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Performance Enhancing Hormone Doping in Sport

ABSTRACT

 

Sport is the organized playing of competitive games according to rules. Hence doping represent drug cheating, a fraud on competitors, the sport, and the public. The charter of the World Anti-doping Agency (WADA) forms a harmonized Code that authorizes an annually updated list of prohibited doping substances and methods as well as accrediting national anti-doping labs around the world. Sports performance has 4 major components: skill, strength, endurance, and recovery, with each sport employing a distinct combination of these elements. These performance characteristics also correspond to the most potent and effective forms of doping. Sports requiring explosive power are most susceptible to androgen doping through their effect on increasing muscle mass and strength whereas sports that require endurance are most enhanced by hemoglobin (blood) doping which increases oxygen delivering capacity to exercising tissues. Performance in contact sports and those involving intense physical activity or training may also be enhanced by growth hormone and its secretagogues through speeding of tissue recovery from injury. Hormones remain the most potent and widely detected doping agents being responsible for about 2/3 of anti-doping rule violations detected by increasingly sophisticated detection methods. At present, the vast majority of positives are still due to a wide variety of androgens, including marketed and illicit nutraceutical, designer, specific androgen receptor modulator (SARM)) synthetic androgens as well as exogenous natural androgens, while the peptide hormones (erythropoiesis stimulating agents, growth hormone and its secretagogues) and autologous blood transfusion remain difficult to detect.

 

INTRODUCTION

 

Across the world, sport is a ubiquitous human social activity that forms an unique intersection of health, recreation, entertainment, and industry (1). It is both a major economic activity as well as a profound influence on social behavior of individuals at home, work, and play. One practical and concise definition of sport is the organized playing of competitive games according to rules. In that context, rule breaking is cheating to achieve an unfair competitive advantage whether it involves using illegal equipment, match fixing, banned drugs, or any other prohibited means.

 

The illicit use of banned drugs (doping) to influence the outcome of a sporting contest, constitutes a fraud against competitors, spectators, sport, sponsors, and the public no different from other personal, professional, or commercial frauds. While performance enhancement is almost invariably the intent of cheating, impairing performance is also well known in horse racing and even, rarely, in human elite sports (e.g. drink-spiking of banned drugs, injurious physical assaults). Rules of sporting contest may change by agreement, but once set, represents the boundaries of fair competition. Nevertheless, fairness is an elastic, socially constructed concept which may change gradually over time. For example, a century ago deliberate training itself was considered an ungentlemanly breach of fairness as competition was then envisaged as a contest based solely on natural endowments. Similarly, some sports once maintained a distinction between amateurs and professionals. The philosophical foundations of the concept of fairness is a deep and complex issue (2,3) where the focus has been mainly on distributive justice with an implicit goal of equality of outcomes. Less attention has been given to the philosophical basis of fair competition in sport where the prior distribution of talent and training and the outcome of contest are intended to provide equality of opportunity, but not of outcome, between contestants.

 

Naïve arguments are made that deny doping is cheating, or unsafe or violates the spirit of sport and asserting that drugs should be freely available or under medical supervision (4-6). However, removing prohibition on doping would immediately render drug taking as pervasive as training in elite sport extending to promising underage and sub-elite athletes. Ensuing demands on doctors to prescribe excessive, often massive, drug doses without medical indications would be unprofessional, unethical, and unsafe. This could convert sporting participation into a potentially dangerous rather than a healthful activity. In practice, creating enforceable boundaries for drugs in sport is unavoidable whether it is prohibition or, even under the most idealistic libertarian scenarios, by age or dosage. Within the limitations of unverifiable self-report regarding an illicit activity (7,8), surveys indicate athletes support antidoping testing mainly to prevent cheating but also to promote safety (9-11). Motivating factors for, and routes by which athletes get involved in doping are complex but include the use of non-banned nutritional supplements as a gateway to doping (12) and the suspicion of athletes or their entourage that their rivals may be using illicit drugs, the so-called “false consensus belief” (7,13-15). These philosophical issues are not considered further here and, recognizing that sport requires concrete, practical decisions, the establishment and enforcement of agreed rules is the basis of fair competition. An excellent discussion of the logic and morality of a decisive antidoping approach from an ethicist with extensive experience in sports anti-doping is recommended (16).

 

It is well understood that individual human genetic endowments are unequal and, among these, sporting prowess is at least partly genetically determined (17). However, little is still known of the genotype-phenotype correlations that underlie beneficial genetic endowments for sports performance. Natural genetic advantages are recognized in height (tallness for basketball, shortness for jockeys and motor-cycle riders) and hereditary erythrocytosis where a high circulating hemoglobin due to a high affinity erythropoietin (EPO) receptor (18)) for endurance sports, or conversely genetic disadvantage such as the common α-actinin-3 deletion genetic polymorphism which limits anaerobic, explosive power (19). More examples of genetic (dis)advantages for sports performance are likely to be identified as genomics continues to expand our understanding of the biological basis of health, including natural human sporting prowess. In the context of sports doping, however, a person’s genetic endowment is a given creating a natural boundary whereby the use of exogenous drugs or chemicals (including DNA) may constitute drug cheating or doping.

 

WORLD ANTI-DOPING AGENCY (WADA) AND THE GLOBAL ANTI-DOPING REGULATORY ENVIRONMENT

 

Cheating is as old as sport itself, yet the present endemic of doping using pharmaceutical drugs to boost sports performance is largely a Cold War legacy. Eastern European national doping programs were established by governments aiming to achieve a short-cut propaganda victory over their Western rivals, a challenge quickly reciprocated and then taken up by individual coaches and athletes. Starting with power sports (20), the epidemic became entrenched as an endemic in sufficiently affluent circles. In 1967, following the introduction of anti-doping rules by some sports federations, the International Olympic Committee (IOC) established its Medical Commission, which published their first list of prohibited substances. During the 1970’s the IOC Medical Commission took an increasingly active role by banning androgens which required developing standardized, valid methods to detect and deter androgen doping. After discarding alternatives such as immunoassays and blood sampling, in the 1980s mass spectrometry (MS)-based tests became (21) and remain the standard for detecting synthetic androgens in urine.

 

In 1999, the IOC established the WADA based in Montreal to be equally supported by governments and sporting organizations with its charter, the WADA Code, representing a harmonized set of global anti-doping rules introduced in 2004, revised in 2009 and 2015 and will be again revised in 2021(22). WADA also publishes an annually updated Prohibited List of Substances and Methods, accredits national anti-doping labs together with their operational anti-doping testing framework, and established the Court for Arbitration in Sport (CAS) to settle anti-doping legal disputes as sport’s “Supreme Court”. The WADA Code has been adopted by over 660 sporting organizations including all Olympic and Paralympic organizations and National Anti-Doping Organizations as well as most non-Olympic International and National Sports Federations. The WADA Code prohibits substances or methods which meet 2 of 3 criteria comprising:

(i) enhance performance (cheating),

(ii) harmful to health (safety) or

(iii) violate the spirit of sport (unsporting).

 

Although the primacy of penalizing cheating is widely understood, implementing these criteria encounter ethical and practical difficulties in proving ergogenic effects of increasing numbers of illicit and/or non-approved substances. These substances have unknown safety so that human testing is not feasible making athlete safety an important consideration. Crucially, the Code imposes strict liability on individual athletes so that a positive anti-doping test (including refusal or avoidance of testing or possession, attempts, trading and tampering with banned drugs) constitutes an anti-doping rule violation (ADRV), regardless of intent or negligence. Sanctions involve suspension from any elite competitive sport and extend to support personnel and teams. Suspensions, once 2 years are now 4 years since adoption of the 2015 Code. This is generally believed to be longer than the ergogenic benefits of doping, although recent evidence suggests that androgen effects on muscle may create durable or even permanent effects (23) which might argue for much longer or permanent banning of androgen doping violators.

 

The Prohibited List bans, at any time either in or out of competition, the use of performance enhancing hormones, including androgens, EPO and growth hormone and related substances or drugs which stimulate endogenous production of these hormones (Table 1). Among the 15 categories of prohibited substances (12) and methods (3), hormones feature prominently in S1 (anabolic agents, mainly androgens), S2 (peptide hormones, growth factors, related substances and mimetics), S4 (hormone and metabolic modulators), and S9 (glucocorticoids) with S1 and S2 having important “catch-all” provision for unnamed but related substances “with similar chemical structure or biological effects”. In addition, the S0 category bans non-approved substances, those without current regulatory approval for human therapeutic use. The prominence of hormones is reinforced by the WADA laboratory statistics for anti-doping tests where hormones remain the most frequently detected banned drugs (Table 2). In 2017, of over 322,000 anti-doping tests ~1.5% were positive with 61% due to hormones, the vast majority (~99%) due to androgens. These findings confirm that the detection of androgen doping is effective whereas the low rate of detection of hemoglobin or growth hormone doping may reflect the limitations of available tests for peptides and peptide hormones which require blood rather than conventional urine sampling and feature low sensitivity and brief windows of detection, rather than their lack of abuse. Further use of out-of-competition testing and blood samples together with more sensitive detection tests with longer windows of detection are required particularly for peptide hormones.

 

Table 2. Performance Enhancing Hormone Tests in WADA Labs

 

2005

2009

2013

2017

ACCREDITED LABS

33

35

33

31

TOTAL TESTS

183,337

277,928

269,878

322,050

POSITIVES

(% OF TOTAL)

3,909

(2.1%)

5,610

(2.0%)

5962

(2.2%)

4756

(1.5%)

HORMONES

(% OF +VE)

55%

73%

57%

61%

ANDROGENS

3893

5541

3352

1813

BLOOD/EPO

16

68

63

85

GH/PEPTIDES

0

1

0

19

Source: WADA website report on laboratory testing figures. See http://www.wada-ama.org/en/Science-Medicine/Anti-Doping-Laboratories/Laboratory-Testing-Figures/

 

The prevalence of doping in elite sports as an illicit activity with drastic consequences for athletes admitting guilt remains difficult to quantify using laboratory-based testing, inference from performance, or self-report questionnaires (24). The most promising methods appear to be questionnaires using the unrelated question random response methodology (25). This methodology was developed to estimate the prevalence of sensitive, disapproved, or illicit activities by asking the sensitive personal questions masked by mixing them with unrelated non-sensitive questions in an anonymized framework. This provides overall prevalence self-report estimates of the target activities without allowing for individual identification of answers. One study of two elite athletic competitions provided estimates of 43.6% and 57.1% for recent (last year) and 70.1% of ever use of banned doping methods (26). However, another study using the same methodology found markedly lower prevalence estimates of 0.7% to 11.9% for recent use of banned doping (27). The discrepancies between these prevalence estimates requires further clarification. A significant limitation of these methods is their reliance on athlete’s perception of banned methods. For example, whether “banned drugs” are interpreted as including the widely used (but non-banned) nutritional supplements which athletes are urged to avoid for fear of adulteration with unlabeled banned substances.

 

Therapeutic Use Exemption (TUE)

 

In rare cases, an elite athlete with a genuine medical need for therapeutic use of a prohibited drug may be granted a TUE (28). This exempts the athlete from the Code’s strict liability provision and permits them to compete during ongoing necessary medical treatment. WADA provides medical guidelines that standardize the evaluation and management of TUE applications for a range of medical illnesses. A TUE is granted by a national anti-doping organization based on an independent, expert review of valid, documented diagnosis, appropriate clinical indications and dose for hormonal treatment with a view to facilitating essential medical treatment but avoiding unjustified use or over-dosage. After stringent review TUE’s may be granted for treatment with testosterone, glucocorticoids, and insulin but there are very rarely any valid medical indications for EPO or, in adults, for growth hormone or IGF-1 in elite athletes. For example, TUE’s are usually justified for young male athletes with genuine androgen deficiency, occurring in ~1:200 men (29),  due to organic pituitary-testicular disorders with an established pathological basis (e.g. bilateral orchidectomy, severe mumps orchitis, Klinefelter’s syndrome, etc.) who require life-long testosterone replacement therapy (30). The TUE will approve, subject to regular review, a standard testosterone replacement regimen, including dosage and monitoring, with changes to regimen requiring approval. TUEs are not granted for men with functional decreases in blood T due to non-reproductive disorders including stress (“over-training”) or ageing (“andropause”, “LowT” “late-onset hypogonadism”), or for women.

 

In principle, detection of prohibited substances is ideally aimed at identifying a xenobiotic substance or its distinctive chemical signature(s) which do not occur naturally in the body, thereby distinguishing it categorically from normal body constituents. Such identification of a non-natural substance that can’t be of endogenous origin is congruent with the strict liability onus in proving an anti-doping rule violation (ADRV). Proving an ADRV is more difficult to achieve with administration of natural hormones or their analogs which must be distinguished from their endogenous counterparts. In this situation, the alternative requires developing valid biomarkers to prove the use of banned substances through their distinctive effects on the body and tissues. It is a formidable challenge to validate an indirect biomarker as proof of an ADRV capable of withstanding vigorous medico-legal challenge when a proven ADRV would prevent an athlete from pursuing their profession. Proof of an ADRV based on a doping detection test requires rigorous standardization and harmonization of every stage of the anti-doping tests from sample collection, chain-of-custody, storage, and analysis including accounting for any fixed (genetic, gender, age, ethnicity) or variable (exercise, hydration, masking vulnerabilities) factors which may impact on proposed test metrics.

 

COMPONENTS OF SPORTS PERFORMANCE AND DOPING

 

Sports performance has 4 major dimensions – skill, strength, endurance and recovery (Figure 1). High performance in any sport requires a characteristic blend of these dimensions although individual sports differ widely in that balance. Similarly, the major ergogenic drug classes have distinctive effects aligned predominantly along one of these dimensions so that the most effective ergogenic drug classes used in doping are dictated by these dimensions of sports performance (Figure 2). While every sport requires an acquired skill, some are largely or solely based on skill and concentration (e.g. board games, target shooting, car driving, and motor-cycle riding) and may benefit from drugs that reduce anxiety, tremor, inattention or fatigue. Sports that are highly dependent on explosive, short-term anaerobic power (sprinting, throwing, boxing, wrestling), typically ones which favor a stocky, muscular build, are most susceptible to androgen-induced increases in muscle mass and strength. Other sports with an emphasis on aerobic effort and endurance (e.g. long distance or duration events), characteristically favored by a lean build, may be boosted by hemoglobin doping (blood transfusion, erythropoietin (EPO) and its analogs or mimetics. Finally, sports that depend on recovery from major injury or recurrent minor injury during intensive training, notably contact sports, may benefit from tissue proliferative and remodeling effects of growth hormone and various growth factors.

Figure 1. Components of Sports Performance

Figure 2. Drugs that Enhance Sports Performance

ANDROGENS

 

Although the ergogenic effects of androgens were discovered empirically soon after the identification of testosterone as the principal male androgen of testicular origin in 1935 (31), their applications to elite sport performance were mainly developed during the Cold War by trial and error experiments undertaken on unknowing elite athletes (20,32,33); however, the scientific basis of androgen doping was only objectively proven in the 1990’s. Until that time, the settled consensus was that exogenous androgens had no effect in eugonadal men whose androgen receptors were already saturated by endogenous testosterone (T) (20,34,35). The then alleged benefits of androgen doping were misattributed placebo responses together with training and nutritional effects. Using an exemplary placebo-controlled, randomized clinical trial design with a wide range of testosterone doses, Bhasin et al showed that T increased muscle mass and strength in eugonadal young men to a similar extent as exercise alone and with additive effects when combined with exercise (36) (figure 3). Subsequent dose-response studies showed that administration of T increased muscle mass and strength by 10% without and 20-37% with exercise (where exercise alone increased them by 10-20%) together with additive effects from 3% increase in circulating hemoglobin. These benefits extended from below to well above physiological T doses or blood levels without evidence of plateau (37,38) and regardless of age (39).

Figure 3. Biological Basis of Androgen Doping

Androgen doping may be either direct or indirect (Table 3, figure 4). Direct androgen doping involves administration of testosterone, natural or synthetic androgens whereas indirect androgen doping includes a variety of non-androgenic drugs which increase endogenous T. Direct androgen doping originally involved all pharmaceutically marketed natural (T, DHT, nandrolone) and synthetic androgens but has extended to non-marketed designer and nutraceutical androgens as well as pro-androgens (androstenedione, DHEA) and the new class of non-steroidal androgens (selective androgen receptor modulators, SARM (40)). Indirect androgen doping involves use of hCG, LH, anti-estrogens (estrogen receptor blockers, aromatase inhibitors), opioid antagonists, and neurotransmitters involved in neuroendocrine regulation of endogenous LH and T secretion (41-44).

Table 3– Direct and Indirect Androgen Doping and Detection Methods

 

Substance

Detection method

Direct

  

Synthetic androgens

L/GC-MS

Natural androgens

L/GC-MS, T/E, CIRMS

Designer & nutraceutical androgens

L/GC-MS (bioassay)

Indirect

  

hCG (urinary or recombinant)

hCG immunoassay or LC-MS

hLH (recombinant)

hLH immunoassay or LC-MS

Anti-estrogens

L/GC-MS

GnRH analogs

L/C-MS

Opioid antagonists & neurotransmitters

L/C-MS

 

Figure 4. Direct and Indirect Androgen Doping

Detection of direct androgen doping using steroids of known chemical structure is highly effective using gas or liquid chromatography MS (45-47). Traces of synthetic androgens or their metabolites may remain detectable for periods up to months after last administration (48). Recent developments including the identification of long-term metabolites has further widened the detection windows for synthetic androgens (49-55). Challenges to detection of synthetic androgens have included the development of non-marketed designer and nutraceutical androgens, the use of natural androgens, and pro-androgens, masking methods, restricting use to out-of-competition training or micro-dosing. Designer and nutraceutical androgens are typically non-marketed synthetic androgens based on structures and synthesis methods recovered from expired patent literature of the 1960-70’s. These are now synthesized by unregulated chemical manufacturers without Good Manufacturing Practice (GMP) licensing advertising and sold over the internet or over-the-counter as nutritional supplements, which may contain undeclared steroids (56). However, once the chemical structures of any synthetic androgens are known, they are easily detectable although the sheer profusion of such chemicals represents an ongoing challenge. Nevertheless, despite their novelty, there is little evidence designer androgens have been used after they are discovered so that there is a high likelihood of detection. As a result, virtually all ongoing androgen ADRVs are still due to conventional marketed synthetic androgens.

 

Distinguishing Between the Exogenous and Endogenous Steroids

 

Administration of natural androgens (T or DHT) or pro-androgens (androstenedione, DHEA), raises the problem of distinguishing between the exogenous and endogenous steroids. Exogenous T administration can be detected by the urine T/E ratio, the ratio in urine of T to its 17α-epimer epitestosterone (E), operating as a sensitive screening test. In males, both T and E are co-secreted by Leydig cells and excreted in urine consistently so that the urine T/E is usually stable for any individual over time, being typically around 1. Administration of exogenous T, which is not converted to E, increases the urine T/E ratio and, when it exceeds a specified threshold, is evidence for administration of exogenous T. The urine T/E ratio thresholds were originally population-based, set initially at 6 and then subsequently lowered to 4. However, the urine T/E ratio is not an effective screening test for testosterone doping in females (57)because, unlike males whose circulating testosterone originates from as single source subject to strong negative hypothalamic feedback, circulating testosterone in females originates from three steroidogenic sources (adrenal, ovary, extraglandular conversion) none of which are subject to strong negative feedback by testosterone. Furthermore, the possibility of false negatives and false positives of population-based thresholds are limitations which may require further analysis to confirm or refute T doping in individual cases. These considerations have led to establishment of the steroid module of the Athletes Biological Passport (ABP), a compendium of serial observation of any individual’s tests which creates adaptive individual-specific T/E ratio threshold (58). This substitution of an individual’s own person-specific, in place of the population-based, thresholds allows for more sensitive and accurate detection of individual deviations in urine T/E ratio as evidence of T doping.

 

One limitation of the urine T/E ratio is a genetic polymorphism of the uridine 5'-diphospho-glucuronosyltransferase(UGT) 2B17 gene which encodes a phase II hepatic enzyme that glucuronidates T rendering it more hydrophilic to facilitate urinary excretion. This polymorphism comprises a genetic deletion which, in homozygotes, produces a non-functional enzyme that reduces urinary T (but not E) excretion to near zero producing an extremely low T/E ratio (<0.1). While this genetic polymorphism has no apparent biological effect on T action, it is unevenly distributed geographically being much more frequent in South East Asian populations (59). This biological false negative means that administration of exogenous T will be greatly reduced and may not exceed the usual population-based T/E ratio thresholds (60). On the other hand, it will exceed any individual’s own specific urine T/E ratio threshold so that genotyping and/or Bayesian profiling of serial T/E ratio in an ABP program provide complementary evidence (61-63).

 

Administration of exogenous T may also be identified by carbon isotope ratio MS (CIRMS) that can distinguish endogenous from exogenous T according to the C13/C12 ratio of urinary T (64,65). Commercially, steroids are manufactured from plant sterols produced by photosynthesis that exhibit distinctly lower C13/C12 ratio (typically, -26‰ to -36‰ relative to the global standard) compared with mammalian T biosynthesis (between -16‰ to -26‰) (66,67). Hence, a significantly lowered (“depleted”) C13/C12 ratio of urinary T, exceeding 3‰ relative to endogenous reference steroids, indicates that urinary T originates at least partly from exogenous chemical manufacture from plant sterols. CIRMS can also be applied to detect administration of other natural androgens or pro-androgens including DHT and DHEA (68), androstenedione, or even attempted masking by administering E (to lower urine T/E ratio) (66). A few T products (<5% (69)) have recently emerged with a lower, more mammalian-like C13/C12 ratio for urine T (70) creating a challenge for CIRMS detection. Nevertheless, extended isotope profiling of other steroid precursors and metabolites provides additional complementary reference biomarkers (71). A longitudinal application of CIRMS along the lines of another module of the ABP has been proposed (72). Furthermore, development of hydrogen ion ratio mass spectrometry has further enhanced the ability to distinguish between endogenous and exogenous steroids even when the carbon isotope ratio is non-informative (73-75). Suppression of urine (or serum) LH excretion may also provide corroborative evidence for the use of exogenous T or other synthetic androgens (63,76-78).

 

While MS is highly effective for detecting specific androgens, it requires knowledge of the chemical structure to be detected and otherwise cannot be applied. This principle applies to never-marketed designer or nutraceutical androgens sold over the internet or in unregulated over-the-counter nutritional supplements with unlabeled steroid content. A potential solution is the modern in vitro androgen bioassay that incorporates the human androgen receptor together with a convenient transactivation chemical read-out signal into a host yeast or mammalian cell (79). This has the generic capacity to detect all bioactive androgens regardless of structure due to their direct activation of the androgen receptor. Constructed in vitro androgen bioassays feature a sensitive dose-response signal proportional to the potency of the bioactive androgen (80-83). Yeast host cells have high specificity for detecting androgens but are less sensitive than mammalian cells, which express native steroid mechanisms including steroidogenic enzymes and/or other steroid receptors. Mammalian in vitro androgen bioassays can also detect pro-androgens, steroids lacking intrinsic androgenic bioactivity but which are converted into androgens within the mammalian cell. Hence, while mammalian host cells sacrifice specificity for higher sensitivity, they can also detect pro-androgens (79). Hence yeast and mammalian in vitro androgen bioassays are complementary in detecting both androgens and pro-androgens. The limitations of in vitro androgen bioassays are their susceptibility to matrix effects and difficulties in standardizing bioassay-based test. Consequently. they are best deployed to characterize products and substances for androgens or pro-androgen content rather than to detect androgens in complex biological samples. Hence the yeast androgen bioassay was decisive in the first conviction for use of a designer androgen by proving that tetrahydrogestrinone (THG) was a potent androgen (84) and has also been used to screen synthetic progestins to show that, unlike the original androgen-derived progestins, the modern generation of progestins are not androgenic (85).

 

Additional underutilized options to detect androgen doping is the use of alternative biological matrices such as hair, skin or nails as well as saliva and exhaled breath (86). Hair has the advantages of minimally invasive sampling with simple, convenient storage and the potential for very long window of detection, according hair growth rates (87). MS-based methods have been reported to detect exogenous (88-102) and endogenous (91,103-106) androgens in human hair following long-term, but not single dose (107), exposure. However, hair analysis tests have yet to undergo the rigorous standardization and validation required to become acceptable anti-doping tests in their medicolegal context. Problems that remain to be fully overcome include matrix effects, low recovery and limited sensitivity as well as the impact of age, hair color, alopecia, and shaving or passive chemical (cosmetic) contamination of hair.  Additionally nails and skin could also provide analogous information on past androgen exposure with relatively long windows of detection but suitably rigorous tests are yet to be convincingly developed (108). Saliva sampling has also been considered (109,110) for anti-doping application analogous to the use of salivary cortisol measurement for diagnosis of hypercortisolism (111). While potentially applicable to xenobiotic drugs, salivary testosterone immunoassay is not sufficiently accurate (112) and is not suitable to detect testosterone doping because even microscopic blood contamination (e.g. gingivitis, chewing hard food, tooth brushing) produces anomalous high readings. The existence of these renders salivary testosterone testing for antidoping purposes as unreliable by providing opportunity for claims of false positive for any adverse findings. Exhaled breath testing has also been investigated for certain small molecular weight chemicals (113). In theory, androgen-induced gene expression in circulating leukocytes might provide an additional biomarker of androgen action if specific and reproducible signatures can be defined (114); however, as direct detection of androgens is feasible and preferable for proving an ADRV, a role for gene expression biomarkers of androgen action remains to be established for anti-doping.

 

Indirect Androgen Doping

 

This doping strategy aims to increase endogenous T production and thereby evades detection by routine screening tests for exogenous T such as urine T/E ratio or CIRMS. Urine hCG is detected by commercial hCG immunoassays using immunoassays specific for intact heterodimeric hCG (including its nicked variant) which, if positive by exceeding a detection threshold (>5 IU/L), requires confirmation by a second immunoassay for intact heterodimeric hCG which is required to prove hCG use. A highly sensitive LC-MS method to detect urine hCG (115) is more specific than immunoassays (116) and has a lower threshold for a positive result in male athletes (117). A key issue is to distinguish a positive hCG urine test, presumptively indicating hCG doping, from early trophoblastic tumor or immunoassay artefacts. As hCG doping is not effective in women and urine hCG screening can detect early pregnancy, an unwarranted privacy intrusion, hCG testing is restricted to male athletes (43). Although direct LH doping is an implausible doping threat (118), suppressed (63,76-78,118) or elevated urine LH may be useful for confirming direct or indirect androgen doping (42,43,76,119). Anti-estrogens (estrogen receptor antagonists) or aromatase inhibitors, which can cause reflex increases in serum and urine LH and testosterone (42), are detected by MS-based chemical detection methods.

 

Overall, detection of direct androgen doping is now so effective that in WADA-compliant elite competitions it is restricted to the ill-informed, often using counterfeit or unlabeled products (120). Yet the potency of androgen doping in power sports continues to prompt development of novel androgen doping strategies. These will include use of undocumented synthetic androgens, novel indirect androgen doping methods and micro-dosing of natural androgens during out of competition training. The retreat to using micro-dosing inherently reduces the dose-dependent ergogenic benefits of doping while maintaining the risk of detection and disqualification. There remains a need to maintain deterrence by effective detection methods for evolving new androgen doping threats.

 

HEMOGLOBIN (BLOOD) DOPING

 

Hemoglobin doping involves either direct blood transfusion or indirect methods of increasing hemoglobin via stimulating erythropoiesis by administration of erythropoietin, its analogs or mimetics (see excellent reviews (121,122)) (Table 4). Boosting hemoglobin is advantageous in aerobic, endurance sports such as road cycling, distance running and cross-country skiing. Maximal oxygen consumption (Vo2), a rate-limiting factor in aerobic exercise, principally determined by cardiac output and blood oxygen transfer with a lesser contribution from tissue oxygen transfer (123). Experiments on exercise tolerance and blood transfusion were first reported in 1945 (124,125)but the scientific basis of hemoglobin doping via enhanced tissue oxygen transfer was firmly established in 1972 by the work of Ekblom et al reporting experiments in healthy volunteers who underwent venesection and/or re-transfusion of 1, 2 or 3 units (400 mL) of blood with repeated testing of maximal exercise-induced oxygen consumption before and after each procedure (126). This proved unequivocally that the maximal oxygen consumption was highly correlated with acute changes in hemoglobin (figure 5). Subsequently, during the 1970-80’s before its banning in 1988, blood transfusion became a prevalent surreptitious practice in road cycling and cross-country skiing and the apparently low prevalence among distance runners may be an underestimate (127). Modelling of historical performance in European road cycling from 1993 onwards shows a unique progression averaging an improvement of 6.4% corresponding closely with the performance enhancement (6-7%) due to rhEPO administration, which is sustained for at least 4 weeks after administration (128-130).

Table 4. Direct and Indirect Hemoglobin Doping and Detection Tests

Doping Mechanism

Detection

Direct (Blood transfusion)

  

Heterologous

Flow cytometry: bimodal population of blood group antigens

Autologous

No direct detection tests.

Athletes Biological Passport Biomarkers:

Urine phthalate excretion

Total hemoglobin mass

Indirect (Erythropoiesis stimulation)

Direct

  

rhEpo & biosimilars (>100) Epo analogs

Urine double immunoblot, (LC-MS)

Indirect

  

Hypoxia altitude training, hypoxic sleep area

Not banned

Hypoxia-mimetics: hypoxia-inducible factor & stabilizers, iron chelation, cobalt, 2,3 DPG analogs

LC-MS/MS

Artificial O2 carriers: HbOC, perfluorocarbons

LC-MS/MS

 

Figure 5. Direct and Indirect Hemoglobin Doping

Blood Transfusion

 

Transfusion may involve either another person’s (homologous) or the athlete’s own (autologous) blood administered prior to a contest to acutely increase circulating hemoglobin. Homologous blood can be transfused at any convenient time to enhance performance in competition but when performed by untrained personal in non-clinical environments risks transfusion reaction, blood-borne infectious disease, and iron overload. By contrast, autologous transfusion reduces health risks but requires complex coordination as venesection itself is detrimental to performance, and it requires balancing recovery from blood withdrawal and loss of erythrocyte viability during long-term cryostorage with training and competition schedules. Although blood transfusion was first banned by the IOC in 1986, the first practical approach to banning blood doping was the introduction of hematocrit testing in 1997 by the international skiing and cycling federations. These regulations excluded athletes on health grounds from entering competition on the day if their hematocrit exceed a safety threshold (0.50). However, this encouraged hematocrit titration to just below threshold and only prevented competing until hematocrit returned under that threshold, which could be quickly accomplished by venesection. The first ADRV’s for blood manipulation involving hematocrit threshold and titration were in 2001.

 

Homologous blood transfusion creates a bimodal population of blood group antigens which is detectable by flow cytometry using a panel of 12 minor blood group antigens (131), from the wider array of blood group antigens (132), which can detect a <5% contamination of exogenous erythrocytes. Subsequent refinements simplified and improved test sensitivity so that a panel of 8 antigens can detect contamination comprising a minor admixture population of 0.3-2.0% with no false positives but high sensitivity (~80%), the latter depending on the magnitude of the minor contaminating mixture (133,134). Alternatives based on genotyping for the admixture population of leukocytes have also been proposed (135,136). As a test proving unequivocally the presence of non-endogenous erythrocytes in the circulation, this method is definitive if performed to the required standard. A remotely hypothetical defense against a positive test, based on stable marrow chimerism from a vanished twin, was raised by a cyclist who subsequently admitted transfusion (135). Based on risk of detection as well as to health risks, homologous transfusion has now largely disappeared in favor of autologous transfusion (137).

 

Autologous Transfusion

 

The biggest gap in current anti-doping tests is the lack of a specific test to detect autologous transfusion (138). Research to identify robust physico-chemical or biological markers for direct identification of a subpopulation of ex-vivo aged erythrocytes is underway using flow cytometry  (139) but the dilution and rapid clearance of effete erythrocytes make for challenging detection problems (140). In the interim, other indirect methods have been developed. These include measuring urinary excretion of phthalates, plasticizers that leach out from the polyvinylchloride blood packs used to store venesected blood (141). This test has brief window of detection (2 day) so will detect auto-transfusion during or immediately before events (characteristic in road cycling, according to convicted dopers) but may miss earlier auto-transfusion. Furthermore, the ubiquity of low-level environmental phthalate exposure requires establishing detection thresholds and non-plastic blood containers can be used. An alternative is the measurement of total hemoglobin mass (142), a measure with good stability and reproducibility even during exercise and circumvents influence of variations in plasma volume such as due to dehydration or dilutional masking (142,143). However, as this requires inhalation of carbon monoxide, which has transient detrimental effects on performance, it is not ideal for routine anti-doping use and its sensitivity may be insufficient to detect all EPO micro-dosing (144,145). Nevertheless, alternative methods for serial measurement of total hemoglobin mass remain attractive. Other hypothetical methods include the detection of microRNA (146) or immune reactions to transfusion (147) but the sensitivity and specificity of these proposed tests remains to be fully evaluated.

 

The best detection test for autologous hemoglobin doping at present is the hematological module of the ABP introduced in 2009 (148). Conceptually, it is a biomarker test which adopts a Bayesian approach of creating serially-adaptive, person-specific reference limits, based on using all prior testing, to supplant population-based thresholds. Combining all of an individual’s previously collected hematological data creates a probabilistic test of whether any new result deviates significantly from that individual’s personal reference limits (149). These person-specific thresholds allow for ongoing refinement and reinforcement by further testing. The thresholds are calculated by a variety of algorithms incorporating routine hematological parameters, notably hematocrit and reticulocyte counts. Those were developed over the last two decades to create the ABP hematological model which is sensitive to both direct and indirect hemoglobin doping (150). The first attempts to regulate hemoglobin doping in the late 1990’s sought to prevent road cyclists or cross-country skiing athletes competing on health risk grounds when their hematocrit exceeded pre-determined, population-based safety criteria (e.g. hematocrit 0.50 or hemoglobin 170 g/L for cycling). However, while this excluded extreme hemoglobin doping only until the short period when the safety threshold was no longer exceeded, it allowed an increase in an athlete’s natural hematocrit, typically averaging ~0.45, up to the permitted ceiling threshold which fostered titrated hemoglobin doping and manipulations like hemodilution by saline or plasma volume expander infusions to avoid detection (151). More sophisticated hematological algorithms were then developed to detect hemoglobin doping initially for the Sydney 2000 Olympics (152,153), the first generation of algorithms developing validated tests for ongoing and for recent cessation of hemoglobin doping, using a combination of biochemical variables related to erythropoiesis physiology. This approach was simplified by a second generation algorithm using only routine hematological parameters (hemoglobin, reticulocytes) (154), and was subsequently combined with the concept of a sequential development of individual-specific reference ranges (155)  into a third generation algorithms (156,157) which were refined for the ABP (148,149). The hematological module of the ABP currently employs an algorithm involving 8 parameters derived from routine hematological profile (hemoglobin, hematocrit, erythrocyte count, reticulocyte count and percentage, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration) (158). This is capable of detecting any form of hemoglobin doping, whether direct or indirect, with good but imperfect sensitivity (143-145) and using only routine hematological tests. The reported increasing use of very low EPO doses (“micro-dosing”) would markedly reduce the magnitude of any dose-dependent ergogenic benefits (145) while still carrying risks of detection, disqualification, and disgrace.

 

Stimulation of Erythropoiesis

 

Indirect methods to increase hemoglobin include administration of recombinant human EPO or its analogs as well as hypoxia-mimetic drugs (hypoxia-inducible factor stabilizers, iron chelation, cobalt, 2,3 diphosphoglycerate analogs) or artificial oxygen carriers (perfluorocarbons, hemoglobin-based oxygen carriers). Related but non-banned methods include altitude training or its simulation by sleeping in hypoxic rooms which are less effective than hemoglobin doping (129).

