitoring or by strict intake and output records
  3. Urine spot Sodium > 40 mEq/L
  4. Central Venous Pressure (CVP) < 6 cm of water
  5. Laboratory evidence of dehydration including an increase in hemoglobin and hematocrit, increase in blood urea nitrogen and increase in albumin than previously.

 

Table 5. Differentiation Between Syndrome of Inappropriate Anti-Diuresis (SIAD) and Cerebral Salt Wasting (CSW)

Parameter

SIAD

CSW

Extracellular Volume

Increased

Decreased

Body Weight

Increased

Decreased

Fluid Balance

Positive

Negative

Tachycardia

-

+

Hypotension

-

+

Hematocrit/Albumin/Blood Urea Nitrogen

Normal

Increased

Central Venous Pressure

Normal or slightly high

Decreased

 

TREATMENT OF CSW ASSOCIATED WITH TBM

 

The primary treatment for CSW is fluid replacement with or without oral salt loading for as long as polyuria continues. Isotonic fluids are preferred for replacement. If the patient has a central venous line then the central venous pressure (CVP) measurements would guide the fluid replacements. In the absence of a CVP line fluid balance is needed by either meticulous intake and output charting or use of daily weight measurements.

 

In patient’s refractory to fluid replacement and oral salt loading, oral fludrocortisone (OFC) has been tried as there is an inhibition of the renin-angiotensin-aldosterone system (RAAS) system in CSW. A recent randomized control trial was conducted in 36 patients with CSW associated with TBM. Half of them received OFC (0.4-1mg/day) plus fluid and oral salt and the other half received only fluids and oral salt. The patients who received OFC in addition had quicker normalization of serum sodium levels (4 days vs 15 days; p 0.04) and lesser cerebral infarctions related to vasospasm (6% vs 33%; p 0.04). However, OFC use was associated with severe hypokalemia and significant hypertension in 2 patients each and in one patient there was an episode of pulmonary edema. OFC had to be withdrawn in 2/18 patients because of these serious adverse events. There was no difference in mortality or disability at 3 and 6 months among patients who received OFC vs the patients who did not (93).

 

CALCIUM ABNORMALITIES IN TUBERCULOSIS

 

Hypercalcemia in Patients with Tuberculosis

 

Hypercalcemia has been known to be associated with a number of granulomatous diseases. The three commonest granulomatous diseases causing hypercalcemia include sarcoidosis, TB, and fungal infection (94). The prevalence of hypercalcemia in patients with TB has ranged from 2-51% in studies done from South Africa (2%), Hong Kong (6%), India (10.6%), Sweden (25%), Malaysia (27.5%), Greece (25% & 48%) and Australia (51%) (13, 95-101). In contrast, prospective studies from the United Kingdom, Belgium, and Turkey did not show any hypercalcemia among patients with newly diagnosed tuberculosis (102-104). The primary determinant in the development of hypercalcemia among patients with TB appears to be their Vitamin D status and nutritional calcium intake. In populations with high nutritional calcium intake and adequate sunlight exposure like in Greece and Australia the prevalence of hypercalcemia is highest. Among countries with good sunlight exposure but poor nutritional calcium intake like most Asian countries there is a more modest prevalence of hypercalcemia. The countries with good nutritional calcium intake but poor sunlight exposure and low Vitamin D levels appear to have the lowest prevalence of hypercalcemia. This has been elegantly explained by Chan et al (105). However, some outliers like the higher prevalence in Sweden and moderate prevalence in India are not completely explained by this hypothesis alone.

 

In a recent paper looking at retrospective records of patients admitted with TB at a tertiary care hospital in Vellore almost 20% of patients were found to have albumin-adjusted hypercalcemia. The authors looked at the risk factors for hypercalcemia by comparing them with the patients without hypercalcemia assuming that background nutritional calcium intake and Vitamin D levels were similar. The primary risk factors for the development of hypercalcemia within this group was presence of renal dysfunction or frank renal failure, use of diuretics, disseminated tuberculosis, and presence of co-morbidities like diabetes and hypertension (106).

 

MECHANISM OF HYPERCALCEMIA WITH TUBERCULOSIS

 

The definitive mechanism that causes hypercalcemia among patients with TB is still unclear. Alternative etiologies for hypercalcemia including adrenal insufficiency, primary hyperparathyroidism, primary hyperthyroidism, milk alkali syndrome have been ruled out in many of the case series. Biochemically several investigators have shown an increase in the levels of 1,25-dihydroxy vitamin D along with low or normal levels of 25-hydroxy vitamin D levels. This suggests an increase in the conversion of 25-hydroxy vitamin D to 1,25-dihydroxy vitamin D (107). This conversion is mediated by the enzyme 1-α-hydroxylase found in the kidney. However, hypercalcemia is even reported in patients with chronic renal failure and among those with absent kidneys (108, 109). This suggests a non-renal site of enzymatic activity. 

 

The tubercular granuloma is suggested as the site for the extra renal 1-α-hydroxylase activity (110). Activated macrophages can express 1-α-hydroxylase activity and in patients with active TB activated macrophages retrieved by broncho-alveolar lavage were able to synthesize 1,25 dihydroxy vitamin D in vitro studies (111). The macrophage production of 1-α-hydroxylase is probably important for the immune response to tuberculous infection. The binding of active Vitamin D (1,25-dihydroxy vitamin D) to Vitamin D receptors within the immune cells stimulates autophagy and production of cytokines that contribute to the clearance of the mycobacterium from the body (112, 113). In addition, active Vitamin D contributes to the downregulation of the inflammatory response of the body to reduce damage to bystander host tissues (114). The increased intestinal absorption of calcium and observed hypercalcemia may be an unintended consequence of this immune-protective phenomenon.

 

This also explains why patients with low levels of substrate (25-hydroxy vitamin D) for the enzyme or among those with poor calcium intake there is less likelihood of the development of hypercalcemia.

 

CLINICAL PRESENTATION

 

Most patients with hypercalcemia related to TB infection are asymptomatic. Rarely patients develop symptoms related to hypercalcemia including polyuria, anorexia, nausea, weakness and lethargy, more serious CNS symptoms like delirium.

 

Patients may develop hypercalcemia later in the course of TB after commencement of therapy with improvements in nutritional and albumin status and improvement in nutritional calcium intake. 

 

TREATMENT

 

A Cochrane review of Vitamin D supplementation in patients with TB did not show any benefits in terms of improved outcomes but there was also no increased risk of developing hypercalcemia (115). Most patients have gradual resolution of hypercalcemia on ATT over 1 to 7 months (96).


Hypocalcemia in Patients Treated with Rifampicin and Isoniazid



Hypocalcemia was noted for the first time in United Kingdom during a randomized control trial of anti-tubercular chemotherapy after several months of therapy. Fourteen out of the 325 patients on the trial developed hypocalcemia. In this trial none of the 325 patients was noted to have hypercalcemia. On the whole as a group the mean calcium levels dropped significantly during the course of the treatment trial. The mechanism is proposed to be the action of both Rifampicin and Isoniazid on vitamin D metabolism (102).

 

When isoniazid is given to normal subjects there is a brisk decline in the levels of active Vitamin D (1,25 dihydroxy vitamin D). There is slower decline in the levels of 25-hydroxy vitamin D accompanied by a compensatory increase in the levels of parathyroid hormones. In the same study isoniazid was shown to inhibit cytochrome p450 related hepatic mixed function oxidase and it is assumed that since the renal 1-α Hydroxylase is also related to cytochrome P450 system there would be decreased conversion of 25-hydroxy vitamin D to 1,25 dihydroxy vitamin D (116).

 

On the other hand, rifampicin is an inducer of hepatic hydroxylase which should in theory lead to an increase in active Vitamin D levels. However, when rifampicin was given to normal volunteers there was a fall in 25-hydroxy vitamin D levels with no changes to the levels of 1,25-dihydroxy vitamin D. The possible explanation for this decline is likely to be that the higher metabolic turnover of active Vitamin D induced by rifampicin is not compensated by an increase in dermal production or increased nutritional provision of vitamin D (117). Regardless, in treatment regimens that include both rifampicin and isoniazid there is a very real possibility of the development of not just hypocalcemia but unmasking of rickets and osteomalacia especially when the patient is poorly nourished.


CONCLUSIONS

 

TB can involve almost all endocrine glands as a primary disease-causing destruction and loss of function. In enclosed spaces like the pituitary fossa and neck the granuloma/tuberculoma/cold abscess can replace vital structures and cause symptoms related to a mass. This chapter did not cover direct tubercular involvement of the ovaries, testes, and the pancreas.

 

Additionally, a whole range of functional hormone abnormalities can accompany the effect on chronic inflammation on the immune-endocrine pathways. Metabolic derangement in calcium and water metabolism are covered in detail. Abnormalities in glucose metabolism are not covered because of the vast amount of information now available on the public health aspects of TB and diabetes mellitus.

 

Fortunately, most abnormalities are self-limited and resolve with successful ATT. However, one needs to consider the rare possibility of a hormonal emergency like an adrenal crisis, hypercalcemic emergency, or pituitary apoplexy in the context of TB.

 

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Environmental Endocrinology: An Expanding Horizon

ABSTRACT

Environment is an important determinant of endocrine health and certain endocrine disorders could also have a significant impact on the surroundings. Environmental endocrinology is an emerging field of medicine which encompasses the bidirectional impact of endocrine disorders and the physical, chemical, biological, and social environment of an individual. As we aim to improve endocrine health, it is also important to address the external environmental factors that may affect a given endocrine condition. As more data is emerging on this subject, it will help to formulate clinical practice guidelines and policies to optimize endocrine disorders in light of a given external environment.

 

INTRODUCTION

Environment with respect to health refers to all the physical, chemical and biological factors extrinsic to a person, even encompassing the related behavioral responses. The given surroundings can have a significant impact on an individual’s health and even health conditions can influence the environment over a period of time (1). Environmental health is an upcoming field and is referred to as the science and practice of preventing human illness while promoting wellbeing, by identifying and evaluating environmental changes. It further helps to identify and limit exposure to hazardous physical, chemical, and biological agents and thereby limiting their exposure to prevent adverse effects on human health (2). Multiple disciplines of medicine are involved in studying this field and research dealing with environmental health often has a direct impact on policy and practice. Amongst various organ systems that are impacted with environmental health, the endocrine system is maximally affected and very relevant in tropical countries (3, 4). This has been shown across different species and in humans across their life span (5-7). The environmental changes can even result in epigenetic alterations that may then transcend across generations (3, 8). In this chapter, we explore the bidirectional relationship of environment and the endocrine system and suggest a future road map for addressing the research gaps identified in this field (Figure 1).

Figure 1. Relationship of the environment and endocrine system

IMPACT OF ENVIRONMENT ON ENDOCRINOLOGY

The Influence of the Physical Environment on the Endocrine System

In the last century several nuclear disasters have happened from time to time. The impact of these on the endocrine system has widely been reported and is a classic example as to how the physical environment can affect the endocrine system (9-11). The unfortunate incidents in Fukushima and Chernobyl have led to a large amount of exposure of radioactive substances that have been released into the environment. In addition to their adverse effects on reproductive health and carcinogenesis, a considerable impact has been noted on many endocrine glands including the pituitary, thyroid and gonads (12).

 

Following the Chernobyl accident in 1986, it was noted that individuals living in the surrounding areas had a 50% lower sympathetic activity and a 36% lower adrenal cortical activity including a significantly lower blood cortisol level. They also were noticed to have increased hypophyseal-thyroid system dysfunction, higher incidence of goiter, and autoimmune thyroiditis (13). An increased level of thyroxine-binding globulin, lower concentrations of free T3, and an increased risk of non-toxic single nodular and multinodular goiters have been reported (14). On the other extreme, an increased secretion of gonadotropic hormones and accelerated sexual development in women was documented. Higher rates of juvenile diabetes were also noticed in those exposed to the radioactive substances. Higher levels of prolactin and renin, with lower progesterone levels have been documented (14).

 

A similar incidence was repeated in Fukushima in 2011, following a tsunami which caused extensive damage to the nuclear reactor situated there. Though the radiation amount released into the environment was relatively less, the exposure was to a significantly larger population (12). Multiple endocrine effects have also been described secondary to the radiation exposure followed by non-accidental deliberate nuclear weapon testing (15). These effects need to be proactively followed and documented as they have strong policy implications. 

 

Another important environmental health domain which has been of concern in recent times, is the health effects of forest fires. Apart from the multitude of environment related effects of vast forest fires, they also are known to affect the endocrine system (16). Polycyclic aromatic compounds (PACs), released during such forest fires are known endocrine disruptors with steroid like actions and their chronic exposure could affect the hypothalmo-pituitary-adrenal axis (17).

 

In addition to these the impact of built environment, limited areas for safe physical activity, and an increased number of fast-food outlets are responsible for an increasing prevalence of obesity in some developing countries (18).

 

Apart from these examples at the macro-environment level, the micro-environment could also have an impact on the endocrine system. A classic example to support this is hypogonadism in men caused by excessive use of sauna, hot tubs, Jacuzzis, heated car seats, and laptop use. The increased testicular temperature caused by excessive exposure to these activities can impair spermatogenesis (19, 20). Apart from direct effect of heat, excessive use of laptops and mobile phones also exposes the body to higher amount of radio frequency electromagnetic radiation, leading to multiple systemic effects including reduced spermatogenesis and increased blood pressure (21).

 

The Effect of Changes in the Chemical Environment on Endocrinology

 

Globally, the endocrine disruptor chemicals (EDCs) are the best-known examples of how the chemical environment can influence the endocrine system. EDCs are defined as exogenous chemicals that may alter any part of the endocrine system which may include interference in hormone synthesis, secretion, circulation, metabolism, receptor interaction, or elimination (22). Based on the site of their origin - EDC’s have been classified as industrial, residential, or agricultural. The common EDC’s used in industries include polychlorinated biphenyls (PCBs) and alkylphenols. Several pesticides, insecticide, herbicides, phytoestrogens, and fungicides that are used in farming are classified as agricultural and those used in household products like phthalates, polybrominated biphenyls, and bisphenol A are considered as residential (23). EDCs have gathered much interest in recent years and are known to affect several endocrine systems especially the gonadal axis (24). A brief summary has been provided in the table below.

 

Table 1. Endocrine disruptors affecting different endocrine organs

Endocrine system

Endocrine disruptor chemical

Impact

Pituitary

Phytoestrogens (i.e., isoflavonoids, cumestans, lignans, stilbens); pesticides (i.e., organophosphates, carbamates, organochlorines, synthetic pyrethroids); Polyvinyl chloride (PVC); phenols, dioxins, heavy metals, perfluorooctanoic acid.

Precocious puberty, delayed puberty, disruption of the circadian rhythm

Adrenal

Xenoestrogens, Hexachlorobenzene

Adrenal biosynthetic defects

Thyroid

Perchlorate, thiocyanates, nitrates

Hypothyroidism

Gonads

Phthalates, vinclozolin, acetaminophen, and polybrominated diphenyl ethers (PBDE)

Phthalates, diethylstilbestrol, bisphenol A (BPA)

PCB, phtalates, atrazine, genistein, BPA, parabens, triclosan, dichlorodiphenyltrichloroethane (DDT), and metoxychloride (MXC)

Phthalates, bisphenol A, biphenyls, and vinclozolin,

Testicular dysgenesis syndrome

 

 

Endometriosis

 

Female infertility

 

 

 

 

 

 

Male infertility

Endocrine gland cancer

PDBE, organochlorides, PCB, DDT, dichlorodiphenyldichloroethylene (DDE), arsenic, and cadmium

Triclocarban, PCB

PCB, dioxins, cadmium, phytoestrogens, DES, furans, ethylene oxide

Testicular Cancer

 

 

 

Thyroid Cancer

 Breast Cancer

 

In addition to EDC’s several other occupational exposures can also cause endocrine disorders. Exposure to cadmium in silversmiths, without proper personal protective equipment could lead to renal tubular acidosis and subsequent hypophosphatemic osteomalacia (25). Chronic exposure to fluoride through drinking water is known to produce a sclerotic bone disease associated with osteomalacia (26). Exposure to other heavy metals like copper has also been associated with different endocrine disorders as seen in Wilson’s disease (27, 28). Altered exposure to certain food items, may also lead to endocrine disorders. While exposure to cow milk and cassava has been associated with development of diabetes, deficiency of iodine containing sea foods in non-coastal areas is associated with the goiter (29, 30).

 

The Impact of Biological Changes in the Environment on the Endocrine Milieu

 

The most recent and a very lucid example of how the biological environment can affect the endocrine system, is that of COVID -19. It has been shown that COVID-19 can have a myriad of effects on different endocrine systems. However, the most pertinent of all has been its association with diabetes (31-33). Interestingly not only COVID-19 can affect diabetes control but presence of diabetes can also have a direct impact on the outcome of COVID-19 (Figure 2).

Figure 2. The bidirectional relationship between COVID-19 and Diabetes

Similar to COVID-19, there have been reports of other communicable diseases intersecting with non-communicable endocrine disorders. A few common examples that are cited in literature include presence of NAFLD (Nonalcoholic fatty liver disease) in individuals with HIV infection, the association of osteoporosis with Hepatitis B infection, cytomegalovirus associated with Paget’s disease of the bone etc. (34, 35).

 

Certain infections may also be responsible for hormonal deficiencies. An example is histoplasmosis that is predominantly spread by bat droppings can result in adrenal insufficiency. This along with adrenal tuberculosis is still the most common cause of primary Addison’s disease in tropical countries unlike the West where autoimmune adrenalitis is the foremost cause. Similar infective etiologies have also been described to cause hypophysitis and resulting hypopituitarism.

 

The after effects of a of vasculo-toxic snake bite on pituitary and other endocrine organs also comes under the domain of biological environment impacting the endocrine system.

 

The Aftermath of Changing Social Environment on the Endocrine System

 

The social environment can influence the endocrine system in several ways. One such impact that has been increasing in recent years, is an increase in road traffic accidents on highways with higher speed limits, leading to traumatic brain injury. This has been associated with both acute and chronic hypopituitarism. Though first described in 1918, it was initially thought to be a rare phenomenon, but over the years has been recognized with increasing frequency (36). It is currently reported in about one third of patients with a traumatic brain injury (37). However, in autopsy studies up to 86% have demonstrated pituitary injury following traumatic brain injury (38).

 

Social norms and religious customs may further have an impact on the endocrine system. From the impact of prolonged periods of fasting on glycemic control to being customarily clad in veils leading to vitamin D deficiency, several such examples have been cited in literature.

 

IMPACT OF ENDOCRINE DISORDERS ON ENVIROMENT

 

The Influence of Endocrine Disorders on the Physical Environment

 

Globally, a rapid increase in the prevalence of obesity has brought about changes in the physical environment ranging from furniture sizes in clinics to more weight friendly gymnasiums. Additionally, operation tables have now become more accommodative of higher weights (39).

 

Another endocrine disorder that has brought about significant changes in the physical environment is osteoporosis. With an increasing life expectancy and consequent increase in the aged population, there has been a remarkable increase in the prevalence and awareness of osteoporosis. Subsequent fall protective arrangements are in place in several public places and transport facilities (40). Separate priority lines have been made available in different areas where prolonged waiting may be required (41). Moreover, battery operated cars are provided for them in airports and railway stations.

 

 

The changes in the chemical environment secondary to endocrine disorders are predominantly due to deficiency of chemical substances leading to hormone deficiencies. Nutritional vitamin D deficiency and iodine deficiency thyroid disorders have led to a massive fortification campaign in several countries. The impact of both these supplementations have seen phenomenal success across different countries especially in the tropical region (42, 43).

 

In a recently published study from Ireland, it was found that almost two-thirds of the mean daily vitamin D intake of adults came from fortified foods like milk and bread. Though individually milk and bread only helped to meet about 30 and 50% of recommended daily allowance, fortifying both simultaneously could help in meeting 70% of the RDA. This shows the impact of how widespread vitamin D deficiency could be managed by altering the chemical environment of commonly available foods (42).

 

Along similar lines a high prevalence of iodine deficiency disorders a few decades ago, has driven the salt iodination movement in several countries. This is another example of how an endocrine disorder can lead to changes in the surrounding chemical environment. This has definitely resulted in a reduction in the prevalence of goiter in many countries (44, 45).

 

The Effect of Endocrine Disorders on the Biological Environment

 

The classic example of how endocrine disorders could change the biological environment of an individual is how presence of diabetes and obesity could alter the clinical course of COVID 19. It is now clear that the presence of these comorbidities could increase the severity, prolong hospitalization and even increase the mortality of COVID-19 infected individuals (31-33, 46).

 

Other biological disorders that could depend on the endocrine milieu of a person include hormone dependent cancers. Elevations of specific hormones that can increase the risk of certain cancers provide a good opportunity to provide novel therapeutic options that may help in the management the hormone sensitive tumors (47). The commonly cited examples of these hormone dependent tumors where the alteration in the hormone levels could affect the biological activity of these disorders include breast, ovarian, and prostate malignancies.

 

The Footprint of Endocrine Diseases on the Social Environment

 

The rapid increase in the prevalence of diabetes and obesity have changed the availability of different foods and beverages available in social gatherings and supermarkets. Now sugar free foods and drinks are available in every gathering, which was not commonly present a few decades ago. Moreover, fat free snacks, low calorie deserts, and high fiber food options have become the new norm in the current day society (48).

 

SUMMARY

 

Even though several examples of the bidirectional impact of environment and endocrine disorders are cited in this chapter, data on this subject are still emerging and more evidence is needed to precisely quantify its impact. This will enable future practice guidelines and polices to improve the quality of life of people affected with endocrine disorders by modifying their environment and also help in positively changing the physical, chemical, biological, and social environment with respect to a given endocrine disorder.

 

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Pancreatic Islet Function Tests

ABSTRACT

 

Objective: To describe testing indications and protocols for the evaluation of pancreatic islet function. Methods: A review of the literature and consensus guidelines concerning testing of pancreatic islet function was performed. Results: Indications for screening for diabetes mellitus are reviewed. Diagnostic criteria for diagnosis are fasting plasma glucose ≥ 126 mg/dl (7.0 mmol/l) or random glucose ≥200 mg/dl (11.1 mmol/l) with hyperglycemic symptoms, hemoglobin A1c (HbA1c) ≥6.5%, and oral glucose tolerance testing (OGTT) 2-h glucose ≥200 mg/dl (11.1 mmol/l) after 75 g of glucose. One-step and two-step strategies for diagnosing gestational diabetes using pregnancy-specific criteria as well as use of the 2-h 75-g OGTT for the postpartum testing of women with gestational diabetes (4-12 weeks after delivery) are described. Testing for other forms of diabetes with unique features are reviewed, including the recommendation to use the 2-h 75 g OGTT to screen for cystic fibrosis-related diabetes and post-transplantation diabetes, fasting glucose test for HIV positive individuals, and genetic testing for monogenic diabetes syndromes including neonatal diabetes and maturity-onset diabetes of the young (MODY). Elevated measurements of pancreatic islet autoantibodies (e.g., to the 65-KDa isoform of glutamic acid decarboxylase (GAD65), tyrosine phosphatase related islet antigen 2 (IA-2), insulin (IAA), and zinc transporter (ZnT8)) suggest autoimmune type 1 diabetes (vs type 2 diabetes). IAA is primarily measured in youth. The use of autoantibody testing in diabetes screening programs are recommended only in first degree relatives of an individual with type 1 diabetes or in research protocols. C-peptide measurements >3 years after clinician diagnosis of type 1 diabetes in adults can be helpful in identifying those who have type 1 diabetes (low or undetectable c-peptide) from those who may have type 2 or monogenic diabetes. Use of OGTTs to examine insulin secretory reserve and intravenous glucose tolerance testing are also reviewed. These tests are primarily used in research studies. Evaluation of glycemic control is discussed, with special attention to hemoglobin A1c (HbA1c) and its correlation with mean blood glucose levels as well as assays of other glycated serum proteins. Finally, protocols used to evaluate hypoglycemia (glucose < 55 mg/dl (3.1 mmol/l)) are described, such as the supervised prolonged fast, during which measurements of glucose, insulin, c-peptide, oral insulin secretagogues, proinsulin, and beta-hydroxybutyrate are obtained. Insulinoma is suggested by elevated insulin, proinsulin and c-peptide levels, beta-hydroxybutyrate < 2.7 mmol/l, and undetectable insulin secretagogues. Use of a modified OGTT in the evaluation of the dumping syndrome is also described, as are the mixed meal test, glucagon tolerance test, c-peptide suppression test and evaluation of autoimmune hypoglycemia.

 

SCREENING FOR DIABETES MELLITUS AND PREDIABETES

 

Early detection and treatment of diabetes mellitus is important in preventing the chronic and acute complications of this disease. Individuals with symptoms suggestive of hyperglycemia, such as polyuria, polyphagia, polydipsia, unexplained weight loss, blurred vision, excessive fatigue, or infections or wounds that heal poorly should be promptly tested.

 

The American Diabetes Association (ADA) recommends routinely screening for type 2 diabetes in adults every three years beginning at age 45. In asymptomatic people, testing for type 2 diabetes should be considered in adults of any age if they are overweight or obese (BMI ≥ 25 kg/m2, or ≥ 23 kg/m2 if Asian), planning pregnancy, and/or if they have additional risk factors as listed below in Table 1. Repeat screening should be performed at least every three years. Patients with prediabetes should be screened yearly (1). The US Preventive Services Task Force recommends glucose screening for all asymptomatic overweight or obese adults ages 40-70 (2); the American Association of Clinical Endocrinologists recommends screening at risk individuals at any age (3).

Table 1. Risk Factors for the Development of Type 2 Diabetes (1)

Physical inactivity

First-degree relative with diabetes

High-risk race/ethnicity (e.g., African American, Latino, Native American, Asian American, Pacific Islander)

Women who delivered a baby weighing >9 lb. or were diagnosed with Gestational Diabetes

Hypertension (≥140/90 mm Hg or on therapy for hypertension)

HDL cholesterol level <35 mg/dL (0.90 mmol/L) and/or a triglyceride level >250 mg/dL (2.82 mmol/L)

Women with polycystic ovary syndrome

HbA1C ≥5.7%, Impaired Glucose Tolerance (IGT), or Impaired Fasting Glucose (IFG) on previous testing

Other clinical signs or conditions associated with insulin resistance (e.g., severe obesity, acanthosis nigricans)

History of cardiovascular disease

HIV

 

Type 2 diabetes is becoming a growing problem in children and adolescents in high-risk populations. To address this issue, the ADA recommends screening overweight [body mass index (BMI) ≥85th percentile] or obese (BMI ≥95th percentile) youth at least every 3 years, beginning at age 10 or at the onset of puberty, if they have 1 or more additional risk factors listed below in Table 2. Repeat testing should be done more frequently if BMI is increasing (1).

Table 2.  Risk Factors for Type 2 Diabetes in Children and Adolescents

Family history of type 2 diabetes (first and second-degree relatives)

High risk ethnicity (Native Americans, African-Americans, Latino, Asian/Pacific Islanders)

Signs of or conditions associated with insulin resistance (acanthosis nigricans, hypertension, dyslipidemia, small-for-gestational-age birth weight, or polycystic ovary syndrome)

Maternal history of diabetes or gestational diabetes during child's gestation

 

DIAGNOSING DIABETES AND PREDIABETES

 

The diagnosis of diabetes can be made using the fasting plasma glucose, random plasma glucose, oral glucose tolerance test, or hemoglobin A1c (HbA1c) (1). Testing should be performed on 2 separate days using one or more of the above tests, unless unequivocal hyperglycemia is present. Alternatively, in the absence of symptoms of hyperglycemia, diabetes can be diagnosed if there are two different abnormal test results from the same sample (1).

 

HbA1c

 

The use of the HbA1c assay was recommended for the diagnosis of diabetes in 2009 by an International Expert Committee (4). HbA1c levels reflect overall glycemic control and correlate with the development of microvascular complications. An HbA1c ≥ 6.5% on two separate occasions can be used to diagnose diabetes. An HbA1c level of 6.0% to less than 6.5% identifies high risk of developing diabetes. The ADA considers individuals with a HbA1c of 5.7% to 6.4% at increased risk for developing diabetes (1). HbA1c should not be used to diagnose gestational diabetes, diabetes in HIV positive individuals, post-organ transplantation, or in people with cystic fibrosis.

Table 3.  ADA Criteria for the Diagnosis of Diabetes (1)

HbA1C ≥6.5%. The test should be performed in a laboratory using a method that is National Glycohemoglobin Standardization Program certified and standardized to the Diabetes Control and Complications Trial (DCCT) assay.

FPG ≥126 mg/dL (7.0 mmol/L). Fasting is defined as no caloric intake for at least 8 h.

2-h plasma glucose ≥200 mg/dL (11.1 mmol/L) during an Oral Glucose Tolerance Test (OGTT). The test should be performed as described by the WHO, using a glucose load containing the equivalent of 75 g anhydrous glucose dissolved in water.

In a patient with classic symptoms of hyperglycemia or hyperglycemic crisis, a random plasma glucose ≥200 mg/dL (11.1 mmol/L) without repeat testing for confirmation.

 

Fasting and Random Plasma Glucose

 

Fasting plasma glucose is one method recommended by the ADA for the diagnosis of diabetes in children and non-pregnant adults (1). The test should be performed after an 8 hour fast. For routine clinical practice, fasting plasma glucose may be preferred over the oral glucose tolerance test because it is rapid, easier to administer, is more convenient for patients and providers, and has a lower cost (1). A random plasma glucose level, which is obtained at any time of the day regardless of the time of the last meal, can also be used in the diagnosis of diabetes in a patient with classic symptoms of hyperglycemia or hyperglycemic crisis.

Table 4.  Fasting Plasma Glucose Criteria
 

Fasting Plasma Glucose

Normal glucose tolerance

<100 mg/dl (5.6 mmol/l)

Impaired fasting glucose (pre-diabetes)

100-125 mg/dl (5.6-6.9 mmol/l)

Diabetes mellitus

≥126 mg/dl (7.0 mmol/l)

 

For the diagnosis of diabetes, standard venous plasma glucose specimens should be obtained. Specimens should be processed promptly, since glucose is metabolized at room temperature. This process is influenced by storage temperature, storage time as well as other factors, and is accelerated in the presence of bacteria or leukocytosis.

 

Whole blood glucose specimens obtained with point-of-care devices should not be used for the diagnosis of diabetes because of the inaccuracies associated with these methods. Capillary and venous whole blood glucose concentrations are approximately 15% lower than plasma glucose levels in fasting specimens.

 

Oral Glucose Tolerance Test (OGTT)

 

OGTTS FOR THE DIAGNOSIS OF DIABETES AND IMPAIRED GLUCOSE TOLERANCE IN NON-PREGNANT INDIVIDUALS

 

Formal oral glucose tolerance tests can be used to establish the diagnosis of diabetes mellitus. They are more cumbersome and costlier than the fasting plasma glucose test, however, the use of only the fasting plasma glucose may not identify a proportion of individuals with impaired glucose tolerance or diabetes (5). A plasma glucose level 2-hours after a glucose challenge may identify additional individuals with abnormal glucose tolerance who are at risk for microvascular and macrovascular complications, particularly in high-risk populations in which postprandial (versus fasting) hyperglycemia is evident early in the disease (6,7).

 

When using an OGTT, the criteria for the diagnosis of diabetes is a 2 h glucose >200 mg/dl (11.1 mmol/l) after a 75-gram oral glucose load (ADA and WHO criteria). The 75-gram glucose load should be administered when the patient has ingested at least 150 grams of carbohydrate for the 3 days preceding the test and after an overnight fast. Dilution of the 75-gram oral glucose load (300-900 ml) may improve acceptability and palatability without compromising reproducibility (8). The patient should not be acutely ill or be taking drugs that affect glucose tolerance at the time of testing, and should abstain from tobacco, coffee, tea, food, alcohol and vigorous exercise during the test.

Table 5.  Oral Glucose Tolerance Test Glucose Criteria
 

2-h Plasma Glucose (after 75-gram Glucose Load)

Normal glucose tolerance

<140 mg/dl (7.8 mmol/l)

Impaired glucose tolerance(pre-diabetes)

140-199 mg/dl (7.8-11.1 mmol/l)

Diabetes mellitus

≥200 mg/dl (11.1 mmol/l)

 

OGTTS FOR THE DIAGNOSIS OF GESTATIONAL DIABETES

 

The prevalence of gestational diabetes (GDM) varies among racial and ethnic groups and between screening practices, testing methods, and diagnostic criteria. The overall frequency of GDM in the 15 centers participating in the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study was 17.8% (9), and regional estimates may vary from 10% to 25 % depending on the population studied (10). The prevalence increases with increased number of risk factors, such that 33% of women with 4 or more risk factors have gestational diabetes (11). This condition is important to diagnose early because of the increased perinatal morbidity associated with poor glycemic control.

 

The US Preventive Task Force recommends screening for gestational diabetes in asymptomatic women after 24 weeks (12); the ADA recommends screening all pregnant women routinely between 24- and 28-weeks’ gestation. If the woman has risk factors, however, screening should be performed at the initial prenatal visit using standard criteria (1).

Table 6.  Risk Factors for the Development of Gestational Diabetes

Overweight or obese

Previous history of impaired glucose tolerance, gestational diabetes, or delivery of a baby weighing >9 lb.

Glycosuria or history of abnormal glucose tolerance

Family history of diabetes (especially first degree relative)

Polycystic ovarian syndrome, hypertension, glucocorticoid use

History of poor obstetric outcome

Age (>25 years)

High risk ethnicity

Multiple gestation

 

Table 7.  Low Risk for the Development of Gestational Diabetes

Age (< 25 years)

Normal weight pre-pregnancy

Low risk ethnicity

No first-degree relatives with diabetes

No history of abnormal glucose tolerance

No history of poor obstetric outcome

 

Table 8.  Time of Initial Testing for Gestational Diabetes

Risk of Development of Gestational Diabetes

Time of Initial Testing for Gestational Diabetes

Low risk

24-28 weeks gestation

Average risk

24-28 weeks gestation

High risk

As soon as feasible; repeat at 24-28 weeks if earlier testing normal

 

More than one method has been recommended for the screening and diagnosis of gestational diabetes. The criteria for the diagnosis of this condition remain controversial because the glucose thresholds for the development of complications in pregnancies with diabetes remain poorly defined. Currently, the ADA suggests screening for GDM with either the “one-step” or “two-step” approach (1). Long term outcome studies evaluating pregnancies complicated by GDM are currently underway and hopefully a uniform approach will be adopted.

 

One-Step Strategy

 

The International Association of Diabetes and Pregnancy Study Group (IADPSG), an international consensus group with representatives from multiple obstetrical and diabetes organizations including the ADA recommend that all women not previously known to have diabetes undergo a 75-gram 2-hour OGTT at 24-28 weeks of gestation. This approach, which has been adopted internationally, is expected to increase the prevalence of GDM as only one abnormal value is sufficient to make the diagnosis (1,13). In 2017, the American College of Obstetricians and Gynecologists (ACOG) stated that clinicians may make the diagnosis of gestational diabetes based on only one elevated blood glucose value if warranted, based on their population, although this organization still supports the “two step” approach for diagnosis of GDM (14).

Table 9.  Oral Glucose Tolerance Test Glucose Criteria for the Diagnosis of GDM

75-gram 2- hour OGTT: Performed at 24-28 weeks gestation in the morning after an overnight fast of at least 8 hours

GDM is diagnosed when any of the following values are exceeded:

Fasting

≥ 92 mg/dL (5.1 mmol/L)

One Hour

≥ 180 mg/dL (10.0 mmol/L)

Two Hour

≥ 153 mg/dL (8.5 mmol/L)

These glucose thresholds were based on outcome data of the Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) study that conveyed an odds ratio for adverse maternal, fetal and neonatal outcomes of at least 1.75 based on fully adjusted logistic regression models (15).

Two-Step Strategy 

The American College of Obstetricians and Gynecologists (ACOG) as well as the National Institutes of Health (NIH) have been in support of the "two step" approach which consists of universal screening of all pregnant women at 24-28 weeks gestation with a 50-gram glucose challenge regardless of timing of previous meals, followed by a 100-gram three-hour OGTT in screen positive patients (14, 16).

 

In the two-step approach, first a 50-gram oral glucose load is administered regardless of the timing of previous meals. The following thresholds have been defined as a positive screen: ≥130 mg/dL, ≥135 mg/dL, or ≥140 mg/dL (7.2 mmol/L, 7.5 mmol/L, or 7.8 mmol/L); the lower threshold has an estimated sensitivity and specificity of 88-99% and 66-77% compared to 70-88% and 69-89% respectively for the higher cutoff values of ≥135 mg/dL or ≥140 mg/dL (1).

Table 10.  Abnormal Glucose Level on Screening Test

50-gram Glucose Load

1-h Plasma Glucose

≥130 mg/dl (7.8 mmol/l)

 

If the screening test is abnormal, the diagnosis of gestational diabetes should be confirmed using a formal 100-gram OGTT. This test should be performed after an overnight (8-14 h) fast. It is generally recommended that the woman ingest at least 150 grams of carbohydrate/day for the 3 days prior to testing to prevent false positive results; however, the necessity of this preparatory diet in normally nourished women has been challenged (17). The ADA recommends using the Carpenter/Coustan criteria (1). At least 2 of the following 4 venous plasma glucose levels must be attained or exceeded to make the diagnosis of GDM (1).

Table 11.  Oral Glucose Tolerance Test Glucose Criteria for the Diagnosis of GDM
 

Carpenter/Coustan

National Diabetes Data Group

Fasting

≥95 mg/dl (5.3 mmol/l)

≥105 mg/dl (5.8 mmol/l)

One Hour

≥180 mg/dl (10.0 mmol/l)

≥190 mg/dl (10.6 mmol/l)

Two Hours

≥155 mg/dl (8.6 mmol/l)

≥165 mg/dl (9.2 mmol/l)

Three Hours

≥140 mg/dl (7.8 mmol/l)

≥145 mg/dl (8.1 mmol/l)

 

OGTTS FOR POSTPARTUM TESTING OF WOMEN WITH GESTATIONAL DIABETES

Women with a history of GDM are at a higher risk of developing type 2 diabetes than women without GDM (18,19). Women at the highest risk are those with multiple risk factors, those who had more severe gestational diabetes, and those with poorer beta cell function (11). The ADA recommends testing women 4-12 weeks after delivery using a two-hour 75-gram OGTT. Women with normal results should be retested at least every 3 years. It is recommended that women with impaired fasting glucose or impaired glucose tolerance be retested on a yearly basis (1).

 

Special Populations

 

 

Diabetes is common in patients with cystic fibrosis and is associated with adverse effects on nutritional status as well as pulmonary function. Annual screening for diabetes is recommended for individuals over age 10 with cystic fibrosis (1). HbA1c and fructosamine can be inaccurate in this population. In a retrospective analysis of the Toronto cystic fibrosis database, screening for diabetes using a HbA1c cutoff of 5.5% had a sensitivity of 91.8% and specificity of only 34.1% (20) but more studies need to be performed before the use of HbA1c is generally recommended for the diagnosis of diabetes in these individuals.

 

The use of the 2-hour 75 gm OGTT is recommended for the screening of healthy outpatients with cystic fibrosis. For patients receiving continuous drip feedings, laboratory glucose levels at the midpoint or immediately after feedings should be obtained. The diagnosis of diabetes is based on glucose levels ≥200 mg/dL on 2 separate occasions. If the patient is acutely ill or ingesting glucocorticoids, a FPG ≥126 mg/dL or 2-hour postprandial glucose ≥200 mg/dL that persists for >48 hours is sufficient to diagnose diabetes (21, 22). 

 

FASTING GLUCOSE FOR DIAGNOSIS OF PREDIABETES AND DIABETES IN PEOPLE LIVING WITH HIV

 

Screening for prediabetes and diabetes by measuring fasting glucose before and 3-6 months after starting or changing antiretroviral therapy is recommended for everyone living with HIV (1).  If normal, a fasting glucose test should be performed yearly.  Screening using a HbA1c test is not recommended for diagnosis due to risk of inaccuracies (1, 23). 

 

OGTTS FOR DIAGNOSIS OF POST-TRANSPLANTATION DIABETES

 

After an individual has had an organ transplant and is on stable immunosuppressive therapy, routine screening for diabetes is recommended.  The recommended screening test is an OGTT post- transplantation (1).

 

ESTIMATING INSULIN SENSITIVITY AND SECRETION

 

The hyperinsulinemic euglycemic insulin clamp procedure is the gold standard for measuring insulin resistance, and the hyperglycemic clamp is the gold standard for measuring insulin secretion.  These are only used in research studies. Fasting data and data from OGTTs are more often used due to ease of performance and lower cost. A simple widely used research method, the Homeostasis Model (HOMA), uses fasting glucose (G) and insulin (I) levels (or c-peptide (C) instead of insulin) to estimate beta cell function and insulin sensitivity. The HOMA calculator as well as additional information concerning this method can be found at: http://www.dtu.ox.ac.uk/homacalculator/. Insulin secretion has also been estimated using the Insulinogenic Index [IGI; ∆I30/∆G30] and the C-peptide Index (∆C30/∆G30). Additional estimates of insulin sensitivity include the Quantitative Insulin Sensitivity Check Index [QUICKI; 1/log(FI) + log (FG)] and the Whole-Body Insulin Sensitivity Index [WBISI]. The Oral Disposition Index is a measure of insulin secretion relative to insulin sensitivity [1/IFx (∆C30/∆G30)]. These measures are not used in routine clinical care.  A surrogate marker of insulin resistance is the lipid accumulation product (LAP) index, which uses information that can be obtained in routine clinical practice (24). It is calculated as follows: females (waist circumference-58) x (triglyceride [mmol/L]); males (waist circumference-68) x (triglyceride [mmol/L]).  The LAP cannot be used if triglycerides are >15 mmol/L.

 

Intravenous Glucose Tolerance Test

 

The short intravenous glucose tolerance test (IVGTT) is used in research studies to assess first phase insulin release. This acute insulin secretory response is typically lost early in the development of both type 1 and type 2 diabetes due to reduction of beta cells and islet cell dysfunction. Abnormal IVGTT results can occur prior to the onset of the diabetes. The test is performed after an overnight 10 h fast, and the patients are instructed to ingest at least 150 grams of carbohydrate for the 3 days preceding the test. A 25-gram glucose bolus (of a 25% glucose solution) is given intravenously, and the acute insulin response calculated from the third to fifth minute after the glucose bolus. The short intravenous glucose tolerance test is sometimes used to assess pancreatic function after pancreatic transplantation.

 

In the Diabetes Prevention Trial Type 1, a glucose load was given intravenously (0.5 g/kg body weight up to a maximum of 35 grams) over 3 minutes, and insulin levels at 1- and 3-minutes post-load were used to estimate acute insulin production (25). Individuals with low insulin response (<100 uU/ml) and positive autoantibodies were at high risk of developing type 1 diabetes. Until effective interventions are established, however, the routine use of this test for the detection of early type 1 diabetes is not recommended.

 

The standard intravenous glucose tolerance test is used in research studies to estimate insulin sensitivity (SI) and glucose effectiveness (SG) using minimal model methodology. The procedure for the standard intravenous glucose tolerance test is to intravenously inject glucose (0.33 g/kg body weight) over 2 minutes and to frequently sample for glucose and insulin over 3-4 hours. Modifications include the addition of a tolbutamide (125 mg/m2) or insulin (20-30 mU/kg) infusion 20-25 minutes after the glucose load. These tests are not used in clinical practice.

 

Additional information can be found in the chapter entitled “Assessing Insulin Sensitivity and Resistance in Humans” in the Diabetes or Endocrine Testing Protocol sections of Endotext.

 

C-Peptide Testing

 

During the processing of proinsulin to insulin in the beta cell of the pancreas, the 31 amino acid connecting peptide which connects the A and B chains, called c-peptide, is enzymatically removed and secreted into the portal vein. C-peptide circulates independently from insulin and is mainly excreted by the kidneys. Levels are elevated in renal failure. Standardization of different c-peptide assays is still suboptimal. C-peptide testing is used to examine insulin secretory reserve in people with diabetes.  Another important use of c-peptide measurements is in the evaluation of hypoglycemia, described below (see Section “Evaluation of Hypoglycemia”).

.

At the time of type 1 diabetes diagnosis, c-peptide levels commonly overlap with those observed in type 2 diabetes, and cannot reliably distinguish between these diabetes types. With longer duration, there is progressive loss of c-peptide, and although c-peptide levels in many individuals with long-standing type 1 diabetes are extremely low or undetectable, there is heterogeneity in residual beta cell function with detectable c-peptide being more common in adult-onset type 1 diabetes (26). In type 1 diabetes, detectable c-peptide is associated with better glycemic control, less hypoglycemia, and decreased microvascular disease (27-28).

 

Type 2 diabetes is heterogeneous, with many individuals having progressive loss of beta cell function over many years evidenced by decreasing c-peptide levels. Fasting and glucose-stimulated c-peptide levels have been used in the past to distinguish type 1 (severe insulin deficiency) from type 2 diabetes with limited success. However, targeted testing may be more discriminatory.  When random c-peptide testing was performed >3 years after clinical diagnosis of type 1 diabetes, 13% had a c-peptide ≥200 pmol/L, and after islet autoantibody and genetic testing, 6.8% of these were reclassified: 5.1% as having type 2 diabetes and 1.6% as having monogenic diabetes (29).

 

C-peptide stimulation using glucagon or a mixed meal such as Sustacal, has also been used to help differentiate between type 1 and type 2 diabetes, and to determine the need for insulin therapy in type 2 diabetes. In the glucagon stimulation test, glucose, insulin and c-peptide levels are measured 6 and 10 min after the intravenous injection of 1 mg of glucagon. Normal stimulation of c-peptide is a 150-300% elevation over basal levels. In the mixed meal tolerance test, Sustacal (6 mg/kg up to a maximum or 360 ml) is ingested over 5 minutes, and glucose and c-peptide are measured 90 min after oral ingestion.

 

These tests have had limited general clinical utility since they do not reliably discriminate between patients who require insulin therapy. They have been used in research studies and in the evaluation of patients after pancreatectomy and pancreatic transplantation. In the Diabetes Control and Complications Trial, a basal c-peptide value of <0.2 pmol/ml and stimulated level of <0.5 pmol/ml were used to confirm the presence of type 1 diabetes at entry (30).

 

PANCREATIC AUTOANTIBODIES

 

Islet autoantibodies can be detected early in the development of type 1 diabetes and are considered markers of autoimmune beta cell destruction. They predict progressive beta cell destruction and ultimately beta cell failure. The autoantibodies for which specific immunoassays are available include the 65-KDa isoform of glutamic acid decarboxylase (GAD65), insulin autoantibodies (IAA), zinc transporter antibodies (ZnT8), islet cell antigen 512 autoantibodies (ICA512), and autoantibodies to the tyrosine phosphatase related antigens islet antigen 2 (IA-2) and IA-2b. Measurements of ICA512, which are autoantibodies to parts of the IA-2 antigen, are no longer recommended. The presence of high levels of 2 or more antibodies is strongly predictive of type 1 diabetes mellitus. These antibodies may be detected before the onset of type 1 diabetes, at the time of diagnosis and for variable amounts of time after diagnosis. They have been used in screening for type 1 diabetes in first-degree relatives of an individual with type 1 diabetes or in research studies related to the early detection, treatment and prevention of type 1 diabetes (www.diabetestrialnet.org). These measurements are not recommended for use in general screening programs in low-risk individuals.

 

Commercially available assays for autoantibodies are sometimes useful in distinguishing type 1 diabetes from type 2 diabetes. The absence of detection of these antibodies, however, does not exclude the diagnosis of type 1 diabetes. Since IAA can form in response to insulin therapy, detection can be the result of insulin injections or autoimmune insulin antibody formation. GAD65 antibodies are frequently observed early in the course of type 1 diabetes. They are also present in the rare neurological disorder, stiff-man syndrome, and in some patients with polyendocrine autoimmune disease.

 

In adults with newly diagnosed diabetes for whom type 1 diabetes is a possible diagnosis, GAD65 is commonly measured first, along with or followed by IA2 and ZnT8. IAA are more commonly detected in young children who develop type 1 diabetes and are generally not measured in adults.

 

Lynam and coworkers (31) developed a clinical multivariable model to help differentiate between type 1 and type 2 diabetes in adults ages 18-50 years.  The model includes age at diagnosis, BMI, islet autoantibodies (GAD, IA-2), and a type 1 diabetes genetic risk score.  The authors define type 1 diabetes by a non-fasting c-peptide <200 pmol/L and rapid insulin requirement within the first 3 years of diagnosis. The definition of type 2 diabetes was not requiring insulin treatment within the first 3 years after diagnosis or, if insulin was used, having a c-peptide measurement of >600 pmol/L at ≥5 years post-diagnosis.  Since the measures of the genetic variants in the type 1 diabetes genetic risk score are not widely available, this model is not used clinically in the United States.

 

GENETIC TESTING FOR MONOGENIC DIABETES SYNDROMES

 

Monogenic diabetes syndromes account for 1%-5% of all individuals with diabetes and have been primarily classified as neonatal diabetes or Maturity-Onset Diabetes of the Young (MODY) based on clinical characteristics. More than 50 affected genes have been described. A Diabetes Care Expert Forum assembled in 2019 to re-consider the classification of monogenic diabetes syndromes. They recommend a classification system based upon molecular genetics, listing the affected gene, inheritance/phenotype, disease mechanism/special features, and the treatment implications (32).

 

When genetic testing is considered, involvement of centers with expertise in the diagnosis and treatment of monogenic diabetes syndromes is recommended (1).  Laboratories performing genetic testing should participate in quality assurance programs (32).  Proper diagnosis is critical since treatment approaches will differ depending upon the gene affected. 

Table 12.  When to Consider Genetic Testing for Monogenic Diabetes Syndromes

Diabetes diagnosed younger than 6 months of age

Diabetes in children and young adults not characteristic of type 1 or type 2 (negative pancreatic auto-antibodies, non-obese, no features of metabolic syndrome) and with a strong family history (diabetes in successive generations suggesting dominant inheritance)

Fasting glucose 100-150 mg/dL, stable A1c (5.6-7.6%), especially if in a non-obese child or young adult

 

The ADA recommends immediate genetic testing for all infants diagnosed with diabetes within the first 6 months of life (1). Common causes of neonatal diabetes include mutations in the following genes: KCNJ11 (potassium inward-rectifying channel, subfamily J, member 11), ABCC8 (ATP-binding cassette, sub-family C, member 8 of the potassium channel), INS (preproinsulin), 6q24 (PLAGL1, HYMA1), GATA6, EIF2AK3, EIF2B1 and FOXP3.

 

MODY most commonly manifests before age 25 years but can be diagnosed in older individuals. The inheritance is typically autosomal dominant. Individuals who have positive islet autoantibody test results and/or low c-peptide concentrations should not be tested for monogenic diabetes syndromes (33). The number of genetic mutations responsible has been increasing each year. Most common forms include: HNF4A-MODY (hepatocyte nuclear factor-4 alpha; MODY 1), GCK-MODY (glucokinase; MODY 2), HNF1A-MODY (hepatocyte nuclear factor 1 homeobox A;MODY 3 ), PDX1-MODY (MODY 4), HNF1B-MODY (hepatocyte nuclear factor-1 beta; MODY 5), NEUROD1-MODY (MODY 6), and INS-MODY (MODY 10).  A MODY risk calculator is available at: https://www.diabetesgenes.org/mody-probability-calculator. 

 

EVALUATION OF GLYCEMIC CONTROL IN DIABETES MELLITUS

 

Hemoglobin A1c

 

Glycosylated hemoglobin, or the hemoglobin A1c (HbA1c) assay, is the most widely accepted laboratory test for the measurement of glycemic control and is recommended for routine use in the management of patients with diabetes mellitus. HbA1c levels reflect average blood glucose levels over the preceding 2-3 months. Although the life span of erythrocytes is approximately 120 days, HbA1c levels represent a weighted average of blood glucose levels, with youngest red blood cells, reflecting mean glucose levels over the past month, contributing to a greater extent than older ones.

 

The International Federation of Clinical Chemistry Working Group on HbA1c defines the HbA1c as the hemoglobin A that is irreversibly non-enzymatically glycosylated at one or both N-terminal valines of the beta-chains of the hemoglobin. Multiple methods have been certified to measure HbA1c. The National Glycohemoglobin Standardization Program, which was started in 1996, has been largely successful in its goal to standardize HbA1c assays throughout the United States to the HPLC method used in the Diabetes Control and Complications Trial. The process has involved certification and proficiency testing, and long-term monitoring of quality control data. Providers should only use laboratories that are certified by the National Glycohemoglobin Standardization Program. Information concerning certified methods and laboratories can be found on their website http://www.ngsp.org/.

 

A consensus statement on the international standardization of HbA1c assays was issued by the American Diabetes Association (ADA), the European Association for the Study of Diabetes, the International Diabetes Federation, the International Federation of Clinical Chemistry and Laboratory Medicine, the International Society for Pediatric and Adolescent Diabetes, the Japanese Diabetes Society and the National Glycohemoglobin Standardization Program (34). HbA1c assays should be calibrated to this reference method and results reported in a standardized manner (A1c (%); A1c (mmol/mol), and estimated average glucose).

 

The ADA recommends determining HbA1c levels every 3 to 6 months to monitor glycemic control (1). Reducing the HbA1c level to as close to normal as possible is directly related to the reduction in the development and progression of the chronic complications of diabetes (31, 35-37). The ADA goal HbA1c is <7% (if this can be accomplished safely) but states that lower goals may be appropriate in individual patients. Higher HbA1c goals may be appropriate for patients with a history of severe hypoglycemia, limited life expectancy, advanced complications, and/or comorbid conditions and those in whom a lower goal is difficult to attain (1). The American Association of Clinical Endocrinologists target HbA1c is 6.5% for otherwise healthy patients at low risk for hypoglycemia. HbA1c targets should be individualized in patients with concurrent illness or those at risk for hypoglycemia (38).

 

The international A1c-Derived Average Glucose Study (ADAG) utilized frequent self-monitoring of blood glucose in adults with type 1 diabetes, type 2 diabetes, and no diabetes. The study described a linear relationship between HbA1c and average glucose level (39).  In the ADAG study, there was no significant difference in the regression lines between HbA1c and mean glucose levels among ethnic and racial groups, although there was a trend toward a difference in regression lines between African/African-American and Caucasian adults. Other studies have shown differences in HbA1c by race and ethnicity, but the reasons for this remain unknown and the individual differences within racial groups are greater than the variation between races (40-42). At this time the recommended HbA1c target does not differ based on race or ethnicity.

 

Table 13.  Correlation of HbA1c with Mean Blood Glucose Concentrations (39, 42-43)

Hemoglobin A1c (%)

Approximate Mean Plasma Glucose (mg/dL)
 

Nathan et al 2008 (ADAG study; reference 31)

Beck et al 2017
(reference 35)

6

100-152

101-163

7

123-185

128-190

8

147-217

155-218

9

170-249

182-249

10

193-282

209-273

 

Table 14. Relationship of HbA1c Levels with Mean Glucose Levels

Hemoglobin A1c (%)

Mean Glucose Concentrations (95% CI; reference 1)

Fasting (mg/dL)

Premeal (mg/dL)

Post meal (mg/dL)

Bedtime (mg/dL)

5.5-6.49

122
(117-127)

118
(115-121)

144
(139-148)

136
(131-141)

6.5-6.99

142
(135-150)

139
(134-144)

164
(159-169)

153
(145-161)

7.0-7.49

152
(143-162)

152
(147-157)

176
(170-183)

177
(166-188)

7.5-7.99

167
(157-177)

155
(148-161)

189
(180-197)

175
(163-188)

8.0-8.5

178
(164-192)

179
(167-191)

206
(195-217)

222
(197-248)

 

Depending upon the assay method being used, certain hemoglobinopathies may interfere with results. This problem is highly method-dependent. Inaccurate results may be obtained in the presence of salicylates, chronic alcohol or opiate use, hyperbilirubinemia, liver or renal disease, iron deficiency, vitamin C, vitamin E, hypertriglyceridemia, lead poisoning, recent blood transfusions, and when there are conditions of abnormal red blood cell turnover such as in anemia, hemolysis, pregnancy, or use of erythropoiesis-stimulating agents. See www.ngsp.org/interf/asp for a full list of interferences for different methods.

 

Because of the improved standardization and reference method for the HbA1c assay, both the ADA and an International Expert Committee suggest that a HbA1c > 6.5% on two occasions is diagnostic of diabetes (1,4). Benefits of the use of HbA1c for the diagnosis of diabetes are that the test is easy to perform, does not have to be performed in the fasting state, and does not require any special preparation. Potential problems include interference by factors associated with abnormal red blood cell turnover and cost (44). The HbA1c range that indicates high risk of developing diabetes is considered 6.0% to <6.5% by the International Expert Committee (4) and 5.7% to 6.4% by the ADA (1).

 

Fructosamine, Glycated Albumin and 1,5-Anhydroglucitol

 

Assays of glycated serum proteins, which mostly measure glycated serum albumin, can reflect short-term glycemic control. The fructosamine assay is most commonly used. Since albumin has a short half-life (14-20 days), this test indicates average blood glucose levels over the past few weeks, which can be helpful in certain conditions such as pregnancy or in patients with hemoglobinopathy or abnormal red blood cell turnover (1,45). These tests may be affected by hypertriglyceridemia, hyperbilirubinemia, hyperuricemia, hypothyroidism and hyperthyroidism, as well as by low serum protein and albumin levels. The relationship between these measures of glycemic control and HbA1c, fasting glucose and mean glucose have been reported in few studies shown below:

Table 15. Relationship Between Tests to Measure Glycemic Control

Hemoglobin A1c (%)

Mean Fasting Glucose (mg/dL)

Mean CGM Glucose (mg/dL)

Mean (Range) Fructosamine (μmol/L)

Mean (Range) Glycated Albumin (%)

Mean (Range) 1,5-anhydroglucitol (μg/mL)

5.4a

102a

  

219 (89-240)

12.2 (5.6-13.5)

  
 

100 b

  

224.9

12.5

27.5

5.7b

    

241.4

13.6

29.1
 

126 b

  

261.7

15.0

17.9

6.1a

    

238

13.3 (7.9-15.6)

  

6.2a

126

  

236
(159-265)

13.6

  
 

126

  

250.5-276.4

15.5-16.9

5.9-15.7

6.5 b

    

270.2

15.6

22.7

6.5c

    

254.7-289.5

16.1-18.3

5.0-15.3

7.4d

  

143

293

19.6

3.4

7.7 a

179

  

305
(266-355)

18.9
(15.7-23.0)

  

7.8d

  

170

312.5

22.8

4.2

8.2d

  

185

344

24.9

6.1

9.4d

  

218

427

30.4

11.6

10.5 a

269

  

445
(356-706)

30.3
(23.1-51.5)

  

a-c References 46-48: ARIC study (adults)

d Reference 49: DirecNet study (youth)

CGM: continuous glucose monitoring

 

Assays of 1,5-anhydroglucitol (1,5-AG) are an alternative measure of hyperglycemia. In the kidney, 1,5-AG is filtered by the glomeruli and reabsorbed in the proximal tubules. This reabsorption is competitively inhibited by glucose. When high glucose levels are associated with glycosuria, there is increased 1,5-AG excretion in the urine and lower serum levels. Concentrations of 1,5-AG reflect hyperglycemia-induced glycosuria over the prior 1-2 weeks. This test may be affected by pregnancy, advanced renal disease (CKD stages 4-5), and by use of SGLT-2 inhibitors.

 

There are few of studies demonstrating the usefulness of the fructosamine, glycated albumin, and 1,5-AG assays in predicting the development of diabetes-related complications (46, 50). Racial differences have also been reported for these assays (50). Since their clinical usefulness is not well established, testing is generally recommended in situations where HbA1c testing is expected to be inaccurate (e.g., abnormal red blood cell turnover).

 

Continuous Glucose Monitoring

 

Glucose data from continuous glucose monitors (CGM) are increasingly being used to assess glycemic control.  These data have the advantage of displaying glycemic patterns, glucose variability, time in target range, and time in hypoglycemia.  CGM is discussed in detail in the chapter entitled “Monitoring Technologies – Continuous Glucose Monitoring, Mobile Technology, Biomarkers of Glycemic Control” in the Diabetes section of Endotext.

 

EVALUATION OF HYPOGLYCEMIA

 

Symptomatic hypoglycemia is defined clinically using Whipple's triad, which includes the presence of symptoms (confusion, lightheadedness, loss of consciousness, seizure, aberrant behavior, sweating, palpitations, weakness, blurred vision, or hunger) at the time of a low plasma glucose level, with improvement of symptoms when plasma glucose levels return to normal (51). The physician should determine if the patient truly has hypoglycemia prior to seeking an etiology. A plasma glucose level < 55 mg/dl (3.1 mmol/l) should raise the suspicion for a hypoglycemic disorder and initiate further evaluation, but many authorities rely on a glucose <40 mg/dl (2.2 mmol/l) as being diagnostic (52). Although symptoms are commonly observed when plasma glucose falls to <55 mg/dl (3.1 mmol/l), levels of <45 mg/dl (2.5 mmol/l) are associated with cognitive dysfunction (neuroglycopenia). Capillary glucose determinations should not be used in the evaluation of hypoglycemic disorders due to their poor accuracy in these situations.

 

The Endocrine Society has published clinical practice guidelines for the evaluation and management of hypoglycemic disorders (53). In persons without diabetes, drugs or critical illnesses, hormone deficiencies, and non-islet cell tumors should be considered based on the clinical findings (54). If the cause of the hypoglycemia is not evident then plasma glucose, insulin, c-peptide, proinsulin, β-hydroxybutyrate, insulin antibodies, and a screen for oral hypoglycemic drugs should be obtained during an episode of spontaneous hypoglycemia. Glucagon 1 mg IV should then be administered with careful follow up of the glucose response. This will help determine if the condition is related to excessive endogenous insulin production. The diagnosis of pancreatic hyperinsulinemic hypoglycemia is supported by the demonstration that insulin secretion is not suppressed normally when the patient develops hypoglycemia. If testing cannot be conducted during an episode of spontaneous hypoglycemia, the prolonged fast or mixed meal test followed by the administration of glucagon is the most useful diagnostic study.

 

Some patients who have had bariatric surgery for the treatment of obesity, most commonly Roux-en-Y gastric bypass, will develop hypoglycemia. This is associated with abnormal OGTTs and mixed meal tests, abnormal transport of food to the small intestine, and, in some cases, hypersecretion of insulin and incretin hormones (55-58). Spontaneous hypoglycemia has been reported after islet auto-transplantation for chronic pancreatitis as well; a deficient glucagon response to hypoglycemia during a mixed meal test has been reported (59).

 

Prolonged Fast

 

The gold standard test in the evaluation of hypoglycemia is the 72-hour supervised fast although a 48-h fast is almost as effective in diagnosing patients with suspected insulinoma (60). The purpose of the fast is twofold. The first is to diagnose hypoglycemia as the cause of the patient's symptoms. The second is an attempt to determine the etiology of the hypoglycemia. Due to the risk of hypoglycemia, patients should be admitted to the hospital to undergo the fast in a monitored setting. The fast could be initiated in a carefully monitored outpatient facility, with the patient entering the hospital if the fast is not terminated prior to the closing of the site. Baseline bloodwork can include cortisol, growth hormone, glucagon, and catecholamines if deficient counterregulation is suspected.

 

During the fast, patients are allowed no food but can consume non-caloric caffeine-free beverages for up to 72 hours. The onset of the fast is the time of the last food consumption. During the fast all non-essential medications should be discontinued. Simultaneous insulin, c-peptide and glucose samples are obtained at the beginning of the fast and every 4-6 hours thereafter. Once the plasma glucose falls to <60 mg/dl, specimens should be taken every 1-2 hours under close supervision. Patients should continue activity when they are awake. The fast continues until the plasma glucose falls below 45 mg/dl (2.5 mmol/l) (plasma glucose <55 mg/dl (3.1 mmol/L) is recommended in the most recent Endocrine Society guidelines (53)) and symptoms of neuroglycopenia develop, at which time, insulin, glucose, c-peptide, oral insulin secretagogues, proinsulin and beta-hydroxybutyrate levels are obtained and the fast is terminated (52). Additional samples for insulin antibodies, anti-insulin receptor antibodies, IGF-1/IGF-2, and plasma cortisol, glucagon or growth hormone can also be obtained if a non-islet cell tumor, autoimmune etiology, or hormone deficiency is suspected. A glucagon tolerance test is then frequently performed to aid in diagnosis (Glucagon, 1 mg intravenously, administered with careful follow up of the glucose response every 10 minutes for 30 minutes. Further details regarding the glucagon tolerance test are below). Patients are fed at the conclusion of the test.

 

The diagnosis of endogenous hyperinsulinism is likely if the patient has neuroglycopenic symptoms, a fall in plasma glucose to <55 mg/dl, inappropriately elevated beta-cell polypeptides (insulin, proinsulin and c-peptide levels; see below table), with undetectable oral insulin secretagogue levels. β-hydroxybutyrate <2.7 mmol/L, and an increase in plasma glucose ≥25 mg/dL (1.4 mmol/L) after intravenous glucagon (53).

 

Table 16. Distinguishing Causes of Symptomatic Hypoglycemia (glucose < 55 mg/dl (3.0 mmol/l)) After a Prolonged Fast

Insulin (µU/mL)

C-peptide (nmol/L)

Proinsulin (pmol/L)

Oral hypoglycemic medication

Interpretation

≥3

<0.2

<5

No

Exogenous insulin

≥3

≥0.2

≥5

No

Endogenous insulina

≥3

≥0.2

≥5

Yes

Oral hypoglycemic (drug-induced)

a Insulinoma, non-insulinoma pancreatogenous hypoglycemia (NIPHS), post gastric bypass hypoglycemia.

Adapted from: Cryer, PE, et al. Evaluation and Management of Adult Hypoglycemic Disorders: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab 94:709-728, 2009

 

Approximately 75% of patients with insulinomas are diagnosed after a 24 hour fast and 90-94% at 48 hours. Although some experts advocate conducting the prolonged fast for only 48 hours (60), others disagree, arguing that prolonging the fast up to 72 hours minimizes misdiagnosis and maximizes the probability of diagnosing an insulinoma (61).

 

Limitations of the prolonged fast:

  • Normal subjects, especially young women, can occasionally have plasma glucose levels of <40 mg/dl (2.2 mmol/l)
  • Rare insulinomas suppress their release of insulin in response to hypoglycemia
  • Insulin levels can sometimes be artificially elevated in the presence of anti-insulin antibodies.

 

OGTT and Mixed Meal Test

 

When the diagnosis of the dumping syndrome is being considered, a modified OGTT has been recommended (62).  After an overnight fast, a 75-gm glucose load is administered.  Glucose levels are measured at baseline and every 30 min up to 180 min.  To diagnose hypoglycemia due to the dumping syndrome, a glucose reading of <50 mg/dL is observed, typically between 60 and 180 min after receiving the glucose load. 

 

For patients with hypoglycemic symptoms several hours after meals, an OGTT or mixed meal test may be performed. The mixed meal test has not been well standardized. This test is typically done after an overnight fast. Patients eat a meal similar to one that provokes their symptoms. If this is not possible then a commercial mixed meal may be used. Patients are then observed for several hours. Samples for plasma glucose, insulin, c-peptide, and proinsulin are collected prior to the meal and every 30 minutes thereafter for 5 hours. If symptoms occur prior to the end of the test then additional samples for the above are collected prior to administration of carbohydrates. If Whipple’s triad is demonstrated, testing for oral hypoglycemic drugs and testing for insulin antibodies should be done. Interpretation of test results is the same as for the 72-hour fast or spontaneous hypoglycemia

 

Glucagon Tolerance Test

 

The glucagon tolerance test serves as a supplemental study to aid in the diagnosis of hypoglycemic disorders when results from the prolonged fast are inconclusive. Following an overnight fast (or at the conclusion of a prolonged fast), 1 mg of glucagon is injected intravenously over 2 minutes. Plasma glucose and insulin levels are measured at baseline, and either 10, 20, and 30 minutes after glucagon, or at 3, 5, 10, 15, 20, and 30 minutes after glucagon injection. In normal patients, maximum insulin response occurs rapidly and usually does not exceed 100 uU/ml (peak insulin 61+19 uU/ml at 3-15 minutes), and the serum glucose levels peak at 20-30 minutes (140 +24 mg/dl) (63).

 

Insulinoma patients demonstrate an exaggerated insulin response to glucagon, with values often exceeding 160 uU/ml within 15-30 minutes of the injection (peak insulin 93-343 uU/ml at 15 minutes) (54). In the hypoglycemic patient at the conclusion of the prolonged fast, an increase in plasma glucose of >25 mg/dl (1.4 mmol/l) post-glucagon suggests an insulin-mediated etiology (63).

 

Patients with malnutrition or hepatic disease may be unable to have a hyperglycemic response to glucagon due to depleted hepatic glycogen stores. Insulin responses in these subjects may be increased but not to the degree seen in subjects with an insulinoma. Drugs such as diazoxide, hydrochlorothiazide and diphenylhydantoin can cause false negative results (62). Patients with non-islet cell tumors such as hemangiopericytomas and meningeal sarcomas can have similar glucose elevations (30 mg/dl) as subjects with insulinomas following glucagon injection (65).

 

Another limitation of the glucagon stimulation test is the failure of some insulinoma patients to hypersecrete insulin following glucagon injection. This problem was reported in 8% of patients with insulinomas in one study (66). In addition, patients with cirrhosis with portocaval anastomosis can have peak insulin levels that are indistinguishable from subjects with insulinomas. Obese subjects and patients with acromegaly can also have exaggerated peak insulin responses, as can patients treated with sulfonylurea drugs and aminophylline.

An additional disadvantage of this test is the danger of causing hypoglycemia after 90-180 min (66) as well as inducing nausea and vomiting. Because of the possibility of severe hypoglycemia, a physician needs to be present during the test.

 

Autoimmune Hypoglycemia

 

The insulin autoimmune syndrome is a rare condition whereby antibodies, either directed against insulin or against the insulin receptor, are responsible for hypoglycemia. Autoimmune hypoglycemia due to insulin antibodies should be suspected when the hypoglycemia is associated with high insulin levels (usually >100 uU/mL) and incompletely suppressed C-peptide levels. Insulin levels are rarely >100 uU/mL in the presence of hypoglycemia due to an insulinoma. Although these elevated insulin levels can be observed with exogenous insulin administration, the associated c-peptide levels are usually extremely low. Autoimmune hypoglycemia is most often seen in people of Japanese descent but has been described in other populations (67). Autoimmune hypoglycemia may also be due to antibodies to the insulin receptor. These patients will have mildly elevated insulin levels (thought to be due to decreased clearance of insulin) and suppressed c-peptide levels, and may have other autoimmune conditions. Antibodies to insulin and/or proinsulin and insulin receptor antibodies can interfere with the measurements of pancreatic hormones using immunoassays (68-69). Insulin, proinsulin and/or insulin receptor antibody testing is needed to confirm the diagnosis of autoimmune hypoglycemia. This testing does not need to be done at the time of hypoglycemia.

 

C-Peptide Suppression Test

 

C-peptide and insulin are secreted in equimolar concentrations in the pancreas, making c-peptide levels a good marker of endogenous insulin secretion. The c-peptide suppression test can be used to test for an insulinoma or to provide supplemental diagnostic information, especially if the results of a supervised fast are not definitive. The c-peptide suppression test must be carefully administered, since the patient is given intravenous insulin to induce hypoglycemia. The advantage of the test is that it is of much shorter duration than the supervised fast.

 

The c-peptide suppression test is performed following an overnight fast. The procedure is to infuse regular insulin, 0.125 U/kg body weight, intravenously over 60 minutes. Blood samples are obtained from the contralateral arm at 0, 30, 60, 90, and 120 minutes for determination of insulin, c-peptide, and plasma glucose levels. An abnormal result is a lower percentage decrease of c-peptide at 60 minutes compared to normative data appropriately adjusted for the patient's body mass index and age (70). For example, an abnormal result for a 45-year-old with a BMI of 25-29 kg/m2 would be <61% suppression of c-peptide at 60 minutes (70). An alternative method (Regular insulin 0.075 IU/kg/hr. infused intravenously over 2 hours) using a different classification plot has been proposed (71) but few data using it have been published.

 

Limitations of this test include the fact that some patients with a documented insulinoma have normal c-peptide levels including normal percent decrease in c-peptide levels. There is also the danger of inducing severe hypoglycemia. In addition, little data concerning the reliability, sensitivity and safety of this test are published.

 

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Pituitary Tumors in Childhood

ABSTRACT

 

The pituitary region in childhood can be mainly affected by two kinds of neoplasia: craniopharyngiomas and pituitary adenomas. Craniopharyngiomas accounts for 1.2 to 4% of all childhood intracranial tumors, at this age adamantinomatous with cyst formation is the most common histological type. Craniopharyngiomas are benign from a histological point of view, but they can be aggressive, invading surrounding tissues and bony structures. Clinical presentation is non-specific with neurological disturbances, such as headache and visual field defects, together with manifestations of endocrine deficiency. Pituitary adenomas constitute less than 3% of supra-tentorial tumors in children, they are less common in pediatric patients than in adolescents or adults. Prolactinoma is the most frequent adenoma type in children, followed by the corticotrophinoma and the somatotrophinoma. Non-functioning pituitary adenomas, TSH-secreting, and gonadotrophin-secreting adenomas are extremely rare in children, accounting for only 3-6% of all pituitary tumors. Presenting symptoms are typically related to endocrine dysfunction, rather than to mass effects. Pituitary adenomas in childhood may have a genetic cause and, in some cases, additional manifestations can occur as part of a syndromic disease. Therapeutic options depend on the tumor type, with surgical approach often remaining the first choice.

 

INTRODUCTION

 

Pituitary function depends on the integrity of the hypothalamo-pituitary axis and the functionality of numerous differentiated cell lines in the anterior pituitary lobe that specialize in specific hormone production. The development of these cell lines is the result of events during pituitary organogenesis that are under the sequential control of transcription factors (1). Any abnormality occurring in the pituitary gland, either congenital (congenital malformations, genetic abnormalities) or acquired (perinatal insults, tumors, infections), will cause profound alterations of the whole endocrine system.

Tumors in the pituitary region can be classified on the basis of topographic criteria as intra-, supra- para- or retrosellar (2). Intrasellar tumors are mostly represented by pituitary adenomas (more than 90% of all intrasellar lesions), while dys-embryogenetic lesions such as Rathke’s pouch cyst or pituitary blastomas are less frequent. The suprasellar tumors are dys-embryogenetic lesions of the midline such as craniopharyngiomas, germinomas, dermoid or epidermoid cysts, lipomas, teratomas, and hamartomas. Other tumors such as meningiomas or gliomas are uncommon during childhood or adolescence. Craniopharyngiomas, the most common cause of hypopituitarism in childhood, and adenomas are the most frequent lesions of the pituitary region in children and adolescents. Virtually all tumors of this region are benign.

 

This chapter aims at reviewing the most recent epidemiological, diagnostic, and therapeutic knowledge on pituitary tumors in childhood and adolescence.

 

CRANIOPHARYNGIOMAS

 

Craniopharyngiomas are rare embryonic malformations of the sellar and parasellar area with an incidence of 0.5 to 2 cases per million persons per year, 30 to 50% of all cases presenting during childhood and adolescence (3-7). They originate from squamous rest cells of the remnant of Rathke’s pouch between the adenohypophysis and neurohypophysis in the region of the pars tuberalis. Rathke’s pouch is a cystic diverticulum from the roof of the embryonic mouth that gives rise to the adenohypophysis and determines the induction of the neurohypophysis. Craniopharyngiomas represent 1.2 to 4% of all childhood intracranial tumors (8-10) and show a bimodal distribution during the first-second decade of life and then in the fifth, apparently without any gender difference (5, 7). The tumor generally originates in the suprasellar region (94-95%), purely suprasellar (20–41%) or both supra- and intrasellar (53–75%), whereas the purely intrasellar forms (5-6%) are less frequent (5). Extremely rare are forms originating in the III ventricle, in the rhinopharynx, in the sphenoid, or in other locations (5). In their pure form, the adamantinomatous form and papillary form are clinicopathologically distinct. In childhood and adolescence, its histological type is usually adamantinomatous with cyst formation (3-7).

 

The pathogenesis of adamantinomatous craniopharyngioma is characterized by the deregulation of the Wnt pathway, in particular by activating mutations in exon 3 of the CTNNB1 gene encoding for β-catenin (11-13). Otherwise, most of papillary craniopharyngioma show a BRAF V600E mutation, resulting into activation of MAPK pathway (13, 14). Papillary forms exhibiting BRAF V600E mutations are rarely found in the pediatric age range (15, 16).

 

Craniopharyngiomas are benign from a histological evaluation but they can be aggressive, invading surrounding bony structures and tissues; they commonly have cystic components that may be multiple and generally cause compression of adjacent neurological structures (3-7). The adamantinomatous form is more locally aggressive and is characterized by a higher rate of recurrence than the papillary form (17). The molecular basis of this phenomenon is still not defined; however, a recent study showed that tissue infiltration could be favored by signaling of tyrosine kinase (18).

 

Clinical Presentation and Diagnosis

 

The diagnosis of craniopharyngioma is often made late, sometimes years after the initial appearance of symptoms. Neurological disturbances, such as headache and visual field defects, together with manifestations of endocrine deficiency such as stunted growth and delayed puberty, are the common presenting symptoms of craniopharyngiomas (3-7). Among adult-onset craniopharyngioma patients, hormonal deficits at the time of diagnosis are much more pronounced when compared with childhood-onset craniopharyngioma patients (3). At diagnosis, endocrine dysfunction is found in up to 80% of patients (3-7). Reduced GH secretion is the most frequent finding, present in up to 75% of patients, followed by FSH/LH deficiency in 40%, and ACTH and TSH deficiency in 25% (3-7). Despite the fact that the tumor is frequently large at presentation, the pituitary stalk is usually not disrupted, and hyperprolactinemia secondary to pituitary stalk compression is found in only 20% of patients (3-7). Diabetes insipidus is also relatively uncommon, occurring in ~17% of patients (3-7, 19). An increase in weight tends to occur as a later manifestation, shortly before diagnosis (3-7). Then, the clinical combination of headache, visual impairment, decreased growth rate, and/or polydipsia/polyuria would be very suggestive of childhood craniopharyngioma in the differential diagnostic process (20).

 

To date, magnetic resonance imaging (MRI) before and after gadolinium application is the standard imaging for the detection for craniopharyngiomas. The neuroradiological diagnosis of craniopharyngiomas is based on the features of the lesion itself and on its relations with the surrounding structures. Particularly, the diagnosis is mainly based on the three characteristic components of the tumor: cystic, solid and calcified (5, 7, 21-23). The cystic component (Fig.1 and 2) constitutes the most important neoplastic part (up to the 70-75% of the total volume), and shows a variable signal depending on the chemical-physical properties of its content (24). A fluid content will appear hypointense in T1 and hyperintense in T2 while a lipid (due to cholesterol), methemoglobin or protein content will appear as hyperintense in T1 and T2 sequences. The solid portion shows an isointense signal in T1 and a hyperintense signal in T2 with enhancement after gadolinium, at variance with the cystic component (Fig. 3 and 4). However, enhancement after paramagnetic contrast is not a consistent feature (24). Computed tomography (CT) imaging is the only way to detect or exclude calcification, which is found in approximately 90% of tumors and therefore a crucial differentiating component for diagnosis (21-23).Calcification appears as areas of low signal in all sequences (23). The radiological appearance of non-homogeneous signal or a prevalent cystic component should not be regarded as a proof of a craniopharyngioma, since macroadenomas can also sometimes be characterized by patterns resembling craniopharyngiomas. Moreover, the craniopharyngioma, without evidence of calcification, could be confused with different neoplasms such as hypothalamic/chiasmatic astrocytomas, germ cell tumors, or Langerhans cell histiocytosis (24).

Figure 1. Resonance imaging T1-weighted sequences on coronal planes. Intra- and suprasellar craniopharyngioma in an 8-year-old boy presenting with reduced growth velocity and headache. This tumor has a total cystic component as shown by the hyper-intense spontaneous signal. (Kindly provided by S. Cirillo, II University of Naples)

Figure 2. Resonance imaging T1-weighted sequences on sagittal planes. Intra- and suprasellar craniopharyngioma in an 8-year-old boy presenting with reduced growth velocity and headache. This tumor has a total cystic component as shown by the hyper-intense spontaneous signal. (Kindly provided by S. Cirillo, II University of Naples)

Figure 3. Resonance imaging T1-weighted sequences on sagittal plane before IV gadolinium chelate administration. Extra-axial craniopharyngioma in the intra and suprasellar space, with non-homogenous signal due to calcifications and cysts, in a 7- year-old boy presenting with reduced growth velocity, sleepiness, and visual loss. (Kindly provided by S. Cirillo, II University of Naples).

Figure 4. Resonance imaging T1-weighted sequences on sagittal plane after IV gadolinium chelate administration. Extra-axial craniopharyngioma in the intra and suprasellar space, with non-homogenous signal due to calcifications and cysts, in a 7- year-old boy presenting with reduced growth velocity, sleepiness, and visual loss. After contrast medium non-homogenous enhancement of the solid component. (Kindly provided by S. Cirillo, II University of Naples).

Treatment Strategy

 

The treatment of craniopharyngioma is individualized on the basis of clinical presentation and several tumoral features such as dimension, location and extension, balancing the therapeutic radicality and the consequent risk of relapse with the onset of neurologic and endocrine complications. In instances where the onset of manifestation is an emergency with symptoms of raised intracranial pressure or rapid deterioration in visual function, to relieve these symptoms and prevent further visual deterioration initial surgical treatment, for hydrocephalus or tumor cyst decompression may be necessary prior to definitive treatment of the tumor (3-7).

 

To date, surgery remains the first treatment option in pediatric craniopharyngiomas. Craniopharyngiomas are characterized by variability of localization and a relationship with important structures like the hypothalamus, optic chiasm, third ventricle, and vessels of the circle of Willis: for this reason, there is no paradigmatic surgical treatment (25). The main purpose of surgery is significant tumoral removal and the operative approach is generally dictated by localization and extent of the craniopharyngioma. Optimal initial localization, without involvement of the hypothalamus and optic chiasm, allows one to aim at radical resection preserving visual and hypothalamic functions; indeed, purely infra-diaphragmatic as well as supra-diaphragmatic/infra-chiasmatic tumors have a favorable surgical outcome with higher gross total resection rates in experienced hands. Otherwise, lesions extending within the third ventricle and lesions beyond 3cm in diameter, independent of their localization, are characterized by a greater complexity of treatment and a worse therapeutic outcome. In effect, radical resection and attempting total neoplastic removal results in significantly impaired functional outcomes (26, 27) , so currently many prefer subtotal removal and subsequent radiotherapy. Aside from the traditional microscopic approach via the subfrontal or pterional craniotomy, transsphenoidal approaches and other minimal invasive surgical methods, e.g., catheter implantation into cystic formations of the tumor, have become popular (26, 27). The transsphenoidal approach is appropriate for infra-diaphragmatic lesions, whereas tumors with suprasellar extensions require a transcranial approach. Nevertheless, the extended transsphenoidal approach has been used in lesion with supradiaphragmatic extension, showing a higher frequency of endocrine and neurological complications compared to the use of the same technique for an intra-diaphragmatic one (28).

 

Radiotherapy is required in case of incomplete tumor removal, which is common for extra-sellar craniopharyngiomas, and can effectively be added to avoid recurrences, determining lower progression rates (21%) compared to subtotal surgery alone (71-90%) (28). In children, however, the benefit of any additional radiotherapeutic treatment should be balanced against the high risk of inducing hypopituitarism later in life. In a retrospective preliminary review aiming at evaluating the efficacy and toxicity of fractionated proton radiotherapy in the management of pediatric craniopharyngioma, local mass control was reported in 14 of 15 patients with few acute side effects and newly diagnosed panhypopituitarism, a cerebrovascular accident (from which the patient recovered), and an out-of-proton-field meningioma in a single patient who received previous radiotherapy as a long-term complications (8, 29).

 

Modern radiotherapy techniques allow a better conformation of the field of action, reducing the dose on the structures adjacent to the craniopharyngioma and the consequent adverse effects, particularly endocrine and visual ones. Currently, intensity modulated radiotherapy (IMRT) and proton beam therapy (PBT) have shown encouraging results in the pediatric population (25).

 

Further, therapeutic options for large cystic craniopharyngiomas are cyst drainage and intracystic instillation of Interferon-alpha, whereas instillation of bleomyicin is no longer used because of neurotoxicity due to leakage.Recently, a multicenter trial on the systemic use of peginterferon alpha-2b, administered subcutaneously, ended prematurely due to a lack of efficacy on the relapse prevention of the solid portion of the neoplasm (28). Relapse of craniopharyngioma occurs in about 35% of patients and the management of recurrence is influenced by previous therapy (30).

 

Currently, attention focuses on the potential of molecular target therapy. Agents that effect the Wnt pathway are not currently available, whereas evaluation of the use of vemurafenib and dabrafenib (BRAF inhibitors) and the combination of dabrafenib and trametinib (a MEK inhibitor) are showing encouraging results (14, 15, 31-33).

 

PITUITARY ADENOMAS

 

Pituitary adenomas are the most common cause of pituitary disease in adults but they are less common in children, becoming increasingly more frequent during the adolescent years (34-37). The estimated incidence of pituitary adenomas in childhood is still unknown since most published series included patients with onset of symptoms before the age of 20 yrs as pediatric patients. Pituitary adenomas constitute less than 3% of supra-tentorial tumors in children, and 2.3-6% of all pituitary tumors treated surgically (34, 35, 38, 39). The average annual incidence of pituitary adenomas in childhood has been estimated to be 0.1/million children (40). Among all supra-tentorial tumors treated during a 25-year period in a center, pituitary adenomas were diagnosed in only 1.2% of children (41). Pituitary carcinomas are rare in adults and extremely rare in children (42). The first, and probably unique, case of pituitary carcinoma in a child was described by Guzel et al. in 2008. A 9-year-old girl, with an history of hydrocephalus treated with ventriculoperitoneal shunt 3 years before, complained of progressive visual and gait disturbance, headache, and speech difficulties. Neurological examination revealed visual loss, papilledema, and dysarthria. Magnetic resonance revealed a large tumor mass in frontal region, multiple lesions in sellar-parasellar region, posterior fossa, and multiple intraspinal metastatic lesions. Gross total resection of frontal mass was performed, and the histopathological and immunohistochemical exams revealed a pituitary carcinoma. Despite of the post-operative use of temozolomide, the patient died after 2 months without response to this therapy (43). There is no consensus on the alleged greater invasiveness of pituitary adenomas in children than in adults, while a slightly greater prevalence in females has been reported (7, 34-36, 40). However, gender distribution reflects the relative contribution of the two main groups, PRL- and ACTH- secreting adenomas, which predominate in most series reported. Prolactinoma is indeed the most frequent adenoma histological type in children, followed by the corticotrophinoma and the somatotrophinoma (44). Non-functioning pituitary adenomas, TSH-secreting, and gonadotrophin-secreting adenomas are very rare in children, accounting for only 3-6% of all pituitary tumors. ACTH-secreting adenomas have an earlier onset and predominate in the pre-pubertal period, where interestingly male cases are more frequent, while GH-secreting adenomas are very rare before puberty, except in XLAG (7). Similar to adults, presenting symptoms are generally related to the endocrine dysfunction, such as growth delay and primary amenorrhea, rather than to mass effects (41, 42, 44-48). Symptoms of pituitary tumor presentation differ according to the tumor type as shown in Table 1 and detailed in the specific sections.

 

Table 1. Prevalence of Clinical Symptoms and Signs in Children/Adolescents with Pituitary Adenomas. Data drawn from ref. 47-53

 

 

PRL-secreting adenomas

 

ACTH-secreting

 adenomas

 

GH-secreting

 adenomas

 

 TSH-secreting

 adenomas

 

 Clinically non-functioning

adenomas

 

Acne

 

–

 

+

 

–

 

–

 

–

 

Delayed/arrest growth

 

-/+

 

+

 

–

 

++

 

++

 

Delayed/Advanced bone age

 

–

 

+

 

+

 

–/+

 

++

 

Delayed puberty

 

++

 

+

 

+

 

+

 

++

 

Early sexual development

 

–

 

++

 

–

 

–

 

–

 

Erythroses

 

–

 

+

 

–

 

–

 

–

 

Fatigue or weakness

 

–

 

+

 

–

 

+

 

–

 

Galactorrhea

 

+++

 

–

 

–/+

 

–

 

–

 

Gigantism/Acromegaly

 

–

 

–

 

++

 

–

 

–

 

Glucose intolerance

 

–

 

+

 

+

 

+

 

–

 

Gynecomastia

 

+

 

–

 

–/+

 

–

 

–

 

Headache

 

++

 

+

 

++

 

+

 

++

 

High school performance

 

–

 

+

 

–

 

–

 

–

 

Hirsutism

 

–

 

+

 

–

 

–

 

–

 

Hypertension

 

–

 

+

 

–/+

 

–/+

 

–

 

Menstrual irregularities

 

++

 

+

 

++

 

+

 

++

 

Mild hyperthyroidism

 

–

 

–

 

–

 

+

 

–

 

Osteoporosis

 

+

 

+

 

–

 

+

 

–

 

Premature thelarche

 

++

 

–/+

 

–

 

–

 

–

 

Primary amenorrhea

 

++

 

+

 

++

 

+

 

++

 

Sleep disturbances

 

–

 

+

 

–

 

++

 

–

 

Striae

 

–

 

+

 

–

 

–

 

–

Visual field defects

 

+++

––/+

 

+++

 

+++

 

+++

 

Weight increase

 

+

 

+

 

–

 

–

 

–

 

– Absent; –/+ rare; ––/+ very rare; + present; ++ frequent; +++ frequent in macroadenomas

 

PRL-SECRETING ADENOMAS

 

Prolactinomas are the most frequent pituitary tumors both in childhood and in adulthood, and their frequency varies with age and sex, occurring most frequently in females between 20-50 years (35, 44, 49-51). Also, pediatric prolactinomas are more frequent in girls, but earlier onset, larger adenoma volume, and higher serum prolactin levels are found in boys (52).

 

Clinical Presentation and Diagnosis

 

PRL-secreting adenomas are usually diagnosed at the time of puberty or in the post-pubertal period, and clinical manifestations vary in keeping with the age and sex of the child (34-36, 44, 50, 51). Pre-pubertal children generally present with a combination of headache, visual disturbance, growth failure, and amenorrhea (Table 1). Growth failure is not, however, a common symptom: in fact, in two different retrospective studies, 4% of 25 patients (51) and 10% of 20 patients (53) were reported to have short stature at the diagnosis of prolactinoma. Weight gain has been reported to occur in patients with hyperprolactinemia (54-56) but never described in children. In a re-evaluation of the young/adolescent patients with hyperprolactinemia admitted to the University Federico II from January 1st 1995 to December 31st 2004 (44, 57), short stature was found in 7 of 50 patients (14%), five girls and two boys, and another two patients, one girl and one boy, had their height below or at the 5th percentile and another 8 (3 girls) had their height between the 5th and 10th percentile. The height percentiles in the patients with extrasellar/invasive macroprolactinomas were lower than in those having smaller tumors (Fig. 5). Additionally, all girls presented with oligomenorrhoea or amenorrhea; most also had galactorrhea; gynecomastia was present in 12 of 21 boys (57.1%). The most common symptoms of prolactinomas in the peripubertal age are those associated with deficiency of the pituitary-gonadal axis. Menstrual irregularities in girls are common in all types of pituitary adenomas, except those causing Nelson’s syndrome (58). Galactorrhea should be carefully investigated by expressing the breast, because teenagers may not spontaneously refer to it as a symptom, and frequently it is not spontaneous. Headache and visual field defects predominate in patients bearing large adenomas (Table 2).

Figure 5. Height (shown as mean percentiles for age) and Body Mass Index in 50 patients with prolactinomas diagnosed before 20 years of age. Data from ref. (57).

 

Table 2. Presentation of Prolactinomas in Children and Adolescents: The Two-Decade Experience of the Department of Endocrinology and Oncology, University “Federico II” of Naples. Data from reference (57)

 

 

Microadenomas

 

Enclosed Macroadenomas

 

Extrasellar and/or Invasive Macroadenomas

 

Number

 

20

 

21

 

9

 

Girls/Boys

 

15/5

 

11/10

 

3/6

 

Age at diagnosis (yrs)

 

14.4±0.5

 

14.8±0.4

 

13.8±1.1

 

Basal PRL levels (μg/L)

 

138.4±21.6

 

671.4±161.9

 

2123±279

 

Tumor volume on MRI (mm3)

 

113.0±15.1

 

1145±145

 

2826±330

 

Symptoms (%)

 

 

 

 

Secondary or Primary Amenorrhea1

 

53.3%

 

72.7%

 

66.7%

 

Oligomenorrhea1

 

46.7%

 

18.2%

 

0%

 

Gynecomastia2

 

100%

 

60%

 

33.3%

 

Galactorrhea

 

42.8%

 

60%

 

33.3%

 

Visual field defects

 

0%

 

50%

 

66.7%

 

Headache

 

33.2%

 

80%

 

66.7%

 

Calculated only in 1girls or 2boys.

 

Impairment of other pituitary hormone secretion was reported to occur in a minority of patients at diagnosis (44, 51, 53, 58), and in some patient’s hypopituitarism developed after surgery. In a more recent analysis (59), we can confirm that only a minority of patients bearing large adenomas had a severe degree of hypopituitarism, while a very few patients with either microadenomas or enclosed macroadenomas had isolated hormone deficiency (Fig. 6). Macroadenomas at presentation are more likely in boys than in girls (37, 38, 44, 53, 60). In our series (57), microprolactinoma and enclosed macroadenomas were more frequent in females with a ratio of 1.7:1 while large macroprolactinomas were 2 times more frequent in males (Table 2).

Figure 6. Prevalence of pituitary deficit according with prolactinoma size in 50 patients at diagnosis. Data from ref. (57).

Hyperprolactinemic patients have a decrease in bone mineral density (BMD), and progressive bone loss has been demonstrated in untreated patients (61). Young hyperprolactinemic men were shown to have a more severe impairment of BMD than patients in whom hyperprolactinemia occurred at an older age (62). In 20 patients with diagnosis of hyperprolactinemia during adolescence, we found (63) significantly lower BMD values in adolescents than in young adult patients with hyperprolactinemia. This finding was confirmed in a large cohort of patients (57). In 22 patients all having a diagnosis of prolactinomas before the age of 18 yrs, the bone mineral density (BMD) in the lumbar spine was significantly lower than in age-matched controls (Fig. 7). The use of drugs to increase bone mass, such as amino bisphosphonates, has not been investigated.

Figure 7. Bone density (BMD) measured as g/cm2 or z-score in 22 patients with prolactinoma (individual data shown as solid circles) and their sex- and age-matched controls (data shown as mean ± SD). Data from ref. 52, modified from ref. (57).

The diagnosis of prolactinoma is based on the measurement of serum PRL levels and neuroradiological imaging. The differential diagnosis of hyperprolactinemia should consider any process interfering with dopamine (DA) synthesis, its transport to the pituitary gland, or its action at lactotroph DA-receptors. A single measurement of PRL levels is unreliable since PRL secretion is markedly influenced by physical and emotional stress. Basal PRL levels greater than 200ng/l are diagnostic, whereas levels between 100 and 200ng/ml and the presence of a mass requires additional investigation to rule out mass an effect of a non-functioning adenoma versus a prolactin- secreting adenoma. Some peculiar conditions should, however, be remembered (64). Serial serum PRL measurements at 0, 30 and 60 min after the needle was inserted into an antecubital vein is a valuable and simple measure to identify stress- related hyperprolactinemia in order to avoid diagnostic pitfalls and unnecessary treatments. It is important to exclude from the assay the monomeric PRL forms, big-prolactin (b-PRL), and big big- prolactin (bb-PRL); the latter may contain immunoglobulin (IgG) (65). These molecular complexes are seldom active but may be measured by the PRL assay. The absence of a clinical syndrome of hyperprolactinemia will suggest the presence of macroprolactin. The ‘high-dose hook effect’ can be a serious problem in the differential diagnosis between prolactinomas and non-functioning adenomas (NFPA): it is mandatory, in these cases and in every patient with a pituitary mass and hyperprolactinemia, to dilute PRL samples routinely (1:10 and 1:100 dilutions) or to use alternative methods to immunoradiometric assays. The difference between macroprolactinomas and ‘pseudoprolactinomas’ is essential to provide a correct treatment approach (66). This problem is, however, of little relevance in children and adolescents, as non-functioning macroadenomas are very rare at this age.

 

Treatment Strategy

 

The goals of prolactinoma treatment are the control of PRL excess and its clinical consequences, and the removal of pituitary adenoma. Today dopamine-agonists (e.g., bromocriptine, quinagolide, or cabergoline) should be considered the first treatment approach for pediatric prolactinomas (34, 44, 51-53, 59, 60). According to a recent study, dopamine-agonists should be started immediately at prolactinoma diagnosis even in case of severe visual impairment. If there are no improvement in visual defects and serum prolactin levels in the first 24-hours, early surgical treatment should be considered to avoid further visual deterioration and radiological signs of progression. The efficient use of dopamine-agonists reduces the necessity of surgical approach (52).

 

Situations requiring first-line neurosurgery typically occur in invasive macroadenomas: in these cases, the aim is resecting the tumor to relief the mass effect. Anyway, in these cases surgical cure usually cannot be obtained so medical therapy after debulking neurosurgery is required, with the benefit of a better response to anti-dopaminergic therapy due to the cytoreduction (67).

 

Treatment with dopamine-agonists is effective in normalizing PRL levels and shrinking tumor mass in the majority of adult patients with prolactinomas (34, 44, 51-53, 59, 60), preserving pituitary function and visual field in most cases (51). In children and adolescents, bromocriptine has been used successfully by several investigators (51, 68-71). In our series, bromocriptine at doses ranging from 2.5-20 mg/day orally normalized prolactin in 38.5% of patients (51). In the remaining patients, 10 with macro- (Fig. 8) and 6 with microprolactinoma (Fig. 9), PRL levels remained above the normal range despite a progressive increase of the dose of the drug. However, the possibility that some patients were indeed not taking bromocriptine appropriately cannot be ruled out as poor compliance to any chronic treatment is a well-known phenomenon in children and adolescents. In addition, some patients required drug discontinuation for intolerable side effects regarding the gastrointestinal tract. Both quinagolide, at doses ranging from 0.075-0.6 mg/day, or cabergoline, at doses ranging from 0.5-3.5 mg/week orally, two selective DA receptor subtype-2 selective agonists, have been reported to be effective in reducing PRL secretion and tumor size in most adult patients with prolactinoma, even in those previously shown to be poorly responsive or intolerant to bromocriptine (57). There are now data on cabergoline, showing that it is more effective and often better tolerated than bromocriptine, due to less and milder side effects. For these reasons cabergoline should be the initial treatment of choice.

Figure 8. Serum PRL response to different dopaminergic drugs, namely bromocriptine (BRC), quinagolide (CV), and cabergoline (CAB) in 15 children with macroprolactinomas. The shaded area represents the normal PRL range. Data are shown as nadir PRL values at diagnosis and during treatment. Data from ref. 51.

Figure 9. Serum PRL response to different dopaminergic drugs, namely bromocriptine (BRC), quinagolide (CV), and cabergoline (CAB) in 11 children with microprolactinoma. The shaded area represents the normal range. Data are shown as nadir PRL values at diagnosis and during treatment. Data from ref. (51).

Of our 50 cases (57), cabergoline induced normalization of PRL levels in all but 3 cases. Two of the three patients had large extrasellar macroprolactinomas (tumor volume of 4579 mm3 and 1983 mm3 respectively) with baseline PRL levels of 3300 μg/L and 1700 μg/L, respectively that progressively decreased but did not normalize after 2-7 years of treatment. Tumor shrinkage by 93.2% and 54.5% was seen in both patients. The third patient had a microprolactinoma (tumor volume=123.6 mm3) with a baseline PRL levels of 500 μg/L that progressively decreased to 88 μg/L at the last follow-up after 6 years of treatment and achieved tumor shrinkage by 53.9% (57). Only one case of pituitary apoplexy following cabergoline treatment in a young patient has been reported so far (72). Twelve of our 50 patients (one with enclosed macroprolactinoma and 11 with microprolactinoma) achieved the disappearance of the tumor so that they were withdrawn from treatment (57). In our former series, tumor shrinkage was observed in most patients with macroadenomas and even in some with microprolactinomas (Fig. 10). The easy weekly administration makes cabergoline an excellent therapeutic approach to children/adolescents with prolactinoma. Cabergoline has been reported to be tolerated, even at rather high doses (73). Relevant safety issues to be considered in patients treated with cabergoline are possible cardiac valve derangement (74-76) and psychiatric adverse effects (mood changes or obsessive behavior including hypersexuality). These phenomena were first described in patients with Parkinson’s disease, who require higher doses of the dopamine agonists than patients with prolactinomas, but has now been documented in patients with pituitary adenomas as well. Cardiac safety of treatment with cabergoline in prolactinomas, even long-term, has been demonstrated in adults (77, 78), so use in children should also be safe, although we need to be aware of cumulative dose builds up if treatment has been started in childhood. Knowledge about psychiatric consequences of dopamine agonists used in pediatric prolactinomas is still scant. Psychotic symptoms during bromocriptine therapy were observed in a child by Hoffman et al. (52). Bulwer et al. also described a case of an adolescent male with a giant prolactinoma who developed impulsive/compulsive sexual symptoms during cabergoline treatment. These were diagnosed as an iatrogenic effect, a hypothesis supported by symptomatic improvement during a one-month trial off cabergoline (79). Despite the rarity of both pediatric prolactinomas and development of psychiatric side effects of dopamine agonists, this important aspect it needs to be further investigated.

 

In patients with tumors resistant to dopamine agonists as well as in those showing severe neurological symptoms at diagnosis, surgery is indicated. Radiotherapy should be limited to the cases with aggressive tumors, non-responsive to dopamine agonists, because of the risk of neurological damage, hypopituitarism, and second malignancies later in the lives of these patients (44, 51-53, 57).

Figure 10. Tumor mass response after bromocriptine, quinagolide, or cabergoline treatment in 15 children with macro- and 11 with microprolactinoma. Data are shown as number of cases with empty sella; greater than 50% tumor shrinkage; 20-50% tumor shrinkage or less than 20% tumor shrinkage shown as unmodified tumor volume. Data from ref. (57).

ACTH-SECRETING ADENOMAS

 

Cushing's disease (CD), caused by an ACTH-secreting pituitary corticotroph adenoma, is the commonest cause of Cushing’s syndrome (CS) in children over 5 years of age (80, 81). CS can occur throughout childhood and adolescence; however, different etiologies are commonly associated with particular age groups with CD being the commonest cause after the pre-school years. The peak incidence of pediatric CD is during adolescence (81). A macroadenoma is rarely the cause of CD in children; pediatric CD is almost always caused by a pituitary microadenoma with diameter <5 mm with a significant predominance of males in pre-pubertal patients (80, 81).

 

The molecular basis of pediatric Cushing’s disease is complex. Recently, pathological variants of USP8 gene have been found in an elevated number of ACTH-secreting adenomas; in the pediatric population USP8 mutated adenomas are clinically distinguished from wild-type adenomas for older age at diagnosis, female preponderance, and more frequent recurrence. In USP8 wild-type adenomas, BRAF and USP48 mutations have been noted. In pediatric corticotrophinomas, the presence of copy number variations, indicating chromosomal instability, has been related to larger size and more frequent invasion of the cavernous sinus (82).

 

There are other extremely rare germline conditions that can predispose to the development of pediatric corticotrophinoma such as DICER1, CABLES1, and CDKN1B mutations. DICER1 syndrome is characterized by pituitary blastomas, and manifest itself in early infancy with a highly deadly Cushing’s syndrome. CABLES1 is another potential ACTH-secreting adenoma predisposition gene, whose mutation has been found in very few pediatric cases. CDKN1B mutation occurs in the MEN4 syndrome, in which pituitary tumors arise usually in adults, as no gene mutations have been found analyzing children bearing a pituitary adenoma (82).

 

Clinical Presentation and Diagnosis

 

The clinical manifestations of CD are mostly the consequence of excessive cortisol production. The clinical presentation is highly variable, with signs and symptoms that can range from subtle to obvious (Table 1). The diagnosis is generally delayed since a decrease in growth rate may be the only symptom for a long time. Growth failure in CD may be due to a decrease of free IGF-I levels and/or a direct negative effects of cortisol on the growth plate (83, 84). In a series of 50 children with CD, Magiakou et al. (85) found that obesity and growth retardation were the most frequent symptoms (in 90 and 83% of patients, respectively). Weight gain and stunted growth were the most frequent symptoms also in the series by Weber et al. (86) and Devoe et al. (87). The skin of the face is plethoric, and atrophic striae can be found in the abdomen, legs, and arms. Muscular weakness, hypertension, and osteoporosis, especially of the spine, are common. Results on BMD or bone metabolism in children with CD have been reported only in a limited number of patients in a few studies (86, 88). Consistent with the findings in adult patients, marked osteopenia was also found in affected children. The bone loss is more evident in trabecular than in cortical bone (89). As compared to patients with adult-onset disease, those with childhood-onset CD have a similar degree of bone loss at the lumbar spine and similar increased bone resorption (90). In a study conducted in 10 patients with childhood-onset and 18 with adulthood-onset CD, BMD at the lumbar spine was significantly lower than in sex and age-matched controls (Fig. 11) (90). Osteoporosis was found in 16 patients (57.1%) (8 adolescent (80%) and 8 adult (44·4%) patients) while osteopenia was found in 12 patients (42.8%) (2 adolescent (20%) and 10 adult (55·6%) patients) (90). Additionally, we have reported that two years of cortisol normalization improved but did recover bone mass and turnover neither in children nor in adult patients with CD (91). This negative finding suggests that a longer period of time is necessary to restore bone mass after the cure of CD and, thus, other therapeutic approaches may be indicated to limit bone loss and/or accelerate bone recovery in these patients (87). In a study Lodish et al. (92) analyzed retrospectively, 35 children with CD; in these patients, vertebral BMD was more severely affected than femoral BMD and this effect was independent of degree or duration of hypercortisolism. BMD for the lumbar spine improved significantly after TSS; osteopenia in this group may be reversible. Complete reversal to normal BMD was not seen.

Figure 11. Z score of bone density at lumbar spine in 10 patients with childhood onset Cushing’s disease compared to 10 healthy adolescents of matched sex- and age and in 18 patients with adult-onset Cushing's disease compared to 18 healthy adults matched sex- and age. Data from ref. (90).

Hypercortisolism leads to decreased bone formation through direct or indirect inhibition of osteoblast function, while bone resorption is normal or increased in patients with CD (90, 93). Hypercortisolism is known to be associated with loss of skeletal mass and can lead to increased vertebral fracture risk (94, 95). It should also be noted that in children with CD the direct negative effect of hypercortisolism on bone formation is further worsened by concomitant hypogonadism and GH deficiency, both of which are associated with decreased BMD. Children with CD often have musculoskeletal weakness and can have decreased weight-bearing activity that may contribute to impaired BMD.

 

Children with CD may also have impaired carbohydrate tolerance, while overt diabetes mellitus is uncommon. Excessive adrenal androgens may cause acne and excessive hair growth, or premature sexual development in the first decade of life. On the other hand, hypercortisolism may cause pubertal delay in adolescent patients. Peculiarly, young patients with CD may present neuropsychiatric symptoms which differ from those of adult patients. Frequently, they tend to be obsessive and are high performers at school.

 

The differential diagnosis of CD includes adrenal tumors, ectopic ACTH production, and the very rare ectopic CRH-producing tumors. However, ectopic ACTH secretion is extremely rare in the pediatric age. In a child/adolescent with suspected CD the diagnosis is based on measurement of basal and stimulated levels of cortisol and ACTH. Measurement of 24-h urinary free cortisol is elevated, and a low dose of dexamethasone (15 μg/Kg) at midnight does not induce suppression of morning serum cortisol concentrations as in normal subjects (96). Loperamide, an opioid agonist, lowers cortisol secretion and has been proposed as a reliable screening test for hypercortisolism in children and adolescents (97) , but has not achieved popular use. Suppression of the spontaneous circadian variations of serum cortisol is another feature of CD. Suppression of cortisol by more than 50% after high-dose dexamethasone (150 μg/kg) given at midnight will confirm that hypercortisolism is due to an ACTH-secreting pituitary adenoma (97). Midnight salivary cortisol measurements have been suggested as an alternative non-invasive screening test in the diagnosis of CS in adults(98), but is there is not much experience of its use in this age group.

 

All patients should undergo pituitary MRI with the administration of gadolinium, but since ACTH- secreting pituitary adenomas are significantly smaller than all other types of adenomas, often having a diameter of 2mm or less (99), pituitary MRI may fail to visualize the tumor. In most instances the diagnosis of CD can be made by initial clinical and laboratory data (Fig.12). Bilateral inferior petrosal sinus sampling has a high specificity, so that no patient with extra-pituitary CS runs the risk of being submitted to transsphenoidal surgery, but it carries a significant number of false negative results (99). This procedure can also be technically difficult in children, and the risk of morbidity from surgery and/or anesthesia must be considered.

 

Lateralization of the adenoma can be of greater help to the surgeon than pituitary scanning (100). Therefore, bilateral venous sampling should only be performed in centers with wide experience in the technical procedure as well as in the interpretation of the results. If a patient without anomalous venous drainage patterns exhibits a lateralizing ACTH gradient of 2:1 or greater (101, 102), removal of the appropriate half of the anterior pituitary gland will be curative in 80% of cases (99). Kunwar and Wilson (99) reported that in the presence of a negative surgical exploration, a guide to the probable location of the adenoma is invaluable, and under the right circumstances, a hemi-hypophysectomy is appropriate and successful in most cases.

Figure 12. The diagnosis of Cushing’s syndrome. LDST, low dose suppression test; HDST, high dose suppression test; CRH, corticotrophin releasing hormone. Data from ref. (36).

Treatment Strategy

 

The goal of the treatment of Cushing’s disease are normalization of cortisol levels, reversion of hypercortisolism-related signs and symptoms, and pituitary adenoma removal. Transsphenoidal adenomectomy is the treatment of choice for ACTH-secreting adenomas in childhood and adolescence, because of the greater prevalence of microadenomas in this population that allows for total tumor removal and thus disease remission. Radiotherapy could be the first-line treatment in children with surgical contraindications (103). Transsphenoidal microsurgery is considered successful when it is followed by remission of signs and symptoms of hypercortisolism and by normalization of laboratory values. Surgical excision is successful in the majority of children, with initial remission rates of 70-98% and long-term cure of 50-98% in most studies (38, 39, 80, 81, 84, 86, 87, 104-109). The success rate decreases when the patients are followed-up for more than 5 years (84, 86, 87), and the outcome cannot be predicted either by preoperative or immediate postoperative tests (87). Surgical cure was found in 59% of 27 patients over a 21-year period, with a higher age favoring cure, as did an identifiable tumor seen at surgery and positive histology (110). Several conditions are indeed predictors of Cushing’s disease recurrence in children: older age at the time of disease symptoms, younger age at the time of surgery, larger tumor diameter, and mutations in USP8 gene in resected tumor tissue (111).The recurrence rate of Cushing’s disease in children in about 40% in 10 years (67).

Noteworthy in pediatric patients there are several technical difficulties with the transsphenoidal surgical approach due to a different anatomic conformation in children compared to adults. In children, the smaller size of the sella and pituitary may interfere with surgical maneuvers, in addition to the difficult identification of surgical landmarks due to different anatomic variations of sellar region such as the shorter intercarotid distance and piriform aperture, and the low pneumatization of sphenoid bone. Furthermore, in children with skull base lesions short nasal-sellar and vomer-clivus distances and smaller transsphenoidal angles than healthy children have been noted (112).

 

Surgery is usually followed by adrenal insufficiency and patients require hydrocortisone replacement for 6-12 months. After normalization of cortisol levels, resumption of normal growth or even catch-up growth can be observed. Generally, final height is compromised compared to target height (68, 85). Johnston et al. (113) have, however, reported that some children do achieve a normal final stature. However, even if catch-up and favorable long-term growth can be achieved after treatment for Cushing's disease, post-treatment GH deficiency is frequent (114). Lebrethon et al. (114) demonstrated that early hGH replacement may contribute to a favorable outcome on final stature (Fig.13). A re-analysis of this series confirmed that pediatric Cushing’s disease patients achieve a normal final stature provided that replacement therapy including GH is correctly performed (115). Normal body composition is more difficult to achieve. Many patients remain obese and BMI SDS was elevated at mean interval of 3.9 years after cure in 14 patients (115).

 

Rarely, surgery may induce panhypopituitarism, permanent diabetes insipidus, and cerebrospinal fluid leak (46); transient diabetes insipidus, and cerebrospinal fluid leak occur more frequently in pediatric patients than adults (116). Probably such a higher prevalence may be due to technical difficulties related to the anatomy of pediatric sellar region. Cure is more likely to be achieved and morbidity is low if the surgery is performed by an experienced neurosurgeon, by analogy with other studies performed in acromegaly (113).

Figure 13. GH treatment in children with Cushing’s disease improves the height gain. Upper graph: Evaluation of growth (change (D) in height SD score) in eight patients during hGH treatment. Bottom graph: Individual changes of height standard deviation score before and after GH replacement. 1= At diagnosis; 2= Before GH treatment; 3= After 1 year of GH treatment; 4= Final height. Data drawn from ref. (114).

In recent times, the endonasal approach, consisting in a direct access of neurosurgeon to the sellar region through the patient’s nares, has also been used in pediatric Cushing’s disease, usually using an endoscope aiming to improve surgical visualization. However, because of small patients’ nares, an expert neurosurgeon is required (112). Nowadays, while there is little evidence of this less invasive technique are available, it seems to be an effective treatment for pediatric CD, without relapses observed in treated patients (109, 117, 118). However, no studies comparing microscopic and endoscopic endonasal technique in pediatric pituitary adenomas are available (67).

 

The treatment modality in patients who have relapses after transsphenoidal adenomectomy is still controversial. Some authors recommend repeat surgery (84, 119), while others favor radiotherapy (120, 121). Transsphenoidal surgery is actually suggested as a second-line treatment in recurrent or persistent CD patients (103), and is required in case of incomplete initial tumor removal, in patients with tumor reappearance after initial complete surgical resection, or even in persistent patients in the days immediately after first TSS aiming to optimize therapeutic efficacy (116). The efficacy of a second surgical treatment in Cushing’s pediatric disease is still unclear, due to the presence in literature of only single case reports, or case series. Interestingly, in one of this case series Lonser et al. reported an initial diseaseremission in 93% of patients (122).

 

Radiotherapy techniques currently used for pediatric corticotrophinomas are divided in two groups: conventional radiotherapy (RT) and stereotactic RT. Conventional RT, in which small, daily radiation doses are delivered to the target tumor over a 25-30 days period, and stereotactic RT, where high radiation doses are delivered to a more precisely identified target area, minimizing radiation exposure to the surrounding central nervous system structures. Stereotactic RT could be performed as a single treatment, called stereotactic radiosurgery such as gamma-knife radiosurgery, or as a fractionated treatment, called stereotactic conformal radiotherapy.

 

Disease remission using conventional radiotherapy is reached by about 80% of pediatric corticotrophinoma (113, 121, 123-127). Conventional RT is a safe treatment, as no nerve damage or other major complications were observed in treated patients. Compared to adults with Cushing’s disease treated with Conventional RT as a second-line treatment, this seems to be slightly more effective in pediatric corticotrophinoma (116). There are only two studies concerning gamma-knife radiosurgery in the pediatric population, reporting a remission rate of 87.5 % and 79.2%, respectively (128, 129). Radiotherapy usually requires time before reaching its maximum result, and for this reason pharmacotherapy could be considered as a temporary treatment until this achievement (103, 116). Of note, hypothalamo-pituitary dysfunction is an early and frequent complication of radiation (87).

 

For the medical therapy of Cushing’s disease, three pharmacological categories are currently available: pituitary-directed agents, adrenal-directed agents, and glucocorticoid receptor antagonists. Scientific evidence regarding their use in pediatrics is scant, and there are no data on the use of mifepristone in pediatric patients.

 

Data on the use of etomidate, an adrenal-directed agent, in the emergency management of severe hypercortisolemia seems to be promising. There are at least 3 case reports demonstrating that intravenous infusion of etomidate at doses ranging from 1 to 3.5 mg/h, with constant dose titration according to serum levels, adding contemporaneous hydrocortisone infusion at 0.25-0.5 mg/kg/h to prevent adrenal insufficiency is a safe and effective approach in patients with very severe Cushing’s disease prior to bilateral adrenalectomy (130-132).

 

Moreover, experience with cabergoline for CD in childhood and adolescence is also limited (133). Bilateral adrenalectomy is actually a third-line treatment, employed in cases of surgical and radiotherapeutic failures. This therapeutic approach has gradually lost his importance in the context of pediatric corticotrophinoma treatment, due to the side effects and the growing evidence of pharmacological treatment as valid therapeutic alternative (116, 119).

 

It is interesting that in pediatric Cushing’s disease patients, in contrast to adult ones, there does not appear to be complete recovery from cognitive function abnormalities despite rapid reversibility of cerebral atrophy (134).

 

GH-SECRETING ADENOMAS

 

GH excess derives from a GH-secreting adenoma in over 98% of cases. In adulthood, these adenomas are relatively rare with an incidence of 1.1 new cases/100,000 individuals per year, and a prevalence from 3 to >13 cases per 100,000 individuals according to the country under study (135) , while gigantism is extremely rare with a little bit more than 400 reported cases to date (136, 137). In childhood, GH-secreting adenomas account for 5-15% of all pituitary adenomas (138). In less than 2% of the cases excessive GH secretion may depend on a hypothalamic or ectopic GH releasing hormone (GHRH)-producing tumor (gangliocytoma, bronchial or pancreatic carcinoid), which causes somatotroph hyperplasia or a well-defined adenoma (139-142).

 

Nowadays, approximately 50% of patients with pituitary gigantism have a known genetic mutation causing the disease, so genetic counselling should be considered (143). In this genetic context, pituitary gigantism could be part of syndromic, or non-syndromic disease. Recently, non-syndromic pituitary gigantism has been described due to aryl hydrocarbon receptor-interacting protein (AIP) gene mutations and Xq26.3 microduplication causing X-linked acrogigantism (XLAG) (144-146). AIP mutations occur in about 40% of gigantism cases, sporadically or in the setting of familial isolated pituitary adenoma (FIPA)., and patients with truncating AIP mutation had a younger age at disease onset and diagnosis, compared to patients with non-truncating AIP mutation (146).

 

Typically, AIP-mutated adenomas bear several features: early disease is manifest usually in the second decade of life, the majority of the cases are GH- or mixed GH/prolactin-secreting pituitary adenomas (144, 146), the tumors are large and invasive, often with suprasellar extension, resistance to first-generation SSA treatment is common, thus require a multimodal treatment and pituitary apoplexy can often occur, especially in pediatric patients (82, 137, 146). Interestingly, AIP-mutated patients with GH excess had been shown to be taller than the non-mutated counterparts (147).

 

XLAG represents 10% of the cases of pre-pubertal gigantism (143). X-LAG is due to a submicroscopic chromosome Xq26.3 duplications that include GPR101 gene, which is differentially overexpressed in the affected pituitary adenoma (82, 148). Duplications are germline in females and somatic in sporadic males with variable levels of mosaicism in the latter (82). Somatic mosaicism occurs in sporadic males but not in females with XLAG syndrome, although the clinical characteristics of the disease are similarly severe in both sexes (148). Three rare case of families in which the germline duplication was transmitted from the affected mother to son have been described, and all carriers of the duplication had gigantism (149). The disease often occurs during the first year of life, mostly in females and as sporadic disease.

 

Regarding the characteristics of the pituitary gland at diagnosis in these patients, most of them harbor macroadenomas, generally mixed GH- and PRL-secreting tumors, while a minority have hyperplasia alone (82, 137, 145). A pattern of multiple microadenomatous foci against a hyperplastic background has also been described (82).Noteworthy is the peculiar presence of acromegalic features in these pediatric patients, and a poor response to SSA treatment such as AIP-mutated somatotrophinomas (82).

 

Concerning syndromic pituitary gigantism, Carney Complex and McCune-Albright syndromes contribute to gigantism approximately in 1% and 5% respectively, where pituitary hyperplasia or a distinct pituitary adenoma could be found in the pituitary gland (82, 143). GH-secreting adenomas may also occur in MEN1 syndrome and cause 1% of cases of pituitary gigantism; the possibility of pituitary hyperplasia due to GHRH hypersecretion from neuroendocrine tumor should be considered in this syndromic context (143).

 

Somatotrophinomas could also be one of the manifestations of the MEN4 syndrome and the pheochromocytoma/paraganglioma and pituitary adenoma association (82) (151).

GH excess and consequent gigantism could be a rare manifestation of NF-1 syndrome, characterized by the presence of optic pathway gliomas but not pituitary adenomas, in addition to the characteristic syndromic manifestations. In this case it can be speculated that GH secretion could be either due to loss of somatostatinergic inhibition or presence of excessive GHRH secretion due to disrupted regulation of GHRH by the optic pathway tumor (82).

 

Clinical Presentation and Diagnosis

 

In adults, chronic GH and IGF-1 excess causes acromegaly, which is characterized by local bone overgrowth, while in children and adolescents leads to gigantism. The associated secondary hypogonadism delays epiphysial closure, thus allowing continued long-bone growth (Fig.14). However, the two disorders may be considered along a spectrum of GH excess, with principal manifestations determined by the developmental stage during which such excess originates (Table 1). Supporting this model has been the observation of clinical overlap between the two entities, with approximately 10% of acromegalics exhibiting tall stature (150), and the majority of giants eventually demonstrating features of acromegaly (151).

 

Gigantism predominantly affects males (78%), is generally characterized at diagnosis by the presence of a macroadenoma, often invading surrounding structures (54.5%), and prolactin co-secretion is present in 34% of pituitary adenomas causing pituitary gigantism (143). As demonstrated, older age at diagnosis, and the consequent longer time of exposure to higher GH and IGF-1 levels than normal, is associated with an increased prevalence of many pathological signs and symptoms, particularly those related to longer-term exposure such as joint disease, facial changes, skin changes, and diabetes mellitus (136, 143). In contrast to adults where there is an increased prevalence of cardiovascular, respiratory, neoplastic, and metabolic complications (136, 141, 152), there is no report of similar complications in childhood.

 

In our study, we did not find any patient with hypertension, arrhythmias, diabetes or glucose intolerance; as expected, however, some degree of insulin resistance and enhanced ß-cell function was observed in our patients at diagnosis (153). In a study conducted in six patients with gigantism, Bondanelli et al. (154) showed that 33% of giant patients had left ventricular hypertrophy and inadequate diastolic filling, 16.7% had isolated intraventricular septum thickening and impaired glucose metabolism. In acromegaly, clinical features develop insidiously and progressively over many years and in modern epidemiological studies the average delay between the onset of symptoms and diagnosis is approximately 5 years (135), while the presentation of gigantism is usually dramatic and the diagnosis is straightforward. All growth parameters are affected although not necessarily symmetrically. Mild-to-moderate obesity occurs frequently (138), and macrocephaly has been reported to precede linear and weight acceleration in at least one patient (155). All patients also had coarse facial features, disproportionately large hands and feet with thick fingers and toes, frontal bossing and a prominent jaw (138). In girls menstrual irregularity can be present (156) while glucose intolerance and diabetes mellitus are rare. Tall stature and/or acceleration of growth velocity was observed in 10 of 13 patients. Headache, visual field defects, excessive sweating, hypogonadism, and joint disorders may also be present (143). Several cases of ketoacidosis have been reported(157, 158).

 

The diagnosis of acromegaly and gigantism is usually clinical, and can be readily confirmed by measuring GH levels, which in more than 90% of patients are above 10 μg/l (139-141). The oral glucose tolerance test (OGTT) is the simplest and most specific dynamic test for both the diagnosis and the evaluation of the optimal control of GH excess (139-141). In healthy subjects, the OGTT (75-100 grams) suppresses GH levels below 1 μg/l after 2 hours, while in patients with GH-secreting adenoma such suppression is lacking, and a paradoxical GH increase is frequently observed. GH excess should be confirmed by elevated circulating IGF-I concentrations for age and gender (159, 160). The assay of IGF-I binding protein-3 is conversely not useful for diagnosis nor for the follow-up of the patients (161, 162). The presence of different GH isoforms in patients with gigantism/acromegaly may represent a diagnostic problem (163). A greater sensitivity of the GH assay may facilitate the distinction between patients and normal subjects, as shown by the use of a chemiluminescent GH assay (164). It might help in demonstrating the persistence of GH hypersecretion after surgery or during medical therapy. In cases of clinical and laboratory findings suggestive of a GH-producing adenoma, pituitary MRI must be performed to localize and characterize the tumor (141-143) (Fig. 15).

 

Figure 14. The patient’s growth and weight chart with normal growth and weight curves (solid lines, 5th, 50th, 75th, and 95th percentile). Measurements subsequent to therapeutic intervention. Reproduced from (165), with permission.

Figure 15. The extent of tumor invasion as visualized with coronal and lateral MRI views and their outlines. Reproduced from (165) with permission.

Treatment Strategy

 

The objectives of treatment of GH excess are tumor removal with resolution of its eventual mass effects, restoration of normal basal and stimulated GH secretion, relief of symptoms directly caused by GH and IGF-1 excess, and prevention of progressive disfigurement, bone expansion, osteoarthritis and cardiomyopathy which are disabling long-term consequences, as well as prevention of hypertension, insulin resistance, diabetes mellitus and lipid abnormalities that are risk factors for vascular damage (139-141). The currently available treatment options for pituitary gigantism include surgery, radiotherapy, and pharmaco-therapeutic suppression of GH levels.

 

For pituitary gigantism treatment, combination therapy is often necessary due to the aggressiveness of the disease, and the consequent low rate of primary control using both surgical and medical first approach, 26 % and 4% respectively (143). Satisfactory results are obtained in the treatment of hyperprolactinemia using dopamine agonists in prolactin co-secreting adenomas.

 

Transsphenoidal adenomectomy is the cornerstone in the treatment of GH-secreting tumors, and is a valid first-line therapeutic option (143). In pediatric patients with gigantism, transsphenoidal surgery was found to be as safe as in adults (166), although some technical difficulties exist due to the different anatomic conformation in children compared to adults as previously reported in the ACTH-secreting adenoma section. The surgical approach can be difficult in McCune-Albright syndrome patients due to the fibrous dysplasia in the surrounding tissue.

 

In patients with intrasellar microadenomas, surgical removal provides biochemical control with normalization of IGF-I in 75–95% of patients (167, 168). In case of macroadenomas, particularly when they exhibit extrasellar growth, transcranial approach might be requested, and persistent postoperative hypersecretion of GH occurs frequently. Despite this, tumor debulking contributes to improving disease control using medical therapy (143). In most surgical series, only about 60% of acromegalic patients achieve circulating GH levels below 5 μg/l (169-173), with better success score when the neurosurgeon is skilled in pituitary surgery (169, 170).

 

Concerning gigantism, for medical treatment it is necessary to consider that several drugs used for acromegaly are not formally studied in children, and for those employed drug-dosing is labelled for adults and might not be directly applicable in pediatric patients. Treatment with somatostatin analogues can be effective in patients with GH excess (150, 174, 175), although limited data are available in adolescent patients. Octreotide given subcutaneously in two patients was shown to inhibit GH levels and reduce growth velocity (176, 177). Of interest, in adolescents, as in adults, we observed tumor shrinkage by 30% on average after first-line treatment with somatostatin analogues. Whether this treatment has facilitated the subsequent surgical approach in this series could not be ruled out because of the limited number of cases studied. Treatment was tolerated very well by all patients (153).

 

 As about one third of patients had concomitant hyperprolactinemia and combined treatment with dopaminergic compounds such as cabergoline and somatostatin analogues, may be necessary.

 

In another case of a 15 yr-old girl with a mixed GH/PRL-secreting adenoma (165), octreotide-LAR (at the dose of 20 mg/28 days) combined with cabergoline (at the dose of 0.5 mg twice/week) normalized serum GH and IGF-I levels, and decreased growth rate from 12 cm/yr to nearly 2.5 cm/yr. This association has be proven to be effective also in an adolescent bearing a somatotropinoma in the context of McCune-Albright syndrome (178). In seven of the eight hyperprolactinemic patients included in our study, combined treatment with octreotide plus bromocriptine or octreotide-LAR or lanreotide plus cabergoline was effective and well tolerated by all patients. Only two patients (15.4%) of the entire series still presented with active acromegaly after treatment with surgery and pharmacotherapy with somatostatin analogues plus dopamine-agonists(153).

 

Although long-acting somatostatin analogues have been shown to be effective and safe in pediatric patients, this therapy often fails to achieve disease control especially in the most frequent genetic forms of pituitary gigantism (AIP-mutated adenomas and X-LAG acrogigantism), which are characterized by poor responses to first generation SSAs. Recently the successful use of pasireotide LAR has been reported in two cases of AIP-mutated gigantism not controlled by surgery and first-generation somatostatin analogues, respectively. Pasireotide LAR allowed not only biochemical control but also the reduction of the pituitary adenoma volume. These patients developed pasireotide-induced diabetes, controlled by drug therapy (179).

 

The GH receptor antagonist pegvisomant is a very potent drug which has been introduced into clinical practice. In patients with resistant acromegaly, the use of the GH-receptor antagonist pegvisomant was followed by normalization of IGF-I levels in more than 80% of patients (180-182). However, there are few data related to pediatric patients. In a 12-year-old girl with tall stature (178 cm), bearing a GH/PRL-secreting macroadenoma inoperable since tumor tissue was fibrous and adherent to the optical nerves, the GH receptor antagonist at a dose of 20 mg/day completely normalized IGF-I levels (183). In a 3.4 year-old girl with a GH/prolactin-secreting adenoma, treatment with pegvisomant and cabergoline was effective to normalize IGF-I levels and height velocity without side effects (184). Combined therapy with the addition of pegvisomant to octreotide LAR rapidly allowed biochemical control in three children with pituitary gigantism, pituitary tumor size did not change despite concomitant therapy with a somatostatin analogue (185). The main limit of pegvisomant is the eventual adenoma size increase during treatment, requiring treatment suspension. Pegvisomant was successfully used also in the youngest known patient with AIP-related pituitary adenoma, in which despite of the previously transsphenoidal surgery, and the medical treatment after surgery with temozolomide, subsequently in addition to bevacizumab, IGF-1 was normalized only after pegvisomant treatment (186).

 

Radiation therapy is rarely used in pediatric patients, and is generally is considered only after the failure of both primary surgical and medical therapies, because of a maximum response is achieved 10–15 years after radiotherapy is administered (187, 188), and the involvement of surrounding structures in the radiation-induced damage. Radiation-induced damage of the surrounding normal pituitary tissue results in hypogonadism, hypoadrenalism, or hypothyroidism in most patients within 10 years (187), whereas complications such as optic nerve damage, cranial nerve palsy, impaired memory, lethargy, and local tissue necrosis have been reduced thanks to improved precise isocentric simulators and accurate dosing techniques. At long term follow-up, about 43% of patients with pituitary gigantism among who undergone to secondary radiotherapy have shown controlled GH and IGF-I levels (136). Noteworthy, as a consequence of the multiple operations and radiotherapy, 64% of patients develop hypopituitarism during long-term follow-up (143).

 

TSH-SECRETING ADENOMAS

 

This tumor type is rare in adulthood and even rarer in childhood and adolescence with only a few cases reported so far (189). Plurihormonal adenomas with GH and TSH co-secretion can also occur. It is frequently a macroadenoma presenting with mass effect symptoms such as headache, visual disturbance, together with variable symptoms and signs of hyperthyroidism (Table 1). TSH-secreting adenomas must be differentiated from the syndrome of thyroid hormone resistance (190). In most cases, the classical criteria of lack of TSH response to TRH stimulation, elevation of serum α-subunit levels, and a high α-subunit/TSH ratio along with a pituitary mass on MRI, are diagnostic of a TSH-secreting adenoma (190).

 

Treatment Strategy

 

Transsphenoidal surgery is the first treatment approach to these tumors. However, since the majority of these adenomas are macroadenomas, which tend to be locally invasive, surgery alone fails to normalize TSH and thyroid hormone levels in most cases. In adults, radiotherapy is recommended as routine adjunctive therapy when surgery has not been curative (190). However, due to the high frequency of post-radiotherapy hypopituitarism, in children pharmacotherapy is the preferred second choice. There is very little success with dopamine agonists for treatment of these tumors (191). In contrast, therapy with somatostatin analogues normalizes TSH levels in the majority of patients, and tumor shrinkage occurs in approximately half of cases (192-195) and shown be useful in children as well. Rabbiosi et al., first used lanreotide successfully as first-line treatment in a pediatric patient bearing a macroadenoma characterized by a low probability of complete surgical eradication due to its antero-superior extension. The response to medical treatment was optimal, with significant tumor shrinkage and development of central hypothyroidism after few months. Thus, suggesting that preoperative somatostatin analogue treatment used for tumor shrinkage may be helpful to prepare a hyperthyroid patient to surgery (189). Before this somatostatin analogue in the pediatric age had only been used in two post-pubertal boys (189). Chronic treatment with SR-lanreotide reduced plasma TSH and normalized fT4 and fT3 levels, suggesting its use in the long-term medical treatment of these adenomas (190, 195).

 

CLINICALLY NONFUNCTIONING ADENOMAS

 

Clinically non-functioning adenomas (NFAs) are extremely rare in childhood, compared with adults (196). Nonetheless, there is in vitro and in vivo evidence that almost all of these tumors synthesize glycoprotein hormones or their subunits (197, 198). In adults, NFAs represent 33-50% of all pituitary tumors, while in pediatric patients they account for less than 4-6% of cases (38, 40, 44), for this reason incidentally discovered adenomas in childhood are rare (199). In a study, 5 out of 2288 patients treated at Hamburg University between 1970-1996 were diagnosed to bear a clinically NFAs (196). In a most recent surgical series, 9 out 85 pituitary adenomas (10.6%) were NFAs in the pediatric group (107). Most silent adenomas arise from gonadotroph cells, the clinical presentation includes visual field defects, headache, and some degree of pituitary insufficiency since invariably all patients had a macroadenoma (Table 1). Recent data show that hypogonadism is the most frequent pituitary deficiency at diagnosis occurring in 71.4% of pediatric patients, followed by TSH deficiency (33.3%), and GH and ACTH deficiency (both 11.1%). No case of diabetes insipidus occurred in this pediatric series (107). Larger macroadenoma could also cause hydrocephalus due to the obstruction of foramen of Monro (199). A modest hyperprolactinemia can also be present due to pituitary stalk compression (196).

 

Treatment Strategy

 

The first approach to these adenomas, silent and even functioning, is transsphenoidal surgery to remove tumor mass and decompress parasellar structures. As in the other adenoma types, surgery has a low morbidity and leads to an improvement of visual symptoms in the majority of cases. Endoscopic endonasal unilateral transsphenoidal approach to the pituitary (204), which has the same indications as the conventional transsphenoidal microsurgery, overcomes many of the potential problems tied to the surgical route, thanks to its minimal invasiveness. This procedure involves no sublabial dissection nor any fracture of the facial bones with dental or naso-sinusal complications. Furthermore, a wider surgical vision of the operating field is obtained, which potentially improves the likelihood of a better and safer tumor removal. In addition, this procedure requires a shorter hospitalization, permits a rapid recovery of the child (205), and maintains neuroendocrine-pituitary integrity, with ensuing normal growth. This approach can also be safely used for the surgical removal of remnant pituitary tumors (206). After surgery these patients partially recover from hypopituitarism. Postoperative radiotherapy can be used in patients with subtotal tumor removal to prevent tumor re-growth and reduce residual tumors, but is burdened by a high prevalence of pan-hypopituitarism (207-209).

 

Medical therapy has poor effects on clinically non-functioning adenomas (197, 210), and data are from adults. A positive response to cabergoline associated with detection of dopamine receptors in vitro has been proven in clinically non-functioning adenomas (211). Positive effects of cabergoline were observed in some patients with α-subunit secreting adenomas, mostly in patients with tumors expressing high number of dopamine D2 receptors (212). Greenman et al. proved that dopamine agonists treatment in adult patients with NFAs is associated with decreased prevalence of residual adenoma growth after neurosurgery. A decrease in residual mass was observed in 38% of patients treated immediately after surgery, while a stable or enlarged residual adenoma was observed in 49% and in 13%, respectively. A significant shrinkage or stabilization of residual mass was achieved (58%) also in patients in which the administration of the same therapy was performed when residual growth was noted during the post-operative follow-up (213). In vitro, chimeric dopamine/SSTR agonists are effective in inhibiting cell proliferation in two-thirds of non-functioning adenomas (214). Somatostatin analogues and dopamine agonists have not been tested in children/adolescents with clinically non-functioning adenomas.

 

Concerning medical therapy for functioning gonadotroph adenomas, there is little published information in the literature about the use of dopamine agonists, somatostatin analogues, GnRH agonists, and antagonists in the pediatric age range (203).

 

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Diabetic Retinopathy

ABSTRACT

Diabetic retinopathy is a significant life-altering complication affecting patients with diabetes. Understanding its pathogenesis, prevention, and treatment is critical to delivering effective and comprehensive care for patients with diabetes at all stages. This review discusses the risk factors, epidemiology, pathogenesis, clinical features, and treatment options for diabetic retinopathy, with an emphasis on practical information useful for endocrinologists and other non-ophthalmologists.

INTRODUCTION

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and a leading cause of blindness worldwide and in the US (1-3). The individual lifetime risk of DR is estimated to be 50–60% in patients with type 2 diabetes and over 90% in patients with type 1 diabetes (4). It is the most frequent cause of blindness in adults between 20-74 years of age in developed countries (5). The same pathologic mechanisms that damage the kidneys and other organs affect the microcirculation of the eye (6). With the global epidemic of diabetes, one expects that diabetes will be the leading global cause of vision loss in many countries (1,2). While DR is specific for diabetes, other eye disorders, such as glaucoma and cataracts, occur earlier and more frequently in people with diabetes (5).

Often, by the time patients seek ophthalmologic examination and treatment, there are significant alterations of the retinal microvasculature. Therefore, it is important for non-ophthalmologists to recognize the importance of eye disease in patients with diabetes so that appropriate referral to eye-care specialists can be a part of their diabetes management program.

EPIDEMIOLOGY 

In the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR), the prevalence of DR in patients with type 1 diabetes was 17% in those with less than 5 years of diabetes vs 98% in those with 15 or more years of diabetes (6). Proliferative diabetic retinopathy (PDR) was absent in patients with type 1 diabetes of short duration but present in 48% of those with 15 or more years of diabetes. In patients with type 1 diabetes, the 25-year rate of progression of DR was 83%, with progression to PDR occurring in 42% of patients (7). Improvement of DR was observed in 18% of patients with type 1 diabetes. In the WESDR, 3.6% of patients with type 1 diabetes were legally blind, and 86% of the blindness was attributable to DR (8). The risk of blindness increases with the duration of diabetes.

In the WESDR, patients with type 2 diabetes of less than 5 years had a prevalence of DR of 28%, while in patients with greater than 15 years of diabetes, the prevalence was 78% (6). A considerable number of patients with type 2 diabetes (12-19%) have DR at the time of the diagnosis of diabetes (1). The prevalence of PDR was relatively low in patients with type 2 diabetes (2%) in patients with less than 5 years duration vs 16% in patients with greater than 15 years duration of diabetes (6). The prevalence of DR and PDR was greater in the patients with type 2 diabetes using insulin. In the patients with type 2 diabetes, 1.6% were legally blind, and one-third of cases of legal blindness were due to DR (8).

Of note, the WESDR cohort is 99% white, and data suggest a higher prevalence of DR in Mexican-Americans and African-Americans with type 2 diabetes (6,9,10). Asians appear to have the same or lower prevalence of DR (1,10). DR occurs in both males and females with diabetes, but males appear to be at a slightly higher risk (9). Diabetic macular edema (DME) occurs more commonly in patients with type 2 diabetes, and with the marked increase in the prevalence of type 2 diabetes, DME is becoming more common (2). DME is over two times more prevalent than PDR (9).

In a pooled analysis of 35 studies between 1980 and 2008, among 22,896 individuals with diabetes, the overall prevalence of DR was 34.6%, PDR 6.96%, DME 6.81%, and vision-threatening DR 10.2% (11). The longer the duration of diabetes, the greater the prevalence of all of these diabetic eye manifestations (11). Moreover, the prevalence of DR, PDR, and DME was greater in patients with type 1 diabetes (77%, 32%, and 14%) compared to patients with type 2 diabetes (32%, 3, and 6%) (1,11).

In developed countries the incidence and the risk of progression of DR have greatly declined in patients with type 1 and type 2 diabetes (1,2,12). The WESDR showed that from 1980 to 2007, the estimated annual incidence of PDR decreased by 77%, and vision impairment decreased by 57% in patients with type 1 diabetes (12). In an analysis of 28 studies with 27,120 patients, the rates of DR and PDR were lower among participants in 1986-2008 than in 1975-1985 (13). Thus, patients with recently diagnosed type 1 or type 2 diabetes in developed countries have a much lower risk of PDR, DME, and visual impairment as compared with patients who developed diabetes in the past (1,12). This marked decrease in the prevalence and incidence of DR and vision impairment is likely due to improved glycemia control, early screening for eye disease, and the more aggressive treatment of blood pressure (1). However, in countries with limited medical resources, this reduced risk of DR and vision impairment is not occurring (2).

In caring for patients with diabetes, health care providers must bear in mind the substantial risks of developing visual loss that these patients face and the treatments that can reduce this risk. For affected patients, diabetes-related visual loss decreases the quality of life and interferes with the performance of daily activities.

RISK FACTORS

Hyperglycemia

The most important treatable risk factor for the development of DR is hyperglycemia. In patients with both type 1 and type 2 diabetes, elevated HbA1c levels are associated with an increased risk and progression of DR (2,7,14-16). Most importantly, randomized controlled trials comparing intensive glycemic control vs. usual care demonstrated a decrease in DR. A meta-analysis of 6 relatively small randomized trials prior to the publication of the Diabetes Control and Complications Trial (DCCT) reported that after 2 to 5 years of intensive therapy the risk of retinopathy progression was significantly reduced (OR 0.49, p = 0.011) (17). Intensive therapy significantly retarded retinopathy progression to more severe states such as PDR or changes requiring laser treatment (OR 0.44, p = 0 018) (17).

The DCCT was a randomized, controlled study of intensive glycemic control (HbA1c approximately 7%) vs. usual care (HbA1c approximately 9%) in 1,441 patients with type 1 diabetes (18). This study found that intensive glucose control reduced the risk of developing retinopathy by 76% compared to usual care (18). In patients with pre-existing retinopathy, intensive control slowed progression of the DR by 54% (18). For every 10% reduction in HbA1c (e.g., 10% to 9% or 9% to 8.1%) the risk of retinopathy progression was reduced on average by 44% (19).The DCCT participants were followed in an observational Epidemiology of Diabetes Interventions and Complications (EDIC) study. During the EDIC study, the mean HbA1c levels became very similar in the intensive and usual care group, with the HbA1c of the intensive treatment group increasing to approximately 8% and the usual care group HbA1c decreasing to approximately 8% (19). Despite the similar A1c levels in the 2 groups over 30 years there continued to be an approximately 50% risk reduction of further DR progression and the development of PDR and DME in the original intensive control group, a phenomenon termed metabolic memory (19). These results indicate the need for early intensive glucose control.

In the Kumamoto study, 110 patients with type 2 diabetes were randomly assigned to a multiple insulin injection treatment group (MIT group) or to a conventional insulin injection treatment group (CIT group) and followed for 6 years (20,21). HbA1c levels were 7.1% in the MIT group and 9.4% in the CIT group. Moreover, the development of DR after 6 years was 7.7% for the MIT group and 32.0% for the CIT group in the primary-prevention cohort (no microvascular disease at baseline) (P = 0.039), and progression of DR occurred in 19.2% of the MIT group and 44.0% of the CIT group in the secondary-intervention cohort (microvascular disease at baseline) (P = 0.049). This study demonstrated that improved glycemic control reduced DR in patients with type 2 diabetes.

In the UK Prospective Diabetes Study (UKPDS), 3,867 newly diagnosed patients with type 2 diabetes were randomized to diet therapy alone or to sulfonylureas or insulin with the goal of achieving a fasting glucose of 108 mg/dL (6mMol/L) in those treated with sulfonylureas or insulin (intensive group). Over 10 years, HbA1c levels were approximately 7.0% in the patients treated with sulfonylureas/insulin therapy compared with 7.9% in the diet group. This study found a 25% reduction in the risk of microvascular endpoints, including the need for diabetic retinal laser treatment, with intensive glucose control (22). A risk reduction of 21% per 1% decrease in HbA1c was observed in this trial. Patients were closely followed after the study ended, and HbA1c levels after one year became similar in the two groups. Similar to the results seen in the DCCT/EDIC study, the benefits on microvascular disease persisted in the intensive control group, confirming the concept of metabolic memory in patients with type 2 diabetes (23).

The ACCORD study was a randomized trial that enrolled 10,251 individuals with type 2 diabetes who were at high risk for cardiovascular disease to receive either intensive or standard treatment for glycemia (HbA1c 6.4% vs. 7.5%). A subgroup of 2,856 individuals were evaluated for the effects of intensive vs. standard care at 4 years on the progression of diabetic retinopathy by 3 or more steps on the Early Treatment Diabetic Retinopathy Study Severity Scale. After 4 years, the rates of progression of diabetic retinopathy were 7.3% in the intensive group vs.10.4% in the standard therapy group (odds ratio, 0.67; P=0.003) (24). It should be noted that in an analysis of the entire ACCORD study cohort, three-line change in visual acuity was reduced in the intensive control group (HR 0.94, CI 0.89-1.00; p=0.05) but no differences in photocoagulation, vitrectomy, or severe visual loss were observed (25). Four years after the ACCORD trial ended, DR progressed in 5.8% of the intensive treatment group vs.12.7% in the standard treatment group (odds ratio 0.42, P < 0.0001) (26), once again confirming the concept of metabolic memory.

It should be noted that two large cardiovascular outcome trials, the ADVANCE trial and the VADT, failed to demonstrate a benefit of intensive glucose control on diabetic retinopathy (27,28). However, a meta-analysis of the four large cardiovascular outcome studies in patients with type 2 diabetes (UKPDS, ACCORD, ADVANCE, and VADT) found that more intensive glucose control resulted in a decrease in HbA1c of -0.90% and a 13% reduction in the need for retinal photocoagulation therapy or vitrectomy, development of PDR, or progression of DR (29). Another meta-analysis of 7 trials with 10,793 participants reported a 20% decrease in DR with intensive glycemic control (0.80, 0.67 to 0.94; P=0.009) (30).

Taken together, these results clearly demonstrate that in patients with both type 1 and type 2 diabetes, improvements in glycemic control will reduce the risk of the development and progression of DR.  

 Rapid Improvement in Glycemic Control

Deterioration of DR, upon initiation of intensive diabetes treatment, was described in the 1980s in patients with type 1 diabetes who were treated intensively with continuous subcutaneous insulin infusions (31-34). In patients with poor glycemic control and DR, rapidly improving glycemic control can worsen DR and, in some instances, result in PDR or DME. This worsening can occur as soon as 3 months after initiating intensive glycemic control. In the DCCT early worsening was observed at the 6- and/or 12-month visit in 13.1% of patients in the intensive treatment group and in 7.6% of patients assigned to conventional treatment (odds ratio, 2.06; P < .001) (35). In the DCCT the most important risk factors for early worsening of DR were a higher HbA1c level and reduction of this level during the first 6 months of treatment (35). It must be recognized that in the DCCT the long-term outcomes in intensively treated patients who had early worsening were similar to or more favorable than outcomes in conventionally treated patients (35). This early worsening of DR with improved glycemic control has also been described in patients with type 2 diabetes treated with insulin or GLP-1 agonists, following bariatric surgery, in pregnant women with diabetes, and following pancreatic transplants in patients with type 1 diabetes (36). The mechanism(s) leading to early worsening of DR with improvements in glycemic control are unknown (36).

While this worsening is distressing, it must be recognized that the long-term benefits of improving glycemic control on DR greatly outweigh the risks of early worsening. Ophthalmologic evaluation should be obtained prior to initiating intensive treatment and close monitoring should occur at 3-month intervals for 6 to 12 months in patients with significant pre-existing DR.

Hypertension

In the WESDR, blood pressure (BP) was not related to incidence or progression of retinopathy in the patients with type 2 diabetes using insulin or the type 2 patients not using insulin, but in the patients with type 1 diabetes systolic BP was a significant predictor of the incidence of DR (37). In contrast, in the UKPDS and other studies high BP in patients with type 2 was associated with the development of DR (2,16,38). In one prospective study the risk of DR increased by 30% for every 10 mm Hg increase in systolic BP at baseline (39).

While observational studies can show an association, randomized controlled trials are required to demonstrate causation and the benefits of treatment. A number of studies have examined the effect of lowering BP in patients with hypertension on the development and progression of DR.

STUDIES IN PATIENTS WITH HYPERTENSION

The UKPDS examined the effect of tight vs. less tight BP control in 1,148 hypertensive patients with type 2 diabetes (40). In the tight BP control group (captopril and atenolol), BP was significantly reduced compared to the less tight group (144/82 mm Hg vs.154/87 mm Hg; (P<0.0001). After nine years the tight BP control group had a 34% reduction in the deterioration of retinopathy (P=0.0004) and a 47% reduced risk (P=0.004) of deterioration in visual acuity. Additionally, patients in the tight BP group were less likely to undergo photocoagulation (RR, 0.65; P = .03), a difference primarily due to a decrease in photocoagulation due to maculopathy (RR, 0.58; P = .02) (41). In contrast to glycemic control, the benefits of lowering BP were not sustained when therapy was discontinued and the differences in blood pressure were not maintained, indicating the absence of metabolic memory (42).

The HOPE study was a randomized study that compared ramipril vs. placebo in 3,577 participants with diabetes who had a previous cardiovascular event or at least one other cardiovascular risk factor (43). The baseline BP was approximately 142/80 mm Hg, and BP decreased by 1.92/3.3 mm Hg in the ramipril group vs a 0.55 mm Hg increase in systolic BP and 2.30 mm Hg decrease in diastolic BP in the placebo group.  This study was not focused on DR but did report that the need for laser was 9.4% in the ramipril group vs. 10.5% in the placebo group (22% decrease; p=0.24).

The ADVANCE study examined the effect of BP control on DR in 1,241 patients with type 2 diabetes (44). Patients were randomized to BP-lowering agents (perindopril and indapamide) or placebo and followed for approximately 4-5 years. Baseline BP was approximately 143/79 mm Hg. In the group randomized to BP medications, a decrease in systolic BP of 6.1 ± 1.2 mmHg and diastolic BP of 2.3 ± 0.6 mmHg was observed (p < 0.001 for both).  Fewer patients on BP lowering therapy experienced new or worsening DR compared with those on placebo (OR 0.78; 95% CI 0.57–1.06; p = 0.12), but the difference was not quite statistically significant. Certain secondary outcomes were significantly reduced (for example DME) in the BP lowering group, but most other eye end points were not significantly decreased compared to the placebo group.

The ACCORD eye study evaluated 2,856 patients with type 2 diabetes for the effect of intensive BP control (BP<120 mm Hg) vs standard BP control (BP<140 mm Hg) on the progression of DR after 4 years of treatment (24). Systolic BP was 117 mm Hg in the intensive-therapy group and 133 mm Hg in the standard-therapy group. The progression of DR was 10.4% with intensive blood-pressure therapy vs. 8.8% with standard therapy (adjusted odds ratio, 1.23; P=0.29).

The Appropriate Blood Pressure Control in Diabetes (ABCD2) Trial was a randomized blinded trial that compared the effects of intensive versus moderate BP control in 470 patients with type 2 diabetes and hypertension (45). The intensive group was treated with either nisoldipine or enalapril, while the usual care BP group received placebo. The mean blood pressure achieved was 132/78 mm Hg in the intensive group and 138/86 mm Hg in the moderate group. Over the 5-year follow-up period, there was no difference in the progression of DR between the intensive and moderate groups.

Thus, in patients with hypertension, randomized trials of lowering BP have not consistently shown beneficial effects on DR.

BASIS FOR VARIABILITY

There are numerous possible explanations for the differences in results between these studies. First, the severity of the hypertension may be important, with greater responses in individuals with higher BP levels. Second, the magnitude of the reduction in BP may be important, with greater benefit with greater decreases in BP. Third, the duration of the study may be an important variable, with the longer the study the greater the chances of benefits. Fourth, the presence of DR at baseline and the severity of DR at baseline may influence the response to BP lowering. Fifth, patient variables such as glycemic control, age, diabetes type, duration of diabetes, etc., may influence results. Finally, the drugs used to lower BP may be a key variable as described below.

STUDIES IN PATIENTS WITH NORMAL BP

Because of the potential benefits of angiotensin converting enzyme inhibitors (ACE inhibitors) and angiotensin receptor inhibitors (ARBs) (Renin-Angiotensin System (RAS) inhibitors) on microvascular disease independent of BP effects, a number of studies have explored the effects of these drugs on DR in patients without elevated BP. Below we briefly describe the largest of these studies.

The EUCLID trial was a randomized double-blind placebo-controlled trial in 354 patients with type 1 diabetes who were not hypertensive and were normoalbuminuric (85%) or microalbuminuric (46). Study participants were randomized to lisinopril or placebo and followed for 2 years. Systolic BP was 3 mm Hg lower in the lisinopril group than in the placebo group. DR progressed in 23.4% of patients in the placebo group and 13.2% of patients in the lisinopril group (p=0.02). Notably progression to PDR was also reduced in the lisinopril treated group.

The Appropriate Blood Pressure Control in Diabetes (ABCD1) trial was a randomized trial in 480 normotensive type 2 diabetic subjects of more intensive vs. usual BP control (47). The intensive group was treated with either nisoldipine or enalapril, while the usual care BP group received placebo. Mean BP in the intensive group was 128/75 mm Hg vs. 137/81 mm Hg in the placebo group (P < 0.0001). After a mean follow-up of 5.4 years, the intensive BP control group demonstrated less progression of diabetic retinopathy (34% vs. 46%, P = 0.019). PDR developed in 0% of patients in the intensive therapy group vs. 3.9% in the placebo group. However, in patients who at baseline did not have DR, the number of patients developing retinopathy was similar in the two groups (39% of patients in the intensive therapy group vs. 42% in the placebo group).