 

The identification of the human EPO gene in 1985 led to the marketing of recombinant human EPO (rhEPO) between 1987-9. Despite the IOC’s prohibition of EPO’s use in sports in 1990, the commercial availability of rhEPO created powerful new opportunities for indirect hemoglobin doping which were soon proven experimentally (159). A drug, which circulates for hours to days, but with potent and long-lasting ergogenic effects after its disappearance due to the 4-month lifespan of erythrocytes, is both attractive for doping and a challenge to anti-doping testing. Expiry of the rhEPO patent in 2004 allowed marketing of a profusion of generic EPO (“biosimilar”) products, estimated globally at over 80 (160), as well as modified EPO analogs (darbepoeitin, pegylated EPO, peginesatide, EPO fusion proteins). A fatal cluster involving deaths of 18 Dutch and Belgian road cyclists, presumably due inadvertent over-dosage during empirical attempts to maximize ergogenic effects of illicit rhEPO, was reported (161), although difficult to verify (162). A similar excess of unexpected deaths of road cyclists was also reported again in 2003-5 when novel EPO analogs and EPO biosimilars were marketed.

 

Detection of EPO in urine is difficult because of the prevailing low concentrations and need to distinguish exogenous recombinant from endogenous EPO. The first effective method for rhEPO in urine was a double immunoblot (163,164)which was capable of detecting urinary excretion of a variety of exogenous EPO products and analogs according to their differences in glycosylation side-chains, and differences in primary amino acid sequence where they exist, while distinguishing them from endogenous EPO. Although further refined (165) and extended to other EPO analogs (166), the immune-electrophoresis test is sensitive but relatively laborious and provides only a short window of detection of up to a week post-administration (167). More sensitive methods based on proteomics (for EPO analogs with differences in primary structure) together with glycomics (for biosimilars and analogs which have host-cell specific variations in side-chain glycosylation but unchanged natural EPO primary structure (168)) are possible but not yet approved. Additional applications to detect EPO and analogs using dried blood spots have been reported (169). Similarly, preliminary investigations have proposed a EPO-induced gene expression signatures as a biomarker to detect EPO administration but specificity relative to exercise and other physiological effects remain to be clarified (170).

 

Other EPO mimetics such as hypoxia mimetic drugs including hypoxia-inducible factor (HIF) stabilizers and related small molecules represent growing threats as potential indirect hemoglobin doping agents (171). These non-peptide chemicals interfere with various steps of the molecular oxygen sensing mechanism to mimic renal hypoxia and thereby induce EPO secretion resulting in increased blood hemoglobin. As a convenient orally active alternative to the lucrative pharmaceutical market for injectable erythropoiesis-stimulating peptides (~$7-8 billion (172)) to counteract anemias of chronic renal failure or marrow failure due to myeloproliferative disease or cytotoxic cancer therapy, they constitute a very active area of pre-clinical patent-based clinical drug development (171). Experience suggests that such innovator products can enter the doping black market before marketing approval (150,171). Despite the profusion of pre-clinical leads, they represent families of related chemical structures disclosed in patents for which LC and/or GC-MS detection tests should, in principle, be effective. Understanding the metabolism of these drugs when they come to market may identify long-lasting metabolites that can extend the windows of detection. Coupled with evidence from the ABP, manipulation of the EPO pathway may be detected in conjunction with corroborative measurement of inappropriately suppressed or elevated endogenous EPO for the prevailing hemoglobin level.

 

HIF is a key generic biological mechanism for tissue sensing of hypoxia and triggering local (neovascularization, angiogenesis) and systemic (EPO) defensive reactions. The promoter of the EPO gene contains enhancer and inhibitor regions with the hypoxia-responsive element which binds HIF and a GATA binding site which enhance and inhibit, respectively, EPO gene transcription. HIF is a heterodimer formed by constitutively expressed subunits with the β subunit in excess and availability of α subunit limiting formation of bioactive HIF. The 3 HIFα subunit isoforms are subject to hydroxylation of specific proline residues by prolyl hydroxylase enzymes which inactivate HIFα by ubiquitination, a tag which targets it to proteasomal degradation. HIFα subunit inactivation is strongly dependent on tissue oxygenation being active during normoxia but reduced during hypoxia when persistence of HIFα stabilizes the HIF heterodimer. Notably, during hypoxia the expression of HIFα in renal cortical cells stimulates EPO gene expression so that HIF stabilization by prolyl hydroxylase inhibitors leads to increased EPO secretion and circulating hemoglobin. Hence inhibiting prolyl hydroxylase activity via blocking its required cofactors (ascorbate, ketoglutarate, iron) using cobalt, nickel, iron chelation, ketoglutarate analogs or mechanism-based chemical inhibitors can result in increased hemoglobin via stimulation of EPO secretion (171). Similarly, small molecule GATA inhibitors potently stimulate circulating EPO, hemoglobin and performance in mice (173) although none have yet been marketed so their human efficacy and safety remain to be determined.

 

Another approach to increase oxygen delivery to muscle has been to exploit the ability of 2,3 diphophoglycerate (2,3 DPG), whose binding to hemoglobin reduces its affinity for oxygen with the left-shift of its oxygen dissociation curve as an oxygen unloading mechanism in tissues. 2,3 DPG analogs, developed as radiation sensitizers for hypoxic radio-resistant tumors, enhance tissue oxygen delivery in vivo (174,175) but would feature only short-term, acute effects readily detectable by mass spectrometry (176,177).

 

Adverse effects from use of rhEPO or its analogs are well known in medicine but poorly recognized in doping. They include immunogenicity (with risk of EPO autoantibody mediated pure red cell aplasia) (178,179), cardiovascular complications (including venous thromboembolism, stroke, hypertension and myocardial infarction) and premature death (180-183). In routine clinical use of EPO to correct renal anemia, the goal is a gradual increase to subnormal hemoglobin targets so that the excessive and/or rapid rises in hematocrit and blood viscosity (184) may explain the excess unexplained deaths among young European road cyclists in the late 1980s. In addition, use of rhEPO may deplete iron stores which limits hemoglobin synthesis so that athletes may also use oral or injectable iron supplements, which carry their own risks such as iv iron supplementation’s potentially adverse effects in enhanced tissue oxidative damage and excess mortality in chronic kidney disease (185). Although clinical safety experience with ESAs is restricted to patients with serious medical disorders, there is evidence from the general community that higher natural hematocrit is associated with worse long-term cardiovascular health outcomes (186-188).

 

GROWTH HORMONE

 

Growth hormone (GH) is a tissue growth promoter in children but after puberty it is predominantly a metabolic hormone although latent tissue growth promoting effects may be unleashed under non-physiological circumstances, such as during recovery from tissue injury. There is consistent anecdotal evidence that GH has been used in elite sports for decades (189). Nevertheless, ergogenic effects of GH remain unproven and largely speculative as discussed in excellent recent reviews (190-192). Claims of GH benefits in sport have included increases in muscle mass and strength, especially in conjunction with androgens, and/or improved tissue healing with more rapid recovery from either major injuries or minor repetitive injuries, such as from intense physical training allowing for more effective training. The biological basis of ergogenic effects of GH have been tested in these two different scenarios with largely inconclusive findings.

 

Evidence for direct enhancement of athletic performance by GH has been investigated in two well controlled RCTs with a primary focus on athletic performance. In one study, 96 recreational sub-elite athletes (63 male, 33 female, mean age 28 years) were administered 8 weeks of daily sc injections of GH or placebo with the men also having weekly im injections of T enanthate or saline placebo for the last 5 weeks (193). GH increased lean (muscle) mass (by +2.7 kg) and reduced fat mass (by -1.4 kg) while T increased lean mass (alone by +2.4 kg, by +5.8 kg with GH). The effects of GH were marginally significant for anaerobic sprint capacity (by +3.9%, p=0.05) when pooling male and female participants but this was due to significant effects in men only (by +5.5% alone and +8.3% with GH). However, there were no significant effects on maximal Vo2 consumption, dead lift, or jump height (193). A second study involved 30 healthy non-athletes (15 male, 15 female, mean age 25 years) who were administered daily sc injections of GH at high (4.6 mg/day) or low (2.3 mg/day) doses or placebo (194). There was no significant effect on muscle mass or maximal Vo2 consumption. Additional controlled studies of GH effects but with less focus on athletic performance have also shown that (a) a single dose of GH (~0.8 mg) in 9 recreational athletes did not affect maximal Vo2 or power output in repeated 30 min bursts of bicycle ergometry (195), (b) short term (6 days), low dose GH (~1.7 mg/day) treatment of 48 male androgen abusers withdrawn from androgens for 12 weeks significantly increased maximal Vo2more than placebo (196), (c) daily sc injections of a GH receptor antagonist (pegvisomant) or placebo for 16 days to 20 sedentary men did not change maximal Vo2 although time to exhaustion at 90% maximal Vo2 was reduced (197)and (d) 4 weeks of daily sc injections of GH (~5 mg/day) increased whole body protein synthesis (198), lipolysis and glucose uptake (199) with uncertain significance for athletic performance. Overall, these studies indicate that GH has, at most, a modest ergogenic effect in men only and through enhancing T effects. That is consistent with the fact that young women have markedly greater growth hormone secretion than young men so that growth hormone cannot explain the sex differences in athletic performance (200).

 

It is also claimed that GH may enhance injury healing, thereby facilitating more intensive training and/or recovery from muscle, connective tissue or bone injury, notably in contact sports. This claim is difficult to evaluate and no well controlled studies of recovery from sports injuries or tolerance of training intensity in elite athletes are reported. The most germane surrogate evidence available arises from investigations on the use of GH in recovery from injuries due to burns, fracture, or for wound healing. A recent Cochrane meta-analysis review of GH treatment effects on recovery from burns injury and healing of donor skin graft sites suggests that GH has a small benefit in skin healing with large burns and reduced hospital stay but there was no benefit in reducing mortality or scarring and adverse effects, notably hyperglycemia, were increased (201). In practice, the increased mortality due to administration of high dose GH in critical illness (202) has led to GH treatment not being widely adopted in clinical practice of treatment of burns. Similarly, the only well controlled study of GH effects on bone healing from fracture reported that, among over 400 patients with tibial fractures treated for up to 16 weeks with GH (1, 2 or 4 mg/day) or placebo, there was no benefit of GH for overall healing (203). Finally, while there are numerous experimental studies of GH or growth factors on wound healing in animal models a wide variety of findings are reported with detrimental, neutral, or beneficial effects but no well-controlled human studies are available. In summary, the available evidence for improved tissue repair or regeneration is minimal.

 

Important caveats on interpreting these few well designed studies are that the effects of higher GH and T doses, as used in doping, have not been studied so that more potent higher dose and/or interactive effects cannot be excluded in the absence of well controlled, high dose, placebo-controlled studies. Nevertheless, the hypothesis that high dose GH exposure would enhance muscular function is inconsistent with the experience of acromegaly in which patients experience much higher (25-100 times) growth hormone exposure than doses that can be ethically administered to healthy human volunteers (204), yet characteristically display muscular weakness rather than increased muscle size or strength (205). Anti-doping science history suggests that caution is required before rejecting evidence for claimed ergogenic effects without investigations replicating the pharmacological doses used.

 

Furthermore, safety analysis is not feasible based on the few, small, short-term studies of GH’s potential ergogenic effects; however, there are significant safety concerns about the long-term risk of cancer following GH administration. Even standard therapeutic GH doses administered to GH deficient children are associated with increased risk of second cancers in some (206-208) but not all (209) follow-up studies although these risks appear largely confined to survivors of childhood cancers and its treatment which render them GH deficient (210-213). Although the significant cancer risk based on uncontrolled observational cohort data using standard GH doses remains contentious (214,215), the long-term risks of much higher GH doses used illicitly by athletes must be viewed with significant concern.

 

Detection of GH doping remains difficult (216). A major challenge is the non-glycosylated primary structure of recombinant and endogenous 22 kDa GH, that lack the distinctive side-chain carbohydrate differences of exogenous glycoproteins EPO or hCG which provide a convenient basis for sensitive molecular detection tests. Nevertheless, minor infidelities in commercial manufacturing of GH may incorporate distinctive non-natural chemical features proving an exogenous origin (217-219) although these findings have not been developed into detection tests. Challenges to the detection of GH doping arise from the physiological pattern of endogenous GH secretion with its intermittent, pulsatile pattern subject to prominent influence of exercise, stress, and nutritional effects together with GH’s brief circulating half-life and low urine concentrations (220,221). Like other major doping classes, there are both direct and indirect forms of GH doping, involving either direct administration of GH or IGF-I or their analogs and indirect GH doping involving drugs that aim to increase endogenous GH and IGF-I secretion (Table 5).

 

Table 5. Growth Factors, Growth Hormone Related and Other Peptides

Growth Factors

Growth Hormone Related Peptides

Other Peptides

 

GHRH analogs

Ghrelin analogs

Other

 

FGFs

GHRH

Lenomorelin (ghrelin)

IGF-1 & analogs (MGF, long R3IGF-1)

Thymosinß4

HGF

CJC-1295

GHRP-1

IGF-2

 

MGF

CJC-1293

GHRP-2 (pralmorelin)

Insulin & analogs

 

PDGF

Sermorelin

GHRP-3

AOD-9604

 

VEGF

Tesamorelin

GHRP-4

hGH 176-191

 

 

 

GHRP-5

 

 

 

 

GHRP-6

 

 

 

 

Hexarelin

 

 

 

 

Ipamorelin

 

 

 

 

Alexamorelin

 

 

 

 

Anamorelin

 

 

 

 

Macimorelin

 

 

 

 

Tabimorelin

 

 

 

 

Examorelin

 

 

 

The first test to detect administration of exogenous GH, the 22kD recombinant form of human GH, was based on blood sampling to measure the ratio of circulating isoforms of GH recognizing the fact that the pituitary secretes not only the major 22 kD isoform (65-80%) but also a variety of minor isoforms including a wide variety of minor isoforms and their multimeric variants (222). Administration of exogenous GH suppresses endogenous pituitary GH secretion leading to a predominance of circulating 22 kD GH. This is the basis for the GH isoform ratio test whereby a serum sample is measured by two different GH immunoassays, one with predominant 22 kD GH specificity (“rec” assay) and the other recognizing the broad spectrum of pituitary GH isoforms (“pit” assay) and the ratio of results (“rec”/”pit” ratio) is an index to detect administration of exogenous recombinant GH (220,223). This ratio test then serves to detect administration of exogenous recombinant human 22kD GH analogous to detection of exogenous T by the urine T/E ratio and exogenous insulin by analysis of serum C peptide (224). The differential GH isoform ratio test has undergone extensive validation involving standardization of the two GH immunoassays with distinctive immunoreactivities to quantify 20kD and 22kD epitopes as well as its application to various populations of elite athletes and evaluating physiological factors which might impact on the validity of test read-out. A strength of this test is that it is aimed at the exogenous doping agent itself, although it cannot definitively distinguish it from its endogenous counterpart. The major limitations of this differential isotope ratio test are its narrow window of detection (24-36 hr post administration) and its inability to detect indirect GH doping. While pituitary-derived human GH might not be detected, human pituitary GH, once obtained from national scale pituitary collection and purification programs, has not been available since 1985 when its risks of Creutzfeldt-Jakob disease were identified (225,226) with recombinant human GH replacing pituitary-extracted GH worldwide. This differential isoform test was first introduced for the 2004 Olympics (227) and led in 2010 to the first successful detection of out of competition GH doping (228).

 

A complementary detection test with a wider window of detection has been developed based on biomarkers of GH action. This uses two serum biomarkers of tissue GH effects, circulating IGF-1 as a short-term marker of hepatic GH action, and N-terminal peptide of procollagen type III (PIII-NP) as a long-term marker of GH-dependent collagen synthesis. In a study of 102 recreational athletes (53 male, 49 female, mean age 25 years, from 4 different European cities) randomly assigned to self-inject 2.7 mg or 5.4 mg GH or placebo once daily, measurement of serum IGF-1 and PIII-NP by specific immunoassays were able to correctly classify 86% of samples from males and 60% of samples from female using an empirical linear discriminant analysis of log-transformed serum IGF-1 and PIII-NP at the specificity of 1:10,000 required for a WADA biomarker threshold (229). Subsequent studies have shown that additional collagen biomarkers, N-terminal propeptide and C-terminal telopeptide of type I collagen, further widen the window of detection for GH administration (230,231). This multiplex biomarker test, based on using standardized immunoassay antibodies, requires establishment of reliable reference range with specificity (false positive detection rate) of no more than 1:10,000 incorporating the impact of gender and age, although exercise, injury, ethnicity and sports type appear not to be confounding influences but is not yet in routine use by WADA anti-doping labs. The two GH doping test, the differential isoform and biomarker approaches, are considered ultimately complementary (232).

 

IGF and Insulin Doping

 

IGF-1 is a circulating marker of hepatic GH effects and mediator of GH action so the marketing in 2005 of recombinant human IGF-I alone, and later with its major binding protein recombinant human IGF binding protein 3 (IGF-BP3) (233), for treatment of diabetes, insulin or GH insensitivity or motor neuron disease, together with the availability of IGF-1 analogs for laboratory use, creates the possibility of IGF doping (234). Time-series analysis of elite sports performance (235) is consistent with the occurrence of IGF-1 doping but its prevalence is unknown (56). As the biological basis for ergogenic effects of IGFs is due to its GH-like effects, this remains largely speculative and accompanied by the same safety concerns. IGF-1, IGF-2 and their analogs (236) as well as insulin and its analogs (237) are all readily detectable by LC-tandem MS and preliminary evidence suggests that biomarkers for IGF-1 administration (IGF-2, IGFBP2) may widen the window of detection (238). However, a specific test to detect IGF doping remains to be established (239).

 

MGF is a splice variant of IGF-I which, although not known to appear in the circulation, have any pharmacological effects, or be approved for human use (240), is advertised on the black-market and internet (241) for alleged anabolic or tissue repair/regeneration benefits. Like other short peptide with known structure, it is readily detectable using LC- tandem MS (241).

 

Insulin has long been used in doping and was prohibited in sports since 1999 (242,243). Other than its proper medical use in diabetics, the use of insulin and its analogs for doping is based solely on its easy availability coupled with anecdotal information from other drug users. There are no clinical studies showing any ergogenic effects of insulin or its analogs in non-diabetic individuals. The doping folklore appears to arise from the classification of insulin as being “anabolic”, in a loose generalization and mistaken analogy to androgens. In healthy non-diabetic individuals, insulin and its synthetic analogs stimulate weight gain via hypoglycemia and increasing appetite, but produce fat rather than muscle gain. The adverse effects include hypoglycemia, hypokalemia, injection-related infections and weight (fat) gain. Doping detection tests for insulin and its analogs continue to evolve and focus on highly sensitive and specific quantitative MS-based proteomics (244).

 

Growth Factors, GH Releasing and Other Peptides

 

For the unscrupulous in pursuit of the unlawful, the increasingly stringent detection of androgen and hemoglobin doping, the two most potent classes of ergogenic drugs, has led to new, highly speculative form of doping involving peptide growth factors and GH releasing peptides. These are within the size range of automated bulk custom peptide synthesis and are marketed cheaply by chemical manufacturers. While notionally sold solely for laboratory research, these unregulated products are available for purchase over the internet. Promoted by speculative fantasies on their mode of action coupled with testimonials to their efficacy but without objective testing or assurance of safety in humans, they are believed to be widely used by gullible and/or desperate athletes and their trainers. As unregistered drugs, this growing range of peptides appears to constitute a greater threat to athlete’s health than a risk of effective cheating.

 

The S2 category of Prohibited Substances lists, in addition to GH and IGF-1, GH fragments and releasing peptides, a wide array of growth factors and modulators and, crucially, a generic catch-all provision for unnamed growth factors and peptides which may affect connective, vascular, muscular, or regenerative tissues or energy utilization and other substances with similar chemical structure or biological effects.

 

The major category of oligopeptides used for doping is the class of GH releasing peptides analogs of the endogenous GH releasing peptides, GHRH and ghrelin, whereby their analogs aim to increase endogenous GH secretion and are therefore banned (table 5). Most of these peptide were developed in the pharmaceutical industry from the 1990s aiming to provide cheaper, orally active, non-peptide agonists with capacity for sustained stimulation of endogenous GH secretion to “rejuvenate the GH/IGF-1 axis” (245), an unusually explicit acknowledgment of the regular nexus between hormonal rejuvenation and doping (246). However, none of these hormonal peptides have been registered for human therapeutic use with only one (pralmorelin) registered for single-dose, diagnostic use (for GH deficiency) in Japan and unacetylated cyclin ghrelin marketed in Europe for Prader-Willi syndrome. Although they may stimulate GH release initially, many failed to achieve sustained GH release due to desensitization and none achieved meaningful clinical improvements in any target diseases. If their unproven ergogenic benefits are due to sustained GH release this renders them unlikely to be beneficial; nevertheless, the caveat on not accepting negative conclusions without direct testing are also relevant to this class of peptides. Like other short peptides, once chemical structures are known, detection is readily feasible using LC-MS (247,248). The illicit nature of this market raises the risks of counterfeit and unsafe products with attendant risks of infection and residual toxic contaminants unlike the purity pharmaceutical product manufacturers are required to demonstrate by batch release testing.

 

PROGRESS, GAPS, AND FUTURE PROSPECTS

 

Anti-doping science continues to make major progress over recent decades especially since the advent of WADA with its harmonization and focus on deterrence through standardized testing. Progress from improved MS-based testing methodologies and instrumentation, summarized annually by the editor (M Thevis) of the major antidoping science journal, Drug Testing and Analysis (249), is evident from the increasing numbers of ADRV findings among frozen stored urine samples now banked for 10 years from previous Olympics. Like any efforts to combat human malfeasance, the quest for drug-free and safe sport requires ongoing vigilance and continual renewal of intelligence-based detection testing. While great progress has been made in the two canonical forms of doping, androgen and hemoglobin doping, human ingenuity continually finds way to challenge the testing just as traditional frauds are supplanted by cyber-crime and ingenious computer hacking. It is important to bear in mind that the winning margin (defined as the difference in performance between gold and silver medals, getting a medal or not, making a final or not in the Olympic athletic or swimming events) is <1% (200) so even small systematic advantages may be important motives and unfair advantages for doping.

 

The major gaps remaining in anti-doping science are (a) the lack of a definitive test for autologous blood transfusion, (b) need for more sensitive detection tests for peptide doping with wider windows of detection and (c) more economical, affordable and robust sample handling and storage procedures including dried blood spot sampling. These challenges must be met by adapting novel technologies such as quantitative proteomics, genomics, and metabolomics as well as implementing more out of competition and blood testing. Such progress depends on innovative applied research which is supported by WADA, Partnership for Clean Competition and certain national anti-doping organizations together with regular peer-review research granting agencies. Finally, the development of effective forensic intelligence investigations, a slow, complex and costly process but which can have salutary effects (e.g. for road cycling in the Lance Armstrong case), is proving a valuable complementary approach as an adjunct to effective laboratory testing.

 

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Laboratory Assessment of Testicular Function

ABSTRACT

 

Since the symptoms of hypogonadism are nonspecific, and the signs of testosterone deficiency can be subtle and slow to develop, the assessment of testicular function relies heavily on laboratory testing. The laboratory diagnosis of hypogonadism is based on a consistent and unequivocally low serum total testosterone level measured in blood samples obtained in the early morning, but normal ranges vary with different methods and among laboratories. Moreover, many men who present with adult onset testosterone deficiency have a low level of sex hormone-binding globulin (SHBG) associated with obesity, insulin resistance, and type 2 diabetes. In these men, tests for the free (or non-SHBG/bioavailable) testosterone fraction testosterone) are helpful for an accurate diagnosis. If testosterone deficiency is confirmed, the next step is to differentiate between primary and secondary hypogonadism by measuring LH and FSH.  With many disorders, however, both the testes and the hypothalamic-pituitary unit are affected. Other tests such as estradiol, inhibin-B, and Mullerian inhibitory hormone, and provocative endocrine tests using hCG, GnRH or its analogs, or antiandrogens or antiestrogens, and semen analysis in the subfertile male are discussed.   

 

INTRODUCTION

 

The evaluation of men for suspected hypogonadism begins with a detailed medical history and a careful physical examination. Laboratory tests are an essential component of almost all evaluations, and proper interpretation of the results obtained requires an understanding of methodology as well as an awareness of the impact of endocrine rhythms, age, race, body composition, drug exposure, co-morbidities, other illnesses, and nutrition on tests of endocrine testicular function. Endocrinologists rely on clinical laboratories to provide accurate and precise, and in some cases highly sensitive assays for accurate diagnoses. We expect the reference ranges to be based on large normal control populations, and for some assays, normative data must be stratified by age. Unfortunately, these expectations are not always fulfilled. Endocrinologists should examine the protocols for each assay, and discuss them with the laboratory director. In recognition of these potential shortcomings, the U.S. Endocrine Society has spearheaded efforts towards standardizing the methods by which testosterone assays are validated (1), and several societies have provided guidelines and recommendations to physicians who order and interpret the results of androgen assays (2-4). The College of American Pathologists and the U.S. Centers for Disease Control havevoluntary Hormone Standardization Programs to help clinical, research, and public health laboratories maintain and enhance the quality and comparability of their results.

 

TESTOSTERONE

 

Testosterone, the major androgen in men, is necessary for fetal male sexual differentiation, pubertal development, and the maintenance of adult secondary sex characteristics and spermatogenesis. Testosterone regulates gene expression in most extra-genital tissues, including muscle and bone, and modulates the immune system. The testes are the source of more than 95% of the circulating testosterone in men although the adrenal cortex produces large amounts of the testosterone precursor steroids, dehydroepiandrosterone (DHEA) and androstenedione.

 

Following birth, there is a period of activation of the gonadotropin-releasing hormone (GnRH) pulse generator that stimulates testosterone secretion to peak levels of 200-300 ng/dl (7-10.5 nmol/L) at ages 1-2 months and lasts until age 4-6 months, and is often called mini-puberty (5). Thereafter, GnRH secretion declines, causing a fall in testosterone to very low levels until puberty begins.  LH and testosterone testing at mini-puberty provides an opportunity to confirm the diagnosis of hypogonadotropic hypogonadism (6).

 

In early puberty, LH and testosterone secretion increase dramatically during sleep which gradually transitions to sustained secretion throughout the day and night (7). The nocturnal rise in testosterone levels in early puberty can be used clinically to evaluate boys with delayed puberty because it may precede pubertal testis growth, and indicates that puberty has begun.  Figure 1 illustrates age-specific median values and ranges for testosterone in morning blood samples from 138 boys (8).  

Figure 1. Serum testosterone levels (median and range) by LC-MS in leftover samples from blood checks before minor surgery or for the exclusion of endocrine diseases. Data from Kulle et al (8). To convert total testosterone levels to nmol/L, multiply by 0.0347

Hypothalamic neurons that express kisspeptin, neurokinin-B, and dynorphin, are thought to activate and up-regulate GnRH and initiate pubertal development (9).  Adult testosterone levels are usually achieved by age 16 years, and generally range from 300-1000 ng/dL (10-35 nmol/L). 

 

The secretion of testosterone is periodic with oscillations occurring over hours, days, and months. Frequent sampling of peripheral blood in adult men reveals small frequent moment-to-moment fluctuations (10) whereas spermatic venous blood sampling reveals robust episodes of testosterone secretion occurring about once per hour (11).  Presumably because of this rapid pulse frequency and a plasma half-life of 60-90 min, only small testosterone fluctuations are generally observed in peripheral blood. Therefore. a single blood sample is usually an adequate assessment of testosterone production on a given day.  There is a substantial diurnal variation in testosterone levels in adult men, however, with highest levels in the early morning, followed by a progressive decline throughout the day, reaching lowest levels in the evening (Figure 2). Thus, the time of day of blood sampling is an important consideration, and a blood sample drawn in the morning between 0800 and 1000h is recommended because reference ranges are generally based on morning values. Nadir evening levels  in young men are generally 15-20% lower than morning values although the differences can be as great as 50% (12). The diurnal testosterone rhythm is blunted as men grow older (13) as well as in young men with primary testicular failure. On the other hand, the diurnal variation in testosterone is exaggerated in hypogonadal men with hyperprolactinemia (14), much like normal adolescents. There may be a seasonable variation in testosterone as well (15).

Figure 2. 24-hour testosterone concentration profiles in eugonadal men (n=5), untreated hypogonadal men with PRL-producing pituitary adenomas, and men with testicular failure (n=5). Adapted from Winters, SJ (14). To convert total testosterone levels to nmol/L, multiply by 0.0347.

There is also a slight decline in testosterone levels following a meal (16), so that some authors suggest obtaining a fasting level.  In general, the results of one abnormal test should be confirmed. In one study in which multiple blood samples were taken from middle-aged men over one year, there was a relatively good correlation (r=0.85) between the plasma testosterone level in the first sample and the mean value of seven subsequent samples; however, as many as 15% of men were misclassified, with either low or normal values that were not reproducible (17).

 

Testosterone deficiency can be an obvious clinical diagnosis, and laboratory tests are often merely confirmatory; however, the diagnosis of hypogonadism is sometimes less straightforward. Experts and international guidelines state that the diagnosis of hypogonadism requires the presence of symptoms of hypogonadism as well as a low serum testosterone level. Symptoms of hypogonadism lack specificity, however, and are subjective. Thus, the diagnosis by laboratory testing may be more objective and less controversial. On the other hand, as discussed below, laboratory test results may be inconsistent and suffer from methodological flaws.  While men with low testosterone levels tend to have symptoms of hypogonadism (18), many studies have shown that symptoms are often unrelated to testosterone levels, and many men with low testosterone levels do not complain of symptoms (19, 20).

 

The level of total testosterone in serum is the best single test to screen for hypogonadism because methodology has been optimized and normative data are widely available.  While early assays extracted plasma steroids into organic solvents and separated them by column chromatography, those research methods generated radioactive waste and are too costly for clinical purposes.  Currently most hospital laboratories use automated platforms with either a competitive or proportional two-site format. These platforms use small sample volumes to avoid matrix effects, and a monoclonal antibody with a chemi-luminescent label for detection. The accuracy of the result is highly dependent on the affinity and specificity of the antibody used, and the survey result summarized in Figure 3 demonstrates that platform immunoassays continue to provide variable results with a coefficient of variation (SD/mean) among methods of 12% at a potency of 256 ng/dL which is close to the lower limit of the usual reference range for normal men. Nevertheless, when the manufacturer provides a reference range which is based on a large number of samples, and results are age-adjusted, automated assays are probably sufficient for most clinical purposes in adult men. Assay modifications have reduced the impact of hemolysis, icterus, and lipemia on measured results.

Figure 3. Total testosterone levels (±SD) in a proficiency sample (Y-04) distributed by the College of American Pathologists, and assayed using 14 different instruments in 1556 participating laboratories in 2018. The mean (±SD) value was 256 ± 51 ng/dL. The most frequent method (#6) was used by 353 laboratories. Method #4 is mass spectrometry, and was 7% lower than the mean of all methods. To convert total testosterone levels to nmol/L multiply by 0.0347

There is a positive bias at low values in most immunoassays so that automated assays are not recommended for clinical purposes in women and children. Reference laboratories are increasingly employing liquid chromatography (LC) - tandem mass spectrometry (MS) methods (21, 22). This approach combines the resolution of chromatography with the specificity of MS, and is now viewed as a gold standard.  Larger aliquots of serum are extracted with organic solvents, steroid hormones are separated by chromatography, in some assays following derivatization, and values are determined by peak area integration of testosterone-containing fractions of the column eluate. Accuracy is gained by adding an internal standard as a stable isotope of the same compound being measured in order to correct for procedural losses.  LC-MS accurately detects testosterone concentrations as low as 1.0 ng/dL (23, 24). In the 2018 CAP survey (Figure 3), the coefficient of variation for the high range sample (419 ng/dL) was 10%, and was a very respectable 11% for both the mid- range (245 ng/dL) and low-range (72 ng/dL) samples among those laboratories (n=35) that used LC-MS methodology. The equipment needed is costly, however, and each LC-MS assay requires substantial analytical development and optimization, as well as highly trained personnel.

 

While one study of men ages 40-80 who self-reported good or excellent health found no age-related change in total testosterone levels (25),  most cross-sectional and longitudinal studies have found that testosterone levels peak in the third decade, and decline as men grow older (e.g. (26, 27). Therefore, most reference laboratories present results for testosterone as a function of age. Small differences based on race and ethnicity have been found (28). One reference range for total testosterone levels in normal volunteers assayed by high-turbulence flow liquid chromatography(HTLC)-MS (22) is shown in Table 1. The decline in total testosterone in this study was roughly 0.4% per year beyond age 20, and mean T levels were 31% lower in men older than age 80 compared to men in their 20’s. 

 

Table 1. Impact of Age on Total Testosterone by HPLC–MS/MS in Normal Men  

 Age (years)

20-29

30-39

40-49

50-59

60-69

70-79

>80

Mean (ng/dl)

590

546

573

534

559

417

404

SD

232

206

209

194

226

177

234

 95th percentile

1052

910

901

909

928

755

716

5th percentile

283

319

310

296

290

168

92

N

61

76

55

51

19

44

19

Results from Quest Diagnostics, San Juan Capistrano, CA, courtesy of M.P. Caulfield. To convert total testosterone levels to nmol/L multiply by 0.0347

 

Table 2 shows a second reference range with combined values from the Framingham Heart Study analyzed by LC-MS, and the European Male Aging Study and Osteoporotic Fractures in Men Study analyzed by GS-MS (29). Mean levels for men in their 80s were 20% lower than for men in their 20s representing a similar decrease of 0.33% per year. Overall values are, however, 20-40% higher than from the reference laboratory in Table 1. Thus age-dependent reference ranges are method-specific, and no generalizable “cut-point” for the diagnosis of testosterone deficiency seems possible.

 

Table 2. Impact of Age on Total Testosterone Levels Measured by LC-MS or GC-MS) from Bhasin et al (29)

Age (years)

20-29

30-39

40-49

50-59

60-69

70-79

80+

Mean (ng/dl)

713

656

617

611

569

567

570

95thpercentile

1104

1084

1001

1059

965

1097

1079

5th percentile

375

343

304

286

276

254

238

N

220

660

872

788

493

289

26

 To convert total testosterone levels to nmol/L multiply by 0.0347

 

SEX HORMONE BINDING GLOBULIN (SHBG)

 

Sex hormone-binding globulin (SHBG) is a glycoprotein of molecular weight 90-100,000 KDa that is produced by the liver. SHBG binds testosterone and other steroids, prolongs their metabolic clearance, and regulates their access to target tissues (24). SHBG had been measured indirectly by radio-ligand binding assays, but two site immuno-radiometric and enzyme-linked assay kits are now widely available, and automated versions have been developed. There is little effect of meals or time-of-day on SHBG

Figure 4. SHBG levels from birth to old age in males and females. Redrawn from Elmlinger et al (30).

A cross-sectional analysis of the impact of age on SHBG levels in males and females is shown in Figure 4. There is an increase following birth to peak values at ages 4-5 years followed by a decline that is greater in males than females (30). SHBG levels in adult male plasma range from 20-100 nmol/L, and rise slightly in old age (28); analysis by race revealed higher levels among younger AA males (31).

 

In normal men, SHBG binds 40-60% of the circulating testosterone with high affinity, and the level of SHBG is a major determinant of the total testosterone level (Figure 5). Single nucleotide polymorphisms in the SHBG gene influence the affinity of SHBG for testosterone and affect the level in plasma (32).

 

Figure 5. Levels of SHBG and total testosterone in 28 normal men whose BMI ranged from 23-40 kg/m2. From Winters SJ et al (33).

Various clinical conditions are associated with reduced or increased SHBG levels, and thereby tend to lower or increase total testosterone concentrations (Table 3).  High SHBG levels in men with HepC or hyperthyroidism may lead to very high total testosterone levels, and can be a diagnostic dilemma. Serum SHBG levels are low in obesity (34), in T2DM or the Metabolic Syndrome (MetS) (35). In fact, low SHBG is frequently used as a biomarker in epidemiology studies to predict the development of the MetS as well as type 2 diabetes mellitus (36).  SHBG levels rise with weight loss (37) and when insulin resistance improves even without weight loss, e.g. with exercise. The molecular mechanism linking low levels of SHBG with insulin resistance and obesity is partly through hyperinsulinemia (38, 39) and through liver fat and cytokines which together regulate the transcription factor HNF4 alpha, a proximate activator of the SHBG promoter (40, 41) and at least 50% of genes expressed in liver.