The DIRECT- Prevent 1 trial was a randomized, double-blind, placebo-controlled trial in 1,421 normotensive, normoalbuminuric individuals with type 1 diabetes without retinopathy (48). Patients were randomized to candesartan or placebo and followed for 4.7 years. Mean systolic and diastolic BP was reduced by 2.6 mm Hg and 2.7 mm Hg, respectively, in the candesartan group vs. the placebo group. DR developed in 25% of the participants in the candesartan group vs. 31% in the placebo group (18% decrease). 

The Direct Protect 1 was a randomized, double-blind, placebo-controlled trial in 1,905 normotensive, normoalbuminuric patients with type 1 diabetes with existing retinopathy (48). Patients were randomized to candesartan or placebo and followed for 4.7 years. Mean systolic and diastolic BP was reduced by 3.6 mm Hg and 2.5 mm Hg, respectively, in the candesartan group versus the placebo group. There was an identical 13% progression of DR in the placebo and candesartan groups, and progression to the combined secondary endpoint of PDR or clinically significant DME, or both, did not differ between the two groups.

The DIRECT-Protect 2 trial was a randomized, double-blind, placebo-controlled trial in 1,905 normoalbuminuric, normotensive, or treated hypertensive people with type 2 diabetes with mild to moderately severe retinopathy (49). Patients were randomized to candesartan or placebo and followed for 4.7 years. The decrease in systolic/diastolic blood pressure was 4.3/2.5 mm Hg greater in the candesartan group than in the placebo group in individuals who were receiving antihypertensive treatment at baseline (p<0·0001 for both), and for those not on anti-hypertensive therapy at baseline the decrease was 2.9/1.3 mm Hg (p=0.0003/p=0.0045). The risk of progression of retinopathy was non-significantly reduced by 13% in patients on candesartan compared to the placebo group (HR 0.87; p=0.20). However, regression on active treatment was increased by 34% (HR 1.34; p=0.009), and overall change towards less severe retinopathy by the end of the trial was observed in the candesartan group (odds 1.17; p=0.003).

The RASS trial was a controlled trial involving 223 normotensive patients with type 1 diabetes and normoalbuminuria and who were randomly assigned to receive losartan, enalapril, or placebo (50). The systolic and diastolic BP during the study were lower in the enalapril group (113/66 mm Hg) and the losartan group (115/66 mm Hg) than in the placebo group (117/68 mm Hg) (P<0.001 for the two systolic and P≤0.02 for the two diastolic comparisons, respectively). After 5 years progression in DR occurred in 38% of patients receiving placebo but only 25% of those receiving enalapril (P=0.02) and 21% of those receiving losartan (P=0.008).   

META-ANALYSIS OF ACE INHIBITORS AND ARBS             

Many of the studies described above used either an ACE inhibitor or an ARB with variable results on DR. To better understand the effect of RAS inhibitors on DR, a meta-analysis has extensively examined these studies and a number of other trials (51). In 7 studies with 3,705 participants without DR, RAS inhibitors reduced the development of DR by 27% (p= 0.00006). This decrease in the development of DR was seen in patients with both type 1 and type 2 diabetes and patients who were hypertensive or normotensive. In 16 studies with 9,580 participants with pre-existing DR, RAS inhibitors decreased the progression of DR by 13% (p=0.00006). This decrease in progression of DR was seen in patients with both type 1 and type 2 diabetes and patients who were normotensive. In hypertensive patients there was a trend (7% decrease) that was not statistically significant. It should be noted that in the hypertensive patients RAS inhibitors were compared to other hypertensive drugs, and the number of hypertensive participants was relatively small (n=839). Therefore, the absence of a decrease in progression of DR in hypertensive patients is not definitive. Six studies with 2,624 participants examined the effect of RAS inhibitors on inducing regression of DR. RAS inhibitors increased the regression of DR by 39% (p=0.00002), and this beneficial effect was seen in patients with type 1 and type 2 diabetes. ACE inhibitors were more effective in reducing the development, progression, and regression of DR than ARBs. Thus, with the data available, RAS inhibitors appear to have benefits on DR above and beyond their effects on BP control.  

CONCLUSION 

Observational studies have shown an association of elevated BP with a higher risk of DR. As should be obvious from the above discussion, the beneficial effects of lowering BP in hypertensive patients on DR have not produced consistent results. Several large carefully carried out studies have failed to demonstrate a beneficial effect of lowering BP on DR (ACCORD, ADVANCE, ABCD2). Potential reasons for this inconsistency were discussed above. It is unlikely that future studies will provide definitive data on this issue, as lowering BP in hypertensive patients with diabetes to prevent cardiovascular disease is essential, and therefore designing clinical trials regarding DR will be very difficult. From the clinician’s viewpoint, treating hypertension in patients with diabetes to prevent cardiovascular disease is standard therapy and may also have beneficial effects DR. Similar to the beneficial effects on renal disease, RAS inhibitors appear to decrease the development and progression of DR, and therefore when treating patients with diabetes who are hypertensive, one should be preferentially consider RAS inhibitors to lower BP in patients with or at high risk of DR. In normotensive patients the available data suggests that RAS inhibition will have beneficial effects on DR, and further studies in this population are possible and would be informative.          

Hyperlipidemia

Observational studies of the association of plasma lipids with DR have been inconsistent (52) with some studies reporting an increased risk of DR with elevated lipid levels (53-57), while other studies have not observed a relationship between lipid levels and DR (10,38,58-60). Of note a Mendelian randomization study did not demonstrate a causal role of total cholesterol, LDL cholesterol, HDL cholesterol, or triglycerides on DR (61). From the clinician’s point of view the key question is whether lowering lipid levels will have a beneficial effect on DR.

FIBRATES

Small studies in the 1960’s presented evidence that treatment with clofibrate improved diabetic retinopathy (62,63). Larger randomized studies have confirmed these observations.

The Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study was a randomized trial in patients with Type 2 diabetes. Patients were randomly assigned to receive either fenofibrate 200 mg/day (n=4895) or placebo (n=4900). Laser treatment for retinopathy was significantly lower in the fenofibrate group than in the placebo group (3.4% patients on fenofibrate vs 4.9% on placebo; p=0.0002) (64). Fenofibrate therapy reduced the need for laser therapy to a similar extent for maculopathy (31% decrease) and for proliferative retinopathy (30% decrease). In the ophthalmology sub-study (n=1012), the primary endpoint of 2-step progression of retinopathy grade did not differ significantly between the fenofibrate and control groups (9.6% patients on fenofibrate vs 12.3% on placebo; p=0.19). In patients without pre-existing retinopathy there was no difference in progression (11.4% vs 11.7%; p=0.87). However, in patients with pre-existing retinopathy, significantly fewer patients on fenofibrate had a 2-step progression than did those on placebo (3.1% patients vs 14.6%; p=0.004). A composite endpoint of 2-step progression of retinopathy grade, macular edema, or laser treatments was significantly reduced in the fenofibrate group (HR 0.66, 95% CI 0.47-0.94; p=0.022).

In the ACCORD Study a subgroup of participants was evaluated for the progression of diabetic retinopathy by 3 or more steps on the Early Treatment Diabetic Retinopathy Study Severity Scale or the development of diabetic retinopathy necessitating laser photocoagulation or vitrectomy over a four-year period (24). At 4 years, the rates of progression of diabetic retinopathy were 6.5% with fenofibrate therapy (n=806) vs. 10.2% with placebo (n=787) (adjusted odds ratio, 0.60; 95% CI, 0.42 to 0.87; P = 0.006). Of note, this reduction in the progression of diabetic retinopathy was of a similar magnitude as intensive glycemic treatment vs. standard therapy.

A double-blind, randomized, placebo-controlled study in 296 patients with type 2 diabetes mellitus and DR evaluated the effect of placebo or etofibrate on DR (65). After 12 months an improvement in ocular pathology was more frequent in the etofibrate group vs the placebo group ((46% versus 32%; p< 0.001).

The MacuFen study was a small double-blind, randomized, placebo-controlled study in 110 subjects with DME who did not require immediate photocoagulation or intraocular treatment. Patients were randomized to fenofibric acid or placebo for 1 year. Patients treated with fenofibric acid had a modest improvement in total macular volume that was not statistically significant compared to the placebo group.

Taken together these results indicate that fibrates have beneficial effects on the progression of diabetic retinopathy (66). The mechanisms by which fibrates decrease diabetic retinopathy are unknown, and whether decreases in serum triglyceride levels plays an important role is uncertain. Fibrates activate PPAR alpha, which is expressed in the retina (67). Diabetic PPARα KO mice developed more severe DR while overexpression of PPARα in the retina of diabetic rats significantly alleviated diabetes-induced retinal vascular leakage and retinal inflammation, suggesting that fibrates could have direct effects on the retina to reduce DR (67).

STATINS

Several large database studies have suggested that statin use reduces the development of DR (68-71). Unfortunately, the number of randomized clinical trials testing the hypothesis that statin therapy reduces DR development or progression is very limited.

In a study by Sen and colleagues, 50 patients with diabetes mellitus (Type 1 and 2) with good glycemic control and hypercholesterolemia and having DR were randomized to simvastatin vs. placebo (72). Visual acuity improved in four patients using simvastatin and decreased in seven patients in the placebo group and none in the simvastatin group (P = 0.009). Fundus fluorescein angiography and color fundus photography showed improvement in one patient in the simvastatin group, while seven patients showed worsening in the placebo group (P = 0.009).

In a study by Gupta and colleagues, 30 patients with type 2 diabetes with clinically significant macular edema, dyslipidemia, and grade 4 hard exudates were randomized to receive atorvastatin or no lipid lowering drugs (73). All patients received laser therapy. Ten (66.6%) of 15 patients treated with atorvastatin and two (13.3%) of 15 patients in the control group showed a reduction in hard exudates (P =.007). None of the patients treated with atorvastatin and five (33.3%) of 15 in the control group showed subfoveal lipid migration after laser photocoagulation (P =.04). Regression of macular edema was seen in nine eyes in the atorvastatin group and five in the control group (P =.27).

In a study by Narang and colleagues, 30 patients with clinically significant macular edema with a normal lipid profile were randomly treated with atorvastatin or with no lipid lowering drugs. All patients received laser therapy. After a 6-month follow-up visual acuity, macular edema and hard exudates resolution was not significantly different in the two groups.

The data on the benefit of statin therapy on DR are not very strong. Given the current recommendations to prevent cardiovascular disease, most patients with diabetes are treated with statins, and therefore it is unlikely that large randomized trials of the effect of statin therapy on DR are feasible.

OMEGA-3-FATTY ACIDS

A Study of Cardiovascular Events in Diabetes (ASCEND) was a randomized, placebo controlled, double blind, cardiovascular outcome trial of 1-gram omega-3-fatty acids (400 mg EPA and 300 mg DHA ethyl esters) vs. olive oil placebo in 15,480 patients with diabetes without a history of cardiovascular disease (primary prevention trial) (74). Total cholesterol, HDL-C, and non-HDL-C levels were not significantly altered by omega-3-fatty acid treatment (changes in TG levels were not reported). After a mean follow-up of 7.4 years the development of retinopathy and the need for laser therapy based on self-report was similar in the omega-3-fatty acid and placebo group. Thus, at this time there is no evidence that omega-3-fatty acids influence DR.

NIACIN

It has been estimated that 0.67% of patients treated with niacin develop macular edema (75). 

Pregnancy

Diabetic retinopathy may progress during pregnancy and up to one year postpartum. For additional information on retinopathy during pregnancy see the chapter in Endotext on “Diabetes in Pregnancy” (76).

Genetics

Some individuals develop DR despite good glycemic control and short duration of disease, while others do not develop DR, even with poor glycemic control and longer duration of diabetes (77). Additionally, the strongest environmental factors (duration of diabetes and HbA1c) only explained about 11% of the variation in DR risk in the DCCT trial and 10% in the WESDR study (12,78). Thus, factors other than glycemic control play an important role.  There is a familial relationship in the development of DR, as twin and family studies indicate a genetic basis (79,80). The differences in the prevalence of DR in different ethnic groups may be related to genetic factors (79). Unfortunately, the identification of genetic susceptibility loci for DR through candidate gene approaches, linkage studies, and GWAS has not provided conclusive results (79-81). From a clinician’s point of view, if there is a family history of DR, one should aggressively control risk factors for DR and ensure close eye follow-up.  

SCREENING

The American Academy of Ophthalmology has recommended screening for diabetic retinopathy 5 years after diagnosis in patients with type 1 diabetes, and at the time of diagnosis in patients with type 2 diabetes. Patients without retinopathy should undergo dilated fundus examination annually. If mild non-proliferative diabetic retinopathy (NPDR) is present, exams should be repeated every 9 months. Patients with moderate NPDR should be examined every 6 months. In severe NPDR, exams should be conducted every 3 months. Patients with a new diagnosis of proliferative diabetic retinopathy should be examined every 2 to 3 months, until they are deemed stable, at which point examinations can be performed less frequently. During pregnancy, patients should be examined every 3 months, since retinopathy can progress rapidly in this setting (2019 AAO preferred practice pattern document for monitoring diabetic retinopathy:  https://www.aao.org/preferred-practice-pattern/diabetic-retinopathy-ppp).

The American Diabetes Association 2020 guidelines (5) recommends the following:

  • Adults with type 1 diabetes should have an initial dilated and comprehensive eye examination by an ophthalmologist or optometrist within 5 years after the onset of diabetes.
  • Patients with type 2 diabetes should have an initial dilated and comprehensive eye examination by an ophthalmologist or optometrist at the time of the diabetes diagnosis.
  • If there is no evidence of retinopathy for one or more annual eye exams and glycemia is well controlled, then screening every 1–2 years may be considered. If any level of diabetic retinopathy is present, subsequent dilated retinal examinations should be repeated at least annually by an ophthalmologist or optometrist. If retinopathy is progressing or sight-threatening, then examinations will be required more frequently.
  • Programs that use retinal photography (with remote reading or use of a validated assessment tool) to improve access to diabetic retinopathy screening can be appropriate screening strategies for diabetic retinopathy. Such programs need to provide pathways for timely referral for a comprehensive eye examination when indicated.
  • Women with preexisting type 1 or type 2 diabetes who are planning pregnancy or who are pregnant should be counseled on the risk of development and/or progression of diabetic retinopathy.
  • Eye examinations should occur before pregnancy or in the first trimester in patients with preexisting type 1 or type 2 diabetes, and then patients should be monitored every trimester and for 1 year postpartum as indicated by the degree of retinopathy.

PATHOGENESIS

Various mechanisms account for the features of diabetic retinopathy. Histopathologic analysis shows thickening of capillary basement membranes, microaneurysm formation, loss of pericytes, capillary acellularity, and neovascularization. Microaneurysms, outpouchings of the capillary wall, serve as sites of fluid and lipid leakage, which can lead to the development of diabetic macular edema. Theories on the biochemistry of these end-organ changes include toxic effects from sorbitol accumulation, vascular damage by excessive glycosylation with crosslinking of basement membrane proteins, and activation of protein kinase C-ß2 by vascular endothelial growth factor (VEGF), leading to increased vascular permeability and endothelial cell proliferation. VEGF, produced by the retina in response to hypoxia, is believed to play a central role in the development of neovascularization (1,82). 

CLINICAL FEATURES 

Nonproliferative Diabetic Retinopathy (NPDR)

Studies have found that retinopathy in both insulin-dependent and non-insulin-dependent diabetes occurs 3 to 5 years or more after the onset of diabetes. In the WESDR, the prevalence of at least minimal retinopathy was almost 100% after 20 years (83). A more recent study has confirmed that at least 39% of young persons with diabetes developed retinopathy within the first 10 years (84). The earliest clinical sign of diabetic retinopathy is the microaneurysm, a red dot seen on ophthalmoscopy that varies from 15 to 60 microns in diameter (Figure 1).

Figure 1. Microaneurysms and intraretinal hemorrhages in nonproliferative retinopathy. (UCSF Department of Ophthalmology)

The lesions can be difficult to distinguish from intraretinal hemorrhages on examination, but with fluorescein angiography microaneurysms can be identified easily as punctate spots of hyperfluorescence (Figure 2, 3). By contrast, hemorrhages block the background fluorescence and therefore appear dark.

Figure 2. Microaneurysms: hyperfluorescent dots in early phase of fluorescein angiogram (arrows). (Zuckerberg San Francisco General Hospital, Dept. of Ophthalmology)

 

Figure 3. Two minutes later, fluorescein leakage from the microaneurysms gives them a hazy appearance. (Zuckerberg San Francisco General Hospital, Dept. of Ophthalmology)

The severity of NPDR can be graded as mild, moderate, severe, or very severe. In mild disease, microaneurysms are present with hemorrhage or hard exudates (lipid transudates). In moderate NPDR, these findings are associated with cotton-wool spots (focal infarcts of the retinal nerve fiber layer or areas of axoplasmic stasis) or intraretinal microvascular abnormalities (vessels that may be either abnormally dilated and tortuous retinal vessels, or intraretinal neovascularization). The “4-2-1 rule” is used to diagnose severe NPDR: criteria are met if hemorrhages and microaneurysms are present in 4 quadrants, or venous beading (Figure 4) is present in 2 quadrants, or moderate intraretinal microvascular abnormalities are present in 1 quadrant. In very severe NPDR, two of these features are present.

The correct evaluation and staging of NPDR is important as a means of assessing the risk of progression. In the ETDRS, eyes with very severe NPDR had a 60-fold increased risk of developing high-risk proliferative retinopathy after 1 year compared with eyes with mild NPDR (85). For eyes with mild or moderate NPDR, early treatment with laser was not warranted, as the benefits in preventing vision loss did not outweigh the side effects (1). By contrast, in very severe NPDR, early laser treatment was often helpful.

Figure 4. Venous beading (arrows) in a case of proliferative diabetic retinopathy. (UCSF Department of Ophthalmology)

Capillary closure can also result in macular ischemia, another cause of vision loss in NPDR. This can be identified clinically as an enlargement of the normal foveal avascular zone on fluorescein angiography (Figure 5).

Figure 5. Capillary dropout around the fovea (white arrow) and in the temporal macula (black arrow). (Zuckerberg San Francisco General Hospital, Dept. of Ophthalmology)

Diabetic Macular Edema (DME) 

Macular edema may be present at all the stages of diabetic retinopathy and is the most common cause of vision loss in nonproliferative diabetic retinopathy. Because of the increased vascular permeability and breakdown of the blood-retinal barrier, fluid and lipids leak into the retina and cause it to swell. This causes photoreceptor dysfunction, leading to vision loss when the center of the macula, the fovea, is affected. In the ETDRS, diabetic macular edema (DME) was characterized as "clinically significant" if any of the following were noted (Figure 6): retinal thickening within 500 microns of the fovea, hard exudates within 500 microns of the fovea if associated with adjacent retinal thickening, or an area of retinal thickening 1 disc diameter or larger if any part of it is located within 1 disc diameter of the fovea (86).

Figure 6. Clinically significant macular edema with hard exudates in the fovea. Cotton-wool spots are present near the major vessels. (UCSF Department of Ophthalmology)

Although the cause of the microvascular changes in diabetes is not fully understood, the deficient oxygenation of the retina may induce an overexpression of vascular endothelial growth factor (VEGF), with a consequent increase in vascular leakage and retinal edema (87). Besides ischemia, inflammation may also play a role in the development of macular edema in diabetic retinopathy. In fact, elevated levels of extracellular carbonic anhydrase have been discovered in the vitreous of patients with diabetic retinopathy (88). Carbonic anhydrase may originate from retinal hemorrhages and erythrocyte lysis and may activate the kallikrein-mediated inflammatory cascade, contributing to the development of DME.

Optical Coherence Tomography (OCT) is a widely used imaging technique that provides high-resolution imaging of the retina (Figure 7) (89). Working as an “optical ultrasound,” OCT projects a light beam and then acquires the light reflected from the retina to provide a cross-sectional image. Most patients with DME have diffuse retinal thickening or cystoid macular edema (presence of intraretinal cystoid-like spaces). In some patients, DME may be associated with posterior hyaloidal traction, serous retinal detachment or traction retinal detachment (90). Cystoid macular edema and posterior hyaloid traction are significantly associated with worse visual acuity (90).

Figure 7. OCT image showing diabetic macular edema (UCSF Department of Ophthalmology).

Proliferative Diabetic Retinopathy (PDR)

In proliferative diabetic retinopathy, many of the changes seen in NPDR are present in addition to neovascularization that extends along the surface of the retina or into the vitreous cavity (Figure 8). These vessels are in loops that may form a network of radiating spokes or may appear disorganized. In many cases the vessels are first noted on the surface of the optic disc, although they can be easily missed due to their fine caliber. Close inspection often reveals that these new vessels cross over both the normal arteries and the normal veins of the retina, a sign of their unregulated growth.

Figure 8. Active neovascularization in PDR. Fibrovascular proliferation overlies the optic disc (white arrow). Loops of new vessels are especially prominent superior to the disc and extending into the macula, where leakage of fluid has led to deposition of a ring of hard exudate around the neovascular net (black arrow). (UCSF Department of Ophthalmology)

New vessels can also appear on the iris, a condition known as rubeosis iridis (Figure 9). When this occurs, careful inspection of the anterior chamber angle is essential, as growth of neovascularization in this location can obstruct aqueous fluid outflow and cause neovascular glaucoma.

Figure 9. Rubeosis iridis in a case of PDR. Abnormal new vessels are growing along the surface of the iris (arrows). (UCSF Dept. of Ophthalmology)

Neovascularization can remain relatively stable or it can grow rapidly; progression can be noted ophthalmoscopically over a period of weeks. Preretinal new vessels often develop an associated white, fibrous tissue component that can increase in size as the vessels regress. The resulting fibrovascular membrane may then develop new vessels at its edges. This cycle of growth and fibrous transformation of diabetic neovascularization is typical. The proliferation occurs on the anterior surface of the retina, and the vessels extend along the posterior surface of the vitreous body. Fibrous proliferation takes place on the posterior vitreous surface; when the vitreous detaches, the vessels can be pulled forward and the thickened posterior vitreous surface can be seen ophthalmoscopically, highlighted by areas of fibrovascular proliferation.

The severity of PDR can be classified as to the presence or absence of high-risk characteristics. As determined in the Diabetic Retinopathy Study, eyes are classified as high-risk if they have 3 of the following 4 characteristics: the presence of any neovascularization; neovascularization on or within 1-disc diameter of the optic disc; a moderate to severe amount of neovascularization (greater than 1/3 disc area neovascularization of the disc, or greater than 1/2 disc area if elsewhere), or vitreous hemorrhage.

Vision loss in proliferative diabetic retinopathy results from three main causes. First, vitreous hemorrhage occurs because the neovascular tissue is subject to vitreous traction. Coughing or vomiting may also trigger a hemorrhage. Hemorrhage may remain in the preretinal space between the retina and the posterior vitreous surface, in which case it may not cause much vision loss if located away from the macula (Figure 10). In other cases, though, hemorrhage can spread throughout the entire vitreous cavity, causing a diffuse opacification of the visual media with marked vision loss (Figure 11, 12).

Figure 10. Preretinal hemorrhage: blood trapped between the retina and the vitreous in a case of incomplete vitreous detachment. Visual acuity is unaffected. (UCSF Department of Ophthalmology)

Figure 11. Left: moderate vitreous hemorrhage; vision = 20/150. Right: 1 year later after spontaneous clearing of the hemorrhage; vision = 20/30. (Zuckerberg San Francisco General Hospital, Dept. of Ophthalmology)

Figure 12. Dense vitreous hemorrhage almost completely obscuring the view of the fundus. (Zuckerberg San Francisco General Hospital, Dept. of Ophthalmology)

Another cause of severe vision loss in PDR is retinal detachment. As the fibrovascular membranes and vitreous contract, their attachments to the retina can cause focal elevations of the retina, resulting in a traction retinal detachment (Figure 13). In other cases the retinal vessels can be avulsed or retinal holes may be created by this traction, leading to a combined traction-rhegmatogenous retinal detachment (Figure 14).

Figure 13. Marked fibrosis with traction exerted on the retina outside the central macula (arrows). The macula does not appear to be elevated centrally. (UCSF Dept. of Ophthalmology)

Figure 14. Traction retinal detachment outside the macula. Note elevation of retinal vessel out of the plane of focus (white arrow). Scatter photocoagulation scars are seen peripherally (black arrow). (UCSF Dept. of Ophthalmology)

Finally, patients with PDR may have macular nonperfusion or coexisting diabetic macular edema that causes vision loss through photoreceptor dysfunction.

TREATMENT

Tight glucose and blood pressure control are critical systemic factors in controlling the progression of diabetic retinopathy. Ocular complications of diabetes are addressed directly through treatment with laser photocoagulation, intravitreal injections, or surgery. Laser treatment has been the primary approach to vision-threatening diabetic retinopathy for decades. Recent randomized clinical trials have demonstrated that intravitreal anti-VEGF agents are more effective than laser under certain conditions.

Laser Photocoagulation for NPDR

Diabetic macular edema is believed to result from fluid and lipid transudation from microaneurysms and telangiectatic capillaries. Focal laser photocoagulation is used to heat and close the microaneurysms, causing them to stop leaking (Figure 15). Macular edema often improves following this form of treatment. Some clinicians apply laser burns in a grid pattern overlying areas of retinal edema without directing treatment to specific microaneurysms; this method can also be effective in reducing retinal thickening. The mechanism by which grid laser treatment achieves these results is not known.

The ETDRS found that the risk of moderate visual loss in eyes with diabetic macular edema was reduced by 50% by photocoagulation (91,92). At 3 years, 24% of untreated eyes experienced a 3-line decrease in vision compared with 12% of treated eyes. Eyes meeting the criteria for clinically significant macular edema in which the edema was closest to the center were most likely to benefit from treatment. Side effects of laser treatment can include scotomata, noticeable immediately after the procedure, if treatment is performed too close to the fovea. Late enlargement of laser scars can also occur, causing delayed visual loss. Inadvertent photocoagulation of the fovea is a risk of the procedure. Since the amount of energy used is minimal, the treatment is performed under topical anesthesia.

In the ETDRS study, only a very small percentage of eyes improved with focal laser treatment, highlighting the fact that the goal of laser treatment is not to improve vision, but rather to stabilize it and prevent worsening. It is also true that inclusion criteria for that study were based on the presence of “clinically significant” macular edema threatening the macula, even if the visual acuity was not yet reduced. For this reason, it has been argued that the study enrolled patients with excellent visual acuity, making it difficult to demonstrate small improvements in vision after laser treatment.

Due to the recent evidence on the efficacy and safety of anti-VEGF therapy for diabetic macular edema, different modalities of laser therapy have been proposed. Laser may be able to stabilize macular edema and reduce the need for multiple anti-VEGF injections. Modified ETDRS laser techniques include lower intensity laser burns, and they take particular care in maintaining a greater space from the center of the fovea (93). Subthreshold laser therapy and minimalistic FA-guided treatment of microaneurysms may also induce less damage to the macula than the classic ETDRS approach (94).

Figure 15. Focal laser scars in the macula following treatment for macular edema (arrow). Edema has resolved. (Zuckerberg San Francisco General Hospital, Dept. of Ophthalmology) 

Laser Photocoagulation for PDR

Scatter laser photocoagulation, also known as panretinal photocoagulation (PRP), is an important treatment modality for PDR and severe NPDR (92). Laser spots are placed from outside the major vascular arcades to the equator of the eye, with burns spaced approximately 1/2 to 1 burn width apart (Figure 16, 17). Although the treatment destroys normal retina, the central vision is unaffected since all spots are placed outside the macula. The theory underlying this treatment is that photocoagulation of the ischemic peripheral retina decreases the elaboration of vasoproliferative factors contributing to PDR. Indeed, VEGF levels in the vitreous are increased in eyes with neovascularization, and they are lower after scatter photocoagulation (95). Other factors such as insulin-like growth factor-1 are similarly elevated in the vitreous of eyes with PDR (96).

Side effects of scatter photocoagulation can include decreased night vision and dark adaptation, and visual field loss. The procedure can be painful, so treatment may be divided into several sessions, and either topical or retrobulbar anesthesia may be used.

Figure 16. Scatter photocoagulation scars in an eye with active PDR. Note that all scatter laser scars are located outside the macula. (UCSF Department of Ophthalmology)

Figure 17. View of laser scars superior to the macula in the same eye. Spots are approximately one-half burn width apart. In the treated area, the retinal vessels are sclerotic (arrows). (UCSF Department of Ophthalmology)

The Diabetic Retinopathy Study evaluated the effects of scatter photocoagulation in over 1700 patients with PDR or severe NPDR. Patients had one eye randomized to treatment and one eye to observation. Treatment was shown to reduce severe visual loss by 50% (97). The ETDRS also found a positive risk-benefit ratio for early scatter treatment in patients with severe NPDR or early PDR. Interestingly, a subsequent study demonstrated that scatter laser performed at a single sitting was not worse than treatment divided over four sessions in terms of inducing macular edema or decreasing visual acuity (98).

Panretinal photocoagulation may induce or aggravate diabetic macular edema, reduce contrast sensitivity and affect the peripheral visual field (85). Macular edema can be approached by focal laser or intravitreal injections before or at the time of panretinal photocoagulation. However, it is not recommended to delay panretinal photocoagulation in high-risk PDR.

The DRCR.net study protocol S has shown that intravitreal anti-VEGF agents may be a substitute for panretinal laser treatment (99). This multicenter randomized clinical trial compared ranibizumab to PRP in patients with PDR. Mean visual acuity letter improvement at 2 years was +2.8 in the ranibizumab group vs +0.2 in the PRP group (P < 0.001). Mean peripheral visual field sensitivity loss was worse, vitrectomy was more frequent, and DME development was more common in the PRP group. Further studies are needed in order to evaluate the long-term implications of using anti-VEGF agents alone. Ranibizumab may be a reasonable treatment alternative to consider for patients with severe NPDR or non-high-risk PDR who can follow-up regularly.

Corticosteroids for DME

It has been demonstrated that corticosteroids stabilize the blood-retinal barrier, inhibiting leukostasis and modulating the expression of VEGF receptor (100). On this basis, periocular and intraocular injections and sustained-release steroid implants have been utilized for the treatment of diabetic macular edema. It should be remembered that any of these different methods to deliver corticosteroids to the macula carry a potential risk of increasing the intraocular pressure (glaucoma) and inducing cataract.

The use of intravitreal triamcinolone acetonide has become accepted as a treatment option for diabetic macular edema. Several formulations are available: Kenalog-40, which has a black box warning against intraocular use, and the preservative-free Triesence. Preliminary data from a randomized clinical trial showed that intravitreal corticosteroids induced a noticeable improvement of visual acuity and foveal thickness in patients with severe, refractory DME (101). However, intravitreal steroids do not appear to be more efficacious than laser treatment in giving a stable, sustained improvement in vision in the long run, as demonstrated by a recent large study (102).

A peribulbar corticosteroid injection is of particular interest for eyes with DME that have good visual acuity where the risks of an intravitreal injection may not be justified. Any intravitreal injection through the pars plana, in fact, may directly damage the crystalline lens or cause a severe, sight-threatening infection of the eye (bacterial endophthalmitis). Unfortunately, in 2007 a randomized clinical trial showed that peribulbar triamcinolone, with or without focal photocoagulation, is not effective in cases of mild DME with good visual acuity (103).

The fact that triamcinolone maintains measurable concentrations in the vitreous cavity for approximately 3 months stimulated further studies on sustained-release or biodegradable intraocular implants that can deliver steroids for a longer period of time.

A fluocinolone acetonide implant (Retisert) was investigated in a multicenter, randomized clinical trial for the treatment of diabetic macular edema. Although the efficacy of this surgically implanted material was demonstrated, it induced cataract in virtually all phakic patients and severe glaucoma needing surgery in 28% of eyes (104,105).

A biodegradable dexamethasone implant (Ozurdex), now approved for the treatment of DME, has demonstrated similar efficacy with more acceptable side effects. At day 90, a visual acuity improvement of 10 letters or more was seen in more eyes in the Ozurdex group (33.3%) than the observation group (12.3%; P = .007), but the statistical significance was lost at day 180 (106). The implant was generally well tolerated.

A smaller device releasing fluocinolone acetonide, implantable suturelessly with an office procedure thorough a 25-gauge needle, has been recently approved for DME in the USA (Iluvien). This implant has been evaluated in the FAME (Fluocinolone Acetonide in Diabetic Macular Edema) study where 956 patients were randomized worldwide (107). At month 36, the percentage of patients who gained ≥15 in letter score was 28% compared with 19% (P = 0.018) in the sham group. In patients who reported duration of DME ≥3 years at baseline; the percentage who gained ≥15 in letter score at month 36 was 34.0% compared with 13.4%. Almost all phakic patients in the insert group developed cataract, but their visual benefit after cataract surgery was similar to that in pseudophakic patients. The rate of glaucoma surgery at month 36 was 5% (108).

Anti-VEGF Drugs for DME 

Vascular endothelial growth factor (VEGF) is an angiogenic factor that plays a key role in the breakdown of the blood–retina barrier and is significantly elevated in eyes with diabetic macular edema (109). Antibody fragments that bind VEGF and inhibit angiogenesis were originally developed as intraocular injection for the treatment of exudative age-related macular degeneration. These anti-VEGF drugs have been tested for the treatment of DME with interesting results.

The first agent that became available was Pegaptanib 0.3 mg (Macugen) (110). A randomized trial demonstrated after 2 years of therapy a gain of 6.1 letters in the pegaptanib arm versus 1.3 letters for sham (P<0.01) (111). Since it is targeted to the isoform VEGF-165 only, it is generally considered very safe but possibly less effective than newer anti-VEGF drugs.

Bevacizumab (Avastin), directed to all the isoforms of VEGF, has been used off-label for the treatment of DME worldwide. The first evidence came from a study on 121 patients with DME followed over 3 months in a phase II randomized clinical trial (112). Recently, the BOLT study demonstrated a mean gain of 8.6 letters for bevacizumab versus a mean loss of 0.5 letters when compared to classic macular laser. The patients received a mean of 13 injections over two years, and the treatment was well tolerated with no progression of macular ischemia (113).

Ranibizumab (Lucentis) binds all isoforms of VEGF and is FDA approved for the treatment of diabetic macular edema. In the Ranibizumab for Edema of the Macula in Diabetes (READ-2) study, ranibizumab-only was superior to laser and to combined therapy (114). The RESTORE study confirmed that ranibizumab monotherapy and combined with laser was superior to standard laser. At 1 year, no differences were detected between the ranibizumab and ranibizumab plus laser arms (115). A larger DRCR study supported ranibizumab plus prompt or deferred photocoagulation as a mainstay of current therapy for patients with DME (116). In the RESOLVE study, at month 12, mean visual acuity improved from baseline by 10.3±9.1 letters with ranibizumab and declined by 1.4±14.2 letters with sham (P<0.0001) (117). The RISE and RIDE studies confirmed the efficacy and the safety of intravitreal monthly injections of ranibizumab with similar results (118).

Aflibercept (Eylea), active against all VEGF-A isoforms, is also FDA-approved for the treatment of DME. In the DA-VINCI study, the different dose regimens of aflibercept demonstrated a mean improvement in visual acuity of 10 to 13 letters versus -1.3 letters for the laser group with a large proportion of eyes (about 40%) gaining 15 or more ETDRS letters at week 52 (119).

More recently, The Diabetic Retinopathy Clinical Research Network Protocol T compared bevacizumab, ranibizumab, and aflibercept in the treatment of center-involving CSME (120). When the initial visual-acuity loss was mild, there were no significant differences among study groups. However, at worse levels of initial visual acuity (20/50 or worse), aflibercept was more effective than bevacizumab. The differences between bevacizumab and ranibizumab and between ranibizumab and aflibercept were not statistically significant.

Currently, on the basis of the above evidence, anti-VEGF therapy is first-line therapy for center-involving macular edema, with possible deferred focal laser treatment. It should be mentioned that adverse side effects associated with intravitreal injections are uncommon but severe and include infectious endophthalmitis, cataract formation, retinal detachment, and elevated IOP. 

Vitreous Surgery for PDR

Surgery may be necessary for eyes in advanced PDR with either vitreous hemorrhage or retinal detachment. In the case of vitreous hemorrhage, many cases will clear spontaneously. For this reason, clinicians often wait 3 to 6 months or more before performing vitrectomy surgery. If surgery is indicated because of persistent non-clearing hemorrhage, retinal detachment involving the macula, or vitreous hemorrhage with neovascularization of the anterior chamber angle (a precursor of neovascular glaucoma), then vitrectomy is performed via a pars plana approach. The vitreous is removed, fibrovascular membranes are dissected away from the retina, retinal detachment is repaired, and scatter laser treatment is applied at the time of surgery via direct intraocular application.

The Diabetic Retinopathy Vitrectomy Study assessed the value of early vitrectomy in patients with severe PDR. The study found that early intervention increased the likelihood of obtaining 20/40 vision or better in eyes with recent severe vitreous hemorrhage or severe PDR. Compared with 15% of control eyes, 25% of treated eyes achieved this level of vision at 2 years (109). In type 1 diabetes, the benefit of early surgery was even more pronounced, with 36% of treated eyes achieving 20/40 vision compared to 12% of control eyes. The importance of this study, performed between 1976 and 1983 when vitrectomy techniques were much less advanced than they are today, was that it showed conventional “watch and wait” management will not necessarily lead to the best visual outcomes in cases of severe PDR. In practice, clinicians evaluate the risks and benefits of each option before proceeding with scatter photocoagulation, vitrectomy, or observation in such cases.

Recently, the DRCR Protocol D evaluated the effects of pars plana vitrectomy in eyes with moderate vision loss from DME and vitreomacular traction. Although retinal thickness was generally reduced, visual acuity results were less consistent (121). Vitrectomy for refractory, chronic diabetic macular edema in the absence of vitreomacular traction should be reserved to selected cases.

Intravitreal ocriplasmin (Jetrea) is able to induce enzymatic vitreolysis and posterior vitreous detachment and could have a role, eventually associated with vitrectomy, in the treatment of vitreomacular traction and macular edema in diabetic retinopathy (122). 

NOVEL THERAPIES FOR DIABETIC RETINOPATHY

Current therapies are limited in their ability to reverse vision loss in diabetic retinopathy. For example, although focal laser photocoagulation can help stabilize vision by reducing macular edema, it rarely improves vision. Corticosteroids induce cataract progression and intraocular pressure elevation. Anti-VEGF agents do not increase cataract formation rates but they generally need more frequent intravitreal injections, carrying the risk of endophthalmitis; they can temporary increase IOP; they might have systemic adverse effects. For addressing these issues, new sustained-release devices are being designed, and studies are ongoing to test new intravitreal medications.

The development of new treatment modalities is being guided by an understanding of the mechanisms of the disease. From this perspective, researchers are now focusing on the role of inflammation on DME. NSAIDs, anti-TNF agents (Etanercept and Remicade), mecamylamine (an antagonist of nACh receptors), and intravitreal erythropoietin are currently under investigation for the treatment of refractory diabetic macular edema (123).

In order to create a national taskforce to study and treat diabetic retinopathy, in 2002 the National Eye Institute instituted the Diabetic Retinopathy Clinical Research Network (www.drcr.net). DRCR is a collaborative network dedicated to design and carry out multicenter clinical trials on diabetic retinopathy and diabetic macular edema. The DRCR network currently includes over 150 participating sites with over 500 physicians throughout the United States.

The DRCR Network has an ongoing project to study genes involved in diabetic retinopathy.

CONCLUSION

Retinopathy remains a challenging complication of diabetes that can adversely affect a patient’s quality of life. Although ophthalmologists can often stabilize the condition or reduce vision loss, prevention and early detection remain the most effective ways to preserve good vision in patients with diabetes. Ensuring tight glucose and blood pressure control and referring patients for ophthalmologic examination are important ways in which internists and other clinicians can help to maximize their patients’ vision and therefore their quality of life. New treatments may offer greater hope for sustained visual improvement in patients with diabetic retinopathy.

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Clinical Strategies in the Testing of Thyroid Function

ABSTRACT

 

In the past decades, emphasis has shifted from testing thyroid function in individuals who are likely to have clinically overt thyroid disorders to a broader population, an approach that includes also identification of so-called subclinical or mild thyroid dysfunctions. The key measurement methods used to detect thyroid dysfunction are still serum thyroid stimulating hormone (TSH) and the main circulating thyroid hormones thyroxine (T4) and triiodothyronine (T3), either as total or estimated free concentrations, and it is indeed the improved assay sensitivities and specificities that have made it possible to diagnose these milder forms. For these key variables, it is preferable for results to be interpretable in relation to population-based reference intervals to use methods that are independent of the particular laboratory assay used. This requirement is satisfied for well standardized assays for serum TSH, total T4 and total T3. However, when free T4 is estimated, assay results often need to be evaluated in relation to method-specific reference intervals or “normal ranges”. Free T3 estimates are even more subject to spurious results and high inter-method variability. This limitation of both assessments is particularly cogent during pregnancy and in the face of critical illness. The widespread search for even minor thyroid dysfunction is influenced by two key questions. How damaging are the effects of subclinical dysfunction? Does treatment confer benefit? The answers are not uniform across populations. The diagnostic imperative is now radically different for the population at large and for women who are pregnant or about to become pregnant.

 

INTRODUCTION

 

In the past decades, emphasis has shifted from testing of thyroid function in individuals who are likely to have clinically overt thyroid disorders to a broader population, an approach that identifies so-called subclinical thyroid dysfunction in up to 10% of women over fifty. The key assays that are used to detect thyroid dysfunction are serum thyroid stimulating hormone (TSH) and the main circulating thyroid hormones thyroxine (T4) and triiodothyronine (T3), either as total or estimated free concentrations. For these key variables, it is preferable for results to be interpretable in relation to population-based reference intervals that use methods that are independent of the particular assay used. This requirement is satisfied for well standardized assays for serum TSH, total T4 and total T3. However, when free T4 is estimated, assay results often need to be evaluated in relation to method-specific reference intervals or “normal ranges”. Free T3 estimates are even more subject to spurious results and high inter-method variability. This limitation of both assessments is particularly cogent during pregnancy and in the face of critical illness (see below).

 

The widespread search for even minor thyroid dysfunction is influenced by two key questions. How damaging are the effects of subclinical dysfunction? Does treatment confer benefit? The answers are not uniform across populations. The diagnostic imperative is now radically different for the population at large and for women who are pregnant or about to become pregnant. Some key practice points that relate to the testing of thyroid function are summarized in Table 1.

 

Table 1. Key Practice Points Related to the Testing of Thyroid Function

Many disorders are associated with increased prevalence of thyroid dysfunction; optimal testing strategy requires information on all co-existing conditions and medications.

The more widely thyroid function is tested, the greater the proportion of abnormal results that show borderline or “subclinical dysfunction”.

Testing of thyroid function is now widely advocated before and early in pregnancy, especially where fertility is impaired, assisted reproduction is used, or where pregnancy complications have occurred.

Except for “subclinical” hypothyroidism in early or impending pregnancy, intervention for subclinical thyroid dysfunction should only be considered after a sustained abnormality has been demonstrated over a minimum of three months.

There is increasing documentation of adverse effects from sustained or progressive subclinical hypothyroidism, although evidence of benefit from treatment is less clear.

The combination of raised serum TSH and positive peroxidase antibody is predictive of likely long-term progression towards overt hypothyroidism.

The relationship between serum TSH and circulating thyroid hormones gives a better index of thyroid status than any single variable. Six key assumptions underpin the diagnostic value of this relationship.

“TSH alone” first line approach to thyroid function testing has important drawbacks and limitations.

Serum TSH is a cornerstone of thyroid diagnosis, but it is not possible to define its “normal” range or reference interval for all clinical circumstances. Age, pregnancy and fertility issues, diurnal variation and pulse secretion, and associated antibody status militate against fixed cut-off points.

Both TSH and T4 show spontaneous biological fluctuations that are much greater than analytical imprecision. A serial change can be inferred with confidence with about 50% alteration in serum TSH and 25% change in the free T4 estimate.

During thyroid hormone therapy, either replacement or suppressive, the optimal target TSH range may differ from the reference interval that is used to establish a new diagnosis.

Interpretation of anomalous test results should take into account the effects of all associated medications, as well as nutraceuticals, e.g. biotin.

No estimate of circulating free thyroxine is impeccable. Especially in situations where assessment is difficult, e.g. late pregnancy and severe associated illness, there are strong arguments for re-establishing total T4 measurement as the preferred “gold standard”.

Identification of marked iodine excess, detected by urinary estimation, may identify reversible thyroid abnormalities, e.g. iodine-induced exacerbation of primary hypothyroidism, atypical thyrotoxicosis with blocked isotope uptake, or resistance to standard doses of antithyroid drugs. Some food sources (e.g. soy, sea weed) and alternative health care products can be heavily iodine-contaminated.

 

WHO SHOULD BE TESTED FOR THYROID DYSFUNCTION?

 

It has long been recognized that the clinical manifestations of hyperthyroidism (thyrotoxicosis), or hypothyroidism are so diverse that diagnosis based on clinical features lacks sensitivity and specificity. Hence, reliance is placed on measurements of circulating thyroid hormones and thyroid stimulating hormone (TSH) to confirm or rule out thyroid dysfunction.

 

After the publication of guidelines from the American College of Physicians in 1998 (1,2), testing for detection of thyroid dysfunction became widely applied, especially in women over 50, the group most likely to have either overt or subclinical thyroid dysfunction. Testing of this group is generally advocated at the time of presentation for medical care, i.e. a case finding strategy, rather than screening of a whole population group.

 

A normal serum TSH value in ambulatory patients without associated disease or pituitary dysfunction has a high negative predictive value in ruling out both primary hypothyroidism and hyperthyroidism (1,2), which has led to a short-cut approach in which free T4 may only be estimated if TSH is above 10 mU/l. However, this ”TSH first” strategy of thyroid function testing has important limitations (see below). If there is no suspicion of pituitary or thyroid disease, a normal TSH concentration does not need to be re-tested for about 5 years (3). To this broad indication has recently been added the still controversial recommendation that universal testing of thyroid function has a place before or as early as possible in pregnancy, although this is very much debated (4-7), and the recent official guidelines from the scientific community are still not in agreement (8,9).

 

In some groups (Tables 2 and 3), known to be at increased risk of thyroid dysfunction, there is a case for routine testing even in the absence of any suggestive clinical features.

 

Almost all developed countries now have routine neonatal screening programs for congenital hypothyroidism using heel prick filter paper blood spots (see below). The value of such programs has long been clear (10), but neonatal screening is not yet routine in numerous developing countries where nevertheless the prevalence of neonatal hypothyroidism may be high, and often associated with iodine deficiency (11) (see below). In terms of benefit from allocation of health care resources in developing countries, the establishment of neonatal screening (12) probably takes precedence over routine testing of adults, even in today’s India (13).

 

Table 2. Groups with an Increased Likelihood of Thyroid Dysfunction

Pregnancy and postpartum (14)

Previous thyroid disease or surgery

Atrial fibrillation (15)

Goiter

Associated autoimmune disease(s) (16-18)

Chromosome 18q deletions (19)

Chronic renal failure (20)

Williams syndrome (21)

Fabry disease (22)

Irradiation of head and neck (23-25)

Radical laryngeal/pharyngeal surgery

Recovery from Cushing’s syndrome (26,27)

Gout (28)

Environmental irradiation (29,30)

Thalassemia major (31)

Primary pulmonary hypertension (32)

Polycystic ovarian syndrome (33)

Morbid obesity (34)

Breast cancer (35,36)

Hepatitis C (pre-treatment) (37)

Down’s syndrome (38)

Turner’s syndrome (39)

Pituitary or cerebral irradiation (25)

Head trauma (40-42)

Very low birth weight premature infants (43,44)

 

Table 3. Drugs with an Increased Likelihood of Inducing Thyroid Dysfunction (45-47)

Inhibit thyroid hormone production

Antithyroid drugs, Amiodarone, Lithium, Iodide (large doses), Iodine-containing contrast media

Alter extra-thyroidal metabolism of thyroid hormone

Propylthiouracil, Glucocorticoids, Propranolol, Amiodarone, Iodine-Containing Contrast Media, Carbamazepine, Barbiturates, Rifampicin, Phenytoin, Sertraline

Alter T4/T3 binding to plasma proteins

Estrogen, Heroin, Methadone, Clofibrate, 5-Fluorouracil, Perphenazine, Glucocorticoids, Androgens, L-Asparaginase, Nicotinic Acid, Furosemide, Salicylates, Phenytoin, Fenclofenac Heparin

Induction of thyroiditis

Amiodarone, Interleukin-2, Interferon-α, Interferon-β, γ-Interferon, Sunitinib, Monoclonal antibody therapy check point inhibitors (Nivulomab, Pembrolizumab, pilimimab)

Effect on TSH secretion

Lithium, Dopamine Receptor Blockers, Dopa Inhibitors, Cimetidine, Clomiphene, Thyroid Hormone, Dopamine, L-Dopa, Glucocorticoids, Growth Hormone, Somatostatin, Octreotide

Impaired absorption of oral T4

Aluminum hydroxide, Ferrous Sulfate, Cholestyramine, Calcium Carbonate, Calcium Citrate, Calcium Acetate, Iron Sulfate, Colestipol, Sucralfate, Soya preparations, Kayexalate, Ciprofloxacin, Sevelamer, Proton pump inhibitors

Other

Thalidomide, Lenalidomide, Chemotherapy for sarcoma

 

THE BASIS FOR A CASE-FINDING STRATEGY

 

Routine laboratory testing of particular population groups becomes well founded if a testing strategy satisfies the following criteria:

  1. An abnormality cannot be identified in a reliable and timely way by standard clinical assessment.
  2. Dysfunction is sufficiently common to justify routine testing, either by case finding or by population screening.
  3. There are adverse consequences of failure to identify an abnormality, including the possibility of progression towards more severe disease.
  4. The laboratory test method is cost-effective and sufficiently sensitive and specific to identify those at risk of adverse consequences.
  5. There are no major adverse consequences of testing
  6. Treatment is safe and effective and prevents some or all of the adverse consequences.
  7. Abnormal findings can be adequately followed-up to ensure an appropriate clinical response. (An early detection program may have little value if this last requirement cannot be met.)

 

While the first five of the above criteria are reasonably established for thyroid dysfunction, the latter two are less secure.

Sensitivity and Accuracy of Clinical Assessment

 

Studies of unselected patients evaluated by primary care physicians show that clinical acumen alone lacks both sensitivity and specificity in detecting previously undiagnosed thyroid dysfunction. In up to one-third of patients evaluated for suspected thyroid dysfunction by specialists, laboratory results led to revision of the clinical assessment (48). Systematic comparison of the standard clinical features of hypothyroidism with laboratory tests (49) showed that clinical assessment identified only about 40% with overt hypothyroidism and classical signs were present only in the most severely affected individuals. Both overt hyper- and hypothyroidism can have important consequences before the usual clinical features are obvious, and clinicians may fail to recognize diagnostic features even when they are present.

 

Boelaert et al. (50) have recently confirmed that the typical multiple classical symptoms of hyperthyroidism become less prevalent with advancing age, with greater importance of weight loss, atrial fibrillation, and shortness of breath as presenting features (51). They proposed a low threshold for assessment of thyroid function in patients older than 60 years who have any of these features.

 

Clinical evaluation remains of central importance to assess severity of thyroid dysfunction, evaluate discordant results, establish the specific cause of thyroid dysfunction and monitor the response to treatment. There is little doubt that repeated laboratory confirmation of normal thyroid function can be wasteful; strategies have been suggested to improve cost-effectiveness (51-53).

 

However, functional disorders of the thyroid (hypothyroidism and hyperthyroidism) are common and, in many cases, managed by primary care providers. In addition to diagnosed cases, there are many patients who present to their provider seeking evaluation of their thyroid status as a possible cause of a variety of complaints including obesity, mood changes, hair loss, and fatigue. There is an ever-growing body of literature in the public domain, whether in print or internet-based, suggesting that thyroid conditions are under-diagnosed by physicians and that standard thyroid function tests are unreliable. Primary care providers are often the first to evaluate these patients and order biochemical testing. This has become a more complex process, with many patients requesting and even demanding certain biochemical tests that may not be indicated (54).

Prevalence

 

In considering the prevalence of thyroid dysfunction, a distinction needs to be made between so-called subclinical and overt abnormalities; paradoxically, this distinction is based on laboratory rather than clinical criteria. There is a trend to replace the term ‘subclinical hypothyroidism’ with the designation ‘mild thyroid failure’ (55,56).

 

In the progressive development of thyroid dysfunction, abnormal values for serum TSH generally occur before there is a diagnostic abnormality of serum T4, because of the markedly amplified relationship between serum T4 and release of TSH from the anterior pituitary (see below). For a two-fold change in serum T4, up or down from the set-point for that individual, the serum TSH will normally change up to 100-fold in the reverse direction (57,58). Thus, TSH becomes recognizably abnormal long before the serum concentrations of T4 or T3 fall outside the population-based reference interval. This was made possible by introduction of immunochemiluminometric methods for measurement of serum TSH with an increased functional sensitivity (59).

 

The more widespread the testing of thyroid function in the absence of suggestive clinical features, the greater the proportion of abnormal results in which only TSH is abnormal. In evaluating serum TSH, typically defined with a normal reference interval of about 0.4-4.0 mU/l, it is important to note that normal values approximate to a logarithmic distribution, with mean and median values at 1.0-1.5 mU/l (57,60-62). While values of 2-4 mU/l lie within the reference range, the likelihood of eventual hypothyroidism increases progressively for values above 2 mU/l, especially if thyroid peroxidase antibodies are present (63).

 

A population study in Colorado (64), of over 25,000 individuals of mean age 56 years, 56% of whom were female, showed TSH excess in 9.5 %, with a 2.2 % prevalence of suppressed TSH; over half the group with suppressed TSH were taking thyroid medication. In women, the prevalence of TSH excess increased progressively from 4% at age 18-24 to 20% over age 74 (64).

 

The National Health and Nutrition Examination Survey (NHANES III) (63), found hypothyroidism in 4.6% of the US population (0.3% overt and 4.3% subclinical) and hyperthyroidism in 1.3% (0.5% overt), with increasing prevalence with age in both females and males (figure 1). Abnormalities were more common in females than males. The prevalence of positive thyroid peroxidase antibodies was clearly associated with both hyper- and hypothyroidism, with important ethnic differences in antibody prevalence.

Figure 1. Percentage of the US population (NHANESIII) with abnormal serum TSH concentrations as a function of age. The disease-free population excludes those who reported thyroid disease, goiter or thyroid-related medications; the reference population excluded, in addition, those who had positive thyroid autoantibodies, or were taking medications that can influence thyroid function. Note the much higher prevalence of TSH abnormalities in the total population, than in the reference population (from reference (63)).

Prevalence data from one region do not necessarily apply in other populations, because of differences such as ethnic predisposition or variations in iodine intake. For example, in Hong Kong, where iodine intake is marginally deficient, only 1.2% of Chinese women aged over 60 years had serum TSH values > 5 mU/l, with a comparable prevalence of suppressed values indicating possible hyperthyroidism (65). Several European studies (66,67) have compared the effect of various levels of iodine intake on the prevalence of thyroid over- and underfunction.

 

Hypothyroidism is generally more common with abundant iodine intake, while goiter and subclinical hyperthyroidism are more common with low iodine intake (66,67). This was illustrated in a random selection of 4649 participants from the Civil Registration System in Denmark in age groups between 18 and 65 years. Thyroid dysfunction was evaluated from blood samples and questionnaires and compared with results from ultrasonography. Median iodine excretion was 53 µg/l in Aalborg and 68 µg/l in Copenhagen. Previously diagnosed thyroid dysfunction was found with the same prevalence in these regions. Serum TSH was lower in Aalborg than in Copenhagen (P=0.003) and declined with age in Aalborg, but not in Copenhagen. No previously diagnosed hyperthyroidism was found with the same overall prevalence in the two regions, but in subjects >40 years hyperthyroidism was more prevalent in Aalborg (1.3 vs 0.5%, P=0.017). No previously diagnosed hypothyroidism was found more frequently in Aalborg (0.6 vs 0.2%, P=0.03). Hyperthyroidism was more often associated with macronodular thyroid structure at ultrasound in Aalborg and hypothyroidism was more often associated with a patchy thyroid structure in Copenhagen. Thus, significant differences in thyroid dysfunction were found between the regions with a minor difference in iodine excretion. The findings are in agreement with a higher prevalence of thyroid autonomy among the elderly in the most iodine-deficient region (68).

 

Thus, thyroid abnormalities in populations with low iodine intake and those with high iodine intake develop in opposite directions: goiter and thyroid hyperfunction when iodine intake is relatively low, and impaired thyroid function when iodine intake is relatively high. Probably, mild iodine deficiency partly protects against autoimmune thyroid disease. Thyroid autoantibodies may be markers of an autoimmune process in the thyroid or secondary to the development of goiter (69).

 

These regional differences may and should influence the choice of diagnostic test and target population. For example, in an iodine replete environment, emphasis could be placed on testing younger or pregnant women for subclinical hypothyroidism by measurement of TSH and thyroid peroxidase antibodies, whereas in an iodine-deficient region there might be additional emphasis on early detection of thyroid autonomy and hyperthyroidism in older people, using a highly sensitive 3rd generation TSH assay.

 

SUBCLINICAL THYROID DYSFUNCTION

 

The proven or presumed importance of subclinical thyroid dysfunction will have a major effect on the extent to which thyroid function testing is applied in any population. The term ‘subclinical’ is used when the serum concentration of TSH is persistently abnormal (however defined), while the concentrations of T4 and T3 remain within their reference intervals. Because results can fluctuate spontaneously, a new diagnosis of subclinical thyroid dysfunction is not warranted on basis of a single laboratory sample. The following five criteria define endogenous subclinical thyroid dysfunction:

  1. TSH increased above or decreased below designated limits (see below)
  2. Normal free T4 concentration (and free T3 for hyperthyroidism)
  3. Abnormality is not due to medication (see below)
  4. There is no concurrent critical illness or pituitary dysfunction.
  5. A sustained abnormality is demonstrated over 3-6 months.

 

Apart from the situation of impending or early pregnancy, where there is clear consensus that subclinical hypothyroidism should be promptly and fully treated, the approach to subclinical thyroid dysfunction remains uncertain (70). Various authorities have expressed divergent views on the importance of detecting the mild TSH abnormalities that reflect subclinical thyroid dysfunction. Extremes of opinion can be summarized as follows. On the one hand, some take the position that subclinical thyroid dysfunction, both hypothyroidism and hyperthyroidism, are disorders that need to be treated in order to avert potential harm (71). To achieve optimal sensitivity, particularly for the diagnosis of hypothyroidism, some have advocated that the upper limit of the TSH reference interval should be lowered (72)because values in the range 2-4 mU/l, usually regarded as normal, are associated with an increased prevalence of future hypothyroidism (62). Active search for subclinical thyroid dysfunction is based on the view that treatment is usually justified, because of potential adverse outcomes, even if proof of benefit is still lacking. At this end of the opinion spectrum, there is support for general community screening for thyroid dysfunction (71), in contrast to a case-finding strategy for women over 50 when they present for medical care (1).

 

Others have taken the view that while there is circumstantial evidence that subclinical thyroid dysfunction can have adverse long-term effects, there is still a lack of strong evidence that treatment of thyroid dysfunction in general confers benefit (73), although more indicative evidence has emerged with time (see later). Thus, treatment of subclinical hyperthyroidism seems to improve bone health (74) and prevent atrial fibrillation (15), while evidence for treatment benefits of subclinical hypothyroidism is much weaker, at least in patients above 65 years of age (see later).

 

A definitive position on this dilemma should ideally emerge from long-term studies focused on outcomes, but if differences are small, studies may be under-powered and the results may still be indeterminate. Other factors to take into account in establishing an approach to widespread thyroid testing include ethnic or environmental predisposition to thyroid dysfunction in various communities, balance with other healthcare priorities that may be more compelling, cost of laboratory testing and the extent to which competent clinical assessment and therapeutic response may be overwhelmed by reliance on laboratory measurements. Yet, even with the aspect of not advocating for universal screening, some guidelines inadvertently include almost all women for testing of thyroid function, even if not stating it clearly (75). Thus, according to the American Thyroid Association (ATA) and American Association of Clinical Endocrinologists (AACE) guidelines, levothyroxine therapy would be considered for 92% of women with subclinical hypothyroidism and TSH ≤10 mU/L (75).

 

Adverse Consequences of Subclinical Thyroid Dysfunction

 

The issues that identify the clinical importance of subclinical thyroid dysfunction are summarized in Table 4; many of these adverse effects relate to the cardiovascular system (73,76). There is still conflicting evidence on whether mild thyroid abnormalities influence cardiovascular mortality and, as yet, no convincing support for the proposition that treatment of subclinical thyroid dysfunction improves survival. In a 12-year follow-up study of women over 65, neither TSH > 5 mU/l, nor <0.5 mU/l were associated with any increase in all-cause or cardiovascular mortality, although a previous history of hyperthyroidism had a minor adverse effect (77). In contrast, another survey of the relationship between serum TSH and all-cause and cardiovascular mortality over a 10-year period in individuals over 60, showed that the group with serum TSH below 0.5 mU/l had a significantly increased mortality, apparently due to cardiovascular disease (78), although an increased serum TSH was not associated with excess mortality (78). More recent studies provide strengthening evidence for adverse effects from minor degrees of thyroid dysfunction. A Scottish study (79) of over 17,000 people followed for an average of 4.5 years, correlated fatal or non-fatal first episodes of cardiovascular disease, arrhythmias, and osteoporotic fractures with ambulatory serum TSH values. With elevated TSH >4 mU/l there was an increased incidence of dysrhythmia, cardiovascular ischemic episodes, and fracture (hazard ratios 1.8-1.95) (77). With a suppressed serum TSH <0.03 mU/l, all three endpoints were also increased in frequency (hazard ratio 1.4-2.0). It is important to note that these authors distinguished between clearly suppressed TSH (<0.03 mU/l) and subnormal-detectable TSH 0.04-0.4 mU/l, the latter of which was not associated with significant adverse effects. These findings appeared to be at odds with the initial evaluation of the Wickham data that showed no adverse cardiovascular effects of subclinical hypothyroidism. However, reanalysis of the Whickham findings (80) did show an adverse effect if the beneficial effect of thyroxine treatment was excluded.

 

Table 4. Reported Effects of Subclinical Thyroid Dysfunction

Subclinical hyperthyroidism (suppressed TSH, normal free T4, and free T3 estimates)

·     Exposure to iodine may precipitate severe thyrotoxicosis (81)

·     Threefold increased risk of atrial fibrillation after 10 years (82)

·     Abnormalities of cardiac function (15,82,83)

·     Osteoporosis risk increased (84,85)

·     Progression to overt hyperthyroidism (86,87)

Subclinical hypothyroidism or mild thyroid failure (increased TSH, normal free T4 estimate)

·     Non-specific symptoms may improve with treatment (88)

·     Progression to overt hypothyroidism (89)

·     Independent risk factor for atherosclerosis (90)

·     Increased risk of coronary artery disease (91-93)

·     Increased frequency of congestive heart failure (91,93,94)

·     Adverse effects on vascular compliance (95-97)

·     Abnormal cardiac function may improve with treatment (98)

·     Beneficial effect of treatment on lipids (99,100)

·     Increased prevalence of depressive illness (101)

·     Impaired fibrinolysis (102)

 

Subclinical Hyperthyroidism

 

NATURAL HISTORY OF SUBCLINICAL HYPERTHYROIDISM

     

Follow up studies suggest that spontaneous progression to overt hyperthyroidism is uncommon and that subnormal-detectable levels of TSH in the range 0.05-0.4 mU/l frequently return to normal within one year (87). Meyerovitch et al. (103) reported the results of sequential tests of thyroid function over a 5-year period in a large community-based cohort. During the follow-up period, test results returned to normal in 27%, 62% and 51% respectively of untreated patients whose initial serum TSH values were >10 mU/l, 5.5-10 mU/l and < 0.35 mU/l (103). While some subjects did show progression, the high chance of resolution towards normal suggests that retesting after follow-up, possibly after at least 6 months, is advisable before considering any intervention. However, quantitative conclusions from that retrospective study may be insecure, as many subjects with abnormal TSH were excluded from follow-up because they were treated after their initial result (103). If those treated were the more severely affected, whether based on symptoms, presence of goiter, or degree of TSH abnormality, the analysis may over-estimate the likelihood of resolution during follow-up. Spontaneous remission of subclinical hyperthyroidism may occur more frequently in Graves’ disease than in nodular thyroid disorders (104). However, in a recent study from UK (105), a third each of 84 patients with subclinical hyperthyroidism due to Graves’ disease progressed, normalized, or remained in the subclinically hyperthyroid state. Older people and those with positive anti-thyroperoxidase antibodies had a higher risk of progression of the disease. These data need to be verified and confirmed in larger cohorts and over longer periods of follow-up. The chance of spontaneous progression to overt hyperthyroidism appears to be no greater than 10% per year (87,104), or even lower, and also seems dependent on the cause of subclinical hyperthyroidism (Graves’ or multinodular) (106). However, it should be noted that the transition from autoimmune subclinical to overt hyperthyroidism can occur more rapidly than is generally the case in immune hypothyroidism (107).

 

CARDIOVASCULAR EFFECTS

 

From the Framingham study it was found that undetected subclinical hyperthyroidism, defined only by suppression of TSH, carried a three-fold increased risk of atrial fibrillation within 10 years (78). As yet, there is no study that shows that treatment given on the basis of low TSH alone, modifies this risk (108), although it is clear that survival is adversely affected by atrial fibrillation (109). In a large cohort study, endogenous subclinical hyperthyroidism is associated with increased risks of total and coronary heart disease mortality, and incident atrial fibrillation, with highest risks of coronary heart disease mortality and atrial fibrillation when the TSH concentration was lower than 0.10 mIU/L (110). A recent meta-analysis of prospective cohorts found an increased risk of coronary heart disease (relative risk (RR) 1.20; 95% confidence interval (CI), 1.02-1.42), total mortality (RR = 1.27; 95% CI, 1.07-1.51), and coronary heart disease mortality (RR = 1.45; 95% CI, 1.12-1.86) from subclinical hyperthyroidism, while this was not the case for subclinical hypothyroidism (111). Another group also analyzed prospective cohorts in a systematic review and meta-analysis (112) and found that higher free T4 levels at baseline in euthyroid individuals were associated with an increased risk of atrial fibrillation in age- and sex-adjusted analyses (hazard ratio, 1.45; 95% confidence interval, 1.26-1.66, for the highest quartile versus the lowest quartile of free T4; P for trend ≤0.001 across quartiles). Estimates did not substantially differ after further adjustment for preexisting cardiovascular disease. Thus, in euthyroid individuals, higher circulating free T4 levels, but not TSH levels, were associated with increased risk of incident atrial fibrillation (112). These results, however, need verification in prospective studies. Finally, impaired left ventricular ejection fraction and reduced exercise capacity have been documented in subclinical hyperthyroidism due to high dosage thyroxine and may be alleviated by beta blockade (113).

 IODINE-INDUCED THYROTOXICOSIS

 

Undiagnosed subclinical hyperthyroidism due to autonomous nodular thyroid disease, a condition especially prevalent in iodine deficient regions, carries the risk of progression to severe overt thyrotoxicosis after iodine exposure (114). An Australian study from a region that is not known to be iodine deficient, was suggestive of recent iodine exposure, most often from radiologic contrast agents, in up to 25% of elderly thyrotoxic patients (81). Prior knowledge of subnormal serum TSH may identify a high-risk group with thyroid autonomy in whom iodine exposure carries the risk of iatrogenic hyperthyroidism (115,116). Prophylactic drugs could be considered in high-risk populations, such as administration of perchlorate and/or a thionamide class drug to elderly patients with suppressed TSH and/or palpable goiter (115).

 

OSTEOPOROSIS

Notably, in a controlled trial of suppressive T4 treatment for multinodular goiter, TSH suppression without clear excess of serum T4 or T3 resulted in a mean 3.6% decrease in lumbar spine density within 2 years (74). Normalization of serum TSH resulted in normalization of the bone mineral density in postmenopausal women.

Subclinical Hypothyroidism

NATURAL HISTORY OF SUBCLINICAL HYPOTHYROIDISM

 

The benchmark study of thyroid epidemiology from Wickham, UK (119), showed that the likelihood of overt hypothyroidism after 20 years was directly related to the initial serum TSH concentration, even when the concentration was in the range between 2 to 4 mU/l, within the upper reference interval. The study by Meyerovitch et al. (103)showed that with the initial serum TSH in the range 5.5-10 mU/l, the chance of normalization after prolonged follow-up was greater than the chance of progression to TSH levels >10 mU/l (see above). The antibody status was unfortunately not assessed in that cohort. Huber et al. (87) followed 82 Swiss women with subclinical hypothyroidism, with normal free T4 and serum TSH >4 mU/l, for a mean of 9.2 years (Figure 2). About half of their cohort had had previous ablative treatment for Graves’ disease. The cumulative incidence of overt hypothyroidism, defined here as low free T4 with TSH >20 mU/l, was directly related to the initial serum TSH, with 55% of women with initial serum TSH >6 mU/l progressing to overt hypothyroidism. Progression was not uniform, and over half of the cohort showed no deterioration of thyroid function, but positive microsomal antibodies (corresponding to the more specific thyroperoxidase antibodies) increased the likelihood of progression. It is now clear that the opposite sequence may also occur, with spontaneous normalization of elevated TSH values (103,120). In a cardiovascular health study (121), subclinical hypothyroidism persisted for 4 years in just over half of older individuals, with high rates of reversion to euthyroidism in individuals with lower TSH concentrations and thyroperoxidase antibody negativity. It was advised that future studies should examine the impact of transitions in thyroid status on clinical outcomes (121). Rosario et al. found that most of 241 women with mild TSH elevations ranging from 4.5 to 10 mIU/l did not progress to overt hypothyroidism and even normalized their serum TSH. However, initial TSH seemed to be a more important predictor of progression than the presence of antibodies or ultrasonographic appearance (122). In a prospective study from China, patients >40 years of age, i.e. a younger mean age than most studies, with higher baseline total cholesterol or positive thyroid peroxidase antibodies, had higher risks of progression to overt hypothyroidism, while those with higher baseline creatinine, higher baseline TSH (≥7 mIU/L, p <0.001), or older age (>60 years vs. ≤50 years, p =0.012), had lower odds of reverting to euthyroidism. They concluded that thyroperoxidase antibodies and total cholesterol seemed to be more important predictors of progression to overt hypothyroidism than the initial TSH concentration, whereas high baseline TSH or creatinine were negatively correlated with reversion to euthyroidism. The prognostic value of total cholesterol and creatinine should therefore be considered in mild subclinical hypothyroidism (123).

Notably, a prospective study showed that the progression of autoimmune subclinical hypothyroidism tends to be slower than for subclinical hyperthyroidism (107). Hence, there is a need for prolonged follow-up and patient education, if the decision to treat is deferred.

Figure 2. Kaplan-Meier estimates of the cumulative incidence of overt hypothyroidism in women with subclinical hypothyroidism (initial serum TSH >4 mU/l) as a function of initial serum TSH, thyroid secretory reserve in response to oral thyrotropin releasing hormone (TRH) and detectable microsomal antibodies. Serum TSH appears to be the strongest of these predictors (from reference (89)).

ATHEROSCLEROSIS AND VASCULAR COMPLIANCE

 

As mentioned earlier, subclinical hypothyroidism, or mild thyroid failure, was shown to be an independent risk factor for both myocardial infarction and radiologically-visible aortic atherosclerosis in a study of Dutch women over 55 years of age (90). This effect was independent of body mass index, total and HDL cholesterol, blood pressure, and smoking status. The attributable risk for subclinical hypothyroidism was comparable to that for the other major risk factors, hypercholesterolemia, hypertension, smoking, and diabetes mellitus. The association was slightly stronger when subclinical hypothyroidism was associated with positive peroxidase antibodies, but thyroid autoimmunity itself was not an independent risk factor (124). A systematic review and meta-analysis of 27 studies demonstrated a significant association of subclinical hypothyroidism and cardiovascular risk with arterial wall thickening and stiffening as well as endothelial dysfunction. However, sustained subclinical thyroid dysfunction did not affect the baseline or development of carotid plaques in healthy individuals (125).

 

In cross-sectional studies, carotid artery-intima media thickness was significantly higher in participants with subclinical hypothyroidism compared to euthyroid controls (126). Small interventional studies suggested that restoring euthyroidism in patients with subclinical hypothyroidism is associated with regression of carotid atherosclerosis (126,127). However, these trials had major limitations, with uncontrolled study designs and/or small sample sizes (the largest included only 45 participants with subclinical hypothyroidism (127).

 

Although well known in overt hypothyroidism (128), the finding of impaired flow-mediated, endothelium-dependent vasodilatation in subjects with borderline hypothyroidism or high-normal serum TSH values (95) was at first unexpected. Baseline artery diameter and forearm flow were comparable, but flow mediated vasodilatation during the period of reactive hyperemia was significantly impaired even in the group with serum TSH of 2-4 mU/l, compared with the group with serum TSH 0.4-2 mU/l (95). The difference could not be attributed to a difference in maximal nitrate-induced vasodilatation, age, sex, hypertension, diabetes, smoking, serum cholesterol, or levels of total T3 and T4 (91). This finding suggested that even a minor deviation from an individual’s pituitary-thyroid set point may be associated with alteration in vasodilatory response. There is no known direct action of TSH that would account for this effect. A Japanese placebo-controlled study of women aged 60-70 with subclinical hypothyroidism with mean pre-treatment serum TSH of 7.3 mU/l showed improvement in pulse wave velocity, an index of vascular stiffness, in response to TSH normalization for 2 months by progressive low dose T4 replacement only up to 37.5 ug/day (96). Thyroxine replacement for 18 months has been reported to improve blood pressure, lipids, and carotid intimal thickness in women with subclinical hypothyroidism (129). In a larger properly powered randomized placebo-controlled trial normalization of TSH with levothyroxine was associated with no difference in carotid intima-media thickness and carotid atherosclerosis in older persons with subclinical hypothyroidism (130); a meta-analysis of 12 studies showed (131) that carotid intima-media thickness was significantly higher among subjects with subclinical hypothyroidism (n=280) as compared to euthyroid controls (n=263) at baseline. This meta-analysis showed that thyroxine therapy in subjects with subclinical hypothyroidism significantly decreased carotid intimal thickness and improves lipid profiles, modifiable cardiovascular risk factors. One of the big differences between these publications was the age difference; the subjects were younger in the meta-analysis (131), while the patients in the other publications were all older and not included in the meta-analysis, which was published earlier. The same was the case in another more recent meta-analysis, including only 3 randomized clinical trials in younger patients with subclinical hypothyroidism (132). They also found a decreasing carotid intimal thickness with levothyroxine therapy. Thyroid hormone replacement in younger subjects with subclinical hypothyroidism may thus play a role in slowing down or preventing the progression of atherosclerosis (131,132), but there is still no such evidence in older individuals.

 

LIPIDS

Overt hypothyroidism is associated with an increase in the serum cholesterol concentration (133) and correction of overt hypothyroidism resulted in a decrease in total and low density lipoprotein (LDL) cholesterol, apolipoprotein A1, apo B and apo E, and serum triglyceride concentrations may also decrease (134,135). A defect in receptor-mediated LDL catabolism, similar to that seen in familial hypercholesterolemia, has been described in severe overt hypothyroidism (136), but there is no evidence to support such an abnormality in mild thyroid failure. At the other extreme, several large population studies reported a positive correlation between serum lipids and serum TSH across its normal range (137), a correlation also associated with increasing blood pressure (138). The clinical impact of these associations remains unknown.

 

The Colorado study including over 25,000 subjects showed a continuous graded increase in serum cholesterol over a range of serum TSH values from <0.3 to >60 mU/l (64). However, there is still no consensus that mild thyroid failure has an adverse effect on plasma lipids, or that T4 treatment sufficient to normalize isolated TSH elevations has a beneficial effect. A meta-analysis suggests that T4 treatment of subjects with mild thyroid failure does lower the mean total and LDL cholesterol, and is without effect on high density lipoprotein (HDL) cholesterol or triglyceride levels (99). In a prospective double-blind, placebo-controlled trial of thyroxine in subclinical hypothyroidism in which the response was carefully monitored with TSH, Meier et al. (100) reported that the decrease in LDL cholesterol was more pronounced with higher initial TSH levels >12 mU/l or with elevated baseline LDL concentrations, but also here the clinical impact remains unknown.

 

It remains uncertain whether the serum concentration of the highly atherogenic Lp(a) particle is increased in overt hypothyroidism and whether T4 treatment sufficient to normalize TSH has a favorable influence. Serum concentrations of Lp(a) have been found to be increased in overt hypothyroidism with normalization after treatment in some studies (139,140) while others fail to confirm this finding (141,142). Finally, in 100 women with subclinical hypothyroidism selected among 87 obese women aged between 50 and 70 years, total cholesterol, LDL-cholesterol and triglycerides concentrations as well as LDL-C/HDL-C ratio and Castelli index were higher in subclinical hypothyroidism than in controls and decreased after levothyroxine substitution. All the calculated atherosclerosisindexes showed significant positive correlations with TSH concentrations in the subclinical hypothyroidism group. Also, in this group the systolic and diastolic blood pressure decreased significantly after treatment. Thus, dyslipidemia in obese subclinical hypothyroidism women is not severe, but if untreated for many years, it is assumed to lead to atherosclerosis. Substitution therapy improved the lipid profile, changing the relations between protective and proatherogenic fractions of serum lipids, and it optimizes blood pressure (143), which by itself does not prove a positive clinical outcome.

CARDIAC FUNCTION  

From echocardiographic studies, there is evidence that mild thyroid failure can significantly increase systemic vascular resistance and impair cardiac systolic and diastolic function (144), as demonstrated by decreased flow velocity across the aortic and mitral valves (98). These changes, which were associated with reduced cardiorespiratory work capacity during maximal exercise, were reversed by T4 treatment sufficient to normalize serum TSH (98). Impairment of both diastolic and systolic function was demonstrable by echocardiography in a subclinically hypothyroid group of patients with TSH in the range 4-12 mU/l (98). Thyroxine treatment sufficient to normalize TSH to a mean of 1.3 mU/l for 6 months was associated with improvement in myocardial contractility (98). Calculation of a global myocardial performance index from the echocardiographic findings also confirmed significantly higher scores in hypothyroid patients in comparison to the control group, showing that regression in global left ventricular functions is an important echocardiographic finding (145).

 

A very recent small study of 20 young patients with autoimmune subclinical hypothyroidism used cardiac magnetic resonance for a myocardial longitudinal relaxation time (T1) mapping technique and demonstrated significant diffuse myocardial injury (146), which may explain results of the cardiac function studies and also provide a novel method for early detection of cardiac dysfunction in subclinical hypothyroidism.

 

Future studies are required to determine the effects of the above finding on long-term cardiovascular outcomes and how these reversible abnormalities relate to cardiovascular prognosis.

 

INSULIN SENSITIVITY AND METABOLIC SYNDROME

 

Several studies suggested that there may be a link between insulin sensitivity, serum lipids, and thyroid function, whether assessed by serum TSH or circulating thyroid hormone levels. Bakker et al. (147) noted that while serum TSH showed no overall correlation with insulin sensitivity or serum lipids, there was a complex interaction between these variables such that the association between TSH and LDL-C was much stronger in insulin- resistant than in insulin- sensitive subjects. The same group showed that low free T4 levels within the reference range are dually associated with LDL-C and insulin resistance (148). Further results will show whether these findings account for the purported link between subclinical hypothyroidism and the metabolic syndrome (149) and whether this link contributes to increased cardiovascular risk in a subgroup of patients with subclinical hypothyroidism. Notably, there is a positive correlation between serum TSH and BMI in euthyroid obese women (9,150). The results of a population study suggest that thyroid function (also within the reference range) could be one of several factors acting in concert to determine body weight in a population. Even slightly elevated serum TSH levels were associated with an increase in the occurrence of obesity (151). Furthermore, a recent cross-sectional study in a sample of 753 subjects (46% males) aged 35-70 years who had no history of diabetes, renal, hepatic, thyroid, or coronary heart disease, and were participants of the Genetics of Atherosclerotic Disease study indicated that subclinical hypothyroidism was associated with fatty liver together with increased odds of metabolic syndrome, insulin resistance, and coronary artery calcification, independent of potential confounders (152).

 

Finally, subclinical hypothyroidism has been suspected to be related to polycystic ovary syndrome, since subclinical hypothyroidism is present in 10-25% of women with polycystic ovary syndrome. However, a recent meta-analysis of 12 studies found that subclinical hypothyroidism did not influence the hormonal profile of women with polycystic ovary syndrome. On the other hand, it resulted in mild metabolic abnormalities, which are, however, not clinically important in a short-term setting (153).

 

The value of routine thyroid testing in the above groups remains uncertain (154). The metabolic syndrome and subclinical hypothyroidism are both highly prevalent in the general population. Cross-sectional epidemiological data suggest that a mutual association exists between the two, although the cause–effect relationship remains poorly elucidated. As subclinical hypothyroidism raises cholesterol, blood pressure, and visceral fat, it is easy to understand why it associates with metabolic syndrome (155). Rather, the reasons whereby patients with metabolic syndrome are at higher risk for subclinical hypothyroidism are less apparent. Some studies have reported that subclinical hypothyroidism is itself characterized by high cardiovascular risk. Therefore, the coexistence of subclinical hypothyroidism and metabolic syndrome may identify subjects at a particularly high risk for future cardiovascular events. Recent data indicated that carotid intima-media thickness, a marker of initial atherosclerosis and a possible predictor of future events, was higher in patients with both subclinical hypothyroidism and metabolic syndrome than in the presence of each condition alone.

 

To date, it remains unclear whether any biological relationship between subclinical hypothyroidism and the metabolic syndrome truly exists and what the underlying mechanisms might be. Nonetheless, given the high prevalence of both conditions, and the observed associations, it is of interest to investigate whether their mutual presence confers a higher cardiovascular disease risk. If confirmed in larger studies, these results may be clinically relevant, suggesting that subclinical hypothyroidism should be investigated in patients with metabolic syndrome to better individualize therapy and counter cardiovascular risk (156).

 

OSTEOPOROSIS

From a previously mentioned study (118), subclinical hypothyroidism was associated with RRs of 1.34 (95% CI 1.14-1.58; I 2 =32%) for hip fracture, 1.27 (95% CI 1.02-1.58; I 2 =51.9%) for any location of fracture, and 1.25 (95% CI 1.04-1.50) for forearm fracture. The authors failed to find any associations between the change in bone mineral density and subclinical hypothyroidism but subclinical hypothyroidism was associated with an increased risk of fractures. Although subclinical hyperthyroidism was related to reduced bone mineral density, there is no evidence of a definite association between subclinical hypothyroidism and the risk of low bone mineral density.

NEUROBEHAVIORAL EFFECTS AND QUALITY OF LIFE

 

It is well known that overt thyroid dysfunction can include psychological or psychiatric symptomatology. A small retrospective study has shown a 2-3 fold increased frequency of previous depression in subjects with mild thyroid failure (101); T4 treatment has been reported to improve neuropsychological responses in this group (157). However, contrary to these findings, more recent controlled studies of unselected patients suggest that subclinical hypothyroidism is not associated with any consistent deficit in quality of life indices or improvement with treatment (158,159). This was very recently confirmed in the TRUST trial using the thyroid patient reported outcome (ThyPRO) questionnaire in older adults with subclinical hypothyroidism; in that study, therapy with levothyroxine did not improve symptoms or tiredness compared with placebo (160).

 

STUDIES IN CHILDREN

 

A study by Cerbone et al. (161) has shown that long-term idiopathic subclinical hypothyroidism did not appear to have an adverse effect on linear growth or intellectual development in children aged 4-18 years. More recently, a meta-analysis including nine studies demonstrated that subclinical hypothyroidism in children is a remitting process with a low risk of evolution toward overt hypothyroidism. Most of the subjects reverted to euthyroidism or remained subclinically hypothyroid, with a rate of evolution toward overt hypothyroidism ranging between 0 and 28.8%, with a rate of 50% in only one study. The initial presence of goiter and elevated thyroglobulin antibodies, the presence of celiac disease, and a progressive increase in thyroperoxidase antibodies and TSH value predict progression toward overt hypothyroidism. Replacement therapy is not indicated in children with subclinical hypothyroidism with TSH 5-10  mU/l, absence of goiter, and negative antithyroid antibodies. An increased growth velocity was observed in children treated with levothyroxine in two studies. Levothyroxine reduced thyroid volume in 25-100% of children with subclinical hypothyroidism and autoimmune thyroiditis in two studies. No effects were seen on neuropsychological functions in one study, and posttreatment evolution of subclinical hypothyroidism was reported in one study (162). Hence, it may be justifiable to follow this group without early recourse to lifelong replacement.

 

On the other hand, the association with Hashimoto’s thyroiditis exerted a negative influence on the evolution over time of mild subclinical hypothyroidism, irrespective of other concomitant risk factors. In children – unlike in adults - with mild and subclinical hypothyroidism related to Hashimoto’s thyroiditis, the risk of a deterioration in thyroid status over time is high (53.1%), while the probability of spontaneous TSH normalization is relatively low (21.9%). In contrast, children with mild and idiopathic subclinical hypothyroidism, had a very low risk of a deterioration in thyroid status over time (11.1%), whereas the probability of spontaneous TSH normalization was high (41.1%) (163).

 

Safety and Effectiveness of Treatment of Subclinical Thyroid Dysfunction

 

The benefits of early diagnosis and treatment are self-evident from the obvious decline in hospitalization and mortality rates for severe thyroid dysfunction over the past decades. Before reliable tests of thyroid function became widely used, severe hyperthyroidism approaching thyroid storm and hypothyroidism with impending myxedema coma occurred quite regularly, but these presentations are now very uncommon (164,165). While the arguments for seeking and treating mild thyroid dysfunction are less compelling, there may be potential benefits for large numbers of people.

 

The points in favor of treating mild thyroid dysfunction relate directly to the adverse consequences listed in Table 4, but for many of these adverse outcomes there is still a lack of long-term studies that show benefit as illustrated in a very recent randomized, double-blind placebo-controlled trial nested within the TRUST trial, which found that normalization of TSH with levothyroxine in people >65 years was associated with no difference in carotid-intima media thickness and carotid atherosclerosis by ultrasound in older persons with subclinical hypothyroidism (130). On the basis of potential benefit from simple straightforward treatment and absence of adverse effects, the argument for active treatment is generally stronger for mild thyroid failure than for subclinical hyperthyroidism. Conservative T4 therapy aimed at normalizing TSH is simple, inexpensive and generally safe (166), although replacement may not be warranted in the older adults with very advanced age (167). Where cardiovascular disease precludes full thyroid hormone replacement, detailed evaluation of the cardiac abnormality is appropriate (168,169). In contrast, treatment of subclinical hyperthyroidism needs to be evaluated in relation to adverse drug effects and the potential for hypothyroidism. It is, however, prudent to take into consideration an effect and consequence of suppressed serum TSH on atrial fibrillation, bone loss, depression, quality of life and mortality when counseling individual patients.

 

In younger patients there may be a benefit of early treatment of subclinical hypothyroidism (170) although the studies supporting this approach were not placebo-controlled and only used surrogate endpoints in small cohorts.

 

USE OF LABORATORY ASSAYS FOR CASE FINDING AND SCREENING

 

If the identification of abnormal thyroid function is to be based on laboratory testing, it is desirable that population reference intervals should not vary between methods. As recently emphasized, serum T3, T4 and serum TSH concentrations are among the many hormone variables where between-assay standardization is crucial to ensure optimal assay specificity (171). At present, that aim is not satisfactory for free T4 estimates, a problem that is particularly troublesome during pregnancy (see below). Analytically, serum TSH, total T4 and total T3 are well standardized, so that considerations of so-called normal ranges relate to the clinically relevant issues. By contrast, the diverse, ingenious manoeuvres involved in the estimation of free T4 and T3 lead to poor standardization between methods, so that method-specific reference intervals need to be used, both for individual clinical diagnosis and population studies. Between-method free T4 variations are especially troublesome in pregnancy and critical illness (see below).

TSH Reference Interval or “Normal Range”

For serum TSH, arguably the most important variable for the diagnosis of primary thyroid dysfunction, no firm consensus range has been agreed, despite reliable standardization between methods. Firstly, a reference interval for the diagnosis of hypothyroidism in adults may be far too broad and lenient to identify women whose fertility or pregnancy outcome might be improved by thyroid supplementation. Second, it has been shown that the reference TSH set-point for each individual can be defined with a narrow band of the broad population range (172,173) (see below). Third, criteria for the new diagnosis of thyroid dysfunction may not be the same as those required for optimal adjustment of therapy. Fourth, for population studies, whether screening or case-finding, a reference interval with higher sensitivity will have lower specificity.

 

Thus, the controversies as to whether the standard TSH reference interval of about 0.4–4.0 mU/l should be narrowed, with lowering of the upper limit (70), or retained (174-176) do not address the needs of individuals. Population studies to define the upper limit of the “normal range” for serum TSH will be influenced by whether those with positive thyroperoxidase antibodies are excluded (see below) (63,177,178). However, even after exclusion of individuals with clinical, antibodies, or sonographic evidence of any thyroid disorder, Hamilton et al. supported an upper reference limit at about 4 mU/l (176), although quite different criteria may apply around pregnancy (see below). Ethnic differences (179), as well as differences and time of sampling in relation to diurnal variation are also important.

 

Terminology for abnormal TSH values has also become inconsistent, as for example in the use of the term suppressed to describe lower-than-normal TSH values. In some studies (180,181) any subnormal value is classified as suppressed, while others reserve this term for lower levels (63) that allow a distinction between undetectable (e.g. <0.03 mU/l) and a subnormal-detectable range below about 0.4 mU/l (76). These two categories may have different diagnostic and prognostic significance. Based on follow-up studies of the probability of progression to overt thyroid dysfunction, there is a strong case for regarding TSH values in the subnormal-detectable range, arbitrarily 0.05-0.4 mU/l, as distinct from the even lower levels that are typical of hyperthyroidism. It is a personal view that the term “suppressed” should be avoided in describing subnormal-detectable values, a semantic point that may affect up to 1% of the population. Since the gradation from normality to severe thyroid dysfunction is a continuum, studies of adverse outcomes or benefits from intervention will be critically dependent on uniform terminology.

 

Choice of Initial Test

 

The definitive diagnosis of thyroid dysfunction should always be made using the typical relationships between trophic hormone and target gland secretion that define endocrine dysfunction. In contrast, case-finding studies of untreated subjects may begin with measurement of TSH alone (182), with T4 and T3 assays added only if TSH is abnormal, or if an abnormality of TSH secretion is suspected. It is self-evident that serum TSH loses its diagnostic value when pituitary function is abnormal (183-186).

 

In the absence of associated disease, a normal serum TSH concentration by a so-called third generation assay (a functional lower limit of sensitivity of about 0.03 mU/l) (57,58), has a high negative predictive value in ruling out primary hypothyroidism and hyperthyroidism. Such immunometric assays, which use two antibodies against different epitopes of the TSH molecule, give a wide separation between the lower limit of the normal reference range at about 0.4 mU/l and the typical suppressed TSH values found in hyperthyroidism. While some subjects with subnormal-detectable TSH values do progress to overt hyperthyroidism, values in this range may also revert to normal (see above).

 

There are some clinical situations in which assessment of thyroid function will give a high prevalence of abnormalities that cannot be interpreted with certainty. Notably, glucocorticoids and dopaminergic agents have a potent effect to suppress TSH secretion (45,187), while TSH is also frequently subnormal in starvation or caloric deprivation (188). Transient increases to above normal can occur in euthyroid subjects during recovery from critical illness (178,189-191). The finding that 33% of serum TSH values fell more than 2 standard deviations (SD) from the geometric mean in acutely hospitalized patients, with 17% of values more than 3 SD from the mean value, indicates that TSH concentrations lack diagnostic specificity in this setting (192). A serum free T4 estimate will generally follow from an abnormal TSH concentration, but during critical illness, free T4 estimates often show non-specific abnormalities (see below) (193). Lack of specificity was the basis for a recommendation against routine assessment of serum TSH and free T4 during acute critical illness in the absence of risk factors, or clinical features suggestive of a thyroid disorder (194).

Testing of thyroid function is appropriate in a wide range of psychiatric disorders, but diagnostic specificity is limited by a high prevalence of transient non-specific abnormalities at the time of acute psychiatric admission (195). Thus, laboratory evaluation should be delayed for 2-3 weeks after acute presentation, unless there are specific risk factors for thyroid dysfunction (196).

 

Potential Adverse Effects of Testing

 

In terms of potential for adverse effects, there may be important differences between screening of unselected populations and the case-finding strategy that is now recommended for thyroid dysfunction. There are no reports of a “labelling effect” (i.e. perception of chronic illness in previously asymptomatic subjects), described in hypertension screening programs (197), when testing for thyroid dysfunction is done at the time of presentation for medical care. Nevertheless, further attention needs to be given to the potential for unwarranted treatment based on false positive results, as well as the cost of follow-up investigations for perceived abnormalities that may not warrant treatment at any stage. In particular, there is a need for clinical consensus as to how marginally abnormal results should be classified. Widespread laboratory testing will lead to an increase in the number of false positive results; the potential for a diagnostic method to give misleading variations from normal may not become known for some years until the full diversity of the non-diseased population is documented (198).

Follow-up of Abnormal Results

 

If serum TSH is used as a single initial test for case-finding, a value outside the reference interval should lead to estimation of serum free T4 on the same sample, if possible, without recall of the patient. This requires an algorithm-based testing protocol, which should also include measurement of serum free T3 if TSH is suppressed, to identify T3 toxicosis. It may also be relevant to measure thyroid peroxidase antibodies if TSH is increased, so as to define an autoimmune mechanism of hypothyroidism, particularly as a raised level of these antibodies is associated with an increased likelihood of progression to overt hypothyroidism (61,178).

 

For screening or case-finding to be effective, patients with unsuspected overt thyroid dysfunction should be actively traced because they will benefit most from treatment and have the most to lose if the abnormal finding is ignored. For mild thyroid dysfunction, a practitioner who has continuing contact with the patient should evaluate the assay result in clinical context and initiate any necessary follow-up. However, there is currently no consensus as to how an appropriate clinical response to abnormal laboratory findings can be assured.

 

A transition towards identification of thyroid dysfunction by laboratory measurement, rather than on clinical criteria, modifies, but does not diminish the role of the clinician. The severity of thyroid dysfunction cannot be judged from the extent of the laboratory abnormality (198,199), which indicates neither the duration of exposure, nor individual susceptibility. Whether a decision is made to treat or to observe, patient education is crucial in establishing effective compliance and rational cost-effective long-term follow-up. Computer based programs can identify affected individuals, but do not replace direct involvement of both patient and clinician. There may be potential medicolegal consequences of failure to respond to abnormal results, if widespread laboratory testing is initiated without an established follow-up plan.

INTERPRETATION OF TSH AND T4 ASSAYS

The TSH-T4 Relationship

 

Definitive assessment of thyroid status requires both a sensitive serum TSH assay and a valid T4 estimate, with interpretation based on the relationship between these two values. It is generally assumed that inverse TSH responses to changes in free T4 are approximately logarithmic (59) (Figure 3). While this concept is useful, recent data suggests that the relationship varies with thyroid status, age, sex, smoking and thyroid autoantibody status (200-203) with progressively greater amplification of the TSH response as thyroid function declines. Diurnal variation in serum TSH, with amplitude about 50% with higher levels between 2100 and 0600, is superimposed on rapid pulse secretion with amplitude about 10%. Basal secretion and pulsatility are both increased in primary hypothyroidism, with retention of diurnal variation (204).

Figure 3. The relationship between serum TSH and free T4 estimate in ambulatory individuals with stable thyroid function and normal hypothalamic-pituitary-thyroid function (adapted from reference (178) with permission).

Figure 4. The relationship between serum TSH and free T4 concentration is shown for normal subjects (N) and in the typical abnormalities of thyroid function: A, primary hypothyroidism; B, central or pituitary-dependent hypothyroidism; C, hyperthyroidism due to autonomy or abnormal stimulation of the gland; D, TSH-dependent hyperthyroidism or thyroid hormone resistance. Note that linear changes in the concentration of T4 correspond approximately to logarithmic changes in serum TSH (178).

The T4-TSH Setpoint

 

Small changes in serum T4 and T3 concentrations, within the normal range, alter the serum TSH concentration, indicating that the inverse feedback relationship between serum free T4 and TSH applies across their normal ranges, as well as in disease states (205,206). Studies of normal subjects demonstrate significant individual variation, independent of sex and age, in the setpoint of the pituitary-thyroid axis (172,173,207), which suggests that the TSH set-point for a particular serum free T4 or free T3 concentration is an individual characteristic. It follows that the concept of “normality” for an individual may be narrower than for a population at-large. Studies of monozygotic and dizygotic twin pairs also suggest that genetic factors influence the serum concentrations of total and free T4 within the normal range (208), as well as the relationship between TSH and free T3 and T4 (209,210). The recent demonstration (211) that multiple genetic factors can influence inter-individual differences in the TSH response to circulating thyroid hormones may ultimately increase the precision and the complexity of interpreting thyroid function are also reviewed recently (212,213).

 

Andersen et al. (173), in a study of normal subjects whose samples were taken monthly between 09:00 and 12:00 for a year, showed that individual references ranges for T4 and T3 were only about half the width of the population reference ranges, indicating that a test result within the population range is not necessarily normal for that individual. Serum TSH showed greater between-sample variation for each individual than serum T4 or T3. Based on the degree of individual variation, it was estimated that a normal serum TSH concentration needed to change by 0.2-1.6 mU/l to be confident of a serial change in thyroid status. Based on this analysis, it was estimated that a single morning sample defined serum T4 and T3 to within 25%, and serum TSH only to within 50%. Since TSH shows diurnal variation and pulsatile secretion in both normal and hypothyroid subjects (204,214), random samples are likely to show even greater variation. This concept can also be described as individuality index (166).

 

It has been suggested that some healthy older adults have normal serum TSH concentrations despite having low serum free T4 values, attributed to resetting of the threshold for TSH inhibition (215). In a large cohort of Danish patients with newly diagnosed hypothyroidism, the increase in serum TSH for a given degree of lowering of serum free T4 was less in the elderly, suggesting that equivalent TSH increases in the elderly may be accompanied by more severe thyroid hormone deficiency (216). It is notable that a higher level of serum free T4 was necessary to normalize serum TSH in children with congenital hypothyroidism than in adults with acquired autoimmune hypothyroidism (217). It is uncertain whether this is a further reflection of age-related difference, or whether this difference reflects a perinatal shift in the central setpoint for regulation of TSH secretion.

 

The TSH-T4 Relationship: Diagnostic Assumptions

 

The precise diagnosis of thyroid dysfunction can generally be established from a single serum sample from the relationship shown in Figure 4, subject to six key assumptions (Table 5). It should be noted that only the last three of these assumptions can be validated in the laboratory; the first three are best verified clinically.

 

 

Table 5. Assumptions Inherent to Diagnostic Use of the T4 -TSH Relationship

(Conditions that may breach these assumptions are shown in italics)

1. Steady-state conditions (note differences in the half-lives of TSH and T4)

·       Early treatment with antithyroid drugs (218)

·       Early response to T4 therapy

·       Evolution of transient thyroid dysfunction (178)

·       Recovery from severe illness (190,191)

2. Normal trophic-target hormone relationship

·       Alternative thyroid stimulators

·       Immunoglobulins (219)

·       Chorionic gonadotrophin (220)

·       Medications that influence TSH secretion (45)

·       T3, triiodothyroacetic acid (221)

·       Other thyroid hormone analogues (222)

·       Glucocorticoids (223)

·       Dopamine (224)

·       Amiodarone (45)

·       Recent hyperthyroidism (218)

·       Recent longstanding hypothyroidism

·       Treated congenital hypothyroidism (225)

·       TSH receptor mutations (226)

·       Variable individual setpoint (173,207,208,213,215,216)

3. Tissue responses proportional to hormone concentration

·       Hormone resistance syndromes (227)(see below)

·       Slow onset/offset of thyroid hormone action

·       Drug effects (45)

·       Amiodarone (45)

·       Phenytoin (228)

4. The assay measurement represents the active hormone

·       Unmeasured agonist in excess (e.g. T3, triiodothyroacetic acid, human choriogonadotropin hormone (178)

·       TSH of altered biologic activity (229,230)

·       Spurious immunoassay results (178)

·       TSH (178)

·       Heterophilic antibodies (231,232)

·       Free T4

·       Abnormal serum binding proteins (233)

·       Autoantibodies (234)

·       Medications that inhibit protein binding (233,235)(227, 229)

·       Heparin artefact (236,237)

5. The assay can reliably distinguish low from normal values

·       Lack of precision at the limit of detection (178,238)

6. Reference ranges are appropriate

·       Influence of age (239,240)(see also above)

·       Associated illness (178)( see below)

 

STEADY-STATE CONDITIONS

 

This first assumption should be questioned whenever anomalous results occur during associated illness, or with medications that perturb the pituitary-thyroid axis. The half-lives of plasma TSH (approximately 1 hour) and plasma T4 (approximately 1 week) differ so widely that acute perturbation of the pituitary-thyroid axis will often result in transient nonsteady state conditions (Figure 5). Due to its much shorter half-life, serum TSH deviates more rapidly from steady state. Other common deviations from steady state relate to short-term pulsatile or diurnal fluctuations in hormone secretion, responses to treatment and spontaneous evolution of disease, as can occur in subacute thyroiditis or postpartum thyroid dysfunction.

 

Figure 5. Measurement of serum T4, rather than serum TSH, is the more reliable single test of thyroid function when steady state conditions do not apply, as in the early phase of treatment for hyperthyroidism or hypothyroidism. (adapted from reference (178)

NORMAL TROPIC-TARGET HORMONE RELATIONSHIP

 

During treatment of prolonged hyperthyroidism, TSH secretion may remain low for several months after serum free T4 becomes normal (218). Conversely, after severe prolonged hypothyroidism, or in some children treated for congenital hypothyroidism (217), TSH hypersecretion may persist despite normalization of serum T4. Serum TSH will then give an inaccurate indication of thyroid status, with the potential for over-treatment if this variable alone is used to assess therapy.

 

TISSUE RESPONSES PROPORTIONAL TO THE HORMONE CONCNETRATION

 

The active or free concentrations of T3 and T4 generally correlate well with clinical features. However, in generalized thyroid hormone resistance due to mutations in the thyroid hormone receptor, serum free T3 and T4 concentrations are elevated and the TSH is inappropriately normal or elevated due to the impaired feedback at the level of the pituitary thyrotrophs.

 

The onset and offset of genomic thyroid hormone action are relatively slow, so that tissue responses may lag behind changes in serum concentrations of free T4 and T3. There is a notable lack of convenient, sensitive, specific, objective indices of thyroid hormone action (see later), so that assessment remains predominantly clinical. Corroborative measurements that can be useful, especially in following the response of individuals to therapy, include measurement of oxygen consumption (241), sex hormone binding globulin (242), and angiotensin converting enzyme (243), as well as several indices of cardiac contractility, although none of them is a very accurate nor specific biomarker for changes in thyroid function.

 

THE ASSAY MEASUREMENT REFLECTS THE ACTIVE HORMONE(S)

 

TSH and iodothyronine assays make comparative, rather than absolute, measurements of hormone concentrations, based on the premise that samples and assay standards differ only in their concentration of the analyte. This assumption fails if there is any other difference between a serum sample and assay standards that influence the measured variable, as, for example, dissimilar protein binding of tracer (244), the presence of binding competitors (235,245), or possible nonspecific interference with enzymatic, fluorescent, or chemiluminescent detection systems. Circulating T3 and T4 autoantibodies may invalidate immunoassays by sequestering the assay tracer (246), while heterophile mouse or sheep (231,232) antibodies and rheumatoid factor can interfere with immunoglobulin aggregation, or with cross linking of the signal and capture antibodies (247,248).

 

If the biologic activity of circulating immunoreactive TSH is increased or decreased, the normal relationship between measured serum TSH and free T4 may be altered. Secreted immunoreactive TSH is heterogeneous, due to differences in its three oligosaccharide side chains. In hypothalamic hypothyroidism, the secreted TSH has decreased bioactivity (229,230), whereas activity may be enhanced in thyroid hormone resistance, primary hypothyroidism, and in some TSH-producing tumors (249,250).

 

THE ASSAY CAN RELIABLY DISTINGUISH LOW FROM NORMAL LEVELS 

 

Assay precision inevitably deteriorates as the limit of detection is approached; this characteristic is crucial in evaluating TSH assays that are used to distinguish hyperthyroidism from normal (57,178). The lower working limit of a TSH assay should be defined in terms of its between-assay reproducibility, defined as functional sensitivity, rather than by the analytical sensitivity of individual assay runs (57,178).

 

REFERENCE RANGES ARE APPROPRIATE 

Since reference ranges of the thyroid related hormone measurements vary substantially according to the methods of measurement, it does not make much sense to provide typical ranges. However, reference ranges show little change with advancing age, except for a possible decline in normal serum T3 with age (215,240). Serum T3 is significantly higher in children (239) and probably also in young adults. For statistical analyses, TSH reference ranges are generally evaluated after logarithmic transformation; the geometric mean is then used to define a realistic lower normal limit. There may be an age-related change in the central setpoint for TSH secretion, with a progressive decline in the TSH setpoint or response to decreased levels in serum T4 (215,216). Associated illness, nutritional changes, and medications frequently cause assay results to fall outside the normal reference ranges as defined in healthy subjects, so that it may be relevant to use wider than normal reference ranges in the face of associated illness (178). Thus, reference ranges are only appropriate and useful in the context of employing the same biochemical laboratory method in establishing the reference range as the one used for routine measurement in patients. Since automated laboratory methods have been extensively implemented globally, where most clinicians rely entirely on the manufacturers’ information on the method quality (sensitivity, accuracy and imprecision), as well as the reference interval, above requirement for one’s own local laboratory to perform these assessments in order to improve local laboratory quality of results has currently left much to desire.

APPLICATION AND INTERPRETATION OF INDIVIDUAL SERUM ASSAYS

Serum TSH as the Initial Test of Thyroid Function

 

Either serum TSH or free T4 can be used as the initial test of thyroid function, with TSH previously assessed as giving better first-line discrimination at slightly higher cost (182). A normal serum TSH concentration has high negative predictive value in ruling out primary thyroid disease, and this assay has become increasingly used as the single initial test of thyroid function, with no further assays done routinely if serum TSH is normal. If serum TSH is increased, free T4 is measured on the same sample to distinguish between overt and subclinical hypothyroidism (Figure 6). If serum TSH is suppressed, i.e.<0.05 mU/l, both free T4 and free T3 should be assessed to distinguish between overt hyperthyroidism, T3-thyrotoxicosis and subclinical hyperthyroidism. The interpretation of subnormal TSH values is influenced by the functional sensitivity of the particular assay (see below).

 

The demonstrated slightly higher costs are probably eliminated by the much better interlaboratory robustness of the TSH assays compared to free T4 estimates, although this has never been properly verified.

Figure 6. An algorithm for the initial assessment of thyroid function, based on initial assay of serum TSH. The limitations of this strategy are summarized in table 5. TPOAb=thyroperoxidase antibodies; TRAb=Thyrotropin receptor antibodies; FT4 and 3=Free thyroxine and -triiodothyronine.

LIMITATIONS OF THE “TSH FIRST” TESTING STRATEGY

 

The rationale for using TSH alone as a first-line test of thyroid function rests on the assumptions that thyroprivic, or primary hypothyroidism is far more common than central or secondary hypothyroidism, and on the fact that serum TSH deviates outside the reference range early in the natural history of thyroid over-function or progressive thyroid failure. The major weaknesses of this approach are the likelihood of missing secondary hypothyroidism (in which the concentration of immunoreactive serum TSH is frequently normal rather than low), and the frequency of abnormal TSH concentrations in the absence of thyroid dysfunction, especially in patients with an associated illness or interference in the TSH assays (Table 6).

 

Beckett and Toft (184) have pointed out the adverse consequences of missing secondary hypothyroidism due to pituitary failure by relying on normal serum TSH to rule out thyroid dysfunction. The diagnosis of this disorder is often difficult, with diverse presentations to a wide range of practitioners who may not be attuned to key clinical features. They estimated that as many as 1500 cases per year of central hypothyroidism could be missed in the UK if the “TSH first” policy were followed inflexibly. This potentially serious diagnostic problem was shown by Wardle et al. (183) who reported an analysis of 56,000 requests for thyroid function testing over 12 months from a population of 471,000. Serum TSH was normal in association with subnormal total and free T4 in 15 patients who, on further investigation, had probable hypopituitarism that would have been missed by assessment of serum TSH alone. Cost benefit analyses would therefore need to balance the savings from not measuring serum T4 routinely, against the cost of further investigation and medical care for the missed diagnoses, in addition to a component for burden of suffering and potential litigation. If advances in technology can reduce the unit cost of measuring T4, the “TSH first” approach may be abandoned.

 

Table 6. Situations in Which Serum TSH Alone can Give a False or Uncertain Indication of Thyroid Status.

Condition

TSH

fT4

fT3

Primary abnormality of TSH secretion

Pituitary-hypothalamic abnormality

L- N

L

 

Extremely premature infants

L- N

L

L

Central TSH excess

N -H

H

H

Hyperthyroidism

T3 toxicosis

U

N

H

Subclinical

U

N

N

Early Treatment

U

H-N-L

H-N-L

TSH assay artefact

L- N -H

H

H

Hypothyroidism

Subclinical

H

N

 

Early Treatment

H

L-N

 

Thyroid hormone resistance

N -H

H

H

Medications

Dopamine

L

N

N

Glucocorticoids

L

N

L-N

Amiodarone (acute)

H

N-H

L

N: normal; L: low; H: high; U: undetectable.

 

Serum Free and Total T4

 

Estimation of free T4, or total T4 linked to a measurement of the thyroid hormone binding ratio, in association with serum TSH, has now become the standard method of assessing thyroid function, except in settings where the TSH-first strategy persists. Assays for total T4 and T3 in unextracted serum include a reagent such as 8-anilinonaphthalene sulfonic acid that blocks T4 and T3 binding to serum proteins, so that total hormone is available for competition with the assay antibody. Assays for free T4 or T3 omit this blocking reagent and use a wide variety of manoeuvres to isolate a moiety that reflects the free hormone concentration. The theoretical basis, practical utility, and validity of the many different approaches to the estimation of serum free T4 and T3, have been considered in detail (251) (see below).

 

Two key assumptions in any method of free T4 estimation are (i) that the dissociation of bound hormone with sample dilution is similar in samples and standards, and (ii) that samples and standards show identical protein binding of the assay tracer. If either of these conditions is breached, the assay is likely to give inaccurate results. Serum free T4 and free T3 can be estimated either by two-step methods that separate a fraction of the free hormone pool from the binding proteins before the assay incubation, or by one-step methods in which the free hormone concentration is measured in the presence of binding proteins (251). Many of the one-step methods become invalid when the sample and standard differ in their binding of assay tracer, but the two-step methods are less prone to non-specific artefacts. Almost all techniques of estimating free T4 give a useful correction for moderate variations in serum thyroxine binding globulin concentration, but no method can yet accommodate the effect of circulating competitors for T4 binding (193,235,252).

 

Indications for Measurement of Serum T3

 

Measurement of serum T3 is indicated, in addition to serum T4, as follows:

  1. In suspected hyperthyroidism with suppressed TSH and normal serum T4, to identify T3-thyrotoxicosis and distinguish this entity from subclinical thyrotoxicosis.
  2. During antithyroid drug therapy to identify persistent T3 excess, despite normal or low serum T4 values (253).
  3. For diagnosis of amiodarone-induced hyperthyroidism, which should not be based on T4 excess alone because of the frequency of euthyroid hyperthyroxinemia during amiodarone treatment (254,255).
  4. To assess the extent of T3 excess in individuals treated with thyroid extracts of animal origin, to assess a potentially damaging hormone excess that is not reflected by the level of T4.
  5. To identify T3-predominant thyrotoxicosis, an entity that is less likely to achieve remission (see below). In some studies this entity is been reported to respond poorly to radioiodine (256), a phenomenon that may relate to rapid iodine turnover within the gland (257).

 

Serum T3 measurements may also be useful:

  1. For estimation of the serum T3-T4 ratio. A high ratio (>0.024 on a molar basis or >20 calculated as ng/µg) that persists during antithyroid drug treatment suggests that patients with hyperthyroid Graves’ disease are unlikely to achieve remission (258). This ratio usually is lower in iodide-induced hyperthyroidism (259) or hyperthyroidism caused by thyroiditis (260) than in Graves’ disease.
  2. To detect early recurrence of hyperthyroidism after cessation of antithyroid drug therapy.
  3. To establish the extent of T3 excess during high-dose replacement or suppressive therapy with T4, or after an accidental or intentional T4 overdose.

 

Low serum T3 concentrations have little specificity or sensitivity for the diagnosis of hypothyroidism. Many patients with nonthyroidal illness have low values, and the serum T3 concentration can remain in the reference range until hypothyroidism is severe. Serum T3 concentrations are usually interpreted together with the concentrations of T4 (Table 7).

 

Table 7.  Relationship Between Serum T4 and T3 in Various Disorders*

 

Serum T4

Serum T3

Low

Normal

High

Low

Severe hypothyroidism

TBG deficiency #

Severe nonthyroidal illness

Euthyroid hypo-thyroxinemia

Nonthyroidal illness

Medications

Fetus

Restricted nutrition

Hyperthyroidism with

     severe nonthyroidal illness

Amiodarone

Normal

Iodine deficiency

T3 treatment

Hypothyroidism

 

T4 treatment

Euthyroid hyper- thyroxinemia

Hyperthyroidism with

     nonthyroidal illness

T4 binding autoantibodies

High

Iodine deficiency

T3 treatment

Antithyroid drugs

T3 toxicosis

T3 binding autoantibodies

Hyperthyroidism

Excess T4 ingestion

Hormone resistance

TBG excess #

* Excludes short term changes related to initiation or cessation of therapy

# Effect on total hormone concentration; free hormone concentration remains normal

TBG, thyroxine binding globulin

 

Indications for TRH Testing

 

The development of high-sensitivity TSH assays has almost eliminated the need for TRH testing in clinical practice. With intact hypothalamic-pituitary function, there is a close correlation between the TSH response to TRH and the basal level of serum TSH, when measured by a highly sensitive assay (261,262). Hence, TRH testing now offers little diagnostic advantage over accurate measurement of the basal serum TSH concentration in the detection of hyperthyroidism (263). However, measurement of serum TSH 20 to 30 minutes after intravenous injection of 200 to 500 µg TRH remains useful for several purposes:

  1. To assess patients whose basal serum TSH values are out of context, in order to identify assay artefacts. For example, a detectable serum TSH concentration that is unresponsive to TRH in a thyrotoxic patient suggests either non-specific assay interference (263), or the presence of a TSH-secreting pituitary tumor where serum TSH is often unresponsive to TRH (250).
  2. To distinguish between cases of thyroid hormone resistance and pituitary-dependent hyperthyroidism. In most instances of hyperthyroidism caused by TSH-secreting pituitary tumors there is no increase in serum TSH in response to TRH (250), while an increased serum TSH response to TRH is seen in the thyroid hormone resistance (227).

 

Serum Thyroglobulin

 

This iodinated 660 kDa dimeric glycoprotein, which is the scaffold for thyroid hormone synthesis, is normally detectable in serum and is released in excess in a wide variety of situations where thyroid tissue is overactive, inflamed, or proliferating (264). Undetectable serum levels suggest absence or suppression of thyroid tissue. In the past decade functional assay sensitivity has improved almost ten-fold from about 1 µg/L to around 0.1 µg/L. As a consequence, thyroglobulin (Tg) is now detectable in serum without TSH stimulation in a larger proportion of patients and has greater diagnostic sensitivity during continuing T4 therapy (265). Nevertheless, undetectable serum Tg in the presence of high TSH remains the benchmark for proof of successful ablation after treatment of differentiated thyroid cancer.

Standard indications for measurement of serum Tg are as follows:

  1. Follow-up of treatment for differentiated thyroid cancer to identify or rule out the presence of residual thyroid tissue, whether normal or metastatic. An undetectable serum thyroglobulin concentration in the presence of high TSH, whether achieved by thyroxine withdrawal, or injection of recombinant TSH, is presumptive evidence that differentiated thyroid tissue has been ablated (266). In contrast, the level of serum Tg that persists when serum TSH is suppressed, can give a useful index of tumor burden (201). In a 16-year follow-up study in differentiated carcinoma, post-ablative Tg concentration was a strong predictor of disease recurrence (267). However, serum Tg measurement is a technically challenging assay and criteria to define a 'highly sensitive' assay may be different, a good knowledge of the technical difficulties and interpretation criteria is of paramount importance for both clinical thyroidologists, laboratory physicians, and scientists involved in the care of differentiated thyroid cancer patients (268). Concurrent measurement of serum TSH and Tg antibody is always required for interpretation of serum Tg values.
  2. Investigation of atypical hyperthyroidism, where there is a suspicion of thyrotoxicosis factitia in which serum Tg is generally very low or undetectable (269).
  3. Assay of Tg in the needle washes after biopsy of extrathyroidal neck masses is useful in identifying metastatic thyroid tissue (270). This technique has higher specificity and sensitivity than cytology from suspect lymph nodes (271).
  4. Serum Tg may serve as a sensitive if non-specific marker of iodine deficiency or ineffective synthesis of thyroid hormone (272).

 

Because elevation of serum Tg concentration occurs in a wide range of thyroid disorders, interpretation of results always requires a knowledge of (i) the clinical context, (ii) the serum TSH concentration and (iii) whether Tg antibodies are present.

 

Interactions between serum Tg and circulating antibodies have the potential to cause false-negative assay results, as discussed below. However, measurement of Tg antibodies in the follow up protocol of treated differentiated thyroid carcinoma can also function as surrogate biomarkers for relapse of the thyroid carcinoma (273-275).

 

Thyroglobulin Antibodies (TgAb)

 

Assessment of thyroglobulin antibodies (TgAb) in serum is indicated:

  1. As an essential component of the interpretation of assays for serum Tg, to establish whether endogenous antibodies could be responsible for spuriously low Tg values by interfering with assay separation, particularly in immunometric assays. It is cause for concern that current assays for TgAb may not give congruent results when cross-matched in the assessment of Tg in antibody-positive samples (178,276,277) (See below).
  2. As secondary follow-up criterion in differentiated thyroid cancer, where a progressive decline in antibody concentration may follow successful ablation (273,274).
  3. In pregnant populations where positive anti-thyroperoxidase antibodies may not reflect the woman’s autoimmune status in all situations (278), perhaps in particular in iodine fortification programs. It may, however, turn out to be more important in future practice than previously thought (278), when previous advice of routine measurements of both thyroperoxidase/microsomal antibodies and TgAb were abandoned (279).

 

Thyroid Peroxidase Antibodies

 

This entity, previously termed antimicrosomal antibody, is closely linked with autoimmune thyroid disease, in particular Hashimoto’s thyroiditis. Measurement of thyroid peroxidase antibody is indicated as follows (278):

  1. To identify an autoimmune cause for primary hypothyroidism.
  2. In individuals with the TSH excess of mild thyroid failure, in whom a positive thyroperoxidase antibody indicates an approximately two-fold increase in risk of progression to overt hypothyroidism (280). A strongly positive result in the presence of mild thyroid failure may influence the decision to commence replacement.
  3. In screening for immune thyroid dysfunction or potential hypothyroidism before or early in pregnancy, especially in high-risk groups and as a predictor of the potential postpartum thyroid dysfunction (see below).
  4. In order to establish whether there is an immune basis for euthyroid goiter, and to evaluate the risk of future hypothyroidism at a stage before there is any increase in serum TSH
  5. In cases suspected of polyautoimmunity, where thyroid autoimmunity is by far the most prevalent autoimmune manifestation (16)

 

TSH Receptor Antibodies (TRAb)

 

Antibodies that interact with the TSH receptor can be measured either by bioassay, or competitive binding techniques. Bioassays that use thyroid cells of human or animal origin, or cells with transfected TSH receptor, generally depend on tissue production of adenylate cyclase for quantitation. These assays allow a distinction to be made between antibodies with thyroid stimulating (TSAb) and blocking (TBAb) activity. Radioreceptor (thyroid binding inhibitor immunoglobulin, TBII) assays that measure competition by circulating immunoglobulin for specific binding of labelled TSH, are widely available, but do not distinguish between blocking and stimulating activity.

 

A competitive binding assay using a recombinant human TSH receptor, showed 98-99% positivity in active Graves’ disease, with positive results in <1% of subjects with nonautoimmune thyroid diseases (281). Impressive sensitivity and specificity have been reported with an assay that uses solubilized native porcine TSH receptor with a biotinylated anti-porcine TSH receptor monoclonal capture antibody (282).

 

Indications for the measurement of TRAb vary in different practice environments, but are clearly applicable to the following situations:

  1. During pregnancy in women with active or previous autoimmune thyroid disease, to assess the risk of neonatal thyroid dysfunction due to transplacental passage of TRAb. Guidelines from the European Thyroid Association (ETA) (283) suggest that TRAb measurements should be made early in pregnancy in women who have received previous ablative treatment, and in the last trimester in women receiving drug treatment for active Graves’ disease. Antibody measurement during pregnancy was not considered necessary for Graves’ disease in remission. Recent guidelines from ETA, however, state that all patients with a history of autoimmune thyroid disease should have their TRAb serum concentrations measured at the first presentation of pregnancy using either a sensitive binding or a functional cell-based bio-assay and if they are elevated, again at 18-22 weeks of gestation (284).
  2. In the differential diagnosis of atypical hyperthyroidism that may be due to Graves’ disease.
  3. In atypical eye disease that may be due to Graves’ orbitopathy.
  4. In Graves’ orbitopathy for risk assessment of deterioration after radioiodine therapy for the hyperthyroidism.
  5. To assess the chance of achieving a drug-induced remission of Graves’ disease and to predict whether an apparent remission of Graves’ disease is likely to be sustained, or whether relapse should be anticipated, although this is still controversial (see below)(285-287).

 

TSH Alpha Subunit

 

Assay of the TSH alpha subunit is indicated where hyperthyroidism appears to be the result of central autonomous TSH excess to distinguish this entity from thyroid hormone resistance (250). Most TSH producing pituitary adenomas show an increase in alpha subunit (250), and this assay may serve as a useful tumor marker after treatment. By contrast, levels generally remain normal in thyroid hormone resistance (227). Values are also high in postmenopausal women, in men with hypogonadism, and in gonadotrophin-producing pituitary tumors; both thyrotrophs and gonadotrophs secrete this subunit.

 

Serum Reverse T3

rT3 levels are altered in some rare forms of impaired sensitivity to thyroid hormone: it is significantly reduced in patients with SBP2 mutations, and substantially increased in patients with MCT8 mutations. However, these syndromes are rare (288). Previous suggestions that it might be useful in distinguishing true hypothyroidism from the hypothyroxinemia of severe illness have not been confirmed (289). 

Urinary Iodine Estimation

 

Both iodine deficiency and excess can lead to important abnormalities of thyroid function. In contrast to the wide-reaching effects of iodine deficiency, especially in relation to pregnancy and the neonate (see below), possible iodine excess should be considered as a precipitating cause of hyperthyroidism in the following situations (290):

  1. Atypical hyperthyroidism with blocked isotope thyroid uptake, with features similar to those of excess thyroid hormone ingestion, or subacute thyroiditis without the expected systemic features.
  2. High dose requirement for antithyroid drug, or failure to respond to these medications. (In some instances, iodine-induced thyrotoxicosis may be extremely resistant to standard therapy, sufficient to require emergency thyroidectomy (291)).
  3. Rapid progression from subclinical to overt hyperthyroidism, especially in patients with autonomous multinodular goiter.

 

Thyrotoxicosis due to iodine excess, which may ultimately be self-limiting, may result from iodine-containing radiographic contrast media or medications, alternative or “natural” health care products and contaminated food products, as observed with soy milk preparations (292). In contrast to subtleties related to urine volume and concentration that are important in assessing urinary iodine excretion in population studies of iodine deficiency, the possibility of marked iodine excess can be assessed from a single urine sample (293). The urinary iodine concentration that can be regarded as excessive is ill-defined and may vary in different populations. Urinary iodine concentrations >1000 ug/l are certainly abnormal, but iodine-induced thyrotoxicosis may also occur at lower levels.

 

THYROID TESTING STRATEGIES IN SPECIAL SITUATIONS

 

Subclinical Dysfunction

 

Follow-up studies suggest that it is inappropriate to commence treatment for subclinical thyroid dysfunction on the basis of a single laboratory result, or even a confirmed assay result within a short period of time (87,103,168,294) (see above). Because both free T4 and TSH show spontaneous fluctuation, it has been suggested that frequent testing may increase the likelihood of an apparent change (295).

 

Hyperthyroidism

 

INITIAL DIAGNOSIS

 

The initial diagnosis of hyperthyroidism is securely established when excess of free T4 and free T3 is associated with an undetectable serum TSH concentration in an assay of appropriate sensitivity. However, the condition can be present without any one of these three criteria. T3-thyrotoxicosis, in which serum T4 remains normal, is more prevalent in iodine deficient regions (296) and can be a premonitory stage of typical hyperthyroidism (297). When hyperthyroidism coexists with another severe illness, serum T3 may be transiently normal or even subnormal (298). Detectable or increased serum TSH concentrations in the face of hyperthyroidism can be due to laboratory artefacts (263), or to autonomous over-secretion of TSH (250). The increase in free T4 and free T3 estimates is usually more marked than the increase in total hormone concentration. Progressive increases in serum total T4, will approach and eventually exceed the limited T4 binding capacity of thyroxine binding globulin (about 300 nmol/l or 24 ug/dl), leading to disproportionate increases in the free serum concentrations of T4 (299) and T3 (300), relative to their total concentrations.

 

TREATED HYPERTHYROIDISM 

 

In the early drug treatment of hyperthyroidism, measurements of serum T4 and T3 are required for dose adjustment because suppression of TSH may persist for months after correction of longstanding hyperthyroidism (298). Hence, failure to decrease thionamide dosage while TSH suppression persists, can result in serious over-treatment during the early phase of therapy. During thionamide therapy, hyperthyroidism may persist due solely to T3 excess (253); assessment of therapy based on serum T4 alone can then result in under-treatment. A daily dose of 15 mg methimazole can result in hypothyroxinemia in about 10 % of previously thyrotoxic subjects within 4 weeks (301); hence a reassessment of both serum free T3 and free T4 is timely after about 3 weeks to allow appropriate dose-adjustment. In contrast to these discrepancies during early treatment, serum TSH generally gives a reliable index of therapy during the long-term drug treatment of hyperthyroidism. The rate of change in serum free T4 and free T3 during treatment of hyperthyroidism with thionamide gives a valuable guide to dose adjustment, even while levels remain increased and serum TSH is still undetectable. For example, if serum T4 decreases by half in the first four weeks of treatment, it is appropriate to decrease the initial thionamide dosage by about half to minimize the possibility of over-treatment.

 

Davies et al. (302) assessed the prognostic significance of various serum TSH levels in a large cohort of patients treated with radioiodine 2-35 years previously, who were receiving no other treatment. After a further two years of follow-up, 83% of those with normal TSH had not changed their diagnostic category, although there was a trend for TSH to increase. An increased TSH was associated with a 14.5 % incidence of hypothyroidism after one year. Notably, spontaneous normalization of subnormal or undetectable TSH values during the follow-up period was more common than recurrence of overt hyperthyroidism (302). Hence, while an increased TSH value might be a pointer towards T4 treatment, there appears to be no basis for further radioiodine therapy solely because TSH remains suppressed. These treatment issues have also been described in guidelines from the ATA (303) and ETA (304).

 

Hypothyroidism

 

INITIAL DIAGNOSIS

 

The initial diagnosis of overt primary hypothyroidism is established by an increase in serum TSH with subnormal free T4; serum free T3 may remain normal except in severe cases. Subclinical hypothyroidism or mild thyroid failure is defined by persistent elevation of serum TSH, while free T4 remains normal (see above). The precise upper limit of the reference range for TSH is difficult to define, because of the approximately logarithmic distribution of this variable. The upper “tail” of normal TSH in the range 2-4 mU/l is thin and subjects with TSH concentrations in this range have an increased likelihood of future hypothyroidism, especially if thyroperoxidase antibodies are positive (280).

 

Serum TSH is the key to the distinction between primary and secondary hypothyroidism. A low serum free T4 in the absence of TSH elevation should always raise the possibility of a pituitary or hypothalamic abnormality, although this combination of findings is also frequent in a number of other situations (Table 8), especially during critical illness (190). It should be noted also that immunoreactive serum TSH is often detectable in secondary or central thyroid deficiency, a phenomenon that appears to result from dissociation between biological and immunological TSH activity (249). Hence, normal or even elevated serum TSH should not be interpreted as conclusive evidence againsthypopituitarism (305).

 

Table 8. Causes of Subnormal Free T4 without TSH Excess

Secondary or central hypothyroidism

Impaired biological activity of TSH

Critical illness

Falsely low free T4 estimate (method-dependent)

Dilution-dependent artefact (see below)

Effect of medications that compete for T4 binding

Impaired TSH response to hypothyroxinemia

Effect of severe illness

Medications e.g. dopamine, glucocorticoids

 

RESPONSE TO TREATMENT

 

A serum TSH value in the low-normal range between 0.5 and 1.5 mU/l, close to the geometric mean, is probably the best single indicator of appropriate thyroxine dosage during standard replacement therapy. In a study of ambulatory patients attending a thyroid clinic, hypothyroid patients taking T4 replacement seldom needed a serum free T4 measurement if the serum TSH was greater than 0.05 mU/L, although at lower TSH values, the magnitude of T4 excess did influence management (306). Numerous studies show that patients taking exogenous thyroxine show higher levels of serum total and free T4 for equivalent levels of serum TSH and T3 when compared with untreated euthyroid control subjects (307,308).

 

It should be noted that in some situations, for example patients with both ischemic heart disease and hypothyroidism, or in very old subjects, the appropriate dose of T4 should be influenced by clinical judgment as well as laboratory findings. The assessment of optimal levothyroxine dosage in patients with secondary or central hypothyroidism remains a challenge because serum TSH does not serve as a reliable marker of under-replacement. Serum TSH may in this context diminish during thyroxine replacement in hypopituitarism (309); while not of primary diagnostic value, the concentration may serve as an index of individual progressive response (186,310)). Since there is no readily available alternative variable of thyroid hormone action, it is generally appropriate to assess replacement clinically and on the basis of both serum T4 and T3. Measured levels of free and total T4 are influenced by the interval between tablet ingestion and blood sampling. In athyreotic subjects who took 0.15-0.2 mg T4 orally, the serum free and total T4 concentrations were increased by about 20% one to four hours later, with return to baseline about nine hours after T4 ingestion; serum TSH and T3 levels showed no time-dependent variation in relation to timing of thyroxine dosage (310).

 

In some situations, the regulation of T4 replacement is both unreliable, unpredictable and very variable. Excluding low patient compliance, this is often due to variable absorption of T4 from the gastrointestinal tract due to e.g. gastric or bowel disease (311), pernicious anemia with achlorhydria (312), a variety of over the counter medications such as antacids (313), laxatives (313), calcium (314), coffee (315), and many other substances. This untoward situation for the patient who is most often very negatively affected by the variable and irregular thyroid function can sometimes be alleviated by changing treatment formulation to a soluble form or soft gel capsules rather than tablets (312).

 

SUPPRESSIVE TREATMENT WITH T4

 

In line with recent guidelines for the stratification of risk in patients with differentiated thyroid cancer (316), there is no single TSH target during long-term postoperative thyroxine therapy (317,318). In high risk patients there may be benefit from adjusting T4 dosage to suppress TSH to undetectable levels (e.g. <0.03 mU/l). However, in many situations it may be appropriate to aim for a TSH target in the lower normal range in order to minimize adverse effects on bone and cardiovascular system. It is also worth noting that the initial ablative treatment of differentiated thyroid carcinoma tends to be less radical with classification into low and high-risk groups according to the guidelines. This may influence the long-term outcome which however remains to be seen in future long-term follow-up studies. When the aim of T4 suppressive therapy is regression of benign thyroid tissue, it may be adequate to give sufficient T4 to reduce serum TSH to 0.1 to 0.3 mU/L (319).

 

TREATMENT WITH T3

 

If currently available T3 formulations are used for replacement, it is difficult to monitor the effectiveness of treatment. Owing to its short plasma half-life, the serum concentration is highly dependent on the interval between dosage and sampling (320). There is also doubt as to whether TSH serves as an accurate index of thyroid hormone action during continuing T3 therapy, with the suggestion that doses of T3 required to normalize TSH could produce tissue hyperthyroidism (321). The difficulty of monitoring T3 replacement by currently available techniques is one of the arguments against the routine use of T3 or thyroid extract (321-323).

 

If the suggestion is confirmed that some genetically-identifiable individuals within the hypothyroid population are better served by combined T4-T3 therapy on the basis of polymorphism of type 2 deiodinase (324,325), or other variations in T4 transport and metabolism, the use of T4-T3 combination therapy will increase. That will require re-evaluation and refinement of monitoring strategies for combined replacement. The development of sustained-release T3 formulations will be advantageous, so that the interval between dosage and sampling can be minimized as a variable (326,327), and the treatment can thereby become more stable.

 

REPLACEMENT WITH UNCERTAIN INDICATION

 

The recent demonstration that fertility and pregnancy outcome may be adversely affected by minor degrees of thyroid dysfunction (see below) has led to the initiation of thyroxine treatment in women of reproductive age, who may lack conclusive criteria for lifelong therapy. It may later be questioned whether continuing replacement is required. Positive thyroperoxidase antibodies may be a basis for continuing therapy, but in the absence of this marker, measurement of serum TSH after 3-4 weeks withdrawal of treatment is likely to be definitive. The alternative of following the TSH response during partial replacement with T4, 50 ug daily, may be preferred. Similar approaches apply where no documented indication exists. Because of the difficulty in establishing the need for treatment during optimal replacement, it is an advantage for hypothyroid patients to retain a permanent record of pre-treatment documentation.

 

Assessment of Thyroid Function During Non-Thyroidal Illness (NTI)

 

Several distinct issues need to be considered when assessing thyroid function during critical illness. First, there is the possibility that an underlying thyroid abnormality might be missed, and second, prolonged severe illness per se may be associated with an abnormality of thyroid hormone secretion or action that may benefit from treatment (328,329). Third, some of the observed abnormalities will reflect the effect of medications or may be methodological artefacts.

 

It is difficult to rule out previously unrecognized or recent thyroid dysfunction by clinical assessment in critically ill patients; current laboratory tests often do little to resolve the problem. During severe illness, one or more of the assumptions that underpin the diagnostic use of the TSH-T4 relationship (see above) may not be valid. For example, acute fluctuations from the steady-state can lead to an anomalous T4-TSH relationship simply because of the marked difference in the plasma half-lives of T4 and TSH.

 

In general, the same sample assayed for TSH by various methods will give similar results during critical illness, while various estimates of free T4 may give widely divergent results (see below). When TSH and T4 changes are considered together, the abnormal results rarely correspond to standard criteria for diagnosis of primary hypothyroidism or hyperthyroidism. In contrast, persistent hypothyroxinemia without the corresponding anticipated rise in serum TSH is a common finding that suggests secondary or central hypothyroidism. These changes appear to be part of a broad neuroendocrine response that also involves the pituitary-adrenal axis, the pituitary-gonadal axis and the IGF binding proteins (329,330). Further study of these responses may eventually lead to therapy that could extend beyond thyroid hormone replacement, for example to substitution of hypothalamic releasing hormones (330).

 

The only reasonably robust method for distinguishing central hypothyroidism from thyroid function variables during critical illness is measurement of the T3 uptake test, which will be high (as opposite to low in central hypothyroidism) (331) and independent of the free T4 estimate which can be affected by a number of drugs used in intensive care patients (see below), and are anyway most often ‘false’ in the extreme situations (here low) where the automated methods for free thyroid hormones are unable to correct properly for binding protein abnormalities, or for abnormal binding to the binding proteins (331) (Figure 7).

 

EFFECTS OF MEDICATION

Interpretation of thyroid function tests during critical illness can be influenced by multiple medications (Table 3) (45), in particular dopaminergics and glucocorticoids, which inhibit TSH secretion, and a wide range of inhibitors of T4 and T3 binding to circulating thyroxine binding globulin (332). Thus, dopamine, dobutamine, glucocorticoids, octreotide, bexarotene, and metformin all inhibit pituitary TSH secretion, while an iodine load such as by using contrast agents, amiodarone or topical iodine preparations modifies hormone synthesis and release in a dual fashion. Lithium, glucocorticoids, and aminoglutethimide are inhibitors of both synthesis and release. Both amiodarone, glucocorticoids and beta-blockers, as well as hepatic contrast agents, inhibit T4-T3 5′ deiodination, and immune function modifications are seen by use of interleukin 1, interferon alpha, interferon beta and monoclonal antibody therapies.

Figure 7. Plots of peripheral thyroid hormone measurements (here T4) in various situations of thyroid dysfunction, protein binding abnormalities and drugs displacing the thyroid hormone from the binding proteins. The degree of distortion of the free T4 estimate depends on the assay methods (measurement platforms) as well as the degree of binding protein abnormalities. T3-test is able to distinguish central hypothyroidism from the effects of critical illness and displacement of the hormone by medicaments.

A number of drugs modify the binding of T4 and T3 to plasma proteins. e.g. estrogen, heroin, methadone, clofibrate, 5-fluouracil, perphenazine, tamoxifen, raloxifene, 5-fluouracil, and perphenazine, increase the concentration of thyroid hormone binding globulin, while glucocorticoids, androgens and l-asparaginase decrease the concentration. A number of drugs may displace T4 and T3 from binding proteins or displace T4 from the tissue pool and others increase the clearance of T4 and T3. A major issue in the serum free T4 measurement is drug and other interference in T4 replacement when absorption of T4 is impaired (333,334). This can happen in cases with impaired gastric activity (311), and coffee intake (315), but also any pharmaceutical drugs (335), many of them sold over the counter (313).

METHODOLOGICAL DISCREPANCIES IN NON-THYROIDAL ILLNESS

 

The Heparin Artifact and Free T4

 

The effect of heparin to increase serum free T4 is an important in vitro phenomenon that can lead to spuriously high estimates of circulating free T4 (236). In the presence of a normal serum albumin concentration, non-esterified fatty acid concentrations >3 mmol/l are required to increase free T4 by displacement from thyroxine binding globulin, but these concentrations are uncommon in vivo. However, in samples from heparin-treated patients, serum non-esterified fatty acids may increase to these levels during in vitro sample storage or incubation as a result of heparin-induced lipase activity (236) (Figure 8). This effect is accentuated by incubation of serum at 37ºC and by increased serum triglyceride or low serum albumin concentrations, particularly if the sample is pre-diluted. If heparin is given in vivo and the sample is then incubated at 37ºC, doses of heparin as low as 10 units may result in non-esterified fatty acid-induced increases in the apparent concentration of serum free T4 (235,237,336). The assay result is analytically correct but does not reflect the in vivo concentration of free T4. Low molecular weight heparin preparations have a similar effect (336).

Figure 8. Heparin-induced release of lipase in vivo can lead to in vitro generation of non-esterified fatty acids during sample incubation or storage. An increase in serum non-esterified fatty acid (NEFA) concentrations to >3 mmol/l is sufficient to displace T4 from thyroxine binding globulin, but such values are uncommon in vivo. This artefact is accentuated by high triglyceride or by low albumin concentrations.

Competitors for Plasma Protein Binding  

 

The accuracy of virtually all methods of free T4 estimation is compromised by medications that displace T4 and T3 from thyroxine binding globulin. Current methods tend to underestimate the concentration of free T4 in the presence of binding competitors because of dilution-related artefacts. Binding competitors are usually less protein-bound than T4 itself so that progressive sample dilution leads to a fall in the free concentration of competitor before the free T4 concentration alters (235). (For a hormone such as T4, with a free fraction in serum of about 1:4000, progressive dissociation will sustain the free T4 concentration up to at least 1:100 dilution. In contrast, 1:10 dilution of serum will result in a marked decrease in the free concentration of a drug that is 98% bound, i.e. has a free fraction in serum of 1:50). Because displacement depends on the relative free concentrations of primary ligand and competitor, the underestimate of free T4 will be greatest in assays with the highest sample dilution. This important dilution-dependent difference between various free T4 methods was shown by the relative ability of three commercial free T4 assays to detect the T4-displacing effect of therapeutic concentrations of furosemide (Figure 9) (337).

 

Similarly, therapeutic concentrations of phenytoin and carbamazepine increase the free concentration of T4 by 40-50% using ultrafiltration of serum that had not been diluted, while the free hormone estimate was spuriously low using a commercial single-step free T4 assay after 1:5 serum dilution (245).

Figure 9. Influence of increasing serum concentrations of added furosemide on estimates of serum free T4 using three commercial free T4 methods that involve varying degrees of sample dilution. The effect of the competitor is progressively obscured with increasing sample dilution (redrawn from (337)).

It is possible that methodologic artefacts have influenced previous descriptions of free T4 changes during critical illness. On the one hand, an apparent increase in free T4 may arise from heparin-induced in vitro generation of free fatty acids during sample incubation (236). On the other hand, estimates of free T4 may be spuriously low in assays that use diluted serum (235,245).

 

Divergent Estimates of Free T4 

 

That estimates of free T4 may show opposite discrepancies by different methods was shown by Sapin et al. (338) in a prospective study of bone marrow transplant recipients. Twenty previously euthyroid subjects were studied on the seventh day after bone marrow transplantation using six commercial free T4 kits, during multiple drug therapy, including heparin and glucocorticoids (Figure 10). Free T4 methods that involved sample incubation at 37ºC showed supranormal free T4 values in 20-40% of these subjects (see heparin effect above), while analog tracer methods that are influenced by tracer binding to albumin gave subnormal estimates of free T4 in 20-30%. By contrast, total T4 was normal in 19 of these 20 subjects. Serum TSH was <0.1 mU/l in half the subjects, independent of the method that was used. Thus, there was the possibility that an erroneous diagnosis of either hyperthyroidism or secondary or central hypothyroidism could be considered, solely as a result of variations in free T4 methodology.

Figure 10. Free T4 estimated by six different kit methods in 20 previously euthyroid patients on the seventh day after bone marrow transplantation. There was a high proportion of abnormal values, either increased or decreased, depending on the type of free T4 method used (see text). Therapy included heparin and glucocorticoids at the time of sampling. The mean for each method has been normalized to 100%, with the limits of the range shown by the box. Serum total T4 remained normal in 19 of the 20 study subjects, while serum TSH was subnormal in 11, independent of assay method (redrawn from (338))

SERUM TSH IN CRITICAL ILLNESS

 

Serum TSH assessment in severe non-thyroidal illness depends on the sensitivity of the particular method. The “third generation” assays with a functional sensitivity below 0.01 mU/L are generally sufficiently sensitive to distinguish the very low values in most thyrotoxic patients from the subnormal but somewhat higher TSH levels of non-thyroidal illness (59,339). Among a group of patients with low serum TSH values (<0.1 mU/L), almost all thyrotoxic patients had values less than 0.01 mU/L, when assessed with a highly sensitive assay, whereas most critically ill euthyroid patients had values between 0.01 and 0.1 mU/L (59). However, in another study, about 4% of patients with non-thyroidal illness had values below the functional sensitivity of a “third generation” assay, indicating that an absolute distinction cannot be made on the basis of TSH alone (339).

 

Differentiated Thyroid Cancer

 

There is now consensus that not all patients with differentiated thyroid cancer require T4 treatment in doses that achieve complete TSH suppression. Thus, based on a general assessment of risk (316,317,340), a TSH target should be determined as a guide to T4 dosage. In the follow-up of differentiated thyroid cancer the interpretation of TSH and serum Tg is inter-dependent. For studies done after initial near-total thyroidectomy, following withdrawal or temporary reduction of suppressive therapy (341) (or with the use of recombinant human TSH (342), serum TSH levels in excess of 30 mU/l appear to achieve adequate stimulation of potential Tg production (343). The failure of endogenous TSH to increase into this range suggests too short a period of T4 withdrawal, a compliance problem, or the presence of a substantial amount of active thyroid tissue.

 

Serum Tg measurement and whole body radioiodine scanning have generally been used in a complementary fashion, but there is now good evidence that current assays for Tg have greater sensitivity than follow-up whole body scanning with 2mCi 131I (316). There has been a clear trend to place greater emphasis on measurements of Tg and to move away from repeated low dose diagnostic whole-body radioiodine scanning, a procedure that has limited sensitivity (344,345). Management of low risk patients now tends to be based on assessment of serum Tg after recombinant TSH stimulation, without the need for diagnostic scanning (345). However, based on reported data the current fashion of obviating RAI based on a negative post-operative US and low serum Tg value is not yet supported by the literature and should be investigated using well designed prospective studies with clear-cut endpoints (346). In the opinion of these authors, the evolution of thyroid cancer management cannot pass through a substitution of RAI by serum Tg measurement and neck US but by an appropriate use of radioiodine theranostics. This will also need to develop basic and clinical research programs that bring together physicians from various specialties.

 

Undetectable serum Tg in the years after ablative treatment has been shown to be a reliable index in ruling out persistent or recurrent disease that requires further evaluation and treatment. Detectable serum Tg, together with detailed ultrasonography for localization has become the mainstay of further investigation (347). There has also been a tendency to deescalate serum Tg measurements by using only unstimulated sensitive serum Tg measurement and not rTSH stimulated assessments (348). Based on ATA’s most recent 2015 guidelines (316), Sunny et al. (349)performed a systematic study of 650 patients followed for papillary thyroid carcinoma who had total thyroidectomy performed and compared the evolution of stimulated and unstimulated serum Tg concentrations. The concentrations were corroborated with tumor burden as determined by additional clinical, ultrasonography neck, and whole-body scintigraphy. The study highlighted the superiority of sSTg over uSTg in the follow-up of papillary thyroid cancerpatients. Follow-up with uSTg alone may result in underestimating the tumor burden.

 

It should not be forgotten, that serum Tg can only be reliably measured in TgAb negative samples, even if measured by high sensitive Tg assays (268,269) so they need to be assessed each time a serum Tg is measured, even if the patient has previously been negative for the antibodies (268,269,350). It also should not be forgotten that measurement of serum TgAb can themselves be used as surrogate markers of relapse/metastases from differentiated thyroid carcinoma (274-276,351).

 

Assay of thyroglobulin on needle washes of suspect lymph nodes has a high degree of sensitivity and specificity, apparently superior to cytological examination (271,272). During long-term follow-up, it is crucial that clinicians are kept informed of changes in serum thyroglobulin methodology that may give a false impression of remission or recurrence (see above).

 

Psychiatric Illness

 

An unusual variety of euthyroid hyperthyroxinemia occurs in some patients hospitalized with acute psychiatric illness (195). Serum T4 is increased, but serum T3 is less frequently elevated; serum TSH is generally normal or slightly high (352). These abnormalities, presumed to be due to central activation of the hypothalamic-pituitary-thyroid axis, often resolve in several weeks (195).

 

Assessment of Thyroid Function Before, During and After Pregnancy

 

Recent advances in the understanding of the importance of optimal maternal thyroid function for fetal brain development in early pregnancy, as well as the influence of thyroid immunity, or thyroid status, on numerous pregnancy outcomes, has greatly increased the frequency of thyroid testing before, during, and after pregnancy. Several guidelines have been published from The Endocrine Society (353), American Thyroid Association (8) – and for subclinical hypothyroidism from European Thyroid Association (9). Projecting this to fertility issues even complicate matters further, and no consensus can currently be obtained (354). There is thus also still controversy regarding whether to treat subtle abnormalities of thyroid dysfunction in the infertile female patient. This guideline document reviews the risks and benefits of treating subclinical hypothyroidism in female patients with a history of infertility andmiscarriage, as well as obstetrical and neonatal outcomes in this population.

 

Clinical guidelines from The Endocrine Society (353) suggest consensus, but significant controversies remain (355). Evidence exists that obstetricians struggle with the diagnosis and treatment of hypothyroidism. According to recent surveys, the management of hypothyroidism during pregnancy is the number 1 endocrine topic of interest for obstetricians. A synopsis of recently published subspecialty guidelines is timely but has not really been done yet. The reproductive effects of abnormal thyroid function and its immune associations are considered in other Endotext chapters. This section will consider the issues from the point of view of testing strategy and assay methodology. From studies of pregnancy outcome, some evidence has become persuasive that miscarriage rate and prematurity (6,356-358) are favorably influenced by maternal thyroid hormone replacement, even when hypothyroidism would be regarded as “subclinical” by standard criteria. Some clinicians advocate early and liberal screening and treatment (356,358), but a recent Cochrane review, however, did not find any clear benefit for universal screening rather than case finding (6).They found based on the existing evidence, that though universal screening for thyroid dysfunction in pregnancy increases the number of women diagnosed with hypothyroidism who can be subsequently treated, it does not clearly impact (benefit or harm) maternal and infant outcomes. While universal screening versus case finding for thyroid dysfunction increased diagnosis and subsequent treatment, they found no clear differences for the primary outcomes: pre-eclampsia or preterm birth. No clear differences were seen for secondary outcomes, including miscarriage and fetal or neonatal death; data were lacking for the primary outcome: neurosensory disability for the infant as a child, and for many secondary outcomes. Though universal screening versus no screening for hypothyroidism similarly increased diagnosis and subsequent treatment, no clear difference was seen for the primary outcome: neurosensory disability for the infant as a child (IQ <85 at three years); data were lacking for the other primary outcomes: pre-eclampsia and preterm birth, and for the majority of secondary outcomes. For outcomes assessed using the GRADE approach the evidence was considered to be moderate or high quality, with any downgrading of the evidence based on the presence of wide confidence intervals crossing the line of no effect. More evidence is needed to assess the benefits or harms of different screening methods for thyroid dysfunction in pregnancy, on maternal, infant and child health outcomes. Future trials should assess impacts on use of health services and costs and be adequately powered to evaluate the effects on short- and long-term outcomes (6).

A key consideration is therefore still whether thyroid function should be universally tested (357) and if so whether it should be done before or early in pregnancy, in view of the fact that women at risk of adverse outcome cannot be identified reliably from their clinical history, even if this is assessed in full detail (359). A recent review (358) has concluded that current guidelines agree that overt hyperthyroidism and hypothyroidism need to be promptly treated and that as potential benefits outweigh potential harm, subclinical hypothyroidism also requires substitutive treatment. The chance that women with thyroid autoimmunity may benefit from levothyroxine treatment to improve obstetric outcome is intriguing, but adequately powered randomized controlled trials are needed. The issue of universal thyroid screening at the beginning of pregnancy is still a matter of debate, and aggressive case-finding is supported. Careful discussion between the physician and patient concerning benefits on the one hand and unnecessary disease burden should be carried out (359).

 

METHODOLOGICAL ISSUES

 

Some methodological issues are relevant before the application of tests is considered (360). There has been almost universal acceptance of free T4 estimates in preference to total T4 measurement in pregnancy because of the well-known estrogen effect to increase thyroxine binding globulin and hence total T4. It is clear that normal pregnancy is associated with a marked increase of about 40% in total T4 concentration, but free T4 is maintained close to normal non-pregnant levels (360). Previous suggestions that free T4 actually declines significantly in late pregnancy (361) are uncertain because of wide method-dependent variations, with strong negative bias in some methods (362-364). In a comparative study of free T4 by seven commercial methods in 23 euthyroid women at term, Roti et al. found that albumin-dependent methods show marked negative bias, with up to 50% subnormal values, while other methods gave values above their non-pregnant reference interval (362). Notably, particularly during pregnancy, methods of free T4 estimation fall far short of desirable assay standardization criteria that aim to minimize between-assay variation (171).

 

A study by Lee et al. confirmed marked negative bias in free T4 estimates during pregnancy and questioned the basis for continuing to rely on free thyroxine estimates during pregnancy (365). In contrast to two kit free T4 methods, totalserum T4 and its derivative, the free T4 index, showed the anticipated inverse relationship with serum TSH, with historically consistent results in numerous reports (365). Thus, because of consistency between methods, total T4 measurement may be superior to free T4 estimation as a guide to therapy during pregnancy, provided that reference values take account of the normal estrogen-induced increase in thyroxine binding globulin during pregnancy (360,366).

 

Serum TSH is generally analytically reliable during pregnancy, but the reference interval is lower at the end of the first trimester, associated with the effect of human choriogonadotropin as a surrogate thyroid stimulator (360). Hence, the general advice that tests of thyroid function during pregnancy should relate to trimester-specific reference intervals (360,367). In the case of free T4, ranges also need to be method-specific (362-365,367). The magnitude of method-dependent variations between non-pregnant and third trimester free T4 estimates is shown in Figure 11.

 

Figure 11. Free T4 estimates in the third trimester of pregnancy show large method-dependent differences from the reference intervals for non-pregnant subjects. Redrawn from ref. (363) and (364) which identify each method studied. The left panel shows values measured by equilibrium dialysis (ED). The solid bars indicate third trimester free T4 estimates, the grey bars, non-pregnant results.

It has recently been demonstrated that mass spectrometry measurement of free thyroid hormones  may alleviate the problems of flaws in the usual free thyroid hormone measurements in pregnancy (368), although these results need to be confirmed on the one hand, and a solution to the must higher costs of this type of methodology has to be solved on the other hand. Until then a pragmatic approach is to use total thyroid hormone measurements multiplied by 1.5 as useful reference interval during pregnancy.

 

There is also during pregnancy, as in the non-pregnant state, a very high individuality index i.e. the intraindividual variability of pregnant women is much higher than the interindividual population- and even trimester-based reference range (369), which further complicates thyroid function assessment in cross sectional measurements. The function variables become much more clinically significant when measurements are used in longitudinal assessments e.g. during pregnancy.