 

Table 3. Factors that Influence the Level of SHBG in Plasma

Increase

Decrease

hyperthyroidism

hypothyroidism

hepatitis C

insulin resistance/NAFLD

GH deficiency

growth hormone excess

alcoholic cirrhosis

glucocorticoids

acute intermittent porphyria

androgens

first generation anticonvulsants

progestins

estrogens, certain SERMS

nephrotic syndrome

mitotane

genetic polymorphisms

thinness

obesity

genetic polymorphisms

 

 

FREE AND NON-SHBG (BIOAVAILABLE) TESTOSTERONE

 

The free hormone hypothesis proposes that the biological activity of a hormone is engendered by its unbound (free) but not its high affinity protein-bound concentration in plasma. The hypothesis remains controversial, and was recently reviewed for testosterone (42).

 

While some authors propose that there is little extra benefit in measuring free testosterone, my view is that the total testosterone level is sometimes inadequate to determine whether testosterone deficiency or excess is present. This occurs with a borderline value, or when the clinical findings and the total testosterone concentration do not agree. I encounter this conundrum most often in obese men, those with type 2 diabetes, NAFLD, or the Metabolic Syndrome among whom total testosterone levels are generally low, and who present because they have symptoms consistent with hypogonadism. In the Boston Area Community Health (BACH) Survey, 57% of men younger than age 50 with low total testosterone levels reported “normal” sexual function compared to 33% of those with low free testosterone levels (19). Likewise, the European Male Aging Study found that men age 40-79 with low free but normal total testosterone levels more often had symptoms and signs of sexual dysfunction while men with low total but normal free testosterone concentrations were more obese but had physical or sexual symptom scores that were similar to men with normal values (20). Thus, free testosterone seemed to be more closely aligned with the symptoms and signs of adult hypogonadism than was the total testosterone level.

 

In older men, on the other hand, the total testosterone level may be within the normal range while free testosterones decline more dramatically. The total testosterone level may also be elevated, as in men with hyperthyroidism, hepatitis-C, and patients treated with first generation anticonvulsants. Each of these situations occurs primarily because the primary condition is associated with a decrease or increase in the circulating SHBG level. 

 

Therefore, an assessment of free or non-SHBG bound testosterone may assist in the accurate diagnosis of androgen deficiency. This approach stems from the hypothesis that the bioactivity of circulating testosterone is due to the small percentage (1-4%) of total testosterone that circulates unbound (free testosterone) as well as the 40-50% of testosterone that circulates loosely bound to albumin and is often designated as “bioavailable-testosterone” (non-SHBG testosterone). The dissociation of albumin-bound testosterone is very rapid, and a short dissociation time is thought to allow the albumin-bound fraction to be available for uptake by cells (43). However, there is considerable debate as to which method for estimating free testosterone most accurately reflects testosterone production (42, 44).

 

Equilibrium dialysis is the benchmark reference method for measuring free testosterone. With the two-step approach, the free testosterone concentration is calculated from the product of the total testosterone level and the percentage that is determined to be free. H3-testosterone is added to the serum sample, and is allowed to partition between two compartments, one containing the tracer and serum sample, and the second containing buffer or an albumin solution. The compartments are separated by a semi-permeable membrane with a low-molecular weight cut-off. The protein-bound testosterone is retained while unbound testosterone, including H3-testosterone, crosses the membrane. It may take several hours to reach equilibrium, and using centrifugal ultrafiltration accelerates dialysis by forcing the unbound steroid thorough the membrane. Overall, the result is dependent on the accuracy of the total testosterone assay, and there is potential error due to temperature effects, sample dilution, and tracer impurities, among other problems. This assay is limited to reference laboratories.

Liquid chromatograph tandem mass spectrometry (LC-MS) can reliably measure hormone concentrations as low 1 pg/mL, and has been adapted for the measurement of free testosterone.  With this approach, equilibrium dialysis of undiluted serum is performed against buffer overnight at 37C, or more rapidly using ultracentrifugation cartridges, and (free) testosterone  is measured directly in the low molecular weight dialysate by LC-MS (45). Many reference laboratories have embraced this approach but it remains too complex and costly for most hospital laboratories.  

 

A published reference range using the two-step equilibrium dialysis method (Table 4) reveals that free testosterone levels decline as men grow older with a difference of 36% between the youngest and oldest reference groups. Thus, the percentage change in free testosterone by this method with age (0.6%/year) exceeds the rate of decline in total testosterone (0.3-0.4%/year).

 

Table 4. Reference for Free Testosterone by Equilibrium Dialysis in Normal Men

 Age (years)

20-29

30-39

40-49

50-59

60-69

70-79

>80

Mean (pg/ml)

102

86

74

84

84

80

65

SD

29

33

22

30

22

27

26

95.00%

148

144

114

136

111

131

101

5.00%

57

45

37

35

55

47

34

N

49

55

48

36

14

36

17

Results from Quest Diagnostics, San Juan Capistrano, CA, courtesy of M.P. Caulfield. 3H-testosterone was added to a sample diluted 1:5 in assay buffer and incubated in a dialysis chamber for 20 h at 37 °C to allow the tracer to reach equilibrium with the endogenous testosterone and binding proteins. To convert total testosterone levels to nmol/L multiply by 0.0347.

 

Calculated Free Testosterone

 

A technically simpler approach used by many hospital laboratories is to calculate the free testosterone level from the levels of total testosterone and SHBG, using experimentally determined testosterone binding constants. The KD for testosterone for SHBG is most often defined as 1 x 109 L/M, and for albumin 3 x 104 L/M.  These are estimates, however, and different constants will produce different results (46). Differences in the level of albumin in the sample have little impact on the calculated free testosterone, and are usually ignored. Free and non-SHBG bound testosterone levels can be computed using an internet program (www.issam.ch/freetesto.htm). There is an excellent correlation between the level of free testosterone obtained by equilibrium dialysis and the calculated free testosterone level (47), and between the calculated free testosterone and non-SHBG testosterone levels. Calculated free testosterone concentrations vary with the binding constants and algorithms employed, however, and SNPs of the SHBG gene may influence the affinity of SHBG for testosterone (32), and thereby the accuracy of the calculated free testosterone level. It has also been suggested that ligand binding to the SHBG dimer is allosteric such that occupancy of one site by a ligand alters the affinity of the second site. Furthermore, because the result is calculated from the levels of testosterone and SHBG, measurement error for either assay impacts directly on the calculated free and non-SHBG testosterone levels. A reference range (21) for free testosterone based on calculation revealed a decline of 61% from young to old adult men (Table 5), representing a rate of decline of 1%/yr.  In a second study (48) the difference between young and old was only 26%, but again far exceeded the fall in total testosterone with aging which was 1.2%.

 

Table 5. Influence of Age on Free Testosterone Levels in Community Men

Age (years)

20-29

30-39

 

40-49

 

50-59

 

60-69

 

70-79

 

>80

 

Mean (pg/ml)

148

132

116

99

82

72

58

5th Percentile

79

70

61

50

45

33

29

95thPercentile

229

212

199

164

135

132

93

n

220

660

872

788

493

289

26

Total testosterone was measured in community dwelling men in Framingham, MA using LC-MS.  SHBG was measured using a two-site immunofluorometric assay from DELFIA-Wallac, Inc. Free testosterone was calculated by the law-of-mass-action equation using of K SHBG-T of 0.998 × 109 L/mol and a K Alb-T of 3.57 × 104 L/mol. Data from Bhasin et al (29). To convert total testosterone levels to nmol/L multiply by 0.0347.

 

Bioavailable Testosterone

 

Non-SHBG-testosterone is called "bio-available” (BAT) because adding SHBG to an androgen-containing sample reduces its androgen receptor binding activity (49). This approach assumes that the testosterone bound with low affinity to albumin is active. The non-SHBG-testosterone (bioavailable) level can be determined by adding a tracer amount of 3H-testosterone to the serum sample, and selectively precipitating the SHBG-bound 3H-testosterone by adding 50% ammonium sulfate or concanavalin-A Sepharose. The 3H-testosterone that remains in the supernatant is presumed to be either free or albumin-bound, and is counted. The percentage of 3H-counts added that is in the supernatant is multiplied by the total testosterone level in order to determine the non-SHBG (bioavailable) testosterone. The assay is not readily automated, requires purified 3H-testosterone, and the complete separation of SHBG from albumin is presumed, but not verified.

 

Free Testosterone Index

 

The free testosterone index (FAI, free androgen index) represents the ratio: total testosterone/SHBG (both in units of nmol/L).  This value is easy to calculate, and may be valid in serum samples from women but is not valid in men (50)because most of the SHBG in men is bound to testosterone. Like the calculated free testosterone, and BAT, the FAI is dependent on accurate values for testosterone and SHBG

 

Direct Free Testosterone

 

The direct free testosterone assay was developed as a single-step, non-extraction method in which an125I-labeled testosterone analog competes with unbound testosterone in plasma for binding to a testosterone-specific antiserum that has been immobilized on a polypropylene assay tube. The basis for the test is that the analog has a low affinity for SHBG and for albumin.   Values for normal men with this method, as a percentage of the total testosterone (0.2-0.64%), are substantially lower than the 1.0-4.0% determined by other methods. While this difference alone does not cause a problem if adequate reference ranges are available, it immediately prompted speculation concerning the accuracy of the method.  Subsequent studies revealed that analog free testosterone assay results are positively correlated with the level of SHBG, much like total testosterone (33), and that free testosterone is un-measurable by analog assays in a dialysate of normal adult male serum (51).  Thus, the free testosterone level determined with analog assays appears to provide essentially the same information as the total testosterone level, is often misleading (tends to over diagnose hypogonadism), and is not recommended. The 2018 College of American Pathologists survey revealed that only 7% of participating laboratories continue to use this method, and it is has nearly disappeared from research articles related to testosterone.

 

Testosterone in Saliva   

 

The level of testosterone in saliva is positively correlated with the plasma free testosterone concentration. Salivary samples are easily collected, usually by a non-stimulated drool.  Both extraction and non-extraction immunoassay methods are available although LC-MS is being increasingly employed (44).   Usual values in adult men are 150-500 pmol/L (40-145 pg/ml).   Salivary testosterone assays are a useful research tool for field studies and other settings in which blood sampling is impossible or impractical.  While androgens are stable for a few days in untreated saliva, method artifacts may occur, and careful assay validation and quality control are essential.  Salivary testosterone assays have not been recommended for clinical purposes.

 

Cell-Based Reporter Bioassays

 

Cell-based reporter bioassays have been developed to analyze androgen bioactivity in biological samples. A stable cell line is created by transfection with plasmids encoding the human androgen receptor and a reporter system containing an androgen-responsive gene such as the mouse mammary tumor virus (MMTV)-luciferase reporter. When cells are stimulated with androgens, luciferase activity is increased dose-dependently (52), These assays remain investigational.

 

DIHYDROTESTOSTERONE

 

5 alpha reduction of testosterone to the more potent androgen dihydrotestosterone (DHT) is essential for fetal male genital development. T is converted to DHT by at least two steroid 5α-reductase (5AR) isoenzymes, 5AR types 1 and 2.   5AR-1 is found in liver, skin, brain, ovary, prostate, and testis whereas 5AR-2 is expressed in prostate, seminal vesicle, epididymis and skin. Approximately 20% of the circulating DHT in men is secreted by the testes, and the remainder is derived from the bioconversion of testosterone in tissues.  Because of a high level of expression of 5AR-2 in prostate, testosterone is effectively converted to DHT in that tissue, in which the level of DHT exceeds the peripheral blood concentration by 5-10-fold. The concentration of DHT in adult male serum is only about 10% of the value for testosterone, however. Therefore, an assay with negligible cross-reactivity with testosterone is needed for an accurate result.  An LC-MS method is now used by most reference laboratories. With gradient elution, the separation is totally complete (53).  Patients with 5α-reductase deficiency type 2 have ambiguous genitalia, and are generally detected as neonates, although a few patients have only microphallus or cryptorchidism.  A rise in testosterone but not DHT following hCG stimulation, producing a ratio of more than 10:1, is characteristic in most of these patients (54).  However, DHT production by 5AR1 can reduce the reliability of the ratio for diagnosis (55). The diagnosis is most often made by urinary steroid profiling by GC-MS, together with mutational analysis. DHT levels are often measured in epidemiological studies and in clinical research on prostate cancer and its treatments, and testosterone treatment results in a dose-dependent increase in serum DHT concentrations (56). Yet DHT levels are probably not useful in most clinical situations.

 

ESTRADIOL AND ESTRONE

 

Estrogens are important male hormones. They regulate the hypothalamic-pituitary-testicular axis, influence the function of  the testes and prostate, increase growth hormone and IGF-1 secretion, modify lipid metabolism and other hepatic proteins, and play an important role in male skeletal health, body fat, and perhaps sexual functioning  (57, 58).  Serum levels of estradiol and estrone are often measured in men with gynecomastia (59) or with unexplained gonadotropin deficiency. These conditions are rarely, but occasionally, due to estrogen-producing tumors, or to acquired (60) or genetic (61)   abnormalities in which estrogen production is increased. Moreover, the accurate measurement of estradiol may be helpful when SERMs, aromatase inhibitors, or hCG are used are used to increase testosterone levels, and in research on the role of estrogens in males (58, 62).  Estradiol is produced from testosterone, and estrone is produced from androstenedione, by aromatase P450, the product of the CYP19 gene. This enzyme is expressed in Leydig cells and in the adrenal cortex, as well as in adipose- and skin-stromal cells, aortic smooth muscle cells, kidney, skeletal muscle cells, and the brain. The promoter sequences of the P450 aromatase genes are tissue-specific, but the translated protein appears to be the same in all tissues. Increased aromatase expression in adipose and skin stroma with obesity is the most common cause for mild estrogen excess in men.  Interestingly, most studies of the age-associated decline in testosterone levels do not find a parallel fall in plasma estradiol levels perhaps because of increasing aromatase activity and fat mass as men grow older (63).

 

Because of low levels in males, traditional immunoassays for estrogens employed large volumes of plasma (2-5 ml) that were extracted with organic solvents. Because those assays are time-consuming and expensive to perform, non-radioactive automated methods that were optimized for the higher values normally found in pre-menopausal women were employed. However, the very small sample volumes used in automated assays may produce unexpectedly high values.  Results for estradiol in male plasma determined in platform assays are unreliable. The lack of agreement between laboratories is shown in Figure 6 which depicts the results for the low potency proficiency sample (mean 199 pg/mL) distributed in 2018 by the College of American Pathologists to 1450 participating laboratories. While many assays produced similar results, overall values ranged from 151 to 415 pg/ml (554-1523 pmol/L), with a coefficient of variation of 33%, compared to the goal of ±12.5%.  The mean value of the sample far exceeds normal levels in males, however, and testing at a lower potency would no doubt have produced even more dramatic differences.

Figure 6. Estradiol levels (±SD) in a mid-range proficiency sample (Y-05) distributed by the College of American Pathologists assayed using 15 different instruments in 1499 participating laboratories in 2018. The most frequently used (#6) was used by 341 laboratories. Mass spectrometry assays (n=11) are represented in column 4.

Mass spectrometry assays for estradiol and estrone, with a limit of detection of 1 pg/ml, have replaced conventional radio-immunoassays in most reference laboratories (24), and are highly recommended. The result with LC-MS is lower than with many immunoassays which presumably also detect interfering substances. Moreover, the between laboratory coefficient of variation for laboratories using MS methods (column 4) was also acceptably low at 10.3%.

 

Most of the circulating estradiol in men is loosely bound to albumin or is unbound (64), and only about 20% is thought to be bound to SHBG. Therefore, the serum level of SHBG was not predicted to appreciably influence the actions of estradiol. Measurement of free estradiol may be useful, however (65). In a series of studies, calculated non-SHBG-bound estradiol levels correlated more strongly with low bone mineral density and with indexes of high bone turnover in older men than did levels of total estradiol (66). On the other hand, increased mortality over 12 years of follow-up was seen among those with either low or high total as well as calculated free estradiol levels at baseline when compared to those in the middle tertile (67).  Akin to testosterone, non-SHBG (bioavailable) estradiol levels can be determined by ammonium sulfate precipitation using 3H-estradiol, or by equilibrium dialysis with or without using LC-MS.  Estimated levels are < 1 pg/mL. Calculated values require an accurate total estradiol assay, affinity constants are less certain than for testosterone, and the influence of testosterone and other steroids that also bind SHBG on the calculated estradiol level is no doubt important.  Age-specific free estradiol levels were reported but total estradiol was measured using an electro-chemiluminescence immunoassay (68); results using LC-MS methods are needed.  

 

GONADOTROPINS

 

FSH and LH, together with TSH and hCG, form a closely related family of heterodimeric glycoprotein hormones. Each consists of a common α-subunit that is non-covalently linked to a specific β-subunit. The α–β dimer is held together by a ‘seatbelt’ structure formed by the C-terminal amino acids of the β-subunit wrapped around the α-subunit.  Both subunits have asparagine-linked carbohydrate chains (2 for human α-subunit, 2 for FSH-β, and 1 for LH-β). The oligosaccharides project from the peptide skeleton, and by shielding of epitopes and altering the tertiary structure of the hormone, the sugars may impact receptor activation and bioactivity as well as antibody binding. Glycosylation also prolongs hormone clearance.

 

Most laboratories utilize fully automated, commercial assay systems for peptide hormones including LH and FSH.  Detection is generally by chemiluminescence, avoiding the use of radioactive tracers. Many assays achieve specificity by utilizing a biotinylated monoclonal antibody to the α-subunit as a capture antibody.  A second monoclonal antibody to the β-subunit is labeled with an organic ester that produces chemiluminescence in the presence of hydrogen peroxide (indicator antibody). Various pituitary and recombinant preparations are used for the standard curve but most assays are calibrated in terms of IU/L of International Reference Preparations (IRP) of highly purified human LH and FSH. The various standards have differing sugar sequences and branch patterns producing some variation in results between laboratories and assays, however, differences are relatively small.  When defined as a level of precision of replicate determinations of <10%, the sensitivity of these assays approximates 0.1 U/L.

Figure 7. Serum LH and FSH levels in normal males measured by an ICMA assay. Redrawn from Resende et al. (69).

Figure 7 shows mean levels of LH and FSH across the stages of pubertal development in boys measured with an automated ICMA (69). Values with an immunofluometric assay were slightly higher. FSH levels tend to exceed LH before puberty in boys, and both gonadotropins rise progressively during puberty with substantial overlap among the various pubertal stages.  

Prepubertal children have low amplitude but discrete pulsatile patterns of LH secretion that are amplified during sleep as puberty begins (70).  In adult men, LH is released in robust pulses every 1-2 h throughout the day and night with within subject variation in pulse height and between-pulse interval (Figure 8). In part because of pulsatility, the normal range for LH is wide, with typical ranges for adult men in terms of 2nd IRP of 1.6–8.0 IU/L for LH and 1.3–8.4 IU/L for FSH. Because of this pulsatility, pooling of 3 samples taken 20–30 min apart may provide a more accurate estimate of a person’s LH value than does a single sample.

 

Figure 8. Pulsatile patterns of LH secretion. Blood samples were drawn every 10 min for 12h starting at 0800 h from a 32-year-old normal man whose testosterone level was 474 ng/dL. Winters, SJ (unpublished).

Men with elevated levels of LH and FSH generally have testicular damage. Testes are usually small, there is oligo or azoospermia, and testosterone levels are low. Circulating estradiol levels are generally normal, however, in part because testicular aromatase is stimulated by the high level of LH. In some men with high LH and FSH levels, testosterone levels remain normal, sometimes because of elevated SHBG. The term “mild” or “compensated” hypogonadism is often applied (71). The diagnosis of gonadotroph adenoma is sometimes entertained, but long-standing infertility and small testes are a clue to the correct diagnosis. Much like the controversy surrounding thyroid hormone replacement in individuals with “subclinical” hypothyroidism, men with compensated hypogonadism who are not seeking fertility, mighty be viewed as candidates for testosterone replacement even though testosterone levels are within the reference range. This is often the case in teenagers with Klinefelter syndrome (72).

 

As men grow older, there is a decrease in sperm quality, the testes tend to be smaller, and there are fewer germ cells with more hyalinization and thickening of the tunica albuginea. Inhibin-B levels fall and FSH levels are often elevated. Mean LH levels also rise, but most often remain within the reference range (73).

 

Men treated with SERMs or aromatase inhibitors (74), and the rare men with inactivating mutations of the estrogen receptor-α (75) often have elevated LH and normal or elevated testosterone levels. These findings imply that estradiol is a major mediator of testicular negative feedback control of GnRH-LH secretion. Elevated LH and testosterone levels are also characteristic of men with androgen insensitivity syndromes (76) and men treated with anti-androgens (77), and reveal that there is additional negative feedback control of gonadotropins through the  androgen receptor.

 

FSH levels may be increased selectively because FSH production is regulated not only by GnRH and gonadal steroids but also by a paracrine control mechanism that involves pituitary activin and follistatin, and gonadal inhibin (see below).  When Sertoli cells fail to function normally, inhibin-B production declines, and the paracrine effects of pituitary activin stimulation of FSH-β gene expression is unopposed. Most men with high FSH levels have small testes and oligo- or azoospermia (78); however, FSH levels may sometimes be within the reference range (note that in some instances the reference range for FSH may be too high and some experts feel that the upper level for FSH is 8IU/L) in infertile men with severe damage to the germinal epithelium (79)  who may have either normal sized or small testes. In these cases, testicular biopsy is sometimes needed for a definitive diagnosis which is most often maturation arrest at the primary spermatocyte stage.

 

An elevated serum FSH level may rarely indicate an FSH-producing pituitary adenoma (80).  These tumors are generally macroadenomas that present with headache and a visual disturbance, and often co-secrete free α-subunit. The testes may occasionally  enlarge (81). The LH level is usually suppressed even when the tumors are LH-β immunoreactive, and testosterone levels are generally low.  LH secretion by a pituitary tumor is very rare (82, 83).  Most pituitary adenomas that are immunoreactive for LH or FSH are clinically silent with little or no hormone secretion.

 

The diagnosis of hypogonadotropic hypogonadism (HH) is based upon finding a low serum testosterone level with inappropriately low/normal LH and FSH concentrations. It is important to verify testosterone deficiency since a low SHBG level may result in the misdiagnosis of testosterone deficiency, and low LH may overlap with the reference range because of the pulsatile nature of LH secretion. Thus, the diagnosis of secondary hypogonadism (84) is sometimes problematic. The differential diagnosis of true hypogonadotropic hypogonadism is broad, and includes congenital HH due to a variety of genetic mutations including Kallmann syndrome (85), and acquired disorders such as prolactinoma, other sellar and suprasellar tumors or cysts, infiltrative diseases and vascular causes, iron overload, head trauma and others. HH in childhood limits pubertal development while in adults HH results in the regression of secondary sexual characteristics, albeit slowly.    

 

Isolated deficiency of FSH due to inactivating mutation in the FSH-β gene is a rare disorder (86-88). Affected men have been azoospermic, with borderline or low testosterone and increased LH levels. Pathological examination revealed narrowed seminiferous tubules, decreased Sertoli cell numbers, absence or aplasia of germ cells, and Leydig cell hyperplasia. Most mutations disrupt the ability of the mutant FSH- β to combine with the alpha-subunit. The low testosterone and increased LH levels may be explained by impaired signaling from the dysfunctional seminiferous tubules to Leydig cells (89). A few hypogonadal men with inactivating mutations of the LH-β gene have also been described (90-92).  Immunoreactive LH may be present or absent in serum depending on the nature of the mutation, but if present is bioinactive (92). Testosterone is low, and FSH is increased.  As placental hCG, rather than LH, stimulates male sexual differentiation, affected males have a normal male phenotype at birth while bioactive LH deficiency results in testosterone deficiency, and prevents normal puberty.

 

Two-site assays can occasionally be "too specific".  Polymorphisms that affect immunoassay detection by monoclonal antibodies may lead to misdiagnoses. For example, there is a relatively common polymorphism in the LH-β gene that is characterized by two point mutations in codons 8 and 15 resulting in two amino acid substitutions and an extra glycosylation site (88). Even though men and women with the LH variant appear to be normal and fertile, the serum LH level is low or undetectable using certain monoclonal antibodies whereas the result with other assays is normal. Clearly, when a man with a low or undetectable LH with a normal testosterone level is encountered, or a disparity between LH and FSH levels cannot be readily explained, a second assay method should be used.   

 

Various glycoforms of LH and FSH with structurally heterogeneous glycans are found in the pituitary and in the circulation, and glycosylation has been shown to influence hormone clearance and biological activity (93). Both sex steroids and activin have been reported to affect gonadotropin glycosylation. Highly sialylated glycoforms, with an acidic pH, tend to have a longer circulating half-life whereas the more alkaline forms tend to exhibit greater bioactivity in vitro. There is evidence that LH and FSH glycosylation is physiologically important. The ideal immunoassay would detect accurately only the total bioactive LH or FSH in the sample, but clearly this goal is difficult to accomplish. As an alternative approach, in vitro bioassays for LH and FSH can be used to assess the function of the gonadotropins. An in vitro bioassay based on the production of testosterone by cultured mouse or rat Leydig cells (94) was developed to assess LH function, and the production of estradiol by rat granulosa cells or immature Sertoli cells can be used to assay FSH bioactivity (95). Bioassays based on cAMP production by cell lines stably expressing gonadotropin receptors, with quantification using cAMP-responsive promoters linked to a luciferase reporter, have also been developed (34).  While useful for the study of the biological properties of recombinant or purified proteins, the clinical use of these assays is limited by the nonspecific effects of serum. In fact, many findings reported in patients using in vitro LH bioassays were found subsequently to be methodological artifacts.

 

Glycoprotein α-Subunit

 

Glycoprotein α-subunit is secreted in bursts that coincide with LH secretory episodes, implying pre-eminent regulation by GnRH (96) Accordingly, serum α-subunit levels increase at puberty (97) and are elevated in men with testicular failure and in postmenopausal women.  α-Subunit is also expressed in thyrotrophs, and levels are increased in patients with primary hypothyroidism (98).

Figure 9. Pulsatile patterns of α-subunit secretion. Blood samples were drawn every 10 min for 12h starting at 0800 h from a 32-year-old normal man whose testosterone level was 474 ng/dL. Winters, SJ (unpublished)

α-Subunit levels are low, but measurable, in normal children and in patients with congenital hypogonadotropic hypogonadism.  Because peak α-subunit levels after GnRH stimulation tend to be lower in IHH patients than in prepubertal boys, this test has been used to distinguish between these two patient groups, although some overlap occurs (99). α-Subunit is produced by as many as 20% of pituitary adenomas (100) often together with FSH or sometimes GH. Because it is cleared by renal excretion, α-subunit levels are high in patients with renal failure (101) .

 

INSULIN-LIKE FACTOR 3

 

Insulin-like factor-3 (INSL3) is a peptide hormone member of the relaxin-insulin hormone family that is secreted by Leydig cells following LH stimulation. INSL3 affects testicular descent through effects on the gubernaculum (102) and plays a role in spermatogonial differentiation. At the time of puberty, INSL3 levels rise in parallel with testosterone (103).  INSL3 concentrations in the blood of normal adult men are approximately 1 ng/mL (0.4-1.5 ng/mL).   Patients with Leydig cell dysfunction have lower levels, and the very low concentrations in men with hypogonadotropic hypogonadism increase following hCG administration (104) but are unaffected by testosterone treatment (105).

 

INHIBIN-B

 

Inhibin, a glycoprotein hormone produced by the testes as well as the ovaries, is responsible for the selective negative feedback control of FSH secretion, and functions as an intra-gonadal regulator (106). Most evidence supports the idea that inhibin decreases FSH-β mRNA levels by blocking pituitary activin-stimulated FSH-b transcription (107).  Inhibin is a 32-kDa heterodimer composed of an α-subunit, and one of two β subunits, βAor βB. Inhibin-B (α-βB) is the form produced by testicular Sertoli cells; inhibin-A (α-βA), produced by the corpus luteum and placenta, is undetectable in adult male plasma. Higher molecular weight forms of the uncombined inhibin α-subunit that lack bioactivity are released into the circulation in excess of dimeric inhibin-B.

 

The Gen II two site inhibin-B ELISA assay uses a capture antibody raised to a peptide from the βB-subunit, and a biotinylated detection antibody raised to a peptide from the inhibin α-subunit (108).  Wells are then incubated with a streptavidin-labeled horseradish peroxidase followed by the substrate tetramethylbenzidine which produces a colorometric signal that is proportional to the amount of inhibin-B in the sample. The assay is not automated and no performance statistics could be located.  Older assays were more complex, and added methionine and an oxidation step with hydrogen peroxide to facilitate capture, and heating with sodium dodecyl sulfate solution to enhance specificity.

 

Inhibin-B is produced by the fetal testis and is measurable in serum at term. Levels increase substantially in newborns coincident with the rise in gonadotropins and testosterone (mini-puberty), remain elevated for 2-4 months and then decline (109).  In contrast to barely detectable levels of gonadotropins and testosterone during childhood, however, circulating inhibin-B is readily measurable in sera from prepubertal boys, implying that production is partly gonadotropin-independent. Serum inhibin-B levels increase to adult values at the time of puberty, and decline as men grow older (110).  Serum inhibin levels are partly determined by gonadotropin stimulation (111) but based on studies in monkeys, also reflect Sertoli cell number (112). With the updated assay, results for normospermic men were (2.5-95thpercentile) 32-416 with a median value of 174 pg/mL (113).

Figure 10. Inhibin B levels in males in relation to age. Reprinted from Andersson A-M (114) with permission of the publisher (Elsevier).

Although there is no remarkable pulsatile fluctuation in circulating inhibin-B levels, there is a diurnal variation in adult men with the highest values in the morning and nadir values approximately 35% lower in the evening. This diurnal pattern parallels that of testosterone (115).

 

Inhibin has been extensively studied as a biomarker of spermatogenesis. In keeping with its function to suppress FSH production, circulating inhibin-B levels are inversely correlated with FSH levels in adult men, and are more strongly correlated inversely when values from men with primary testicular failure are included in the analysis (111).  Inhibin-B levels are higher in fertile than infertile men but there is substantial overlap between the groups (113, 116). There is also a demonstrable, albeit weak, positive correlation with sperm count (117), and with the germ cell score in testicular biopsy specimens among infertile men (118). Inhibin-B levels are low in men with testicular failure, and are very low in men with Klinefelter syndrome in whom seminiferous tubules are hyalinized and Sertoli cells are essentially absent (119). Low but measurable inhibin-B levels in men following chemotherapy (120) or testicular irradiation (121), some of whom develop germinal cell aplasia, suggest that germ cell factors regulate SC inhibin production. Low inhibin-B levels predict persistent azoospermia in men with testicular cancer who undergo orchidectomy, chemotherapy and irradiation (122). Serum inhibin levels changed little in normal men who participated in a male contraceptive clinical trial of testosterone together with a progestin, and developed azoospermia or severe oligospermia (123).

 

Inhibin-B levels have been studied as a biomarker in the male partners of infertile couples undergoing testicular spermextraction (TESE) as a predictor of success of intra-cytoplasmic sperm injection (ICSI). While mean inhibin B levels tend to be lower (and FSH higher) in men with no sperm found at TESE, there is no level of inhibin-B that reproducibly predicts either the presence or absence of spermatozoa in TESE samples, or successful in vitro fertilization (124). Thus, measurement of inhibin-B is not recommended in the decision making for fertility potential among azoospermic men undergoing TESE and ICSI. Further, very large doses of FSH are needed to increase circulating inhibin-B levels in men, so that an FSH stimulation test for inhibin-B is also not helpful in a clinical evaluation of hypospermatogenesis.

 

Inhibin-B levels are reduced in gonadotropin-deficient men (125).  In those with the complete form of congenital hypogonadotropic hypogonadism, values are lower than in men with partial gonadotropin deficiency who have some spontaneous pubertal development. This quantitative difference is partly due to a presumed larger mass of Sertoli cells (as reflected by larger testicular size) in the latter group of men. Low inhibin-B levels have been reported to differentiate boys with constitutional delay of puberty (CDP) from those with congenital hypogonadoptropic hypogonadism (CHH) with 80-100% sensitivity and specificity (126). While plasma inhibin B concentrations in CHH and CDP may overlap, in one study of boys age 14-18 years with delayed puberty, among those who were genital stage 1 (testis volume ≤ 3ml), inhibin-B levels <35 pg/ml predicted persistent hypogonadism (testosterone levels < 3 nmol/L), and presumably the diagnosis of CHH, after 2 years of follow-up (127).  Inhibin-B is undetectable in most boys with congenital anorchia as in castrates, and is therefore a useful test to help distinguish these patients from boys with intra-abdominal testes (128).

 

High inhibin-B levels have been reported in a few boys with FSH-producing pituitary tumors (129), in patients with Sertoli cell tumors (130) and in boys with McCune Albright syndrome with macro-orchidism and autonomous function of Sertoli cells (131).  Adrenal tumors often express the inhibin α-subunit, and there is one report of high serum dimeric inhibin level in an adult man with an adrenal neoplasm (132).

 

ANTI- MULLERIAN HORMONE

 

Anti-Mullerian hormone (AMH, also known as Mullerian inhibitory hormone) is a 140 KDa homodimeric member of the TGF-β family of growth and differentiation factors (133). It is produced by fetal Sertoli cells and causes regression of Mullerian structures during male sexual development (134). AMH production increases in response to FSH and is inhibited by androgens. AMH is readily detectable in the serum of prepubertal boys in concentrations of 10-70 ng/ml, and declines to levels of 2-5 ng/ml with entry into adolescence (135). AMH remains detectable at low values throughout adulthood (104) but how AMH functions in males beyond fetal life is uncertain. AMH levels are elevated in untreated men with congenital hypogonadotropic hypogonadism and decline following treatment with hCG (136). AMH is absent from the plasma of most prepubertal boys with congenital anorchia but is generally detectable in boys with bilateral cryptorchidism (134). Therefore, measuring AMH is useful in evaluating boys with non-palpable gonads. AMH levels are low in men with seminiferous tubular failure but overlap with normal in men with oligospermia, and do not predict testicular sperm retrieval for ICSI (137).  Levels tend be low in obesity (138),

Figure 11. Serum concentrations of AMH in healthy males from birth (cord blood) to age 69 years. From Aksglaede L et al (139). Red dots are values from men with bilateral anorchia. Republished with permission of The Endocrine Society.

FUNCTIONAL TESTS

 

hCG Stimulation

 

Human chorionic gonadotropin (hCG) can be used as a test agent to examine Leydig cell steroidogenesis in prepubertal boys who secrete little or no endogenous gonadotropins. A variety of protocols have been used. In one study, serum testosterone levels rose to greater than 300 ng/dL (10.5 nmol/L) in healthy prepubertal boys administered hCG 1500 IU intramuscularly every other day for seven doses. The effect of hCG to increase circulating levels of testosterone, precursor steroids and DHT can be used in conjunction with mutation analysis to help evaluate patients with a 46,XY karyotype who have ambiguous genitalia which may be due to androgen insensitivity, defects in testosterone biosynthesis, or 5α-reductase deficiency (140). When the basal and hCG-stimulated profile indicates accumulation of steroid precursors upstream of an enzymatic defect, sequencing can be performed to identify the disorder. A raised serum testosterone to DHT ratio would suggest the diagnosis of 5α-reductase deficiency type 2. However, DHT production by 5α-RD1 can reduce the reliability of the ratio for diagnosis (55).

 

Testosterone levels are generally unaffected by hCG in boys with congenital bilateral anorchia but increase in boys with bilateral intra-abdominal testes (141). Therefore, this test, together with measurement of inhibin-B and AMH, help establish which boys have intra-abdominal testes and should undergo laparoscopy, and orchidopexy or orchiectomy (128).