 

BEFORE PREGNANCY OR WHEN PREGNANCY IS CONFIRMED

 

Because of the adverse effects of maternal thyroid deficiency, there is agreement that pre-pregnancy testing is now mandatory to optimize thyroxine replacement and to assess thyroid status in women who have an increased risk of thyroid dysfunction (8,9,353,370-372), for example a history of recurrent miscarriage, impaired fertility requiring assisted reproductive technology, type 1 diabetes, or any other factors known to be associated with increased risk of thyroid dysfunction (Table 2). Notably, in one key study, about a third of women with subclinical or overt hypothyroidism in the first trimester would have been missed if testing had been confined to those assessed as being at higher risk, based on detailed questioning about potential risk factors, in particular a personal or family history of thyroid or other autoimmune disorder, or previous thyroid-related treatment (373). A further study confirms that about half of the women at risk for pregnancy-related thyroid dysfunction would be missed by testing confined to the group assessed to be at high risk (374). Hence, the view that routine testing may now be warranted (14). Because the major influence of thyroid hormone on fetal brain development is early in pregnancy and the onset of thyroid hormone action is slow, effective testing needs to be done earlier than would follow the first routine obstetric visit at 10-12 weeks.

 

While there is now good evidence that any level of TSH elevation that suggests maternal hypothyroidism should be treated, there is currently no consensus about responses to other test abnormalities. An important finding is that thyroxine treatment of peroxidase antibody-positive euthyroid women from about the end of the first trimester may improve obstetrical outcome, as judged by rates of miscarriage and premature delivery (375). A recent meta-analysis confirms that thyroid autoantibodies are associated with miscarriage and preterm delivery and suggests that thyroxine treatment diminishes these risks (376). Pre-partum assessment of antibody status may also provide information that improves prediction of post-partum thyroid dysfunction (377).

 

A different aspect is related to women already on thyroxine replacement therapy before pregnancy. It has been shown that almost 50% of thyroxine treated women were not optimally replaced in early pregnancy (TSH <0.4 or >4.0 mU/l) with the potential for significant adverse effect on pregnancy outcome (370). In thyroxine-treated women it should now be standard practice to assess optimal replacement prior to pregnancy and to re-evaluate this when pregnancy is confirmed, with a prompt 25-30% increase in replacement dosage (371), with two extra daily doses per week as an approximation. The required increase is larger in women who have had thyroid ablation than in those with spontaneous hypothyroidism (371).

 

A special situation arises in thyroxine-treated women who have in vitro fertilization. Ovulation-induction cycles, even in the absence of ongoing pregnancy, can be associated with 2-10 fold increases in serum TSH (378), indicating that previously adequate replacement may not accommodate estrogen-induced thyroxine requirements induced by a very rapid increase of thyroid hormone binding proteins. This suggests that women who lack endogenous thyroxine should receive increased dosage from the time of ovulation induction, to accommodate increased thyroxine requirement, whether associated with a subsequent pregnancy, or not. The need to routinely test thyroid function before in vitro fertilization and institute or increase thyroxine treatment seems self-evident.

 

ASSESSMENT DURING PREGNANCY

 

In women treated for hypothyroidism, some guidelines still recommend that replacement thyroxine dosage should be progressively adjusted in pregnancy in response to increasing serum TSH, a response that is likely to be too late to assure optimal first trimester maternal T4 levels for optimal fetal brain development. Hence, the advice to increase dosage by two daily doses per week as soon as pregnancy is confirmed, followed by a check of serum TSH about 4 weeks later (353,371).

 

In women treated for Graves’ disease with antithyroid drug, dose reduction should be the aim, keeping TSH in the lower normal range. In contrast to the adverse effects of even the mildest grades of hypothyroidism, an extensive study has shown no adverse effect of mild overactivity during pregnancy (379). It is thus often possible to cease antithyroid drug late in pregnancy, with prospective follow-up to detect post-partum recurrence. In contrast to immune thyroid disorders, nodular thyroid disorders do not appear to show marked fluctuation during and after pregnancy. Recent guidelines recommend the use of propyluracil in the first trimester (380), and thiamazole (methimazole) thereafter due to the risk of birth defects by thiamazole (380,381), and general side effects to propylthiouracil (382,383).

 

In the event of uncontrolled or severe maternal hyperthyroidism, careful assessment should be made of fetal growth and thyroid size followed by fetal blood sampling in some cases as a guide to optimal therapy (384,385). Guidelines for the assessment of TRAb to gauge the likelihood of neonatal thyrotoxicosis have been discussed above (283).

 

POSTPARTUM ASSESSMENT

 

Among women who were euthyroid prior to pregnancy, autoimmune thyroid dysfunction not due to Graves' disease occurs in approximately 5–8% in the first year post-partum (386). Postpartum thyroiditis is an autoimmune disorder that causes lymphocytic inflammation of the thyroid. It is more frequent in women with elevated first-trimester serum thyroperoxidase antibody concentrations (387) and in women with other autoimmune disorders, such as type 1 diabetes mellitus (388,389).

 

The thyroid inflammation in postpartum thyroiditis initially leads to transient thyrotoxicosis as preformed thyroid hormones are released from the damaged gland. This phase usually occurs 1–3 months post-partum and lasts for 6–9 weeks. Symptoms are typically mild, but rarely florid, transient thyrotoxicosis can be seen. Serum T4 levels are proportionally higher than T3 levels, reflecting the ratio of stored hormone in the thyroid gland (in contrast to Graves' disease where T3 is often preferentially elevated) (390).

 

In prospective studies there is a high prevalence of thyroid dysfunction during the post-partum period (8,353), and a high percentage of those women with subclinical postpartum thyroiditis proceeded to permanent thyroid failure (391,392). In an Australian study, this effect was most pronounced in women with low iodine intake (393). Hence, women with postpartum hypothyroidism should be treated on the basis of confirmed TSH elevation even if the indication for lifelong replacement has not been firmly established. This strategy is especially important because untreated maternal hypothyroidism has critical implications for fetal development in subsequent pregnancies. Also, recent findings of psychiatric disorders coinciding with autoimmune thyroid disorders in the postpartum period (394)indicates that these women should be screened for thyroid dysfunction, and very likely both conditions should be followed on a longer term basis if significant in the postpartum period. By contrast, almost all women with suppressed or subnormal TSH values 6 months postpartum, showed normal TSH 12 and 18 months later, indicating that postpartum hyperthyroidism is likely to be a temporary phenomenon (8,353).

 

In women with established hypothyroidism, replacement dosage of thyroxine can generally be decreased to the pre-pregnancy dose with testing deferred for several months to review dosage. In Graves’ hyperthyroidism, where antithyroid drug has been ceased during pregnancy, resumption of treatment is generally deferred, based on symptoms and the results of testing at two-three monthly intervals. Where thyroperoxidase antibody testing early in pregnancy has been positive in the absence of frank thyroid dysfunction, prospective testing may be warranted in the 12 months post-partum.

 

Knowledge of maternal thyroid status should be taken into account in assessing the significance of neonatal screening for congenital hypothyroidism, because of reports of temporary effects of maternal blocking antibody in the infant.

 

IODINE STATUS  

 

The critical importance of adequate iodine nutrition during pregnancy is widely acknowledged. The increased pool of thyroxine that follows from the increase in serum thyroxine binding globulin, as well as increase in urinary iodine excretion that will aggravate any degree of iodine deficiency, with potential for adverse effects on fetal development. While assessment of iodine status by measurement of urinary iodine excretion in individual women does not have wide support, data on iodine nutrition within each geographical area should be an important public health issue, with appropriate supplementation to achieve estimated urinary iodine excretion in the range 150-300 microgram daily in pregnant women.

 

THE PHYSICIAN-LABORATORY INTERFACE

 

Because of the diverse clinical presentations of thyroid dysfunction, initial requests for assessment of thyroid function are often made by clinicians who, while alert to the possibility of thyroid dysfunction, may not be familiar with the limitations of current assays, or with medication effects. Clinical decisions can be assisted by comments from the laboratory, based on detailed knowledge of current immunoassay limitations (263,395-398). The quality of this assistance will depend on two key components: the training and experience of the reporter and the available clinical information. It is therefore paramount that clinicians and clinical biochemistry specialist communicate, since a practical and useful approach to optimal patient management can only be achieved by collaboration (178).

 

Historical

 

The competitive binding assays that are used for thyroid diagnosis were initially developed 30-35 years ago by clinical investigators who used ‘in house’ assay reagents and were often closely involved in patient care. This nexus between laboratory and clinical investigation allowed deficiencies in early assays to be readily appreciated, but advanced diagnostic technology was available to only a few practitioners, with results sometimes available only after long delay. In recent decades there has been a strong trend away from ‘in house’ reagents which have been replaced by kits that incorporate highly sophisticated standardized reagents and automated instrumentation (e.g. solid phase antibodies, magnetic separation systems, chemiluminescent detection systems). Assay turnaround is much faster and up-to-date techniques are widely available to almost all practitioners, most of whom are inexperienced in endocrinology or laboratory methodology. Non-specialist users of endocrine assays are most likely to benefit from laboratory assistance in the interpretation of results, but as assay automation has increased, laboratory professionals have become more distant from the bedside. As clinicians receive less assistance, they tend to provide less relevant information and vice versa. Laboratory personnel in turn, see results that are uninterpretable or ambiguous without the relevant clinical background. Potential assay imperfections may be ignored simply because clinical correlation is not possible (see below).

 

Diagnostic Approach to Discordant or Apparently Anomalous Results

 

The relationship between laboratory results and clinical findings may be either concordant or discordant. With discordant results, a distinction needs to be made between a previously unsuspected diagnosis, subclinical disease, and anomalous assay results. If the assay result is confirmed, the fundamental assumptions that underpin the diagnostic use of tropic-target hormone relationships should be considered (see above).

 

The following steps may be helpful in evaluating anomalous assay results:

  1. Re-evaluation of the clinical context, with particular attention to pre-diagnostic probability, long-term features suggestive of thyroid disease and medication history.
  2. Assessment of whether serum TSH is markedly suppressed (<0.05 mU/l) or simply in the subnormal-detectable range.
  3. Further sampling to establish whether the anomalous result is persistent or transient.
  4. Estimation of serum free T4 by an alternative method, preferably a two-step technique that removes binding proteins from the assay system before quantitation.
  5. If hyperthyroidism is suspected, measurement of the serum free T3, or total T3 with appropriate binding correction.
  6. Measurement of serum total T4 to establish whether the serum free T4 is disproportionately high or low, possibly due to a preanalytical or method-dependent artefact.
  7. Possible evaluation of propositus and family members for evidence of unusual binding abnormalities or hormone resistance.

 

In some circumstances, the cause of an apparently anomalous or unusual laboratory result may become obvious from the clinical context (table 10). By taking account of drug therapy, or acute perturbation of the pituitary-thyroid axis, assay validity can be affirmed and unnecessary further investigation may be avoided.

 

Table 10.  Situations in Which Interpretation of Unusual Laboratory Results Depends on Relevant Clinical Information

Clinical background

Assay Results

fT4

fT3

TSH

Pregnancy

L*

L,N

N

Antithyroid drug treatment, initial months

H,N,L

H,N,L

U

Recent T4 commencement for hypothyroidism

N

N

H

Hypothyroidism, appropriate T4 dose

H

N

N

Hypothyroidism, intermittent compliance

H,N

 

H

Appropriate T4 suppressive therapy

H

N

U,L

Recombinant TSH, suppressive T4

H

N

H

Hypopituitarism

L

 

L,N

Phenytoin

L*

 

L,N

Critical illness

L*

L

L

Heparin effect in critical illness

L,N,H*

L

L

Recovery phase of critical illness

L,N

 

H

Drugs that inhibit T4/T3 binding to TBG

L*

L*

L,N

Amiodarone effect in euthyroid subject

H

L

N

Acute T4 overdose

HH

H,N

N

Rheumatoid arthritis

N

N

H

U undetectable; L low; N normal; H high; * effect dependent on assay method

 

Clinical Feedback, Quality Assurance, and Cost Effectiveness

 

Clinical feedback will remain a key aspect of quality assurance in laboratory testing. While assay precision or reproducibility can be evaluated solely in the laboratory, diagnostic accuracy requires clinical correlation. As with any diagnostic test, the non-specificity of a procedure may not become apparent until the full diversity of the non-diseased population is appreciated. Premarketing evaluation of assays may fail to include the critical samples that probe the diagnostic accuracy of an assay, so that non-specific interference is not appreciated until procedures have been in use for some time.

 

In studies of “cost effectiveness” it is hard to evaluate the human and financial costs that result from needless duplication, unnecessary testing and inappropriate management. Diagnostic inaccuracy of immunoassays may account for substantial unnecessary expenditure on laboratory resources (263,397,398). Dilution effects and binding protein abnormalities that affect free T4 estimation, the multiple effects of medications in non-thyroidal illness and the problems associated with thyroglobulin autoantibodies will continue to challenge both clinical chemists and clinicians; further technological development will not substitute for collaboration between these two groups.

 

Clinical issues may influence the selection of thyroid tests that will best serve a particular population. For example, the effects of severe illness on free T4 estimates may be of minor importance for a laboratory that serves predominantly ambulatory patients. A different assay profile, with emphasis on measurement of total hormone as the “gold standard” for T4 assessment may be required in a laboratory that evaluates thyroid function during critical illness (328-330) or in obstetric practice (8,353). In the future, hopefully a more sophisticated methodology such as tandem mass spectrometry may be able to eliminate the many confounders in thyroid hormone measurements.

 

ACKNOWLEDGEMENTS

 

The chapter was updated from a previous comprehensive version by the late Jim Stockigt M.D., FRACP, FRCPA, Monash University and Alfred and Epworth Hospitals, Melbourne, Australia.

 

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Glucocorticoid Receptor

ABSTRACT

 

The glucocorticoid receptor (GR) is an evolutionally conserved nuclear receptor superfamily protein that mediates the diverse actions of glucocorticoids as a ligand-dependent transcription factor. This receptor is a protein that shuttles from the cytoplasm to the nucleus upon binding to its ligand glucocorticoid hormone, where it modulates the transcription rates of glucocorticoid-responsive genes positively or negatively. Tremendous efforts have been made to reveal the molecular signaling actions of the GR, including intracellular shuttling, transcriptional regulation and interaction with other intracellular signaling pathways. Glucocorticoids are essential for both maintenance of the resting state and the stress response, and are pivotal in the treatment of many disorders, including autoimmune, inflammatory, allergic, and lymphoproliferative diseases. Thus, pathologic or therapeutic implications of the GR, including genetic alterations in the human GR gene, disease-associated GR regulatory molecules, and development of GR ligands with selective GR actions, are of great importance. This chapter provides an overview on such GR-related research activities.

 

INTRODUCTION

 

Glucocorticoids are steroid hormones secreted by the adrenal glands. They are important for the maintenance of basal and stress-related homeostasis by acting as end products of the stress-responsive hypothalamic-pituitary-adrenal (HPA) axis (1). Glucocorticoids regulate a variety of biologic processes and exert profound influences on many physiologic functions (2,3). In pharmacologic doses, glucocorticoids are used as potent immunosuppressive agents in the therapeutic management of many inflammatory, autoimmune and lympho-proliferative diseases (4). At the cellular level, the actions of glucocorticoids are mediated by an intracellular receptor protein, the glucocorticoid receptor (GR) (its gene name is “nuclear receptor subfamily 3, group C, member 1: NR3C1”), which belongs to the steroid/sterol/thyroid/retinoid/orphan receptor superfamily of nuclear transactivating factors with over 200 members in general and over 40 in mammals currently cloned and characterized across species (5). Human GR consists of 777 amino acid residues (5). GR is ubiquitously expressed in almost all human tissues and organs including neural stem cells (6). GR functions as a hormone-dependent transcription factor that regulates the expression of glucocorticoid-responsive genes, which probably represent 3-10% of the human genome and can be influenced by the ligand-activated GR directly or indirectly (7).

 

EVOLUTION OF GR

 

Nuclear hormone receptors (NRs) form a highly conserved protein family observed even in simple metazoans. They are phylogenically differentiated into 7 subfamilies under the evolutional selection pressure, and are still active in the current human population (8). GR is a member of the steroid hormone receptor (SR) subfamily (subfamily 3) of NRs. This receptor family of vertebrates consists of six evolutionarily related SRs: two for estrogens (estrogen receptor (ER) a and ERβ) and one each for androgens (androgen receptor: AR), progestins (progesterone receptor: PR), glucocorticoids (GR), and mineralocorticoids (mineralocorticoid receptor: MR) (Figure 1). These steroid receptors are also categorized as type I receptors, based on their functional characteristics, such as cytoplasmic localization in the absence of ligand with association to the heat shock proteins, homo-dimerization and recognition of their target DNA sequence (see below), while the other NRs belong to type II to IV (5).

Figure 1. Steroid hormone receptors (SRs: class I receptors) and their homologies expressed as percent identity to the protein sequence of human GR. AR; androgen receptor, ER: estrogen receptor, ER: estrogen receptor, GR; glucocorticoid receptor, MR: mineralocorticoid receptor, PR-A: progesterone receptor-A. Modified from (9).

SRs evolved in the chordate lineage after the separation of deuterostomes and protostomes, prior to or at the base of the Cambrian explosion about 540 million years ago (10,11) (Figure 2A). The receptor phylogeny suggests that two serial gene duplications of an ancestral SR gene occurred before the divergence of lamprey and jawed vertebrates (Figure 2B). The first gene duplication (duplication #1 in Figure 2B) created an estrogen receptor (ER) and a 3-ketosteroid receptor, whereas the second duplication (duplication #2 in Figure 2B) of the latter gene produced a corticoid receptor and a receptor for 3-ketogonadal steroids (androgens, progestins, or both). Therefore, the ancestral vertebrates (e.g., lamprey) had three SRs: an estrogen receptor (ER), a receptor for corticoids (corticosteroid receptor: CR) and a receptor that bound androgens, progestins or both (ancestral PR). At some later time within the gnathostome lineage, each of these three receptor genes were duplicated again (duplications #3, #4 and #5 in Figure 2B) to yield the six SRs currently found in jawed vertebrates: the ER creating ERa and ERβ, CR yielding the GR and MR, and the 3-ketogonadal steroid receptor (ancestral PR) producing the PR and AR. Therefore, the genome of ‘higher’ vertebrates is thought to be the result of three genome duplication events that occurred early in chordate evolution (10,12). Although the timing of these events is not entirely clear, it is most likely that the first 2 duplications occurred before the lamprey-gnathostome divergence and one after (10,13).

Figure 2. Evolution of SRs including GR. A: Appearance of the SR member receptors through evolution of the chordate lineage. The first ancestral SR, which is close to the current ER, appeared ~540 million years ago. At lamprey, 3 receptors, ER, PR and CR, emerged. From the ray-finned fishes, all SR members, ER, PR, AR, GR and MR, appeared. Modified from (14). B: Phylogeny of the SR family genes. Current human SRs including GR were generated through several gene duplications (shown as orange squares). Appearance of the ancestral (Anc) SR1, SR2 and CR are shown with arrows in the phylogeny tree. Blue lines indicate the lamprey-gnathostome divergence. Modified from (10).

The GR and its closest family member MR, both descend from duplication of the ancestral CR (AncCR) gene, and emerged in the vertebrate lineage approximately 450 million years ago (12,15) (Figure 2B). The GR is activated by cortisol, while the MR is activated by aldosterone in tetrapods and by deoxycorticosterone (DOC) in teleosts. The MR is also sensitive to cortisol, though considerably less so than to aldosterone and DOC (12,15). Like the MR, the AncCR is sensitive to aldosterone, DOC and cortisol, indicating that the specificity of GR for cortisol is evolutionarily derived (12,15).

 

To determine how the preference of the GR for cortisol evolved, Ortlund et al. identified substitutions that occurred during the same period as the shift in GR function (16). Using maximum likelihood phylogenetics, he revealed that GR retained AncCR’s sensitivity to aldosterone, DOC and cortisol, from the common ancestor of all jawed vertebrates, but the GR from the common ancestor of bony vertebrates obtained a phenotype like that of the current GRs that respond only to cortisol. These findings indicate that the specificity of GR for cortisol evolved during the interval between these two speciation events, approximately 420 to 440 million years ago (16). Amino acid substitutions found in the modern GR compared to AncGR are not a consequence of the direct introduction of corresponding nucleotide changes, but supported by permissive mutations that enabled the intermediate receptor to tolerate insertion of the final substitutions (17).

 

Teleosts, one of the 3 subgroups of ray-finned fishes that covers most of the living fishes today, underwent an additional gene duplication event about 350 million years ago (18). Thus, all fishes that belong to this subclass, including carp and rainbow trout, have 2 GR genes (GR1 and 2 in rainbow trout). However, zebrafish has only one GR gene in contrast to the other teleost families, because this species lost the 2nd GR gene sometime during the last 33 million years (18).

 

STRUCTURE OF THE HUMAN GR GENE AND PROTEIN

 

All SRs including GR display a modular structure comprised of five to six regions (A-F): the amino-terminal A/B region, also called immunogenic or N-terminal domain (NTD), and the C and E regions, which correspond to the DNA- (DBD) and ligand-binding (LBD) domains, respectively (Figure 3). D region represents the hinge region (HR), while F region is located in the C-terminal end of the NRs with high variability. GR does not have a F region. The GR cDNA was isolated by expression cloning in 1985 (19). The human GR gene consists of 9 exons and is located in the long arm of the chromosome 5 (5q31.3) in an inverse orientation and spanning ~160 kbs. Alternative splicing of the human GR gene in exon 9 generates two highly homologous receptor isoforms, termed a and b. These are identical through amino acid 727, but then diverge, with human GRa having an additional 50 amino acids and human GRb having an additional, nonhomologous 15 amino acids (20). The molecular weights of these receptor isoforms are 97 and 94 kilo-Dalton, respectively. Human GRa is expressed virtually in all organs and tissues, resides primarily in the cytoplasm, and represents the classic glucocorticoid receptor that functions as a ligand-dependent transcription factor. Human GRb, also expressed ubiquitously, does not bind glucocorticoid agonists and functions as a dominant negative receptor for GRa-induced transcriptional activity (see Section 7. THE SPLICE VARIANT GR-beta ISOFORM) (21).

Figure 3. Genomic and complementary DNA and protein structures of the human (h) GR with its functional distribution, and the isoforms produced through alternative splicing.
The hGR (NR3C1) gene consists of 10 exons. Exon 1 is an untranslated region (UTR), exon 2 encodes for NTD (A/B), exon 3 and 4 for DBD (C), and exons 5-9 for the hinge region (D) and LBD (E). GR does not have an F region in contrast to the other steroid hormone receptors. The GR (NR3C1) gene contains two terminal exons 9 (exon 9 and 9) alternatively spliced to produce the classic GR and the nonligand-binding GR isoform. C-terminal gray-colored domains in GR and GR show their specific portions. Locations of several functional domains are also indicated. AF-1 and -2: activation function-1 and -2; DBD; DNA-binding domain; HD: hinge region; LBD: Ligand-binding domain; NTD: N-terminal domain, NL1 and 2: Nuclear translocation signal 1 and 2.

The N-terminal domain (NTD) of GRa contains a major transactivation domain, termed activation function (AF)-1, which is located between amino acids 77 and 262 of the human GRa (22,23). AF-1 belongs to a group of acidic activators, such as VP16, nuclear factor of kB (NF-kB), p65 and p53, contains four a-helices, and plays an important role in the communication between the receptor and molecules necessary for the initiation of transcription, including coactivators, chromatin modulators and basal transcription factors [RNA polymerase II, TATA-binding protein (TBP) and a host of TBP-associated proteins (TAFIIs)] (24). GRa AF-1 is relatively unfolded at the basal state, while it forms a significantly complex helical structure in response to binding to cofactors, such as TBP and p160 coactivators (25,26). TBP-induced conformational change in AF-1 facilitates association of this domain to a p160 coactivator (27).

 

The DNA-binding domain (DBD) of the human GRa corresponds to amino acids 420-480 and contains two C4-type zinc finger motifs through which GRa binds to specific DNA sequences, the glucocorticoid-responsive elements (GREs) (28,29). The DBD is the most highly conserved domain throughout the NR family. It has two similar zinc finger modules, each nucleated by a zinc ion coordination center held by four cysteine (C) residues and followed by a-helix (Figure 4A). The N-terminal’s first a-helix lies in the major groove of the double-stranded DNA, while the C-terminal part of the second a-helix is positioned over the minor groove (Figure 4B).

Figure 4. Structure of GR DBD and its interaction with DNA GRE. A: Zinc finger structures in DBD of hGR. Numbered eight cysteine (C) residues chelate Zn2+ to form two separate finger structures. Red-colored amino acid residues form -helical structures. Box with bold line indicates lever arm, while that with dashed line shows D-box. Modified from (30). B: 3-Dimensional model of the physical interaction between the GR DBD and DNA GRE. The N-terminal’s first -helix of the GR DBD lies in the major groove of the double-stranded DNA, while the C-terminal part of the second -helix is positioned over the minor groove. The image was created and donated by Dr. D.E. Hurt (National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD). Box with bold line indicates lever arm, while that with dashed line shows D-box. Modified from (30).

The ligand-binding domain (LBD) of the human GRa corresponds to amino acids 481-777, binds to glucocorticoids, and plays a critical role in the ligand-induced activation of GRalpha. The crystal structure of the GRalpha LBD was successfully analyzed by using a point mutant containing a single replacement of phenylalanine at amino acid 602 by serine, and is comprised of 12 a-helices and 4 small β-sheets that fold into a three-layer helical domain (31) (Figure 5). Helices 1 and 3 form one side of a helical sandwich, while helices 7 and 10 form the other side. The middle layer of the helices (helices 4, 5, 8, and 9) is present in the top but not the bottom half of the protein. This arrangement of helices creates a cavity in the bottom half of the LBD, which is surrounded by helices 3, 4, 11 and 12, and functions as a ligand-binding pocket (31-33). Interaction of the LBD with the heat shock protein (hsp) 90 contributes to the maintenance of the protein structure that allows LBD to associate with ligand. Ligand-binding induces a conformational change in the LBD and allows GRa to communicate with several molecules, such as importin a of the nuclear import system, components of the transcription initiation complexes and other transcription factors that mediate the ligand-dependent actions of GRa. The LBD also contains one transactivation domain, termed AF-2. The activity of AF-2 is ligand-dependent.

Figure 5. Structure of the GR LBD. The GR consists of 12 -helices and 4 small β-sheets that fold into a three-layer helical domain. Helices 1 and 3 form one side of a helical sandwich, while helices 7 and 10 form the other side. The middle layer of helices 4, 5, 8, and 9 are present in the top but not in the bottom half of the protein, thus creating a ligand-binding pocket (shown as yellow star) in the bottom half of the LBD, surrounded by helices 3, 4, 11 and 12. The image was created with the MacPyMOL software using 3K22 of the RCSB Protein Data Bank (PDB) (http://www.rcsb.org/pdb/home/home.do).

TRANSCRIPTIONAL AND TRANSLATIONAL REGULATION OF GR ISOFORMS

 

As described above, the human GR gene expresses two mRNAs through alternative use of exon 9a and 9b, and produces two splice variants. The human GRa mRNA further expresses multiple isoforms by using at least 8 alternative translation initiation sites (34) (Figure 6). Since human GRb shares a common mRNA domain that contains the same translation initiation sites with human GRa (19), the human GRb variant mRNA seems also to be translated through the same initiation sites to a similar host of b isoforms. They are produced by ribosomal leaky scanning and/or ribosomal shunting from their alternative translation initiation sites located at amino acids 27 (GRa-B), 86 (GRa-C1), 90 (GRa-C2), 98 (GRa-C3), 316 (GRa-D1), 331 (GRa-D2) and 336 (GRa-D3), C-terminally from the classic translation start site (1: for the GRa-A) (34). Thus, they have different lengths of NTDs but the same DBDs and LBDs. Compared to GRa-A, GRa-C2 and GRa-C3 isoforms have stronger transcriptional activities on a synthetic GRE-driven promoter, while GRa-D1, GRa-D2 and GRa-D3 demonstrate weaker activities (34). GRa-B and GRa-C1, however, possess transcriptional activities similar to that of GRa-A (34). Absence of AF-1 in GRa-D isoforms may explain their reduced transcriptional activity, while ~100 amino acids (particularly 3 polar amino acids) located in the N-terminal portion of AF-1 appear to support increased transcriptional activity of GRa-C isoforms (35). All human GRa isoforms translocate into the nucleus in response to ligand, while they are differentially distributed in the cytoplasm and/or the nucleus in the absence of ligand and display distinct transactivation or transrepression patterns on global gene expression examined by cDNA microarray analyses (34). Such isoform-specific transcriptional activity is in part explained by their distinct chromatin modulatory activity, which is evident in the different potencies of the translational isoforms to induce apoptosis in T-cell Jurkat cells (36).

Figure 6. GR isoforms produced through alternative splicing or use of different translational initiation sites. The human GR (NR3C1) gene contains two terminal exons 9 (9alpha and 9beta) alternatively spliced to produce the classic GR (GRalpha-A) and GRbeta-A. C-terminal dark yellow-colored domains in GRalpha-A and GRbeta-A show their specific portions. Using at least 8 different translation initiation sites located in NTD, the human GR (NR3C1) gene produces multiple GR isoforms termed A through D (A, B, C1-C3 and D1-D3) with distinct transcriptional activities on glucocorticoid-responsive genes. Since GRalpha and GRbeta share a common mRNA domain that contains the same translation initiation sites, the GRbeta variant mRNA appears to be also translated through the same initiation sites and to produce 8 isoforms with different lengths of NTD. Modified from (20,37). AF-1 and -2: activation function-1 and -2; DBD; DNA-binding domain; HD: hinge region; LBD: Ligand-binding domain

Translational Human GRa isoforms are differentially expressed in various cell lines, tissues and at different developmental stages (34). For example, GRa-D isoforms are predominant in immature bone marrow-derived dendritic cells (DCs), while GRa-A is a main isoform in mature DCs, and this characteristic expression explains maturation-specific alteration of glucocorticoid sensitivity in these cells (38). GRa-A is highly expressed in brains of toddlers to teenagers, whereas peak expression of GRa-D is observed in those of neonates (39). Thus, these N-terminal human GRa isoforms may differentially transduce glucocorticoid hormone signals to tissues, depending on their selective expression and inherent activities.

 

The human GR gene has eleven different promoters with their alternative first exons (1A1, 1A2, 1A3, 1B, 1C, 1D, 1E, 1F, 1H, 1I and 1J) (40,41) (Figure 7). Therefore, the human GR gene can produce eleven different transcripts from different promoters that encode the same GR proteins sharing a common exon 2 containing the translating ATG codon. 1A1, 1A2, 1A3 and 1I are located in the distal promoter region spanning ~32,000-36,000 bps upstream of the translation start site, while 1B, 1C, 1D, 1E, 1F, 1H and 1J position in the proximal promoter region located up to ~5,000 bps upstream of such a site (40). Through differential use of these promoters, expression levels of GR protein isoforms can vary considerably among tissues and disease states (40,42). DNA methylation of the human GR gene promoter area is one of the mechanisms that regulate the activity of specific GR gene promoters. Indeed, childhood trauma, which influences development of the borderline personality disorder by affecting the stress-responsive HPA axis, contributes to the alteration of DNA methylation levels of the human GR gene promoter in the brain (43). Elevated DNA methylation in the human GR gene promoter is also found in the brain hippocampus of the patients with major depression (44). Furthermore, the methylation status of the human GR gene promoter in the peripheral blood is highly altered during the perinatal period. Interestingly, preterm infants demonstrate significantly lower levels of the DNA methylation compared to full-term infants, explaining in part relative glucocorticoid insensitivity observed in preterm babies (45).

 

In addition to selective use/activation-inactivation of the specific GR gene promoters, alternative untranslated 1st exon transcripts differentially control stability and translational efficiency of their existing GR mRNA, and contribute to differential tissue expression of the GR proteins (46). By employing many splice/translational GR isoforms expressed from different promoters, human GR appears to form at least 256 different combinations of homo- and hetero-dimers with varying expression levels and transcriptional activities. This marked complexity in the transcription/translation of the human GR gene allows cells/tissues to respond differentially to the circulating concentrations of glucocorticoids depending on the needs of respective tissues (20).

Figure 7. The human (h) GR (NR3C1) gene has 11 different promoters with specific exon 1 sequences. The hGR (NR3C1) gene has 11 different promoters harboring specific exon 1 sequences. Alternative exon 1s are shown as yellow arrows or arrowheads. The 5’ flanking region of the hGR (NR3C1) gene has proximal and distal promoter regions, which respectively span from ~-37,000 to ~-32,000 and from ~-5,000 to ~0, upstream of the translation initiation site located in the exon 2 (shown as “ATG” and arrowhead), and contain exons 1A1, 1A2, 1A3, and 1I, and 1B, 1C, 1D, 1E, 1F and 1H, respectively. Modified from (40,41).

ACTIONS OF GR

 

Nucleocytoplasmic Shuttling of GRa

 

In the absence of ligand, GRa resides primarily in the cytoplasm of cells as part of a large multiprotein complex, which consists of the receptor polypeptide, two molecules of hsp90, and several other proteins (28,47-49) (Figure 8). Following ligand binding, the receptor dissociates from the hsps and translocates into the nucleus. The GRa contains two nuclear translocation signals (NL), NL1 and NL2 (Figure 3): NL1 contains a classic basic-type nuclear localization signal (NLS) structure that overlaps with and extends C-terminally from the DBD of GRa (50). The function of NL1 is dependent on importin a, a protein component of the nuclear translocation system, which is energy-dependent and facilitates the translocation of the activated receptor into the nucleus through the nuclear pore. NL2 spans over almost the entire LBD. In the absence of ligand, binding of hsps with the LBD of GRa masks/inactivates NL1 and NL2, thereby maintaining GRa in the cytoplasm. Inside the nucleus, GRa binds to GREs in the promoter regions of target genes. The interaction of GRa with GREs is dynamic, with the GRa binding to and dissociating from GREs in the order of seconds, while the GRE-bound receptor helps other GRas to bind DNA by increasing chromatin accessibility (the mechanism called “assisted loading”), and ultimately up-regulates their steady state association on glucocorticoid-responsive gene promoters (51,52). The above findings were obtained using the multi-copy GREs artificially inserted into the host cell chromatin, but a recent report confirmed them by examining endogenous GREs using a single molecule imaging technique (53). GRa also modulates transcriptional activity of other transcription factors by physically interacting with them. After modulating the transcription of its responsive genes, GRa dissociates from the ligand and slowly returns to the cytoplasm as a component of the heterocomplexes with hsps (54-56). The ubiquitin-proteasomal pathway degrades ligand-bound GRa in the nucleus, facilitating clearance of the receptor from GREs; thus, this system regulates the transcriptional activity of GRalpha in a negative fashion (57,58).

Figure 8. Intracellular circulation of GR. Circulation of GR between the cytoplasm and the nucleus, and its transcriptional regulation on the glucocorticoid-responsive genes in the nucleus are shown in the panel. GR translocates into mitochondria or lysosomes as well. GREs: glucocorticoid responsive elements; TFREs: transcription factor responsive elements; HSPs: heat shock proteins; TF: transcription factor. From (59).

Several mechanisms have been postulated for the regulation of GRa nuclear export [27]. The Ca2+-binding protein calreticulin plays a role in the nuclear export of GRa, directly binding to the DBD of this receptor (60-62). In contrast, the CRM1/exportin and the classic nuclear export signal (NES)-mediated nuclear export machinery does not appear to be functional directly on GR (50,60,63). Rather, NES-harboring and phospho-serine/threonine-binding protein 14-3-3s can bind the human GR phosphorylated at serine 134, and segregates the nuclear GRa into the cytoplasm (64,65) (see also Section FACTORS THAT MODULATE GR ACTIONS, B. Epigenetic Modulation of GRa, Phosphorylation).

 

In addition to translocating into the nucleus, GRa was reported to shuttle into mitochondria upon ligand activation and to stimulate mitochondrial gene expression by binding to their own DNA (66) (Figure 8). Exposure of rats to stress or corticosterone induces translocation of GRa to mitochondria and modulates mitochondrial mRNA expression (67), indicating that this activity of GRa is evident at an animal level. GRa was also shown to move into the lysosome, which leads to the negative regulation of its transcriptional activity (68).

 

Mechanisms of GRa-mediated Activation of Transcription

 

Classically, GRa exerts its transcriptional activity on glucocorticoid-responsive genes by binding to GREs located in the promoter region of these genes (69). The optimal tandem GREs is an inverted hexameric palindrome separated by 3 base pairs, PuGNACANNNTGTNCPy, on which each GRa molecule binds one of the palindromes, forming a homodimer on this binding site through multiple contacts between the 2 receptors (70,71). Recent research indicated that sequence variation of GREs, including 3 non-specific spacer nucleotides, influences the 3-dimensional structure of DBD and modulates the transcriptional activity of whole GRa molecule (72,73); Binding of GRa DBD to GRE DNA sequence induces conformational changes in the dimerization surface located in D-loop through the lever arm, which positions itself between the first a-helix and D-loop (Figure 4A). Two receptors bound on each GRE half site then communicate with each other with their GRE sequence-specific dimerization surfaces, and ultimately develop net transcriptional activity. These findings suggest that DNA GRE is a sequence-specific allosteric modulator of GRa transcriptional activity through alteration of its protein conformation, explaining in part gene-specific transcriptional effects of this receptor.

The GRE-bound GRa stimulates the transcription rates of glucocorticoid-responsive genes by facilitating formation of the transcription initiation complex on the GREs-containing promoter of these genes via its AF-1 and AF-2 transactivation domains (74) (Figure 3). Actions of AF-1 located in NTD of GRa is ligand-independent, while AF-2 is created on GRa LBD upon ligand-binding (75).

 

The transcription initiation complex attracted and formed on DNA-bound GRa is a mega protein structure that include over 100 proteins with different activities, such as RNA polymerase II and its ancillary factors, general transcription factors and numerous co-regulatory molecules with/without enzymatic activities (74). Research studies aimed to identify molecules interacting with GRa AF-2 have led to several proteins and protein complexes, called coactivators or cofactors, that form a bridge between DNA-bound GRa and the transcription initiation complex, and assist enzymatically with the transmission of the glucocorticoid signal to RNA synthesis promoted by the RNA polymerase II (76) (Figure 9). These include: (1) p300 and the homologous cAMP-responsive element-binding protein (CREB)-binding protein (CBP), which also serve as macromolecular docking “platforms” for transcription factors from several signal transduction cascades, including NRs, CREB, activator protein-1 (AP-1), NF-kB, p53, Ras-dependent growth factor, and signal transducers and stimulators of transcription (STATs) (77). Because of their central position in many signal transduction cascades, the p300/CBP coactivators are also called co-integrators; (2) p300/CBP-associated factor (p/CAF), originally reported as a human homologue of yeast Gcn5, which interacts with p300/CBP and is also a broad transcription coactivator (78,79); and (3) the p160 family of coactivators, which preferentially interact with SRs (80). These include the steroid receptor coactivator-1 (SRC-1), SRC-2, which consists of transcription intermediate factor-II (TIF-II) and the glucocorticoid receptor-interacting protein-1 (GRIP1), and SRC-3, which consists of the p300/CBP/co-integrator-associated protein (p/CIP), activator of thyroid receptor (ACTR) and the receptor-associated coactivator-3 (RAC3) (76,80,81). These 3 subclasses of p160 family coactivators are also called, respectively, as nuclear receptor coactivators (NCoA) 1, 2 and 3.

Figure 9. Schematic model demonstrating the interaction and activity of HAT coactivators and other chromatin modulators, which are attracted by GR to the promoter region of glucocorticoid-responsive genes. Promoter-associated GR is cleared by the ubiquitin-proteasomal pathway, which regulates turnover of GR on DNA. Modified from (82). AF-1 and -2: activation function-1 and -2; CBP: cAMP-responsive element-binding protein (CREB)-binding protein; DRIP: vitamin D receptor-interacting protein; GREs: glucocorticoid response elements; p/CAF: p300/CBP-associated factor; SWI/SNF: mating-type switching/sucrose non-fermenting; TRAP: thyroid hormone receptor-associated protein.

The p160 coactivators are the first to be attracted to the DNA-bound NRs and help accumulating p300/CBP and p/CAF proteins to the promoter region, indicating that p160 proteins play a pivotal role in NR-mediated transactivation. A study using the cryoelectron microscopy demonstrated detailed attraction modes of p160 proteins and p300/CBP to DNA-bound and ligand-activated ERa (83); Each of the tandem ER response elements (EREs)-bound receptors independently attracts one p160 molecule via the transactivation surface of the receptor created by their AF-1 and AF-2. Then, the two NCoAs attracted to the receptors recruits one p300/CBP molecule to the DNA/receptors/p160s complex through multiple contacts mediated by different portions of the p160 proteins.

 

In addition to physical interaction and subsequent formation of the transcriptional initiating complex on the DNA-bound receptors by these coactivators (that is assembly of transcriptional initiation complex), these molecules have intrinsic histone acetyltransferase (HAT) activity through which they acetylate specific lysine residues of chromatin-bound histones, loosen the tightly assembled chromatin structure and facilitate access of other transcription factors and transcriptional complexes to the promoter region (76). These HAT coactivators also acetylate specific lysine residues of their own molecules, NRs and other transcription factors, and modulate their mutual protein-protein interaction and/or association to attracted promoters (84-86). The p160 family coactivators and p300/CBP protein contain one or more copies of the coactivator signature motif sequence LxxLL, where L is leucine and x is any amino acid (80,87). LxxLL forms a helical structure, and develops multiple hydrophobic bonds with its leucine residues to the AF-2 surface, which is created by helixes 3, 4 and 12 of the GRa LBD upon binding to ligand glucocorticoid (Figure 10A). p160-type coactivators contain 3 LxxLL motifs in its nuclear receptor-binding box (NRB) located in their central portion (76) (Figure 10B). Each of these motifs demonstrates different affinity to various NRs, indicating that specific p160 proteins participate in the transcriptional activity of particular NRs through preferential use of LxxLL motifs (88). For example, GRa preferentially interacts with GRIP1 p160 protein through C-terminally located 3rd LxxLL motif of this coactivator (89).

Figure 10. p160 coactivators physically interact with its multiple LxxLL motifs to the AF-2 surface of GR. A: 3-dimensional interaction image of GR AF-2 and the LXXLL peptide. The GR AF-2 surface has three large pockets into which core leucines (L745, L748 and L749) of the LXXLL peptide deeply bury themselves. There are additional intermolecular contacts that are important for peptide binding, including the electrostatic bonds created between (i) R746 (LXXLL peptide) and D590 (receptor), (ii) D750 (LXXLL peptide) and R585 (receptor) and (iii) D752 (LXXLL peptide) and K579 (receptor). From (89). B: p160-type coactivators (NCoAs) have 3 LxxLL motifs in their NR-binding box (NRB). Linearlized GRIP1 (NCoA2) molecule with NRB located in the middle portion is shown as a representative of the p160-type coactivators (NCoAs). In addition to NRB, GRIP1 has the basic helix-loop-helix (bLHL) and the PAS domains in its N-terminal portion, and p300/CBP-binding domain and one transactivation domain containing the HAT domain in the C-terminus.

The AF-2 transactivation domain of GRa also attracts several other distinct chromatin modulators, such as the mating-type switching/sucrose non-fermenting (SWI/SNF) complex and components of the vitamin D receptor-interacting protein/thyroid hormone receptor-associated protein (DRIP/TRAP) complex (76). The SWI/SNF complex is an ATP-dependent chromatin remodeling factor with a multi-subunit structure in which the ATPase domain functions as the catalytic center (90). Depending on the energy of ATP hydrolysis, the SWI/SNF complex introduces superhelical torsion into DNA. One of its components, SNF2 binds to AF-2 of GRa directly, functioning as an interface between the GRa and the SWI/SNF complex (91). The DRIP/TRAP complex is also a multiprotein conglomerate, which consists of over 10 different proteins, including DRIP205/TRAP220/PBP and components of SMCC (76). The DRIP/TRAP complex may modulate transcription through interaction and modification of general transcription factors, such as TFIIH or the C-terminal tail of the RNA polymerase II. DRIP205/TRAP220 contains two LxxLL motifs through which it binds to the ligand-activated AF-2 directly (92). Since the DRIP/TRAP complex and p160 coactivators use the same motif for interaction with NRs, they may bind to the same site of these receptors and sequentially interact with them for full activation of transcription. Alternatively, they may interact with a particular set of NRs, or have a different effect on different tissues (76,81).

 

In contrast to the mechanisms of transactivation by AF-2, those of AF-1 are not as well elucidated yet. Using the yeast system, the Ada complex may act on AF-1-mediated transcriptional activation through direct interaction to this module (93). The SWI/SNF complex, TBP and the HAT coactivators, such as p160 and p300/CBP, also physically interact with AF-1 and mediate its transcriptional activity (94-97). In addition, DRIP150, a component of the DRIP/TRAP complex, and the tumor susceptibility gene 101 (TSG101) interact with the AF-1 of the GRa in a yeast two-hybrid screening (98). The RNA coactivator, steroid RNA activator (SRA), also interacts with AF-1 (99). Given that any of these molecules and complexes interact with both AF-1 and AF-2, it is likely that concurrent activation of AF-1 and AF-2 by their common and/or distinct binding partners may be necessary for optimal activation of GRa-mediated transcriptional activity (100).

 

Several coactivators supporting the particular actions of glucocorticoids have been identified for GRa. The PPARg coactivator-1a (PGC1a) is a ~800 amino acid single polypeptide molecule originally identified as a cofactor physically interacting with PPARg in the yeast two-hybrid screening using a brown adipocyte cDNA library (101). PGC1a has an essential role in thermogeneration and energy metabolism by controlling mitochondrial biogenesis (101). It also regulates gluconeogenesis and cholesterol metabolism, as well as blood pressure and muscle fiber determination through physical interaction with various NRs, transcriptional factors and coactivators, such as PPARa, hepatocyte nuclear factor 4, CREB, nuclear respiratory factors, and p160 and p300/CBP coactivators (101). GRa also interacts physically with PGC1a through the latter’s LxxLL motif and this interaction is important for stimulation of gluconeogenesis through transcriptional stimulation of the 2 key genes respectively encoding the glucose-6-phosphatase (G6P) and the phosphoenolpyruvate carboxykinase (PEPCK) (101). It is known that longevity-associated histone deacetylase Sirt1 regulates PGC1a activity through its deacetylase activity-dependent or -independent manner (102,103). Sirt1 is shown to interact physically with GRa as well, and PGC1a and Sirt1 cooperatively enhance GR-induced transcriptional activity of glucocorticoid-responsive genes (104).

 

The CREB-regulated transcription coactivator 2 (CRTC2) is a coactivator previously known to be specific to CREB, and plays a central role in the glucagon-mediated activation of gluconeogenesis in the early phase of fasting (105). This coactivator functions also as a coactivator of GRa by physically interacting with its LBD outside of AF-2, and is required for glucocorticoid-mediated early phase gluconeogenesis by supporting the transcriptional activity of GRa on the G6P and PEPCK genes, while PGC1a cooperates with GRa for maintaining a late phase of fasting-induced gluconeogenesis (106).

 

Presence of numerous transcriptional cofactors that interact with GRa and influence its transcriptional activity indicate that they may have functional redundancy and/or activities specific to each of them, regulating particular sets of GRa-responsive genes. A study employing knockdown of GRalpha cofactors, such as CCAR1, CCAR2, CALCOCO1 or ZNF282, has addressed this important issue: it revealed that knockdown of any of these cofactor molecules resulted in specific impact on the expression of a particular set of glucocorticoid-responsive genes (107), suggesting that each cofactor molecule has distinct transcriptional regulatory activity on GRa, thus their expression profiles in tissues/organs potentially influence the transcriptional activity of GRa in respective tissues.

 

Emerging Concept on GRa-mediated Transcriptional Repression

 

GRa has long been believed to exert its transcriptional activity by binding to the classic GREs, which consists of inverted hexameric palindrome separated by 3 base pairs. However, Surjit, et al. identified unique DNA sequences also targeted by the GRa DBD, called “negative” GREs (nGREs), which play substantial roles in gene transrepression caused by GRa (108). The consensus sequence of nGREs is an inverted quadrimeric palindrome separated by 0-2 nucleotide pairs (CTCC(N)0–2GGAGA). In the structural study employing the prototype nGREs found in the thymic stromal lymphoprotein (TSLP) promoter as a model, 2 GRa molecules bound each palindrome as a monomer with different affinity in a head-to-tail fashion, in contrast to GRa-classic GREs where 2 receptors bind DNA in a head-to-head fashion (109) (Figure 11). nGREs are ubiquitously present in the genes repressed by glucocorticoids throughout several animal species, facilitating access of the silencing mediator for retinoid and thyroid hormone receptors (SMRT)/nuclear receptor corepressor (NCoR)-repressing complexes on the agonist-associated GRa bound on these sequences. This is a new concept, indicating that direct binding of GRa through its DBD to DNA sequences distinct from those of the classic GREs mediates glucocorticoid-induced transcriptional repression. However, a genome-wide study revealed that classic GREs and the “new” nGREs both contribute to transactivation and transrepression of glucocorticoid-responsive genes, suggesting that GRa-targeting DNA sequences per se are insufficient to confer direction of transcriptional regulation, but epigenetic factors and subsequent chromatin modification may play critical roles (110).

Figure 11. GR binds nGREs as a monomer. GR binds nGREs as a monomer at each of its half site (A) in contrast to its binding as a homo-dimer to classic GREs (B). nGREs of the mouse TSLP gene is used as an example. Images are from the PDB Website (www.rcsb.org). Image data for GR interaction with nGREs and classic GREs are DOI: 10.2210/pdb4hn5/pdb and DOI: 10.2210/pdb3g9m/pdb, respectively.

Interaction of GRa with Transcription Factors

 

Glucocorticoids exert their diverse effects through its single receptor protein module, the GRa. In addition to direct regulation of gene expression through GRa/DNA interaction, these hormones affect other signal transduction cascades through mutual protein-protein interactions between specific transcription factors and GRa, influencing the former’s ability to stimulate or inhibit the transcription rates of the respective target genes.

 

The protein-protein interaction of GRa with other transcription factors may take place on the promoters that do not contain GREs (tethering mechanism), as well as on those having both GRE(s) and responsive element(s) of the transcription factors that interact with GRa (“composite promoters”) (111) (Figure 12). Repression of the transactivation activity of other transcription factors through protein-protein interaction may be particularly important in suppression of immune function and inflammation by glucocorticoids (112,113). Substantial part of the effects of glucocorticoids on the immune system may be explained by the interaction between GRa and NF-kB, AP-1 and probably STATs (114-116). It was also reported that GRa directly interacts with the transcription factors “T-box expressed in T-cells” (T-bet) and GATA-3, which play key roles respectively in the differentiation of T helper-1 and T helper-2 lymphocytes (117,118). GRa also influences indirectly the actions of the interferon regulatory factor-3 (IRF-3) through the p160 nuclear receptor GRIP1, by competing with this factor for binding to the coactivator (119). These transcription factors are important for the regulation of immune function and the above interactions may explain some GR actions on the immune system. The following subsections will discuss GRa-interacting transcription factors and their effects on GRa-induced transcriptional activity.

Figure 12. Three different modes of transcriptional regulation of the glucocorticoid-responsive promoters by GR. GR may interact with other transcription factors directly or indirectly. Protein(s) or protein complex(es) may intermediate their interaction in the latter case. GREs: glucocorticoid responsive elements; TF: transcription factor; TFREs: transcription factor responsive elements

Nuclear Factor-kB (NF-kB)

 

NF-kB is one of the most important transcription factors that regulate inflammation and immune function. NF-kB is stimulated by many inflammatory signals and cytokines (115,120). It is a dimer of various members of the NF-kB/Rel family, including p50 (and its precursor p105), p52 (and its precursor p100), c-Rel, RelA and RelB in mammalian organisms. The heterodimer p65/p50 is a major and the most abundant form of NF-kB. In its inactive form, NF-kB creates a trimer with an additional regulatory protein, IkB in the cytoplasm. A variety of extracellular signals, such as bacterial and viral products (like lipopolysaccharide (LPS)) and several proinflammatory cytokines, induces phosphorylation of IkB by activating a cascade of kinases. The phosphorylated IkB then dissociates from NF-kB and is catabolized, while the liberated NF-kB enters into the nucleus where it binds to the kB-responsive elements in the promoter regions of its responsive genes. Ligand-activated GRa directly binds NF-kB p65 at its Rel homology domain through its DBD and suppresses the transcriptional activity of NF-kB, while NF-kB suppresses GRa-induced transactivation through GREs. Interaction with GRa inhibits binding of NF-kB to its responsive elements or neutralizes its ability to transmit an effective signal (121-124). The LBD of GRa is necessary for this suppressive action (125). GRa also suppresses NF-kB-induced transactivation by an additional mechanism, in which the GRa tethered to the kB-responsive promoters attracts histone deacetylases (HDACs) and/or modulates the phosphorylation of the RNA polymerase II C-terminal tail (126,127). In addition, ligand-activated GRa increases the synthesis of IkB by stimulating its promoter activity through classic GREs, thus segregating active NF-kB from the nucleus by forming inactive heterocomplexes with IkB in the cytoplasm (128). A study further indicated that attraction of the p160 coactivator GRIP1 together with GRa to NF-kB is required for glucocorticoid-induced repression of NF-kB-mediated cytokine gene expression in mouse primary macrophages (129).

 

Activator Protein-1 (AP-1)

 

AP-1 is a transcription factor, which regulates diverse gene expression involved in cell proliferation and differentiation (114,130,131). It acts as a dimer of the bZip protein family members, in which c-Fos and c-Jun heterodimers are most abundant. AP-1 transduces biological activities of phorbol esters, growth factors and pro-inflammatory cytokines, such as IL-1 and tumor necrosis factor (TNF) a. These compounds/cytokines stimulate different members of the mitogen-activated protein kinase (MAPK) family, e.g., p38 MAPK, extracellular signal-regulated kinase (ERK) and Jun N-terminal kinase (JNK). Once these kinases are activated, they stimulate the synthesis of specific transcription factors involved in the induction of fos and jun gene transcription, as well as enhance the transcriptional activity of both pre-existing and newly synthesized c-Fos/c-Jun proteins. AP-1 and GRa mutually interact and repress each other’s transcriptional activity on their respective responsive promoters. The LBD and DBD of GRa and the leucine zipper domain of c-Jun are necessary for this interaction (29). Inhibition of the binding of AP-1 to DNA may be one of the underlying mechanisms of GRa-induced suppression of AP-1-mediated transcriptional activity. Furthermore, GRa competes with AP-1 for the p300/CBP coactivator, which has a limited reserve, therefore, AP-1 may not have access to adequate amounts of this coactivator to exert its transcriptional activity fully (132).

 

cAMP Response Element-binding Protein (CREB)

 

CREB functions downstream of many hormones and bioactive molecules, which bind to the cell surface-located G-protein-coupled receptors that employ cAMP as their second messenger. CREB is also a member of the bZip transcription factors (133). It forms homo- and hetero-dimers with other proteins of the same family and binds to the cAMP-responsive element (CRE). Stimulation of the above receptors induces the accumulation of cAMP that leads to activation of the cAMP-dependent protein kinase A (PKA). This kinase then phosphorylates CREB at a specific serine residue and promotes recruitment of the transcriptional co-integrator CBP and its specific coactivator CRTC2 to stimulate the transcription of cAMP-responsive genes. GRa and CREB mutually repress the transcription from their simple responsive promoters (134,135). Although direct association of GRa and CREB has been reported in vitro, their physical interaction is still unclear (134,136). CRTC2 might act as a bridging factor between CREB and GRa, particularly in their synergistic activation of the composite promoters, such as that of G6P, PEPCK and the somatostatin gene, which contain both GREs and CRE sequences (106,136,137) (see also Section 5. ACTIONS OF GR, B. Mechanisms of GRalpha-mediated Activation of Transcription).

 

Transforming Growth Factor (TGF) b Downstream Smad6

 

Members of the Smad family of proteins transduce signals of transforming growth factor (TGF) b superfamily members, such as TGFb, activin and bone morphogenetic proteins (BMPs), by associating with the cytoplasmic side of the type I cell surface receptors of these hormones (138). Nine distinct vertebrate Smad family members have been identified, which are classified into three groups: receptor-regulated Smads (R-Smads), such as Smad1, 2, 3, 5 and 8, a common-partner Smad (Co-Smad), Smad4, and inhibitory Smads (I-Smads) like Smad6 and Smad7 (138).

 

Upon binding of TGFb, activin or BMP to their receptors, cytoplasmic R-Smads are phosphorylated by the receptor kinases, translocate into the nucleus and stimulate the transcriptional activity of TFGb-, activin- or BMP-responsive genes by binding to their response elements located in their promoter region as a hetero-trimer with Co-Smad (138). I-Smads, such as Smad6 and Smad7, act as inhibitory molecules in the TGFb family signaling, by forming stable associations with activated type I receptors, which prevent the phosphorylation of R-Smads (138). Smad6 also competes with Smad4 in the heteromeric complex formation induced by activated Smad1 (139). In addition, I-Smads directly suppress the transcriptional activity of TGFb family signaling by binding to promoter DNA and attracting HDACs and/or the C-terminal binding protein (CtBP) (140-142). Since I-Smads are produced in response to activation of the TGFb family signaling (143), they literally function in the negative feedback regulation of the Smad signaling pathways. Smad6 preferably inhibits BMP signaling, while Smad7 is a more general inhibitor, repressing TGFb and activin signaling, in addition to that of BMP (144).

We found that Smad6 physically interacts with the N-terminal domain of the GRa through its Mad-homology 2 domain and suppresses GRa-mediated transcriptional activity in vitro (145). Adenovirus-mediated Smad6 overexpression also inhibits glucocorticoid action in rat liver in vivo, preventing dexamethasone-induced elevation of blood glucose levels and hepatic mRNA expression of PEPCK, a well-known rate-limiting enzyme of hepatic gluconeogenesis (145). Smad6 suppresses GRa-induced transactivation by attracting HDAC3 to DNA-bound GRa and by antagonizing acetylation of the histones H3 and H4 induced by p160 HAT coactivators (145). Thus, Smad6 regulates glucocorticoid actions as a corepressor of GRa. It appears that the anti-glucocorticoid actions of Smad6 may contribute to the neuroprotective, anti-catabolic and pro-wound healing properties of the TGFb family of proteins through cross-talk between TGFb family members and glucocorticoids (145).

 

C2H2-type Zinc Finger Proteins (ZNFs)

 

C2H2-type ZNFs constitute the largest class of putative human transcription factors consisting of over 700 member proteins (146,147). In addition to C2H2-type zinc fingers (ZFs), these proteins harbor several structural modules, such as the Broad-Complex, Tramtrack, and Bric-a-brac (BTB)/Poxvirus and zinc finger (POZ), Krüppel-associated box (KRAB) and SCAN domains (147). These modules are usually located in the N-terminal portion, and function as platforms for protein-protein interactions, whereas ZFs are positioned in the C-terminal area and function mainly as a DNA-binding domain (147). The BTB/POZ and KRAB domains have transcriptional regulatory activity (mostly repressive), whereas the SCAN domain does not (148). Among human C2H2-type ZNFs, about 7% have a BTB/POZ domain (BTB/POZ-ZNFs), 43% harbor a KRAB domain (KRAB-ZNFs) and 7% contain a SCAN domain (SCAN-ZNFs) (146). Sixty-seven % of the human C2H2-type ZNFs have only ZFs without any of these domains (thus, they are “poly-ZNFs”) (146). Some poly-ZNFs, such as members of the specificity protein (SP)/Krüppel-like factor (KLF) family transcription factors (e.g., SP1, KLF4 and KLF11) cooperate with GRa for regulating the transcriptional activity of specific glucocorticoid-responsive genes in distinct biological pathways, such as monoamine oxidase A expression in CNS and glucocorticoid-mediated skin barrier formation in prenatal fetus (147). Furthermore, GRa stimulates the transcriptional activity of the KLF9 gene through the GREs located in the promoter region of this gene, and expressed KLF9 plays important roles in glucocorticoid-mediated survival of the newly differentiated hippocampal granule neurons (147). One poly-ZNF called CCCTC-binding factor (CTCF) is an architectural protein playing a major role in the formation of chromatin looping, which governs enhancer-gene promoter communication, and ultimately contributes to the tissue/phase-specific expression of glucocorticoid-responsive genes (149). Although direct evidence of its interaction to GRa is still missing, CTCF interacts with ERa and the thyroid hormone receptors (TRs) and regulates their transcriptional activity (150,151), thus it is highly possible that this molecule also plays roles in the regulation of GRa transcriptional activity. One KRAB-ZNF, the zinc finger protein 764 (ZNF764), which composes of a N-terminally located KRAB domain and seven C2H2-type ZF motifs in the C-terminal area, was identified as a coactivator of several SRs including GRa, possibly cooperating with other NR coactivators (152). Indeed, haploinsufficiency of the ZNF764 gene by microdeletion was associated with partial tissue insensitivity to glucocorticoids and developmental abnormalities of androgen-dependent organs in an affected boy (152). In a genome-wide binding study using ChIP-sequencing, ZNF764- and GRa-binding sites are found in close proximity, indicating that ZNF764 modulates GRa transcriptional activity by incorporated in the transcriptional complex formed on DNA-bound GR (153).

 

Forkhead Transcription Factors

 

Forkhead transcription factors are characterized by their DNA-binding domain called “Forkhead Box”, and consist of over 100 family members classified from FOXA to FOXR (154). Among them, FOXO subgroup proteins (FOXO1, 3, 4 and 6 in humans) mediate biological actions of the insulin/PI3K/Akt signaling pathway through phosphorylation of several serine/threonine residues of this subgroup proteins, acting on cell proliferation, cell cycle regulation, oxidative stress, DNA repair, energy and glucose metabolism (154). Some of forkhead transcription factors (e.g., FOXA1) can act as pioneer factors for other transcription factors including NRs, by opening DNA-binding sites of the latter molecules on the chromatin (see also Section 5. ACTIONS OF GR, E. New Findings on Genome-wide Transcriptional Regulation by GRa) (155). FOXA3 acts as a pioneer factor for GRa by facilitating the latter binding to DNA possibly through modulation of the chromatin accessibility and is required for glucocorticoid-mediated fat accumulation in adipose tissues (156).

 

Other Transcription Factors

 

Functional interaction of GRa has also been reported with other transcription factors, including the chicken ovalbumin promoter-upstream transcription factor II (COUP-TFII), HNF-6, NR4A orphan receptors (neuron-derived orphan receptor-1 (NOR-1), nuclear receptor-related 1 (NURR1) and Nur77), liver X receptors (LXRs), farnesoid X receptor (FXR), p53, T-bet, GATA-1 and -3, Oct-1 and -2, NF-1 and C/EBPb. COUP-TFII is an orphan nuclear receptor, which plays important roles in neurogenesis as well as glucose, lipid and xenobiotic metabolism. This NR physically interacts with the hinge region of GRa and suppresses GRa-induced transcriptional activity by attracting the corepressor SMRT (157). Mutual protein-protein interaction of GRa and COUP-TFII was necessary for glucocorticoid-induced enhancement of the promoter activity and the endogenous mRNA expression of the COUP-TFII-responsive PEPCK, suggesting that COUP-TFII may participate in some of the metabolic effects of glucocorticoids through direct interactions with GRa (157). The hepatocyte nuclear factor 6 (HNF6) is a transcription factor that consists of 2 different DNA binding domains (CUT and homeobox) and plays an important role in the hepatic metabolism of glucose. It represses GRa-induced transactivation by directly binding to GRa DBD (158). Interaction of another orphan nuclear receptor Nur77 and GRa is critical for the regulation of proopiomelanocortin (POMC) gene expression (159). LXRs consist of 2 isoforms LXRa and LXRb, and play a central role in the regulation of cholesterol/fatty acid metabolism by binding to their metabolites as a ligand, while FXR acts on bile acid metabolism. GRa and these NRs modulate each other’s transcriptional activity by communicating through direct protein-protein interaction (160-162). p53, a transcription factor functioning as a tumor suppressor, physically interacts with GRa in the cytoplasm along with an additional protein Hdm2. GRa and p53 mutually repress each other’s transcriptional activity by increasing their degradation rates (163,164). GRa also interacts with Oct-1 and -2 on the mouse mammary tumor virus (MMTV) promoter and the gonadotropin-releasing hormone promoter (165-169). The POU domain of Oct-1 and the DBD of GRa interact with each other in vitro. NF-1, which also stimulates the MMTV promoter, interacts with GRa and cooperatively modulates the activity of this promoter (169,170). The transcriptional activity of GATA-1, a transcription factor that plays an essential role in the erythroid differentiation is repressed by GRa at the experimental cellular levels. NTD of GRa is necessary for the interaction with GATA-1 (171). The CAAT/Enhancer-binding Protein (C/EBP) is one of the bZip family transcription factors that have diverse effects on proliferation, development and differentiation of embryonic cells/fetus, and influence functions of the liver, adipose, immune and hematopoietic tissues in adults (172). C/EBPb, also known as the nuclear factor IL-6 (NF-IL6), synergistically stimulates transcription of GRa on the composite promoter that contains both C/EBPb- and GRa-binding sites (173). GRa, on the other hand, enhances C/EBPb activity on its simple responsive promoter (173,174). Direct in vitro binding of these proteins has been reported.

 

GENOME-WIDE TRANSCRIPTIONAL REGULATION BY GRa

 

Chromatin-based Regulation of GRa Transcriptional Activity

 

GRa regulates expression of glucocorticoid-responsive genes by influencing their transcriptional activity through direct or indirect interaction with their enhancer/promoter regions. In eukaryotic cells, DNA is packed into chromatin by associating with numerous nuclear proteins, such as histones and chromatin-modifying factors (175,176). Double-stranded DNA wraps by 1.67 turns around a histone octamer that consists of 2 copies of each core histones H2A, H2B, H3 and H4, and forms the smallest structural unit called “nucleosome”, which is further compacted into a higher order chromatin. Nucleosome-associated histones possess a highly flexible N-terminal tail whose chemical modifications, such as acetylation and methylation at specific lysine (K) residues, modulate accessibility of GRa to its target DNA sequences residing in chromatin. Chromatin is further packed into the 3-dimensional structure called topologically associated domains (TADs) in which several protein-coding gene bodies, promoters and regulatory elements interact with each other through formation of chromatin looping, and their modes of interaction alter in different cellular circumstances. A poly-ZNF protein CTCF plays a central role in the formation of chromatin looping by cooperating with the cohesion protein complex and other accessory factors, including the transcription factor IIIC (TFIIIC), ZNF143, PR domain zinc finger protein 5 (PRDM5) and chromodomain helicase DNA-binding protein 8 (CHD8) (147,149) (Figure 13). In addition to CTCF, interaction of transcription factors, such as between GRa and NF-kB, influences formation of chromatin looping possibly through cooperation with CTCF (177). A study using a new technique called Hi-C (high throughput 3C) further revealed that even chromosomes are packed into the nucleus with some orders shared by many tissues/organs (178,179).

Figure 13. Organization of the topologically associated domain (TADs) and chromatin looping promoted by CTCF for differential expression of glucocorticoid-responsive genes. CTCF organizes 3-dimensional chromatin interaction for the formation of TADs and chromatin looping, in cooperating with the cohesion protein complex and other component proteins. Chromatin loop-forming activity of CTCF is essential for differential use of enhancers/promoters by GR-responding genes, and underlies organ/tissue-specific actions of glucocorticoids. Modified from (147).

In rat liver, more than 11,000 GR-binding sites (GBSs) are identified primarily at intergenic distal and intronic regions, but only ~10% of GBSs are located in the promoter area (~2.5 kbs from the transcription start site: TSS), consistent with the fact that distantly located enhancer regions can communicate with the gene promoter through gene looping (180,181). Interestingly, ~80-90% of GRa-accessible sites exists prior to glucocorticoid addition/GRa stimulation, while their distribution is highly tissue-specific, indicating that local tissue factor(s) mainly determine(s) the sets of genes responsive to glucocorticoids by regulating chromatin accessibility (180). Indeed, some transcription factors, such as C/EBPb, AP-1 and FoxA1, have their binding sites close to GBSs (thus, composite sites) and act as tissue-specific priming factors (or pioneer factors) for the access of GRa to GBSs, respectively in murine mammary epithelial cells, rat liver and human prostate cancer cells (181-183). These pre-existing GBSs are enriched with CpG islands and are generally demethylated, further suggesting that DNA methylation also contributes negatively to the opening of GBSs (184). However, a study revealed that GRa can act as a pioneer factor for several other transcription factors previously reported to be pioneer factors for GRa (185). This report indicates that GRa can function both as a pioneer and a dependent factor based on the composition of the binding sites in the regulatory elements and/or local chromatin conditions.

 

Influence of Gene Variation (Single Nucleotide Polymorphisms: SNPs) to Tissue Glucocorticoid Responsiveness

 

Humans demonstrate variation in their responsiveness to glucocorticoids (sensitivity to glucocorticoids), which then influences the development of numerous disorders, such as hypertension, obesity, diabetes mellitus, osteoporosis and ischemic heart diseases, asthma and acute lymphoblastic leukemias. However, genetic background(s) that explain(s) such difference in glucocorticoid responsiveness among human subjects is(are) not known. To access this problem, variation of the single nucleotide polymorphisms (SNPs) in over 100 individuals was compared with glucocorticoid-induced mRNA expression profiles in subjects’ EBV-transformed lymphocytes and their secretion of some cytokines (186). The results revealed that the SNPs located close (~100 kbps) to the glucocorticoid-responsive genes were associated with variation in glucocorticoid responsiveness of their own mRNA expression, while SNPs located in the transcription factors known to regulate GRa transcriptional activity did not show statistically significant differences. These results suggest that the genetic areas close to glucocorticoid-responsive genes, possibly containing enhancer regions and/or other gene regulatory sequences, influence primarily the responsiveness of mRNA expression of their associated genes to glucocorticoids, rather than those found in the protein-coding sequence of GRa, its partner molecules or glucocorticoid-responsive genes themselves. The above results on the genetic factors determining individual glucocorticoid sensitivity are consistent with recent findings obtained in the genome-wide association studies (GWAS) in which ~70% of SNPs associated with susceptibility to common disorders and traits (thus individual variation) are found in the gene regulatory regions but not in the protein-coding sequences (187).

 

Tissue/Organ-specific Actions of GRa Revealed by GR Gene Knockout/Knockin Studies

 

Modifications of gene expression with gene knockout (deletion of existing genes) are tremendously helpful for understanding physiologic actions of endogenous GRa in glucocorticoid-target tissues. Whole body GR gene knockout revealed that GR deficient pups die just after birth due to respiratory insufficiency caused by lack of lung surfactant, indicating that GR action is essential for survival (188). By using the same mice, GR is also shown to be required for gluconeogenesis upon fasting and erythropoiesis under stress (such as erythrolysis or hypoxia) (189,190). Mice harboring forebrain-specific GR gene knockout developed a phenotype mimicking major depressive disorder in humans, including hyperactivity and impaired negative regulation of the HPA axis, indicating that alteration of GRa actions in the forebrain plays a causative role in the disease onset of major depressive disorder (191). Paraventricular nucleus (PVN) of the brain hypothalamus is the central component of the HPA axis (1), thus GR gene knockout mice in this brain region was developed and their HPA axis was evaluated. The results indicated that PVN GR is required for negative regulation of the HPA axis at a basal condition and under stress (192). GR gene knockout mice specific in the noradrenergic neurons, components of the neural circuit mediating the adaptive stress response together with the HPA axis, were also created (1,193). These mice demonstrated depressive- and anxiety-like behavior upon stress with specificity to duration and gender, indicating that GR in the noradrenergic neurons plays an important role in stress response and associated behavioral changes in addition to its actions in the HPA axis. In mice with cardiomyocyte/vascular smooth muscle cell-specific GR gene knockout, fetal heart function is impaired and causes generalized edema in embryonic day (E) 17.5. Histologically, disorganized myofibrils and cardiomyocytes are found in fetal heart, while altered expression of the genes involved in contractile function, calcium handling and energy metabolism are observed. These results suggest that GRa actions in the cardiomyocytes and vascular smooth muscle cells are important for proper functioning and maturation of the fetal heart (194). GR gene knockout specific in the vascular endothelial cells revealed that GRa in this tissue mediates a tonic effect of glucocorticoids on blood pressure, possibly by supporting autocrine or paracrine activity of this tissue for releasing vasoactive mediators in response to glucocorticoid treatment (195). GRa in this tissue is also required for the protective response against sepsis by conferring glucocorticoid-mediated suppression of cytokine and nitric oxide production (196). Challenge of vascular endothelial cell-specific GR knockout mice with LPS also revealed that GRa in this tissue is required for survival of animals against this compound by appropriately suppressing circulating levels of inflammatory cytokines (TNFa and IL-6) and release of the nitric oxide (197), indicating the important actions of the vascular endothelial cell-residing GRa for controlling otherwise overshooting inflammatory response. T-lymphocyte-specific GR gene knockout mice revealed that GRa-mediated immune suppression mainly through Th1 lymphocytes is also necessary for survival of the mice against Toxoplasma gondii infection (198). Uterine-specific GR knockout mice generated with the Cre-recombinase expressed under the PR gene promoter revealed that uterine GRa is required to establish the local cellular environment necessary for maintaining normal uterine biology and fertility (199). The GR gene knockout specific to testicular Sertoli cell identified that GRa in these cells is required to maintain normal testicular Sertoli/germ cell numbers and circulating gonadotropin levels, as well as optimal Leydig cell maturation and steroidogenesis, thus GRa in these cells is required for supporting normal male reproduction (200).

 

By using a knockin procedure (replacement of wild type genes with their mutants), physiologic importance of the specific GRa functions associated with introduced mutations was evaluated. For example, knockin of the mutant GRa defective in binding to classic GREs (GRdim harboring A458T replacement, which is inactive in transactivation of glucocorticoid-responsive genes harboring GREs, but active in transrepression through protein-protein interaction with other transcription factors), revealed that transactivational activity of GRa is not essential for survival (112). Indeed, mice harboring GRdim demonstrated partially active HPA axis, full activity in glucocorticoid-mediated development of adrenal medulla, and defective glucocorticoid-mediated thymocyte apoptosis. However, the GRdim mutant receptor was subsequently shown to bind GREs of the N-methyltransferase (PNMT) gene, which is a rate-limiting enzyme for the production of catecholamines in the adrenal medulla, and to activate strongly the expression of this gene (201). Thus, the GRdim mutation cannot completely abolish transactivational activity of GRa, further suggesting that this activity of GRa may be required for survival. In addition, the effect of this mutant receptor on recently identified nGREs is not known, making the original conclusion elusive.

 

FACTORS THAT MODULATE GR ACTIONS

 

New Ligands with Specific Activities

 

Glucocorticoids have two major activities on the transcription of glucocorticoid-responsive genes, namely transactivation and transrepression (202). The former activity is mainly mediated by binding of GRa to its DNA responsive sequences in the promoter region of glucocorticoid-responsive genes and stimulating the transcription of downstream protein-coding sequences. Mechanisms underlying the latter activity are more complex, mostly mediated by suppression of other transcription factor activities by GRa. At pharmacologic levels, the transactivation activity is well correlated with side effects of glucocorticoids, such as glucose intolerance and overt diabetes mellitus, central obesity, osteoporosis and muscle wasting (202). On the other hand, the transrepressive activity of glucocorticoids is associated mostly with their beneficial therapeutic effects, such as suppression of the inflammation and immune activity, and induction of apoptosis of several neoplastic cells/tissues. Thus, significant efforts have been put to produce dissociated glucocorticoids with transrepression but no transactivation activity (202).

 

RU24858, RU40066 and RU24782 were the first steroids reported to have such selectivity, having an efficient inhibitory effect on AP-1- and NF-kB-mediated gene induction with reduced transactivation activity in vitro (203). However, they did not have any therapeutic advantage when they were used in vivo. Compound Abbott-Ligand (AL)-438, a derivative of a synthetic progestin scaffold, binds GRa with similar affinity to that of prednisolone and shows the activity equivalent to prednisolone in suppressing paw-edema in a rat experimental model (204). AL-438 does not increase circulating glucose levels and bone absorption in contrast to prednisolone, indicating that this compound is a promising selective glucocorticoid. ZK216348, the (+)-enanitomer of the racemic compound ZK209614, binds GRa and demonstrates anti-inflammatory activity comparable to that of prednisolone under both systemic and topical applications with much less unwanted effects on blood glucose and skin atrophy (205). This compound, however, binds PR and AR in addition to GRa, and does not show clear selectivity between transactivation and transrepression in vitro. C108297 functions as a GRa modulator through induction of unique interaction profiles of GRa to some splice variants of the p160 coactivator SRC1. This compound potently enhances GR-mediated memory consolidation, partially suppresses hypothalamic expression of the corticotropin-releasing hormone (CRH), and antagonizes to GR-mediated inhibition of hippocampal neurogenesis (206). Cortivazol, a pyrazolosteroid, induces nuclear translocation of GRa and stimulates GRa-induced transcriptional activity (207). Another compound, AL082D06 (D06), the tri-aryl methane, specifically binds GRa with a nano-molar affinity and acts as an antagonist for GRa but not for other SRs, in contrast to RU 486 (208). CORT-108297 acts also as a competitive GRa antagonist with high affinity to GRa (Ki 0.9 nM), but almost 1000-fold lower affinity to other SRs, PR, ER, AR and MR (209,210).

 

Two new non-steroidal GRa ligands, GSK47867A and GSK47869A, act as potent agonists with prolonged effects (211). These compounds bind the ligand-binding pocket of GRa with high affinity and induce both transactivational and transrepressional activities at concentrations ~10-50 times less than those of dexamethasone. Interestingly, GSK47867A and GSK47869A induce very slow GRa nuclear translocation and prolonged nuclear retention that leads to delayed but prolonged activation of the receptor. In computer-based structural simulation, these compounds induce unique GRa LBD conformation at its hsp90-binding site, which may underlie their extended GRa activation by causing defective interaction to hsp90 and altered intracellular circulation of GRa. By employing high throughput screening of 3.87 million compounds with the GR fluorescence polarization binding assay, heterobiaryl sulfonamide 2 was recently identified as a potent non-steroidal GR antagonist (212). Non-steroidal compounds mapracorat (also known as BOL-303242 and ZK245186) and the plant origin ginsenide Rg1 function as selective agonists with strong anti-inflammatory effects and a better side effect profile (213,214).

 

Compound A (CpdA), a stable analogue of the hydroxyl phenyl aziridine precursor found in the Namibian shrub Salsola tuberculatiformis Botschantzev, exerts anti-inflammatory activity by down-regulating TNFa-induced pro-inflammatory gene expression through inhibition of the negative effects of GRa on NF-kB, but demonstrates virtually no stimulatory activity on GRa-induced transactivation (215). This compound also suppresses similarly to dexamethasone the transcriptional activity of the T-bet transcription factor, a master regulator of Th1-mediated immune response, and reduces production of the Th1 cytokine interferon g from murine primary T-cells (216). By sparing AP-1-induced transcriptional activity and subsequent activation of the JNK/MAPK signaling pathway, CpdA does not influence epithelial cell restitution, an indicator of wound healing, in contrast to regular glucocorticoids (217,218). Thus, CpdA appears to be a dissociated compound of plant origin retaining the beneficial anti-inflammatory effect of glucocorticoids, being in part devoid of some of the known side effects of these compounds. CpdA also preserves the anti-cancer effect of glucocorticoids in human T-, B- and multiple myeloma cells, and cooperates with the anti-leukemic proteasome inhibitor Brtezomib in suppressing growth and survival of these cells (219). This compound is also beneficial for the treatment of bladder cancer by suppressing cell growth by promoting transrepressive actions of GRa and partially by acting as an AR antagonist (220). CpdA does not allow GRa to bind single GRE (half-site) sites in contrast to glucocorticoids, and this activity of CpdA is beneficial for its use in the treatment of triple-negative breast cancer, as single GRE-mediated gene regulation by glucocorticoids is associated with development of chemotherapy resistance (221).

 

Industrial chemicals are known to influence actions of several SRs, and are major threats for the life of living organisms including humans by interfering with the physiological actions of these receptors (222,223). Recent screening of these compounds using MDA-kb2 human breast cancer cells identified bisphenol Z and its analog bis[4-(2-hydroxyethoxy(phenyl)sulfone (BHEPS) as GR agonists, binding to the ligand-binding pocket of GRa and by shifting the helix-12 to the antagonist conformation in the structural simulation (224). Phthalates, ubiquitous environmental pollutants known for their adverse effects on health, bind GRa and other ketosteroid receptors, such as AR and PR, with high binding potencies comparable to natural ligands, suggesting that they may alter transcriptional activities of these receptors (225). Although underlying mechanism(s) are still unknown, chronic low doses of ingested petroleum can alter tissue expression levels of GRa in house sparrows, and modulates the glucocorticoid-signaling system and the HPA axis (226). Tolylfluanid, a commonly detected fungicide in Europe can induce biological changes that recapitulate many features of the human metabolic syndrome in part through modulating the GRa signaling pathway in male mice (227).

 

In addition to the above-explained compounds with agonistic or antagonistic actions on GRa, expanding numbers of new compounds with such activities have been identified, including: 2-aryl-3-methyloctahydroohenanthrene-2,3,7-trils (228), C118335 (229), 6-(3,5-dimethylisoxazol-4-yl)-2,2,4,4-tetramethyl-2,3,4,7,8,9-hexahydro-1H-cyclopenta[h]quinolin-3-one 3d (QCA-1093) (230), several compounds containing “diazaindole” moieties (231), heterocyclic GR modulators with a 2,2-dimethyl-3-phenyl-N-(thiazol or thiadiazol-2-yl) propanamide core (232), LLY-2707 (233), trierpenes (alisol M 23-acetate and alisol A 23-acetate) (234), GSK866 analogs UAMC-1217 and UAMC-1218 (235), AZD9567 (236), 1,3-benzothiazole analogs (237), 20(R, S)-protopanaxadiol and 20(R, S)-protopanaxatriol (238) and β-Sitosterol (239).

 

EPIGENETIC MODULATION OF GRa

 

Acetylation and CLOCK-mediated Counter Regulation of Target Tissue Glucocorticoid Action against Diurnally Fluctuating Circulating Glucocorticoids

 

All SRs including GRa are acetylated by several acetyltransferases, such as p300, p/CAF and Tip60, and have common acetylation sites in a consensus amino acid motif, KXKK, located in their hinge region (240-242). The human GRa is acetylated at lysine 494 and 495 within an acetylation motif also located in its hinge region, and was reported to be deacetylated by the HDAC2, an effect that is required for suppression of NF-kB-induced transcriptional activity by the activated GRa (243) (Figure 14). This finding indicates that acetylation of the GRa at these lysine residues attenuates the repressive effect of GRa on this transcription factor. In agreement with these results, we recently found that the Clock transcription factor acetylates GRa at the multiple lysine cluster that includes lysines 494 and 495, and represses GRa-induced transcription of several glucocorticoid-responsive genes (244). Clock, the “circadian locomotor output cycle kaput”, and its heterodimer partner “brain-muscle-arnt-like protein 1” (Bmal1), belong to the basic helix-loop-helix (bHLH)-PER-ARNT-SIM (PAS) superfamily of transcription factors, and play an essential role in the formation of the diurnal oscillation rhythms of the circadian CLOCK system (245). The CLOCK system, located in the suprachiasmatic nucleus (SCN) of the brain hypothalamus, acts as the “master” oscillator and generator of the body’s circadian rhythm, while the peripheral CLOCK system, virtually distributed in all organs and tissues including the CNS outside the SCN, acts generally as a “slave” CLOCK under the influence of the central SCN CLOCK. The Clock transcription factor shares high amino acid and structural similarity with the activator of thyroid receptor (ACTR), a member of the p160-type nuclear receptor coactivator family with inherent histone acetyltransferase activity, and thus, has such an enzymatic function (246).

Figure 14. Distribution of the amino acid residues of the human GR susceptible to acetylation, phosphorylation, ubiquitination or SUMOylation. Human GR has 4 acetylation sites (lysines: K at amino acid position 480, 492, 494 and 495, shown with “A”), at least 5 phosphorylation sites (serines: S at amino acid position 45, 203, 211, 226 and 395, shown with “P”), 1 ubiqitination site (Lysine: K at amino acid position 419, shown with “U”) and 3 SUMOylation sites (Lysines: K at amino acid position 277, 293 and 703, shown with “S”).

Clock physically interacts with GRa LBD through its nuclear receptor-interacting domain (NRID) in its middle portion, and acetylates human GRa at amino acids 480, 492, 494 and 495. Acetylation of GRa attenuates binding of the receptor to GREs, and hence, represses GR-induced transactivation of the GRE-driven promoters (244) (Figure 15). Since the lysine residues acetylated by Clock are located in the C-terminal extension (CTE) that follows DBD and plays a role in DNA recognition by SRs (247), it is likely that acetylation of these residues reduces binding of GRa to GREs by altering the action of CTE. The part of the hinge region acetylated by Clock also overlaps with the nuclear localization signal (NL)-1 (50,244), thus it is also possible that acetylation of GRa alters nuclear translocation of this receptor. It is well known that the central master CLOCK located in SCN creates diurnal fluctuation of circulating cortisol, therefore peripheral CLOCK-mediated repression of GRa transcriptional activity in glucocorticoid target tissues functions as a local counter regulatory mechanism for oscillating circulating cortisol (248).

Figure 15. Clock/Bmal1 suppresses GR-induced transcriptional activity through acetylation. Clock physically interacts with GR LBD through its nuclear receptor-interacting domain and suppresses GR-induced transcriptional activity by acetylating with its intrinsic HAT activity a lysine cluster located in the hinge region of the GR (A) through which Clock reduces affinity of GR to its cognate DNA GREs (B). A: acetylation; Bmal1: brain-muscle-arnt-like protein 1; DBD: DNA-binding domain; GREs: glucocorticoid response elements; HR: hinge region; K: lysine residue; LBD: ligand-binding domain; NTD: N-terminal domain. From (244).

In addition to the above findings obtained in in vitro cellular systems, we examined the acetylation status of human GRa and the expression of Clock-related and glucocorticoid-responsive genes in vivo and ex vivo, using peripheral blood mononuclear cells (PBMCs) from healthy adult volunteers (249). The levels of acetylated GRa were higher in the morning and lower in the evening, mirroring the fluctuations of circulating cortisol in reverse phase. All known glucocorticoid-responsive genes tested responded as expected to hydrocortisone, however, some of these genes did not show the expected diurnal mRNA fluctuations in vivo. Instead, their mRNA oscillated in a Clock- and a GRa acetylation-dependent fashion in the absence of endogenous glucocorticoid ex vivo, indicating that circulating cortisol might prevent circadian GRa acetylation-dependent effects in some glucocorticoid-responsive genes in vivo. These findings indicate that peripheral CLOCK-mediated circadian acetylation of GRa functions as a target tissue- and gene-specific counter regulatory mechanism to the actions of diurnally fluctuating cortisol, effectively decreasing tissue sensitivity to glucocorticoids in the morning and increasing it at night (36). Indeed, in another study where we measured mRNA expression of ~190 GRa action-regulating and glucocorticoid-responsive genes in subcutaneous fat biopsies from 25 obese subjects, we found that the levels of evening cortisol were much more important than those in the morning to regulate mRNA expression of glucocorticoid-responsive genes in this human tissue (250). It appears that higher sensitivity of tissues to circulating glucocorticoids in the evening due to reduced GRa acetylation by CLOCK underlies stronger impact of evening serum cortisol levels to glucocorticoid-regulated gene expression compared to morning levels.

 

The circadian CLOCK system and the HPA axis regulate each other’s activity through multilevel interactions in order to ultimately coordinate homeostasis against the day/night change and various unforeseen random internal and external stressors (251,252). For example, one CLOCK transcription factor Cry2 interacts with GRa and represses its transcriptional activity (253). Furthermore, GRa binds GREs located in the promoter region of the Per1, Per2 and other CLOCK components and stimulate their expression, an effect that contributes to resetting of the circadian rhythms by glucocorticoids (254,255). The peripheral CLOCK system residing in the adrenal glands contributes to the creation of circadian glucocorticoid secretion from this organ in addition to diurnally secreted ACTH from the pituitary gland (256). An important study further revealed new local factors, which also regulate circadian production of glucocorticoids in the adrenal glands: the intermediate opioid peptides secreted from the adrenal cortex influence in a paracrine fashion the amplitude of the serum corticosterone oscillations in mice through the C-X-C motif chemokine receptor 7 (CXCR7), a b-arrestin-biased G-protein-coupled receptor expressed on the adrenocortical cells (257).

 

Based on the above-indicated multilevel interaction between the CLOCK system and the HPA axis, uncoupling of or dysfunction in either system alters internal homeostasis and causes pathologic changes virtually in all organs and tissues, including those responsible for intermediary metabolism and immunity (248,251,252). Disrupted coupling of cortisol secretion and target tissue sensitivity to glucocorticoids may account for (1) development of central obesity and the metabolic syndrome in chronically stressed individuals, whose HPA axis circadian rhythm is characterized by blunting of the evening decreases of circulating glucocorticoids, as a result of enhanced input of higher centers upon the hypothalamic PVN’s secretion of CRH and arginine vasopressin (AVP); and (2) increased cardiometabolic risk and increased mortality of night-shift workers or subjects exposed to frequent jet-lag because of traveling across time zones (248,258). In addition, given that tissue sensitivity to glucocorticoids is increased in the evening as mentioned above (thus, evening cortisol levels have stronger impact to gene expression than those in the morning), supplemental administration of high-dose glucocorticoids at night for the treatment of adrenal insufficiency or congenital adrenal hyperplasia may increase a possibility of glucocorticoid-related side effects. Furthermore, administration of glucocorticoids at a specific period of the circadian cycle might increase their pharmacological efficacy, while at the same time reducing their unwanted side effects, because CLOCK differentially regulates transactivational and transrepressive actions of glucocorticoids, which are respectively correlated with side-effects and beneficial anti-inflammatory activities of these compounds used at pharmacological concentrations (258).

 

Phosphorylation

 

GRa has several phosphorylation sites and all of them are located in the NTD (20,259) (Figure 14). Classically, GRa is phosphorylated after binding to its ligand and this may determine target promoter specificity, cofactor interaction, strength and duration of receptor signaling and receptor stability (259,260). There are several kinases that phosphorylate GRa in vitro and in vivo (261). Yeast cyclin-dependent kinase p34CDC28 phosphorylates rat GRa at serines 224 and 232, which are orthologous to serines 203 and 211 of the human GRa, with the resultant phosphorylation enhancing rat GRa transcriptional activity in yeast (262). These residues are also phosphorylated after binding of the GRa with agonists or antagonists and the phosphorylated receptor shows reduced translocation into the nucleus and/or altered subcellular localization in mammalian cells (259,263). The p38 MAPK phosphorylates serine 211 of the human GRa, enhances its transcriptional activity and mediates GRa-dependent apoptosis (264). p38 MAPK and JNK also phosphorylate serine 226 of the human GRa and suppress its transcriptional activity by enhancing nuclear export of the receptor (63). Modulation of the molecular interactions between GRa AF-1 and cofactors through phosphorylation of these serine resides underlies in part the transcriptional regulation of this receptor by these kinases, as these serines are located within the AF-1 domain (265). Threonine 171 of the rat GRa is also phosphorylated by p38 MAPK and glycogen synthase kinase-3 (GSK3): phosphorylated rat GRa demonstrates reduced transcriptional activity in yeast and human cells, however, the human GRa does not have a threonine residue equivalent to that of the rat GRa (266,267). On the other hand, one GSK3 family protein, GSK3b, phosphorylates human GRa at serine 404 and modulates hGRa transcriptional activity including its repressive effect on NF-kB (268).

 

Several serine/threonine phosphatases, such as the protein phosphatase 2A (PP2A) and protein phosphatase 5, dephosphorylate human GRa at serine 203, 211 and/or 226, possibly through their association with GRa LBD (269,270). Stimulation of A549 human respiratory epithelial cells with b2 adrenergic receptor agonists increases PP2A, which in turn increases glucocorticoid sensitivity by dephosphorylating GRa at serine 226 (271). However, PP2A also regulates indirectly GRa phosphorylation by increasing dephosphorylation of JNK and subsequent activation of this kinase, as JNK directly phosphorylates GRa (272).

 

The cyclin-dependent kinase 5 (CDK5) physically interacts with the human GRa through its activator component p35, phosphorylates GRa at multiple serines including those at 203 and 211, and modulates GRa-induced transcriptional activity by changing accumulation of transcriptional cofactors on GRE-bound GRa (273). CDK5 and p35 are expressed mainly in neuronal cells and play important roles in embryonic brain development. Aberrant activation of CDK5 in CNS also plays a significant role in the pathogenesis of neurodegenerative disorders, such as Alzheimer’s disease and amyotrophic lateral sclerosis (274). We reported that, in addition to GRa, CDK5 phosphorylates MR and strongly suppresses its transcriptional activity (275). In brain regions, such as hippocampus and amygdala, which do not express 11b-HSD2, MR functions as a physiologic receptor for circulating glucocorticoids, and activation/suppression of MR plays an important role in glucocorticoid-related memory deficits and alterations in mood and cognition (276). Indeed, MR mediates enhancement of neuronal excitability, stabilization of synaptic transmission, and stimulation of long-term potentiation (LTP) in CA1 hippocampal cells, while MR activation is protective to hippocampal granular cell neurons. Thus, it is possible that CDK5-mediated regulation of MR might underlie development of glucocorticoid-associated pathologic conditions, such as neurodegenerative disorders and mood disorders (277,278). We examined changes of the CDK5 activity in mice under stress, and found that acute and chronic stressful stimuli differentially regulate the kinase activity together with contemporaneous alteration of the GRa phosphorylation in a brain region-specific fashion, indicating that CDK5 and its regulatory effects on GRa is an integral component of the stress response and mood disorders (279).

 

We also found that adenosine 5’ monophosphate-activated protein kinase (AMPK), a central regulator of energy homeostasis that plays a major role in appetite modulation and energy expenditure, indirectly phosphorylates human GRa at serine 211 through activation of p38 MAPK (280). Through phosphorylation of GRa, AMPK regulates glucocorticoid actions on carbohydrate metabolism, modifying transcription of glucocorticoid-responsive genes in a tissue- and promoter-specific fashion. Indeed, activation of AMPK in rats reverses glucocorticoid-induced hepatic steatosis and suppresses glucocorticoid-mediated stimulation of glucose metabolism. These findings indicate that the AMPK-mediated energy control system modulates glucocorticoid action at target tissues, and activation of AMPK could be a promising target for developing pharmacologic interventions in metabolic disorders in which glucocorticoids play major pathogenetic roles.

 

The v-akt murine thymoma viral oncogene homolog 1 (AKT1) or protein kinase B, another serine-threonine kinase known to regulate cell proliferation and survival, and aberrantly activated in various malignancies including acute leukemia, also phosphorylates human GRa at serine 134, which is located in NTD of this receptor (281). This phosphorylation of GRa retains the receptor in the cytoplasm through which activated AKT1 develops glucocorticoid resistance in acute leukemic cells, a major determinant for the prognosis of leukemic patients (281). AKT1 cooperates with phospho-serine/threonine-binding proteins 14-3-3s for regulating the transcriptional activity of GRa with 2 distinct mechanisms, one through segregation of GRa in the cytoplasm upon phosphorylation of serine 134 by AKT1 and subsequent association of 14-3-3 to GRa, and the other through direct modulation of GRa transcriptional activity in the nucleus (65). For the latter, AKT1 and 14-3-3 are attracted to DNA-bound GRa, accompanied by AKT1-dependent p300 phosphorylation, histone 3 (H3) serine (S) 10 (H3S10) phosphorylation and H3K14 acetylation at the DNA site in which 14-3-3 acts as a molecular scaffold (65). The above findings suggest that specific inhibition of the AKT1/14-3-3 activity on the cytoplasmic retention of GR but sparing the activity inside the nucleus may be a promising target for the treatment of glucocorticoid resistance observed in acute leukemia. Furthermore, they may also provide an explanation to somewhat conflicting findings previously reported for the actions of 14-3-3s on GRa (64,268,281,282).

 

Ubiquitination

 

The ubiquitin/proteasome pathway plays important roles in transcriptional regulation promoted by numerous trans-acting molecules. NRs, including GRa, ERs, PR, TRs, RARs and PPARs, as well as other transcription factors, such as p53, cJun, cMyc and E2F-1, are ubiquitinated and subsequently degraded by the proteasome (57,283). The transcriptional intermediate molecules, such as NR coactivators, chromatin remodeling factors, and some chromatin components, such as histone H1 and high mobility group (HMG) proteins, are also ubiquitinated and lysed by the proteasome (57,283,284). Moreover, the proteasome interacts with the C-terminal tail of the RNA polymerase II and is directly associated with the promoter regions of several genes, influencing their transcriptional activities (285). Thus, ubiquitination and subsequent processing of these molecules by the proteasome appear to regulate the transcriptional activity of GRa, possibly by facilitating rapid turnover of promoter-attracted and -associated GRa, ultimately down-regulating the transcriptional activity of this receptor. Indeed, mouse GRa contains a PEST motif at amino acids 407-426 (399-419 in human GRa) through which the ubiquitin-conjugating enzyme E2 and the ubiquitin-ligase enzyme E3 recognize their substrates (286). The lysine residue of the mouse GRa located at amino acid 426 (419 in human GRa) appears to be ubiquitinated, as inhibition of ubiquitination by compound MG-132 enhances the transcriptional activity of wild type GRa, while the mutant receptor with lysine to alanine replacement at amino acid 426 demonstrates elevated transcriptional activity and is insensitive to MG-132 (286) (Figure 14). Ubiquitination of GRa also influences motility of the receptor inside the nucleus, which was evaluated with the fluorescence recovery after photobleaching (FRAP) technique, possibly by changing association of the receptor to the nuclear matrix through ubiquitination (58,287,288).

 

SUMOylation

 

GRa is also SUMOylated. SUMOylation is the reaction conjugating the small ubiquitin-related modifier (SUMO) peptide (~100 amino acid peptide with molecular mass of ~11 kDa) to substrate proteins and conducted by an enzymatic cascade similar to those of ubiquitination but specific to SUMOylation (289). The human GRa has three SUMOylation sites, at lysines 277, 293 and 703 (290) (Figure 14). The first 2 sites are located in the NTD and act as major SUMOylation sites, while the last site is positioned in the LBD. SUMOylation of the former 2 sites (K277 and K293) suppresses GRa-induced transcriptional activity of a promoter containing multiple GREs, possibly by influencing the synergistic effect of multiple GRs bound on this promoter (291-293). In contrast, SUMOylation of the 3rd site (K703) enhances GRa-induced transcriptional activity, which is further enhanced by RSUME (RWD-containing SUMOylation enhancer) by changing attraction of the GRIP1 coactivator (294).

 

The death domain-associated protein (DAXX), a protein mediating the Fas-induced apoptosis through interacting with the death domain of Fas, was postulated to mediate SUMOylation-induced repression of GRa transcriptional activity (295). Other molecules, such as HDACs and the protein inhibitors of activated STAT (PIAS) family, which interact with SUMOylated proteins including GRa (296,297), might also participate in SUMO-mediated repression of GRa transcriptional activity, as the DAXX effect appears to be cell type- and/or cellular context-specific (298). SUMOylation of GRa is necessary for GRa-induced transrepression through the nGREs (an inverted quadrimeric palindrome separated by 0-2 nucleotide, see Section 5. ACTIONS OF GR, C. Emerging Concept on GRa-mediated Transcriptional Repression) by facilitating the formation of a complex consisting of SUMOylated GRa, SMRT/NCoR1 and HDAC3 (299). It is known that phosphorylation of rat GR at amino acid position 246 (226 in the human GRa) by JNK facilitates SUMOylation of the receptor and regulates GRa-induced transcriptional activity in a target gene-specific fashion (291).

 

11b-Hydroxysteroid Dehydrogenases (11b-HSDs)

 

There are 2 types of 11b-hydroxysteroid dehydrogenases (11b-HSDs), type 1 and 2 (11b-HSD1 and 2). 11b-HSD1 catalyzes the conversion of the inactive cortisone to active cortisol, thus increases intracellular cortisol levels potentially contributing to tissue hypersensitivity to glucocorticoids. 11b-HSD1 is widely expressed, particularly in the liver, but also in the lung, adipose tissue, blood vessels, ovary and CNS (300). The transgenic mice over-expressing 11b-HSD1 in adipose tissues develop insulin-resistant diabetes mellitus, significant accumulation of visceral fat and hyperlipidemia, and increased systemic blood pressure, indicating that this enzyme may play a role in the development of visceral obesity-related metabolic syndrome by increasing availability of local cortisol in adipose tissues (301,302). 11b-HSD2, on the other hand, catalyzes the conversion of active cortisol into inactive cortisone, and is expressed in the classic mineralocorticoid-responsive tissues, such as kidney, colon and sweat glands (300). This enzyme enables these tissues to respond to the circulating mineralocorticoid aldosterone, protecting MR from binding to the excess amounts of circulating cortisol (300).

 

Chaperones and Co-chaperones

 

GRa forms a heterocomplex with several heat shock proteins (hsps), including hsp90, hsp70, hsp40 and hsp23 (69). These proteins bind many proteins and help their correct assembly and folding, therefore they are called as chaperones. In addition to hsps, there is an additional protein group called co-chaperones, such as Hop (hsp70-hsp90 organizing protein), SGTA (small glutamine-rich tetratricopeptide repeat-containing protein a), FKBP51 (FK506-binding protein 51) and FKBP52, which support folding function of hsps by forming a protein complex with the latter molecules (69). Hsps modulate the transcriptional activity of GRa by influencing maintenance, activation and intracellular circulation of this receptor (303). Specifically, hsp90, hsp70 and hsp40 organize proper folding of the GRa protein, and are required for the maintenance of its high affinity state against ligand where interaction of hsp90 to Hop as well as that between hsp70 and SGTA are required (304,305). Upon binding of the GRa to glucocorticoids, hsp90 helps the receptor to translocate close to the nuclear pore in the side of the cytoplasm by facilitating GRa’s association to microtubules through FKBP52. After GRa goes through the nuclear pore complex and enters into the nucleus, hsp90 regulates GRa-induced transactivation negatively, possibly by reducing the association of GRa to DNA GREs (306). However, there are conflicting reports indicating that hsp90 stabilizes the association of ligand-bound GRa to DNA and helps GRa stimulating the transcriptional activity of glucocorticoid-responsive genes (307). These chaperones also protect GRa from the degradation mediated by the ubiquitin-proteosomal pathway in the nucleus (308). Receptor-associating protein 46 (RAP46), another co-chaperone associated with several hsps, synergizes with hsp70 to regulate GRa transactivation negatively (309). Impact of co-chaperones on in vivo actions of GRa was evaluated in humans and mice. FKBP51 is a co-chaperon known as a negative regulator of GRa activity by reducing the latter’s affinity to glucocorticoids, and nucleotide variations in its encoding gene FKBP5 are associated with development of mood disorders and anxiety in humans possibly by skewing the GRa-signaling system (310,311). FKBP51 knockout mice demonstrate reduced basal activity of the HPA axis, a blunted response to acute stress and an enhanced recovery from this challenge (312).

 

Chemical Compounds

 

There are several chemical compounds that modulate GRa activity. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a wide-spread environmental contaminant that produces adverse biologic effects, such as carcinogenesis, reproductive toxicity, immune dysfunction, hepatotoxicity and teratogenesis, suppresses GRa-induced transactivation possibly by reducing the ligand-binding affinity of GRa (313-315). Geldanamycin, a benzoquinone ansamycin, which specifically binds hsp90 and disrupts its function, suppresses GRa-induced transactivation by inhibiting the translocation of GRa into the nucleus (308,316). GRa is also regulated by the cellular redox state. Thioredoxin, a compound accumulated during oxidative stress, enhances GRa transactivation, most likely due to functional replenishment of GRa (317). Ursodeoxycholic acid (UDCA), one of the hydrophilic bile acids, which acts as a bile secretagogue, cytoprotective agent and immunomodulator, and is used for the treatment of various liver diseases including primary biliary cirrhosis, induces translocation of GRa into the nucleus and causes GRa-mediated inhibition of NF-kB transactivation (318). Mizoribine (4-carbamyl-1-b-D-ribofurano-sylimidazolium-5-olate), an imidazole nucleotide with immunosuppressive activity binds to 14-3-3 and enhances 14-3-3/GRa interaction, which may further potentiate 14-3-3’s effect on GRa transactivation (319). For more details of the GRa/14-3-3 interaction, please see Section FACTORS THAT MODULATE GR ACTIONS, Epigenetic Modulation of GRa, b Phosphorylation.

 

NON-CODING RNAS

 

Human genome expresses numerous non-protein-coding RNAs in addition to protein-coding mRNAs. Indeed, over half of the genome sequence expresses RNAs in both directions either as single- or double-stranded RNAs (320,321). Classic examples of the former RNAs are ribosomal RNAs and transfer RNAs, while several distinct new members have been identified recently (322). Depending on their size, non-coding (nc) RNAs are empirically categorized as short (~200 bs) or long (>200 bs) ncRNAs. The former family includes micro (mi) RNAs, small interfering (si) RNAs, small nucleolar (sno) RNAs, piwi (pi) RNAs and transcription start site (TSS)-associated RNAs, while the latter consists of long intergenic (linc) RNAs, enhancer-associated (e) RNAs, exon-encoding long ncRNAs, circular (c) RNAs, promoter-associated RNAs and others. ncRNAs are also produced from protein-encoding mRNAs through nuclease digestion, such as 3’ UTR RNAs (323). Recently, some of these distinct classes of ncRNAs have been revealed to regulate mRNA/protein degradation and transcriptional activity of GRa and other NRs. In this subsection, new findings on miRNAs and long ncRNAs will be discussed.

 

Micro (mi) RNAs

 

miRNAs are single-stranded, ~22 b-long RNAs transcribed mainly by the RNA polymerase II either from their own genes or from the intronic sequence of the protein-coding/non-coding genes (324). Transcribed precursor miRNAs are processed by multiple reactions including digestion by the RNase III enzyme Dicer, and are liberated as mature forms into the cytoplasm. miRNAs are incorporated as binding modules for target mRNAs into the RNA-induced silencing complex (RISC), a multi-protein machinery containing Dicer, Argonaut (AGO), human immunodeficiency virus (HIV)-transactivating response RNA (TAR)-binding protein (TRBP) and the protein activator of the interferon-induced protein kinase (PACT). Binding of the RICS complex mainly to 3’UTR of the target mRNAs through the complemental 6-8 nucleotides of miRNAs leads to degradation of associated mRNAs or to inhibition of their translation to proteins. In addition to functioning inside the produced cells, miRNAs are secreted into extracellular space/circulation as components of the exosome, and act as “hormones” by influencing the functions of distant organs and tissues (325). This unique action of miRNA was confirmed in vivo using adipocyte-specific Dicer knockout mice supplemented with exosomes obtained from normal animals (326). Human genome contains over 1,000 miRNAs and some of them are known to regulate expression of the GRa protein, while glucocorticoids/GRa regulate expression of other miRNAs.

 

In a study exploring the miRNAs that mediate ACTH-dependent downregulation of GRa in mouse adrenal glands, 4 miRNAs, miR-96, -101a, -142-3p and -433 induced by ACTH injection, suppress GRa protein expression by ~40% (327). miR-142-3p reduces GRa expression by directly interacting with its 3’UTR region, and attenuates responsiveness to glucocorticoids in T-cell leukemia cells (328). miR-124a, -18, -18a and -124 also attenuate GRa protein expression and regulate GRa-induced transcriptional activity in various cells and tissues (329-331). miR-29a mitigates glucocorticoid-induced bone loss in part by reducing GRa expression (332). Glucocorticoids, on the other hand, modulate expression of some miRNAs, such as miR-449a, -98, and miR-155, which in turn mediate hormonal effects of these steroids (333,334). Systematic screening of glucocorticoid-responsive miRNAs in rat primary thymocytes identified over 200 miRNAs responsive to this hormone, and some validated miRNAs regulate cell death pathway (335). In myeloma cells, glucocorticoids induce miR-150-5p, which changes expression of the genes involved in cell death and cell proliferation pathways, thus this miRNA mediates in some part the therapeutic effects of glucocorticoids on multiple myeloma (336). miR-119a-5p is also glucocorticoid-responsive miRNA that mediates anti-proliferative effects of glucocorticoids on osteoblasts by affecting the WNT signaling pathway (337).

 

Long Non-coding (lnc) RNAs

 

Several lncRNAs regulate the transcriptional activity of GRa and/or other SRs. The steroid RNA coactivator (SRA) is a prototype lncRNA that regulates the transcriptional activity of several SRs (99). SRA was originally cloned in the yeast two-hybrid assay by using NTD of the PR as bait. It enhances ligand-induced transcriptional activity of AR, ER, GRa and PR. It is found in the complex containing the p160 coactivator SRC1, and regulates transcriptional activity in part by associating with the SRA stem-loop-interacting RNA binding-protein (SLIRP) and the RISC complex (99,338,339). Recently, SRA was shown to function also as a repressor of transcription, acting as a scaffold for a repressor complex (340).

 

The growth arrest-specific 5 (Gas5), which is a multi-exon-containing ncRNA with a poly-A tail, is accumulated in cells whose growth is arrested due to lack of nutrients or growth factors (341). Gas5 functions as a repressor of the GRa and some other SRs (342). Gas5 sensitizes cells to apoptosis by suppressing glucocorticoid-mediated induction of several responsive genes, including those encoding the cellular inhibitor of apoptosis 2 and the serum/glucocorticoid-responsive kinase. Gas5 binds GRa DBD and acts as a decoy “GRE”, thus, it competes with DNA GREs for binding to GRa (Figure 16). These findings indicate that Gas5 is a ribo-repressor of the GRa, influencing cell survival and metabolic activities during starvation by modulating the transcriptional activity of GRa. Accumulation of Gas5 upon growth arrest or starvation was previously demonstrated in a cellular context, but a study revealed that fasting of mice also accumulates Gas5 in their metabolic organs, such as liver and adipose tissues, through modulation of the mammalian target of rapamycin (mTOR) signaling pathway, but not in the brain and immune organs including thymus and spleen (343). Since basal expression levels of Gas5 in the immune organs are much higher than those of the metabolic organs, Gas5 may have a regulatory activity on GRa in the immune system independent to the nutrient/energy availability, as evidenced by the fact that Gas5 is differentially expressed in blood leukocytes of the patients with autoimmune, inflammatory or infectious diseases (343). Moreover, Gas5 has been shown to be implicated in glucocorticoid response in children with inflammatory bowel disease (344), in multiple sclerosis (345-347), in human beta cell dysfunction (348), as well as in hematologic malignancies (349,350). Similar to Gas5, PRNCR1 (also known as PCAT8) and PCGEM1 bind AR DBD and enhance the transcriptional activity of this receptor (351). These lncRNAs are highly expressed in the prostate gland, and play a role in the androgen-dependent development of prostate cancer. Their effects on GRa have not been tested as yet.

Figure 16. Interaction model of the Gas5 RNA “GRE” to GR DBD and the molecular actions of Gas5 on GR-induced transcriptional activity. A: 3-Dimenstional structure of Gas5 “GRE”-mimic and its interaction model with GR DBD. From (342). B: Schematic model of Gas5 molecular actions on GR-induced transcriptional activity. Gas5 accumulated in response to growth arrest/starvation binds GR DBD and attenuates GR-induced transcriptional activity by competing with DNA GREs located in the promoter region of glucocorticoid-responsive genes.

THE SPLICING VARIANT GRbeta ISOFORM

 

The GRb isoform, which is expressed from the human GR gene through alternative use of its specific exon 9b, is known to have a dominant negative activity on classic GRa-induced transcriptional activity (21,352). This isoform was originally identified in humans, and was also reported in zebrafish, mice and rats (19,353-355). Since human (h) GRb shares the first 727 amino acids from the N-terminus with hGRa (19,356) (Figure 3), hGRb shares the same NTD and DBD with hGRa, but has a unique “LBD”. The divergence point (amino acid 727) of hGRa and hGRb is located at the C-terminal end of helix 10 in the hGRa LBD, therefore the hGRb “LBD” does not have helices 11 and 12 of the hGRa. As these helices are important for forming the ligand-binding pocket and for the creation of the AF-2 surface upon ligand binding (31), GRb cannot form an active ligand-binding pocket, does not bind glucocorticoids, and so, does not directly regulate GRE-containing, glucocorticoid-responsive gene promoters. In the absence of the hGRb “LBD”, the truncated hGR consisting of the NTD and DBD is transcriptionally active on GRE-containing promoters (357), thus the hGRb “LBD” somehow attenuates the transcriptional activity of the other subdomains of the molecule on GRE-driven promoters.

 

The dominant negative activity of GRb was first demonstrated in transient transfection-based reporter assays using GRE-driven reporter genes (21,358), but was subsequently confirmed on endogenous, glucocorticoid-responsive genes, such as the mitogen-activated protein kinase phosphatase-1 (MPK-1), myocilin and fibronectin (359,360). Further, GRb was shown to attenuate glucocorticoid-induced repression of the TNFa and interleukin (IL)-6 genes (359). We also confirmed this negative effect of GRb on GRa-mediated transrepression using microarray analyses (361). Several mechanisms explaining this GRb function have been reported, including (1) competition for GRE binding through their shared DBD, (2) heterodimerization with GRa and (3) coactivator squelching through the preserved AF-1 domain (21,357,358). All these different mechanisms of actions appear to be functional, depending on the promoters and the tissues affected by this GR isoform. Recently, the human GRb was shown to possess intrinsic transcriptional activity independent to its dominant negative effect on GRa-induced transcriptional activity, while the physiologic role(s) of this activity remain(s) to be examined (342,361,362) (see below). Inside the cells, hGRb can localize both in the cytoplasm and in the nucleus (363,364).

 

Similar to the human GR gene, the zebrafish (z) GR gene consists of 9 exons and produces zGRa and zGRb proteins, which contain 746 and 737 amino acids, respectively (353) (Figure 17). zGRa and zGRb share the N-terminal 697 amino acids, whereas they have specific C-terminal portions, which contain 47 and 40 amino acids, respectively. In contrast to hGRa and hGRb, which are produced through alternative use of specific exon 9a and 9b, zGRa and zGRb are formed as a result of intron retention (353). zGRa and zGRb use exon 1 to exon 8 for their common N-terminal 697 amino acids. zGRa uses exon 9 for its specific C-terminal portion, while zGRb continuously employs the rest of exon 8 and uses a stop codon located at the 3’ portion of this exon to express its specific C-terminal peptide (353). Protein alignment comparison of hGRb and zGRb indicated that these two molecules employ exactly the same divergence point, while their b isoform-specific C-terminal peptides show little sequence homology (353). These pieces of molecular information indicate that hGRb and zGRb evolved independently. Mouse (m), and recently, rat (r) GRb are also shown to produce in the same fashion as zGRb, indicating that intron retention may be a general mechanism for expressing this receptor isoform in organisms, while splicing-mediated expression employed by hGRb is rather unique (355,365). Nevertheless, zebrafish, mouse and rat GRb demonstrated the same functional properties as those of hGRb, namely, inability to bind glucocorticoids, a dominant negative activity on respectively zGRa-, mGRa- and rGRa-induced transactivation of GREs-drive promoters, and a strikingly similar tissue distribution as hGRb (353,365). Thus, hGRb, mGRb, rGRb and zGRb were produced through convergent evolution, most likely developed through a strong requirement of this type of GR isoform in the survival of these species. Indeed, the presence of nonligand-binding C-terminal variants is not unique to the GR. Similar to the human, mouse, rat and zebrafish GR, several other human NRs, e.g. ERb, TRa, vitamin D receptor (VDR), constitutive androstane receptor (CAR), dosage-sensitive sex reversal-1 (DAX-1), NURR-2, NOR-2, PPARα and PPARγ, all have C-terminally truncated receptor isoforms that are defective in binding to cognate ligands and have a dominant negative activity on their corresponding classic receptors (366-375). This suggests that evolution has allowed the development and retention of such alternative NRs, probably because they play important biologic roles.

Figure 17. Genomic and complementary DNA and protein structure of the zebrafish GR isoforms. The zebrafish (z) GR gene consists of 9 exons. The zGR gene expresses zGRa and zGRb splicing variants through intron retention (353). C-terminal gray colored and shaded domains in zGRa and zGRb show their specific portions. They are respectively encoded by exon 9 and the 3’ portion of exon 8, which are also shown in the same labeling in the genomic and complementary DNA models. Mouse and rat GR are produced with the same mechanism (355). Modified from (352). DBD: DNA-binding domain; LBD: Ligand-binding domain; NTD: N-terminal domain; UTR: untranslated region.

Biological actions of GRb and associated molecular mechanisms have been examined further during the last years. Using adeno-associated virus-based transfer of GRb to mouse liver, this isoform modulates mRNA expression of many genes in this organ including those related to endocrine system disorders, cancer, gastrointestinal diseases and immune diseases/inflammatory response both in a GRa-dependent and -independent fashions (376). Specifically, GRb attenuates GRa-dependent expression of the hepatic PEPCK gene and hepatic gluconeogenesis, while GRb stimulates expression of STAT1 through GREs located in the intergenic area close to the latter gene. The latter finding suggests that GRb can regulate gene expression by binding to classic GREs, in contrast to the previous findings obtained with GRE-driven reporter genes. In addition, GRb antagonizes to GRa-mediated suppression of bladder cancer cell migration and myogenesis of cardiomyocytes (377,378). GRb suppresses PTEN expression and enhances insulin-stimulated growth by stimulating the phosphorylation of AKT1 in a GRa-independent fashion (379). Further, GRb acts as a coactivator of T-cell factor-4 and enhances glioma cell proliferation also in a GRa-independent manner (380).

Several clinically oriented investigations suggest that GRb is responsible for the development of tissue-specific insensitivity to glucocorticoids in various disorders, most of them associated with dysregulation of immune function. They include glucocorticoid-resistant asthma, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), ankylosing spondylitis, chronic lymphocytic leukemia and nasal polyps (381-387). In these studies, various immune cells express elevated levels of GRb, which correlate with reduced sensitivity to glucocorticoids. Viral infection also stimulates GRb expression: for example, its expression in the peripheral mononuclear cells is strongly stimulated in the infants with bronchiolitis caused by the respiratory syncytial virus infection, and its expression levels are correlated with severity of the disease (388). Elevated levels of pro-inflammatory cytokines, such as IL-1, -2, -4, -7, -8 and -18, TNFa, and interferons a and g, might be responsible for increased GRb expression in cells from patients with these pathologic conditions, as these cytokines experimentally stimulate the expression of GRb in lymphocytes, neutrophils or airway smooth muscle cells (389-394). Further, presence of a single nucleotide polymorphism in the 3’ UTR of the hGRb mRNA (rs6198G allele), which increases its stability, and thus, causes elevated expression of the GRb protein, was associated with increased incidence of RA, SLE, high blood pressure, ischemic heart disease and nasal carriage of Staphylococcus aureus (382,395-397), possibly through inhibition of glucocorticoid actions by the increased concentrations of GRb. These pieces of clinical evidence further support that GRb has a dominant negative activity on GRa-induced transcription inside the human body, functioning as a negative regulator of glucocorticoid actions in local tissues.

 

PATHOLOGIC MODULATION OF GR ACTIVITY

 

Natural Pathologic GR Gene Mutations that Cause Familial/Sporadic Generalized Glucocorticoid Resistance or Chrousos Syndrome

 

Mutations in the human GR gene result in familial/sporadic generalized glucocorticoid resistance syndrome [see reviews (398-402)]. Since this syndrome was first reported by Chrousos et al. (403), we now call it as “Chrousos syndrome” (398,404). The condition is characterized by hypercortisolism without Cushingoid features (403,405). To overcome reduced sensitivity to glucocorticoids in tissues, affected subjects have compensatory elevations in circulating cortisol and ACTH concentrations, which maintain circadian rhythmicity and appropriate responsiveness to stressors, and resistance of the HPA axis to dexamethasone suppression, but no clinical evidence of hypercortisolism (404). Instead, the excess ACTH secretion causes increased production of adrenal steroids with mineralocorticoid activity, such as deoxycorticosterone (DOC) and corticosterone and/or androgenic activity, such as androstenedione, dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S); The former accounts for symptoms and signs of mineralocorticoid excess, such as hypertension and hypokalemic alkalosis. The latter accounts for the manifestations of androgen excess, such as ambiguous genitalia and precocious puberty in children, acne, hirsutism and infertility in both sexes, male-pattern hair-loss, menstrual irregularities and oligo-anovulation in females, and adrenal rests in the testes and oligospermia in males. The clinical spectrum of the condition is broad and a large number of subjects may be asymptomatic, displaying biochemical alterations only (404).

 

An increasing number of kindreds and sporadic cases with abnormalities in the GR number, affinity for glucocorticoid, stability, and translocation into the nucleus have been reported (406-414). The molecular defects that have been elucidated are presented in Figure 18 and Table 1. The propositus of the original kindred was a homozygote for a single nonconservative point mutation, replacing aspartic acid with valine at amino acid 641 in the LBD of GRa; this mutation reduces the binding affinity of the affected receptor for dexamethasone by three-fold and causes loss of transactivation activity (411). The proposita of the second family had a 4-base deletion at the 3’-boundary of exon 6, removing a donor splice site. This results in complete ablation of one of the GR alleles in affected members of the family (412). Recent research employing mice with GR haploinsufficiency confirmed that ablation of one GR allele is sufficient to develop generalized glucocorticoid resistance (415). The propositus of the third kindred had a single homozygotic point mutation at amino acid 729 (valine to isoleucine: V729I) in the LBD, which reduced both the affinity and the transactivation activity of GRa (414). Several pathologic heterozygotic or homozygotic mutations of the GRgene have been recently identified in the patients as listed in Table 1 (403,411-444).

Figure 18. Location of the known human GR mutations causing Chrousos syndrome or its mirror image, sporadic glucocorticoid hypersensitivity, in the human GR (NR3C1) gene (A) and in the linearized hGR protein molecule (B). Nucleoside numbers of the mutated sites are determined by the definition employing adenine of the translation initiation site as number 1.

Table 1. Mutations in the NR3C1 Gene Causing Familial/Sporadic Generalized Glucocorticoid Resistance (Chrousos) or Hypersensitivity Syndromes

Authors

cDNA*

Amino acid

Molecular Defects

Genotype

Phenotype

Chrousos et al. (403)
Hurley et al.

(411)

1922A>T

Asp641Val

Transactivation: Decreased
Affinity to ligand: Decreased (x3)
Nuclear translocation: 22 min
Abnormal interaction with GRIP1

Homozygous

Hypertension
Hypokalemic alkalosis

Karl et al.

(412)

4bp deletion in exon-intron 6

  

GRa number: 50% reduction
Inactivation of affected allele

Heterozygous

Hirsutism
Male-pattern hair-loss
Menstrual irregularities

Malchoff et al. (414)

2185G>A

Val729Ile

Transactivation: Decreased
Affinity to ligand: Decreased (x4)
Nuclear translocation: 120 min
Abnormal interaction with GRIP1

Homozygous

Precocious puberty
Hyperandrogenism

Karl et al. (413)
Kino et al. (416)

1676T>A

Ile559Asn

Transactivation: Decreased
Transdominance (+)
Decrease in GR binding sites
Nuclear translocation: 180< min
Abnormal interaction with GRIP1

Heterozygous

Hypertension
Oligospermia
Infertility

Ruiz et al. (418)
Charmandari et al. (419)

1430G>A

Arg477His

Transactivation: Decreased
No GREs binding
Decrease in GR binding sites
Nuclear translocation: 20 min

Heterozygous

Hirsutism
Fatigue
Hypertension

Ruiz et al. (418)
Charmandari et al. (419)

2035G>A

Gly679Ser

Transactivation: Decreased
Affinity to ligand: Decreased (x2)
Nuclear translocation: 30 min
Abnormal interaction with GRIP1

Heterozygous

Hirsutism
Fatigue
Hypertension

Mendonca et al. (417)

1712T>C

Val571Ala

Transactivation: Decreased
Affinity to ligand: Decreased (x6)
Nuclear translocation: 25 min
Abnormal interaction with GRIP1

Homozygous

Ambiguous genitalia
Hypertension
Hypokalemia
Oligo-amenorrhea

Vottero et al. (420)

2241T>G

Ile747Meth

Transactivation: Decreased
Transdominance (+)
Affinity to ligand: Decreased (x2)
Nuclear translocation: Decreased
Abnormal interaction with GRIP1

Heterozygous

Cystic acne
Hirsutism
Oligo-amenorrhea

Charmandari et al. (421)

2318T>C

Leu773Pro

Transactivation: Decreased
Transdominance (+)
Affinity to ligand: Decreased (x2.6)
Nuclear translocation: 30 min
Abnormal interaction with GRIP1

Heterozygous

Fatigue
Anxiety
Acne
Hirsutism
Hypertension

Charmandari et al. (431)

2209T>C

Phe737Leu

Transactivation: Decreased
Transdominance (+)
Affinity to ligand: Decreased (x1.5)
Nuclear translocation: 180 min

Heterozygous

Hypertension
Hypokalemia

Charmandari et al. (430)

1201G>C

Asp401His

Transactivation: Increased­
Transdominance (-)
Affinity to ligand: no change
Nuclear translocation: Normal

Heterozygous

Hypertension
Diabetes mellitus
Accumulation of visceral fat

McMahon et al. (426)

2bp (TG) deletion at 2318 and 2319

Phe774Serfs⃰

No transactivation activity
No ligand-binding activity

Homozygous

Severe hypoglycemia developed 1 day after birth
Hypertension
Fatigues with feeding

Nader et al. (422) (443)

2141G>A

Arg714Gln

Transactivation: Decreased
Transdominance (+)
Affinity to ligand: Decreased (x2.0)
Nuclear translocation: 20 min
Abnormal interaction with GRIP1

Heterozygous

 

 

 

 

 

 

 

 

 

 

 

 

 

Heterozygous

Hypoglycemia developed at age 2 years and 10 months
Hypertension
Accelerated bone age
Mild clitoromegaly

 

 

 

 

 

 

 

 

Infertility

Bouligand et al. (429)

1405C>T

Arg469Ter

Transactivation: Decreased
Affinity to ligand: Decreased
Nuclear Translocation: No

Heterozygous

Bilateral adrenal hyperplasia
Hypertension
Hypokalemia

Zhu et al.
Nicolaides et al. (423) (424)

1667C>T

Threo556Ile

Transactivation: Decreased
Transdominance: No
Affinity to ligand: Decreased
Nuclear translocation: 50 min

Heterozygous

Bilateral adrenal hyperplasia

Roberts et al.

(428)

1268T>C

Val423Ala

Transactivation: Decreased
Transdominance: No
Affinity to ligand: no change
Nuclear translocation: 2.6-fold delay

Heterozygous

Hypertension

Nicolaides et al. (425)

2177A>G

His726Arg

Transactivation: Decreased
Transdominance: No
Affinity to ligand: Decreased
Nuclear translocation: 60 min

Heterozygous

Hirsutism
Acne
Alopecia
Fatigue
Anxiety
Irregular menstrual cycle

Lin et al. (432)

26C>G

Pro9Arg

Not performed

Heterozygous

Hypertension

Paragliola et al. (433)

367G˃T

Glu123Ter

Not performed

Heterozygous

Chronic fatigue

Anxiety

Hirsutism

Irregular menstrual cycles

Infertility

Tatsi et al. (434)

592G˃T

Glu198Ter

Not performed

Compound heterozygous

Hypertensive encephalopathy

Al Argan et al. (435)

1392delC

Ile464Ilefs⃰

Not performed

Heterozygous

Low body weight

Hyperandrogenism

Severe anxiety

Adrenocortical hyperplasia

Vitellius et al. (436)

1429C˃A

Arg477Ser

Cytoplasm to nuclear translocation: Decreased

DNA binding: (-)

Dominant negative effect: (-) Transactivation: (-)

Heterozygous

Obesity

Velayos et al. (437)

1429C˃T

Arg477Cys

Not performed

Heterozygous

Mild hirsutism

Vitellius et al. (436)

1433A˃G

Tyr478Cys

Cytoplasm to nuclear translocation: Decreased

DNA binding: Weak and delayed

Dominant negative effect: (-)

Transactivation: Decreased

Heterozygous

Adrenal mass

Vitellius et al. (438)

1471C˃T

Arg491Ter

Transactivation: (-)

Heterozygous

Bilateral adrenal hyperplasia

Vitellius et al. (438)

1503G˃T

Gln501His

Transactivation: Decreased

Heterozygous

Bilateral adrenal hyperplasia

Ma et al. (439)

1652C˃A

Ser551Tyr

Ligand binding: Decreased

Cytoplasm to nuclear translocation: Decreased

Transactivation: Decreased

Homozygous

Fatigue

Hypokalemia

Hypertension

Polyuria

Velayos et al. (437)

1762_1763insTTAC

His588Leufs⃰

Not performed

Heterozygous

Hirsutism

Chronic fatigue

Anxiety

Cannavò et al. (440)

1915C˃G

Leu595Val

Not performed

Not available

Hirsutism

Amenorrhea

Hypertension

Trebble et al. (441)

1835delC

Ser612Tyrfs⃰

Protein expression: (-)

Ligand binding: (-)

Cytoplasm to nuclear translocation: (-)

Dominant negative effect: Yes

Transactivation: (-)

Heterozygous

Fatigue

Vitellius et al. (442)

1980T˃G

Tyr660Ter

Transactivation: (-)

Heterozygous

Hypertension

Vitellius et al. (436)

2015T˃C

Leu672Pro

Protein expression: Decreased

Ligand binding: (-)

Cytoplasm to nuclear translocation: (-)

DNA binding: (-)

Dominant negative effect: No

Transactivation: (-)

Heterozygous

Adrenal mass

Donner et al. (444)

2317_2318delCT

Leu773Valfs⃰

Protein expression: slightly reduced

Transactivation: Decreased

Dominant negative effect: No

Ligand binding: (-)

Heterozygous

Hypertension

 

 

We examined the impact of 10 pathologic GRa point mutations (559N, V571A, V575G, D641V, G679S, R714Q, V729I, F737L, I747M and L773P) to the molecular structure of the GRa LBD focusing on its ligand-binding pocket and AF-2 surface by using computer-based molecular simulation, and found some rules on the molecular disruption of these structural units by the mutations (89); (1) Topology of the peptide backbones is highly preserved in pathologic GRa mutant LBDs (Figure 19A). This result suggests that alteration in property and/or positioning of the side chain of replaced amino acids is rather crucial for developing molecular defects. (2) Defects in the ligand-binding pocket of the mutant receptors are driven primarily by loss/reduction (indirectly through structural changes in LBD induced by the mutations) of the electrostatic interaction formed by arginine 611 and threonine 739 of the receptor to glucocorticoid and a subsequent conformational mismatch (Figure 19B). (3) Defects of the AF-2 surface that reduce affinity to the LxxLL motif are caused mainly (also indirectly) by disruption of the electrostatic bonds to the non-core leucine residues of this peptide that determine the peptide’s specificity to GRa LBD (Figure 19C), as well as by reduced non-covalent interaction against core leucines and subsequent exposure of the AF-2 surface to solvent.

Figure 19. Impact of pathologic GR point mutations to the molecular structure of GR LBD. A: Distribution of the pathologic GR point mutations in its LBD and their overall impact on the 3-dimensional LBD peptide backbone. Thickness and color of the overlaid C-traces of the GR mutant receptor LBDs and the wild type GR LBD indicate the areas of least (thin and blue) to most (thick and red) motion over the course of simulation. Locations and side chains of the mutated amino acids are indicated, whereas dexamethasone (shown with the white and red spheres of space-filling model) is located inside LBP. B: Alteration of the electrostatic bond formed by arginine (R) 611 and threonine (T) 739 of pathologic GR mutants to dexamethasone may largely explain the reduced affinity of many pathologic GR mutants to this steroid. The left panel demonstrates superimposed 3-dimensional interaction images of dexamethasone and the key residues of all pathologic GRa mutants. Among the key amino acids of pathologic mutants participating in interaction with dexamethasone, R611 is largely deviated in these mutant receptors, which underlies reduced/disappeared electrostatic interaction between this residue and the carbonyl oxygen at carbon-3 of dexamethasone. Q570 and N564 are omitted from these panels. Major changes observed in the electrostatic bond formed by R611 and T739 are indicated with a purple dotted circle. The right panel shows schematic molecular interaction between wild type GRa and dexamethasone. Purple and orange arrows indicate electrostatic and non-covalent bonds, respectively. DEX: dexamethasone. C: Defective non-covalent bonds formed between Q597, D590, K579 and R585 of the pathologic GRa mutants and N742, R746, D750 and D752 of the LxxLL peptide mainly explain reduced interaction of the mutant receptor AF-2s to this peptide. The panel demonstrates 3-dimensional image of the molecular interaction between the LXXLL peptide and key residues of the wild type GRa. The LxxLL peptide forms important electrostatic bonds with its non-core leucine residues (N742, R746, D750 and D752) against the receptor residues (Q597, D590, R585 and K579, respectively) as marked with purple dotted boxes. Pathologic GRa mutants demonstrate significant shift of the side chains of some of these receptor residues among which the side chain of R585 shows the most significant deviation (shown in square inserts). Modified from (89).

GR Gene Mutation-Mediated Hypersensitivity Syndrome

 

Only one mutation has been reported in the GRa NTD that replaces aspartic acid at amino acid 401 by histidine (D401H) (G to C replacement at nucleotide position 1201) (430). The patient harboring this heterozygous mutation presented with manifestations consistent with glucocorticoid hypersensitivity, in accordance with the in vitro results showing that the mutant receptor hGRaD401H demonstrated a 2.4-fold increase in its ability to transactivate the glucocorticoid-responsive promoters. This condition represents the mirror image of the Chrousos syndrome. Although not in humans, one porcine heterozygotic substitution that replaces alanine at amino acid 610 with valine (A610V) in LBD of the porcine GR causes a gain-of-function phenotype, shifting the titration curve of GR-transcriptional activity to leftward (this suggests increase of the receptor affinity to glucocorticoid) (445).

 

GR Gene Polymorphisms

 

Polymorphisms of the human GR gene have also been reported (446). A heterozygous polymorphism replacing aspartic acid to serine at amino acid 363 (N363S) that mildly increases transcriptional activity of the affected receptor in vitro is associated with increased sensitivity to glucocorticoids, weakly correlating with the development of central obesity, and thus, influencing the metabolic profile and the longevity of humans in a negative fashion (447-449). This polymorphism found at amino acid 363 was first described by Karl et al. (412).

 

The polymorphism in the human GR gene that causes arginine to lysine replacement at amino acid 23 (ER22/23EK: GAG AGG to GAA AAG) is associated with relative glucocorticoid resistance by altering the expression levels of GRa translational isoforms (450). This polymorphism increases muscle mass in males and reduces waist to hip ratio in females, and is associated with greater insulin sensitivity, and lower total and low-density lipoprotein cholesterol levels, indicating that this polymorphism causes beneficial effects on longevity by reducing glucocorticoid actions (451,452).

 

One recent study examined influence of N363S and ER22/23EK polymorphisms to intelligence quotient (IQ) and behavior of 344 young subjects who have been followed up from their birth (453). The study found that N363S is not associated with IQ, while ER22/23EK showed significantly higher IQ scores. Both polymorphisms did not show any effects on the behavior scores. Antenatal glucocorticoid treatment reduces IQ scores in the subjects carrying N363S or ER22/23EK polymorphism.

 

The BclI GR polymorphism comprises a C to G nucleotide substitution at 646 bp downstream of exon 2 in intron B of the human GR gene that creates a cutting site for the BclI restriction enzyme. G-allele of this polymorphism increases tissue sensitivity to glucocorticoids as shown by greater suppression of serum cortisol levels after dexamethasone administration (454). This polymorphism is associated with development of mood disorders, psychopathology, bronchial asthma, hypertension, hyperinsulinism and obesity (455,456). It is also associated with increased bone resorption in patients receiving glucocorticoid replacement therapy (457). Although a large study employing adolescents (15-17 years old) did not confirm the association of the BclI polymorphisms to changes in several stress-related neurological parameters (458), a study employing 460 subjects with post-traumatic stress disorder (PTSD) found that this polymorphism and another polymorphism rs258747, located in the 3’-flanking region of the human GR gene and potentially influencing stability of GR mRNA, significantly increase a risk for developing PTSD (459). In one study, the BclI GR polymorphism was associated with lower frequency of insulin resistance in the women with polycystic ovary syndrome (PCOS) in contrast to the findings obtained in normal subjects (460).

 

A single nucleotide polymorphism that replaces A with G at the nucleoside 3669 (A3669G) located in the 3’ end of exon 9b has been described in a European population (461). This polymorphism does not change the amino acid sequence but increases the stability of GRb mRNA and increases GRb protein expression, leading to greater inhibition of GRa-induced transcriptional activity and causing glucocorticoid resistance in tissues. The presence of the A3669G allele is associated with reduced central obesity and a more favorable lipid profile in affected subjects (461).

 

Viral Infection

 

HUMAN IMMUNODEFICIENCY VIRUS TYPE-1

 

Patients with the Acquired Immunodeficiency Syndrome (AIDS), which is caused by infection of the Human Immunodeficiency Virus type-1 (HIV-1), have several manifestations compatible with increased activity of GRa. They develop reduction of innate and Th1-directed cellular immunity, which is also seen in the conditions of glucocorticoid excess. Patients with AIDS often develop symptoms and signs that manifest in hypercortisolemic states, such as muscle wasting, myopathy, dyslipidemia and visceral obesity-related insulin resistance (462-466). Therefore, it is possible that some HIV-1-related factor(s) may modulate the function of GRa in patients with AIDS. Please see for more details the chapter on AIDS and the HPA Axis in the Adrenal Section of Endotext.

 

We have shown that one of the HIV-1 accessory proteins, Vpr, a 96-amino acid virion-associated protein with multiple functions (467,468), enhances GRa transactivation by functioning as a coactivator (469) (Figure 20). Indeed, Vpr contains a NR coactivator motif LxxLL at amino acids 64-68. This motif is used by host NR coactivators to bind NRs (80) (see Section ln ACTIONS OF GR, Mechanism of GRa-mediated Activation of Transcription). Similarly, through this motif, Vpr directly binds GRa and cooperatively enhances its activity on its responsive promoters along with host NR coactivators SRC-1 (p160-type protein, NCoA1) and p300/CBP (469). Vpr directly binds p300 at its C-terminal amino acids 2045-2191, where the p160 coactivators (NCoAs) also bind (470). Since Vpr circulates at detectable levels in HIV-1-infected individuals and is able to penetrate the cell membrane, its effects may be extended to cells not infected by HIV-1 (471,472). Indeed, extracellularly administered Vpr polypeptide regulates glucocorticoid-responsive genes, such as IL-12 p40, in the same way as the potent glucocorticoid, dexamethasone (473). In addition to regulating GRa activity, Vpr modulates the transcriptional activity of PPARb/d and PPARg, the NR family proteins important for fatty acid metabolism (474,475). Through modulating activities of GRa and the PPARs, Vpr appears to participate in the development of the characteristic AIDS-related lipodystrophy syndrome, which is quite prevalent among AIDS patients (476,477).

Figure 20. Linearized Vpr, Tat, E1A, p300 and CtBP1 molecules and their mutual interaction domains. Vpr interacts with GR and several other NRs through its LxxLL motif located at amino acids 64 to 69. Binding sites of Vpr and p160-type HAT coactivators overlap with each other on p300. Since Vpr has a LxxLL motif similar to p160 coactivators, Vpr mimics host p160 coactivators and enhances GR transcriptional activity. Tat also binds both p300 and p160 coactivators. p300 facilitates attraction of many transcription factors, cofactors and general transcription complexes, and loosens the histone/DNA interaction through acetylation of the histone tails by its histone acetyltransferase (HAT) domain. E1A binds p300 at the latter’s C-terminal portion, while it physically interacts with the N-terminal portion of CtBP1 through its C-terminal end. The N-terminal portion of CtBP1 physically interacts with HDAC5 and Retinoblastoma protein (Rb), which have repressive activity on transcription. CtBP1 regulates its interaction to binding partners by sensing cellular NAD+ levels through its NAD+-binding domain. The HAT domain of p300 and the NAD+-binding domain of CtBP1 are indicated in grey. Modified from (478). CREB: CRE-binding protein, HAT: histone acetyltransferase, HDAC5: histone deacetylases 5, NF-B: nuclear factor-B, NAD: nicotinamide adenine dinucleotide, NR: nuclear hormone receptor, p/CAF: p300/CBP-associating factor, pTEFb: positive-acting transcription elongation factor b, Rb: retinoblastoma protein, SF-1: steroidogenic factor-1, STAT2: signal transducer and activator of transcription 2, TFIIB: transcription factor IIB.

Another HIV-1 accessory protein, Tat, which functions as a major transactivator of the HIV-1 long terminal repeat promoter (479) also potentiates GRa activity moderately by increasing accumulation of the positive transcription elongation factor b (pTEFb) (480-482) (Figure 20). Like Vpr, Tat readily penetrates the cell membranes (483) and may, therefore, modulate the transcriptional activity of GRa in the cells/tissues not infected by HIV-1.

 

Through Vpr and Tat, HIV-1 may facilitate the transcription of genes encoding its own proteins by directly stimulating viral proliferation. On the other hand, by enhancing transactivation of GRa and other NRs, these proteins may contribute to the viral proliferation possibly by suppressing the host immune system, while they participate in the development of several pathologic conditions associated with HIV-1 infection (480,484).

 

ADENOVIRUS

 

Adenoviruses cause illness of the respiratory system, such as common cold syndrome, pneumonia, croup and bronchitis, as well as illnesses of other organs, such as gastroenteritis, conjunctivitis and cystitis. They encode the E1A protein, which is expressed just after the infection and is necessary for the transcriptional regulation of the adenovirus-encoded genes (485). In addition to the viral genes, E1A regulates the transcriptional activity of a variety of host genes through interaction with the host transcriptional integrator p300 and its homologous molecule CBP (77,486) (Figure 20). In an in vitro system, E1A, in contrast to Vpr, blocks the actions of glucocorticoids on the transcriptional activity of genes, producing resistance to glucocorticoids (470).

 

E1A also interacts with the C-terminal tail-binding protein 1 (CtBP1), which functions as a transcriptional repressor for numerous transcription factors, by communicating with the class II HDACs and other inhibitory molecules like the retinoblastoma protein (Rb) (487) (Figure 20). E1A suppresses functions of p300/CBP and CtBP1 by binding to their functionally critical domains (77,487). Although there is no supportive clinical evidence, it is highly possible that adenovirus changes the peripheral action of glucocorticoids as well as of other bioactive molecules that activate NRs and directly regulates the transcriptional activity of their target genes, ultimately contributing to the pathologic states observed in adenoviral infection.

 

OTHER VIRUSES

 

We examined the impact of viral infection (murine cytomegalovirus: mCMV) on glucocorticoid-mediated modulation of gene expression in dendritic cells (488). Among 96 genes examined, the viral infection significantly enhanced dexamethasone-induced IL-10 expression. Activation of the toll-like receptors (TLRs) by the virus stimulates the extracellular signal-regulated kinase (ERK) 1/2, which in turn increases phosphorylation of the human GRa at serine 203, resulting in the enhancement of GRa transcriptional activity on the IL-10 gene promoter. Since IL-10 is a potent anti-inflammatory cytokine, it appears that the virus stimulates its own infection/propagation by enhancing GRa activity on this cytokine. Respiratory syncytial virus (RSV), which is one of the major causes of lower respiratory tract infection and hospital visits during infancy and childhood, is reported to repress the anti-inflammatory action of glucocorticoids through GRa (489-491).

 

ACKNOWLEDGEMENTS

 

This literary work was supported by the intramural fund of the Sidra Medical and Research Center to T. Kino.

 

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HPA Axis and Sleep

ABSTRACT

 

Sleep is an important component of mammalian homeostasis, vital for our survival. Sleep disorders are common in the general population and are associated with significant adverse behavioral and health consequences. Sleep, in particular deep sleep, has an inhibitory influence on the hypothalamic-pituitary- adrenal (HPA) axis, whereas activation of the HPA axis or administration of glucocorticoids can lead to arousal and sleeplessness. Insomnia, the most common sleep disorder, is associated with a 24­hour increase of ACTH and cortisol secretion, consistent with a disorder of central nervous system hyperarousal. On the other hand, sleepiness and fatigue are very prevalent in the general population, and studies have demonstrated that the pro­ inflammatory cytokines IL­6 and/or TNF-alpha are elevated in disorders associated with excessive daytime sleepiness, such as sleep apnea, narcolepsy and idiopathic hypersomnia. Sleep deprivation leads to sleepiness and daytime hypersecretion of IL­6, whereas daytime napping following a night of total sleep loss appears to be beneficial both for the suppression of IL­6 secretion and for the improvement of alertness. These findings suggest that the HPA axis stimulates arousal, while IL­6 and TNF-alpha are possible mediators of excessive daytime sleepiness in humans. It appears that the interactions of and disturbances between the HPA axis and inflammatory cytokines determine whether a human being will experience deep sleep/sleepiness or poor sleep/fatigue.

NORMAL SLEEP

Sleep is an important component of mammalian homeostasis, vital for the survival of self and species. We, humans spend at least one third of our lives asleep, yet we have little understanding of why we need sleep and what mechanisms underlie its capacities for physical and mental restoration. Sleep has been proposed to play a fundamental role in the conservation, utilization, and reallocation of energy to maintain cellular homeostasis, anabolism, proper immune function, and normal neural plasticity (1, 2). In addition, sleep contributes substantially to the achievement of synaptic homeostasis by re-normalization of the potentiation of synaptic connections that occurs during wakefulness, in order to support learning and memory functions (2-4). In spite of this though, there has been a significant increase of empirical knowledge that is useful in the evaluation and management of most sleep complaints and their underlying disorders (5). The interaction of circadian effects, i.e., usual time to go to sleep, and amount of prior wakefulness (homeostatic response), determines the onset and amount of sleep (6). Regulated by a strong circadian pacemaker, free running natural sleep­wake rhythms cycle at about 25 hours rather than coinciding with the solar 24-hour schedule (7). However, cues from the environment (zeitgebers) entrain sleep's rhythm to a 24-hour schedule. As a result, persons depend on external cues to keep their diurnal cycle "on time." The normal diurnal clock resists natural changes in its pattern by more than about 1 hour per day, which explains the sleep difficulties that usually accompany adaptation to new time zones or switches in work shifts.

Individuals differ considerably in their natural sleep patterns. Most adults in non-tropical areas are comfortable with 6.5 to 8 hours daily, taken in a single period. Children and adolescents sleep more than adults, and young adults sleep more than older ones. Normal sleep consists of four to six behaviorally and electroencephalographically (EEG) defined cycles, including periods during which the brain is active (associated with rapid eye movements, called REM sleep), preceded by four progressively deeper, quieter sleep stages graded 1 to 4 on the basis of increasingly slow EEG patterns (8) (Figure 1). Deep sleep or slow wave sleep (SWS) (stages 3 and 4) gradually lessens with age and usually disappears in the elderly.

Figure 1. Effects of age on normal sleep cycles. REM sleep (darkened area) occurs cyclically throughout the night at intervals of approximately 90 minutes in all age groups. REM sleep decreases slightly in the elderly, whereas stage 4 sleep decreases progressively with age, so that little, if any, is present in the elderly. In addition, the elderly has frequent awakenings and a notable increase in wake time after sleep onset.

SLEEP DISORDERS

Sleep disorders are common in the general population and are associated with significant behavioral and health consequences (9, 10). Insomnia, the most common sleep disorder, is often associated with psychologic difficulties (11-13) and significant cardiometabolic morbidity and mortality (14-24). Excessive daytime sleepiness is the predominant complaint of most patients evaluated in sleep disorders clinics and often reflects organic dysfunction. Sleep apnea, narcolepsy, and idiopathic hypersomnia are the most common disorders associated with excessive daytime sleepiness. Sleep apnea occurs predominantly in middle-aged men and post­menopausal women and is associated with obesity and cardiovascular complications, including hypertension, while narcolepsy and idiopathic hypersomnia are chronic brain disorders with an onset at a young age (5). In the general population, excessive daytime sleepiness and fatigue are frequent complaints of patients with obesity (13, 25, 26), depression (13, 27, 28) and diabetes (27). The parasomnias, including sleepwalking, night terrors, and nightmares, have benign implications in childhood, but often reflect psychopathology or significant stress in adolescents and adults and organic etiology in the elderly.

SLEEP AND THE STRESS SYSTEM

In mammalian organisms, including human beings, the stress system consists of central and peripheral components, whose main function is to maintain homeostasis, both in the resting and stress states (29-31). The central components include: (i) the paraventricular nuclei (PVN) of the hypothalamus, which secrete corticotropin-releasing hormone (CRH and arginine vasopressin (AVP); ii) the CRH neurons of the paragigantocellular and parabranchial nuclei of the medulla and (iii) the noradrenergic nuclei of the medulla and pons, including the locus caeruleus (LC), regulating arousal, and other nuclei mostly secreting norepinephrine (NE) and regulating the systemic sympathetic and adrenomedullary, as well as the parasympathetic nervous systems. The peripheral components include (i) the neuroendocrine hypothalamic-pituitary-adrenal (HPA) axis; (ii) the efferent systemic sympathetic and adrenomedullary (SNS) and parasympathetic nervous systems (31).

Normal Sleep and the Hypothalamic­Pituitary­Adrenal (HPA) Axis

Although the association between sleep and stress has been noted for hundreds of years, a more systematic approach in the relation between several features of sleep and stress system activity has only taken place in the last two decades (Figure 2). In 1983, Weitzman and colleagues reported that sleep, in particular SWS, appears to have an inhibitory influence on the HPA axis and cortisol secretion (32). Since then, several studies have replicated this finding. In turn, central (intracerebroventricular) administration of CRH (33, 34) or systemic administration of glucocorticoids (35) can lead to arousal and sleeplessness. In normal individuals, wakefulness and stage 1 sleep (light sleep) accompany cortisol increases (36), while slow wave sleep or deep sleep is associated with declining plasma cortisol levels (37). In addition, in normals, induced sleep disruption (frequently repeated arousals) is associated with significant increases of plasma cortisol levels (38). Furthermore, mean 24-hour plasma cortisol levels are significantly higher in subjects with a shorter total sleep time than those with a longer total sleep time (39).

Figure 2. A simplified, heuristic model of the interactions between central and peripheral components of the stress systems with sleep and REM sleep. ACTH: corticotropin; AVP: arginine vasopressin; CRH: corticotropin­releasing hormone; GH: growth hormone; LC/NE: locus caeruleus­norepinephrine/sympathetic nervous system. A solid green line denotes promotion/stimulation; a dashed red line denotes suppression.

The amount of REM sleep, a state of central nervous system activation that resembles unconscious wakefulness (paradoxic sleep), appears to be associated with a higher activity of the HPA axis. An early study showed that 24­hour urinary 17­hydroxycorticoids were increased during REM epochs in urological patients (40). More recently, a study in healthy, normal sleepers showed that the amount of REM sleep was positively correlated with 24-hour urinary free cortisol excretion (41). These results are consistent with the co­existence of HPA axis activation, and REM sleep increases in patients with melancholic depression (30).

Corticotropin Releasing Hormone and ACTH in Sleep/Wake Regulation

CRH produced and released from parvocellular neurons of the paraventricular nucleus is the key regulator of the HPA axis (31). Release of CRH is followed by enhanced secretion of adrenocorticotropin hormone (ACTH) from the anterior pituitary and cortisol from the adrenal cortex. In addition, CRH exerts various influences on behavior, including cerebral activation and waking maintenance, through activation of CRH receptors, which are expressed in the basal prosencephalic areas, thalamus, hypothalamus, mesencephalus, brainstem, and pons (42).

 

In animals, central (intracerebroventricular) administration of CRH induces increased waking (34, 43, 44), decreased NREM and REM sleep (44, 45), and altered locomotor activity (44, 46). Also, specific CRH antisense oligodeoxynucleotides caused a reduction in spontaneous wakefulness during the dark period, but not during the light period in rats (47). Several reports indicate that CRH is excitatory in the locus caeruleus (LC), amygdala, hippocampus, cerebral cortex, and some portions of the hypothalamus (30, 31). Spontaneous discharge rates in the LC are highest during arousal and lowest during sleep.

In humans, the majority of the studies suggest that the sleep of young individuals is rather resistant to the arousing effects of CRH (48-51). In contrast, middle-aged individuals responded to an equivalent dose of CRH with significantly more wakefulness and suppression of slow wave sleep compared to baseline (51). Based on these findings, we concluded that middle-aged men show increased vulnerability of sleep to stress hormones, possibly resulting in impairments in the quality of sleep during periods of stress. These findings suggest that changes in sleep physiology associated with middle­ age play a significant role in the marked increase of prevalence of insomnia in middle­age. Also, peripheral administration of CRH is associated with a REM suppression, which is stronger in the young than in the middle aged.

The administration of ACTH and its analogues in humans has been associated with general CNS activation consisting of a decreased sleep period time and sleep efficiency, and an increase of sleep latency (39, 44). Continuous administration of ACTH produced a marked reduction in NREM sleep (44).

Glucocorticoid Effects On Sleep

The administration of glucocorticoids causes a robust suppression of REM sleep (44, 52). In addition to the well-established decrease of REM sleep in some studies, the continuous or pulsatile nocturnal administration of cortisol was paradoxically associated with a modest increase of SWS (48,53). It has been suggested that this effect of cortisol on SWS compared to CRH may be mediated by a feedback inhibition of CRH by cortisol.

 

A study in Addisonian patients demonstrated that an evening replacement dose of hydrocortisone was necessary for proper expression of REM sleep, (vide infra) suggesting that glucocorticoids have some permissive action for this sleep parameter, possibly reflecting an inverse u-shaped dose response curve (54).

In clinical practice, the use of pharmacologic doses of glucocorticoids is associated with sleep disturbance. In fact, in a multicenter, placebo­controlled study in which steroids were used on a short-term basis, insomnia was one of the most common side effects (35).

In addition to their direct effects on sleep, glucocorticoids might also modulate sleep indirectly by influencing the activity of the circadian clock system (from the Latin “circa diem” meaning “approximately a day”), a highly conserved timekeeping system that creates internal rhythmicity under the influence of day/night cycles (55-57). At the cellular and molecular level, glucocorticoids influence peripheral clocks at multiple sites through their intracellular receptor, the glucocorticoid receptor (GR), which functions as a ligand-activated transcription factor (58). Besides this molecular cross-talk, accumulating evidence suggests that glucocorticoids might play a fundamental role in the entrainment of wake-sleep cycle rhythmicity through regulation of behavioral adaptation to phase shifts, possibly through an indirect feedback to the SCN (44, 59).