 

Adult men with primary testicular failure have elevated endogenous serum LH concentrations, and hCG will predictably increase serum testosterone levels less in these men than in eugonadal men. In gonadotropin-deficient men, the testosterone response to short-term administration of hCG is also blunted because Leydig cell steroidogenic enzymes are down-regulated. Overall, hCG testing nowadays provides little clinically useful information in either group of adult men.

 

Blockade of Steroid Hormone Biosynthesis and Action

 

Lowering circulating sex steroid levels with pharmacological inhibitors, or blocking steroid hormone action with receptor antagonists, can be used to assess the integrity of the hypothalamic-pituitary-testicular unit as a research tool, and clinically as a treatment for adult men with adult onset hypogonadism (142).

 

Ketoconazole, a competitive inhibitor of cytochrome P450 cholesterol side chain cleavage (P450scc) and C17/20 lyase in the biosynthetic pathway to testosterone is used in the treatment of endemic mycoses and off-label in other conditions including Cushing syndrome, but causes a dose-dependent reduction in circulating testosterone and estradiol levels. (143). Because ketoconazole also lowers cortisol production, glucocorticoids are co-administered with ketoconazole to prevent the symptoms of cortisol deficiency.

 

Estrogens produced by the testes, peripheral tissues, and the CNS play an important role in the physiological feedback regulation of gonadotropin secretion in men. Selective estrogen response modulators (SERMs) bind to estrogen receptors (ER-α and/or ER-β) and exert estrogen-like effects, or inhibit estrogen effects, in a tissue-specific manner. SERMs also have non-classical extra-nuclear (membrane) signaling mechanisms in certain cells. By blocking estradiol negative feedback, most SERMS, such as clomiphene (144) and tamoxifen, increase plasma LH and FSH levels. Similarly, aromatase inhibitors (145) such as anastrazole, reduce circulating estradiol levels, and increase LH in men.  Dosages of 25 mg/day of clomiphene produced a mean two-fold increase in serum testosterone levels in adult men (Helo 2015). (142)., and while higher doses are more effective, estradiol levels may increase, and gynecomastia and galactorrhea may occur (144).  Two-fold increments in LH were produced by anastrazole 10 mg daily within 3-4 days.  Both blocking estrogen negative feedback and decreasing estradiol production increased LH pulse frequency indicating an effect of estradiol on the GnRH pulse generator. A normal response implies functional integrity of GnRH-LH-testosterone pathways; however, responses among normal subjects are variable, and diagnostic tests using SERMS are not thought to be useful clinically.  So far, these agents are not FDA-approved for any use in men.

 

Nonsteroidal antiandrogens increase serum LH levels in men by blocking the androgen negative feedback effect on GnRH secretion (146) and can be used to test GnRH-LH integrity.

 

GnRH Test

 

GnRH is used as a research tool to examine the responsiveness of gonadotrophs to their physiological stimulus. In normal adult men, the intravenous administration of 100 µg of GnRH increases serum LH levels three- to- six-fold while serum FSH levels rise by about 50%. Generally, the total and incremental release of LH and FSH following GnRH administration is directly proportional to the basal hormone level, although exceptions do occur.

 

The GnRH test was introduced as a method to diagnose hypogonadism, and to distinguish hypogonadism due to hypothalamic from pituitary disorders. The LH and FSH response to GnRH is subnormal when gonadotrophs are damaged or destroyed by pituitary tumors or by other pathologies. However, the gonadotropin response to stimulation with GnRH is also diminished in patients with GnRH deficiency since GnRH up-regulates its receptor on gonadotrophs, as well as the expression level of each of the gonadotropin subunit genes and thereby LH and FSH production. Thus, the LH response to GnRH stimulation is attenuated in these patients even though the pituitary is essentially normal. Therefore, when evaluating gonadotropin deficient adult men, the GnRH test generally provides little information beyond that of the basal testosterone, LH and FSH levels, and is not recommended for clinical purposes.

 

GnRH analog (naferelin, leuprolide, buserelin, triptorelin) testing may help distinguish prepubertal boys with constitutional delay of puberty (CDP) from those with congenital hypogonadotropic hypogonadism (147-149). These agents bind the GnRH receptor with higher affinity and have a longer circulating half-life than does native GnRH. The LH and testosterone responses at 4h and 24h in boys with CDP generally exceed those of HH.  In one study, prepubertal boys age 13.7-17.5 years with testicular volume ≤ 4ml whose LH level at 4h following 0.1 mg sc triptorelin exceeded 5.3 U/L all progressed to testicular enlargement, testis size ≥ 8 ml, over 18 months. Those boys with lower LH basal ≤ 2U/L and stimulated ≤5.3 mIU/ml values did not progress to this level. The CHH group also had low levels of inhibin-B (<111 pg/ml) at baseline (127). Thus, the test seems to be of use in evaluating prepubertal boys with delayed puberty. In patients with CHH who are homozygotes or compound heterozygotes for mutations of the GnRH receptor gene, GnRH binding to its receptor may be either absent or reduced in affinity, or receptor signal transduction may be impaired, and the LH response to GnRH stimulation in these patients may be absent or reduced. The LH response in those with partial hypogonadotropic hypogonadism overlaps with the normal response (150), and while these tests are helpful so far there is no gold-standard diagnostic test to fully differentiate boys with CHH from those with CDP.

 

SEMEN ANALYSIS

 

Laboratory testing for men who present for an evaluation of infertility begins with the semen analysis (151). Most laboratories report the semen volume, the sperm density (million/mL), motility (% with progressive motion on the microscopic slide), and the sperm morphology (% with oval-shaped sperm heads), and follow WHO guidelines in their methods and reports (152). The test helps predict fertility potential but is not a test of fertility since there is substantial overlap between results for fertile and infertile men (153). Moreover, lack of complete adherence to standardized methods, and insufficiently skilled laboratory staff, may substantially limit the diagnostic value of the semen analysis. The sample can be collected by masturbation, or by interrupted intercourse, generally after 2-3 days of abstinence from ejaculation. Most of the sperm are in the first portion of the ejaculate so it is important to collect a complete sample. Condom collection is less desirable since condoms contain spermicides, but if so, the sample should be immediately placed into a sterile jar. It is important to analyze the sample within 60 min of collection, as motility decreases over time. In cold climates, the sample should be kept warm in transit by holding it within outer clothing adjacent to the body. Even with optimized collection methods, there is a physiological day-to-day variation in sperm production, so 2-3 samples may provide more insight that a single sample. A man can be considered to be sterile if there are no sperm in the ejaculate (azoospermia).

 

Table 6. WHO Guidelines for an Adequate Semen Analysis

Semen volume

 ≥1.4 mL

Sperm concentration

≥15 million/mL

Total sperm output

≥40 million sperm

Sperm progressive (total) motility

≥32 (≥40%)

Sperm morphology

≥4%

 

GENETIC TESTING

 

Assisted reproductive techniques have enabled azoospermic and severely oligospermic (<5 million/mL) men to reproduce. Therefore, there is a need to identify and discuss the consequences of possible mutations with the infertile couples. Genetic abnormalities are found in 3% to 5% of men with severe oligosperma, and 14% to 19% of men with non-obstructive azoospermia (NOA). The peripheral blood karyotype should be analyzed in these men unless there is a likely cause for hypospermatogenesis (e.g. cancer chemotherapy or orchitis). Of those with azoospermia, 2/3 have Klinefelter syndrome (47,XXY and mosaics) (154). Rarely, azoospermic men have a 46,XX chromosome constitution with Y-chromosome material detectable on the X-chromosome by FISH analysis using an antibody to the transcription factor encoded by the SRY gene. In studies of various ethnic groups, 4-15% of men with idiopathic non-obstructive azoospermic and 1–10% of men with severe oligospermia have microdeletions of the AZF regions of the Y chromosome (155, 156). Thresholds of 0.5-5 million sperm/mL have been suggested for genetic testing for higher specificity and overall lower cost (157) although some authors would like to see all infertile men tested.  Microdeletions (undetectable in standard karyotypes; YCMD) are detected by PCR and may partially or completely eliminate the azoospermia factor (AZF)a or the AZFb/c region including genes that are important for spermatogenesis. YCMD are detected by PCR. Couples are counselled that male offspring will inherit the AZF microdeletion, and will be at risk for infertility. In azoospermic men Y deletion testing is essential prior to considering surgical sperm retrieval because AZFa , AZFb or AZFB+c deletion are associated with very severe spermatogenic defects such as germ cell arrest or Sertoli cell only syndrome with an essentially zero prospect of success (160). On the other hand, even azoospermic men with AZFc deletions have ~ 50% chance of surgical sperm recovery (160).

 

Obstructive azoospermia may be recognized by a low-volume ejaculate with low pH. The testes of these men are generally normal in size, and FSH and inhibin-B levels are in the reference range. Obstruction can be demonstrated by ultrasound (158). If bilateral absence of the vas deferens (CBAVD) is found, approximately 75% of men will have cystic fibrosis, sometimes with little respiratory or pancreatic disease (159). When Cystic Fibrosis is diagnosed by genetic testing, the next step is to screen the female partner for Cystic Fibrosis gene abnormalities in order to assess the risk to any offspring.  

 

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Overview of Endocrine Hypertension

ABSTRACT

Endocrine hypertension typically is referred to disorders of the adrenal gland including primary aldosteronism, glucocorticoid excess, and the pheochromocytoma-paraganglioma syndromes. Rare conditions in patients with congenital adrenal hyperplasia and glucocorticoid resistance (Chrousos syndrome) can also lead to hypertension. Nonadrenal endocrine disorders, such as growth hormone excess or deficiency, thyroid dysfunction, primary hyperparathyreoidism, testosterone deficiency, vitamin D deficiency, obesity-associated hypertension, insulin resistance and metabolic syndrome are also linked to hypertension. In this chapter, we provide an overview of endocrine hypertension including rare syndromes of mineralocorticoid excess.

INTRODUCTION

Hypertension is the most common diagnosis in USA as it affects approximately 31% of Americans (1,2) and approx. 33% of the Mozambican population using a blood pressure cutoff

of 139/89 mm Hg (3). The assignment of a diagnosis of hypertension is dependent on the appropriate measurement of blood pressure, the level of blood pressure (BP) elevation, and the duration of follow-up (4). The secondary causes of hypertension include mostly renal as well as endocrine diseases. An accurate diagnosis of endocrine hypertension offers clinicians the chance to achieve an optimal treatment with either specific pharmacologic or surgical therapy (5). Herein, the different causes of endocrine hypertension with a focus on prevalence, clinical presentation, and currently diagnostic tools.

How to Measure BP

Manual measurement using a mercury sphygmomanometer and a stethoscope remains the Gold Standard. However due to environmental issues regarding mercury, this technique tends to be abandoned. Automatic devices have substituted them, but a standardised procedure of obtaining comparable measurements is poor and their validity in clinical practice is limited (6). The device should have an upper arm cuff and should be properly validated and calibrated. A correct cuff size that encircles 75%–100% of the arm should be used. Blood pressure assessment should be based on the mean of 2 or more properly measured seated BP readings on each of 2 or more office visits. Optimally, the measurement of the blood pressure can take place in the office, with the patient seated comfortably with legs uncrossed or in supine position for 3–5 minutes without talking or moving around. It is recommended to avoid caffeine, smoking as well as exercise before the measurement. Clothes covering the cuff location of the upper arm should be removed (7). At the first visit, BP should be recorded in both arms and the higher reading must be considered and repeated measurements after 1-2 minutes can be done. During the measurement, the patient’s arm needs to be supported, and upper arm must be at the level of right atrium. Regarding auscultatory determinations, radial pulse obliteration can be palpated to estimate systolic blood pressure. Korotkoff sounds must be recorded, with readings of SBP and DBP at the onset of the first and the last audible sound, respectively (7).

Classification of BP

Based on recommendations of the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC 7)(8), the classification of BP for adults aged 18 years or older has been as follows:

  • Normal: Systolic lower than 120 mm Hg, diastolic lower than 80 mm Hg
  • Prehypertension: Systolic 120-139 mm Hg, diastolic 80-89 mm Hg
  • Stage 1: Systolic 140-159 mm Hg, diastolic 90-99 mm Hg
  • Stage 2: Systolic 160 mm Hg or greater, diastolic 100 mm Hg or greater

 

The 2017 ACC/AHA guidelines eliminate the classification of prehypertension and divides it into two levels (9):

  • Elevated blood pressure with a systolic pressure between 120- and 129-mm Hg and diastolic pressure less than 80 mm Hg
  • Stage 1 hypertension, with a systolic pressure of 130 to 139 mm Hg or a diastolic pressure of 80 to 89 mm Hg

Figure 1 provides an overview of classification of BP for adults 18 years and older.

Figure 1. Classification of Hypertension. AHA, American Heart Association; ACC, American College of Cardiology; ESC, European Society of Cardiology; ESH, European Society of Hypertension; DHL, German Hypertension League; NICE, National Institute for Health and Care Excellence of the United Kingdom. DBP, diastolic blood pressure; SBP, systolic blood pressure. Modified from: Jordan J, Kurschat C, Reuter H. Arterial hypertension. Dtsch Arztebl Int. 2018 Aug 20;115(33-34):557-568(10)

Prevalence of Hypertension

Several studies have previously reported prevalence of hypertension among different populations worldwide, but these data depend on different classification systems used. Hypertension affects 28.6% of adults in United States Data from the National Health and Nutrition Examination Survey 2011-2012 showed an increase in the prevalence of hypertension in all age groups compared to 1991 (11). Among adults with hypertension in that survey, 52% achieved a BP of less than 140/90 mm Hg with 76% taking antihypertensives, and with 83% being aware of their hypertension. The prevalence of hypertension increases with age and most individuals with hypertension are diagnosed with primary (essential) hypertension. Hypertension is a major risk factor for stroke, ischemic heart disease, and cardiac failure. It is the second most common reason for office visits to physicians in the United States. Analysis of the Framingham study data suggested that individuals from age 40 to 69 years have an increasing risk of stroke or coronary artery disease mortality with every 20 mm Hg increment in SBP.

Prevalence of Secondary Hypertension

In most people, hypertension is primary, but approximately 15-30% of hypertensive population has secondary hypertension (12). Among children presenting with hypertension, 50% have a secondary cause (13). Young adults (<40 years old), are reported to have a prevalence of secondary hypertension 30% (14). The secondary causes of hypertension include primarily causes such as primary renal disease, oral contraceptive use, sleep apnea syndrome, congenital or acquired cardiovascular disease (i.e. coarctation of the aorta) and excess hormonal secretion. Endocrine Hypertension was previously reported to account for approx. Recent studies suggest an overall prevalence of >5% and possibly > 10% for endocrine hypertension among the hypertensive population (15,16), but several authors have suggested that this prevalence is probably underestimated. The most common causes of endocrine hypertension are excess production of mineralocorticoids (i.e. primary hyperaldosteronism), catecholamines (pheochromocytoma), thyroid hormone, and glucocorticoids (Cushing syndrome) (17). Table 1 lists the most common causes of Secondary hypertension.

Table 1. Etiology of Secondary Hypertension

Endocrine Causes

Other Causes

Adrenal-dependent causes 

Renal causes (2.5-6%)

Pheochromocytoma and sympathetic paraganglioma 

Polycystic kidney disease

Primary aldosteronism 

Chronic kidney disease

Hyperdeoxycorticosteronism 

Urinary tract obstruction

Congenital adrenal hyperplasia 

Renin-producing tumor

 11β-Hydroxylase deficiency 

Liddle syndrome

 17α-Hydroxylase deficiency 

Renovascular causes

 Deoxycorticosterone-producing tumor 

renal artery stenosis fibromuscular dysplasia or atherosclerosis

 Chrousos syndrome

Vascular causes 

   Cushing syndrome 

Coarctation of aorta

   Apparent mineralocorticoid excess/11β- hydroxysteroid dehydrogenase deficiency 

Vasculitis

Parathyroid-dependent causes 

Collagen vascular disease

  Hyperparathyroidism 

Neurogenic causes 

Pituitary-dependent causes 

Brain tumor

 Acromegaly 

Autonomic dysfunction

 Cushing disease 

Sleep apnea

Secondary hyperaldosteronism 

Intracranial hypertension

 Renovascular hypertension 

Drugs and toxins

Thyroid-dependent causes 

Alcohol

 Hypothyroidism 

Cocaine

 Hyperthyroidism 

Cyclosporine, tacrolimus

Vitamin D deficiency

NSAIDs
 

Erythropoietin

Adrenergic medications

Decongestants containing ephedrine

Herbal remedies containing licorice or ephedrine

Nicotine

 

Prevalence of Resistant Hypertension and Typical Causes

The prevalence of resistant hypertension is high: 53% of patients in NHANES had a BP < 140/90 mm Hg vs. 48% in the Framingham Heart Study. In NHANES participants with chronic kidney disease, 37% had a BP < 130/80 mm Hg. In ALLHAT, 34% of patients

remained uncontrolled after 5-year follow-up on at least 2 antihypertensive drugs (5).

One important question in this regard is when to screen for secondary causes. Some patients with hypertension, but without primary aldosteronism, demonstrate ACTH-dependent aldosterone hypersecretion by stress (18). The clinician should carefully screen for cardinal signs and symptoms of Cushing syndrome, hyper- or hypothyroidism, acromegaly, insulin resistance (acanthosis nigricans), or pheochromocytoma (flushing and excessive sweating). Hypertension in young patients and refractory hypertension (characterized by poorly controlled blood pressure on > 3 antihypertensive drugs) should alert the physician to screen for secondary causes (14). The importance of endocrine-mediated hypertension resides in the fact that in most cases, the cause is clear and can be traced to the actions of a hormone, often produced in excess by a tumor, such as an aldosteronoma, in a patient with hypertension due to primary aldosteronism. More importantly, once the diagnosis is made, a disease-specific targeted antihypertensive therapy can be implemented, and, in some cases, surgical intervention may result in complete cure, obviating the need for life-long antihypertensive treatment.

As in other causes of hypertension, the clinician should question the patient about dietary habits (salt intake etc.), weight fluctuations, use of over the counter drugs and health supplements including teas and herbal preparations, recreational drugs, and oral contraceptives. Moreover, a detailed family history may provide valuable insights into familial forms of endocrine hypertension. The review of systems should include disease-specific questions. Many patients harboring a pheochromocytoma are symptomatic. Symptoms may include headaches, palpitations, anxiety-like attacks and profuse sweating, similar to symptoms of hyperthyroidism. The triad headache, palpitations, and sweating in a hypertensive patient was initially found to have a sensitivity of 91% and specificity of 94% for pheochromocytoma (19). More recent studies suggest that this typical triad of symptoms is found much less frequently, for instance, in only 10% of cases (20). Ten or more percent of patients with pheochromocytoma may not have any clinical symptoms and may be normotensive (19,21,22).

Patients with Cushing’s syndrome often complain of weight gain, insomnia, depression, easy bruising and fatigue. Acne and hirsutism (in women) can also be observed. The challenge these days is to recognize patients with evolving Cushing’s syndrome amongst the many obese and often poorly controlled diabetic individuals. An Endocrine Society Clinical Practice Guideline can assist in this task (23). Primary hyperaldosteronism is manifested by mild to severe hypertension. Hypokalemia can be present, but it is not a universal finding and there is normokalemic and normotensive primary aldosteronism (24,25). Polyuria, myopathy and cardiac dysrhythmias may occur in cases of severe hypokalemia. A thorough physical exam with attention to evidence of target organ injury and features of secondary hypertension should be conducted.

Low renin is often associated with several causes of hypertension. Figure 2 lists conditions with low renin levels.

Figure 2. Low Renin Conditions

Despite the increasing understanding of the pathophysiology of hypertension, control of the disease is often difficult and far from optimal. Recent large meta-analyses and genotype studies have identified some “risk genes” for hypertension (15). Surendran and colleagues found a low frequency nonsense variant in the gene ENPEP, which codes for the enzyme aminopeptidase A that converts angiotensin II into angiotensin III and therefore being part of the regulation of the renin-angiotensin-aldosterone system  (26). Liu and colleagues observed associations for the aggregation of rare and low frequency missense variants in the genes NPR1, DBH, and PTPMT1 (27). The gene DBH codes for the enzyme dopamine beta-hydroxylase, which catalyzes the conversion of dopamine into noradrenaline and, thereby, influences the autonomic nervous system. The gene PTPMT1 codes for the mitochondrial protein tyrosine phosphatase 1, which influences insulin production (27).

CLINICAL DIAGNOSIS OF ENDOCRINE HYPERTENSION

The first step when evaluating a patient with suspected endocrine-related hypertension is to exclude other causes of secondary hypertension, particularly renal disorders. A detailed medical history and review of systems should be obtained. The onset of hypertension and the response to previous anti-hypertensive treatment should be determined. Consideration of adherence to prescribed antihypertensive regimen should be given. A history of target organ damage (i.e. retinopathy, nephropathy, claudication, heart disease, abdominal or carotid artery disease) and the overall cardiovascular risk status should also be explored in detail (28).

The prevalence of resistant hypertension is high: 53% of patients in NHANES had a BP < 140/90 mm Hg vs. 48% in the Framingham Heart Study. In NHANES participants with chronic kidney disease, 37% had a BP < 130/80 mm Hg. In ALLHAT, 34% of patients remained uncontrolled after 5-year follow-up on at least 2 antihypertensive drugs (16). Table 2 presents clinical history, physical exam findings, and routine labs that suggest specific endocrine causes of hypertension.

Table 2: Endocrine Causes of Hypertension. Clinical Presentation. Diagnostic Tools

Etiology

Clinical presentation

Diagnostic tools

Adrenal-dependent causes 

Pheochromocytoma and sympathetic paraganglioma 

Headaches, palpitations, anxiety-like attacks, and profuse sweating

Free plasma or fractionated urinary metanephrines

Primary aldosteronism 

Polyuria, myopathy, and cardiac dysrhythmias may occur in cases of severe hypokalemia

Increased Aldosterone/Renin Ratio. Suppressed PRA, Increased aldosterone. Low potassium.

Congenital adrenal hyperplasia 

 11β-Hydroxylase deficiency 

Androgen production is increased and may lead to prenatal virilization with resulting pseudohermaphroditism in females. Males may develop pseudoprecocious puberty, short stature, and sometimes prepubertal gynecomastia

Increased 17 OH PRG, DOC, 11-deoxycortisol, androstenedione, testosterone, and DHEA-S

Germline mutation testing

 17α-Hydroxylase deficiency 

Pseudohermaphroditism in XY males, and sexual infantilism and primary amenorrhea in females

Low/low normal blood levels of androstenedione, testosterone, DHEA-S, 17-hydroxyprogesterone, aldosterone, and cortisol

Germline mutation testing

Deoxycorticosterone-producing tumor 

Hypertension, adrenal tumors usually large and malignant. Women may present virilization and men feminization.

Low renin and low/normal aldosterone. Increased DOC

Chrousos syndrome

Children may present with ambiguous genitalia and precocious puberty. In women, hirsutism and oligo-amenorrhea. Men may be infertile and/or oligospermic. No features of Cushing’s syndrome. Hypertension.

Hypokalemic alkalosis

Increased DOC, costisol, ACTH. Increased adrenal androgen secretion.

Liddle syndrome

Hypertension and spontaneous hypokalemia

Low potassium and low levels of aldosterone and renin

Cushing syndrome 

Weight gain, insomnia, depression, easy bruising, fatigue, acne, hirsutism, hyperglycemia

24-h urinary free cortisol excretion on at least 2 occasions. Suppressed ACTH

1mg overnight dexamethasone suppression test

Apparent mineralocorticoid excess/11β-hydroxysteroid dehydrogenase deficiency 

Congenital: Growth retardation/short stature, hypertension, hypokalemia, diabetes insipidus renalis, and nephrocalcinosis polyuria and polydipsia.

Acquired form is  attributed to licorice root ingestion and presents with hypertension and hypokalemia

Hypokalemia, metabolic alkalosis, low renin, low aldosterone, normal plasma cortisol levels

Abnormal urinary cortisol-cortisone metabolite profile

Parathyroid-dependent causes 

  Hyperparathyroidism 

Hypercalcemia, hypercalciuria, nephrocalcinosis, cortical bone loss, proximal myopathy, weakness and easy fatigability, depression, inability to concentrate

PTH intact, increased serum calcium concentration

Pituitary-dependent causes 

 Acromegaly 

Enlargement of the lower lip and nose, prognathism, mild hirsutism (in women), sweating, oily skin, diabetes mellitus, acanthosis nigrigans

IGF-1

 Cushing's disease

Weight gain, insomnia, depression, easy bruising, fatigue, acne, hirsutism, hyperglycemia

24-h urinary free cortisol excretion on at least 2 occasions

High normal/increased plasma ACTH

1mg overnight dexamethasone suppression test

MRI pituitary

Thyroid-dependent causes 

 Hypothyroidism 

Fatigue, weight gain, bradycardia, loss of appetite

Increased TSH

Low FT3, FT4

 Hyperthyroidism 

Nervousness, anxiety, palpitations, hyperactivity, weight loss, tachycardia

Low TSH

Increased FT3, FT4

 

PRIMARY ALDOSTERONISM

Prevalence of Primary Aldosteronism

In a community-based study (Framingham Offspring) comprising 1688 nonhypertensive participants, increased plasma aldosterone concentrations within the physiologic range predisposed persons to the development of hypertension (29). Previous studies have reported a prevalence of primary aldosteronism (PA) of 1-2 %. Newer data suggest an overall prevalence of >5% and possibly > 10% among the hypertensive population (15,16). In patients with mild to moderate hypertension without hypokalemia, the prevalence of PA has been reported to be 3% (30). In patients with resistant hypertension, the prevalence ranges between 17 and 23 % (31). In a study involving 1616 patients with resistant hypertension, 21% (338 pts) had an Aldosterone/Renin Ratio of > 65 with concomitant plasma aldosterone concentrations of > 416 pmol/L (15 ng) (25). After salt suppression testing, only 11% (182 pts) of these patients had primary aldosteronism (25). In patients with adrenal incidentaloma and hypertension, the prevalence of aldosteronism is low at 2% (31). Many (up to 63%) patients with PA may not present with hypokalemia but are rather normokalemic (31,32). Low renin hypertension is not always easy to differentiate from PA (33). Born-Frontsberg and colleagues found that 56% of 553 patients with primary aldosteronism had hypokalemia and 16% had cardio-and cerebrovascular comorbidities (32). In addition to the patient group with resistant hypertension, screening for primary aldosteronism is recommended for those patients with diuretic-induced or spontaneous hypokalemia, those with hypertension and a family history of early-onset hypertension or cerebrovascular accident at young age, and those with hypertension and an adrenal incidentaloma (27,34).

Etiology of Primary Aldosteronism

PA can be a sporadic or familial condition. Many cases of sporadic PA are caused by an aldosterone-producing adrenal adenoma. However, bilateral zona glomerulosa hyperplasia is much more common in apparently sporadic primary hyperaldosteronism than previously thought and is an important differential diagnosis, since it is treated medically with aldosterone antagonists, rather than by adrenalectomy (35). Selective use of adrenal venous sampling is helpful in this setting (36,37). Very rarely, PA can be caused by an adrenal carcinoma, or unilateral adrenal cortex hyperplasia (also called primary adrenal hyperplasia) (36).

Familial aldosteronism is estimated to affect 2% of all patients with primary hyperaldosteronism and is classified as type 1, type 2, type 3, and type 4 (38–40). Patients with familial aldosteronism type 3 produce amounts of 18-OHF and 18-oxoF 10-1,000 times higher than patients with familial aldosteronism type 1 (approx. 20 times normal) or patients with familial aldosteronism type 2 or sporadic aldosteronism (41). Patients with familial aldosteronism type 3 have a paradoxical rise of aldosterone after dexamethasone, atrophy of the zona glomerulosa, diffuse hyperplasia of the zona fasciculata, and severe hypertension in early childhood (around age 7 years) that is resistant to drug therapy but curable by bilateral adrenalectomy (42).

In familial hyperaldosteronism type 1, an autosomal dominantly inherited chimeric gene defect in CYP11B1/CYPB2 (coding for 11beta-hydroxylase/aldosterone synthase) causes ectopic expression of aldosterone synthase activity in the cortisol-producing zona fasciculata, making mineralocorticoid production regulated by corticotropin (24,43). The hybrid gene has been identified on chromosome 8. Under normal conditions, aldosterone secretion is mainly stimulated by hyperkalemia and angiotensin II. An increase of serum potassium of 0.1 mmol/L increases aldosterone by 35%. In familial hyperaldosteronism type 1 or glucocorticoid-remediable aldosteronism, urinary hybrid steroids 18-oxocortisol and 18-hydroxycortisol are approx. 20-fold higher than in sporadic aldosteronomas. Intracranial aneurysms and hemorrhagic stroke are clinical features frequently associated with familial hyperaldosteronism type 1 (35). The diagnosis is made by documenting dexamethasone suppression of serum aldosterone using the Liddle’s Test (dexamethasone 0.5 mg q 6h for 48h should reduce plasma aldosterone to nearly undetectable levels (below 4 ng/dl) or by genetic testing (Southern Blot or PCR) (44). In contrast, familial hyperaldosteronism type 2 is not glucocorticoid-remediable and is caused by mutations in the inwardly rectifying chloride channel CLCN2 (42).

Familial aldosteronism type 3 is caused by heterozygous gain-of-function mutation in the potassium channel GIRK4 (encoded by KCNJ5) leading to an increase in aldosterone synthase expression and production of aldosterone (35). Familial aldosteronism type 4 results from germline mutations in the T-type calcium channel subunit gene CACNA1H (45). Germline mutations in CACNA1D (encoding a subunit of L-type voltage-gated calcium channel CaV1.3) are found in patients with primary aldosteronism sometimes associated with seizures, and neurological abnormalities (46) . Table 3 shows genetic and clinical characteristics of familial aldosteronism.

Table 3. Classification of Familial Hyperaldosteronism

Type

Gene Mutation

Treatment

Clinical manifestations

FH-1

CYP11B2/CYP11B1 Chimeric

Low-dose dexamethasone

Intracranial aneurysms and hemorrhagic stroke

FH-2

CLCN2 (R172Q, M22K, G24D, S865R, Y26N)

MRA

Primary aldosteronism

FH-3

KCNJ5 (T158A, I157S, E145Q)

Bilateral adrenalectomy

Primary aldosteronism

 

KCNJ5 (G151E, Y152C)

MRA

Primary aldosteronism

FH-4

CACNA1H (M1549V, S196L, P2083L, V1951E)

MRA

Primary aldosteronism

 

Diagnosis – Screening and Confirming Tests

Primary aldosteronism is screened for by measuring plasma aldosterone (PA) and plasma renin activity (PRA) or direct renin concentration. There are various assays for measuring aldosterone, which can prove to be problematic(47,48). Measuring PRA is complicated and includes generating angiotensin from endogenous angiotensinogen. Quantification of renin’s conversion of angiotensinogen to angiotensin is performed utilizing radioimmunoassays for PRA, which are not standardized among laboratories. Measuring plasma renin molecules directly by an automated chemiluminescence immnoassay as direct renin concentration also is feasible. A PA/PRA-ratio > 30 with a concomitant PA > 20 ng/dl has a sensitivity of 90% and specificity of 91% for primary aldosteronism. Because low renin hypertension can be difficult to distinguish from PA, an upright plasma aldosterone of at least 15 ng/dl may be helpful (30).

As hypokalemia can reduce aldosterone secretion, it should be corrected before further diagnostic work-up. Also, if a patient with hypertension treated with an ACE inhibitor or ARB, calcium channel blocker, and a diuretic (all of which should increase PRA, thereby lower the PA/PRA-ratio or ARR), still has a suppressed renin and 2-digit plasma aldosterone level, primary aldosteronism is likely. False-positive ARRs may occur in premenopausal women during the luteal phase of the menstrual cycle as well as in those who are on medication with estrogen-containing contraceptive agents (14). Because of medication interference, it is commonly recommended to withdraw betablockers, ACE inhibitors, ARBs (angiotensin receptor blockers), renin inhibitors, dihydropyridine calcium channel blockers, nonsteroidal anti-inflammatory drugs, and central alpha 2-agonists approx. 2 weeks before PA/PRA-ratio or ARR testing, and to hold spironolactone, eplerenone, amiloride, and triamterene, and loop diuretics approx. 4 weeks before ARR testing. Licorice root products should also be withheld 4 weeks prior to testing (49). Confirmatory testing can be done by different techniques (31,50) [Table 4(50)]. A study including 148 hypertensive patients found that a new overnight diagnostic test using pharmaceutical renin-angiotensin-aldosterone system blockade with dexamethasone, captopril and valsartan, has low cost, is rapid, safe and easy to perform with an estimated sensitivity of 98% and specificity of 100% (51).

To clinically distinguish hyperplasia from unilateral adenoma, imaging with computed tomography and magnetic resonance imaging are helpful.

Table 4. ConfirmatoryTests (50)

Confirmation Method

Protocol

Interpretation of Results

Oral Salt Suppression Test

·Increase sodium intake for 3-4 days via supplemental tablets or dietary sodium to >200 mmol/day
· Monitor blood pressure
· Provide potassium supplementation to ensure normal serum levels
· Measure 24h urinary aldosterone excretion and urinary sodium on 3rd or 4th day

· PA confirmed: if 24h urinary aldosterone excretion >12 mcg in setting of 24h sodium balance >200 mmol
· PA unlikely: if 24h urinary aldosterone excretion <10mcg

Intravenous Saline Infusion Test

· Being infusion of 2L of normal saline after patient lies supine for 1 hour.
· Infuse 2L of normal saline over 4 hours (500 mL/h)
· Monitor blood pressure, heart rate, potassium
· Measure plasma renin and serum aldosterone at time=0h and time=4h

· PA confirmed: 4h aldosterone level > 10 ng/dL
· PA unlikely: 4h aldosterone level < 5 ng/dL

Captopril Challenge Test

· Administer 25-50mg of captopril in the seated position
· Measure renin and aldosterone at time=0h and again at time=2h
· Monitor blood pressure

· PA confirmed: serum aldosterone high and renin suppressed*
· PA unlikely: renin elevated and aldosterone suppressed*
*varying interpretations without specific validated cut-offs

Fludrocortisone Suppression Test

· Administer 0.1 mg fludrocortisone q6h for 4 days
· Supplement 75-100 mmol of NaCl daily to ensure a urinary sodium excretion rate of 3 mmol/kg/body weight
· Monitor blood pressure
· Provide potassium supplementation to ensure normal serum levels
· Measure plasma renin and serum aldosterone in the morning of day 4 while seated

· PA confirmed: Seated serum aldosterone > 6 ng/dL on day 4 with PRA< 1ng/mL/h
· PA unlikely: suppressed aldosterone < 6 ng/dL

Oral Salt Suppression Test

·Increase sodium intake for 3-4 days via supplemental tablets or dietary sodium to >200 mmol/day
· Monitor blood pressure
· Provide potassium supplementation to ensure normal serum levels
· Measure 24h urinary aldosterone excretion and urinary sodium on 3rd or 4th day

· PA confirmed: if 24h urinary aldosterone excretion >12 mcg in setting of 24h sodium balance >200 mmol
· PA unlikely: if 24h urinary aldosterone excretion <10mcg

Intravenous Saline Infusion Test

· Being infusion of 2L of normal saline after patient lies supine for 1 hour.
· Infuse 2L of normal saline over 4 hours (500 mL/h)
· Monitor blood pressure, heart rate, potassium
· Measure plasma renin and serum aldosterone at time=0h and time=4h

· PA confirmed: 4h aldosterone level > 10 ng/dL
· PA unlikely: 4h aldosterone level < 5 ng/dL

 

Localization

Despite imaging studies, adrenal venous sampling (AVS) with cosyntropin (ACTH) infusion is often essential if the patient desires surgery in case of a unilateral adenoma: cutoff for unilateral adenoma > 4 “cortisol-corrected” aldosterone ratio (adenoma side aldosterone/cortisol: normal adrenal gland aldosterone/cortisol); cutoff for bilateral hyperplasia < 3 “cortisol-corrected” aldosterone ratio (high-side aldosterone/cortisol: low-side aldosterone/cortisol) (36,52).

Medical Treatment

The 2016 Endocrine Society clinical practice guideline for the management of primary aldosteronism suggests that patients with hypertension, spontaneous hypokalemia, undetectable renin, and a plasma aldosterone concentration above 20 ng/dl (550 pmol/L) may not need to undergo further confirmatory testing but instead proceed with further imaging and/or adrenal vein sampling or (if unable or unwilling to undergo surgery/adrenalectomy) treatment with a mineralocorticoid antagonist (36). Bilateral adrenal hyperplasia is treated with spironolactone, eplerenone, and/or amiloride (50).

Spironolactone is a nonselective, competitive mineralocorticoid receptor antagonist and is generally considered first-line therapy for patients with BAH at doses ranging between 12.5-400 mg/d (usually 12.5-50 mg/d). It also acts as antagonist of the androgen receptor, a weak antagonist of the glucocorticoid receptor, and an agonist of the progesterone receptor. These actions are associated with adverse effects, including hyperkalemia, hyponatremia, gynecomastia, menstrual disturbances and breast tenderness and decreased libido in women, and gynecomastia in men, occuring in a dose-dependent manner.

Eplerenone, is a more expensive but selective mineralocorticoid receptor blocker with fewer antiandrogenic effects, but also with lower affinity for the mineralocorticoid receptor and less effectiveness than spironolactone with respect to BP lowering in patients with moderate hypertension (53); Generally, higher doses of eplerenone are prescribed for similar effects as spironolactone (usually 25-50 mg twice daily) (50).

Currently under investigation are aldosterone synthase inhibitors, which may not have any nongenomic/non-mineralocorticoid receptor-mediated adverse effects (54). In cases of familial hyperaldosteronism type 1, dexamethasone is effective in suppressing ACTH and, hence, aldosterone overproduction (55).

Surgical Treatment

Adrenal adenomas producing aldosterone should be removed. Nearly all patients with such endocrine hypertension have improved blood pressure control and up to 60% are cured (normotensive without antihypertensive therapy) from hypertension (56–59). This outcome is influenced by various factors including age, duration of hypertension, coexistence of renal insufficiency, use of more than 2 antihypertensive drugs preoperatively, family history of hypertension, and others. Parameters of insulin sensitivity can be restored to normal with treatment of PA (60). A cross-sectional study including 460 pts with primary aldosteronism and 1363 controls with essential hypertension found no significant difference between pre- and postoperative levels of fasting plasma glucose and serum lipids (61). This topic has been extensively reviewed from a pro and contra perspective. If a patient does not desire surgery/adrenalectomy for a unilateral aldosteronoma/hyperplasia (Figure 3), medical therapy should be initiated (54). AVS and CT/MRI of the adrenal glands show a unilateral abnormality in 60.5% and 56%, respectively, but were congruent on the involved side in the same patient in only 37% in a recent systematic review (62). If a patient is older than age 40 years, the risk for an adrenal incidentaloma increases (62). Unilateral adrenalectomy can be helpful in some patients with primary aldosteronism and bilateral adrenal hyperplasia (56).

Figure 3. Conn adenoma. Appearance of a 1 cm right-sided adrenal nodule (arrow) on contrast-enhanced computed tomography in a middle-aged man with hypertension treated for 20 years, initially only with a betablocker before becoming medically refractory and hypokalemic with inappropriate kaliuresis. After laparoscopic right adrenalectomy, the patient required only one antihypertensive drug to control his blood pressure.

PHEOCHROMOCYTOMA (PPGLS)

Prevalence

Pheochromocytomas are rare neoplasms, with an estimated occurrance of approximately 0.2 percent of patients with hypertension. It has been reported that the annual incidence of pheochromocytoma is nearly 0.8 per 100,000 person-years (63). Pheochromocytomas may occur at any age, however they are commonly presented  in the fourth to fifth decade (64) .

Etiology

Pheochromocytoma and paragangliomas (PPGLs) rare neuroendocrine tumors are composed of chromaffin tissue containing neurosecretory granules (65). Most pheochromocytomas are sporadic but as of known today, approx. 40% of patients with pheochromocytoma or paraganglioma irrespective of age at onset and family history harbor a germline mutation (66,67). At present, there are 10 currently clinically relevant syndromes known: multiple endocrine neoplasia type 2, von Hippel-Lindau syndrome, neurofibromatosis type 1, paraganglioma syndromes 1 through 5, caused by mutations of the succinate dehydrogenase genes SDHD (syndrome 1), SDHAF2 (syndrome 2), SDHC (syndrome 3), SDHB (syndrome 4), and SDHA (syndrome 5), and the hereditary pheochromocytoma syndromes resulting from germline mutations in the genes coding transmembrane protein 127 (TMEM127) and MYC-associated factor X (MAX). Further susceptibility genes include EGLN1 (PHD2), EGLN2 (PHD1), DNMT3A, IDH1, FH, MDH2, SLC25A11, KIF1B, and HIF2A (68–70). There is controversy when genetic testing should be obtained in patients with pheochromocytoma, especially considering cost effectiveness.

Clinical Features

The clinical presentation of patients with PPGLs shows a wide variety from no or minor symptoms, to dramatic life-threatening manifestations. Asymptomatic patients present mostly incidentally discovered adrenal masses. Normotensive patients may also have sporadic pheochromocytomas (71). It appears that approx. 15% of patients with pheochromocytoma are normotensive (19,21). The classic triad of pounding headache, profuse sweating, and palpitations occurs sporadically with a duration from several minutes to 1 hour. Paroxysmal hypertension occurs commonly in 35-50% of patients. The patients show a complete relief of symptoms between episodes. The high BP surges and the other symptoms are associated with the underlying tumoral catecholamine release, which is the major cause for the high prevalence of cardiovascular emergencies, such as myocardial infarction, stroke, and heart failure. Pheochromocytoma (PHEO) and PPGLs may be the prevalent cause of acute Takotsubo-like catecholamine cardiomyopathy (TLC) (72–75). This association has been reported in in up to 3% of patients with secreting PPGL. The real prevalence of PPGL in TTC remains to be determined. The biochemical profile of pheochromocytomas associated with the a forementioned hereditary syndromes varies (76). Patients with MEN 2 and VHL syndrome may have clinically “silent” pheochromocytomas. Blood pressure does not correlate with circulating catecholamines in patients with pheochromocytoma. Sipple syndrome for multiple endocrine neoplasia type 2 first described by Max Schottelius and Felix Fraenkel in 1886 (77).

Diagnosis – Screening

The diagnosis can be established by measuring free plasma or fractionated urinary metanephrines (metanephrine and normetanephrine) (22). When plasma free metanephrines cannot be measured by HPLC with electrochemical detection or high-throughput automated liquid–chromatography-tandem mass spectrometry (LC-MS/MS), measuring plasma free metanephrines by RIA or measuring plasma chromogranin A may represent good markers for pheochromocytoma. In rare circumstances, pheochromocytomas release large O-methylated dopamine metabolite methoxytyramine, which can be elevated in extra-adrenal tumor location (in particular, neck and skull-base paragangliomas) and the presence of metastatic disease (78). In patients with renal failure, plasma concentrations of free metanephrines can be increased several folds (79). For optimal diagnostic accuracy, established reference values for plasma free and 24-hour urinary fractionated metanephrines should be btained, according to age and sex. The upper cutoff level of plasma free normetanephrine, but not for metanephrine or methoxytyramine is higher in older patients (80).

Several medications can cause false-positive biochemical testing. Thus, plasma normetanephrine levels may increase in patients treated with tricyclic antidepressants, antipsychotics, buspirone, MAO inhibitors, sympathomimetics, cocaine, levodopa, phenoxybenzamine, acetaminophen, alpha- methyldopa, and sulphasalazine. Plasma metanephrine levels may increase in patients treated with buspirone, MAO inhibitors, sympathomimetics, cocaine, and levodopa. Urine normetanephrine levels may be higher in patients receiving all the above-mentioned substances, as well as labetalol and sotalol. Urine normetanephrine levels may be increased by buspirone, MAO inhibitors, sympathomimetics, cocaine, levodopa, labetalol and sotalol (14). People who eat biogenic amines may have false-positive urinary metanephrine results. However, for measuring plasma free metanephrines and the O-methylated dopamine metabolite methoxytyramine, no specific dietary requirements are needed, but fasting state (14,81).

The 2014 Endocrine Society clinical practice guideline recommends that all patients with pheochromocytoma-paraganglioma should be engaged in shared decision making for genetic testing (22). All patients with paraganglioma should undergo testing for succinate dehydrogenase (SDH) mutations and those patients with metastatic disease should be tested for SDHB mutations. Recognizing the distinct genotype-phenotype presentations of patients with hereditary tumors, the guideline recommends a personalized approach to patient management. Of note is that SDHD and SDHAF2 are maternally imprinted and therefore one or more generations can be skipped. During the first 2 decades of life (before the age of 20 years), the most common hereditary pheochromocytoma-paraganglioma syndromes found are related to von Hippel Lindau disease, paraganglioma syndrome type 4 (SDHB), and neurofibromatosis type 1. Pheochromocytomas related to multiple endocrine neoplasia type 2 occur most frequently between the third and fifth decade of life and should first be considered in a patient presenting with bilateral pheochromocytomas. The mean penetrance of pheochromocytoma or paraganglioma in individuals carrying a RET germline mutation is 50% by the age of 44 years (82).

Approximately 35% of extra-adrenal pheochromocytomas are considered “malignant” (metastasizing) as opposed to approximately 10% of those arising in the adrenal gland. The 2017 WHO classification of endocrine tumors replaced the term “malignant” with “metastatic”. The risk for metastases increases when the tumor exceeds 5 cm in size and when there is a germline mutation in the SDHB gene (83,84).

Localization

CT or MR imaging can localize the tumor in approx. 95 % of cases. For metastatic pheochromocytomas, 18F-Fluorodopamine and 18F-FDG PET appears to be more helfpul than 123I-MIBG or 131I-MIBG scintigraphy (85,86). In fact, MIBG scintigraphy should nowadays only been used in selected patients (85,87). Many medications can interfere with 123I-MIBG or 131I-MIBG uptake (for instance, calcium channel blockers, antipsychotics) and should be discontinued before the scan/imaging. The 2014 Endocrine Society guideline recommends the use of 123I-MIBG in patients with metastatic pheochromocytoma-paraganglioma when radiotherapy with 131I-MIBG is planned and occasionally in some patients with an increased risk for metastatic disease (large tumor size, extra-adrenal tumor, multifocal or recurrent disease) (22). For patients with head and neck paragangliomas, 111In-octreotide has a very good sensitivity (88). Newer functional imaging techniques such as 68Ga-labeled 1,4,7,10-tetraazacylododecane-1,4,7,10-tetraacetic acid-octreotate (DOTATATE) of 18F-labeled L-dihydroxyphenylalanine (L-DOPA) have excellent resolution in detecting pheochromocytomas and paragangliomas.

Medical Treatment

The Endocrine Society, the American Association for Clinical Chemistry, and the European Society of Endocrinology have released clinical practice guidelines recommended preoperative blockade of hormonally functional PPGL to prevent cardiovascular complications, along with medication for normalization of blood pressure as well as heart rate. Alpha-adrenergic blockade (i.e., doxazosin, prazosin or terazosin) followed by a β-adrenergic blockade (i.e., propranolol, atenolol) is recommended for preoperative preparation (89). It is also suggested to administer high-sodium diet and fluid intake to prevent low blood pressure after surgery. Approx. 50% of patients with metastatic pheochromocytomas respond to 131I-MIBG therapy by partial remission or at least stable disease. Selective alpha1 blocking agents, such as prazosin (Minipress), terazosin (Hytrin), and doxazosin (Cardura), have more favorable adverse effect profiles and are used when long-term therapy is required (metastatic pheochromocytoma). Newer therapy options of metastatic pheochromocytoma-paraganglioma include 90Y-DOTATATE and 177Lu-DOTATATE. Chemotherapy is usually administered according to the so-called Averbuch protocol from 1988. New therapies may include tyrosine kinase inhibitors in selected patients (90).

Surgical Treatment

For tumors exceeding 5 cm in size, open adrenalectomy has long been considered the suggested procedure for tumor removal rather than laparoscopic or retroperitoneoscopic minimally invasive tumor removal, to ensure complete tumor resection, prevent tumor (capsule) rupture, and avoid local recurrence (22) (Figure 4 and 5)  

Figure 4. Computed tomography showing recurrence of a right adrenal pheochromocytoma. unpublished observation in a patient with MEN2-related bilateral pheochromocytomas and unilateral tumor recurrence 11 years after bilateral adrenalectomy, photo: courtesy of Prof. Andrea Tannapfel).

Figure 5. Macroscopic photo of a right adrenal pheochromocytoma removed from the above patient with multiple endocrine neoplasia type 2.

For pheochromocytomas less than 6 cm, a recent cohort study from a multicenter consortium-based registry for 625 patients treated for bilateral pheochromocytomas between 1950 and 2018 compared patients undergoing total vs. cortical-sparing adrenalectomy and found that patients undergoing cortical-sparing adrenalectomy did not demonstrate decreased survival, despite development of recurrent pheochromocytoma in 13%. The authors recommend cortical-sparing adrenalectomy should be considered in all patients with hereditary pheochromocytoma (91). A retrospective, multicenter, international study in patients carrying the Met918Thr RET variant with no age restrictions who were followed from 1970 to 2016 based on registry data from 48 centers globally, found that adrenal-sparing surgery in multiple endocrine neoplasia type 2B can preserve normal adrenal function. In that study, three (10%) of the 31 patients in whom adrenal-sparing surgery had been performed, developed long-term recurrence, while normal adrenal function was mantained in 16 (62%) of patients (92). Apparently one third of one functioning adrenal gland is sufficient for normal glucocorticoid and mineralocorticoid secretion (93).

Cushing’s Syndrome

Hypercortisolemia is associated with hypertension in approximately 80% of adult cases and half of children (94,95). A workshop consensus paper attempted to rationalize the treatment of hypertension in patients with Cushing’s syndrome (95). In patients with Cushing’s disease, night-time blood pressure decline is significantly lower than that in patients with essential hypertension (96). After cure of Cushing’s syndrome, approximately 30% of patients have persistent hypertension (97). In children and adolescents, blood pressure normalization occurs in most patients within a year and seems to be dependent on the degree and duration of presurgical hypercortisolemia (94). In patients with Cushing’s disease, renin and DOC levels are usually normal, whereas in ectopic corticotropin syndrome, hypokalemia is common and related to an increased mineralocorticoid activity with suppressed renin and elevated DOC levels (98).

There are several mechanisms of blood pressure elevation in Cushing’s syndrome: increased hepatic production of angiotensinogen and cardiac output by glucocorticoids, reduced production of prostaglandins via inhibition of phospholipase A, increased insulin resistance, and oversaturation of 11beta-Hydroxysteroid dehydrogenase activity with increased mineralocorticoid effect through stimulation of the mineralocorticoid receptor (99). Screening studies for Cushing’s syndrome include measuring 24-h urinary free cortisol excretion on at least 2 occasions, performing a 1 mg dexamethasone suppression test, checking a midnight salivary cortisol and diurnal rhythm of cortisol secretion, and others listed in the recent Endocrine Society Clinical Practice Guideline (100). Therapy should be directed at removing glucocorticoid excess (101). Hypokalemia (especially in patients with ectopic ACTH production) can be treated with mineralocorticoid receptor antagonists such as spironolactone or eplerenone. Thiazide diuretics may also be helpful.

Given the increasing improvement in imaging and laboratory (assays etc.) techniques/modalities, one can expect an increasing number of incidentally discovered tumors and nodules in various organs including the adrenal glands. The future challenge will be when and to which extent to test individuals for disease conditions (102,103). For those individuals with adrenal incidentalomas but clearly lack of clinical features of Cushing’s syndrome, subclinical hypercortisolism may be detected biochemically depending upon which cutoff values and assays will be used (104). For the latter population, the American Association of Clinical Endocrinologists recommend using a cutoff for (8 AM) serum cortisol of 5 mcg/dl after 1 mg overnight (11 PM) dexamethasone which reveals approx. 58% sensitivity at a 100% specificity (105). A lower cutoff for serum cortisol suppression, i.e. 1.8 mcg/dl, usually rules out Cushing’s syndrome (102). A prospective, randomized study including 45 patients with subclinical hypercortisolism and adrenal incidentalomas was divided into 23 pts who underwent adrenalectomy and 22 pts under surveillance. Monitoring included glycemic control, blood pressure, lipid profile, obesity, and bone mineral density. In the surgical group, diabetes mellitus improved in 62% and hypertension in 67% of pts, whereas the conservative group showed worsening of glycemic control, blood pressure and lipid profiles (37).

To better understand the sequelae of disturbed adrenal hormone synthesis, please refer to Figure 6 and related Endotext chapters (106,107).

Figure 6. Adrenal Steroid Synthesis. Z Glom = zona glomerulosa; Z Fas = zona fasciculata; Z Ret = zona reticularis; 19-H = 19-Hydroxylase; HSD = Hydroxysteroid dehydrogenase; P450aro = aromatase; 5alpha-Red = 5alpha-Reductase. The 3 adrenal cortex zones Z Glom, Z Fas, and Z Ret stand above the “column” of hormones that are produced in the respective zone. The steroidogenic enzymes on the left starting with P450scc (Desmolase) are listed in order for “vertical and horizontal reading”, i.e. Desmolase converts cholesterol to pregnenolone, 3beta-OH-Steroid Dehydrogenase I/II convert pregnenolone to progesterone, 17-OH-Pregnenolone to 17-OH-Progesterone, and P450c11 converts deoxycorticosterone to 18-OH-Corticosterone and 11-Deoxycortisol to cortisol, etc. (modified from ref. 35: Koch CA. Encyclopedia of Endocrine Disease, 2004)

GLUCOCORTICOID RESISTANCE (CHROUSOS SYNDROME)

This autosomal recessive or dominant inherited disorder is rare and caused by inactivating mutations of the glucocorticoid receptor gene (108,109). Cortisol and ACTH are elevated but there are no clinical features of Cushing syndrome. Permanent elevation of ACTH can lead to stimulation of adrenal compounds with mineralocorticoid activity (corticosterone, DOC), along with elevated cortisol secretion may lead to stimulation of the mineralocorticoid receptor, resulting in hypertension. In women, hirsutism and oligo-amenorrhea may develop through stimulation of androgens (androstendione, DHEA, 5-androstendiol). Clinically, children may present with ambiguous genitalia and precocious puberty. Men may be infertile and/or oligospermic. Women may have acne, excessive hair, menstrual irregularities with oligo- anovulation, as well as infertility (108–110).

Treatment entails suppression of ACTH secretion with high doses of dexamethasone (1-3 mg/day). Mineralocorticoid receptor-dependent hypertension may be treated with blockade of the receptor, with spironolactone or eplerenone.

Congenital Adrenal Hyperplasia

11Beta-Hydroxylase Deficiency

The most common cause of congenital adrenal hyperplasia (CAH) is 21-hydroxylase deficiency. Hypertension per se has not been regarded as a component of this syndrome. Recent data have suggested that hypertension may be more prevalent in this patient population than previously thought (111–113).

Approx. 5% of all cases of CAH care caused by 11beta-hydroxylase deficiency. 11beta-hydroxylase is responsible for the conversion of deoxycorticosterone (DOC) to corticosterone (precursor of aldosterone) and 11-deoxycortisol to cortisol. In approximately 2/3 of individuals affected by a deficiency of this enzyme, monogenic low renin hypertension with low aldosterone levels occurs caused by accumulation of 11-deoxycortisol and DOC (114,115). The earliest age of onset of hypertension was reported at birth (116). The inheritance mode is autosomal recessive. The responsible gene CYP11B1 is located on chromosome 8 and is mutated (40,117,118). Since corticotropin (ACTH) is chronically elevated and precursors such as 17-OH progesterone and androstendione accumulate, androgen production is increased and may lead to prenatal virilization with resulting pseudohermaphroditism in females. Males may develop pseudoprecocious puberty, short stature, and sometimes prepubertal gynecomastia (119,120). Usually, glucocorticoid replacement reduces hypertension in these patients. In selected patients, bilateral adrenalectomy may be safe and effective in managing high blood pressure (121).

17Alpha-Hydroxylase Deficiency

This enzyme deficiency is rare and leads to diminished production of cortisol and sex steroids. Chronic elevation of ACTH causes an increased production of DOC and corticosterone with subsequent hypertension, hypokalemia, low aldosterone concentrations with suppressed renin as well as pseudohermaphroditism in XY males (122), and sexual infantilism and primary amenorrhea in females (123,124). Diagnosis may be delayed until puberty. Plasma adrenal androgen levels are low as are cortisol, aldosterone, plasma renin activity, and 17alpha-hydroxyprogesterone. DOC, corticosterone, and 18-hydroxycorticosterone are elevated. Blood pressure is reduced by glucocorticoid replacement. The responsible gene for cytochrome P450C17 is located on chromosome 10q24.

Deoxycorticosterone-Producing Tumor

Deoxycorticosterone-producing tumors are rare adrenal tumors presented mostly large and malignant (125). Along with deoxycortisone, androgens and estrogens may be cosecreted. Women may present virilization and men feminization. Hypertension and hypokalemia may manifestate with rapid onset. Renin and aldosterone are often low.

Apparent Mineralocorticoid Excess

Low-renin hypertension (undetectable aldosterone, hypokalemia) can present in various forms, one of them is apparent mineralocorticoid excess (AME), an autosomal recessive disorder caused by deficiency of the 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) enzyme (49,126,127). This enzyme converts cortisol to the inactive cortisone in renal tubular cells.

In 1977, New et al. (128) first described this syndrome and in 1995 Wilson et al. (129) first reported that mutations in the 11beta-HSD2 gene located on chromosome 16q22 cause AME. The 11beta-HSD2 enzyme is co-expressed with the mineralocorticoid receptor in renal tubular cells and leads to conversion of cortisol to cortisone (130,131). Cortisone does not bind to the mineralocorticoid receptor. Cortisol and aldosterone bind with equal affinity to the mineralocorticoid receptor, but normal circulating concentrations of cortisol are 100 to 1000 fold higher than those of aldosterone (132). If 11beta-HSD2 is oversaturated or defective, more cortisol will be available to bind to the mineralocorticoid receptor (133). Diminished 11beta-HSD2 activity may be hereditary or acquired. Acquired deficiency of this enzyme may result from inhibition by glycyrrhhetinic acid which may occur with use of licorice, chewing tobacco, and carbenoloxone. In childhood, AME often causes growth retardation/short stature, hypertension, hypokalemia, diabetes insipidus renalis, and nephrocalcinosis. Diminished 11beta-HSD2 activity may play a role in the pathogenesis of preeclampsia (134). The diagnosis of AME can be established by measuring free unconjugated steroids in urine (free cortisol/free cortisone ratio), and/or steroid metabolites (tetrahydrocortisol + allotetrahydrocortisol/tetrahydrocortisone) (135). Affected individuals have low renin and aldosterone levels, normal plasma cortisol levels, and hypokalemia. Treatment of AME consists of spironolactone, eplerenone, triamterene, or amiloride. Renal transplant is an option for patients with advanced renal insufficiency.

Constitutive Activation of The Mineralocorticoid Receptor (Geller Syndrome)

The Mineralocorticoid (MC) receptor can be mutated leading to the onset of hypertension before age 20 (136). In vitro experiments demonstrate that progesterone and spironolactone, usually antagonists of the (MC) receptor, become agonists in Geller syndrome, suggesting “gain of function” mutations in the MC gene on chromosome 4q31. The inheritance pattern is autosomal-dominant.

Liddle Syndrome

In 1963, Liddle (137) described patients with severe hypertension, hypokalemia, and metabolic alkalosis, who had low plasma aldosterone levels and plasma renin activity. An improvement of the hypertension occurred after salt restriction and triamterene therapy. Spironolactone is ineffective in this autosomal-dominant inherited syndrome. So-called “gain of function” mutations in the genes coding for the beta- or gamma-subunit of the renal epithelial sodium channel, located at chromosome 16p13, lead to constitutive activation of renal sodium reabsorption and subsequent volume expansion. The 24-h urine cortisone/cortisol ratio is normal.

Pseudohypaldosteronism Type 2

Pseudohypoaldosteronism type 2 or Gordon’s syndrome (138) is a rare Mendelian disorder, transmitted in an autosomal dominant fashion, and can cause low renin hypertension (139). It has an unknown prevalence, since many patients remain undiagnosed. Published families with this condition (hypertension, hyperkalemia, metabolic acidosis, normal renal function, low/normal aldosterone levels) are predominantly from Australia or the United States (138). Hypertension in these patients may develop as a consequence of increased renal salt reabsorption, and hyperkalemia ensues as a result of reduced renal K excretion despite normal glomerular filtration and aldosterone secretion (140). The reduced renal secretion of potassium makes this condition look like an aldosterone-deficient state, thus the term “pseudohypoaldosteronism”.

These features are chloride dependent. Infusion of sodium chloride instead of sodium bicarbonate corrects the abnormalities, as does the administration of thiazide diuretics, which inhibit salt reabsorption in the distal nephron. Gordon and coworkers found that all features could be reversed by very strict dietary salt restriction (138). Gordon syndrome is an autosomal, dominantly inherited disorder with genes mapping to chromosomes 1, 12, and 17 (141,142). Mutations have been identified in WNK kinases WNK1 and WNK4 on chromosomes 12 and 17, respectively (141,143). Abnormalities such as activating mutations in the amiloride-sensitive sodium channel of the distal renal tubule are responsible for the clinical phenotype (144,145). Severe dietary salt restriction, antihypertensives, with preferably use of thiazide diuretics, can control the hypertension in this syndrome. Interestingly, common variants in WNK1 contribute to blood pressure variation in the general population (146).

Insulin Resistance

The metabolic syndrome is characterized by hypertension, abdominal/visceral obesity, dyslipidemia, and insulin resistance (147). At least 24% of adults in the United States meet the criteria for the diagnosis of metabolic syndrome, and this number may even be higher for individuals over the age of 50 years (148). Insulin resistance is significantly associated with hypertension in Hispanics and can cause vascular dysfunction (16,149). Patients with essential hypertension often are insulin resistant (150). Interestingly, not all insulin resistant patients are obese. Excess weight gain, however, accounts for as much as 70% of the risk for essential hypertension and also increases the risk for end stage renal disease (16). In insulin-sensitive tissues, insulin can directly stimulate the calcium pump leading to calcium loss from the cell (151). In an adipocyte, elevated cytosolic calcium concentrations can induce insulin resistance. In a cell resistant to insulin, the insulin-induced calcium loss from cells would be decreased. With the subsequent increase in intracellular calcium, vascular smooth muscle cells respond more eagerly to vasoconstrictors and thus lead to rising blood pressure. Other mechanisms possibly explaining the association of insulin resistance and hypertension are increased sodium retention and increased activity of the adrenergic nervous system. In obesity, increased production of most adipokines (bioactive peptides secreted by adipose tissue) impacts on multiple functions including insulin sensitivity, blood pressure, lipid metabolism, and others (152,153).

Primary Hyperparathyroidism

Parathyroid hormone levels in hypertensive patients usually are in the normal range and appropriate for the serum calcium concentration. However, patients with essential hypertension excrete more calcium compared to normotensive people, suggesting an enhanced parathyroid gland function (154). When infused, PTH is a vasodilator, although chronic infusion of PTH raises blood pressure in healthy subjects (155,156). High-calcium intake may lower blood pressure (157,158). However, hypercalcemia is associated with an increased incidence of hypertension (1). In patients with primary hyperparathyroidism, hypertension is observed in approximately 40% of cases. The mechanisms of these observations/associations are unclear. Hypertension is usually not cured or better controlled after parathyroidectomy (159). In patients with asymptomatic primary hyperparathyroidism, surgery/parathyroidectomy did not show any benefit regarding blood pressure or quality of life when compared to medical management (160). On the other hand, severe hypertension may improve in patients with primary hyperparathyroidism who undergo parathyroidectomy. Arterial stiffness measured in the radial artery seems to be increased in patients with mild primary hyperparathyroidism (161). Patients with primary hyperparathyroidism have carotid vascular abnormalities (162). In normotensive patients with primary hyperparathyroidism, SBP variability is increased and is reduced by parathyroidectomy (163,164). Furthermore, parathyroidectomy in patients with primary hyperparathyroidism may decrease risk of cardiovascular diseases by lowering total cholesterol levels, although ambulatory diastolic BP increases in response to surgery (165). Another contributory factor to hypertension in patients with primary HPT may be endothelial dysfunction (166). In MEN syndromes, hypertension in patients with hyperparathyroidism may be related to an underlying pheochromocytoma or primary aldosteronism. Criteria for parathyroidectomy have recently been revisited at the Fourth International Workshop on the management of asymptomatic primary hyperparathyroidism, including now skeletal and/or renal involvement (nephrocalcinosis on imaging) (167).

Hyperthyroidism

Hyperthyroidism increases systolic blood pressure by increasing heart rate, decreasing systemic vascular resistance, and raising cardiac output (168–171). In thyrotoxicosis, patients usually are tachycardic and have high cardiac output with an increased stroke volume and elevated systolic blood pressure (172,173). Approx. one third of patients with hyperthyroidism have hypertension which often resolves after achieving euthyroidism (174). Subclinical hyperthyroidism may contribute to left ventricular hypertrophy and thereby lead to hypertension , although it has not yet been found to be associated with hypertension (174).

Hypothyroidism

Hypothyroid patients have impaired endothelial function, increased systemic vascular resistance, extracellular volume expansion, and an increased diastolic blood pressure (89,171,175). Hypothyroid patients have higher mean 24-h systolic blood pressure and BP variability on 24-h ambulatory BP monitoring (176). In 32% of hypertensive hypothyroid patients, replacement therapy with thyroxine leads to a fall in diastolic blood pressure to 90 mm Hg or less (177). There is a positive association between serum TSH and blood pressure within the normal serum TSH range, statistically significant for diastolic hypertension (177). Subclinical hypothyroidism may or may not to be associated with hypertension. Hypothyroidism can lead to volume-dependent blood pressure elevation with low plasma renin concentrations (178–180).

Acromegaly

The prevalence of hypertension in patients with growth hormone excess is approximately 46% and more frequent than in the general population (181,182). Growth hormone has antinatriuretic actions and may lead to sodium retention and volume expansion (181,182). Increased systolic output and high heart rate as manifestations of a hyperkinetic syndrome may lead to congestive heart failure (181,183). Blood pressure values are increased in patients with acromegaly associated with reduced glucose tolerance or diabetes compared to those with normal glucose tolerance (181). The RAAS system appears to be implicated in the pathogenesis of hypertension in patients with growth hormone excess (181,183–185). Comorbidities in acromegalics, such as hypertension, hyperlipidemia, diabetes mellitus, and cardiomyopathy, all may improve even with partial biochemical control of growth hormone excess (184,186). However, in some patients, hypertension and diabetes mellitus may persist after attempting biochemical cure/remission (187).

Other Potential Endocrine Conditions Causing Endocrine Hypertension

There is accumulating evidence that vitamin D deficiency may be linked to an increased cardiovascular risk and hypertension (188). Potential mechanisms in this setting are concurrent insulin resistance and direct vitamin D action through the renin-angiotensin-aldosterone system (Figure 7).

Figure 7. Pathway of vitamin D metabolism and its relationship with PTH and the renin-angiotensin-aldosterone system (modified from Ullah et al., 2009)(188)

Testosterone deficiency is frequently identified in obese individuals and those with diabetes mellitus and/or metabolic syndrome including hypertension. Replacement therapy in selected patients may be beneficial not only related to their symptomatology of androgen deficiency such as low libido, poor erections, fatigue, and others, but also in regards to their metabolic profile and blood pressure (189,190).

Similarly, individuals with growth hormone deficiency may be at risk for developing hypertension, mostly because of their body composition being more “fat” and “inflamed” when compared to subjects with growth hormone sufficiency, as assessed by serum IFG-1 levels matched to gender and age. The key in such patients will be to replace them with growth hormone individually to an IGF-1 level at which no features of growth hormone excess develop and to increase physical activity. In obese subjects who are willing to take on major lifestyle changes with the goal to lose weight, eat and live healthier, temporary medication assistance (phentermine, topiramate, liraglutide, lorcaserin, orlistat, naltrexone-bupropion) including administration of growth hormone may be acceptable (191–193).

Individual tissue-dependent sensitivity of the glucocorticoid receptor and actions of endogenous glucocorticoids may play a major role in the development of hypertension, obesity, and diabetes mellitus (119,194,195).

Similarly, individuals with growth hormone deficiency may be at risk for developing hypertension, mostly because of their body composition being more “fat” and “inflamed” when compared to subjects with growth hormone sufficiency, as assessed by serum IFG-1 levels matched to gender and age. The key in such patients will be to replace them with growth hormone individually to an IGF-1 level at which no features of growth hormone excess develop and to increase physical activity. In obese subjects who are willing to take on major lifestyle changes with the goal to lose weight, eat and live healthier, temporary medication assistance (phentermine, topiramate, liraglutide, lorcaserin, orlistat, naltrexone-bupropion) including administration of growth hormone may be acceptable (191–193).

Individual tissue-dependent sensitivity of the glucocorticoid receptor and actions of endogenous glucocorticoids may play a major role in the development of hypertension, obesity, and diabetes mellitus (119,194,195).

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Existing and Emerging Molecular Targets for The Pharmacotherapy of Obesity

ABSTRACT

 

Obesity is pandemic and a multidisciplinary approach is critical for its management. Anti-obesity treatment includes lifestyle modifications combined with anti-obesity medications. Anti-obesity drugs target either central nervous system pathways, which regulate sensations of satiety and fullness, or peripheral modulators of digestion, metabolism and lipogenesis. Combined anti-obesity agents is a novel, promising field, especially the co-administration of gut hormone analogues with centrally acting molecules. Consequently, it is hoped that in the near future, individualized pharmacological management of obesity could be meaningfully achieved by targeting different pathways governing energy homeostasis and weight regulation.  This chapter reviews potential molecular targets of the energy homeostasis system along with new anti-obesity drugs currently under investigation.

INTRODUCTION                                                                                                                      

 

The pathophysiology that leads to obesity is considered a novel field for research. Understanding human metabolism and the homeostatic mechanisms of weight regulation includes comprehension of the interaction between central nervous system and peripheral modulators of weight maintenance. Current anti-obesity molecular pharmacotherapy is based on single molecule anti-obesity drugs that act either via enhancement of satiety feeling, inhibition of hunger, or triggering of catabolism. However, on average, the weight-lowering effects of these medications are modest at best and side effects are common.

 

According to current clinical practice guidelines for pharmacological management of obesity published in 2015 by The Endocrine Society, if a patient’s weight is not responsive to lifestyle intervention, weight loss pharmacotherapy can be offered for a BMI ≥27kg/m2 when an obesity-related comorbidity is present, or when the BMI is ≥30kg/m2 (1). In fact, pharmacologic weight management should be considered in patients who meet these weight criteria and have any of a number of chronic conditions in which obesity is considered to play a major role, including type 2 diabetes mellitus (T2DM), cardiovascular disease, hypertension, dyslipidemia, obstructive sleep apnea, nonalcoholic fatty liver disease, certain cases of malignancies (i.e. endometrial, breast, colon) (2), osteoarthritis, depression (3), and infertility (4).

 

Currently, there are six anti-obesity medications that have received US Food and Drug Administration (FDA) approval: orlistat, phentermine, phentermine/topiramate extended release (ER), lorcaserin, naltrexone sustained release (SR)/bupropion SR, and liraglutide (the only injectable formulation). At the same time, the European Medicines Agency (EMA) has approved only three of these: orlistat, bupropion/naltrexone and liraglutide.

 

Considering the extent to which obesity impairs health alone or through expression of one or more of these comorbidities, the need for new molecular pharmaceutic agents is crucial. As detailed below, future weight-loss medications will be based on our knowledge of key regulatory sites of weight regulation and energy homeostasis so as to achieve greater efficacy while minimizing off-target side effects, characteristics that are necessary for approval by both American and European drug regulatory agencies.