AGING, HPA AXIS AND SLEEP

Old age is associated with marked sleep changes consisting of increased wake, minimal amounts of SWS, declining amounts of REM sleep, and earlier retiring and rising times. Some studies have shown that older adults have elevated cortisol levels at the time of the circadian nadir and have higher basal cortisol levels than younger adults (60-62). It is difficult to discern whether the latter changes are associated with aging or increased medical morbidity common in this group. It has been suggested that the effect of aging on the levels and diurnal variation of human adrenocorticotropic activity could be involved in the etiology of poor sleep in the elderly (60). Higher evening cortisol concentrations are associated with lower amounts of REM sleep (60, 62), and increased wake (62). More recently, it was shown that older women without estrogen replacement therapy (ERT), when subjected to mild stress, showed greater disturbances in sleep parameters than women on ERT (63).

SLEEP DEPRIVATION AND HPA AXIS

If sleep is important for our sense of well­being, then it is conceivable that sleep deprivation represents a stressor to human bodies and should be associated with activation of the stress system (64). However, several studies that have assessed the effects of one night's sleep deprivation on the HPA axis have shown that cortisol secretion is either not or minimally affected by sleep following prolonged wakefulness (65-67). More recent studies have reported somewhat antithetical results, with some studies showing that cortisol secretion is elevated the next evening following sleep deprivation (68-71) and the other studies showing a significant decrease of plasma cortisol levels the next day (72-74). Additionally, the study by Vgontzas et al (72) indicated that this inhibition of the HPA axis activity was associated with an enhanced activity of the growth hormone axis. Also, similarly to these inconsistent results from studies of total sleep deprivation, partial sleep loss (4 hours of sleep for 6 days) has been reported to be associated with evening cortisol elevation (75) while a modest restriction of sleep to 6 hours per night for one week was associated with a significant decrease of the peak cortisol secretion (76). Newer studies using different sleep restriction experimental protocols have also found inconsistent results, with the majority of them reporting no effect of sleep restriction on the HPA axis (77-82). Methodological differences primarily related on the way subjects were handled during deprivation may explain these opposing findings. The finding that sleep deprivation leads to lower cortisol levels post­ deprivation (primarily during the subsequent night of sleep) suggests that lowering the level of HPA activity, which is increased in depression, may be the mechanism through which sleep deprivation improves the mood of depressed individuals. In one study, we assessed the effects of a 2-hour midafternoon nap following a night of total sleep deprivation on sleepiness, proinflammatory cytokines (IL­6, TNFα) and cortisol levels. Parameters of interest (subjective feeling of sleepiness, psychomotor vigilance­ PVT, IL­6, TNFa and cortisol levels) were measured on the fourth (predeprivation) and sixth days (postdeprivation). We observed a marked and significant drop of cortisol levels during napping, which was followed by a transient increase during the postnap period (83). These findings suggest that sleep and particularly SWS has an inhibiting effect on cortisol secretion and that wake and alertness are associated with higher levels of cortisol.

Prolonged sleep deprivation in rats results in increased plasma norepinephrine levels, higher ACTH and corticosteroid levels at the later phase of sleep deprivation (84). It is postulated that these increases are due to the stress of dying from septicemia rather than to sleep loss. The effects of prolonged sleep deprivation on the HPA axis in humans have not been studied. It is possible that there is activation of stress response when a certain tolerability threshold has been reached.

SLEEP DISORDERS AND HPA AXIS

Although sleep disorders/disturbances with their various physical and mental effects on the individual should be expected to affect the stress system, information regarding the effects of sleep disorders/disturbances on this system is limited.

Insomnia and HPA Axis

Insomnia, a symptom of various psychiatric or medical disorders, may also be the result of an environmental disturbance or a stressful situation. When insomnia is chronic and severe, it may itself become a stressor that affects the patient's life so greatly that it is perceived by the patient as a distinct disorder itself. Either way, as a manifestation of stress or a stressor itself, insomnia is expected to be related to the stress system. Few studies have measured cortisol levels in "poor" sleepers or insomniacs, and those results are inconsistent. The majority of these studies reported no difference between controls and poor sleepers in 24-hour cortisol or 17­hydroxysteroid excretion (85). In a 1998 study in 15 young adult insomniacs, 24-hour urinary free cortisol (UFC) excretion levels were positively correlated with total wake time (86). In addition, 24-hour urinary levels of catecholamines and their metabolites DHPG and DOPAC were positively correlated with percent stage 1 sleep and wake time after sleep onset. However, the total amount of the 24-hour UFC or catecholamine excretion was not different from normative values.

These preliminary findings were confirmed and extended in a controlled study in which objective sleep testing and frequent blood sampling was employed; the 24-hour ACTH and cortisol plasma concentrations were significantly higher in insomniacs than matched normal controls (87). Within the 24-hour period, the greatest elevations were observed in the evening and first half of the night (Figure 3). Also, insomniacs with a high degree of objective sleep disturbance (% sleep time < 70) secreted a higher amount of cortisol, compared to those with a low degree of sleep disturbance. Pulsatile analysis revealed a significantly higher number of peaks per 24h in insomniacs than in controls (p < 0.05), while cosinor analysis showed no differences in the circadian pattern of ACTH or cortisol secretion between insomniacs and controls. Thus, insomnia is associated with an overall increase of ACTH and cortisol secretion, which, however, retains a normal circadian pattern. Also, this increase relates positively to the degree of objective sleep disturbance. These findings are consistent with a disorder of CNS hyperarousal not only during the night but during the day as well, rather than one of sleep loss, which is usually associated with no change or a decrease in cortisol secretion, or a circadian disturbance. Increased evening and nocturnal cortisol peripheral concentrations have been reported in one study (88), while another study that included insomniacs without evidence of objective sleep disturbance did not report differences between insomniacs and controls (89).

Figure 3. Twenty-four-hour plasma cortisol concentrations in insomniacs (■) and controls (O). The thick black line indicates the sleep recording period. The error bar indicates SE, P < 0.01.

It appears that the difference between these two groups of studies is the degree of polysomnographically documented sleep disturbance. For example, in the study by Rodenbeck et al (88) the correlation between the area under the curve (AUC) of cortisol and % sleep efficiency was ­0.91 suggesting that high cortisol levels are present in those insomniacs with an objective short sleep duration. In contrast, in the study by Riemann et al (89), in which no cortisol differences were observed between insomniacs and controls, the objective sleep of insomniacs was very similar to that of controls (88.2% Sleep efficiency vs 88.6%). In another study that applied constant routine conditions, all indices of physiological arousal were increased but not to a significant degree due to lack of power and controls not being selected carefully (90). Interestingly, in the latter study a visual inspection of cortisol data suggested an elevation of cortisol values of 15% to 20% in the insomnia group, a difference similar to that reported in the study by Vgontzas et al. (87) and which should be considered of clinical significance. These preliminary findings on the role of objective sleep disturbance, were recently corroborated by newer studies in experimental or community samples of insomnia patients using polysomnography or actigraphy to objectively assess nighttime sleep (91-93).  In all these three studies, insomnia coupled with objective short sleep duration was associated with higher cortisol levels both in adults (91, 92), as well as in children (93). 

 

Based on our observations from the studies on Insomnia and HPA axis, that cortisol levels are higher in those with objective short sleep duration (Figure 4) we expected that insomnia with short sleep duration should be associated with significant medical morbidity and mortality. A study, which used a large general random sample of men and women (n=1,754), demonstrated that insomnia with objective short sleep duration (<5h nighttime sleep) entailed the highest risk for hypertension, followed by the insomnia group who slept 5­6 hours, compared to the normal sleeping and >6h sleep duration group (14). In the same population sample, chronic insomnia with objective short sleep duration was also found to be associated with increased odds for type 2 diabetes (15), as well as neuropsychological deficits in speed processing, attention, visual memory and verbal fluency (16). In order to examine the mortality risk in this sample, we followed up men and women for 14 and 10 years respectively. After controlling for several confounders, the mortality rate in insomniac men with objective short sleep duration was four times higher than in control normal sleepers (17).

Figure 4. Twenty-four-hour plasma cortisol concentrations in insomniacs, with low total ST (■) vs. those with high total ST (O) (MANOVA). The thick black line indicates the sleep recording period. The error bar indicates SE, P < 0.01.

More recently, from the same random, general population sample of the Penn State Cohort, 1395 adults were followed up after 7.5 years (22). All of the subjects underwent 8-hour polysomnography. We used the median polysomnographic percentage of sleep time to define short sleep duration (i.e. < 6 hours). Compared with normal sleepers who slept ≥6 hours, the highest risk for incident hypertension was in chronic insomniacs with short sleep duration. This study was the first longitudinal study to have examined the association of insomnia with objective short sleep duration with incident hypertension using polysonomnography (22). 

Finally, another cross-sectional study on a research sample on chronic insomniacs, showed that chronic insomnia (based on standard diagnostic criteria with symptoms lasting ≥6 months) when associated with physiological hyperarousal, (as defined by long MSLT values) is associated with a high risk for hypertension (19). Collectively, the above data further support that objective sleep measures in insomnia are an important index of the medical severity of the disorder, and they also point out the need for validation of practical, feasible, inexpensive methods, such as actigraphy, to measure sleep duration outside of the sleep laboratory. From a clinical standpoint, these data suggest that the therapeutic goal in insomnia should not be just to improve the quality or quantity of nighttime sleep. Rather, they suggest that the common practice of prescribing only hypnotics for patients with chronic insomnia at most is of limited efficacy. Furthermore, the focus of psychotherapeutic and behavioral modalities, including sleep hygiene measures, should not be to just improve the emotional and physiological state of the insomniac pre­ or during sleep, but rather to decrease the overall emotional and physiologic hyperarousal and its underlying factors, present throughout the 24-hour sleep/wake period.

It is possible that medications that suppress the activity of the HPA axis, such as antidepressants (94), could be a promising tool in our pharmacologic approaches. The effects of antidepressants on sleep, as well as on the daytime function and well­being of insomniacs, have not been assessed systematically yet some preliminary studies have reported improvement on sleep (95, 96). The potential usefulness of antidepressants in insomnia was further supported by a study by Rodenbeck et al. (97), who demonstrated the beneficial role of doxepin (a TCA) on both sleep and cortisol secretion in patients with primary insomnia without a clinically diagnosed depression. Moreover, two other studies have also underlined the efficacy and the safety of small doses of doxepin in adults suffering from primary insomnia, as well as in a model of transient insomnia (98, 99).

 

In conclusion, more studies are needed in order to confirm the possible therapeutic role of antidepressants on chronic insomnia as well as to clarify their underlying sleep­promoting mechanisms.

DISORDERS OF EXCESSIVE DAYTIME SLEEPINESS AND THE HPA AXIS

Obstructive sleep apnea (OSAS), the most common sleep disorder associated with excessive daytime sleepiness and fatigue, is accompanied by nocturnal hypoxia and sleep fragmentation. The latter conditions should be expected to be associated with an activation of the stress system. Indeed, it has been shown that urinary catecholamines, as well as plasma catecholamines measured during the nighttime, are elevated in sleep apneics compared to controls (100). Also, using microneurography, it has been shown that obstructive apneic events are associated with a surge of sympathetic nerve activity (101). In addition, another study lately demonstrated the beneficial effect of CPAP treatment on the stress system of sleep apneic patients (102). It has also been proposed that sympathetic activation in sleep apnea is one of the mechanisms leading to the development of hypertension, a condition commonly associated with sleep apnea. This proposal was supported by a study which showed that 3-month CPAP therapy could moderate hypertension in obese apneic men, an effect that may be attributed to the normalizing actions of CPAP on the stress system by eliminating chronic intermittent hypoxia and repetitive microawakenings (103).

Similarly, it was expected that sleep apnea would also be associated with an activation of the HPA axis. However, findings from different studies are inconsistent. Few studies that have assessed the plasma cortisol levels in sleep apneics have failed to show any differences between sleep apneics and controls (104-107), or more recently, sleep apnea was even reported to be associated with relative hypocortisolemia (108). In addition, no differences were reported in the plasma or urinary free cortisol levels following the abrupt withdrawal of CPAP, the most commonly recommended treatment for sleep apnea (109). However, another study reported that CPAP corrected preexisting hypercortisolemia, particularly after prolonged use (110). In accordance with the latter study, a more recent one demonstrated an association between OSAS and a mild but significant at night elevation of cortisol levels, in obese apneics compared to obese nonapneic controls. The increased levels of cortisol were corrected after the 3­month use of CPAP (111). These results were confirmed later by Henley et al., who showed that untreated compared to treated OSAS was associated with marked disturbances in ACTH and cortisol secretory dynamics (112). Two other studies lately reported increased cortisol levels in sleep apnea (113, 114). This latter study by Kritikou and collaborators showed that sleep apnea in non-obese men was associated with HPA axis activation, similar albeit stronger compared with obese individuals with sleep apnea. The same study was also the first to show that women, similarly to men, suffer from the same degree of the HPA axis activation. Finally, similarly to our previous study in obese men (111), short-term CPAP use had a significant effect on cortisol levels compared with baseline. 

Of interest is also that, in nondepressed, normally sleeping, nonapneic obese men, plasma levels of cortisol were lower than those in nonobese controls and exogenous administration of CRH provoked an enhanced ACTH response (111). These results suggest that in obesity there is hyposecretion of hypothalamic CRH, associated with hypotrophic adrenal cortices requiring compensatorily elevated amounts of ACTH to produce normal amounts of cortisol (111).

The association of the two other major organic disorders of excessive daytime sleepiness, narcolepsy and idiopathic hypersomnia, with the stress system has not been assessed. Preliminary data from a study that assessed the responsiveness of plasma cortisol and ACTH to the exogenous administration of ovine CRH in idiopathic hypersomniacs was associated with a normal or reduced plasma cortisol response, while the ACTH response to CRH tended to be significantly higher in the patients than controls (115). These preliminary findings suggested a subtle hypocortisolism and an inferred hypothalamic CRH deficiency in patients with idiopathic hypersomnia. A central CRH deficiency in these patients is consistent with their clinical profile of increased daytime sleepiness and deep nocturnal sleep (generalized hypoarousal). Consistent with these findings, it was reported that narcolepsy is associated with reduced basal ACTH secretion and a putative reduction of central CRH (116).

FATIGUE, SLEEPINESS AND SLEEP APNEA, AND ADRENAL FUNCTION

Patients with Cushing's syndrome frequently complain of daytime fatigue and sleepiness. In one controlled study, it was demonstrated that Cushing's syndrome was associated with increased frequency of sleep apnea (117). In that study, about 32% of patients with Cushing's syndrome were diagnosed with at least mild sleep apnea, and about 18% had significant sleep apnea (apnea/hypopnea index > 17.5 events per hour). Interestingly, those patients with sleep apnea were not more obese or different in any craniofacial features compared to those patients without sleep apnea. These findings are interesting in light of the new findings that visceral obesity, which is prominent in Cushing's syndrome, is a predisposing factor to sleep apnea (118). Also, Cushing's syndrome in the absence of sleep apnea was associated with increased sleep fragmentation, increased stage 1 and wake, and decreased delta sleep (119).

Adrenal insufficiency or Addison's disease is associated also with chronic fatigue. A study indicated that untreated patients with adrenal insufficiency demonstrated increased sleep fragmentation, increased REM latency, and decreased amount of time in REM sleep, findings that may explain the patients' fatigue (54). These sleep abnormalities were reversed following treatment with a replacement dose of hydrocortisone. These results suggest that cortisol secretion may be needed to facilitate both initiation and maintenance of REM sleep. It should be noted that in normal individuals, exogenous glucocorticoids have been found to reduce REM sleep (52). The authors interpreted their findings that the inhibitory role of glucocorticoids on REM sleep in normals, along with their permissive role in Addison's patients, demonstrate that some cortisol is needed for REM sleep, with excess cortisol inhibiting REM sleep, perhaps indirectly by suppression of CRH.

Also, fatigue and excessive daytime sleepiness are prominent in patients with secondary adrenal insufficiency, e.g., steroid withdrawal, and African trypanosomiasis. The latter conditions are associated with decreased adrenocortical function and elevated TNFα and/or IL­6 levels (120, 121), which may be the mediators of the excessive sleepiness and fatigue associated with primary or secondary adrenal insufficiency, as well of the profound somnolence of patients with African trypanosomiasis.

SLEEP AND CYTOKINES

Research has shown a strong interaction between the HPA axis and the immune system (29). Cytokines have a strong stimulating effect on the HPA axis, whereas cortisol, the end­product of the HPA axis, suppresses secretion (Figure 2). In this section, we will describe what is known on the role of cytokines in sleep regulation and sleep disorders, as well as the interaction effect of cytokines and HPA axis on sleep and its disorders.

Feelings of fatigue and sleepiness are common symptoms associated with infectious diseases and many other physical and mental pathologic conditions. Physicians for millennia have advised their patients to sleep during the course of an illness. However, it is only within the past 20 years that there has been a systematic study on changes in sleep that occur with infection or following microbial product­induced cytokine production (reviewed in 122).

The first reports of the effects of IL­1 on sleep in animals were published in 1984 (123, 124). Following the observation that astrocytes (neuroglia) produce IL­1 (125), which provided the basis to explore whether IL­1 was somnogenic, several studies by Krueger and collaborators established the role of this cytokine in the sleep physiology of the rabbit. These studies (123, 126, 127) indicated that: 1) administration of IL­1 increased electroencephalogram (EEG) slow wave activity in the delta frequency band; 2) the effects of IL­1 on sleep were dose­related; and 3) the enhancement of induction of slow wave sleep (SWS) by IL­1 was not merely a byproduct of fever because pretreatment of animals with anisomycin abolished IL­1­induced fever but not IL­1­induced SWS. Since then, IL­1 has proven to be somnogenic in species other than rabbits, including rats, mice, cats, and monkeys.

The same group of investigators demonstrated that TNFα induced SWS in several species (126, 128), whereas TNFα mRNA (129) and protein in brain (130), exhibit circadian rhythms that coincide with sleep­wake activity. Also, direct intervention with the TNF system by the use of antibodies, binding proteins, or soluble receptors or receptor fragments reduced SWS in otherwise normal animals (131). In addition, mice that lack the 55 kDa TNF receptor sleep less than background strain controls (132). The effects of IL­6 on sleep in animals have not been assessed systematically.

Normal Sleep in Humans and Circadian Secretion of Cytokines

There are several reports that in normal people plasma levels of cytokines are related to the sleep­wake cycle. Moldofsky et al. first described such relations in human beings, showing that IL­1 activity was related to the onset of slow­wave sleep (133). Subsequently, other investigators showed that plasma levels of TNFα vary in phase with EEG slow wave amplitudes (134), and that there is a temporal relation between sleep and IL­1b activity (135, 136). Other studies, using indirect ex vivo methods or direct in vivo measures of plasma concentrations, have demonstrated that IL­6 and TNFα levels in young healthy individuals peak during sleep (103, 136, 137).

Specifically, in the study by Vgontzas et al. (137), at baseline, IL­6 is secreted in a biphasic circadian pattern, with two nadirs at 08.00 and 21.00 h and two zeniths at about 19.00­20.00 and 04.00­05.00 h, with the stronger peak at 05.00 h (Figure 5). Previous studies noted the circadian pattern of IL­6 secretion and its late-night peak (138-140). That these studies did not report on the daytime zenith of IL­6 at about 19.00­20.00 h could be attributable to their using infrequent sampling or a small number of subjects.

Figure 5. Twenty-four-hour plasma IL­6 concentrations pre­ and post­sleep deprivation in eight healthy young men. Each data point represents the mean + SE. *, P < 0.05 indicates statistical significance from the peak value within 24 h for each condition (MANOVA followed by Dunnett, post hoc test). The darkened area indicates the sleep recording period.

In the same study, we demonstrated that daytime IL­6 levels are negatively related to the amount of nocturnal sleep (137). Thus, decreased overall secretion of IL­6 is associated with a good night's sleep and a good sense of well­being the next day, and good sleep is associated with decreased exposure of tissues to the proinflammatory and potentially detrimental actions of IL­6 on the cardiovascular system, insulin sensitivity, and bones (141, 142).

These findings on the circadian pattern of IL­6 secretion were also illustrated in two studies that assessed the effects of modest sleep restriction (76) (Figure 6) as well as the effects of a 2­hour midafternoon nap following a night of total sleep loss (83) (Figure 7) on sleepiness, psychomotor performance and finally plasma levels of proinflammatory cytokines (IL­6, TNFα) and cortisol.

Figure 6. Twenty-four-hour circadian secretory pattern of IL­6 Before (◊) and after (▪) partial sleep restriction. Bar indicates SE. The thick black bar on the abscissa represents the sleep recording period during baseline. The open bar on the abscissa represents the sleep recording period during partial sleep restriction. *, P<0.05.

Figure 7. Twenty-four-hour IL­6 values pre­( ♦ ) and post­(□) sleep deprivation in the no­nap group (top) and the nap group (bottom). Thick black lines on the abscissa indicate nighttime and nap recording periods. Significant reduction of IL­6 during the nap period (1400­1600), P<0.05 

Recently, we assessed the effects of recovery sleep after one week of mild sleep restriction on IL-6, sleepiness and performance (79). Serial 24-h IL-6 plasma levels increased significantly during sleep restriction and returned to baseline after recovery sleep (Figure 8). Subjective and objective sleepiness increased significantly after restriction and returned to baseline after recovery. In contrast, performance deteriorated significantly after restriction and did not improve after recovery.

Figure 8. Serial 24-h IL-6 values at baseline (♦), restriction (■), and recovery (▲). Thick white, gray, and black lines on the abscissa indicate the nighttime sleep recording period at baseline, restriction, and recovery, respectively.

 

The view that IL­6 is involved in sleep regulation is further supported by the observation that exogenous administration of IL­6 in humans caused profound somnolence and fatigue (143), while in another study, its administration was associated with an increase of SWS in the second half of the night, suggesting a direct action of IL­6 on central nervous system sleep mechanisms (144). The sleep­disturbing effect of exogenous IL­6 noted in the first half of the night might be attributed to increased secretion of CRH, ACTH and cortisol induced by IL­6 during the early part of the night. An alternative, not mutually exclusive, hypothesis is that high levels of IL­6 per se may compromise early nighttime sleep.

Sleep Disturbance, Cytokines and Normal Aging

In a study in which we compared older adults to young subjects, the mean 24­h IL­6 and cortisol secretion was significantly higher in older adults (P < 0.05) (62) (Figure 9). IL­6 secretion in older adults was increased both during the daytime and nighttime, whereas cortisol secretion was more pronounced during the evening and nighttime periods. TNFα secretion in young adults showed a statistically significant circadian rhythm with a peak close to the offset of sleep; such a rhythm was not present in older adults. Both IL­6 and cortisol levels were positively associated with total wake time. The effect of IL­6 on wake time was markedly stronger for the older group than for the young group. The combined effect of cortisol and IL­6 on wake time was additive. IL­6 had a negative association with REM sleep only in the young, while cortisol was associated negatively with REM sleep both in the young and old, with a stronger effect in the young. These results suggest that in healthy adults, age-related alterations in nocturnal wake time are associated with elevation of both plasma IL­6 and cortisol concentrations, while REM sleep declines with age is primarily associated with cortisol increases.

Figure 9. Twenty-four-hour plasma concentrations of IL­6 (top), TNFα (middle), and cortisol (bottom) in healthy young (□) and old (■) individuals. Each data point represents the mean ± SE. The darkened area indicates the sleep recording period.

 

It has been previously suggested that increased HPA axis activity associated with aging is a result of the "wear and tear" of lifelong exposure to stress (53, 54). An alternative, not mutually exclusive, explanation is that the significant alteration of HPA axis activity associated with age is at least partially secondary to the hypersecretion of IL­6, whose peripheral levels are a good marker of increased morbidity and mortality (145). The source of IL­6 hypersecretion in the elderly is not known. However, we know that IL­6 peripheral levels correlate negatively with sex­steroids levels, positively with the amount of adipose tissue, are decreased after a good night's sleep, and are elevated in chronic pain/inflammatory syndromes (62,137,142,146-148). Old age is associated with decreased sex­steroid concentrations, increased proportional body fat, decreased quantity and quality of sleep, and frequent chronic pain/inflammatory conditions. Reducing the secretion of IL­6 in elderly, either by (a) administration of sex steroids, (b) decreasing fat through diet and exercise, (c) improving nighttime sleep, and (d) controlling adequately chronic pain and inflammation with nonsteroidal anti­inflammatory agents, may improve sleep, daytime alertness, and performance, and decrease the risk of common ailments of old age, e.g., metabolic and cardiovascular problems, cognitive disorders, and osteoporosis (118, 149, 150).

Cytokines as Potential Mediators of Pathological or Experimentally Induced Excessive Daytime Sleepiness

Excessive daytime sleepiness (EDS) occurs in about 5­9% of the general population (9, 13, 151) and is the chief complaint of the majority of patients evaluated at sleep disorders centers. EDS is one of the major physiological consequences of obstructive sleep apnea. Besides the obvious effects of daytime sleepiness on patients' occupational and social life, daytime sleepiness appears to be a major concern of public safety.

There has been a number of studies of cytokine profiles in patients with excessive daytime sleepiness (pathologic or experimentally­ induced) within the last 20 years. In one of the first studies in 1996, Entzian et al. studied 10 hospitalized patients requiring therapy for obstructive sleep apnea (104). Blood samples were collected every 4 hours during the day (08.00 to 20.00 h) and at 2­h intervals during nighttime sleep. Whole blood cultures stimulated with lipopolysaccharide were used to determine cytokine release. The circadian rhythm of TNFα was significantly altered in sleep apnea patients; the peak concentrations that occurred during the night in normal control subjects were not present in sleep apnea patients. Rather, sleep apnea patients exhibited increased TNFα concentrations in the afternoon, the time period during which concentrations in normal control subjects are at a minimum. It is also interesting to note that in spite of a lack of statistical differences due to inherent inter­individual variability, infrequent sampling and a small sample size, absolute IL­1 concentrations in the sleep apnea patients were more than twice those obtained from normal controls, and IFN concentrations were more than three times those of normal controls. Finally, IL­6 in sleep apneics reached maximum concentrations in the evening, in contrast to normal subjects where IL­6 peaked at about 2.00 a.m.

In 1997, we published a study in which cytokine profiles were obtained from several patient populations with disorders of excessive daytime sleepiness (152). Three populations were studied; those with obstructive sleep apnea (n = 12); narcoleptics (n = 11); and idiopathic hypersomniacs (n = 8). Single blood samples were drawn in the morning after the completion of the nighttime sleep laboratory recordings. Plasma concentrations of IL­1β, TNFα, and IL­6 were determined by ELISA. Relative to control subjects, plasma IL­1 concentrations did not differ between the three groups. TNFα was elevated in sleep apnea patients and narcoleptics, and IL­6 was elevated only in sleep apnea patients. Correlational analyses indicated that TNFα and IL­6 correlated positively with measures of excessive daytime sleepiness; TNF was positively correlated with the degree of nocturnal sleep disturbance, and the degree of hypoxia, whereas IL­6 concentrations were correlated with degree of nocturnal sleep disturbance, degree of hypoxia, and body mass index. The potential role of IL­6 as a mediator of daytime sleepiness was further suggested in a study by Hinze­Selch et al. that showed that IL­6 secretion by monocytes was higher in narcoleptics than controls and plasma levels of IL­6 were non-significantly higher in patients compared to controls (153).

The results of our first study prompted us to study further the role of IL­6 and TNFα as potential mediators of EDS in disorders of EDS, i.e., sleep apnea, and in conditions of experimentally­induced daytime sleepiness following sleep deprivation. Those preliminary findings were later corroborated by several studies by us or other researchers that showed that: (a) Single and 24­hour TNFα and IL­6 plasma levels are elevated in adults and children with sleep apnea independently of obesity (103, 118,154-158) (Figure 10); (b) body mass index (BMI) positively correlates with both TNFα and IL­6 levels, suggesting that these two cytokines may play a role in daytime sleepiness experienced by obese individuals in the absence of sleep apnea (25); (c) daytime levels of IL­6 and TNFα are elevated in healthy humans experiencing somnolence and fatigue as a result of total sleep deprivation (137) or even after a modest sleep loss by restricting sleep to 6 hours a night per week (76,159); and (d) a midafternoon nap following a night of total sleep deprivation is beneficial for both the suppression of IL­6 secretion and for the improvement of alertness (83). In the latter studies, greater pre­sleep deprivation amounts of slow wave (deep) sleep rendered the subjects resistant to the effect of sleep deprivation. It is common experience that individuals differ significantly in terms of their ability to sustain sleep loss or curtailment. Those with greater amounts of SWS are inherently more capable of tolerating sleep loss, possibly avoiding exposure to the potentially harmful effects of increased IL­6 secretion. Other studies have confirmed these findings by showing that IL­6 and TNFα are elevated following an 88­hour period of wakefulness (160) and that the nighttime rise of IL­6 levels is delayed during partial sleep deprivation (161). TNFα plasma levels are also elevated in other diseases associated with excessive daytime sleepiness, such as chronic fatigue syndrome, post­dialysis fatigue, HIV patients (162). In 2004, a pilot, placebo­ controlled, double­blind study further supported the somnogenic actions of IL­6 and TNFa. In this study, researchers tested the results of etanercept, a TNFα antagonist in eight obese male apneics suffering from excessive daytime sleepiness. Both sleepiness and AHI (number of apneas/hypopneas per hour) were reduced significantly by the drug compared to the placebo. IL­6 levels were also significantly decreased (163).

Figure 10. Plasma TNF, IL­6, and leptin levels in sleep apneics and BMI­matched obese and normal weight controls. A *, P <0.01 vs. normal weight (nl wt) controls, B *, P < 0. 5 vs. nl wt controls, C *, < 0.05 vs. obese and lean controls.

These studies collectively provide evidence that cytokines are elevated in individuals suffering from a disorder of EDS, e.g., apnea, or healthy individuals experiencing EDS secondary to acute or short­term sleep loss and support the hypothesis that EDS (pathologic or experimentally-induced) may be mediated in part by somnogenic cytokines.

Insomnia and Cytokines

Chronic insomnia, by far the most commonly encountered sleep disorder in medical practice, is characterized by long sleep latencies or increased wake time during the night and increased fatigue during the day, although in objective daytime sleep testing, insomniacs are unable to fall asleep (164, 165).

In a 2002 study, we demonstrated that the mean 24­hour IL­6 and TNF secretions were not different between insomniacs and controls. However, mean IL­6 levels were significantly elevated in insomniacs compared to controls in the mid­afternoon and evening pre­sleep period (15.00­23.00, P < 0.05) (146) (Figure 11). Furthermore, cosinor analysis showed a significant shift of the major peak of IL­6 secretion from early morning (05.00) to evening (20.00) in insomniacs compared to controls. Also, TNFα secretion in controls showed a statistically significant circadian rhythm with a peak close to the offset of sleep; such a rhythm was not present in insomniacs (Figure 12).

Figure 11. Twenty-four-hour circadian secretory pattern of IL­6 in insomniacs (○) and controls (●). The thick black line on the abscissa indicates the sleep recording period. Error bar indicates SE, * P < .05.

Moreover, the daytime secretion of TNF in insomniacs was associated with a regular periodicity of about 4 hours, and its amplitude was significantly different from zero. Controls showed a similar rhythm, which, however, was not significant.

Figure 12. Twenty-four-hour circadian secretory pattern of TNFα in insomniacs (○) and controls (●). The thick black line on the abscissa indicates the sleep recording period. Error bar indicates SE.

Based on these findings, we concluded that chronic insomnia was associated with a shift of IL­6 and TNF secretion from nighttime to daytime, which may explain the daytime fatigue and performance decrements associated with this disorder. The daytime shift of IL­6 and TNF secretion, combined with a 24h hypersecretion of CRH and cortisol, both arousal hormones, may explain the insomniacs' daytime fatigue and difficulty falling asleep during the daytime and/or the nighttime.

In conclusion, despite the fact that the above findings show abnormal secretion patterns of TNFα and IL­6 in insomnia, further studies are needed in order to get better insight into the association between cytokine secretion pattern and chronic insomnia.

Sleepiness vs. Fatigue: The Role of the Interaction of HPA Axis with Cytokines

From our previous studies, it became evident that cytokines are elevated both in disorders of deep sleep/EDS as well as in disorders of poor sleep/fatigue. These seemingly inconsistent findings can be better understood if we clarify the terms sleepiness vs. fatigue and understand the effects of cytokines on sleep/sleepiness in terms of their interaction with HPA axis.

“Sleepiness” and “fatigue” have been considered to be either the same state, different states on a continuum or finally fundamentally different states. In medical practice and literature, these terms are often used interchangeably; however, there is enough clinical evidence to propose a separate definition for these 2 terms in sleep disorders medicine. Sleepiness is a subjective feeling of physical and mental tiredness associated with increased sleep propensity. Fatigue is also a subjective feeling of physical and/or mental tiredness; however, it is not associated with increased sleep propensity. Based on these definitions, sleep disorders or conditions associated with sleepiness include sleep apnea, narcolepsy, and sleep deprivation. On the other hand, sleep disorders associated with fatigue include chronic insomnia, sleep disturbances in the elderly, and psychogenic hypersomnia. This distinction between “sleepiness” and “fatigue” was adopted unanimously as useful for the field of insomnia research by an expert panel of 25 sleep researchers who convened in Pittsburgh on March 10­11, 2005 (166).

Based on our studies, we propose that daytime cytokine hypersecretion and/or circadian shift of cytokine secretion not associated with HPA axis activation leads to sleepiness and deeper sleep, and a good example of this is sleep deprivation. On the other hand, we suggest that daytime cytokine hypersecretion and/or circadian alteration of cytokine secretion associated with HPA axis activation, e.g., insomnia, leads to fatigue and poor sleep.

Such a model, which combines cytokine secretion and HPA axis function to explain sleepiness and increased sleep versus fatigue and poor sleep, is supported by experiments on the effects of exogenous activation of the host defense system on sleep in humans. For example, exogenous administration of IL­6 in healthy humans in the evening was associated with both fatigue and a sleep disturbing effect in the first half of the night, most likely due to increased secretion of corticotrophin­releasing hormone, ACTH, and cortisol, during the early part of the night, induced by IL­6 (144). Also, in dose­response experiments using endotoxin, it was shown that subtle host defense activation not associated with HPA axis activation and increased body temperature enhanced the amount of non­REM sleep, whereas higher doses associated with increased cortisol secretion and increased body temperature, resulted in reduced non­REM sleep and increased wakefulness (167).

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Aldosterone Deficiency and Resistance

ABSTRACT

 

Aldosterone is crucial for regulating sodium conservation in the kidney, salivary glands, sweat glands, and colon. This adrenal steroid hormone acts via the mineralocorticoid receptor (MR) to promote active transport of sodium and potassium excretion in its target tissues, through activation of specific amiloride-sensitive sodium channels (ENaC) and a Na-K ATP-ase pump. Defective aldosterone biosynthesis or action results in various clinical and laboratory test manifestations, such as hypotension, hyponatremia, hyperkalemia, and acidosis. Primary adrenal insufficiency and congenital adrenal hypoplasia are discussed in other chapters. In this chapter the mechanisms underlying aldosterone-deficient conditions, such as hyporeninemic hypoaldosteronism, primary hypoaldosteronism, including aldosterone synthase deficiency (ASD), acquired forms of the disease, and pseudohypoaldosteronism, an aldosterone resistance syndrome due to insensitivity of target tissues to aldosterone, are reviewed. 

INTRODUCTION

Aldosterone is crucial for sodium conservation in the kidney, salivary glands, sweat glands, and colon. Aldosterone is synthesized exclusively in the zona glomerulosa of the adrenal gland. Destruction or dysfunction of the adrenal gland in conditions such as primary adrenal insufficiency, congenital adrenal hypoplasia, isolated mineralocorticoid deficiency, acquired secondary aldosterone deficiency (hyporeninemic hypoaldosteronism), acquired primary aldosterone deficiency, and inherited enzymatic defects in aldosterone biosynthesis cause clinical symptoms and laboratory characteristics owing to aldosterone deficiency. Pseudohypoaldosteronism is an aldosterone resistance syndrome i.e. a condition due to the insensitivity of target tissues to aldosterone. In this chapter, aldosterone-deficiency conditions other than primary adrenal insufficiency and congenital adrenal hypoplasia are reviewed.

ALDOSTERONE BIOSYNSTHESIS

All human steroid hormones are derived from cholesterol. Aldosterone is synthesized in the zona glomerulosa of the adrenal cortex through four enzymes, cholesterol desmolase (CYP11A1), 21-hydroxylase (CYP21A2), aldosterone synthase (CYP11B2), and 3β-hydroxysteroid dehydrogenase (3β-HSD) (Figure 1). CYP11A1, CYP21A2 and CYP11B2 are cytochrome 450 enzymes (CYP), which are membrane-bound, heme-containing enzymes that accept electrons from NADPH through accessory proteins and use molecular oxygen to perform hydroxylation or other oxidative conversions (1). CYP11A1, which is a side-chain cleavage enzyme, cleaves the side chain from C21 of cholesterol, converting cholesterol to pregnenolone in adrenal mitochondria and this is the first step in steroidogenesis. The CYP11A1 gene is located on the long arm of human chromosome 15q24-q25 (2). Pregnenolone is returned to the cytosolic compartment and is converted to progesterone by 3β-HSD. Progesterone is then hydroxylated at C21 by CYP21A2, an enzyme located in the smooth endoplasmic reticulum, to yield deoxycorticosterone (DOC). The CYP21A2 gene is located on the short arm of human chromosome 6 (3). Only CYP21A2 is active in humans, the other, CYP21A1P is a pseudogene (4). CYP11B1, which is a mitochondrial enzyme, catalyzes β-hydroxylation at C11 and converts DOC to corticosterone. The terminal two steps in the conversion of corticosterone to aldosterone (18-hydroxylation and 18-methyloxidation) are catalyzed by CYP11B2 (aldosterone synthase) (5) which was previously named corticosterone 18-hydroxylase/18-methyloxidase (CMO I/CMO II) or 18-hydroxylase/isomerase. These two steps previously proposed to be catalyzed by separate enzyme, CMO 1 and II, are known to involve only one enzyme substrate interaction, aldosterone synthase encoded by CYP11B2 gene (6). The CYP11B1 and CYP11B2 genes are located on the long arm of chromosome 8 and the amino acid sequence of CYP11B2 shares more than 90% homology with that of CYP11B1 (7). In humans, the expression of CYP11B1 and CYP11B2 in the adrenal glands is spatially separated. While expression of CYP11B1 takes place in the zona reticularis/fasciculata, CYP11B2 expression and aldosterone synthesis are restricted to the zona glomerulus (8).

Figure 1. Aldosterone Biosynthesis. Aldosterone is derived from cholesterol. Biosynthetic pathway of aldosterone and structure of adrenal steroids and their biosynthetic precursors are shown in the figure. The enzymes that catalyze each step are listed in the adjacent box at the right side of the figure.

Epigenetic Regulation Of Cyp11b2 Expression

CYP11B2 gene expression is epigenetically controlled. DNA methylation at CpG dinucleotides alter gene expression by affecting transcription factor binding activity (9). Cyclic AMP responsive element binding protein 1 (CREB 1) /ATF family members and nuclear receptor subfamily 4, group A (NR4A) members bind the CYP11B2 promoter at Ad1  (cAMP response element at -71/-64) and Ad5 (cAMP response element at -129/-114), respectively, leading to activation of transcription. DNA methylation at CpG1 greatly decreased CREB 1 binding to Ad1 in the promoter lesion of CYP11B2  gene (10). In addition, DNA methylation at CpG2 reduced basal binding activities of NR4A1 and NR4A2 with Ad5 by 30% and 50%, respectivly (10). Ang II infusion in the rat decreased the methylation ratio of CYP11B2 gene  and increased gene expression in the adrenal gland (10). A low-salt diet induced hypomethylation of rat CYP11B2 and increased CYP11B2 mRNA levels parallel with aldosterone synthesis (10).

REGULATION OF ALDOSTERONE SECRETION

Aldosterone secretion is regulated by multiple factors. The renin-angiotensin system and potassium ion are the major regulators, whereas ACTH and other POMC peptides, sodium ion, vasopressin, dopamine, ANP, β-adrenergic agents, serotonin and somatostatin are minor modulators.

The Renin-Angiotensin System

Renin is a 430 amino acid enzyme that cleaves renin substrate or angiotensinogen, which is a 453 amino acid alpha-globulin product of the liver, to produce the decapeptide, angiotensin I. Angiotensin I is rapidly cleaved by angiotensin-converting enzyme (ACE) in the lung and other tissues to form the octapeptide, angiotensin II. Moreover, angiotensinase cleaves the NH2-terminal Asp residue from angiotensin II and produces the heptapeptide, angiotensin III, then to the hexapeptide angiotensin IV. The circulating levels of angiotensin III are 15 to 25% of those of angiotensin II. Angiotensin II, III and IV stimulate aldosterone secretion and vasoconstriction, while angiotensin II is more potent for vasoconstriction. The angiotensins are inactivated within minutes by tissue and plasma peptidase. The levels of the circulating renin are the rate-limiting factor in this process.

Renin is synthesized by the juxtaglomerular cells in the renal cortex and its secretion is controlled by renal arterial blood pressure, sodium concentrations of tubular fluid sensed by the macula densa, and renal sympathetic nervous activity (11). Factors that decrease renal blood flow, such as hemorrhage, dehydration, salt restriction, upright posture, and renal artery narrowing, increase renin levels. In contrast, factors that increase blood pressure, such as high salt intake, peripheral vasoconstrictors, and supine posture, decrease renin levels. Hypokalemia increases and hyperkalemia decreases renin release.

The effect of angiotensin II and III on the adrenal glomerulosa is initiated by binding to G-protein coupled receptors. The first mechanism of the intracellular signal transduction is activation of phospholipase C, which hydrolyzes PIP2 to IP3, which then releases intracellular calcium ions (12). Interestingly, angiotensin II does not stimulate adenylate cyclase activity. Angiotensin II stimulation leads to increased transfer of cholesterol to the inner mitochondrial membrane and increased conversion of cholesterol to pregnenolone and corticosterone to aldosterone (13).

Potassium

Potassium directly increases aldosterone secretion by the adrenal cortex and aldosterone then lowers serum potassium by stimulating its excretion by the kidney. High dietary potassium intake increases plasma aldosterone and enhances the aldosterone response to a subsequent potassium or angiotensin II infusion (12). The primary action of potassium for stimulating aldosterone secretion is to depolarize the plasma membrane, which activates voltage-dependent calcium channels, that permit influx or efflux of extracellular calcium (12–14), leading to the activation of calmodulin and calmodulin-dependent kinase, subsequently. The activated kinase phosphorylates both activating transcription factor and members of CRE-binding protein family which bind to 5’ flanking promotor regions of the CYP11B2 gene and trigger gene transcription in the zona glomerulosa, followed by increased aldosterone biosynthesis (13,14).

Pituitary Factors

ACTH and possibly other POMC-derived peptides, including α-MSH, α-MSH, β-LPH, and β-END, influence aldosterone secretion, however, the role of ACTH in aldosterone secretion is minor (12). ACTH increases aldosterone secretion by binding to glomerulosa cell-surface melanocortin-2 receptor, by activating adenylate cyclase, and increasing intracellular cAMP (15). Like other agents, ACTH stimulates the same two early and late steps of aldosterone biosynthesis.

Vasopressin has a modest and transient stimulatory effect on aldosterone secretion from zona granulosa cells in vitro. This effect is probably mediated via V2 receptors and phospholipase C generating IP3 and diacylglycerol (16).

Sodium

Sodium intake influences aldosterone secretion by an indirect effect through renin and to a minor extent by direct effects on zona glomerulosa responsiveness to angiotensin II. High sodium intake increases vascular volume, which suppresses renin secretion and angiotensin II generation and decreases the sensitivity of aldosterone response to angiotensin II.

Inhibitory Agents

Dopamine inhibits aldosterone secretion in humans by a mechanism that is independent of the effects of prolactin, ACTH, electrolytes, and the renin-angiotensin system (17,18). This inhibitory effect may involve binding to D2 receptors on glomerulosa cells (19). Atrial natriuretic peptide (ANP) directly inhibits aldosterone secretion and blocks the stimulatory effects of angiotensin II, potassium and ACTH, at least in part, by interfering with extracellular calcium influx (20).

MECHANISMS OF ALDOSTERONE ACTION

Effect of Aldosterone

Aldosterone is crucial for sodium conservation in the kidney, salivary glands, sweat glands, and colon. Aldosterone promotes active sodium transport and excretion of potassium in its major target tissues. It exerts its effects via the mineralocorticoid receptor (MR) and the resultant activation of specific amiloride-sensitive sodium channels (ENaC) and the Na-K ATP-ase pump (21). Aldosterone and the MR may be involved in the regulation of genes coding for the subunits of the amiloride sensitive sodium channel and the Na-K ATP-ase pump, serum and glucocorticoid regulated kinase (SGK), channel-inducing factor, as well as of other proteins (22,23). Activated SGK1 phosphorylates the neural precursor cell-expressed, developmentally down-regulated protein 4-2 (Nedd4-2) which allows binding of 14-3-3 proteins (24). Then, the interaction of Nedd4-2 and ENaC causes an accumulation of ENaC at the plasma membrane and enhances epithelial sodium transport by increasing open probability of ENaC. In a later phase translation and allocation of ENaC, basolateral Na-K ATP-ase and apical K channel (ROMK) are enhanced in its target tissues (25–27).

On the other hand, rapid effects in response to aldosterone but independent of the MR were described as so-called non-genomic or rapid signaling of aldosterone. The G protein-coupled estrogen receptor (GPER) [previously known as G protein-coupled receptor 30 (GPR30)], a member of the seven transmembrane domain family of cell surface receptors, has been reported to be a membrane receptor for aldosterone (28). The expression of GPER is ubiquitous, including in vascular cells (both endothelial cells and smooth muscle cells) and is required for rapid MR-independent effects of aldosterone in vascular smooth muscle cells (28). Aldosterone has both vasodilator and vasoconstrictor effects. The effect of aldosterone on endothelial function would vary depending on the balance between GPER and MR expression. In vascular endothelial cells, aldosterone activation of GPER mediates vasodilation, while activation of endothelial MR has been linked to enhanced vasoconstrictor and/or impaired vasodilator response (28–30).

Mineralocorticoid Receptor

The mineralocorticoid receptor (MR) is found in the cytoplasm and nucleus and the sodium channels are expressed in the apical membrane of epithelial cells of the distal convoluted tubule as well as in cells of other tissues involved with conservation of salt, such as colon, sweat glands, lung, and tongue. MR is a member of the nuclear receptor superfamily. Together with the glucocorticoid, progesterone, and androgen receptors, MR forms the steroid receptor subfamily (30). Steroid receptors display a modular structure comprised of five regions (A-E). The N-terminal A/B region harbors an autonomous activation function. The central C region, corresponding to the DNA-binding domain, is highly conserved and is composed of two zinc fingers involved in DNA binding and receptor dimerization. The D region is a hydrophilic region and it forms a hinge between DNA-binding domain and ligand-binding domain. The E region corresponds to the C-terminal ligand-binding domain and mediates numerous functions, including ligand binding, interaction with heat-shock proteins, dimerization, nuclear targeting, and hormone-dependent activation (31) (Figure 2). The human MR (hMR) and human glucocorticoid receptor (hGR) have almost identical DNA-binding domains (94% homology in the amino acid) and very similar ligand-binding domains (57%), but divergent N-terminal A/B regions (<15%) (32). The hMR gene was mapped on chromosome 4q31.1-31.2 (33,34) and hMR cDNA encodes a 107 kilodalton polypeptide with 984 amino acids (32). The hMR gene consists of 10 exons, including two exons 1 that encode different 5'-untranslated sequences (35). Expression of the two different hMR variants is under the control of two different promoters that contain no obvious TATA element, but multiple GC boxes. Both hMRα and hMRβ mRNAs are expressed at approximately the same level in the mineralocorticoid target tissues (36).

Figure 2. The linearized structures of the mineralocorticoid receptor gene, mRNAs and protein. The MR gene consists of 10 exons. The MR has two exons 1 (exon 1α and exon 1β), each with an alternative promoter; however, the finally translated MR protein is the same. Exons 1 are untranslated regions, exon 2 codes for the immunogenic domain (A/B), exons 3 and 4 for the DNA-binding domain (C), and exons 5-9 for the hinge region (D) and the ligand-binding domain (E) (37)

Molecular and Cellular Mechanisms of the Aldosterone Action

MRs in its unliganded state is located in the cytoplasm, as part of hetero-oligomeric complexes containing heat shock proteins 90, 70 and 50 (38). Upon binding with their ligand, the receptor-ligand complex dissociates from the heat shock proteins, homo- or heterodimerizes and translocates into the nucleus. Homodimers or heterodimers of the MR interact with hormone-responsive elements (HRE) and/or other transcription factors in the promoter regions of target genes, including the subunits of the ENaC or other proteins related to this channel and sodium transport in general, and modulates the transcription rates of these genes (39) (Figure 3).

Figure 3. Mechanism of aldosterone action on sodium reabsorption at the distal convoluted tubule of the nephron. Aldosterone binds to the MR, which is located in the cytoplasm in complex with heat shock proteins 90, 70 and 50. After binding, the receptor-ligand complex translocates into the nucleus, binds to hormone-responsive elements (HRE) of target genes where it modulates their transcription rate. Amiloride-sensitive sodium channel (ENaC) subunits or other related proteins may be targets of such regulation (40).

Pre-Receptor Regulation

Since cortisol circulates at plasma concentrations several orders of magnitude higher than those of aldosterone does, and since it has a high affinity for the MR, it would be expected to overwhelm this receptor in mineralocorticoid target tissues and cause mineralocorticoid excess. A local enzyme, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), however, converts active cortisol to inactive cortisone, and protects the MRs from the effects of cortisol (40) 11β-HSD catalyzes the inter-conversion of hormonally active C11-hydroxylated corticosteroids (cortisol in humans or corticosterone in rodents) and their inactive C11-keto metabolites (cortisone in humans or 11-dehydrocorticosterone in rodents). Two isozymes of 11β-HSD have been identified, 11β-HSD type 1 (11β-HSD1) and 11β-HSD2, which differ in their biological properties and tissue distributions. 11β-HSD2, a potent NAD-dependent 11β-hydrogenase, rapidly inactivates glucocorticoids. The human 11β-HSD2 gene encodes 405 amino acids and its molecular weight is approximately 40-kilodalton (41). 11β-HSD2 has a hydrophilic N-terminal domain that is thought to anchor the protein into membranes (42). 11β-HSD2 is localized as a dimer in the nucleus and cytoplasm of cells of the cortical collecting duct and colon (42,43). Prednisolone and prednisone are substrates for both 11β-HSD isozymes (44,45) and dexamethasone is metabolized slightly by 11β-HSD2 (46). Licorice derivatives, such as glycyrrhizic acid, and the hemisuccinate derivative carbenoxolone are inhibitors of 11β-HSD2. Inhibition of 11β-HSD2 with such agents, confers mineralocorticoid potency to physiologic concentrations of endogenous glucocorticoids in the kidney and colon (47). Thus, in normal physiology, 11β-HSD2 protects the MR by converting cortisol to the inactive cortisone and allows aldosterone-selective access to the inherently nonselective MR in mineralocorticoid target tissues.

Amiloride-Sensitive Sodium Channel (Epithelial Sodium Channel; ENaC)

The cDNA of the α-subunit of the ENaC (αENaC) was cloned from the rat colon in 1993 (48) and soon after the cDNAs of the β- and γ-subunits of this channel were cloned for the same species (49). The human α-, β- and γ-subunits of ENaC were also cloned (50,51). In vitro studies demonstrated that the α subunit of the ENaC itself had the majority of Na channel function, while, the β- and γ- subunits alone were not shown to play as major a role in sodium transport (48). However, the β- and γ-subunits enhanced the function of the α-subunit and all subunits are required for full ENaC activity (52). It appears then that this channel consists of the α-, β- and γ-subunits and an amiloride-binding protein (Figure 4). Aldosterone increases transcription of αENaC but not β- and γ-subunits, resulting enhanced channel assembly and transported from endoplasmic reticulum to Golgi (53). In Golgi, furin proteolytically cleaves specific sites in the extracellular domains of α- and γ-ENaC, resulting in channel activation. At the cell surface, Nedd4-2 binds to ENaC, increasing endocytosis and degeneration (54).The proline-rich region of the C-terminal of the αENaC is important for binding to α-spectrin and for stabilization of the sodium channel in the membrane (55). Recently, several studies demonstrated abnormalities of the β- and γ-subunits of the ENaC in patients with Liddle's syndrome, characterized by mineralocorticoid excess (hypertension and hypokalemic alkalosis), and suppressed aldosterone secretion (56–59). The truncation caused by these mutations influenced the PY motif at the N-terminal of the molecule. This motif is responsible for the binding of the channel subunits with NEDD4, a carrier protein facilitating clearance of the channel (60). Moreover, a point mutation of the αENaC gene, located close to the N-terminal of the protein, was reported to cause a decrease of the probability of an open sodium channel, resulting in defective reabsorption (40,61).

The ENaC-Regulatory Complexes in Aldosterone-Mediated Sodium Transport

Aldosterone-induced trans-epithelial Na+ transport via ENaC involves the coordinate functioning of stimulatory signaling proteins such as serum- and glucocorticoid-induce kinase-1 (SGK1) (23,62), glucocorticoid-induced leucine zipper protein-1 (GILZ1) (63) and connector enhancer of kinase suppressor of Ras 3 (CNK3) (64), with inhibitory proteins, such as neural precursor cell expressed, developmentally downregulated protein (Nedd4-2) (24) and extracellular signal-regulated kinase (ERK) 1/2 (23,24,62,65).

 

SGK1 is an aldosterone-regulated protein kinase that stimulates renal ENaC through many mechanisms. First, SGK1 phosphorylates the E3 ubiquitin ligase and Nedd4-2, and inhibits its actions. Nedd4-2 interacts with the C-terminal tail of ENaC subunits, decrease surface expression of the channel via channel ubiquitinoylation (23,24,62). Second, SGK1 phosphorylates kinase with no lysine (WNK) 4 and prevents ENaC endocytosis (66). Third, SGK1 directly phosphorylates alpha ENaC and transforms silent ENaC channels to active ones (67). Then, SGK1 alters ENaC expression, trafficking and activity, and stimulates Na+ transport in the kidney cortical collecting duct (CCD) (68). However, SGK1 is a short-lived protein. Following synthesis, SGK1 is rapidly targeted to the endoplasmic reticulum (ER), where ER-associated ubiquitin ligases CHIP and HRD1 aid in its ubiquitinoylation and subsequent proteasome-mediated degradation (69). Another aldosterone-induced ENaC-regulator, GILZ, which protects SGK1 from rapid ER-associated degradation by controlling protein-protein interaction (53.6). In kidney CCD, GILZ1 is robustly induced by aldosterone (70). GILZ1 stimulates ENaC cell surface expression and activity at least in part by inhibiting ERK1/2, which abrogates ENaC function (65,71,72).

The recently identified MR target gene CNKSR3 (connector enhancer of kinase suppressor of Ras 3), commonly referred as CNK3, is highly expressed in the connecting tubule (CNT) and the CCD (73). CNK3, like SGK1 and GILZ1, is rapidly induced by physiological concentrations of aldosterone (64). CNK3 acts to assembly various ENaC-regulatory components in close vicinity of the channel and thereby exerts its stimulatory effects on channel function (74).

Epigenetic Control of  ENaC Transcription by Aldosterone-Sensitive Dot1A-Af9 Complex

Chromatin regulates gene transcription by the post-translational modification of histone N-terminal tails such as acetylation and methylation. The histone H3 Lys 79 methyltransferase disruptor of telomeric silencing alternative splice variant a (Dot1a) methylates histone H3 Lys79, which resides in the globular domain (75). ALL-1 fused gene from chromatin 9 (Af9), putative transcription factor, physically and functionally interact with Dot1a to form a nuclear repressor complex that directly or indirectly binds specific site of the alpha ENaC promoter. Aldosterone reduces the level of Af9 mRNA and protein. Then, Af9 overexpression induces hypermethylation of histone H3 Lys 79 and repression of alpha ENaC transcription (76). Aldosterone impairs the formation of Dot1a -Af9 complex associated with alpha ENaC promoter by 1) decreasing abundance of Dot1a and Af9; 2) attenuating the interaction between Dot1a and Af9 via Sgk-1-catalyzed phosphorylation of Af9 at Ser 435; 3) counterbalancing the repression through binding to mineralocorticoid receptor (MR) and facilitating its translocation into the cell nucleus, where MR and Dot1a compete for binding to Af9.  These are aldosterone-dependent and -independent mechanisms for Dot1a-Af9-mediated repression of alpha ENaC transcription. While aldosterone -independent de-repression achieved through the action of ALL-1 fused gene from chromatin 17 (Af17), Af17 upregulates alpha ENaC transcription by decreasing Af9 binding to Dot1a and relieving Dot1a-Af9-mediated repression of ENaC (77). 4) SGK1 phosphorylates Af9, thus, down-regulating Dot1a-Af9 complex, and relieving the basal repression on alpha ENaC transcription (67,78).

Figure 4. Model of a putative amiloride-sensitive sodium channel (ENaC). The amiloride-sensitive sodium channel appears to consist of the α-, β- and γ- subunits and an amiloride-binding protein. This channel is located at the apical site of the renal epithelium and plays a role in passive sodium transport, which is mainly regulated by mineralocorticoids (79).

THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM IN NEWBORNS AND INFANTS                

Aldosterone secretion rate of newborns and infants was similar to that of older children and adults. Therefore, the aldosterone secretion rate corrected by body surface was much higher in infancy than later in life (80). Urinary aldosterone at birth depends on gestational age and increases progressively, concurrently with the levels of plasma aldosterone. Plasma renin activity, plasma aldosterone and urinary excretion rate of aldosterone decrease with age (81). At  birth, human kidneys display tubular immaturity leading to sodium wasting and impaired ability to reabsorb water. Past studies showed that plasma potassium concentrations were significantly higher in newborns than in their respective mothers, while neonatal and maternal plasma sodium concentrations were closely related. Aldosterone and renin levels in newborns differs significantly from the corresponding maternal concentrations (82). The aldosterone-renin ratio significantly increases with gestational age. Thus, neonatal partial aldosterone resistance was previously suggested because of the high urinary sodium loss in the presence of hyperactivity of the renin-angiotensin-aldosterone system (83). Previous study found that the highest aldosterone levels detected in the cord blood originated from de novo synthesis by the fetal adrenal glands (84). In addition, neonatal aldosterone resistance was associated with weak or undetectable renal MR expression at birth. MR mRNA is transiently expressed between 15 and 24 weeks of gestation, but it is undetectable in late gestational age and neonatal kidney (85). 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD2) and alpha ENaC are closely correlated with cyclic MR expression.

CLASSIFICATION OF HYPOALDOSTERONISM

Various syndromes are characterized by or associated with hypoaldosteronism. Hypoaldosteronism is classified in three large categories, defective stimulation of aldosterone secretion, primary defects in adrenal synthesis or secretion of aldosterone, and aldosterone resistance, according to their pathophysiology and summarized in Table 1.

Table 1. Causes of Hypoaldosteronism and Hormonal Profiles

Causes of Hypoaldosteronism

Hormonal Profiles

DEFECTIVE STIMULATION OF ALDOSTERONE

v  Congenital keep tablehyporeninemic hypoaldosteronism

v  Acquired hyporeninemic hypoaldosteronism

Ø Associated with diabetes mellitus

Ø Associated with nephropathy

Ø Glomerulonephritis

Ø Gouty nephritis

Ø Pyelonephritis

Ø Nephropathy associated with multiple myeloma

Ø Nephropathy associated with systemic lupus erythematosa

Ø Mixed cryoglobulinemia

Ø Nephrolithiasis

Ø Analgesic nephropathy

Ø Renal amyloidosis

Ø Iga nephropathy

v  Associated with autonomic insufficiency

v  Associated with liver cirrhosis

v  Associated with sickle cell anemia

v  Associated with acquired immune deficiency syndrome

v  Associated with polyneuropathy, organomegaly, endocrinopathy, m protein and skin changes syndrome

v  Lead poisning

v  Excess sodium bicarbonate

v  Sjogren's syndrome

v  Drugs interfering with renin production

Ø Β-blocker

Ø Prostaglandin synthetase inhibitors

Ø Non-steroidal anti-inflammatory drugs

Ø Calcium channel blocker

v  Other drugs

Ø Cyclosporin a

Ø Mitomycin c

Ø Cosyntropin

Low plasma renin;

Low plasma and urinary aldosterone

Drugs interfering with angiotensin ii production

Ø  Angiotensin ii converting enzyme inhibitors

High plasma renin; low plasma aldosterone; low angiotensin ii

PRIMARY DEFECTS IN ADRENAL SECRETION OF ALDOSTERONE

Combined with defective cortisol synthesis

a) Congenital causes

Ø Congenital adrenal hypoplasia (dax-1 mutation)

Ø Congenital adrenal hyperplasia

§  Cholesterol desmolase deficiency (lipoid adrenal hyperplasia)

§  3β-hydroxysteroid dehydrogenase deficiency

§  21-hydroxylase deficiency

§  11β-hydroxylase deficiency

 

Adrenoleukodystrophy, adrenomyeloneuropathy

Low plasma renin; low plasma aldosterone; low plasma cortisol

 

 

 

 

 

 

High plasma deoxycorticosteorne

b) Acquired causes

Ø  Autoimmune adrenal destruction

·       Addison's disease

·       Multiple autoimmune endocrinopathy

Ø  Infectious adrenal destruction

·       Bacterial infection

·       Fungal infection

Ø  Infiltration of adrenal glands

·       Amyloidosis

·       Hemochromatosis

·       Sarcoidosis

·       Metastatic or infiltrative malignant disease

Ø  Bilateral adrenalectomy

Ø  Drug induced

§  Mitotane

§  Aminoglutethimide

§  Torilostane

§  Ketoconazole

Low plasma renin; low plasma aldosterone; low plasma cortisol

v  Isolated deficiency of aldosterone secretion

Ø Congenital causes

§  Cyp11b2 (aldosterone syntase) deficiency

¨     Corticosterone methyloxidase type i (cmo i) deficiency

 

¨     Corticosterone methyloxidase type ii (cmo ii) deficiency

¨      

High plasma renin; low plasma aldosterone

 

Normal plasma 18-hydroxycorticosterone/aldosterone ratio

High plasma 18-hydroxycorticosterone/aldosterone ratio

Ø Acquired causes

§  Critically ill patients associated with hypotension or hypovolemia

¨     Sepsis

¨     Pneumonia

¨     Peritonitis

¨     Cholangitis

¨     Liver failure

·       After removal of mineralocorticoid secreting adrenal tumor

·       Discontinuation of agents with mineralocorticod activity

·       Heparin or chlorbutol administration

 

Low plasma aldosterone concentration; inappropriate elevated plasma renin

DEFECTIVE ALDOSTERONE ACTION

v  Pseudohypoaldosteronism (pha) type 1

Ø Renal (autosomal dominant pha)

Ø Systemic pha (autosomal recessive pha)

v  Secondary pseudohypoaldosteronism

§  Associated with urinary tract infection

§  Associated with medication that blocks epithelial sodium channel (enac)

¨     Amiloride

¨     Triamterene

¨     Trimethoprim

¨     Pentamidine

§  Administration of aldosterone antagonists

¨     Spironolactone

¨     Progesterone

¨     17-hydroxyprogesterone

¨     Synthetic progestin

§  Drugs that may lead to aldosterone resistance

                Caludinerin inhibitor (cyclosporin a, tacrolimus)

High plasma renin; high plasma and urinary aldosterone

 

Defective Stimulation of Aldosterone

The first category of conditions, which is characterized by defective stimulation of aldosterone secretion, includes the syndromes of congenital and acquired hyporeninemic hypoaldosteronism. One of these conditions is due to a defect of renin secretion such as hyporeninemia resulting from β-blockers, prostaglandin synthetase inhibitors, and calcium channel blockers. Another condition is due to decrease in the conversion of angiotensin I to angiotensin II mediated by converting enzyme inhibitor medications and is associated with hyperreninemia.

Primary Defects in Adrenal Biosynthesis or Secretion of Aldosterone

The second category of conditions, which are characterized by primary defects in adrenal synthesis or secretion of aldosterone, includes all causes of primary adrenal insufficiency and primary hypoaldosteronism caused by aldosterone synthase (CYP11B2) deficiency or as an acquired state. Primary adrenal insufficiency causes include congenital adrenal hypoplasia, congenital adrenal hyperplasia, adrenoleukodystrophy/ adrenomyeloneuropathy, acquired adrenal insufficiency due to autoimmune, infectious and infiltrative disease, bilateral adrenalectomy and use of adrenolytic agents and enzyme inhibitors that block cortisol and aldosterone biosynthesis. These conditions are usually combined with defective cortisol synthesis. Aldosterone synthase (CYP11B2) deficiency (ASD) leads to reduced aldosterone production associated with low or high levels of 18-hydroxycorticosterone, referred to as CMO I or CMO II deficiency, respectively. Several conditions may be associated with aldosterone biosynthetic activity. Heparin suppresses aldosterone synthesis. Critically ill patients with persistent hypovolemia and hypotension also have inappropriately low plasma aldosterone concentrations in relation to the activity of the renin-angiotensin system. Isolated primary hypoaldosteronism in occasionally associated with metastatic cancer of the adrenal gland.

Defective Aldosterone Actions

The third category which is characterized by defective aldosterone action includes syndromes of aldosterone resistance such as pseudohypoaldosteronism type 1 and sodium-wasting states resulting from excessive amounts of circulating mineralocorticoid antagonists, such as spironolactone and its analogues, and synthetic progestin or natural agonists, such as progesterone or 17-hydroxyprogesterone. These mineralocorticoid antagonists may antagonize aldosterone at the levels of mineralocorticoid receptor (86) and frequently, these states are compensated for by elevated concentrations of plasma aldosterone.

HYPORENINEMIC HYPOALDOSTERONISM

The most common form of isolated hypoaldosteronism is caused by impaired renin release from the kidney. Hudson et al. first described this syndrome in 1957 (87), however, hyporeninemia was first recognized in 1972 (88) (89). The typical patient is 50 to 70 years old and usually presents with chronic and asymptomatic hyperkalemia and mild to moderate renal insufficiency with a 40-70% decrease in the glomerular filtration rate when compared to that of age matched healthy subjects. Hyperchloremic metabolic acidosis is seen in approximately half of the patients. This acidosis is classified as a renal tubular acidosis type IV (90). The acidosis is a consequence of decreased renal ammonia neogenesis, reduced hydrogen ion-secretory capacity in the distal nephron, and mild reduction in the proximal tubular threshold for bicarbonate reabsorption. Occasionally, muscle weakness or cardiac arrhythmias are present in some patients. More than a half of the patients have diabetes mellitus (91). Other frequently associated states include autonomic neuropathy, hypotension, and various nephropathies such as glomerulonephritis, gouty nephropathy, and pyelonephritis. Also, this syndrome is associated with nephropathies associated with multiple myeloma and systemic lupus erythematosus, mixed cryoglobulinemia, nephrolithiasis, analgesic nephropathy, renal amyloidosis, IgA nephropathy, cirrhosis, sickle cell anemia, acquired immune deficiency syndrome (AIDS), polyneuropathy, organomegaly, endocrinopathy, M protein and skin changes (POEMS) syndrome, lead poisoning, excess sodium bicarbonate, and Sjogren’s syndrome (90,92–101) . Moreover, this syndrome occurs transiently in association with use of non-steroidal anti-inflammatory drugs, cyclosporin A, mitomycin C, cosyntropin, and other agents in susceptible individuals (102–104).

Pathophysiology

Urinary aldosterone excretion is low under basal conditions and fails to increase after sodium restriction. Plasma renin activity is also low and does not increase appropriately during sodium restriction, periods of prolonged upright posture, or diuretic administration (88). Interstitial renal disease and damage to the juxtaglomerular apparatus seems the most likely cause for the primary defect in renin generation or release and secondary deficiency of aldosterone. However, in some patients with this syndrome there is an absent or blunted aldosterone response to angiotensin II (94,104), suggesting a coexisting primary defect in aldosterone secretion or it reflects atrophy of the zona glomerulosa caused by chronic renin deficiency.

There are various mechanisms to be explained for the hyporeninemia. First possible mechanism is the hypervolemia. The expanded extracellular fluid volume due to hypertension may suppress renin. In fact, long-term sodium restriction and diuretic administration increase plasma renin activity in these patients, however, the increments of plasma renin activity are less than those of normal subjects (97). A second possible mechanism is insufficiency of the autonomic nervous system, particularly in patients with diabetic neuropathy. Impaired adrenergic response to postural change may contribute to insufficient renin release. Besides, these patients exhibit decreased sensitivity to β-adrenergic agonists, suggesting defects in both production and action of catecholamines (96). A third proposed mechanism is secretion of abnormal forms of renin, such as a defect in the conversion of prorenin to renin. Insufficiency of autonomic nervous system may be associated with impaired conversion of prorenin to renin. Indeed, patients with diabetes mellitus and autonomic neuropathy have elevated plasma levels of prorenin (105). A fourth possibility is prostaglandin deficiency. Production of prostaglandin I2 (prostacyclin), which mediates renin release, is apparently diminished in patients with hyporeninemic hypoaldosteronism as assessed by measurement of the stable urinary metabolite 6-keto-prostaglandin F1α (95). Furthermore, the prostaglandin I2 in these patients was unresponsive to the potent stimulator’s norepinephrine and calcium. Prostaglandin I2 deficiency may cause hyporeninemic hypoaldosteronism by causing defects in the conversion of prorenin to renin and renin release (106).

Diagnosis

The diagnosis of hyporeninemic hypoaldosteronism must be considered in any patient with unexplained hyperkalemia. Excess potassium intake from food or drugs does not cause sustained hyperkalemia, if renal function is normal. Renal function should be evaluated and drugs that impair renal potassium excretion should be excluded as a cause. The clinical diagnosis is confirmed by low plasma renin activity and low plasma concentrations or urinary aldosterone excretion under conditions that activate the renin-angiotensin-aldosterone axis by maintenance of upright posture and/or furosemide administration. A low random plasma renin concentration associated with a normal ratio of aldosterone to plasma renin activity is also useful for the diagnosis (94).

Therapy

The therapeutic approach should be chosen after taking into consideration the age of the patients and other concurrent disorders. Only monitoring potassium concentrations is enough for patients with moderate hyperkalemia and without electro-cardiographic changes. Drugs that promote hyperkalemia, such as β-adrenergic antagonists, cyclooxygenase inhibitors, angiotensin-converting enzyme inhibitors, heparin, and potassium-sparing diuretics, should be avoided. Dietary potassium intake should be reduced, if possible. Diuretics are the initial treatment for patients who have disorders associated with sodium retention, such as hypertension and congestive heart failure. Mineralocorticoid replacement with fludrocortisone is reserved for patients with severe hyperkalemia without hypertension and congestive heart failure.

PRIMARY HYPOALDOSTERONISM- ALDOSTERONE SYNTHASE DEFICIENCY (ASD)

Congenital hypoaldosteronism is a rare inherited disorder transmitted as either an autosomal recessive or autosomal dominant trait with mixed penetrance. This disorder was previously termed "corticosterone methyloxidase (CMO)” deficiency and subdivided into two types according to the relative levels of aldosterone and its precursors in an affected person. Patients with "corticosterone methyloxidase I (CMO I)" deficiency have elevated serum levels of corticosterone and low levels of 18-hydroxycorticosterone and aldosterone. In contrast, patients with "corticosterone methyloxidase II (CMO II)" deficiency have high levels of 18-hydroxycorticosterone, the immediate precursor of aldosterone (107). With greater understanding of structure-activity relationships in the CYP11B2 enzyme, this disorder may be better considered a spectrum of hormonal deficiencies, depending on the nature of the CYP11B2 gene defect (108). Two steps of aldosterone biosynthesis from corticosterone previously proposed to be catalyzed by separate enzymes, CMO I and II, previously, are known to involve only one enzyme substrate interaction (6). Isolated aldosterone deficiency results from loss of activity of aldosterone synthase encoded by CYP11B2 gene (109–118). Therefore, the term aldosterone synthase deficiency type 1 (ASD1) and type 2 (ASD2) reflects more appropriately the molecular basis of this disease. In both ASD1 and 2, glomerulosa zone corticosterone is increased and aldosterone decreased, but 18-hydroxycorticosterone is increased in ASD2 (108).  ASD1 is associated with loss of both 18-hydroxilation and 18-oxidation enzyme activities. In ASD2, the ability to convert corticosterone (B) to 18-hydorxytetrahydro11-dehydrocorticosterone (18-OH-B) is preserved with failure of further oxidation of 18-hhdroxicorticosrerone to aldosterone (119). The deficiency of aldosterone is much more severe in ASD1. In contrast, aldosterone may reach normal levels under intense stimulation of renin-angiotensin system in ASD2 (108). The clinical presentations of these deficiencies are otherwise similar.

Clinical Presentation

The clinical presentation is typical of aldosterone deficiency, including electrolyte abnormalities such as a variable degree of hyponatremia, hyperkalemia and metabolic acidosis, with poor growth in childhood, but there are usually no symptoms in adults (107,120). Miao et al. reviewed 45 ASD patients (20 of ASD1, 12 of ASD2, 13 of undefined subtype) (121).  From their review, 95% of the patients having ASD1 and all of having ASD2 and an undefined subtype had hyponatremia, while 89% showed hyperkalemia. In infants, it is characterized by recurrent dehydration, salt wasting and failure to thrive. These symptoms are present generally within the first 3 months of life, and most often after the first 5 days of life. A modest uremia with a normal creatinine level reflects dehydration in the presence of intrinsically normal renal function. Plasma renin activity might vary, while elevated plasma renin activity levels were more likely to be found in the ASD1 (121).

Diagnosis and Therapy

The diagnosis can be established by measuring the appropriate corticosteroids or their major metabolic products, such as 11-deoxycorticosterone (DOC), corticosterone, 18-hydroxycorticosterone, 18-hydroxy-DOC, and aldosterone levels in plasma. The ratio of plasma 18-hydroxycorticosterone to plasma aldosterone differentiates the two disorders; it is less than 10 in ASD1 (CMO I deficiency) and more than 100 in ASD2 (CMO II deficiency) (121,122). Patients with ASD2 (CMO II deficiency) tend to have increased plasma cortisol levels that may result from increased adrenal sensitivity to ACTH induced by the increased plasma angiotensin II levels in response to sodium depletion (123).

Both forms of the syndrome are treated by replacement of mineralocorticoid with the usual dosage of fludrocortisone (0.1-0.3 mg/ day). Almost infants and children require oral sodium supplementation (2 g/day as NaCl alone or in combination with NaHCO3), although some infants with severe symptom need intravenous fluids. Oral sodium supplementation may be discontinued once plasma rennin activity has decreased to normal, but mineralocorticoid replacement is usually maintained through childhood.

Molecular Mechanism of CYP11B2 Deficiency

ASD has been identified in Jews of European, North American, and Iranian descent (119). In Asians, it was reported in the Thai (124), Indian (124), Japanese (125) and Chinese populations (120,126).

To date, approximately 40 mutations, such as missense and nonsense mutations, splicing mutations, small insertions/deletions, gross deletions, and complex rearrangements, in the CYP11B2 have been reported in cases of ASD; the most common mutations were missense and nonsense (121). Some variants, such as p.Q170X, p.E198D, c.1398+2T>A, p. F233fsX*295, p.L462R, p.Q337X and p.Q272W, were identified in patients without an ASD classification subtype (121). A majority of mutations led to complete loss of enzyme activity, while in some mutations, such as V386A and R181W, double homozygosity was required for clinical phenotype (112,113,121).

Some patients with ASD1 (CMO I deficiency) have a homozygous 5 nucleotide deletion in exon 1 which leads to a frameshift and premature stop codon, resulting in the complete lack of enzyme production (109,110). A male Caucasian patient with ASD1 (CMO I deficiency) had a homozygous point mutation causing a R384P substitution, resulting in complete loss of 11 β- and 18-hydroxylase activity (111) (Figure 5). This suggests that the arginine-384 in aldosterone synthase is highly conserved and apparently quite important for enzyme activity.

A male infant of Turkish parents who presented with ASD1 had a homozygous missense mutation (L451F) in exon 8 of CYP11B2 gene. The L451F mutant protein in vitro showed complete aldosterone deficiency with 11-deoxycirticosterone or corticosterone as substrates. The L451F mutation located immediately adjacent to the highly conserved heme-binding C450 of the cytochrome P450 (117). Computer modeling of the molecule suggested that this substitute my lead a steric effect resulting in preventing the activity of CYP11B2 (117).

Three siblings of Pakistan origin who presented with ASD1 had a homozygous mutation (S308P) in exon 5 of CYP11B2 gene. The S308P mutant protein in vitro showed complete loss of enzyme activity. This mutated residue is likely to locate within the a-helix I, close to the heme-binding, active site of the enzyme. This structural change may be the cause of this disorder in this family (118). 