 

TARGETS OF PHARMACOTHERAPY IN THE MANAGEMENT OF OBESITY

 

Novel insights provided by pathophysiology indicate the presence of a complex homeostatic system in which information about the energy reserve status and the meal quality and content is relayed from the periphery (gastrointestinal tract, pancreas, and adipose tissue) via specific orexigenic and anorexigenic peptides and hormones to the central nervous system (CNS). Peripheral peptide hormones are released postprandially and travel in the circulation to bind to their receptors in the homeostatic regulatory centers in the CNS, notably the arcuate nucleus (ARC) of the hypothalamus and the dorsal vagal complex (DVC) in the brainstem medulla. The ARC contains neurons expressing key orexigenic neurotransmitters, agouti-related peptide (AgRP) and neuropeptide Y (NPY), as well as anorexigenic neurotransmitters, proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART). Food intake is thus modulated by complementary mechanisms so as to maintain energy and weight homeostasis. New drug therapies have begun to focus on combination therapy using medications that target more than one of these central pathways, thereby achieving more favorable weight loss outcomes. In addition, combining treatments may provide a better safety profile given that lower doses of each drug when used together may achieve better weight loss than higher doses of a single agent (see Figure 1 below).

 

Factors That Influence Appetite

 

The regulation of satiety and appetite depends on the interaction of three major factors: biological systems, modern macro-environmental exposures, and micro-environmental influences. Biological systems are shaped by genetic and epigenetic influences from early-life events that govern development of orexigenic and anorexigenic neuro-hormonal pathways involved in the pathophysiology of obesity. Modern macroenvironment (food production, consumption, availability, social structure, weather influencing physical activity, television and technology, cultural norms, endocrine disruptors) and microenvironment (nutrition, exercise, sleep, stressful lifestyle, circadian rhythm) play an important role in the conformational development of cognitive and emotional brain regions, thus predisposing to the obese phenotype.

 

Genetic Factors of Physical Activity

 

Specific genes predict to what extent adults remain active. This is evidenced in a study examining identical twins in which environmental factors shared by children at age 13 accounted for 78% to 84% of sport participation, whereas genetic differences provided no contribution at all. At the age of 17 to 18 the genetic influences represented 36% of the variance in the level of participation in sports, and by age 18 to 20, genetic factors were responsible for almost all (85%) of the differences in participation in sports.

Figure 1. Sites of Action of the Most Important Anti-Obesity Drugs

CENTRALLY-ACTING ANTI-OBESITY DRUGS

 

Monoamine Neurotransmitter Modulators

 

With the exception of the glucagon-like peptide 1 (GLP-1) receptor agonist liraglutide, currently available weight loss medications act on the central nervous system to enhance dopamine, norepinephrine, and serotonin action to enhance satiety, diminish hunger, and consequently affect weight loss. Drug combinations have opened new horizons as they use multiple neural pathways, leading to better results with less adverse events. Recently, a review of fifty reports involving 43,443 subjects compared the efficacy of the central acting anti-obesity drugs lorcaserin (5HT2c receptor agonist), naltrexone-bupropion (opioid receptor antagonist combined with a norepinephrine releasing agent that stimulates POMC neuronal firing), phentermine-topiramate (a norepinephrine and dopamine modulator plus a carbonate anhydrase inhibitor), and liraglutide. It was found that the maximal mean weight loss relative to placebo was -3.06, -6.15, -7.45, and -5.5kg after 1 year with mean weight regain +0.48kg, +0.91kg, +1.27kg, +0.43kg the following year, respectively.  In these studies, the one-year drop-out rate was 40.9%, 49.1%, 34.9%, 24.3%, respectively (5).

 

Leptin, Leptin Analogues and Leptin Sensitizers

 

Leptin is a protein secreted primarily by white adipose tissue (WAT). It directly stimulates anorexigenic POMC neurons and inhibits adjacent orexigenic NPY neurons in the ARC of the hypothalamus, thus promoting satiety, increasing energy expenditure, and resulting in weight loss (6). Circulating levels of leptin increase with adiposity and decline following body weight reduction; the latter might be implicated in the total and resting energy expenditure reduction seen after weight loss. The discovery of leptin in 1994 was a seminal event in obesity research. It helped to establish that body weight should be viewed as a disorder with a strong biological basis rather than simply the result of poor lifestyle choices. Studies with congenitally leptin-deficient, severely obese subjects revealed that administration of physiological doses of leptin decreased food intake and body weight (7). Obese individuals, however, are leptin-resistant and have increased circulating leptin levels. Whether administration of leptin could overcome leptin resistance and exert an anti-obesity effect was tested in a placebo-controlled study with 47 obese men and women given varying doses of recombinant human leptin (0.03 mg/kg and 0.30 mg/kg, respectively) for 24 weeks and advised to eat 500 kcal less than body requirements each day. A dose-dependent decrease in body weight was shown, ranging from -1.3 kg in the placebo group to -1.4 kg in the 0.03 mg/kg leptin-treated group, and to -7.1 kg in the 0.30 mg/kg leptin-treated group (8). These results suggested that leptin resistance can be overcome with high doses of leptin but resulting in only modest weight loss similar to currently approved medications.  In addition, whether these effects can be sustained long-term is not known. Reports were similar from animal studies testing the effect of leptin sensitizers targeting the protein tyrosine phosphatase-1B (PTP1B)(9)(10) or the chemical chaperones that repair ER stress, including 4-phenyl butyric acid (PBA) and tauroursodeoxycholic acid (TUDCA) (11), each of which demonstrated reduced food intake and body weight. Like leptin treatment, sustainability of these anti-obesity effects is still not clear.

 

Weight loss is associated with reduction in energy expenditure, which makes long term weight loss maintenance difficult (12). Furthermore, 6 days of high fat diet in mice suffice to dramatically decrease the levels of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in the arcuate nucleus (13) while short term overfeeding of normal weight mice can lead to an increase of leptin resistance (14). Besides the inefficiency of leptin analogues as monotherapy, combinations of leptin with amylin (15), fibroblast growth factor 21(FGF21), exendin4, (16), or a GLP-1/glucagon co-agonist (17) were proposed. Only the combination with the GLP-1/glucagon co-agonist has shown improvement of leptin sensitivity (18). Apart from diet, stress of endoplasmic reticulum contributes to leptin resistance (19). Several plant-derived substances, such as celastrol (20) and withaferin (21) have been tested in diet-induced obese rodents for improvement of this pathway that leads to leptin resistance.

 

METRELEPTIN

 

Metreleptin (MYALEPT) is an injectable human recombinant leptin analogue approved in Japan for metabolic disorders including lipodystrophy and in USA as first-line treatment for non-HIV related forms of generalized lipodystrophy (leptin deficiency, congenital/acquired lipodystrophy) (22). A previous indication for hypothalamic amenorrhea has been withdrawn (23). (see Table 1)

 

Table 1. Metreleptin (MYALEPT)

FDA approved/Phase

Approved in Japan for lipodystrophy disorders and in USA for non-HIV lipodystrophy

Mechanism of action

Human recombinant leptin injectable analogue

Clinical Benefits

↓blood glucose, triglycerides, hepatic fatty steatosis

Adverse events

Headache, hypoglycemia, decreased weight, abdominal pain

-previous indication for hypothalamic amenorrhea discontinued

 

PRAMLINTIDE/METRELEPTIN

 

The combination of amylin-leptin (pramlintide-metreleptin) has been shown to be effective in the treatment of obesity. The anti-obesity properties of the combined treatment with pramlintide and metreleptin (pramlintide/metreleptin) were tested and showed a significant weight reduction of 12.7 ± 0.9% (11.5 ± 0.9 kg) without plateauing in obese patients during a 20-week trial period (24). The sponsors subsequently announced positive results from a 28-week proof-of-concept study with pramlintide and metreleptin combination treatment in overweight or obese subjects. The combination treatment reduced body weight on average by 12.7%, significantly more than treatment with pramlintide alone (8.4%), which is interpreted as 10 pounds more weight loss with the combined treatment. Remarkably, subjects receiving pramlintide/metreleptin continued to lose weight until the end of the study, compared to those treated with pramlintide alone, whose weight loss had stabilized towards the end of the study. The magnitude of weight loss was found to be dose-dependent and baseline BMI-dependent. Patients with a starting BMI less than 35 kg/m2 experienced the best weight loss efficacy with the combined treatment. A year later, the results of the 52-week blinded, placebo-controlled Phase II extension study of pramlintide/metreleptin were announced. The results indicated sustained and robust weight loss through the combined treatment; again, the most robust efficacy was seen in patients with a BMI less than 35 kg/m2 (25). Although the pramlintide/metreleptin combination seemed to be the next promising anti-obesity drug to be marketed, the sponsors discontinued its development in 2011, following commercial reassessment of the program (26).

 

Melanocortin-4 Receptor Agonists

 

The melanocortin system has a highly significant role in the hypothalamic regulation of body weight and energy expenditure. Leptin inhibits the release of the orexigenic neuropeptides orexin and melanocortin-concentrating hormone (MCH) in the lateral hypothalamic area (LHA) through the release of CART and melanocyte-stimulating hormone (α-MSH). The latter derives from the cleavage of POMC by prohormone convertase-1 and acts via melanocortin-3 and -4 receptors (MC3R, MC4R) activation. α-MSH emerged as a promising novel anti-obesity drug, and intranasal administration of the melanocortin sequence MSH/ACTH4-10 to normal-weight subjects was shown to acutely increase subcutaneous WAT lipolysis (27) and decrease body fat by 1.7 kg, when administered for six weeks (28). It eventually proved not to induce any significant reduction in body weight or body fat when compared with placebo in a 12-week study of 23 overweight men.

 

In preclinical studies, obese primates treated for eight weeks with the MC4R agonist RM-493 (Setmelanotide) lost an average of 13.5% of their body weight, with significant improvements in both insulin sensitivity and cardiovascular function. In June 2014, the results from the first human Phase II trial were released, testing the hypothesis that an MC4R agonist increases resting energy expenditure in obese subjects. A total of 12 obese but otherwise healthy individuals were randomized and completed both RM-493 and placebo periods in this double-blind, placebo-controlled, two-period crossover study. Analysis of the data indicates that short-term treatment with RM-493 increased resting energy expenditure significantly (by 6.4% vs placebo), thus suggesting RM-493 may be clinically effective for treating obesity. In 2015, administration of Setmelanotide to obese individuals for a limited time increased resting energy expenditure (REE) by 6.4% and shifted substrate oxidation to fat (29). Currently, Setmelanotide is being tested as a therapeutic option for rare genetic disorders of obesity such as POMC deficiency, heterozygous deficiency obesity, and POMC epigenetic disorders (30-32). (see Table 2)

 

Table 2. Setmelanotide (RM-493)

FDA approved/Phase

Phase II

Mechanism of action

MC4R-agonist

Weight loss vs placebo

13.5%

Clinical Benefits

↑insulin sensitivity, cardiovascular function, energy expenditure, ↓ body weight

-tested for POMC deficiency, heterogenous deficiency obesity, POMC epigenetic disorders

Adverse events

Headache, arthralgia, nausea, spontaneous penile erection, female genital sensitivity

 

Melanin-Concentrating Hormone (MCH) Antagonists

 

The melanocortin-concentrating hormone (MCH) is an important orexigenic neuropeptide in the LHA. Its release is stimulated by NPY and inhibited by leptin, exerting its orexigenic effects through the MCH1 receptor (MCHR1) (33). Like NPY, MCH exerts pleiotropic effects on locomotor activity, sensory processing, anxiety, aggression, and learning. Thus, despite the role of MCH in hunger stimulation, MCHR1 blockade as an anti-obesity target is questionable because such inhibition could elicit undesirable side effects. In animal models, MCH antagonists have consistently demonstrated efficacy in reducing food intake acutely and in inhibiting body weight gain when given chronically (34). Five compounds have reached testing in human subjects. Although they were reported as well-tolerated, none has proceeded to Phase II studies. A major issue with many lead compounds is increased cardiovascular risk due to drug-induced QTc prolongation (35). Among others, the MCHR1 antagonist AMG 076 entered Phase I safety and tolerability testing in 2004, but there have been no subsequent reports of its status since 2005. The MCHR1 antagonist GW-856464 also entered Phase I studies in 2004; however, in 2010 it was reported that low bioavailability precluded further development. The MCHR1 antagonist NGD-4715 was safe and well-tolerated in a Phase I clinical trial, but its development ceased in 2013. Similarly, despite the reported tolerability and indication of efficacy of the MCHR1 antagonist ALB-127158, its development was terminated before the initiation of Phase II studies. Finally, the longest (28-day) Phase I study with BMS-830216, a pharmacological antagonist of MCH signaling (36) produced no indications of weight loss or reduced food intake and the compound did not proceed to Phase II studies.

 

Subtype-Selective Serotonin-Receptor Agonists

 

Central serotonin participates in feeding behavior and energy balance modulation, reducing food intake in animals and human beings.  This finding was supported by reports of two selective serotonin reuptake inhibitors (SSRIs) developed to treat depression, fluoxetine and sertraline, being associated with non-sustained weight loss in obese subjects. Thus, agonists to appropriate serotonin receptors are potentially valuable drugs. The serotonin (5-HT) system directly modulates the hypothalamic POMC (anorexigenic) and NPY (orexigenic) networks, enhancing satiety and causing hypophagia. These effects are mediated by 5-HT2C and 5-HT1B receptors, located on hypothalamic POMC and NPY neurons, respectively. Through the 5-HT1B receptors, serotonin inhibits the NPY/Agrp neurons, thereby decreasing the GABAergic inhibitory input to POMC cells; while through the 5-HT2C receptors it directly activates the anorexigenic POMC neurons. Via these actions, serotonin increases α-MSH and decreases AgRP release into the hypothalamic melanocortin system, promoting satiety. Between 1973 and 2000 there was an explosion in the pharmaceutic industry regarding central acting anti-obesity drugs. Three non-selective serotonin-receptor agonists were approved by FDA: fenfluramine (1973-1997), the combination phentermine-fenfluramine (1992-1997), and dexfenfluramine (1996-1997). These were all 5-HT1b agonists characterized for their ability to inhibit food consumption, but also had effects on other serotonin receptors that lead to unacceptable side effects (cardiac valvular thickening) and were voluntarily withdrawn from the market.

 

In 1997, when fenfluramine and dexfenfluramine were discontinued by the manufacturer, sibutramine, a serotonin and norepinephrine reuptake inhibitor emerged. Sibutramine has only little clinical relevance as an antidepressant but enhances weight loss due to an increase in energy expenditure and inhibition of food intake (37). In addition to weight loss, sibutramine was found to improve fasting levels of insulin, triglycerides, and high-density lipoprotein cholesterol. Sibutramine was also associated with increase of blood pressure, cardiovascular events, and cardiac arrhythmias (38). For these reasons, FDA withdrew it in 2010.

 

LORCASERIN

 

As activation of the 5-HT1B receptor has been implicated in both primary pulmonary hypertension (39) and valvopathy (40), the 5-HT2C receptor subtype has been proposed as a target for therapeutic intervention to allow weight loss. Several potent and selective 5-HT2Creceptor agonists proved to be effective in suppressing food intake and inducing weight loss in rodents, including WAY-163909 (41), CP-809101 (42), and vabicaserin (43). However, only lorcaserin (APD356) moved into clinical testing. Lorcaserin (Belviq) is a selective 5-HT2c receptor agonist, which belongs in the third generation of 5-HT-based anti-obesity drugs (44). It activates hypothalamic POMC neurons to induce satiety and decrease food intake but does not affect energy expenditure. Through actions on midbrain dopaminergic tone, it has been shown to suppress binge- food behaviors. Its action in addictive disorders is currently under investigation (45). Based on the outcome of the BLOOM (46) and BLOSSOM trials (47), in 2012 the FDA approved lorcaserin as an addition to a reduced-calorie diet and exercise for eligible patients (48). The efficacy of lorcaserin appears similar to that of orlistat (mean difference in weight loss between active and placebo treated groups approximately 3 to 4 kg) and perhaps slightly less than that of phentermine-topiramate. The impact of lorcaserin in patients with T2DM and BMI: 27-45kg/m2 was examined in the BLOOM-DM trial which showed a reduction of body weight by approximately 5kg versus 1.6kg in the placebo group, as well as significant decreases in heart rate, HDL levels, and waist circumference. Valvopathy was shown not to occur in excess with treatment and lorcaserin was generally well tolerated, with a low incidence of side effects such as headache, dizziness, fatigue, nausea. After the results of BLOOM-DM trial, a potential combination of GLP-1RA and 5-HT2A/C is now under investigation (49).

 

In a multicenter, randomized, double-blind, placebo-controlled, parallel-group study involving12,000 overweight and obese patients with cardiovascular disease or multiple cardiovascular risk factors (CAMELLIA-TIMI 61), the effect of long-term treatment with lorcaserin on major cardiovascular events and conversion to T2DM over a 5-year period were examined. After one year of treatment, 5% weight loss was observed in 38.7% and 17.4% in the lorcaserin and the placebo groups, respectively. Regarding cardiac risk, the lorcaserin group was non-inferior to the placebo group with slightly better values in cardiac risk factors (blood pressure, heart rate, glycemic control, lipid profile). Adverse events were rare in both groups, apart from the incidence of serious hypoglycemia in the lorcaserin group in those with diabetes managed using insulin or sulfonylureas (50, 51). In addition, lorcaserin administration decreased the incidence of T2DM by 19% in patients with prediabetes and by 23% in patients without diabetes. In patients with T2DM, lorcaserin resulted in a reduction of 0.33% in HbA1c compared with placebo at 1 year from a mean baseline of 7.0%. (see Table 3, 4)

 

Table 3. Lorcaserin (Belviq)

FDA approved/Phase

2012

Mechanism of action

Selective Serotonin 2C agonist

Weight loss vs placebo

3-4kg

Clinical Benefits

↓food intake, heart rate, HDL levels, waist circumference, HbA1c

Adverse events

Headache, dizziness, fatigue, nausea, dry mouth, constipation, heart valvopathy

-In diabetics: hypoglycemia, headache, back pain, cough, fatigue, risk of serotonin syndrome/neuroleptic malignant syndrome, valvular heart disease

 

 

Table 4. Clinical Trials of Lorcaserin

Clinical trial

Patients

Dose

Treatment, placebo from baseline

% of patients losing ≥5% of baseline weight

Comment

 

BLOSSOM

1-year randomized, double-blind, placebo-controlled trial

(2011)

4008 patients (18-65 y.o., BMI- 30-45kg/m2 or 27-29.9kg/m2 with comorbidity) randomized in a 2:1:2 ratio

i.10mg x2 po

 

ii.10mg x1 po

 

iii.placebo

i.-5.8kg

 

 

ii.-4.7kg

 

 

iii.-2.9kg

i.47.2%

 

 

ii.40.2%

 

 

iii.25%

Exclusion criteria: recent cardiovascular events, diabetes mellitus, BP >150/95mmHg

BLOOM

2-year randomized, double-blind, placebo-controlled trial

(2010)

 

3182 adults (mean BMI-36.2kg/m2) randomized to lorcaserin twice daily or placebo group. After 52 weeks, the placebo group continued placebo and lorcaserin group selected placebo or lorcaserin for 52 weeks

i.10mg x2 po

 

ii. placebo

i.-5.8kg

 

 

ii.-2.2kg

i.47.5%

 

 

ii.20.3%

Weight loss was greater in the group which continued lorcaserin for the second year

BLOOM-DM

1-year randomized, double-blind, placebo-controlled trial

(2012)

604 patients (HbA1c: 7-10%, BMI-27-45kg/m2, treatment with metformin, sulfonylurea or both)

i.10mg x2 po

 

ii.10mg x1 po

 

iii.placebo

i.-4.7kg

 

 

ii.-5.0kg

 

 

iii.-1.6kg

i.37.5%

 

 

ii.44.7%

 

 

iii.16.1%

↓heart rate, HDL levels, waist circumference in lorcaserin treated groups

NO valvopathy was statistically significant

CAMELLIA-TIMI 61

3.3-year randomized, placebo-controlled trial

(2018)

 

12,000 patients overweight/obese-three subgroups

A. diabetes

B. prediabetes

C. normoglycemic

i.10mg x2/day

 

ii. placebo

At 1 year the mean treatment difference:

 A: -2.6kg

 B: -2.8kg

 C: -3.3kg

 

 

At 1 year compared with placebo:

A: 37.4%

B: 39.7%

C: 42.3%

↓ BMI, waist circumference, waist-to-hip ratio, HbA1c, reduced microvascular complications

 

Bupropion

 

Bupropion is a dopamine and norepinephrine-reuptake inhibitor that has been marketed as an anti-depressant and for smoking cessation. Previous animal studies have clearly shown a dose-dependent satiety effect of bupropion following intraperitoneal injection (52). The acute effects of dopamine and noradrenaline reuptake inhibition on energy homeostasis demonstrated their additive effects on short-term food intake (53). Bupropion increases dopamine activity and POMC neuronal activation, thereby reducing appetite and increasing energy expenditure (54). Whether the acute meal terminating effects of bupropion documented in animal studies could be translated into long-term weight loss efficacy in humans was addressed by three clinical trials with overweight and obese adults (55, 56, 57) using different treatment doses (100 to 400 mg/d) and duration (up to 24 weeks). They have all shown bupropion to have dose-dependent modest weight reducing efficacy, plus a safe profile. One study that assessed the anti-obesity efficacy of bupropion over two years reported maintenance of weight loss during the continuation phase, while another demonstrated its efficacy even in depressed patients. Although the weight loss effect of bupropion was superior in non-depressed patients compared to those suffering from depression, the fact that bupropion was well-tolerated and effective in this group of patients provides a potential valuable adjunctive therapy to elevate mood in depressed subjects in whom weight gain secondary to antidepressant therapy is an issue. Cardiovascular effects, such as a rise in blood pressure and tachycardia, were usually mild, while the risk of seizure, which was high with the original bupropion formulation, has been significantly reduced with the advent of bupropion-SR and bupropion-ER.

 

An interesting finding of the previous studies was that the rather modest weight loss effect of bupropion reached a plateau by 24 weeks of treatment. This could be explained by the molecular pathophysiology of the weight reducing effects of bupropion, which directly stimulates the hypothalamic POMC neurons that in turn release α-MSH and β-endorphin. α-MSH mediates the anorectic effect of POMC activation, whereas β-endorphin exerts negative feedback on POMC neurons via opioid receptors (58). The latter possibly points to one of the compensatory mechanisms that limits long-term efficacy of bupropion and other weight loss modalities.

 

Naltrexone

 

Naltrexone is an opioid receptor antagonist. By blocking opioid receptors on the POMC neurons, feedback inhibition is prevented further increasing POMC activity. Monotherapy with opioid antagonists to decrease short-term food intake has been tested (59). Naltrexone failed to produce consistent or clinically meaningful weight loss, even at large doses (300 mg/d) (60), implying that a single opioid mechanism is unlikely to explain all aspects of ingestive behavior.

 

Bupropion/Naltrexone Sustained Release (SR)

 

The combined bupropion/naltrexone (NB) therapy induced significantly greater weight loss on a diet and exercise program over 56 weeks compared to monotherapy and placebo (61). In 2014, the FDA approved this combination (Contrave, Mysimba) for body weight management in adults who are overweight and obese. This combined therapy of opioid antagonist and aminoketone antidepressant is titrated over four weeks to the maximum dose. NB has shown remarkable benefit in patients with binge-eating disorder (BED) and concomitant alcohol abuse, but this result needs further evaluation (62). Four major 56-week phase III randomized, double-blind, placebo-controlled trials have shown the therapeutic effect of ΝΒ SR (COR-I, COR-II, COR-BMOD, COR-DIABETES) in different dosage combinations (see Table 6). In COR-I, the weight loss ratio on NB 16/360mg, NB 32/360mg or placebo was -5.0%, -6.1%, -1.3% (P<0.00) respectively. In COR-II, the weight loss ratio on NB 32/360mg or placebo was -6.4%, -1.2% (P<0.001) (63). In COR-BMOD, NB SR 32/360mg plus intensive behavioral modification was compared with the behavioral modification alone as a therapeutic option. The weight loss ratio was -11.5% versus -7.3% (P<0.001), respectively (64). Recently, COR-Diabetes has included patients with T2DM with or without antidiabetic treatment. The NB SR 32/360mg treatment resulted in -5.1% weight loss versus -1.8% in the placebo group (P<0.001). NB treatment resulted in a HbA1c reduction, cardiovascular benefit, and lipid profile improvement (65). Due to FDA request for further investigation of the effect of NB on major cardiovascular events, the LIGHT trial was created. Unfortunately, this trial terminated early following recommendation by the academic leadership of the study because confidential interim data were publicly released by the sponsor (66). (See Table 5, 6)

 

Table 5. Bupropion/Naltrexone Sustained Release (Contrave, Mysimba+

FDA approved/Phase

2014

Mechanism of action

Aminoketone antidepressant/Opioid antagonist

Weight loss vs placebo

4.8kg

Clinical Benefits

↓ appetite

Adverse events

Nausea, constipation, headache, vomiting, dizziness, insomnia, dry mouth, suicidal ideation, increase blood pressure/heart rate, hepatotoxicity, angle-closure glaucoma Uncontrolled hypertension, seizures, anorexia nervosa/bulimia, chronic opioid use, coadministration with MAO inhibitors

 

Table 6: Clinical trials of Naltrexone/Bupropion SR

Clinical trial

Patients

Dose

Treatment, placebo from baseline

% of patients losing ≥5% of baseline weight

Comment

 

COR I

1-year randomized, double-blind, placebo-controlled trial

(2010)

1742 patients randomly categorized in a 1:1:1 ratio

i.16/360mg po

 

ii.32/360mg po

 

iii. placebo

i.-5.0%

 

 

ii.-6.1%

 

 

iii.-1.3%

 

i.39%

 

 

ii.48%

 

 

iii.16%

 

COR II

1-year randomized, double-blind, placebo-controlled trial

(2013)

1496 patients randomly categorized in a 2:1 ratio to NB 32/360mg or placebo; patients on NB with <5% weight loss in 28-44 week were reassigned to continue 32/360mg or increase daily dose to NB 48/360mg

i.32/360mg (or increased daily dose 48/360mg)

 

ii.placebo

i.-6.4%

 

 

 

 

 

ii.-1.2%

 

i.50.5%

 

 

 

 

 

ii.17.1%

Random reassignment to higher dose did not change weight loss results

COR-BMOD

1-year randomized, double-blind, placebo-controlled trial

(2011)

793 patients with obesity randomly categorized in a 1:3 ratio

i. BMOD+ NB (32/350mg)

 

ii. BMOD+ placebo

i.-11.5%

 

 

 

ii.-7.3%

i.66.4%

 

 

 

ii.42.5%

The efficacy of NB is obvious, and a lifestyle change can increase weight loss

COR-DIABETES

1-year randomized, double-blind, placebo-controlled trial

(2013)

505 patients overweight/obese and T2DM with/without oral anti-hypoglycemic agents randomly categorized in a 2:1 ratio

i.32/360mg

 

ii. placebo

i.-5.0%

 

ii.-1.8%

i.44.5%

ii.18.9%

↓HbA1c, certain improvements in CVD risk factors.

↑ nausea, constipation, vomiting

 

 

Zonisamide

 

Given the pathophysiology behind the anti-obesity efficacy of the selective serotonin-receptor agonists and the dopamine-reuptake inhibitors, an ideal drug would combine serotonergic and dopaminergic activity. This is exactly the case of Zonisamide, a marketed antiepileptic drug that exerts dose-dependent biphasic dopaminergic (67) and serotonergic (68) activity. Its weight loss efficacy was investigated by a double-blind, placebo-controlled trial which reported a 32-week mean weight loss of 9.2 kg (1.7 kg) (9.4% loss) for the Zonisamide group (dose administered up to 600 mg/d) compared with 1.5 kg (0.7 kg) (1.8% loss) for the placebo group (P<0.001); Zonisamide was generally well-tolerated with only a few adverse effects (69). The findings were similar when the long-term effectiveness and tolerability of Zonisamide for weight control was examined in psychiatric outpatients using various psychotropic medications; the mean BMI reduction achieved was 0.8±1.7 kg/m2 and ranged from -2.9 kg/m2 to 4.7 kg/m2 (p<0.001), while the drug was generally safe and well-tolerated (70). Zonisamide was also assessed in the treatment of binge-eating (BE) disorder where it proved to be effective in reducing binge-eating frequency, severity of illness, and weight; however, the reports regarding its tolerability were conflicting (71). (see Table 7).

 

Table 7. Zonisamide

Mechanism of action

Selective serotonin-receptor agonist and dopamine-reuptake inhibitor

Weight loss vs placebo

7.8kg

Clinical Benefits

Assess in the treatment of binge-eating disorder

Adverse events

Nausea, headache, insomnia

 

Zonisamide/Bupropion SR

 

Whether the anti-obesity efficacy of Zonisamide is increased when combined with bupropion (dopamine and norepinephrine -reuptake inhibitor) has been evaluated in a few Phase II clinical trials with different combined doses; the bupropion SR/Zonisamide SR combination is marketed under the trade name Empatic. In its 24-week, double-blind, placebo-controlled Phase IIb trial (72), patients completing 24 weeks of bupropion SR 360 mg/Zonisamide SR 360 mg therapy lost 9.9% of their baseline body weight, or 22 pounds, compared to 1.7% for placebo patients (p<0.001). Of patients who completed 24 weeks of therapy, 82.6% lost at least 5% of their baseline body weight and 47.7% lost at least 10% of their baseline body weight compared to 18.9% and 5.7% of placebo patients, respectively (p<0.001 for both). Patients experienced significant weight loss as early as by their first post-baseline visit at week four. Importantly, patients continued to lose weight until the end of the trial period with no evidence of a weight loss plateau. Early results showed that patients lost an average of 14% over 48 weeks. Improvements were observed in key markers of cardiometabolic risk such as waist circumference, triglycerides, fasting insulin, and blood pressure. The most commonly reported adverse events for all patients were headache, insomnia, and nausea. The most common adverse events leading to discontinuation were insomnia, headache, and urticaria (hives). There were no serious adverse events attributed by investigators to the study drug. There were no statistically or clinically meaningful differences between the drug and placebo on measures of cognitive function, depression, suicidality or anxiety. These reports revealed a significant weight-reduction effect for the combination Bupropion/Zonisamide. However, the safety concerns (73) will need to be addressed in the upcoming Phase III studies before firm conclusions about its safety profile can be drawn. (see Table 8)

 

Table 8. Zonisamide/Bupropion (Empatic)

FDA approved/Phase

Phase II completed

Mechanism of action

Selective serotonin-receptor agonist and dopamine-reuptake inhibitor/dopamine and norepinephrine reuptake inhibitor

Weight loss vs placebo

9.9% of their baseline weight

Clinical Benefits

↓cardiometabolic risk

Adverse events

Headache, insomnia, nausea, urticaria

 

Topiramate

 

Topiramate is another anticonvulsant agent associated with weight loss. It is a sulphamate-substituted fructose that is approved as an antiepileptic/antimigraine agent and has multiple effects on the CNS, including action on the orexigenic GABA systems causing appetite suppression (74). A 6-month dose-ranging study in obese human subjects addressing its anti-obesity efficacy at doses of 64, 96, 192, and 384 mg/day (in divided twice-daily dosing) concluded that all doses produced significantly greater weight loss compared to placebo, and that weight loss in the 192 mg/day group was similar to the 384 mg/day group (75). This is important as topiramate has been associated with several neuropsychiatric effects, especially when administered at high doses (of 192 mg/d or more). Another study investigating the weight loss efficacy and safety of topiramate doses of 96, 192, and 256 mg/day over a 1-year period in obese subjects using the immediate release form tablets (before the development of the controlled-release formulation). Clinically significant weight loss (7.0, 9.1, and 9.7% of their baseline body weight for the doses of 96, 192, and 256 mg/day, respectively), was reported compared to 1.7% body weight loss in the placebo group (P<0.001) plus improvements in blood pressure and glucose tolerance (76). Finally, several other studies investigated the therapeutic effect of topiramate in patients with BED and bulimia (77) that are both associated with obesity; the results were very promising regarding control of symptoms in both disorders. (see Table 9)

 

Table 9. Topiramate

FDA approved/Phase

Phase II completed

Mechanism of action

Sulphamate-substituted fructose acts on GABA system

Weight loss vs placebo

7.0%(96mg),9.1%(192mg), 9.7% (256mg/day)

Clinical Benefits

Assess in the treatment of binge-eating, bulimia

Adverse events

Headache, insomnia, nausea, urticaria

 

Phentermine

 

Phentermine is a sympathomimetic amine, which has anorexigenic action, that also releases insignificant quantities of dopamine. Thus, it is characterized by lower abuse potential (78). Its main mechanism of action involves catecholamine release in the hypothalamus resulting in enhanced satiety feeling and reduction of food intake (79). The most common side effects of phentermine as a sympathomimetic drug is heart rate increase, hypertension, dizziness, dry mouth, insomnia, irritability, and gastrointestinal disorders (80). Phentermine was the first FDA approved anti-obesity drug in 1959 for those aged >16 years old, but for only short-term use (maximum 3 months). The reason for the time limit is because the pharmaceutic industry had not updated labeling since 1959. In 1968, in a double-blind, placebo-controlled trial, 108 overweight or obese women were categorized into three groups that received continuously or intermittently (every 4 weeks) dosed phentermine or placebo, respectively. The weight loss was -12.2kg, -13.0kg or -4.8kg, respectively (81).

 

Currently, the off-label long-term use of phentermine is indicated only if there is clinical benefit, stable blood pressure and pulse rate in the absence of cardiovascular history or substance abuse disorders. In a recently published retrospective cohort study, 13,972 patients were observed for 6, 12 and 24 months after phentermine initiation. They were categorized in five groups based on the time of phentermine administration: short-term use, short-term intermittent use, medium-term continuous use, medium-term intermittent use, long-term continuous use. Weight-loss, changes in blood pressure, heart rate, and incidence of cardiovascular events (myocardial infarction, stroke, angina, coronary artery bypass grafting, carotid artery intervention, death) were examined. Weight loss was greater among off-label groups than referent group of short-term use with results depending on the duration of phentermine initiation. Specifically, at six months, short-term intermittent patients lost 1.8% further body weight relative to short-term single patients and medium-term intermittent patients lost 5.6% further body weight relative to short-term single patients. At twelve months, the medium-term intermittent use group lost further 5.6% body weight relatively to the short-term use group. At twenty-four months, long-term the continuous use group lost 7.4% additional body weight in comparison with the short-term use group. Concerning safety of phentermine, changes in heart rate and diastolic blood pressure were insignificant at six, twelve, and twenty-four months. Interestingly, although the referent group showed a slight increase in systolic blood pressure (+0.5-3.2 mmHg) at twenty-four months, all groups had slightly lower systolic blood pressure than the referent group at twelve- and twenty-four-months follow-up period. Lastly, the incidence of major cardiovascular events was low. So, it was shown that the off-label over three months therapy with phentermine was superior to short–term administration with greater weight-loss effect and cardiovascular safety. More studies with fewer limitations should follow in order to support these findings (82). In 2013, a clinical trial comparing phentermine as monotherapy or as part of a combined therapy, took place and resulted in a weight loss of 5.1% at 28 weeks follow-up period in favor of the combined phentermine/topiramate group.(see Table 10)

 

Table 10. Phentermine

FDA approved/Phase

1959

Mechanism of action

Norepinephrine release and minor dopamine release

Weight loss vs placebo

0.23kg/week

Clinical Benefits

Lower abuse potential

Adverse events

Stimulation, insomnia, dry mouth, constipation, primary pulmonary hypertension

Contraindicated in cardiovascular disease, coadministration with MAO inhibitors, hyperthyroidism, glaucoma, drug abuse

 

Phentermine/Topiramate ER

 

Because of dose-related side effects seen with topiramate treatment including suicidality, metabolic acidosis, acute myopia, and secondary angle closure glaucoma, a lower dose of topiramate was used (in a special controlled release formulation) in a novel anti-obesity drug called Qsymia. The main mechanism of action of Phentermine/Topiramate extended release(ER) is possibly the alteration of various neurotransmitters, including inhibition of voltage-dependent sodium channels, glutamate receptors, and carbonic anhydrase as well as potentiation of γ-aminobutyrate activity (83).Two large randomized, double-blind, placebo-controlled trials took place (EQUIP and CONQUER) followed by a 2-year extension trial (SEQUEL). In the EQUIP trial 1,267 patients with BMI>35kg/m2were allocated in two groups and received phentermine/topiramate ER 3.75/23mg and 15/92mg, respectively, once daily. With 59.9% of the patients discontinuing, this trial found no statistically significant difference between the two groups regarding weight reduction (84). In the CONQUER trial 2,487 patients were allocated in three groups and received phentermine/topiramate ER 7.5/46mg, phentermine/topiramate ER 15/92mg, and placebo, respectively. The results were in favor of the combined therapy while the greater dosage resulted in greater weight loss with mean weight loss -7.8kg, -9.8kg, and -1.2kg in the three respective groups (85). Patients who completed the CONQUER trial entered the SEQUEL trial for 52 weeks. The weight loss as percentage of the initial weight was -9.3%, -10.5% and -1.8% in the three respective groups. A statistically significant improvement of lipid profile, glycemic control, and waist circumference in the phentermine/topiramate ER groups was reported (86). Based on the positive results from three Phase III studies, in 2012 FDA approved topiramate/phentermine extended-release as an adjunct to a reduced-calorie diet and increased physical activity for chronic weight management in eligible adults. Meanwhile however, approval was denied by European regulatory authorities, who cited potential risk to the heart and blood vessels, psychiatric side effects, and cognitive side effects in explaining their decision (see Table 11, 12).