A large number of kindreds with ASD2 (CMO II deficiency) have been identified among Jews originally from Isfahan, Iran. Such patients are all homozygous for two mutations, R181W in exon 3 and V386A in exon 7 (109,112,113) (Figure 5). These mutations together reduce aldosterone synthase activity to 0.2 % of normal without affecting 11 β-hydroxylase activity (112,113). However, one non-Iranian patient with ASD2 (CMO II deficiency) carries mutations in the paternal allele, including V386A and T318A mutations, and maternal allele, including R181W and a deletion/frameshift mutation, resulting in complete loss of enzyme activity (113). This suggests that the high levels of 18-hydroxycorticosterone seen in ASD2 (CMO II deficiency) can be synthesized by CYP11B1, which has some 18-hydroxylase activity, and not by CYP11B2. A patient with apparent ASD 1 was homozygous for the mutations E198A and V386A, yet when assayed in vitro the double mutant enzyme behaved similarly to the mutant enzyme found in the Iranian Jewish ASD 2 patients (127). Thus, a difference in expression of CYP11B1 rather than allelic variation of CYP11B2 may be involved in the mechanism underlying the different levels of 18-hydroxycorticosterone between ASD1 and 2 (CMO I and CMO II deficiency). The distinction between ASD 1 and ASD 2 is not precise, and these disorders should be regarded as different degrees of severity on a continuous clinical spectrum.

 A male Japanese patient with ASD1 (CMO I) was a compound heterozygous for W56X in exon 1 and R384W in exon 7. W56X was inherited from his mother and R384X was from his father. Since both alleles contain nonsense mutations, a lack of CYP11B2 activity was speculated to cause his condition (125).

Two male Japanese patients with ASD2 (CMO II) had homozygous missense mutation (G435S) in the exon 8 of CYP11B2 gene. The expression studies indicated that the steroid 18-hydroxylase/oxidase activities of mutant enzyme were substantially reduced.

A female infant of Albanian origin with ASD2 (CMO II) revealed homozygosity for a pathogenic T185I mutation in Exon 3 of the CYP11B2 gene and two other homozygous polymorphisms F168F and K1738 in Exon3 (128). Both healthy parents revealed heterozygous for all three substitutions.

Another female Italian Caucasian patient was diagnosed with a compound heterozygous mutation located in exon 4 causing a premature stop codon (E255X) and a further mutation in exon 5, also causing a premature stop codon (Q272X). The patient’s CYP11B2 encoded two truncated forms of aldosterone synthase predicted to be inactive because they lack critical active site residues as well as the hormone-binding site. However, this case displays biochemical features intermediate between those of ASD1 and 2 (CMO I and II).

Some cases of ASD without causative mutations in CYP11B2 have also been reported (116,119).

Figure 5. Relative positions of CYP11B1 and CYP11B2 on chromosome 8 and mutations of CYP11B2. A, The relative positions of CYP11B1 and CYP11B2 on chromosome 8q22. Arrows indicate direction of transcription. B, Mutations of CYP11B2 in reported patients with CYP11B2 deficiency are summarized in the figure (109,121,126,128).

ACQUIRED FORMS OF PRIMARY HYPOALDOSTERONISM  

Several conditions may be associated with aldosterone biosynthetic defects. The administration of heparin causes natriuresis and hyperkalemia (129). Heparin preparations suppress aldosterone synthesis, leading to a compensatory rise in plasma renin activity. However, it has been demonstrated that this suppression of enzyme activity is attributable to chlorbutol (1,1,1-trichloro-2-methyl-2-propanol), the preservative used in commercial heparin, rather than to pure heparin (130).

Persistently hypotensive, critically ill patients with sepsis, pneumonia, peritonitis, cholangitis and liver failure, also have inappropriately low plasma aldosterone concentrations in relation to elevated plasma renin activity (131). The defect is at the level of the adrenal but has not been associated with any particular disease or therapy. Plasma cortisol levels are high, reflecting the stressed state. The response to angiotensin infusion is impaired, and the ratio of plasma 18-hydroxycorticosterone to aldosterone is increased, suggesting selective insufficiency of CMO II. It is possible that the hypoxia causes a relative zona glomerulosa insufficiency (132).

ALDOSTERONE RESISTANCE

Pseudohypoaldosteronism (PHA) Type 1

Mineralocorticoid resistance (pseudohypoaldosteronism type 1, PHA1) results from inability of aldosterone to exert its effect on its target tissues and was first reported by Cheek and Perry as a sporadic occurrence in 1958 (133). This disease, usually presents in infancy with severe salt-wasting and failure to thrive, accompanied by profound urinary sodium loss, severe hyponatremia, hyperkalemia, acidosis, hyperreninemia, and paradoxically markedly elevated plasma and urinary aldosterone concentrations. Usually, renal and adrenal functions are normal. This disease has been reported in over 70 patients (134). The prevalence, as estimated from recruitment through a genetic laboratory at the Hôpital Européen Georges Pompidou in France, which is a national reference center for a rare disease, is ~1 per 80,000 newborns (135)(136). Approximately one fifth of these cases are familial, and both an autosomal dominant and a recessive form of genetic transmission have been observed. A previous study found that all patients had renal tubular unresponsiveness to aldosterone, while some had involvement of other mineralocorticoid target-tissues, including the sweat and salivary glands, and the colonic epithelium, as well. Autosomal recessive PHA1 presents in the neonatal period with hyponatremia caused by multi-organ salt loss, including kidney, colon, and sweat and salivary glands. Autosomal recessive PHA1 persists into adulthood and shows no improvement over time. However, literature regarding follow-up of these patients after diagnosis is insufficient.  In contrast, autosomal dominant PHA1 is characterized by an isolated renal resistance to aldosterone, leading to renal salt loss. Particularly autosomal dominant form of PHA1 typically shows a gradual clinical improvement during childhood, allowing the cessation of sodium supplementation. 

PATHOPHYSIOLOGY

The mechanism(s) by which aldosterone controls sodium transport in its target tissues involves the mineralocorticoid receptor (MR) and proteins that are associated with the amiloride-sensitive sodium channel (ENaC). The latter proteins are expressed in the apical membrane of epithelial cells of the distal convoluted tubule and in the membranes of cells of other tissues involved in the conservation of salt, such as colon, sweat gland, lung and tongue. Thus, the MR and the ENaC were considered as potential candidate molecules for the pathogenesis of PHA1. In fact, mutations of α- and β-subunits of the ENaC were reported in PHA patients from autosomal recessive kindreds (61,137). Mutations of the MR were also reported in the patients with autosomal dominant PHA1 (138,139). However, no molecular defects were found in either MR or ENaC in some patients with PHA1, especially in those with the sporadic form PHA1, which suggests molecular heterogeneity in PHA1 (79,140–144).

DIAGNOSIS

Electrolyte profiles suggest mineralocorticoid deficiency or end-organ resistance, along with hyperkalemia, hyponatremia and metabolic acidosis associated with profound urinary salt loss. Renal and adrenal function is normal. The diagnosis is confirmed by the markedly elevated plasma aldosterone concentrations and plasma renin activity.

The differential diagnosis of PHA1 includes salt-wasting states due to hypoaldosteronism, including several forms of congenital adrenal hyperplasia, isolated hypoaldosteronism due to corticosterone methyloxidase (CMO) I and II deficiencies and congenital adrenal hypoplasia. Normal cortisol and excessive aldosterone responses to adrenocorticotropin (ACTH) are expected in patients with congenital PHA.

THERAPY         

The standard treatment of PHA has been replacement with high doses of salt, with a variable response among patients (134). Recently, carbenoxolone, an 11β-hydroxysteroid dehydrogenase inhibitor, was employed as therapy in PHA1 and an ameliorating effect was observed which was attributed to mediation by the MR (140). We studied a 17-yr-old male patient with congenital multifocal target-organ resistance to aldosterone. We examined his clinical response to carbenoxolone, expected to increase the intracellular level of cortisol in the kidney by preventing local conversion of cortisol to cortisone, and to high doses of fludrocortisone, a synthetic mineralocorticoid. Subsequently, and for a brief period of time, we administered dexamethasone, which has no intrinsic salt-retaining activity, in addition to carbenoxolone, to suppress endogenous cortisol, along with its intrinsic mineralocorticoid activity.

Figure 6. Effect of carbenoxolone, carbenoxolone plus dexamethasone, and fludrocortisone (top panel) on the serum sodium (middle panel) and potassium (bottom panel) concentrations of a patient with PHA. Carbenoxolone normalized plasma electrolytes, addition of dexamethasone reversed this effect, while fludrocortisone at high doses also normalized plasma electrolytes (140).

Carbenoxolone normalized the patient's serum electrolyte concentrations and decreased his urinary excretion of sodium within a week (Figure 6). Subsequent long-term therapy of this patient with carbenoxolone (450 mg/day p.o.) maintained his electrolyte concentrations within the normal range. His urinary 24 h free cortisol was increased during carbenoxolone therapy. Addition of dexamethasone suppressed his urinary free cortisol excretion and reversed the beneficial effect of carbenoxolone on serum and urinary electrolytes (Figure 6). These data suggest that an increase in urinary free cortisol observed during carbenoxolone therapy was due to a localized effect of this drug on the kidney rather than on tissues involved in the negative feedback effect of glucocorticoids. The effect of carbenoxolone does not seem to be mediated by GR but seems to be exerted purely via the MR (Figure 7). There were no adverse effects of long-term carbenoxolone therapy in this patient. He also reported increased stamina, a better ability to concentrate and less anxiety. On treatment, the patient grew 6 cm/y and progressed from -4SD to -3SD scores for mean height for age. He also progressed in his pubertal development from Tanner stage III to IV for pubic hair, while his bone age advanced from 12 to 14 y.

Figure 7. Mechanism of the effect of carbenoxolone. Carbenololone inhibits of conversion of cortisol to cortisone in the kidney, resulting in the enhancement of the effect of cortisol as a ligand for MR. Dexamethasone suppressed cortisol production and reversing the beneficial effect of carbenoxolone in our patient with PHA1.

Both carbenoxolone and fludrocortisone normalized the serum electrolytes of our patient, suggesting the presence of a functional, albeit possibly defective, renal MR. Interestingly, the same patient was unresponsive to intravenous infusion of aldosterone and fludrocortisone (up to 3 mg/day) when studied in infancy (145), suggesting that the clinical improvement that has been noted in the majority of PHA patients with age may be related to changes in their responsiveness to mineralocorticoid.

On the other hand, another study reported that carbenoxolone did not show any significant salt-retaining effect in two patients with multiple PHA, while carbenoxolone significantly suppressed the renin-aldosterone system in a patient with renal-form PHA (146).  This difference of responsiveness to carbenoxolone may be due to an age-dependent change on mineralocorticoid responsiveness. Additionally, the different mineralocorticoid responsiveness of renal and multisystem PHA patients indicates a difference in their MR function. The partial response to carbenoxolone in renal PHA suggests that there is at least a partly functional MR. This is also supported by the observation that spironolactone, a mineralocorticoid antagonist, aggravated sodium loss in several patients with renal PHA (147).

MOLECULAR MECHANISM(S) OF PSEUDOHYPOALDOSTERONISM TYPE 1          

In 1996, a study reported homozygous mutations introducing a stop codon or frame shift in the αENaC gene of affected members of families with autosomal recessive PHA (61).  To date, worldwide more than 40 different mutations have been described in the coding region of ENaC subunit genes (148–150). The majority of mutations appear in the αENaC gene SCNN1A, most frequently in exon 8 (61,150–152). Mutations are nonsense, single base deletions or insertions, or splice-site mutations, leading to abnormal length of mRNA and protein. Few missense mutations in αENaC gene have also been reported (149,153). Only a few cases of mutations in β and gamma ENaC genes have been reported (149,154,155). Phenotype and genotype correlations have been noted with more severe phenotype in nonsense, frameshift, and abnormal splicing mutations than patients with missense mutations (148,154,155).

A Swedish study regarding families with autosomal recessive PHA, homozygous or compound heterozygous mutations showed that a stop codon or a frame shift in the αENaC gene was associated with pulmonary disease as well (150). The truncation caused by these mutations influenced the PY motif at the N-terminal region of the molecule. This motif is responsible for the binding of the channel subunits with Nedd4, a carrier protein facilitating clearance of the channel (60). Moreover, a point mutation of the αENaC gene, located close to the N-terminal of the protein, was reported to cause a decrease of the probability of an open sodium channel, resulting in defective reabsorption (61,153). In the other four families with autosomal recessive PHA, insertion of a T in exon 8 and nonsense mutation (R508X) in exon 11 of the αENaC gene, resulting in a truncated αENaC subunit, was found (156). A splice site mutation in intron 12 of the βENaC gene, which preventing correct splicing of the mRNA was found in a Scottish patient (156). Also, other autosomal recessive families with PHA had a homozygous splice-site mutation in the γENaC, while a Japanese sporadic patient with the systemic form of PHA was a compound heterozygote for mutations in the αENaC, which resulted in the generation of a truncated channel subunit (137,157) . Compound heterozygous mutations (Q217X in exon 4 and Y306X in exon 6) of βENaC have been reported in the patient with multi-organ PHA1 of Ashkenazi family in Israel (154). These mutations produce shortened βENaC subunits with 253 and 317 residues respectively instead of the 640 residues present in βENaC subunit. Expression of cRNA carrying these mutations in Xenopas oocytes showed that the either mutation drastically reduced to only 3% of normal ENaC activity (154).  An African American female with PHA, who had persistent and symptom hyperkalemia, had compound heterozygous mutation in the βENaC gene: c.1288delC in exon 9, a one-base deletion that generated a frameshift mutation, and c.1466+1 G>A, an intronic base substitution in intron11 that leaded to a splice site mutation (158).

To date more than 50 different mutations in the human MR gene (NR3C2) causing autosomal dominant PHA1 have been described. NR3C2 mutations were found in 62% of patients with renal PHA1 referred to a genetics laboratory at the Hôpital Européen Georges Prompidou in France (135). Nonsense mutations, frameshift mutations, splice site mutations, and deletions of whole or part of the gene lead to gross change of the MR protein. Nonsense mutations are found in all exons and lead to truncated MR protein. A past study. reported families with autosomal dominant PHA, who had molecular defects of the MR resulting in non-expression of one of the 2 alleles (138) (Figure 8). In addition, another study reported a sporadic patient with PHA who had a heterozygous mutation in exon 9 of the MR that introduced a premature stop codon (144) (Figure 8). These results, may suggest that expression of only one allele of the MR is insufficient to prevent salt loss. Another case study did not identify any abnormalities of the MR in PHA patients from two families with the autosomal dominant form of the disease (144), while other authors reported a heterozygous missense mutation in exon 8 of the MR gene identified in PHA patients from a Japanese autosomal dominant family (139) (Figure 8). A heterozygous nonsense mutation in exon 2 (S163X, C436X) and in exon 9 (R947X) of the MR, leading to a premature stop codon of the MR gene were found in other patients with autosomal dominant PHA (159–161). It was previously reported a heterozygous splice acceptor site mutation, which results in exon 7 skipping and subsequently in premature termination in exon 8 of MR with Japanese female patients with PHA1 (162). This study showed that RT-PCR products of mRNA with that patient showed both wiled-type and mutated mRNA, suggesting that haploinsufficiency due to nonsense mediated mRNA decay with premature termination is not sufficient to give rise to the PHA phenotype (162). It was also reported that Q776R mutation in exon 5 or L979P mutation in exon 9, which is located in the ligand-binding domain of the MR, presented reduced or absent aldosterone binding, respectively (163). Three-dimensional structure of MR suggests that the residue Q776 is located in helix 3 and is locking aldosterone in the ligand-binding pocket (163). A study examined patients with PHA1 presenting isolated renal salt loss from six families in Italy and Germany and found one nonsense mutation (E378X), one frameshift mutation (A958R) and two missense mutations (S818L and E972G) (164). S818L does not bind aldosterone or activate transcription or translocate into the nucleus. Three-dimensional molecular structure showed that S818 was located in helix H5 and S818 was speculated to be necessary to stabilize helix H5 and the -sheet 1 via hydrogen bond to Y828. E972G mutation showed a significantly lower ligand-binding affinity and only 9% of wild-type transcriptional activity. Three-dimensional molecular structure showed that E972 is involved in a hydrogen-bond network with R947 anchoring helix H12 to H10. Thus, substitute of E972G suggested to be open up the hydrophobic core and displace helix H10, causing the decreased ligand-binding ability (164).

A Japanese study reported four sporadic patients and two siblings with a renal form of PHA (165). Two siblings and one sporadic patient had R651X of NR3C2 (MR) gene. One sporadic patient had R947X, another two patients had 603A deletion and 304-305CG deletion, respectively, both resulting in frameshift mutations (165).

Another study reported two female Japanese infants with the renal form of PHA1 and identified two heterozygous mutations. One had a c.4932_493insTT in Exon 2, resulting in a premature stop codon (p.Met166 LeufsX8) and another had a nonsense mutation of R861X in exon 7 (166).  These mutations resulted in haploinsufficiency of the MR and were the cause of aldosterone resistance in the kidney.

From the study of the genetics laboratory at the Hôpital Européen Georges Pompidou in France, 20 mutations were found in exon 2; all of them led to truncated receptors, Of the 22 mutations identified in exon 3 and 4, coding for the MR DBD, 11 were nonsense or frameshift mutations, the reminder missense mutations. Thirty variants were located in exon 5-9 and affected LBD; the majority were missense mutations. Nine were splice variants in different introns, 19 were large deletions encompassing single or multiple exons and the flanking intronic regions of the NR3C2 gene (135) (figure 8).

These studies suggest major molecular heterogeneity in PHA.

Figure 8. Mutations of the MR in patients with PHA1. Mutations of the MR that have been reported in patients with PHA1 are summarized in the figure (135,138,139,144,166). 

Another study investigated 5 unrelated cases of sporadic PHA (79,140,143). The researchers found a nonconservative homozygous mutation (A241V) in the MR of 4 of the patients and a conservative heterozygous mutation (I180V) in one of these patients and his asymptomatic father, while no abnormalities were found in the DNA- or ligand-binding domains of the MR. The Val241 and Val180 substitutions were found also in the norm 6al population. The heterozygosity and homozygosity frequencies of the Val241 and Val180 mutations were 48%, 38%, 22% and 1.5%, respectively. Another finding was a nonconservative amino acid substitution (T663A) in the αENaC, which was located close to the C-terminal (79). Of the 5 patients, 2 were homozygous and 3 heterozygous for this variation, respectively. This amino acid substitution was also present at high frequency in apparently normal controls. The homozygosity and heterozygosity frequencies of the αENaC Ala663 were 31% and 64%, respectively. Three of the 4 (75%) patients with multiple tissue resistance to aldosterone had both αENaC (heterozygous or homozygous) and MR (homozygous) mutations as described above, while only 7% of our controls with apparently normal salt conservation had the same concurrent abnormalities (Table 2, p < 0.025).

Table 2. MR and aENaC Polymorphisms in PHA and Normal Subjects

 

 MR

 αENaC

 Target organ

 

 I180V

 A241V

 T663A

 

 

 Homo

 hetero

 homo

 Hetero

 homo

 Hetero

 

 

 Pt.1

 

 

 

+

 

+

 

 

 

 

 

+

 

 Multiple

 Pt.2

 

 

 

 

 +

 

 Multiple

 Pt.3

 

 

 +

 

 +

 

 Multiple

 

 Pt.4

 

 

 

 

 

 

 

 

 

 

 

+

 

 Multiple

 

 Pt.5

 

 

 

 

 

+

 

 

 

 

 

+

 

 Isolated

 

controls

 

1.5%

 

22%

 

38%

 

48%

 

31%

 

64%

 

 

 

controls

 

 

 

+

 

+

 

 

 

 

 

+

 

 

 

controls

 

 

 

 

 

+

 

 

 

+

 

 

 

 

 

controls

 

 

 

 

 

+

 

 

 

 

 

+

 

 

                (79) with permission

The researchers identified, in a Japanese patient with sporadic PHA, three homozygous substitutions in the MR gene: G215C, I180V or A241V, which had previously reported to occur in healthy populations. Luciferase activities induced by MR with either G215C, I180V or A241V substitution were significantly lower than those for wild-type MR with aldosterone at concentrations ranging from 10-11 to 10-9 M, 10-8M, or 10-11 to 10-6M, respectively. A homozygous A to G substitution of the donor splice site of αENaC intron 4 was found in the patient. These results suggest that each of three MR polymorphisms identified in our patient is functionally and structurally heterogeneous (167).

The authors suggested that the above polymorphisms may confer vulnerability in salt conservation, which might be expressed fully only when concurrently present with other genetic defects of the MR or other proteins that participate in sodium homeostasis, such as Nedd4 (168). This hypothesis, if true, would be compatible with a sporadic presentation or a digenic or multigenic expression and heredity as previously described in retinitis pigmentosa (169). In this case, hereditary transmission might be complex and appear either as a dominant and/or recessive trait with variable penetrance.

Secondary Pseudohypoaldosteronism (PHA)

Secondary PHA is a form of renal resistance to aldosterone. The cause of secondary PHA is either renal disease or medication. The clinical and laboratory findings resemble those of a transient PHA. Since Rodriguez-Soriano et al. reported the first case in 1983 (169), more than 68 cases have been reported. Secondary PHA may occur mainly in neonates and young infants with urinary tract infections, such as pyelonephritis, and/or malformation of urinary system causing obstructive uropathy, tubulointerstitial nephritis, sickle cell nephropathy, and systemic lupus erythematosus(170). Secondary PHA has been also related to drugs like non-steroidal anti-inflammatory agents and potassium-sparing diuretics (170–172). This state occurs in male infants more frequently than female infants because of the higher incidence of urinary tract infections and obstructive uropathy in male infants rather than in female infants(169). Patients present poor feeding, poor weight gain or failure to thrive, vomiting, diarrhea, polyuria, and dehydration. Acute worsening of their general condition may occur, with severe weight loss, peripheral circulatory failure, rise in serum urea and creatinine levels, and occasional life-threatening hyperkalemia (169). The laboratory features are hyponatremia, hyperkalemia, metabolic acidosis, elevation of plasma aldosterone concentrations and plasma renin activity, and inappropriately increased sodium and decreased potassium excretion in urine (173).  The aldosterone resistance of secondary PHA is transient and usually reverts with the resolution of the infection.

PATHOPHYSIOLOGY

The very high ratio of plasma aldosterone to potassium, together with diminished urinary K/Na values, strongly suggests that hyponatremia and hyperkalemia result from a lack of response of the renal tubule to endogenous mineralocorticoids (174). The intrarenal expression of several cytokines, such as tumor necrosis factor alpha, interleukin (IL) 1, IL-6, transforming growth factor beta-1, angiotensin II, endothelin, thromboxane A2, and prostaglandins, are increased in cases of urinary tract infections. These changes result in inhibition of aldosterone action through reduction of its expression and/or impairment of its receptor, vasoconstriction and reduction of glomerular filtration rate, increased natriuresis and/or decreased Na+-K+-ATPase activity(173) . A past study found that the number of mineralocorticoid receptors in obstructive uropathy were low in the acute phase but returned to normal after successful surgical correction of the obstruction (175). This suggests that a reduced aldosterone effect can also reflect down-regulation of the receptor sites, due to highly elevated aldosterone levels (175).

THERAPY

The clinical and laboratory findings improve within one or two days and disappear after the completion of medical treatment of urinary tract infection and/or surgical correction of obstructive uropathy, usually within a few days to one week after beginning of treatment (173). However, in some patients, sodium bicarbonate and/or sodium chloride supplementation may be necessary for a week or month (173)

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AIDS AND HPA Axis

ABSTRACT

The Acquired Immunodeficiency Syndrome (AIDS), caused by infection with the Human Immunodeficiency Virus Type-1 (HIV), is characterized by profound immunosuppression, particularly of the innate, and T-helper (Th) 1-directed immunity. AIDS causes multisystem dysfunction, including impairment of the hypothalamic-pituitary-adrenal (HPA) axis, a major system coordinating the resting state and the adaptive response to stress. This neuroendocrine axis consists of three components: the hypothalamus, the pituitary gland, and the adrenal cortex with its end-effector molecules, the glucocorticoids. AIDS/HIV influence the HPA axis directly, through modulation of the host immune activity and alterations of the cellular biological pathways via HIV-encoded proteins, as well as indirectly, through immunodeficiency-associated opportunistic infections and various side effects of the therapeutic compounds employed, including those used in the highly active antiretroviral therapy (HAART). In this chapter, the interaction between AIDS/HIV and the HPA axis is reviewed and discussed.

INTRODUCTION

Patients with Acquired Immunodeficiency Syndrome (AIDS), caused by infection with the Human Immunodeficiency Virus Type-1 (HIV), develop profound immunosuppression, particularly of their innate and T-helper (Th) 1-directed cellular immunity (1). These patients may also present with dysfunction of many organ systems, including the hypothalamic-pituitary-adrenal (HPA) axis (2). During the last 25 years, numerous reports have provided evidence for alterations of the HPA axis and its influence on target tissues in HIV-infected patients (Table 1). Indeed, AIDS has been associated with adrenalitis caused by opportunistic infections, adrenal dysfunction secondary to neoplastic infiltration into the adrenal cortices, and changes related to circulating cytokines and other bioactive molecules known to influence functions of the HPA axis (3). Glucocorticoid hormones secreted from the adrenal cortex act as end-effectors of the HPA axis and have strong anti-inflammatory effects (4). Thus, these hormones were considered for reversing HIV-mediated depletion of circulating CD4+ lymphocytes and slowing progression to AIDS, as well as to subside complications associated with HIV infection (5) (Table 2).

Table 1. Impact of HIV infection on the HPA Axis/Glucocorticoid/GR Signaling System

Manifestations

Virus-mediated

Treatment-mediated

Adrenalitis (Common) and adrenal insufficiency (Rare)

Ö

 

Pituitary (corticotroph) dysfunction

Ö

 

GR affinity-dependent generalized glucocorticoid resistance

Ö?

 

Modulation of glucocorticoid metabolism

Ö

Ö

Modulation of GR activity

Ö

Ö

AIDS-related insulin resistance/lipodystrophy syndrome

Ö

Ö

Fatigue/muscle wasting

Ö?

 

 

Table 2. Conditions/Manifestations in which Glucocorticoid Treatment is Considered in HIV-Infected Patients

Conditions/manifestations

Types of conditions/manifestations

AIDS-related lymphoma (Hodgkin and non-Hodgkin)

Complication

HIV-associated nephropathy

Complication

Kaposi sarcoma*

Complication

Appetite loss/fatigue

Complication

Opportunistic infections (mycobacterium tuberculosis, cryptococcus)

Complication

HIV-associated immune reconstitution inflammatory syndrome

Complication

Slowing of AIDS progression (increase of CD4+ counts)

Direct effect on HIV replication

*Acceleration of Kaposi sarcoma by glucocorticoids (110)

 

Although development of HIV vaccines targeting components of the viral particles is still challenging, establishment and clinical introduction of the highly active antiretroviral therapy (HAART) that employs combinatory use of the three different types of antiretroviral drugs, such as nucleoside and non-nucleoside analogues acting as reverse transcriptase inhibitors, non-peptidic viral protease inhibitors (PIs) and the compounds blocking entry of HIV to CD4+ lymphocytes, efficiently suppress HIV replication in infected patients and have dramatically improved clinical course and life expectancy of AIDS patients (6-9). However, the prolongation of lives with long-term use of the above antiretroviral agents have generated novel morbidities and complications, which influence the patients’ quality of life and add new risk factors for premature death. Central among them is the quite common AIDS-related insulin resistance and lipodystrophy syndrome (ARIRLS), which is characterized by a striking phenotype and marked metabolic disturbances that are reminiscent of Cushing syndrome (10). In agreement with above-indicated clinical background, acquired alterations in the sensitivity of tissues to glucocorticoids were originally hypothesized in AIDS patients, and this concept was further extended to other nuclear receptor (NR) family proteins. In addition, some PIs inhibit the cytochrome p450 enzyme CYP3A4, which is necessary to metabolize glucocorticoids into inactive forms (11). Thus, the pharmacologic action of glucocorticoids used in the AIDS patients treated with these compounds is pronounced due to slowing of their metabolism, and “iatrogenic” Cushing syndrome is subsequently developed in these patients (12).

AIDS patients frequently develop several different types of malignancies, such as lymphoma and Kaposi sarcoma, in part due to profound destruction of host immune system by HIV (13,14). Glucocorticoids are among the central compounds for the treatment of the patients harboring these malignancies (13,15). Glucocorticoids are also pivotal for the treatment of HIV-associated nephropathy, which is observed in about 10% of AIDS patients (16). Use of glucocorticoids is further considered for the patients with HIV-associated tuberculosis and other opportunistic infections as part of the immunoadjuvant therapy (17,18).

In this chapter, we will explain known interactions between HIV infection and the HPA axis, particularly focusing on glucocorticoid hormones. We also present our understanding on some emerging concepts of such interactions, and discuss their possible mechanisms and relevance to HIV pathogenesis. 

HPA AXIS AND GLUCOCORTICOID ACTIONS

Humans face unforeseen short- and long-term environmental changes called “stressors”, which can be external (e.g. excessive heat or cold, food deprivation, trauma and invasion by pathogens) or internal (e.g. hurtful memories, splachnic injuries, neoplasia’s) (19-22). To adapt to these changes, humans have the stress-responsive system, which senses such stressors through various peripheral sensory organs, processes them in the central nervous system (CNS), and adjusts the CNS and peripheral organ activities (19-22). The hypothalamic-pituitary-adrenal (HPA) axis with its end-effectors glucocorticoids is one of the two arms of this regulatory system, together with the locus caeruleus/norepinephrine-autonomic system and their end-effectors, norepinephrine and epinephrine (19,21,22). At baseline, activity of the HPA axis and circulating glucocorticoid levels are in a typical diurnal rhythm, reaching their zenith in the early morning and their nadir in the late evening in diurnal animals including humans through input from a circadian rhythm center, the suprachiasmatic nucleus (SCN), and they participate in the maintenance of internal homeostasis (20,23,24). Upon exposure to stressors, the HPA axis is liberated from this regular circadian rhythm, and is strongly activated to modulate many biological activities including those of the CNS, intermediary metabolism, immunity and reproduction via highly elevated circulating glucocorticoids (4,19-25). However, this stress-induced activation of the HPA axis may also exert an array of adverse effects when its response is not properly tailored to the stressful stimuli (25). For example, acute hyper-activation of the HPA axis has been associated with development of post-traumatic stress disorder, while chronic activation of the HPA axis, and consequently prolonged elevation of serum glucocorticoid levels, induce visceral-type obesity and insulin resistance/dyslipidemia, which are represented as metabolic syndrome (19,21-25).

The HPA axis consists of the hypothalamic PVN parvocellular corticotropin-releasing hormone (CRH)- and arginine vasopressin (AVP)-secreting neurons, the corticotrophs of the pituitary gland, and the adrenal gland cortex (3,21-24) (Figure 1A). The PVN neurons release CRH and AVP into the hypophyseal portal system located under the median eminence of the hypothalamus in response to stimulatory signals from higher brain regulatory centers (3,21-24). Secreted CRH and AVP reach the pituitary gland and synergistically stimulate secretion of the adrenocorticotropic hormone (ACTH) from corticotrophs (19,21-24,26). ACTH released into systemic circulation finally stimulates both production and secretion of glucocorticoids (cortisol in humans and corticosterone in rodents) from the zona fasciculata of the adrenal cortex (25). Secreted glucocorticoids modulate activity of virtually all organs and tissues to adjust their functions. In addition, these hormones suppress higher regulatory centers of the HPA axis, the PVN and the pituitary gland, ultimately forming a closed negative feedback loop that aims to reset the activated HPA axis and restore its homeostasis (19).

Figure 1. The HPA axis and intracellular actions of GR

Organization of the HPA Axis

The HPA axis consists of 3 components: the PVN of hypothalamus, the anterior pituitary gland and the adrenal cortex. Neurons residing in PVN produce CRH and AVP and release them into the pituitary portal vein under the control of upper centers, including the central circadian rhythm center, hypothalamic suprachiasmatic nucleus (SCN). Released CRH and AVP stimulate secretion of ACTH from corticotrophs of the anterior pituitary gland. ACTH then stimulates the production and secretion of glucocorticoids (cortisol in humans and corticosterone in rodents) from adrenocortical cells located in zona fasciculata of the adrenal gland. Circulating glucocorticoids suppress upper regulatory centers including PVN and pituitary gland, forming a closed regulatory loop.

Intracellular Actions of GR

In the absence of glucocorticoids, GR resides in the cytoplasm forming a heterocomplex with several heat shock proteins (HSP). Upon binding to glucocorticoids, GR releases HSPs and translocates into the nucleus. In the nucleus, GR directly binds its specific sequence called glucocorticoid response elements (GREs) located in the promoter/enhancer region of glucocorticoid-responsive genes as a homodimer, and stimulates transcription by attracting many transcriptional cofactors and the RNA polymerase II complex. GR also modulates transcriptional activity of other transcription factors through physical protein-protein interaction without associating directly to DNA. After regulating transcription of glucocorticoid-responsive genes, GR moves back into the cytoplasm with help of the nuclear export system and returns to its ligand friendly condition by reforming a heterocomplex with HSPs. This complex regulatory system for the GR intracellular activity is sensitive to many inputs from other intracellular regulatory systems in order to adjust net GR actions upon local needs. [modified from (27)]

Infection of pathogens including HIV potently activates the HPA axis and induces subsequent secretion of glucocorticoids from the adrenal cortex (28,29). Pathogens generally stimulate central part of this regulatory system (e.g., brain hypothalamus and pituitary corticotrophs) directly with their structural and genetic components, and indirectly with cytokines and inflammatory mediators, such as the tumor necrosis factor a (TNFa), interleukin (IL)-1 and IL-6, secreted from activated immune cells and/or infected tissues (30). Secreted glucocorticoids in turn subside inflammation, functioning as a counter regulatory mechanism for otherwise overshooting immune response (31). Glucocorticoids do this mainly by suppressing release of humoral inflammatory mediators, granulocyte migration, cellular immunity and production of Th1 cytokines, such as IL-12, TNFa and the interferon (IFN) g, while they stimulate humoral immunity and secretion of Th2-inducing anti-inflammatory cytokines, including IL-4, IL-10 and the transforming growth factor b (4,32,33).

Glucocorticoids exert profound influences on many physiologic functions by virtue of their diverse roles in growth, development, and maintenance of cardiovascular, metabolic and immune homeostasis (4,34,35). Excess amounts of glucocorticoids have strong effects on intermediary metabolism, developing insulin resistance/overt diabetes mellitus and hyperlipidemia (especially triglycerides and free fatty acids) through modulation of their broad target regulatory systems/molecules (4). As glucocorticoids possess potent anti-inflammatory and immunosuppressive activities, they are used as invaluable therapeutic means in inflammatory and autoimmune diseases (36). In addition, glucocorticoids are central components of the anti-cancer treatment especially for hematologic malignancies, such as leukemia and lymphoma (4).

Glucocorticoids exert their effects on their target cells through the glucocorticoid receptor (GR), a ligand-specific and -dependent transcription factor, ubiquitously expressed in almost all tissues and cells (21-24,28). There are two 3’ splicing variants, GRa and GRb, through alternative use of a different terminal exon termed 9a or 9b. GRa is the classic glucocorticoid receptor, which binds glucocorticoids and transactivates or transrepresses glucocorticoid-responsive genes (37). GRa shuttles between the cytoplasm and the nucleus; Binding of glucocorticoids to GRa causes it to dissociate from the cytoplasmic hetero-oligomer containing heat shock proteins and to translocate into the nucleus through the nuclear pore (28) (Figure 1B). Ligand-bound and nucleus-translocated GRa binds as a homo-dimer to specific DNA sequences called glucocorticoid response elements (GREs) located in the promoter/enhancer regions of glucocorticoid-responsive genes to modulate their transcription (28). On the other hand, GRb does not bind glucocorticoids and functions as a dominant negative inhibitor of GRa on GRE-containing glucocorticoid-responsive promoters, together with its intrinsic transcriptional activity on the genes not related to glucocorticoid transcriptional activity (37,38). However, its physiologic and pathophysiologic roles have not been fully determined as yet (37).

The GRE-bound GR (hereafter for GRa) attracts to the promoter regions of glucocorticoid-responsive genes numerous “coactivator complexes”, including those with histone acetyltransferase (HAT) activity, such as the NR coactivator [p160, p300/CREB-binding protein (CBP) and p300/CBP-associated factor (p/CAF)] complex, the SWI/SNF and the vitamin D receptor-interacting protein/thyroid hormone receptor-associated protein (DRIP/TRAP) chromatin-remodeling complexes (39). Among them, p160-type NR coactivators bind first to the ligand-activated and DNA-bound GR through their coactivator LxxLL motifs, and attract other coactivators and chromatin modulatory complexes including p300/CBP to the promoter/enhancer region of glucocorticoid-responsive genes. Through these proteins and protein complexes, GR alters chromatin structure and facilitates access of other transcription factors, RNA polymerase II and its ancillary factors to the promoter region of glucocorticoid-responsive genes, and ultimately changes their transcription rates (28). In addition to these protein molecules, recent research identified that several long non-protein-coding RNA molecules, such as the steroid receptor RNA coactivator (SRA) and the growth arrest-specific 5 (Gas5), regulate the transcriptional activity of GR (40,41).

Complexity of the GR-signaling system residing in glucocorticoid target organs/tissues suggests that it provides potential regulatory “windows” to the GR-induced transcriptional network, which further indicates that glucocorticoid activity is under the tight regulation of numerous factors to adjust its activity upon local needs (25,28) (Figure 1B). This peripheral modulation of the glucocorticoid-signaling system is referred to as “sensitivity of tissues to glucocorticoids”, which determines effectiveness of circulating glucocorticoids in local tissues (30). Depending on its directions -decreased or increased-, it is categorized into two subgroups; resistance and hypersensitivity. Both states may be generalized or tissue-specific, as well as congenital or acquired. The generalized, congenital form of glucocorticoid resistance, namely the familial/sporadic glucocorticoid resistance syndrome or Chrousos syndrome, was described and established approximately 30 years ago (42-45). It is characterized by partial, relatively well-compensated resistance of all tissues to glucocorticoids and is mostly caused by inactivating mutations in the GR gene (42-45). On the other hand, tissue-specific, acquired forms of glucocorticoid resistance/hypersensitivity have been inferred but not fully confirmed or elucidated (46). Such states may be limited to certain tissues, as for instance leukocytes or adipocytes, and present with the manifestations associated with deficiency or excess glucocorticoids specific to respective tissues (25). Allergic, inflammatory or autoimmune diseases, such as glucocorticoid resistant asthma, Crohn’s disease, rheumatoid arthritis and systemic lupus erythematosus, may be glucocorticoid resistant states found in the components of the immune system (25,46). Conditions associated with chronic deprivation of energy resources, such as severe lean and anorexia nervosa, are considered as glucocorticoid resistance specific in the liver, fat and/or muscles, in part through activation of several kinases including the AMP-activated protein kinase and subsequent cytoplasmic segregation of a newly-identified GR coactivator, the CREB-regulated transcription coactivator 2 and/or induction of the repressive molecules, such as the RNA corepressor Gas5 (41,46-49). In contrast, the conditions associated with excess energy resources, such as central obesity-associated insulin resistance, hyperlipidemia and hypertension, may be glucocorticoid hypersensitivity states in adipose and/or vascular tissues (46).

INTERACTION OF THE HPA AXIS AND HIV INFECTION

Pathologic Conditions Associated with the Adrenal Glands in AIDS Patients

The adrenal gland is one of the organs frequently found damaged by HIV infection at autopsy, mostly caused by the opportunistic infection of other pathogens due to immunodeficiency of AIDS patients (50-52). Incidence of adrenalitis has significantly dropped recently, because the immune function of AIDS patients is much better preserved and the incidence of opportunistic infection has been dramatically reduced due to introduction of HAART (53). Pathologic findings of AIDS-associated adrenalitis are intra-adrenal inflammatory lesions with or without necrosis, thrombosis, and/or fibrosis, as well as metastases of Kaposi sarcoma. Cytomegalovirus adrenalitis is the most common cause of the adrenal insufficiency seen in AIDS patients (51,52), while cryptococcus, mycobacteria, histoplasma, Toxoplasma gondii, and Pneumocystis carinii also affect the adrenal glands (50,53,54). Pathologic findings vary from mild focal inflammation to extensive hemorrhagic necrosis. Although several cases with extensive adrenal necrosis and profound adrenal dysfunction have been reported (55-57), infectious adrenalitis does not usually cause clinical adrenal insufficiency in most of the AIDS patients (2). Indeed, 17% of 74 hospitalized AIDS patients demonstrated abnormal response of serum cortisol against ACTH injection in one early study, whereas only 4% of these patients developed adrenal insufficiency (58). However, a report on 60 advanced AIDS patients with less than 50 cells/ml of peripheral CD4+ lymphocyte counts demonstrated that over 25% of these patients had abnormally low and high levels of respectively serum cortisol and plasma ACTH, reduced excretion of urinary free cortisol and/or blunted response of serum cortisol to exogenous ACTH (59). Thirty-eight (63.3%) patients had cytomegalovirus antigenemia. Furthermore, 16 out of the 36 patients followed up for at least one year developed overt adrenal insufficiency and half of them were treated with corticosteroid replacement. In conclusion, pathologic findings of the adrenal glands are frequently encountered at autopsy, yet these are mild and are not associated with overt primary adrenal insufficiency in the majority of cases. Presence of adrenal insufficiency, and hence, glucocorticoid replacement therapy should be considered in some end-stage AIDS patients with special caution. Indeed, one fatal case with severe adrenal insufficiency due to cytomegalovirus infection even under the treatment with pharmacologic doses of glucocorticoids was reported (60).

Change of the HPA Axis/Pituitary Gland in AIDS Patients

           
Because the adrenal glands are frequently affected in AIDS patients and common manifestations of these patients, such as weakness, fatigue and body weight loss, mimic those of adrenal insufficiency, many studies have examined basal and/or reserve activity of the HPA axis (2,61-63). A majority of publications indicates that basal levels of serum cortisol and plasma ACTH are normal or slightly elevated and their circadian rhythm is preserved in AIDS patients (54,64-68). Elevations of serum cortisol have been reported both in the early stages of AIDS and in severely affected, terminal patients (63,69,70). Twenty four-hour urinary free cortisol excretion was increased depending on severity of the AIDS-associated manifestations (71). The adrenocortical reserve capacity evaluated with a standard ACTH stimulation test is preserved in the majority of patients, while it is reduced in advanced cases (59). In a large study of 350 patients with HIV infection, 30.9% of participants displayed serum cortisol levels below 100 µg/L with a median value of 55.48 µg/L (11.36-99.96 µg/L); however, only 16.3% of participants had stimulated serum cortisol levels below 180 µg/L with median of 118 µg/L (19.43-179.62) (60). Importantly, the authors found a high prevalence of hypocortisolism among HIV patients, especially in those who had been on ART for a longer time (72). Secretion of ACTH in response to CRH is blunted, especially in terminal-stage AIDS patients (62,63,73,74). Altered profiles of circulating cytokines are suggested as a cause of low responsiveness of the pituitary gland to CRH (62). Significant blunting of the ACTH response in AIDS patients was also reported in the cold immersion stress test (66).

Focal to widespread necrosis and/or fibrosis of the anterior pituitary gland was observed at autopsy in 10 out of the 88 AIDS patients; 5 showed apparent signs of cytomegalovirus infection in the absence of apparent inflammatory reaction, and one demonstrated severe cryptococcus infection (75). Based on the above evidence, it appears that the function of the pituitary gland (corticotrophs) for secretion of ACTH is generally preserved in AIDS patients. Hyponatremia and hypovolemia observed in AIDS patients at the end-stage of their disease is likely to be a result of the adrenal insufficiency due to dysfunction of the adrenal gland caused by specific adrenal lesions, such as infectious adrenalitis or neoplastic infiltration (51).

GLUCOCORTICOIDS IN THE TREATMENT OF AIDS PATIENTS

Protease Inhibitor-Mediated Inhibition of Glucocorticoid Metabolism and Development of Iatrogenic Cushing Syndrome

PIs, which inhibit activity of the viral-encoded protease and are widely used as part of HAART, act as inhibitors of one of the cytochrome P450 (CYP) enzymes, CYP3A4, which is necessary for metabolizing glucocorticoids into inactive forms in the liver (11). Ritonavir is the strongest suppressor of CYP3A4-mediated 6b-hydroxylation of steroids, while indinavir and nelfinavir are moderate suppressors and saquinavir is the weakest (11). All these PIs cause full-blown Cushing syndrome in AIDS patients treated even with inhaled or intranasal synthetic glucocorticoids (e.g., fluticasone, budesonide, mometasone and belclomethasone) by extremely reducing their metabolic clearance (12,76-82). Duration of the glucocorticoid-PI co-administration prior to the development of iatrogenic Cushing syndrome is highly variable, from 10 days to 5 years (mean: 7.1 years), while mean doses of administered glucocorticoids (e.g., fluticasone) are around 200-800 mg/day (mean: 400mg/day) in adults (12). Thus, glucocorticoids, even applied topically, should be used with caution in the patients treated with PIs. Changing ritonavir to other PIs or use of different classes of anti-viral drugs may help reducing this characteristic side effect.

Other Therapeutic Compounds That Potentially Affect Glucocorticoid Metabolism in AIDS Patients

Some other medications used for the treatment of AIDS patients are known to affect glucocorticoid metabolism and contribute to the development of adrenal insufficiency or Cushing syndrome. Ketoconazole, an anti-fungal compound frequently used for fungal skin infections especially in immunocompromised patients, such as those with HIV infection and those on chemotherapy, can suppress steroidogenesis by inhibiting the steroidogenic enzymes P450 side-chain cleavage enzyme and 17b-hydroxylase, and cause cortisol deficiency (83,84). This effect of ketoconazole is not observed with other similar compounds, such as fluconazole and itraconazole, and imidazole derivatives. Phenytoin and rifampicin, which are respectively an anticonvulsant and an antibiotic used for the treatment of tuberculosis, can accelerate cortisol metabolism, and thus, potentially cause adrenal insufficiency particularly in AIDS patients with reduced adrenal reserve (53). Megestrol acetate, a progesterone derivative also known as 17α-acetoxy-6-dehydro-6-methylprogesterone, is often used at relatively high doses to boost appetite and to induce weight gain in AIDS patients with cachexia (85). This compound has some glucocorticoid actions, therefore, causes glucocorticoid excess and subsequent adrenal insufficiency upon its withdrawal or under stress (86).

Potential Use of Glucocorticoids for Slowing AIDS Progression and Treatment of AIDS Complications

Current therapeutic regimens, including HAART, have enabled us to control viremia and viral replication in HIV-infected patients, and thus, have expanded their life expectancy significantly (6,8). However, these therapeutic regimens are expensive and their adherence rates are sometimes low (87-89). In addition, compounds used for the treatment of AIDS often have chronic toxic side effects, such as the characteristic AIDS-related insulin resistance and lipodystrophy syndrome (ARIRLS), which will be discussed in a later section, as well as mitochondrial toxicity, lactic acidosis, hepatotoxicity, and cardiomyopathy (90). Thus, other antiretroviral agents have been developed, including inhibitors of viral integrase, host CXCR4 and CCR5, and fusion of HIV to CD4+ lymphocytes (91). In addition to these compounds that directly interfere with viral activities, immunosuppressive agents, such as glucocorticoids and cyclosporine A, have been tested in HIV-infected patients, as these agents may suppress HIV-mediated immune activation, which is one of the major factors for AIDS progression and reduction of peripheral CD4+ lymphocytes (5,92-95); The synthetic glucocorticoid prednisone at 0.3-0.5 mg/kg/day successfully increases peripheral CD4+ lymphocyte counts and prevents their reduction for up to 10 years (5,96). It also suppresses circulating levels of TNFa and IL-6, known indicators of HIV-mediated host immune activation and possible causative agents for AIDS-associated wasting syndrome (92,97,98). These cytokines may also participate in HIV replication by potentiating Tat-mediated activation of the HIV long terminal repeat (LTR) promoter via stimulation of the nuclear factor-kB (NF-kB) (99). This beneficial effect of glucocorticoids is more obvious in patients whose immune system is less damaged (5,95). Glucocorticoids do not alter peripheral viral load in the patients who have already been treated with antiretroviral drugs, and thus, have low viral load before initiation of therapy (5,94,95). However, one case report indicated that high doses of prednisone (100 mg for 9 consecutive days) demonstrated extremely strong suppression on the circulating virus titer of the patient infected with multi-drug-resistant HIV (100). The synthetic glucocorticoid dexamethasone inhibits elimination of CD4+ lymphocytes by macrophages isolated from HIV-infected patients in vitro (101). Glucocorticoids reduce circulating mature monocytes in monkeys (sooty magabey) infected with the simian immunodeficiency virus, a model virus of HIV used in animal studies (102). These monocytes act as the HIV reservoirs due to their ability to transfer the virus to CD4+ lymphocytes and their relatively long life (103). Furthermore, reduced diurnal amplitude of circulating cortisol in HIV-infected patients is correlated with their greater T cell immune activation, which is a known risk factor for immunologic and clinical progression of AIDS (104). This evidence suggests that healthy diurnal cortisol production is beneficial for slowing down the AIDS progression. Thus, at treatment-naïve or equivalent states, glucocorticoids appear to inhibit viral replication by suppressing HIV-mediated inflammation, subsequent production of inflammatory cytokines and viral transmission from monocytes to CD4+ lymphocytes. However, glucocorticoids are also risk factors for AIDS-associated complications, including sarcopenia, osteoporosis and/or osteonecrosis of the hip, and are reported to accelerate development of human herpes virus-8 (HHV8)-associated Kaposi sarcoma in the patients with pleural tuberculosis, interstitial pneumonia and glomerulonephritis (105-113). Indeed, HHV8 encodes the latency-associated nuclear antigen (LANA), which functions as a coactivator of GR through direct physical interaction (114). Glucocorticoids are also risk factors for elective hip surgery (total hip arthroplasty and resurfacing), and may be a potential factor for the development of CD8 encephalitis in HIV-infected patients (111,115).

 

Thus, the therapeutic use of glucocorticoids in AIDS patients appears to be quite limited by several factors, particularly in the era of improved HAART, which can control viral replication with less side effects. Selective glucocorticoids or other non-steroidal compounds, with immunosuppressive actions but not metabolic side effects, might be beneficial in the treatment of AIDS patients. Indeed, some of such compounds (e.g., Compound Abbott-Ligand (AL)-438, ZK216348 and the hydroxyl phenyl aziridine precursor analogue Compound A) are under investigation for their selective glucocorticoid effects (116) (please see Endotext chapter in the Adrenal Diseases and Function section entitled “Glucocorticoid Receptor”).

In addition to the effect on circulating CD4+ lymphocyte counts, glucocorticoids act as central components in the treatment regimens for HIV-associated lymphoma (such as Hodgkin and non-Hodgkin lymphoma and latter’s subtypes Burkitt lymphoma and plasmablastic lymphoma), multi-centric, HHV8-associated Castleman’s disease (also known as giant or angiofollicular lymph node hyperplasia, lymphoid hamartoma, angiofollicular lymph node hyperplasia) and HIV-associated nephropathy (13,16,117-120). Glucocorticoids are also used to subside some complications of opportunistic infections, such as those by Pneumocystis carinii and mycobacteria (pleuritis and pericarditis), and those associated with immune reconstitution inflammatory syndrome (IRIS), which sometimes happens in AIDS patients upon recovery of their immune system with antiretroviral treatment (121-124). One clinical study examining the beneficial effects of glucocorticoids for the treatment of AIDS-associated cryptococcal meningitis was performed (18). Moreover, a recent double-blind, placebo-controlled, cross-over study investigated the effects of a single low-dose administration of hydrocortisone (10 mg oral) on cognition in 36 HIV-infected women (125). The authors found that this low dose had beneficial effects in verbal learning and delayed memory, working memory, visuospatial abilities and behavioral inhibition (125). Further larger studies are clearly needed to verify these promising results. Finally, glucocorticoids are prescribed empirically for AIDS patients to treat their fatigue and appetite loss (126-130).

Adverse Effects of the Contraceptive Medroxyprogesterone Acetate for Increasing the Chance of HIV Infection through GR Activation

It is important for the HIV endemic area whether contraceptives increase/reduce the chance of HIV infection, therefore several clinical studies were previously performed to address this possibility (131). These compounds, regularly mixtures of progestins and estrogens, stimulate the progesterone (PR) and estrogen receptor for mimicking the hormonal profiles of pregnancy (132). There are 2 types of contraceptives with regard to their routes of administration; injection and oral intake (131). Recent studies revealed that one of the injectable contraceptives, medroxyprogesterone acetate (MPA), a compound widely used in sub-Saharan Africa, increases a chance of HIV infection particularly in young women with high exposure to this virus (131). Subsequent research revealed that MPA can bind GR in addition to PR with high affinity in contrast to other progestins, such as progesterone and norethisterone acetate, and strongly suppresses inflammatory response in endocervical cells by activating local GR (131,133). Moreover, medroxyprogesterone acetate was found to increase HIV-1 replication in human peripheral blood mononuclear cells through mechanisms involving the glucocorticoid receptor, increased CD4/CD8 ratios and CCR5 levels (134). Further, this compound enhances Vpr-mediated apoptosis of human CD4+ lymphocytes by cooperating with GR, which further affects clinical course of HIV-infected patients (133).

 

GLUCOCORTICOIDS RESISTANCE/HYPERSENSITIVITY ASSOCIATED WITH AIDS PATIENTS

Glucocorticoid Resistance with Reduced GR Affinity to its Ligands

Norbiato et al. reported a distinct subgroup of AIDS patients who showed apparent adrenal insufficiency with fatigue, weakness, body weight loss, hypotension, and skin and mucosal hyperpigmentation associated with markedly elevated levels of serum cortisol and moderately increased levels of plasma ACTH (135). In these patients, affinity of the GR to its ligand was markedly decreased in peripheral leukocytes with concurrent elevations of receptor numbers, suggesting that the apparent adrenal insufficiency seen in these patients might be caused by decreased sensitivity of peripheral tissues to glucocorticoids. This research group estimated that up to 17 % of AIDS patients are likely to have altered GR actions (136).

Pathologic mechanism(s) underlying this characteristic condition with markedly reduced receptor affinity has(have) not been elucidated as yet. A similar glucocorticoid resistance state associated with reduced receptor affinity was previously reported in glucocorticoid resistant asthma patients. In the latter patients, the affinity change is limited to immune tissues, such as peripheral leukocytes, and is progressively reverted to normal when cells are incubated ex vivo (137). Since incubation of patients’ peripheral lymphocytes with IL-2 and IL-4 preserves the decrease in receptor affinity (137,138), and since elevation of these cytokine levels is generally observed in asthma patients (139), it is likely that cytokine-related mechanisms are involved in the development/maintenance of the receptor affinity change observed in AIDS patients. It was subsequently reported that glucocorticoid resistant asthma was also associated with increased expression of the GRb isoform, suggesting that this splicing variant receptor might participate in the pathogenesis of the glucocorticoid resistance of AIDS patients as well (140). Because many kinases and other molecules important for the cytokine and growth factor signaling potentially modulate GR activity (28,30), and cytomegalovirus alters GR transcriptional activity by phosphorylating this receptor through activation of the extracellular signal-regulated kinases (141), it is possible that some of such molecules might also contribute to the alteration of the receptor affinity in AIDS patients.

The exact prevalence of this glucocorticoid resistance associated with reduced receptor affinity observed in AIDS patients is not known. Although similar patients were reported by another group just after appearance of the initial cases (142), very few reports followed subsequently, suggesting that this characteristic AIDS-related pathologic condition may be rare and/or associated with some special condition of AIDS patients, which may be disappeared after introduction of HAART. In this instance, severe uncontrollable immune dysregulation and/or inflammation by HIV observed at an early and/or specific period may be required for developing this characteristic phenotype. 

In late ’90s, an acquired form of lipodystrophy, which partially mimics the clinical presentation of Cushing syndrome, was reported in AIDS patients (10,143-146). The patients had a characteristic redistribution of their adipose tissue, with an enlargement of their dorsocervical fat pad (“buffalo hump”), axial fat pads (bilateral symmetric lipomatosis), lipomastia, and expansion in their abdominal girth ("Crix-belly" or "protease paunch") [lipohypertrophy in trunk and abdomen]. Since these manifestations are reminiscent of the typical phenotype of chronic glucocorticoid excess or Cushing syndrome, this condition was initially referred as a pseudo-Cushing state, a term reserved for obese, depressive or alcoholic patients with biochemical hypercortisolism who are frequently hard to differentiate from true Cushing syndrome (31). In addition to these initial characteristic manifestations, some patients develop lipoatrophy in face, buttocks and limbs (147). Furthermore, they frequently demonstrate metabolic complications, such as severe insulin resistance, hyperlipidemia and hepatic steatosis, similar to some of the congenital lipodystrophy syndromes (29,46,147). Taken together, this AIDS-related characteristic syndrome has 3 major components in its manifestations, lipohypertrophy, lipoatrophy and metabolic complication, such as insulin resistance and dyslipidemia.

Pathologic causes of ARIRLS are not known, but appear to be multifactorial. ARIRLS patients demonstrate manifestations shared with or district from those of other lipodystrophies unrelated to HIV infection, suggesting that it is caused by the pathologic mechanisms somewhat different from the latter conditions (148). Alteration of the HPA axis and/or the glucocorticoid/GR signaling system appear(s) to be involved in the development of certain part of this syndrome, as we will discuss below. 

Factors Contributing to the Development of ARIRLS

ANTIRETROVIRAL DRUGS

Protease Inhibitors (PIs)

Possible mechanisms contributing to this characteristic syndrome are listed in Table 3 and summarized in Figure 2. As several previous reports indicated, one of the earlier suggestions was that the syndrome was outcome of adverse effects of antiretroviral drugs including PIs, nucleoside reverse transcriptase inhibitors (NRTIs) and/or non-nucleoside reverse transcriptase inhibitors (NNRTIs) (147,149). PIs interfere with viral replication by efficiently inhibiting the activity of the viral-encoded protease, which normally digests the Gag-Pol p160 kDa precursor protein, producing several polypeptide fragments with distinct functions (149,150). NRTIs and NNRTIs, on the other hand, inhibit viral replication by suppressing the activity of the reverse transcriptase also encoded by HIV (149). The effects of various antiretroviral drugs on the development of lipodystrophy and metabolic complications are listed in Table 4. Since prototype drugs were significantly associated with the development of ARIRLS, new compounds with less association were subsequently developed.

Figure 2. Major proposed mechanisms in the genesis of ARIRLS. Three major components, antiretroviral drugs, viral factors and host factors differentially contribute to the development of ARIRLS by respectively modulating adipogenesis, lipogenesis, and tissue insulin action through induction of/responsiveness to inflammatory cytokines, damage to adipocytes (e.g. by mitochondrial toxicity and reactive oxygen species) and/or through modulation of host cellular mechanisms, such as NR (GR, PPAR, PXR and LXR) signaling systems and inhibition/modulation of p450 enzyme activity (such as CYP3A and steroidogenic enzymes). Some changes can also alter tissue glucocorticoid action (glucocorticoid sensitivity) through expression of the GR and/or 11bHSD1 that converts inactive cortisone to active cortisol. As sum of these changes, major manifestations, lipohypertrophy, lipoatrophy and insulin resistance/dyslipidemia are finally developed in which modulation of the glucocorticoid metabolism/signaling system play a significant part. Their specific actions on visceral and subcutaneous fat may contribute to the development of lipohypertrophy and lipoatrophy in different body areas. [from (29,46,147,151)]

Table 3. Potential Contributing Factors to AIDS-Related Insulin Resistance and Lipodystrophy Syndrome (ARIRLS) Before and After Treatment with Antiretroviral Drugs

 

Before Rx

After Rx

Nonspecific, disease-related

 

 

Sickness-related starvation

+

Refeed

Sickness-related change in body composition

Lean body mass loss*

Fat mass gain*

Infection-induced hypercytokinemia

+

 

Cytokine-induced adipose tissue 11bHSD1 stimulation

+

-

Stress- and starvation-induced hypercortisolism          

+

-

Specific, HIV-related

 

 

Virally-induced muscle, liver, and fat glucocorticoid hypersensitivity

+

+

Virally-induced adipose tissue PPARg inhibition

+

+

Virally-induced adipose tissue 11bHSD1 stimulation

+

+

Antiretroviral drug-related

 

 

Rx-induced-insulin resistance/dyslipidemia

-

+

Alteration of glucocorticoid clearance through hepatic CYP3A inhibition

-

+

Modulation of NR activity (PXR and LVR) by acting as ligands

-

+

Genetic/constitutional predisposition      

+

+

+: presence, -: absence, ?: unknown, * During stress and starvation, both fat and lean body mass are lost. Post stress and starvation body weight gain is primarily due to fat accumulation. 

Table 4. Differential Effects of Antiretroviral Drugs on Fat and Metabolism Associated with AIDS-Related Insulin Resistance and Lipodystrophy Syndrome (ARIRLS)*

Class of drugs

Name of drug

Abbreviation

Lipo-atrophy

Lipo-hypertrophy

Dyslipidemia

Insulin

resistance

PIs

Ritonavir

RTV

+/-

+

+++

++

 

Indinavir

IDV

+/-

+

+

+++

 

Nelfinavir

NFV

+/-

+

++

+

 

Lopinavir

LPV

+/-

+

++

++

 

Amprenavir Fosamprenavir

APV FPV

+/-

+

+

+/-

 

Saquinavir

SQV

+/-

+

+/-

+/-

 

Atazanavir

ATV

-

++

+/-

-

 

Darunavir

DRV

-

+

+/-

+/-

 

 

 

 

 

 

 

NRTIs

Stavudine

D4T

+++

++

++

++

 

Zidovudine

AZT, ZDV

++

+

+

++

 

Didanosine

ddI

+/-

+/-

+

+

 

Lamivudine

3TC

-

-

+

-

 

Abacavir

ABC

-

-

+

-

 

Tenofovir

TDF

-

-

-

-

 

Emtricitabine

FTC

-

-

-

-

 

 

 

 

 

 

 

NNTRIs

Efavirenz

EFV

+/-

+/-

++, increased HDL

+

 

Nevirapine

NVP

-

-

++, increased HDL

_

 

 

 

 

 

 

 

CCR5 inhibitor

Maraviroc

MVC

?

?

-

-

 

 

 

 

 

 

 

Integrase inhibitor

Raltegravir

RAL

?

?

-

-

 

 

 

 

 

 

 

Fusion inhibitor

Enfuvirtide

T20

?

?

-

-

Modified from (147) (Permission for re-use was obtained from Elsevier with the license number: 3012541054207)

* These data should be considered with caution because discrepancies exist among studies that cannot be presented in one table. 

Mechanistically, PIs act as inhibitors of the CYP3A4 enzyme, which metabolizes and inactivates glucocorticoids as we discussed above (11). Thus, these compounds may slightly increase circulating levels of endogenously produced cortisol by reducing its clearance in the liver, and participate in the development of ARIRLS. PIs also decrease hepatic lipase activity and modulate differentiation of pre-adipocytes (152-154). A possible underlying mechanism for this PI-mediated modulation of adipocyte activity is that these compounds change the expression levels of the peroxisome proliferation receptor (PPAR) g and the CAAT/enhancer-binding protein (C/EBP) a (148). PPARg is a NR family protein and acts as a pivotal regulator of glucose and lipid metabolism and development/differentiation of adipocytes (155). C/EBPa is a bZip family transcription factor, and plays also a key role in adipogenesis and adipocyte differentiation (156). In addition, PIs increase IL-6 and TNFa production by activating the NF-kB pathway in subcutaneous fat (157). These cytokines are known to play important roles in local inflammation and lipid accumulation in adipose tissue (158). The adverse effect of PIs may also result from induction of the endoplasmic reticulum stress or inhibition of the proteosomes (159,160).

NRTIs and NNRTIs

In addition to PIs, these classes of antiretroviral drugs are also associated strongly with development of ARIRLS. Among them, thymidine NRTI (tNRTI) stavudine and zidovudine cause severe lipoatrophy in AIDS patients, thus they were removed from the list of the first-line antiretroviral compounds in Western countries (161). These compounds demonstrate mitochondrial toxicity by inhibiting the mitochondrial DNA polymerase g, facilitating generation of the reactive oxygen species in adipose tissues and possibly causing lipoatrophy in AIDS patients (147). Although weak, NNRTIs, such as efavirenz and nevirapine, also have an activity to develop lipodystrophy and dyslipidemia (147).

Modulation of NR Activity by Antiretroviral Drugs

In addition to above-indicated potential actions of antiretroviral drugs on the development of ARIRLS, some of these compounds can modulate the transcriptional activity of several NRs, such as the pregnane X receptor (PXR), constitutive androstane receptor (CAR), liver X receptors (LXRs) and the estrogen receptor a (ERa), and directly stimulate their transcriptional activity. Interestingly, these antiretroviral drugs were demonstrated to act potentially as ligands for the receptors in in silico structural analysis on the ligand-binding pocket of these receptors (154). PXR and CAR act as xenobiotic sensing receptors and induce drug metabolizing enzymes with broad ligand specificity for many chemical compounds, and several PIs can stimulate CYP3A4 and CYP2B6 promoter activity through activation of these receptors (154). Activation of PXR, either by its known ligands or transgenic expression of PXR, increases production of glucocorticoids in the adrenal glands by stimulating expression of the steroidogenic enzymes, such as CYP11A, CYP11B1, CYP11B2 and 3b-hydroxysteroid dehydrogenase, and develops Cushingoid manifestations in rodents (162), suggesting that PIs may increase cortisol production and participate in the development of ARIRLS indirectly through activation of PXR. Furthermore, PIs (ritonavir, atazanavir and darunavir) and maraviroc (CCR5 antagonist) activate the transcriptional activity of LXRa and/or LXRb, while NNRTIs (tenofovir and efavirenz) stimulate ERa (but not ERb) (154). Since LXRs are the receptors for regulating cholesterol/fatty acid metabolism and insulin actions, activation of these receptors by antiretroviral drugs may underlie pathophysiology of ARIRLS (163). In addition, LXRs and ERa cooperate with GR for expression of glucocorticoid-responsive genes, thus it is likely that these antiretroviral drugs enhance glucocorticoid actions indirectly through stimulating these NRs (164,165).

VIRAL FACTORS

Although antiretroviral drugs are generally accepted for causing ARIRLS, a small percentage of HIV-infected patients develop characteristic features of this syndrome prior to their introduction; HIV-infected patients who are not receiving antiretroviral therapy often have lipid abnormality, including elevated triglyceride levels, a high proportion of small and dense LDL particles, and low HDL cholesterol levels, similar to ARIRLS patients (166). Furthermore, different classes of chemical compounds that target different components of HIV/adipocyte biological pathways can develop similar ARIRLS manifestations in AIDS patients (147). These pieces of evidence thus suggest that the HIV infection itself could nonspecifically, -in part via inflammatory cytokine elevations and stress induced cortisol hypersecretion-, induce an insulin resistant phenotype (31). Pro-inflammatory cytokines, such as TNFa, IL-1 and IL-6, which are released from the HIV-infected macrophages localized in adipose tissues, do cause resistance to insulin and fat accumulation in neighboring adipocytes (158). In addition, these cytokines indirectly activate GR in adipose tissues by stimulating expression of the 11b-hydroxysteroid dehydrogenase-1 (11bHSD1), which converts inactive cortisone into active cortisol (167). Moreover, increased expression of GR is also reported in subcutaneous fat of zidovudine-treated AIDS patients (168). In this context, antiretroviral drugs might just exacerbate already present, smoldering insulin resistance and lipodystrophy, not expressed because of the known malnutrition of sick AIDS patients and the absence of sufficient calories to build visceral and other fat deposits (10,30,169). As manifestations of the sickness syndrome subside with treatment, the emaciated patient goes through refeeding with body weight gain of mostly fat, tilting the ratio of fat to lean body mass upward, further worsening insulin resistance.

HIV, in its 9.8 kb genomic information, encodes and produces 3 precursor proteins, the Gag, RNA polymerase and envelope polypeptides, whose processed products are the reverse transcriptase, protease, integrase, matrix, and capsid, as well as 6 accessory proteins, Tat, Rev, Nef, Vif, Vpr and Vpu (170) (Figure 3). Some of these polypeptides are virion-associated proteins incorporated in the viral particle and others are expressed in host cells where they direct viral replication and gene expression and several host cell functions. Since infection with HIV has a dramatic impact on host target cells, it is quite possible that some of these viral proteins modulate host cell glucose and lipid metabolism by changing the activity of GR in local tissues, such as in adipose tissue, skeletal muscles and liver, and participate in the development of ARIRLS. Indeed, there are several pieces of evidence indicating that AIDS patients have altered tissue sensitivity to glucocorticoids. First of all, they all develop reduction of innate and Th1-directed cellular immunity. Levels of plasma IL-2, IL-12 and IFN-g, which direct cellular immunity, are suppressed in AIDS patients, while levels of IL-4 are increased (171,172). All changes can be induced by exogenously introduced glucocorticoids and are seen in hypercortisolemic patients with classic Cushing syndrome (173). AIDS patients also frequently present with muscle wasting and myopathy, as well as dyslipidemia and visceral obesity-related insulin resistance (174-176). Therefore, some unknown viral factor(s) might modulate tissue sensitivity to glucocorticoids in AIDS patients in a tissue-specific fashion, sparing their HPA axis preserving normal negative feedback sensitivity to glucocorticoids.

Figure 3. Lineralized structure of the HIV genome and localization of vpr and tat coding region (shown in black boxes). HIV, in its 9.8 kb genomic information, encodes and produces 3 precursor proteins, the Gag (gag), RNA polymerase (pol) and envelope polypeptides (env), whose processed products are the reverse transcriptase, protease, integrase, matrix, and capsid, as well as 6 accessory proteins, Tat, Rev, Nef, Vif, Vpr and Vpu. LTR: long terminal repeat [modified from (170,177)]

In agreement with these reported findings, one of the HIV proteins, Vpr, which is a 96-amino acid virion-associated accessory protein with multiple functions, including influencing transcriptional activity and having a cell cycle-arresting effect, increases the action of GR by several fold, functioning as a potent GR coactivator (178). The GR coactivator activity of Vpr is biologically evident in the suppression of IL-12 production from monocytes and the expression of activated NF-kB ligand (RANKL) in lymphocytes (179,180). Similar to host p160 type coactivators, Vpr contains one LxxLL coactivator motif through which it binds to the ligand-activated and promoter-bound GR (178). GR-bound Vpr then attracts p300/CBP, and ultimately potentiates the transcriptional activity of GR by acting as a molecular adaptor between GR and p300/CBP (177,181) (Figure 4). p300/CBP are HAT coactivators also known as integrators or regulatory “platforms” for many signal transduction cascades by providing docking sites for many transcription factors, including NRs, CRE-binding protein (CREB), activator protein-1 (AP-1), NF-kB and the signal transducers and activators of transcription (STATs) (182) (Figure 4). Vpr easily penetrates the cell membrane to exert its biologic effects (183,184), thus its effects may be extended to tissues not infected with HIV.

Figure 4. Linearized Vpr, Tat and p300 molecules and their mutual interaction domains. Vpr interacts with cellular molecules, such as NR, p300/CBP coactivators and 14-3-3, while Tat is physically associated with pTEFb elongation factor through its component Cyclin T1. Tat also binds p300/CBP and p160 type coactivators. Numerous transcription factors, transcriptional regulators and viral molecules bind the transcriptional coactivator p300. Binding sites of p160 NR coactivators and Vpr overlap with each other and they both bind NRs and p300/CBP. Thus, Vpr mimics the host p160 NR coactivators and enhances NR transcriptional activity. p300 facilitates attraction of transcription factors, cofactors and general transcription complexes by loosening the histone/DNA interaction through acetylation of histone tails by its histone acetyltransferase (HAT) domain. [modified from (29,30)]. CREB: CRE-binding protein, HAT: histone acetyltransferase, NF-kB: nuclear factor-kB, NR: nuclear hormone receptor, p/CAF: p300/CBP-associating factor, pTEFb: positive-acting transcription elongation factor b, Rb: retinoblastoma protein, SF-1: steroidogenic factor-1, STAT2: signal transducer and activator of transcription 2, TFIIB: transcription factor IIB.

Another HIV accessory protein, Tat, the most potent transactivator of the HIV long terminal repeat promoter, also moderately potentiates GR-induced transcriptional activity, possibly through accumulation of the positive-acting transcription elongation factor b (pTEFb) complex, that is comprised by the cyclin-dependent kinase 9 and its partner molecule cyclin T, on glucocorticoid responsive promoters (185) (Figure 4). Because Tat, like Vpr, also circulates in blood and exerts its actions as an auto/paracrine or endocrine factor by penetrating the cell membrane (186), it is possible that Tat modulates tissue sensitivity to glucocorticoids irrespectively of a cell’s infection by HIV. Concomitantly with Vpr, Tat may induce tissue hypersensitivity to glucocorticoids that might contribute to viral proliferation indirectly, by suppressing local immune system activity and by altering the host’s metabolic balance, with both functions being governed by glucocorticoids (30,46).

Vpr reduces tissue sensitivity to insulin not only through potentiating the actions of glucocorticoids, but also by modulating insulin’s transcriptional activity via interaction with the protein of the 14-3-3 family, which participates in the cell cycle arrest activity of Vpr (29,187). Insulin uses the forkhead transcription factors (FoxOs) to control gene induction; baseline unphosphorylated FoxOs are active, reside in the nucleus, and bind to their responsive sequences in the promoter region of insulin-responsive genes; in contrast, insulin activates Akt kinase, which phosphorylates specific serine and threonine residues of FoxOs rendering it inactive (188). Indeed, once FoxOs are phosphorylated at specific residues, they lose their transcriptional activity, by binding with 14-3-3 through phosphorylated residues and subsequently segregated into the cytoplasm (188). We found that Vpr moderately inhibited insulin-induced translocation of FoxO3a into the cytoplasm through inhibiting its association with 14-3-3 (187). Thus, Vpr may participate in the induction of insulin resistance by interfering with the insulin signaling through FoxOs/14-3-3 (29,151,177). 

We further found that Vpr-mediated insulin resistance might be compounded by the ability of the viral protein to interfere with the signal transduction of PPARg (183). Indeed, Vpr suppresses the c-Cbl associating protein (CAP) mRNA expression in pre-adipocyte cells and associated with the PPAR-binding site located in the promoter region of this gene. CAP is predominantly expressed in insulin-sensitive tissues and positively regulates insulin action, directly associating with both the insulin receptor and the c-Cbl proto-oncogene product (189). Vpr delivered either by exogenous expression or as a peptide added to media suppresses PPARg agonist-induced adipocyte differentiation (183). Thus, circulating Vpr, or alternatively Vpr produced as a consequence of direct infection of adipocytes, may suppress differentiation of preadipocytes by acting as a corepressor of PPARg-mediated gene transcription (29,183,190). We further found that Vpr regulates the transcriptional activity of PPARb/d as well, and alters cellular energy metabolism organized by mitochondria (191). Vpr disturbs the insulin signaling and induces hepatic steatosis by disrupting the transcriptional program of PPARs in the liver and adipose tissue in the animal models, such as the transgenic mice expressing Vpr specifically in these organ and tissue and the mice inoculated with the pump that continuously releases the synthetic Vpr peptide into circulation (192). Moreover, Vpr was demonstrated to induce fatty liver in mice via LXRα and PPARα dysregulation (193). Taken together, based on these pieces of evidence, Vpr may be a key factor for the development of lipodystrophy, insulin resistance and hyperlipidemia observed in HIV-infected patients through modulation of the GR/PPARs/LXR and FoxOs/14-3-3 activities.

HOST FACTORS

Several host factors may influence susceptibility and manifestation of ARIRLS. Variant alleles of APOC3, APOE contribute to an unfavorable lipid profile in patients with HIV infection, while application of antiretroviral therapy further worsens it (194). Another study identified that APOE polymorphism is also associated with the dyslipidemia seen in AIDS patients treated with PIs (195). One recent study demonstrated that polymorphisms of the genes involved in apoptosis and adipocyte metabolism are significantly related to the development of ARIRLS (196). Among the polymorphisms examined, ApoC3-455 variant is associated with lipoatrophy, while two variants of the adrenergic receptor b2 influence fat accumulation in ARIRLS patients (196). A polymorphism in the TNFa gene promoter is associated with development of lipodystrophy in one study, while this association was not confirmed in larger studies (194). Stavudine-induced lipoatrophy is associated with the HLA-B100*4001 allele among the genetic variants of HLA-A, HLA-B HLA-C, HLA-DRB1, HLA-DQB1 and HLA-DPB1 (190). A newly identified polymorphism (Tth111I) in the GR gene is negatively associated with the development of some manifestations of ARIRLS in the African-American population (197). Finally, toxicity of antiretroviral drugs depends on their metabolism in each patient, which is partly determined genetically (196).

Summary for ARIRLS

Above pieces of evidence indicate that ARIRLS is most likely caused by multiple factors, including the infection itself, - via nonspecific inflammatory cytokine - and stress-induced hypercortisolism causing insulin resistance-, several HIV products disturbing the cellular functions of the host, and antiretroviral drugs, all acting on a genetic and constitutional background of variable predisposition to the syndrome. It is highly possible that alteration of glucocorticoid/GR signaling system by any of the above indicated factors contributes to the development of ARIRLS. Further studies are necessary to characterize this syndrome further, to better define the mechanisms involved in its development, and devise ways to prevent it from occurring or for reversing it.

ACKNOWLEDGEMENTS

This literary work was supported by the intramural fund of the Sidra Medical and Research Center to T. Kino.

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