 

Table 11. Topiramate/Phentermine Extended Release (ER) (Qsymia)

FDA approved/Phase

2012

Mechanism of action

Norepinephrine release, GABA modulation, voltage-gated ion channel modulation, stop of AMPA/kainite excitatory glutamate receptors and carbonic anhydrase

Weight loss vs placebo

6,6kg

Clinical Benefits

↓ lipid profile, HbA1c, waist circumference

Adverse events

Paresthesia, dizziness, dysgeusia, insomnia, constipation, dry mouth, fetal toxicity, metabolic acidosis, cognitive impairment

Contraindicated in: Glaucoma, hyperthyroidism, coadministration with MAO inhibitors

 

Table 12. Clinical Trials of Phentermine/Topiramate ER

Clinical trial

Administration

N

Treatment, placebo from baseline

% of patients losing ≥5% of baseline weight

Comment

 

CONQUER

Double-blind, placebo-controlled trial over 1 year

(2011)

4-week titration+ 52 weeks of treatment:

15/92mg po

or 7.5/46mg po

2487 patients (BMI:27-45kg/m2 with 2+ risk factors

i.15/92mg

 

ii.7.5/46mg

 

iii. placebo

 

 

 

 

 

 

i.-9.8kg

 

ii.-7.8kg

 

iii.-1.2kg

 

 

 

 

 

 

i.70%

 

ii.62%

 

iii.21%

↑improvement in blood pressure, waist circumference, lipid levels, fasting glucose and insulin

SEQUEL

2-year study overall;1-year extension of CONQUER

(2012)

227 patients completed the original blinded treatment

 

 

i.15/92mg

 

ii.7.5/46mg

 

iii. placebo

i.-10.9kg

 

ii.-9.6kg

 

iii.-2.1kg

i.79.3%

 

ii.75.2%

 

iii.30%

 

 

Neuropeptide Y (NPY) Inhibitors

 

The ARC NPY neurons inhibit the anorexigenic POMC neurons (via NPY Y1 and Y5 receptors) and promote the release of the orexigenic neuropeptides orexin and MCH in the LHA, thus promoting food intake. Therefore, NPY blockade could be a promising target for body weight management. Animal experiments (in mice) have shown that pharmacologic blockade or genetic deletion of the Y1- and Y5-receptors reduces food intake and weight, with Y1-receptor signaling appearing to be the major mediator of the orexigenic effects of NPY. However, NPY is the most abundant central neuropeptide and regulates many functions beyond feeding; thus, targeting NPY neurons/Y receptors specifically for obesity is not easy and could result in unacceptable side effects. In addition, experimental medical blockade of NPY signaling with the Y5-receptor antagonist MK-0577 failed to cause any significant weight loss in a 1-year clinical trial (87). On the other hand, the oral, once-daily, centrally acting selective Y5-receptor antagonist velneperit, previously known as S-2367, induced a mean placebo-adjusted weight loss of 5.0% from initial weight (p <0.0001) over 54 weeks of therapy and was accompanied by improvement of lipid profile and waist circumference reduction (88).Nevertheless, velneperit did not proceed in markets due to disappointing results in phase IIb trials. However, the combined Y1/Y5-receptor antagonism may prove more effective, though we are not aware of any Y1/Y5-receptor antagonist in development to date. In contrast to Y1 and Y5, the Y2- and Y4-receptors are the targets of the satiety hormones PYY and pancreatic polypeptide (PP), respectively, and, as mentioned below, two drugs, a Y2/Y4-receptor agonist (obinepitide and a selective Y4-receptor agonist (TM30339)) are in Phase I/II clinical trials and are yielding results that appear quite promising as regards weight loss. A combined anti-obesity medication of velneperit/orlistat is under way (phase II clinical trial), also with promising results (89).

 

Dopamine antagonists

 

The mesolimbic dopamine system was proven to play a critical role in compulsive overeating or binge eating, which is one of the main reasons why people become overweight or obese. There is some evidence that blocking the action of dopamine in animals can reduce food intake, particularly of foods that are high in fat and sugar. GSK 598809 is a D3 antagonist that blocks dopamine. Preliminary data from human studies failed to show any significant effect on body weight control (90).

 

Tesofensine

 

Tesofensine (TE) is a presynaptic inhibitor of norepinephrine, dopamine, and serotonin. Like sibutramine, it suppresses appetite and may result in significant weight loss, as this was shown when given for the treatment of Parkinson’s disease, but also in a multi-dose, dose-ranging trial where 203 obese patients were randomly assigned to Tesofensine (0.25, 0.5, and 1.0 mg) or placebo once daily. Phase II testing of the drug has been completed. After 24 weeks, mean weight reduction was greater in the Tesofensine groups (-6.7, -11.3, -12.8 kg, for the three doses, respectively) compared with placebo (-2.2 kg). Additionally, an improvement in lipid profile and glycemic control was observed. A dose-dependent increase in blood pressure was observed along with a 7.4bpm increase in pulse rate in the 0.5mg/day group. Adverse events such as headache and mood alterations were also present in all groups especially in the 1mg/day group (91). In another trial, 32 males were allocated in two groups and received 2mg/day Tesofensine and placebo, respectively. The interesting point in this trial was that the patients were free to consume their usual amounts levels of food and exercise as usual. However, in the Tesofensine group they lost 1.8kg over 2 weeks because Tesofensine increased visual analog scale ratings of satiety and 24h fat oxidation in comparison with placebo. Even if an increase in total energy expenditure was not observed, an increase in sleeping energy expenditure was found. Altogether, Tesofensine induces weight loss by promoting the satiety feeling and slightly increasing metabolic rate (92). The effect of Tesofensine in appetite sensations was evaluated in another phase II trial, in which patients were allocated in 4 groups and received 0.25mg, 0.5mg, 1mg and placebo, respectively, for 24 weeks. For the first 12 weeks, a dose-dependent increase in the satiety feeling was noticed even though this feeling faded away as the trial was in progress (93). In 2010, a study on the abuse effect of Tesofensine, bupropion, atomoxetine, and placebo in comparison to d-amphetamine took place and concluded that the studied substances had no abusive action (94). Tesofensine has been shown to increase both blood pressure and pulse rate. In 2018, a phase III clinical trial was powered by the pharmaceutic industry producing Tesofensine. In this study 372 patients were allocated in three groups and received Tesofensine 0.25mg, 0.5mg and placebo. Furthermore, a combination of Tesofensine/metoprolol is recently being examined against hypothalamic injury-induced obesity and Prader-Willi syndrome (95). (see Table 13)

 

Table 13. Tesofensine

FDA approved/Phase

Phase III

Mechanism of action

Triple monoamine reuptake inhibitor of dopamine, norepinephrine, serotonin

Weight loss vs placebo

4.5-10.6%

Clinical Benefits

Pharmacological similar to sibutramine

↓ appetite, body weight, lipid profile, blood glucose

Adverse events

Headache, mood alterations, potentially increase heart rate, blood pressure, psychiatric disorders

.

Lisdexamfetamine dimesylate

 

Another sympathomimetic, Lisdexamfetamine dimesylate, at certain doses appears effective in decreasing binge-eating days in patients with BED compared with placebo, according to a study published online by JAMA Psychiatry (96). The study included 259 and 255 adults with BED in safety and intention-to-treat analyses, respectively. Patients received lisdexamfetamine 30, 50 or 70 mg/day or placebo. BE days per week decreased in the 50 mg and 70 mg groups but not in the 30 mg group compared with placebo. Confirmation of these findings in ongoing clinical trials may result in improved pharmacologic treatment for moderate to severe BED.

 

Cannabinoid-1 Receptor (CB1) Antagonists

 

Among other neurotransmitter systems, the cannabinoid system modulates the hypothalamic melanocortin and NPY feeding networks. It has been shown that administration of cannabinoid-1 receptor (CB1) agonists and antagonists induces hyperphagia and hypophagia, respectively. These observations led to development of rimonabant, a cannabinoid-1 receptor antagonist, for the treatment of obesity, which was shown quite effective in promoting weight loss; however, it increased the incidence of mood-related disorders (97). As a result, in 2009, rimonabant was withdrawn from the market and the development of other cannabinoid-1 receptor antagonists for the treatment of obesity has also been discontinued. Before withdrawal, rimonabant was shown to have advantages in glycemic control and cardiovascular events (98). In 2010, another CB1 antagonist (AM6545) was found to have less psychological side effects and to induce satiety feeling and weight loss in animal studies (99). (see Table 14)

 

Table 14. Cannabinoid Type-1 Receptor Antagonists (SR141716, AM251, AM6545)

Mechanism of action

Antagonism of cannabinoid type-1 receptors stimulates anorexigenic signaling

Clinical Benefits

↓ body weight, blood glucose, cardiovascular events

-AM6545: has limited CNS penetration

Adverse events

Mood alterations

 

Human Chorionic Gonadotropin (hCG)

 

Human chorionic gonadotropin (hCG) in the form of subcutaneous injection and oral or sublingual diet drops has been advertised as aiding in weight loss of one to two pounds daily, absence of hunger, and maintenance of muscle tone. Clinical trials, however, failed to support this claim (100). In fact, FDA recommended avoiding buying over-the-counter weight loss products which contain hCG. One might ask why the hCG diet has so many enthusiastically supporting it. The reason may be that this diet needs to be accompanied by severe calorie restriction, to only 500-800 calories per day. Anyone following such recommendations is bound to lose weight, if only short-term. Most crucially, though, since hCG has been reported to induce serious side effects, this drug should not be used for the treatment of obesity. In addition, very low-calorie diets have not been shown to be superior to conventional diets for long-term weight loss, plus they have risks, such as gallstone formation, irregular heartbeat, and an imbalance of electrolytes. Therefore, if weight loss is the goal, there are safer ways to make it happen.

 

Nesfatin-1

 

Nesfatin-1 is a satiety molecule, which was first described in rats and is derived from its precursor molecule nucleobindin2 (NUCB2) (101). It is expressed both centrally in hypothalamic food intake-regulatory nuclei, the nucleus paraventricular and the arcuate nucleus, and peripherally, in the stomach, pancreas, adipose tissue, and testis. In the gastric oxyntic mucosa, nesfatin-1 is co-expressed with the orexigenic peptide ghrelin in X/A-like cell in rats and humans. The anorexigenic action of nesfatin-1 is based on its ability to cross the blood-brain barrier. It is notable that NUCB2/nesfatin-1 not only decreases food intake, gastric emptying, and small intestine motility, but also reduces glucose and increases insulin levels (102). Intracerebroventricular (icv) injection of full length nesfatin-1 caused a significant reduction of food intake in rats and mice (103). These findings suggest that downstream signaling might be altered, a hypothesis to be further investigated. The fact that nesfatin-1 acts in a leptin-independent way, indicates that it might be a new molecular target in the pharmacotherapy of obesity. The identification of the yet unknown nesfatin-1 receptor will allow the development of nesfatin-1 agonists and antagonists. Whether peripheral nesfatin-1 is primarily involved in the regulation of food intake is questionable and should be further investigated.

 

GASTROINTESTINAL AND PANCREATIC PEPTIDES THAT REGULATE FOOD INTAKE

 

The gut-brain axis plays an important role in food consumption regulation. During food intake, information regarding meal quality and content and short-term alterations in nutrient status is relayed from the gastrointestinal (GI) tract and pancreas to the brain which in turn determines meal size. Apart from feeding, a few satiation signals optimize these processes by influencing gastrointestinal motility and secretion. Several peptides have been identified that mediate this GI system-brain communication including satiety signals such as gastrin releasing peptide (GRP), cholecystokinin (CCK), peptide YY (PYY), glucagon-like peptide-1 (GLP-1), pancreatic polypeptide, glucagon, and amylin, as well as the orexigenic peptide ghrelin. While the anorexigenic peptides are secreted during feeding, ghrelin is secreted before meals and acts to increase hunger and meal initiation. Some of the GI and pancreatic peptides implicated in the regulation of food intake act directly on regions of the brain involved in the regulation of food intake, including the ARC in the hypothalamus and the area postrema, while others act outside of the CNS.  For example, modulating the activity of neurons such as the vagus nerve, which projects to the nucleus of the solitary tract in the brain stem.

 

CCK and CCK1R Agonists

 

CCK is the first described intestinal satiation peptide (104). It is produced by the mucosal I cell (105) of the duodenum and jejunum, and the enteric nervous system, in response to luminal nutrients, especially lipids and proteins. Through endocrine and/or neural mechanisms, CCK regulates numerous GI functions, including satiation, by acting on two CCK-specific receptors: the CCK receptor 1 (CCK1R), expressed mainly in the GI system, and the CCK2R that predominates in the brain. The vagus nerve plays a critical role in CCK-induced satiation as it contains CCK1R, indicating the afferent pathway through which CCK relays satiation signals from the GI to the hindbrain region. Corroborating this hypothesis is the well-documented attenuation of CCK-induced satiation following abdominal subdiaphragmatic vagotomy (106). In addition, CCK inhibits gastric emptying, thereby augmenting gastric distention and mechanoreceptor stimulation, which in turn augments the anorectic effects of CCK (107). Despite the satiety effect of CCK, its potential as an anti-obesity target is questionable. Human studies with intravenously infused CCK carboxy-terminal octapeptide (CCK-8) have shown decreases in meal size and duration in a dose-dependent manner (108). However, the CCK satiating effects were very short-lived, usually not lasting more than 30 minutes, which raises issues as to its importance in long-term body weight regulation. In an animal study, chronic CCK administration with up to 20 peripheral injections per day, although reducing meal size, was associated with increased meal frequency, leaving body weight unaffected (109). Finally, the reports from trials testing CCK1R agonists as potential anti-obesity drugs were disappointing (110). It is currently suggested that there might be a role for CCK in body weight regulation not as a monotherapy but possibly as an adjunctive/synergistic therapy to long-term adiposity signals, such as leptin (111).

 

Glucagon-Like Peptide-1 Analogues

 

The dominant role of GI in satiation (112) is mediated not only by the gastric mechanoreceptors and upper intestinal neuropeptides such as CCK, but also by gut satiation peptides that are secreted from lower-intestine enteroendocrine cells in response to ingested food. They in turn diffuse through interstitial fluids to activate nearby nerve fibers and/or enter the bloodstream to function as hormones and augment the perception of GI fullness by acting in specific parts of the CNS. There is a well-defined duodenal-ileal communication (the ileal brake) via which the proximal intestine informs the distal intestine as to meal quality and content so that the latter modulates/restricts feeding duration, proximal GI motility, and gastric emptying, while it also regulates metabolic responses to nutrient intake. GLP-1 appears to engage such a mechanical and behavioral brake effect on eating and is produced primarily by L cells in the distal small intestine and colon. Along with glucagon and oxyntomodulin, GLP-1 is cleaved from proglucagon, which is expressed in the gut, pancreas, and brain. The GLP-1 equipotent bioactive forms GLP17–36 and GLP17-37 are rapidly inactivated in the circulation by dipeptidyl peptidase-4 (DPP4). Therefore, GLP-1 analogues that have a slightly different molecular structure, but a significantly longer duration of action compared to wild GLP-1 have been used for therapeutic interventions in patients with diabetes, in whom they significantly improved glycemic control, fasting plasma glucose, β-cell function, and probably β-cell regeneration. Currently, the GLP-1 analogues used in clinical practice for diabetes control are exenatide, lixisenatide, dulaglutide, liraglutide, and semaglutide. Beyond the improved glycemic control achieved, clinical studies have also demonstrated anorectic effects and significant weight loss via these agents (113, 114). Although the exact mechanisms by which GLP1 induces anorexia are not yet fully known, it is suggested that vagal and possibly direct central pathways are involved (115). The GLP-1 receptor R (GLP1R) is the principle mediator of the anorectic effects of GLP-1 (116) and is expressed by the gut, pancreas, brainstem, hypothalamus, and vagal-afferent nerves (117).

 

LIRAGLUTIDE

 

Its mechanism of action is both central and peripheral targeting satiety centers of the brain and regulating glucose metabolism. It is the only injectable medication for obesity and is titrated from 0.6mg to 3.0mg over 4 weeks. The most common side effects of liraglutide and generally of GLP1 analogues are gastrointestinal (nausea, diarrhea, constipation, vomiting, dyspepsia, abdominal pain) and rarely pancreatitis. The product has a boxed warning stating that thyroid C-cell tumors have been seen in rodents but the relevance of this in humans is uncertain. The drug should not be used in patients with a personal or family history of medullary thyroid carcinoma (MTC) or in patients with multiple endocrine neoplasia syndrome type 2. Three major trials, SCALE-Obesity, Prediabetes, SCALE-Diabetes, SCALE-Maintenance, have established the therapeutic benefit of liraglutide for weight loss. The SCALE-Obesity, Prediabetes evaluated liraglutide in patients who were overweight and obese but did not have diabetes. The study included 3,731 individuals who were assigned to treatment with liraglutide 3 mg or a placebo. Patients were also counseled on diet and exercise. At the end of the 56-week trial, the liraglutide group lost an average 8% (7.2kg) of their body weight compared to 2.6% (2.8kg) in the placebo group (118); net weight loss was 4.4kg. In the SCALE-Diabetes trial, 846 adults who were overweight or obese and had T2DM were allocated to receive either daily 3.0mg liraglutide or placebo, with mean weight loss -6.0% and -2.0%, respectively (119). In the SCALE-Maintenance trial, 422 adults who were overweight or obese and had lost >5% of initial body weight with a calorie restriction diet were allocated to receive either liraglutide or placebo, respectively, with mean weight loss -6.2% and -0.2%, respectively (120).Recently, Saxenda (liraglutide 3.0mg) has asked for a label update based on the results of the LEADER trial, which studied the effects of the lower dose version of liraglutide (1.8 mg) used to treat diabetes. According to this trial, which examined a population with T2DM and established cardiovascular disease,1.8mg liraglutide daily showed statistically significant reduction of cardiovascular death, of non-fatal myocardial infarction (heart attack), and of non-fatal stroke by 13% versus placebo, when added to standard care. (121) (see Table 15, 16).

 

Table 15. Liraglutide (Saxenda)

FDA approved/Phase

2014

Mechanism of action

Glucagon-like peptide-1 agonist

Weight loss vs placebo

4.4kg

Clinical Benefits

↓cardiovascular death, non-fatal myocardial infarction, non-fatal stroke

Adverse events

Nausea, hypoglycemia (serious if co-administrated with insulin), gastrointestinal disorders, fatigue, dizziness, abdominal pain, increased lipase, acute pancreatitis, acute gallbladder disease, increase heart rate, suicidal ideation, thyroid c-cell tumors seen in mice

Contraindicated in: History of medullary thyroid carcinoma or multiple endocrine neoplasia 2

 

Table 16. Clinical Trials of Liraglutide

Clinical trial

Patients

Dose

Treatment, placebo from baseline

% of patients losing ≥5% of baseline weight

Comment

 

SCALE-Obesity+Prediabetes

1-year randomized, double-blind, placebo-controlled trial

(2015)

3731 patients overweight/obese without DM (61.2% had prediabetes) randomly divided into two groups

i.3.0mg sc once daily

 

ii. placebo

i.-8.4kg

 

 

ii.-2.8kg

i.63.2%

 

 

ii.27.1%

Improvement of body weight, glycemic index, blood pressure, waist circumference

SCALE-Diabetes

1-year randomized, double-blind, placebo-controlled trial

(2015)

846 adults with T2DM overweight/obese

i.3.0mg sc once daily

 

ii. placebo

i.-6.0%

 

 

ii.-2.0%

i.54.2%

 

 

ii.21.4%

More GI disorders in the liraglutide group. No pancreatitis was reported

SCALE-Maintenance

1-year randomized, double-blind, placebo-controlled trial

(2013)

422 adults overweight/obese who had lost ≥5% of initial body weight during a calorie-restriction period were randomized

i.3.0mg sc once daily

 

 

ii. placebo

i.-6.2%

 

 

 

ii.-0.2%

i.81.4%

 

 

 

ii.48.9%

A combination of liraglutide, diet, exercise induced further weight loss and improvement in certain cardiovascular risk factors

 

SEMAGLUTIDE

 

Semaglutide is a novel long-acting GLP1 analogue indicated for T2DM and awaiting approval for obesity at higher doses. The efficacy of this anti-obesity drug has been proven by the SUSTAIN 1-6 trials. In these trials, patients who were overweight or obese, with and without T2DM, with or without antidiabetic medications, were allocated in groups which received semaglutide in two different dosages (0.5mg or 1.0mg) or placebo or another anti-diabetic therapy. The superiority of semaglutide 1.0mg against semaglutide 0.5mg or placebo or another anti-diabetic agent was obvious (122). In SUSTAIN 7, Semaglutide administered in subcutaneous injections once weekly was compared with Dulaglutide. Mean weight loss was greater in the group which received 1.0mg semaglutide (-4.9kg) vs the groups that received 0.5mg semaglutide (-3.6kg), 1.5mg Dulaglutide (-3kg), and0.75mg Dulaglutide (-2.3kg). Additionally, oral semaglutide is currently approved for the treatment of T2DM. In order to avoid malabsorption, semaglutide is administrated 30 minutes before breakfast. Apart from semaglutide, other oral GLP-1 agonists, such as TTP054/TTP-054 and ZYOG1, are under investigation (122). Two other trials, STEP, which studies the effects of semaglutide in patients with obesity, and SELECT, which investigates the cardiovascular effects of semaglutide in patients with obesity are currently underway (123). PIONEER, which examines the cardiovascular safety of oral administration of semaglutide in patients with T2DM, recently showed the non-inferiority of this medication to placebo (124). (see Table 17).

 

Table 17. Clinical Trials of Semaglutide

Clinical trial

Study Design

Dose

Treatment, placebo from baseline

% of patients losing ≥5% of baseline weight

SUSTAIN 1

Double-blinded

For 30 weeks

i.0.5mg sc once weekly

ii.1.0mg sc once weekly

iii. placebo

i. -3.7kg

ii. -4.5kg

iii. -1.0kg

i.37%

ii.45%

iii.7%

SUSTAIN 2

Double-blinded

Duration: 56 weeks

i.0.5mg sc once weekly

ii.1.0mg sc once weekly

iii. sitagliptin 100mg per po once daily

i.-4.3kg

ii.-6.1kg

iii.-1.9kg

i.46%

ii.62%

iii.18%

SUSTAIN 3

Open-label

Duration:56 weeks

i.1.0mg sc once weekly

ii. exenatide extended release 2.0mg

i.-5.6kg

ii.-1.9kg

i.52%

ii.17%

SUSTAIN 4

Open-label

Duration: 30 weeks

i.0.5mg sc once weekly

ii.1.0mg sc once weekly

iii. insulin glargine

i.-3.5kg

ii.-5.2kg

iii.+1.2kg

i.37%

ii.51%

iii.5%

SUSTAIN 5

Double-blinded

Duration:30 weeks

ii.0.5mg sc once weekly

ii.1.0mg sc once weekly

iii. placebo

i.-3.7kg

ii.-6.4kg

iii.-1.4kg

i.42%

ii.66%

iii.11%

SUSTAIN 7

Open-label

Duration: 40 weeks

i.0.5mg sc once weekly

ii.0.75mg dulaglutide sc once weekly

iii.1.0mg sc once weekly

iv.1.5mg dulaglutide sc once weekly

i.-4.6kg

ii.-2.3kg

 

iii.-6.5kg

iv.-3.0kg

i.44%

ii.23%

 

iii.63%

iv.30%

SUSTAIN 6

(CVD outcomes)

Double-blinded

Duration:104 weeks

i. 0.5mg sc once weekly

ii.1.0mg sc once weekly

iii. placebo 0.5mg

iv. placebo 1.0mg

i.-3.6kg

ii.-4.9kg

iii.-0.7kg

iv.-0.5kg

Non inferior

SUSTAIN 8

Phase 3b

Semaglutide vs canagliflozin

 

 

 

SUSTAIN 9

Semaglutide as an add-on to SGLT2 monotherapy or in combination with either metformin or sulfonylurea

 

 

 

 

OTHER LONG-ACTING GLP-1 ANALOGUES

 

Other long-acting GLP-1 analogues are currently being investigated for weight loss in addition to diabetes treatment. Once-daily 13-week treatment with 20 μg or 30 μg of lixisenatide reduced body weight significantly more compared to placebo (-3 kg for lixisenatide 20 μg; p<0.01, -3.47 kg for lixisenatide 30 μg; p<.01, -1.94 kg for placebo) (125). Current findings regarding CJC-1134-PC, which is a conjugate of exendin-4 and recombinant human albumin and represents a once-weekly glucagon-like peptide-1 receptor agonist, suggest that it provides similar reduction in body weight compared with exenatide twice-daily. It may have a more favorable adverse event profile which might improve patient compliance and probably total weight loss in the long-term (126). Finally, albiglutide and taspoglutide are two novel GLP-1 analogues currently being investigated. A recent review that examined the efficacy, safety, and perspective for the future of the once-weekly GLP-1 receptor agonists exenatide, taspoglutide, albiglutide, LY2189265 and CJC-1134-PC, and compared them to the currently available agonists, exenatide BID and liraglutide QD, concluded that the long-acting agonists are not superior compared to the currently used exenatide BID and liraglutide QD regarding weight loss (127).In a separate development, an orally administered PYY3-36 and GLP-1 combination has been formulated using a sodium N-[8-(2-hydroxybenzoyl) amino] caprylate (SNAC) carrier (127). Early studies revealed that the neuropeptides delivered orally in this way had a pharmacodynamic profile consistent with the reported pharmacology, were rapidly absorbed by the gastrointestinal tract, and reached concentrations several-fold higher than those seen naturally postprandially (128). Oral GLP-1 (2-mg tablet) alone and in combination with PYY3-36 (1-mg tablet) showed enhanced fullness at meal onset and induced a significant reduction in energy intake. Exenatide-CCK (129) and Liraglutide-Setmelanotide (130) have been also introduced as different combined anti-obesity therapies which act synergistically on POMC-deficient patients.

 

Single Molecule Multi-Agonists

 

The main therapeutic idea of this category is based on the concept that a single molecule could target multiple receptors (at least two; multi-agonist), thus allowing synergistic action of both pharmaceutical agents.

 

GLUCAGON-LIKE PEPTIDE 1/GLUCAGON

 

As mentioned before, GLP-1 analogues are effective anti-obesity medications and improve glucose intolerance. Glucagon has direct action on the liver by stimulating gluconeogenesis and glycogenolysis (131). It can even result in hyperglycemia and T2DM. Of note, patients with T2DM are characterized by impaired glucagon secretion. However, glucagon in CNS decreases food intake, increases energy expenditure via brown fat thermogenesis, decreases fat accumulation via lipolysis and lipid synthesis inhibition, improves cardiac performance, inhibits gastric motility, and stimulates autophagy. In 2009, the first human study announced that low-dose co-infusion of GLP-1 and glucagon could decrease food intake and increase energy expenditure (132). Therapy with a GLP-1/glucagon multi-agonist was created when amino acids 17, 18, 20, 21, 23 of glucagon were substituted in the glucagon molecule by the respective GLP-1 residues (133). The alanine at position 2 of the peptide was substituted with Aminoisobuturic acid (Aib) to protect the molecule from DDP-IV inactivation, and a lactam bridge was introduced between amino acids 16 and 20 to stabilize the secondary structure to ensure glucagon receptor potency. Once weekly administration of this pharmaceutical agent, for 4 weeks, in diet-induced obesity in mice, resulted in improvement of obesity, hepatic steatosis, glucose control, and lipid profile. Increase in energy expenditure was observed only with the multi-agonist therapy, but not with the glucagon monotherapy. Moreover, it was found that therapy with the multi-agonist improved leptin sensitivity in DIO mice (134). Different GLP-1/glucagon multi-agonists are currently under investigation (135). Interestingly, an oxyntomodulin multi-agonist was under investigation concurrently with the GLP1/glucagon multi-agonist.

 

OXYNTOMODULIN

 

Oxyntomodulin (OXM) is a 37-amino acid anorexigenic peptide hormone produced in the L-cells of the distal small intestine and colon, where it co-localizes with GLP-1 and PYY. Animal studies have shown weight reduction and improved glucose metabolism following chronic OXM injections beyond that explained by food intake restriction, suggesting an additional effect of OXM on energy expenditure. Just like GLP1, OXM is a product of proglucagon gene believed to modulate energy homeostasis at least in part via GLP1R, although its GLP1R binding affinity is about 100 times lower than that of GLP1 (136). Centrally however, GLP1 and OXM have different targets, as OXM activates neurons in the hypothalamus (137), whereas GLP1 acts in the hindbrain and other autonomic control areas (138). In human studies, acute anorectic effect of OXM was demonstrated by intravenously infused OXM (139). A reduction in food intake was also seen and retained during chronic administration in a 4-week trial with OXM injections three times a day 30 minutes before meals in a group of overweight and obese volunteers (n = 14). OXM reduced nutrient intake (35% ± 9%) resulting in significant weight loss compared to placebo (2.3 ± 0.4 kg vs 0.5 ± 0.5 kg, respectively). The findings of another study with twelve overweight or obese human volunteers who underwent a randomized, double-blinded, placebo-controlled study were similar; an ad libitum test meal was used to measure energy intake during intravenous infusions of either PYY3-36 or OXM or combined PYY3-36/OXM. Again, OXM significantly reduced energy intake compared to placebo, although the combined treatment had superior effects compared to PYY3-36 or OXM monotherapy. Human studies have also clearly demonstrated the direct effect of OXM on energy expenditure (140); this effect was later confirmed by indirect calorimetry (141). These modest but favorable results suggest significant promise for OXM-based therapies for obesity. In addition to the established action of OXM on appetite, another mechanism that potentially plays a role in energy intake and glucose metabolism is gastric emptying. Intravenous infusion of OXM reduced gastric emptying in humans (142). Whether reduction in gastric emptying is involved in the acute and long-term metabolic effects of OXM is not yet clear. Nevertheless, the immediate future will reveal OXM’s role in obesity management. However, as for other peptide hormones, their clinical application is limited by their short circulatory half-life, a major component of which is cleavage by DPP-IV. Therefore, structurally modified analogues with an altered OXM pharmacological profile have been produced with longer duration of action, good safety profile, and positive effects on body weight (and glucose metabolism) management in animal studies (143). These findings bring closer their usage in human clinical trials. Furthermore, the crystal structure of OXM has been determined, and this advance should facilitate the rational design of oxyntomodulin peptidomimetics to be tested as oral anti-obesity pharmaceuticals. Even so, despite the promising weight reduction efficacy of OXM, only a small number of development projects appears to be at an advanced stage. TKS1225 is an OXM analogue. The present status of this molecule is unknown. OXY-RPEG has been engineered via its proprietary reversible pegylation technology to increase its half-life and increased potency. In preclinical testing, OXY-RPEG was significantly superior to twice daily injections of OXM in the reduction of food intake and the degree and durability of weight loss. In 2009, an oxyntomodulin-based multi-agonist peptide with glucagon and GLP-1 agonistic actions were created. This multi-agonist had advanced action comparing to the one that Day et al had introduced at the same year (144). A 2-weeks trial in DIO mice showed weight loss and glucose control improvement. This beneficial action was obvious even in mice without GLP1 or glucagon receptor confirming the superiority of this analogue. Oxyntomodulin functions endogenously as a physiologic co-agonist, but regarding its small bioactivity, it is mainly characterized by its function as biosynthetic precursor to glucagon.

 

GLUCAGON-LIKE PEPTIDE 1/AMYLIN

 

In 2010, salmon calcitonin-exendin-4 combined therapy achieved reduction of food intake and weight in non-human primates (145). Of note, the human amylin receptor subtypes consist of calcitonin receptor and receptor activity-modifying proteins. This observation was the first step in the development of multi-agonist molecules targeting GLP-1 and Amylin (146). Two of these peptide hybrids (phybrids) had a C-terminally truncated Exenatide, which was covalently linked to the N-terminus of an amylin analogue (Davalintide) through either a repeat β-ala-β-ala dipeptide, or through triple-glycine linear repeat. Administration of phybrids resulted in greater weight loss in non-human primates than monotherapy, although similar to that achieved by a physical commixture of the single hormones. Another GLP1/Amylin phybrid was introduced, which used a full-length Exenatide sequence linked to Davalintide viaan intervening 40-kDa PEG. This phybrid reduced both blood glucose and body weight in a dose-dependent fashion.    

 

GLUCAGON-LIKE PEPTIDE 1/GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE

 

This single-molecule multi-agonist was quite controversial. Glucose-Dependent Insulinotropic Polypeptide (GIP), is a 42-amino acid peptide, produced by K-cells in the duodenum and jejunum and released into the general circulation upon stimulation by dietary lipids (147). The investigation following the discovery of this new peptide, showed that GIP is the first incretin hormone. It acts directly on the pancreas augmenting glucose-stimulated insulin secretion (148). It is worth mentioning that GIP has the ability to enhance both insulin secretion in hyperglycemia and glucagon release in hypoglycemia (149). A few years later, the role of GIP in obesity development became apparent. GIP acts on adipocytes enhancing adipogenesis, inhibition of lipolysis, stimulation of de novo lipogenesis (150) and on chylomicrons stimulating triglyceride release. It also affects adipocyte glucose and fatty acid uptake and adipocyte lipoprotein lipase enzyme activity (151). It is remarkable that although GIP was regarded as an obesogenic hormone, mice overexpressing GIP showed improved β-cell function and improved glycemic control and were resistant to DIO (152). Additionally, in studies with mice, it was shown that the chronic GIP agonist administration improves glucose metabolism without body weight changes (153). In 2013, two single-molecule multi-agonists GLP1/GIP were introduced, whose action was based on the insulinotropic action of both components (153). GIP agonist enhanced GLP1 action upon glucose metabolism and GLP1 could mitigate obesogenic effect of GIP via its anorectic effect. The biochemical structure of multi-peptide was similar to GLP1/glucagon multi-agonist i.e. a single peptide with potency at both receptors (GIP residues were introduced in the median and the C-terminal part of peptide; certain modifications that increased activity on the glucagon receptor were removed; the C-terminus of the peptide ended with the nine amino acid extension found in exendin-4 and an Aib was added at position 2 to protect against DPP-IV inactivation) (154). Several clinical trials in mice, rodent models, non-human primates and humans were performed, concluding that the GLP1/GIP multi-agonist therapy reduced food intake, and consequently body weight, improved glycemic control, lipid profile and lipolysis but without any improvement in energy expenditure.

 

GLUCAGON LIKE PEPTIDE 1/GLUCAGON/GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE

 

The creation of this single-molecule multi-agonist was based on the biochemical structure of GLP1/GIP and GLP1/glucagon multi-peptides. An Aib at position 2 both protected the molecule from DPP-IV inactivation and decreased its potency at the glucagon receptor; an amino acid lysine at position 10 was fatty-acylated via a γ-glutamic acid linker to palmitic acid; amino acids at positions 16,17, 20, 27, 28 replaced balanced glucagon bioactivity; a C-terminal exendin-4 extension sequence (CEX) succeeds agonism at all three receptors 10-fold greater than native hormones (155). The main mechanism of action is based on the combination of the anorectic effect of GLP-1, the lipolytic and thermogenic characteristics of glucagon, and the action of GIP on β-cell function and glycemic control. Contrary to GLP-1/GIP multi-agonist, which doesn’t affect energy expenditure, this triple agonist increases energy expenditure. (see Table 18)

 

Table 18. Single Molecule Multi-Agonists

Drug name

Clinical benefits

Adverse events

Glucagon-like peptide 1/glucagon

oxyntomodulin, MED10382, G530S (Glucagon analogue/Semaglutide), GC-co-agonist 1177

↓ food intake, obesity, hepatic steatosis, HbA1c, lipid profile

↑energy expenditure

 

Glucagon-like peptide1/amylin co-agonism

↓ blood glucose and body weight dose-dependently

 

Glucagon-like peptide 1/glucose-dependent insulinotropic polypeptide

↓ blood glucose, lipid profile, food intake, body weight,

↑ lipolysis

No improvement in energy expenditure

Glucagon-like peptide 1/glucagon/glucose-dependent insulinotropic polypeptide

↓body weight, HbA1c, hepatosteatosis, cholesterol, ↑energy expenditure, lipolysis

 

 

Peptide-Mediated Delivery of Nuclear Hormones

 

The use of nuclear hormones as an agent of GLP-1 and Glucagon is a novel promising therapy in the treatment of obesity. Nuclear hormones are characterized by high potency and pleiotropic action as well as unwanted adverse effects. The basic idea involves a linkage of a nuclear hormone to a peptide, usually through a linker that would allow metabolism of the nuclear hormone only within the targeted cell reducing the undesirable effects in other tissues. However, in the cell types that possess the specific peptide receptor, its activation should lead to internalization of the ligand-nuclear hormone receptor complex. In this case, the peptide receptor plays the role of a gateway into the cell. Upon internalization, biological processing of a suitably designed linker would release the nuclear hormone and allow activation of its intracellular receptor. Although a promising option, not all nuclear hormones can be used as peptide-mediated agents. They should have high tissue selectivity, ability to be internalized and compatibility to peptide wanted. Estrogens, tri-iodothyronine, and dexamethasone are the nuclear hormones that have been tested.

 

GLUCAGON-LIKE PEPTIDE 1-MEDIATED DELIVERY OF ESTROGEN

 

Glucagon-Like Peptide 1-mediated delivery of estrogen was first introduced in 2012. The use of estrogens was indicated by the fact that estrogen replacement therapy in postmenopausal women improved multiple cardio metabolic parameters (156). Furthermore, estrogens have anabolic, insulinotropic, and anorectic effects (157). The combination of estrogen and GLP-1 was found to improve body weight and glycemic control in rodent models with the metabolic syndrome (158). The weight-lowering effect was due to appetite suppression, while the GLP-1/E2 combination showed greater potency comparing to GLP-1 analog or E2 alone. Further clinical trials enhanced this finding showing an influence on feeding behavior. Additive contribution of GLP-1/E2 on pancreatic islet function, cytoarchitecture and protection from deleterious insults such as lipotoxicity was found in 2015 (159). Despite the powerful metabolic benefits associated with estrogen action, effects on the reproductive endocrine system and oncogenic potential have restricted their clinical use in postmenopausal women. Furthermore, many aspects of molecular pharmacology and mechanism of action remain unresolved. Specifically, neither the precise intracellular processing of the GLP-1/E2 conjugate, which results in active estrogen cargo release, nor the molecular identity that delivers estrogen activity, have been determined. It is possible that estrogens enhance brain penetration and alter the bio-distribution of the conjugate to more privileged sites for central nervous action.

 

GLUCAGON-MEDIATED DELIVERY OF TRI-IODOTHYRONINE

 

Glucagon and thyroid hormone can separately lower body weight and LDL cholesterol in humans. Thyroid hormones act both on liver, regulating hepatic lipid metabolism and hepatosteatosis, and in adipose cells, increasing energy expenditure and enhancing lipolysis (160, 161). On the other hand, they can cause cardiac hypertrophy, tachycardia, muscle catabolism, and bone deterioration. Glucagon receptors are highly concentrated not only in the liver, which is the preferred site for T3 action, but also in adipose tissues, kidney, and the cardiovascular system resulting in metabolic enhancement along with toxicity risk. Considering all of the above, a glucagon/T3 conjugate was created. A native T3 combined with a DPP4-protected C-terminally extended glucagon analog via a peptide spacer (162). Several control compounds were also generated to permit appropriate pharmacological comparisons. These additional peptides included a conjugate with selective chemical substitution in the peptide to suppress glucagon activity, a compound with a linker that proved metabolically stable and was incapable of intracellular T3 release, and a third control conjugate that carried a metabolically-inert thyroid hormone. Finan found that the conjugate glucagon/T3 corrected lipid metabolism in rodent models with dietary-induced metabolic syndrome. The above findings showed that the body-weight effect of the conjugate can partially be governed by actions in adipose depots because glucagon receptors exist in rodent adipocytes, less than in liver. Moreover, the glucagon/T3 conjugate effect is supported by the uncoupling protein 1-mediated thermogenesis, enhanced FGF21 secretion and biased by PGC-1 cofactor signaling. Interestingly, the combination of glucagon/T3 seems to decrease arterial plaque area in LDL receptor -/- mice and fibrosis in mice with advanced fatty liver disease.  Although the above data demonstrate the cardiovascular benefit of this conjugate, further chemical improvements should be made in order to be safe for chronic use in higher mammals and especially humans.

 

GLUCAGON-LIKE PEPTIDE 1-MEDIATED DELIVERY OF DEXAMETHASONE

 

It is widely known that dietary-induced obesity causes chronic peripheral and central inflammation (163). Glucocorticoids are widely known for their anti-inflammatory characteristics, but due to their ubiquitous action profile, their therapeutic use can lead to off-target effects. In 2017, DiMarchi and Tschop created a GLP1/dexamethasone conjugate which managed to improve body weight in DIO mice, in a superior way to GLP1 or dexamethasone alone.  This combination improved hypothalamic inflammation, astrocytosis, microgliosis, and insulin sensitivity. The targeted delivery of dexamethasone to GLP1R-positive cells prevented typical dexamethasone off–target effects on glucose metabolism, bone density, and the hypothalamus-pituitary-adrenal axis activity (164). (see Table 19)

 

Table 19. Peptide-Mediated Delivery of Nuclear Hormones

Drug name

Clinical benefits

Glucagon-like peptide 1/estrogen

↓ food intake, body weight, HbA1c

Glucagon/tri-iodothyronine

↓ lipid profile, arterial plaque and fibrosis in advanced fatty liver disease

Glucagon-like peptide 1/dexamethasone

↓ hypothalamic inflammation, astrocytosis, microgliosis, ↑insulin sensitivity

 

Peptide Y (PYY)

 

PYY is a 36-amino acid anorexigenic peptide with a hairpin-like U-shaped fold secreted from the entero-endocrine L-cells of the ileum and colon in response to feeding. PYY presents in two major forms, PYY1-36 and PYY3-36. More specifically, PYY is a member of the pancreatic polypeptide-fold (PP-fold) family which also includes NPY and PP and interacts with a family of receptors (mainly Y2R). It is produced postprandially, in response and proportionally to caloric load, by the distal-intestinal L cells along with oxyntomodulin (OXM) and GLP-1. Just like GLP-1 and OXM, PYY1-36 is rapidly proteolyzed by DPP4. However, unlike the other two neuropeptides, the cleaved product PYY3-36, is bioactive. Human studies have shown that PYY delays gastric emptying and promotes satiety (165), while short-term intravenous administration of PYY3-36 , at doses generating physiologic postprandial blood excursions, was shown to decrease calorie intake by approximately 30% in lean and obese subjects, without causing nausea, affecting food palatability, or altering fluid intake, nor was it followed by compensatory hyperphagia (166). Another study confirmed the above findings, reporting dose-dependent reductions of food intake (maximal inhibition, 35%; P<0.001 vs control) and calorie intake (32%; P<0.001) after intravenous infusions of several different concentrations of PYY3-36 (167). Sloth et al. first showed the significantly higher energy expenditure following PYY3-36 intravenous infusion compared with PYY1-36 or control. In a recent study, the effect of infused PYY3-36 on energy intake was compared to that of OXM or the combined PYY3-36/OXM treatment; the results demonstrated that energy intake was significantly less with the combined treatment compared to PYY3-36 or OXM monotherapy (168). Whether these findings pointed to a weight loss efficacy of PYY was evaluated in a 12-week trial of 133 obese patients who were randomly assigned to intranasal PYY3-36 (200 or 600 mcg three times a day before meals) or placebo, in conjunction with diet and exercise. At the 200 mcg dose, PYY3-36 failed to reduce body weight, while 60% of patients treated with the high PYY3-36 dose (600 mcg three times a day) dropped out due to nausea and vomiting, so that no meaningful inference could be drawn from the few patients who completed the study on 600 mcg. These findings contrast with those in rodents (169, 170) and nonhuman primates (171) where PYY3-36 preparations reduce body weight. One suggested explanation is that the PYY3-36 effect is critically modulated by the time of injection. As the main anorexigenic effect of PYY is by Y2R-mediated NPY inhibition, PYY is obviously more effective at times that the orexigenic NPY is increased. In accordance with this theory is the reported weight loss effect of PYY3-36 when injected in rodents in the fasting state or in the early dark cycle — times when NPY is naturally induced (172).

 

PYY3-36 is structurally similar to pancreatic polypeptide (PP); PYY3-36 acts mainly through Y2R, while PP acts through Y4R. Obinepitide (TM30338), a synthetic dual-analogue of PYY3-36 and PP that stimulates both Y2/Y4-receptors, has been developed. Pre-clinical studies have shown that obinepitide efficiently reduces weight in obese mice. Furthermore, initial studies in humans have shown that once-a-day subcutaneous administration of obinepitide in obese human subjects inhibited food intake, at a statistically significant level, up to at least nine hours after dosing (173). Various PYY analogues have been created including intravenous, oral or nasal formulations. Interestingly, the combined therapy of PYY3-36 and GLP-1 receptor agonist (exendin-4) was found to decrease food intake and body weight in an additive manner in animal models and humans. Specifically, this synergistic result was attributed to the enhancement of c-fos reactivity in special cerebral nuclei (174). (see Table 20)

 

Table 20. PYY

Mechanism of action

Anorexigenic peptide which decreases gastric motility, increases satiety, inhibits NPY receptors

Clinical Benefits

↓ appetite, decreases food intake, ghrelin levels

Adverse events

Short-time action

 

Ghrelin Vaccines and Ghrelin Inhibitors

 

Ghrelin is a 28-amino acid peptide produced primarily by the stomach and proximal small intestine (175). It is the only known circulating orexigenic hormone and signals both on vagal afferents and in the arcuate nucleus where it powerfully enhances NPY orexigenic signaling (176, 177). Its levels increase before meals and are suppressed by ingested nutrients, with carbohydrates being the most effective ones (compared to proteins and lipids). Ghrelin’s suppression results from neutrally transmitted (non-vagal) intestinal signals, augmented by insulin. An experimental ghrelin vaccine, CYT009-GhrQb, was discontinued in 2006 as it did not have the expected effects on weight loss. A novel one conjugated to the hapten, keyhole limpet hemocyanin (KLH), tested in rodent models, was shown to decrease feeding and induce weight loss (178). NOX-B11 is a ghrelin-neutralizing RNA spiegelmer that attaches to the active form of ghrelin and blocks its ability to bind to its receptor thus blocking the orexigenic activity of exogenously administrated ghrelin in rats (179). However, NOX-B11 did not affect basal food intake in nonfood-deprived rats, thus this treatment may only be efficacious when plasma ghrelin levels are high, such as before a meal or during times of food restriction (dieting).Since the discovery that the effects of ghrelin are primarily mediated by the GH secretagogue receptor (GHSR) 1a, there have been multiple potent, selective, and orally bioavailable ghrelin antagonists produced with good pharmacokinetic (PK) profiles that are currently in preclinical testing. An amide derivative 13d (Ca2+ flux IC50 = 188 nM, [brain]/[plasma] = 0.97 @ 8 h in rat), for example, showed a 10% decrease in 24-hour food intake in rats, and over 5% body weight reduction after 14-day oral treatment in diet-induced obese (DIO) mice (180).

 

Moreover, the discovery of ghrelin O-acyltransferase (GOAT) as the enzyme that catalyzes ghrelin octanoylation, revealed several therapeutic possibilities including the design of drugs that inhibit GOAT and block the attachment of the octanoyl group to the ghrelin third serine residue; such GOAT inhibitors could potentially prevent or treat obesity (181). Octanolyation of ghrelin by GOAT on its third amino acid (serine-3) is necessary for the hormone’s biological functions. Octanoylated ghrelin enhances hyperphagia and increases gastrointestinal motility. Furthermore, it reduces insulin secretion causing glucose dysfunction, enhances thermogenesis, adipogenesis and liver lipogenesis, limiting lipolysis at the same time (182). So, inhibiting GOAT could impede the production of acyl-ghrelin and increase desacyl-ghrelin, thus improving glucose homeostasis. In 2010, GO-CoA-Tat was created. A peptide-based bi-substrate analog which inhibited GOAT activity. The chronic treatment with GO-CoA-Tat, resulted in body weight stabilization in vehicle-treated mice fed MCT-rich HFD. Additionally, a decrease of fat mass was shown, but not of lean mass (183). Another study on Siberian hamsters also resulted in improvement in ingestive behavior. Remarkably, after 48h food deprivation, GO-CoA-Tat attenuated food foraging, food intake, and food hoarding post-refeeding relative to animals treated with saline. GO-CoA-Tat treated mice improved their blood glucose (184).

 

Another promising anti-obesity agent against ghrelin is a brain penetrant CAMKK2 inhibitor. Generally, CAMKK2 has been identified as the hypothalamic AMPK kinase that transduces Ca2+-mediated ghrelin signaling, inhibiting selectively hypothalamic AMPK and NPY’s downstream orexigenic effect. 4t, a 2,4-diaryl 7-azaindole, was created in order to inhibit AMPK phosphorylation in a hypothalamus-derived cell line. When this agent was tested in rodents, it managed to reduce ghrelin-induced food intake (185) (see Table 21).

 

Table 21. Ghrelin Vaccine (NOX-B11)

Mechanism of action

Ghrelin vaccine

Clinical Benefits

↓ food intake, hypothalamic orexigenic signals, ↑energy expenditure

Adverse events

No weight loss seen in human trials

 

Fat-Specific Satiation Peptides

 

ENTEROSTATIN AND APOLIPOPROTEIN A-IV

 

Enterostatin and apolipoprotein A-IV appear to be GI peptides that are specifically stimulated by fat ingestion and subsequently regulate intake and/or metabolism of lipids. Although peripheral and central enterostatin administration decreases dietary fat intake in animals (while enterostatin-receptor antagonists did the opposite) (186), its administration to humans has shown no effects on food intake, appetite, energy expenditure, or body weight (187). Similarly, apolipoprotein A-IV, which is synthesized and secreted exclusively by the small intestine (primarily by the jejunum, but also by the duodenum and ileum), acts as a satiety factor that is downregulated by leptin (188) and upregulated by insulin and PYY in both rodents and humans (189). Although exogenous administration of apolipoprotein A-IV was quite effective concerning meal size, food intake, and weight gain reduction in rats (190), data is lacking regarding apo A-IV therapeutic administration in humans and its effects on body weight.

 

Pancreatic Satiation Peptides

 

PANCREATIC POLYPEPTIDE (PP)

 

Pancreatic polypeptide (PP) is a 36-amino acid peptide that is structurally similar to PYY. It is primarily produced in the pancreas in response to ingestion of food and in proportion to caloric load (191). Animal studies have shown that peripheral administration of PP decreases feeding (through Y4R in the area postrema), whereas centrally administrated PP increases it (through Y5R deeper in the brain) (192). In humans, intravenous infusion of PP (10 pmol/kg/min) (supra-physiological levels of PP) in ten healthy volunteers (men and women of normal body weight) caused a sustained decrease in both appetite and cumulative 24-hour energy intake by 25.3 +/- 5.8% (193). The findings of another study studying the anorexigenic effect of a lower infusion rate of PP (5 pmol/kg/min) in lean fasted volunteers were similar, holding promise for potential use as an anti-obesity agent (194). Another trial studying whether combined treatment with PP/PYY3-36 is superior regarding weight loss compared to either agent alone concluded that PP and PYY3-36 do not inhibit feeding additively in humans (195). Again, this study was conducted on lean subjects. Conversely, as previously mentioned, a synthetic analogue (TM30338) of both PYY3-36 and PP, which acts as an agonist of both the Y2 and Y4 receptors, yielded very promising results as concerns early meal termination when administered once-a-day subcutaneously in obese human subjects. Similarly, initial reports of a selective Y4-receptor agonist (TM30339) currently under development were also quite promising inducing reduction of food intake and promoting weight loss.

 

AMYLIN AND AMYLIN ANALOGUES

 

Amylin is a 37-amino acid neuroendocrine peptide hormone co-secreted postprandially with insulin by pancreatic β-cells. Among other properties, amylin is characterized by centrally mediated glucoregulatory and anorexigenic actions (196). It inhibits gastric emptying and glucagon secretion as well as decreases meal size and calorie intake (fat specific) (197) in a dose-dependent manner. These are vagus-independent actions and are exerted via binding to specific amylin receptors in the hindbrain area postrema (198), which is in contrast with the peripheral neural mechanisms engaged by most other gut peptides involved in energy homeostasis system regulation. The anorectic efficacy of amylin along with its glucoregulatory actions were investigated in human studies with the usage of pramlintide, a subcutaneous injectable amylin analogue which differs from amylin by only three amino acids. Studies in patients with type 1 and type 2 diabetes have shown great improvement in glycemic control plus sustained reductions in food intake and meal size, as well as mild progressive weight loss, following acute and long-term adjunctive pramlintide treatment (120 μg) (199). The most common adverse event associated with pramlintide usage was transient, mild-to-moderate nausea. This weight loss is noteworthy because it occurred in subjects with type 2 diabetes, on concomitant insulin therapy, and in the face of a significant A1C reduction, factors that all favor weight gain. Similar to the GLP-1 analogues discussed previously, pramlintide is currently approved for the treatment of type 1 and type 2 diabetes.

 

Whether pramlintide could constitute a potent anti-obesity agent was investigated in well-designed trials addressing this issue. In such a study (16-week randomized, double-blind, placebo-controlled), 204 individuals with obesity but not diabetes were treated with self-administered subcutaneous injections of pramlintide (nonforced dose escalation ≤ 240 μg) or placebo three times a day, 15 minutes before meals without concomitant lifestyle intervention (200). Pramlintide was generally well-tolerated and approximately 90% of the pramlintide-treated subjects were able to escalate to the highest dose of 240 μg three times a day. In contrast to the placebo-treated subjects who experienced minimal changes in body weight over the 16-week treatment period, the pramlintide-treated subjects attained significant weight loss from baseline as early as week 2, which was progressive up to week 16, with no evidence of a plateau. At week 16, the placebo-corrected reduction in body weight after pramlintide treatment was statistically significant compared with placebo (3.7 ± 0.5%, P < 0.001; 3.6 ± 0.6 kg, P < 0.001). Furthermore, the reduction in weight in pramlintide-treated subjects was accompanied by a significant reduction in waist circumference compared with placebo-treated subjects after 16 weeks of treatment (evaluable 4.3 ± 0.6 vs. 0.7 ± 0.9 cm, P < 0.01). At the end of the 16-week trial, 31% of the subjects treated with pramlintide achieved ≥ 5% weight loss compared to just 2% of the placebo group (P < 0.001). Interestingly, 8 weeks after treatment cessation, the pramlintide-treated subjects had on average regained one third of the overall weight loss observed by week 16. These findings constitute a proof of concept that pramlintide may have therapeutic use as an anti-obesity agent. Remarkably, at this higher dose (240 μg three times a day), the mean reduction in body weight with pramlintide treatment over 16 weeks was approximately twice that previously observed over a similar time-frame in insulin-treated subjects with type 2 diabetes who were treated with lower pramlintide doses (120 μg). This could suggest that higher doses of pramlintide might be necessary to achieve significant weight loss, although it is not yet clear whether concurrent insulin treatment was the main cause of that difference.

 

AMYLIN/PRAMLINTIDE COMBINATIONS

 

Previous animal studies have shown that amylin treatment significantly enhanced hypothalamic anorexigenic leptin signaling, while the combination treatment with amylin and leptin led to marked, synergistic reductions in food intake (up to 45%) and fat-specific weight loss (up to 15%). Recently, the weight-lowering effect of combined amylin/leptin agonism in human obesity was evaluated using the analogues pramlintide/metreleptin, respectively. As previously discussed, (see leptin), three trials addressing the weight loss efficacy of the combined treatment over 20, 28, and 52 weeks, respectively) reported sustained and robust weight loss by the combined treatment. Development was discontinued following commercial reassessment of the program. A Phase II study of davalintide, a second-generation analogue of amylin, for the treatment of obesity has also completed. In this study however, the weight loss efficacy and tolerability profile of davalintide was not superior to pramlintide, and was inferior to the pramlintide/metreleptin combination, thus resulting in deciding to halt further development of davalintide.

 

The anti-obesity effect of the combined treatment amylin/PYY3-36 was evaluated in an animal study, given that they both may have the potential for short-term signals of meal termination with anorexigenic and weight-reducing effects (201, 202). Statistical analyses revealed that food intake suppression with the combined treatment was synergistic, whereas body weight reduction was additive; this combination has not yet been studied in humans.  Additional preclinical studies looking at the safety and efficacy of the combined treatment with pramlintide/phentermine and pramlintide/sibutramine was evaluated in a randomized placebo-controlled study with 244 obese or overweight nondiabetic subjects (203). The results suggested that the weight loss achieved at week 24 with either combination treatment was greater than with pramlintide alone or placebo (P < 0.001; 11.1 +/- 1.1% with pramlintide + sibutramine, 11.3 +/- 0.9% with pramlintide + phentermine, -3.7 +/- 0.7% with pramlintide; -2.2 +/- 0.7% with placebo; mean +/- s.e.), without any major adverse events.

 

As mentioned above, the human amylin receptor subtypes consist of calcitonin receptor and receptor activity-modifying proteins. Because of their mechanism of action, amylin mimetics coupled with calcitonin receptor agonists, are known as dual action amylin and calcitonin receptor agonists (DACRA). DACRA KBP-088 showed greater efficacy relative to davalintide regarding in vitro receptor pharmacology and in vivo efficacy of food intake and body weight (204). DACRA KBP-088 and KBP-042 improved body weight, glycemic control and adipose hypertrophy in high-fat diet-fed rats (205). A long acting amylin analogue is also in phase I clinical trial as a once daily anti-obesity treatment (206). (Table 22)

 

Table 22. Amylin/Pramlintide Combinations

Drug name

FDA approved/Phase

Mechanism of action

Clinical Benefits

Adverse events

pramlintide

Approved for DM1, DM2

Amylin analogue

-in DM1, DM2: ↓ blood glucose, food intake, body weight, waist circumference

Nausea

Davalintide (AC2307)

Phase II

Amylin analogue

↓food intake, body weight, HbA1c

hypoglycemia

DACRA KBP-088, KBP-042

 

Dual amylin and calcitonin receptor agonist

↓body weight, glycemic control, adipose hypertrophy

 

 

PERIPHERAL MODULATORS OF THE EFFICIENCY OF DIGESTION, METABOLISM, AND LIPOGENESIS

 

Lipase Inhibitors

 

Apart from early termination of food intake augmented by the centrally acting appetite suppressants, another potential therapeutic anti-obesity approach is the induction of a negative energy balance through the inhibition of nutrient, particularly fat, absorption. Lipase inhibitors inhibit gastric and pancreatic lipases in the lumen of the gastrointestinal tract that decrease systemic absorption of dietary fat. Orlistat is currently the only marketed anti-obesity drug of this category licensed for the treatment of obesity (including weight loss and weight maintenance). Additionally, it has been proven to improve glucose metabolism and nonalcoholic fatty liver disease. The most common adverse events are gastrointestinal system and include oily spotting, flatus with discharge, diarrhea, fecal urgency, and vitamin malabsorption (207).

 

The only other pancreatic and gastrointestinal lipase inhibitor currently in clinical development is Cetilistat (ATL-962). A short-term (12-week) randomized, placebo-controlled study of weight reduction addressing the efficacy, safety, and tolerability of Cetilistat in obese patients reported that Cetilistat produced a clinically and statistically significant weight loss in obese patients to similar extents at all doses examined compared to placebo (60 mg t.i.d. 3.3 kg, P<0.03; 120 mg t.i.d. 3.5 kg, P=0.02; 240 mg t.i.d. 4.1 kg, P<0.001), plus it significantly improved other obesity-related parameters including waist circumference, serum cholesterol and low-density lipoprotein cholesterol levels. Cetilistat treatment was also well-tolerated and the common orlistat-induced GI adverse events, such as flatus with discharge and oily spotting, occurred in only 1.8-2.8% of subjects in the Cetilistat-treated group (208). The combined results from three Phase I clinical studies designed to investigate the efficacy, pharmacodynamics, and tolerability of a range of Cetilistat doses [50 mg t.i.d. (n = 7), 60 mg t.i.d. (n = 9), 100 mg t.i.d. (n = 7), 120 mg t.i.d. (n = 9), 150 mg t.i.d. (n = 16), 240 mg t.i.d. (n = 9) and 300 mg t.i.d. (n = 9)] compared with placebo or orlistat [120 mg t.i.d. (n = 9)] in healthy volunteers were published (209). They reported that Cetilistat is equipotent with orlistat regarding fecal fat excretion; it however achieves a much better tolerance profile, as the number of episodes of steatorrhea per subject in the orlistat group (4.11) was 2.5-fold greater than that in the Cetilistat-treated group. The different tolerance profile between the two lipase inhibitors, seems to be related to the physical form of the fat in the intestine (rather than the amount of fat) resulting from each medication. Thus, Cetilistat acts more like a detergent, whereas orlistat may promote the coalescence of micelles, leading to oil-drops and increased gastrointestinal adverse events. Finally, a 12-week trial compared the efficacy and safety of Cetilistat (40, 80 or 120 mg three times daily) and orlistat (120 mg t.i.d.) relative to placebo in obese patients with type 2 diabetes on metformin (210). In this study similar reductions in body weight were observed in patients receiving Cetilistat (80 or 120 mg t.i.d.) or orlistat; these reductions were significant compared to placebo (3.85 kg, P = 0.01; 4.32 kg, P = 0.0002; 3.78 kg, P = 0.008). Furthermore, treatment with Cetilistat (80 or 120 mg t.i.d.) or with orlistat significantly improved glycemic control relative to placebo; again, Cetilistat was well-tolerated and showed fewer discontinuations due to adverse events than in the placebo and orlistat groups. Based on the above findings, this novel lipase inhibitor is currently at the furthest stage in the clinical development of new drugs of this class (see Table 23).

 

Table 23. Lipase Inhibitors

Drug name

FDA approved

Mechanism of action

Weight loss vs placebo

Clinical Benefits

Adverse events

Orlistat (Xenical)

1999

Lipase inhibitor

2.6%

↓ HbA1c, nonalcoholic fatty liver disease

Gastrointestinal side effects, vitamin malabsorption

Contraindicated in:Chronic malabsorption syndrome, cholestasis

Cetilistat (ATL-962)

 

Pancreatic and gastric lipase inhibitor

 

↓body weight, lipid profile, waist circumference

Gastrointestinal (less than orlistat)

 

Growth Hormone (GH) and GH Lipolytic Domain Synthetic Analogues

 

Besides its growth effects, GH also possesses significant metabolic properties, including lipolysis induction. On the other hand, GH dynamics change with increasing adiposity and GH circulating levels and response to stimuli are repressed in obesity (211, 212). Taken together, it could be hypothesized that GH administration is an effective therapeutic option for weight loss and fat mass reduction in obese individuals. However, the majority of the 16 clinical trials of GH administration in obesity indicated little or no beneficial effects of GH treatment on body weight (213). There is a report from an Australia-based biotechnology company of the development of a modified fragment of amino acids 177-191 of GH (hGH177-191) (AOD-9604) that mimics the lipolytic effects of GH without producing growth effects. AOD-9604 however failed to induce significant weight loss in a 24-week trial of 536 subjects and its development as an anti-obesity agent was terminated (214). In 2018, it was announced that GH not only promotes lipolysis, but also enhances the creation of beige adipose tissue through activation of STAT5 and induction of ADRB3. Consequently, it promotes the adrenergic action of WAT.

 

β3-Adrenoreceptor Agonists

 

The β3-adrenergic receptor is expressed in adipocytes; its activation by cognate β-agonists cause lipolysis and increase thermogenesis. Thyroid hormones increase thermogenesis via the thyroid hormone receptor β subtype; however, to date, every attempt to develop selective thyroid hormone receptor agonists which are effective in adipose tissue without systemic side-effects has failed. In 2000, a selective human β3-agonist, L-796568, was developed (215). Although its acute (4-hour period) administration in overweight human subjects was associated with significant increase in energy expenditure (by ~8%) (216), a 28-day clinical trial investigating the efficacy of chronic use of L-796568 in overweight and obese non-diabetic men receiving the drug (350 mg/d) failed to display any significant changes in body composition or 24-hour energy expenditure (217). The ineffectiveness of β3-adrenreceptor activation to induce significant and sustained lipolysis in humans may be explained by the fact that human WAT expresses minimal levels of β3-adrenoreceptors; similarly, their expression is also low within human brown adipose tissue.

 

11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitors

 

Previous studies have shown enhanced conversion of inactive cortisone to active cortisol through the expression of 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) in cultured omental adipose stromal cells (218); the autocrine action of cortisol may be crucial in the pathogenesis of central obesity and features of the metabolic syndrome, such as insulin resistance. The reports relating to effectiveness of carbenoxolone (nonselective 11β-HSD inhibitor) in reducing central obesity are conflicting (219). Currently, several pharmaceutical companies are developing selective 11β-HSD1 inhibitors that are effective in adipose tissue and may be more effective in improving insulin sensitivity and reducing body weight. Preliminary data from animal studies evaluating the weight-loss benefit of T-BVT, a new 11β-HSD1 pharmacological inhibitor with specificity for WAT, are very promising regarding its anti-obesity effectiveness and amelioration of multiple metabolic syndrome parameters (220). CNX-010-49, is another selective tissue-acting 11β-HSD1 inhibitor under investigation. Animal studies showed that this inhibitor acts on glucocorticoids and isoproterenol resulting in lipolysis in mature 3T3-L1 adipocytes. It not only enhances muscle glucose oxidation and mitochondrial biogenesis, but also reduces proteolysis and gluconeogenesis in primary mouse hepatocytes. As a result, it improves glucose control, lipid metabolism, and inhibits body weight gain without affecting feed consumption. A potential cardiovascular benefit was found because of the action of CNX-010-49 on plasminogen activator inhibitor-1 (PAI-1), interleukin-6 (IL-6), and fetuin-A (221). (see Table 24)

 

Table 24. 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitors

Drug name

Mechanism of action

Clinical Benefits

T-BVT

Selective to white adipose tissue 11β-HSD1

 

CNX-010-49

Selective to white adipose tissue 11β-HSD1

↑lipolysis,

↓ HbA1c, lipid metabolism, inhibits body weight gain without affecting feed consumption

 

Angiogenesis Inhibitors

 

Increasing adiposity is associated with expansion of the adipose capillary bed. Several vascular growth factors are produced by enlarged adipocytes, for example, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and angiogenin, which may in turn facilitate the expansion of adipose tissue. Thus, anti-angiogenesis may eventually participate in the treatment of obesity. This hypothesis is strengthened by studies where the experimental administration of anti-angiogenic agents in mice from different obesity models resulted in significant weight reduction and adipose tissue loss (222). Remarkably, there were benefits on food intake, metabolic rate, and preferred energy substrate. These findings appeared to modulate fat tissue by altering vasculature. Although there are many foods and beverages containing naturally occurring inhibitors of angiogenesis (e.g. green tea, oranges, strawberries, lemons, red wine, ginseng, garlic, tomato, olive oil, etc.), no convincing clinical trials have been conducted investigating their anti-obesity effect so far. Currently, a Phase II trial using the anti-angiogenic/anti-MMP drug ALS-L1023 for the treatment of obesity is underway (223). Similarly, endostatin was found to have both anti-adipogenic and anti-angiogenic action protecting mice against dietary-induced obesity (224).

 

Sirtuin 1 (SIRT1) Activators

 

Sirtuin 1 (SIRT1) is a member of the Sirtuin family of proteins that comprises seven members in mammals (SirT1-T7). Sirtuin proteins have gained considerable attention due to their importance as physiological targets for treating diseases associated with aging. They contribute to cellular regulation interacting with metabolic pathways and may serve as entry points for drugs. SIRT1 has gained popularity as it has been linked with the French Paradox and the calorie restriction-mediated longevity and delayed incidence of several diseases associated with aging, such as cancer, atherosclerosis, and diabetes. The calorie restriction-induced modulations have been demonstrated in organisms ranging from yeast to mammals. White adipose tissue seems to be a primary factor in the longevity brought about through calorie restriction, as mice engineered to have reduced levels of WAT live longer (224). Corroborating this, it was found that food withdrawal is followed by SIRT1 binding and repression of genes controlled by the fat regulator PPAR-γ (peroxisome proliferator-activated receptor-γ), including genes mediating fat storage. This, in turn, activates fat mobilization and lipolysis and reduces WAT mass (225). In addition to PPAR-γ, SIRT1 also interacts with PGC-1α, inducing the expression of mitochondrial genes involved in oxidative metabolism and fatty acid oxidation, while it also enhances leptin sensitivity by repressing PTP1B. The weight restricting effects of SIRT1 were further supported by experiments with resveratrol (RSV), a potent allosteric SIRT1 activator, which was shown to protect mice from diet-induced obesity (226). Furthermore, mice treated with SRT1720, a potent, selective synthetic activator of SIRT1, were resistant to diet-induced obesity due to enhanced oxidative metabolism in skeletal muscle, liver, and brown adipose tissue, indicating the positive metabolic consequences of specific SIRT1 activation (227). Currently, several pharmaceutical companies are investigating specific SIRT1 activators in Phase I and Phase II trials for the treatment of type II diabetes and obesity (228) to define their utility in the treatment of obesity and metabolic diseases.

 

Cyclic-GMP Signaling in Anti-Obesity Pharmacotherapy

 

Cyclic nucleotides, including 3-5-cyclic guanosine monophosphate (cGMP) and 3-5-cyclic adenosine monophosphate (cAMP), are second messengers important in many biological processes. Knowledge of the role of cAMP in the regulation of energy homeostasis has been extended, thanks to its intimate relationship with AMPK (AMP-activated protein kinase) signaling; intracellular cAMP activates the AMPK signaling pathway. AMPK regulates energy balance at both cellular and whole-body levels (229). Activation of AMPK facilitates fatty acid oxidation and mitochondria biogenesis, which promotes energy expenditure (230). Interestingly, activation of AMPK in the hypothalamus promotes food intake behavior (231). e.g. physiologic processes in the same direction and induces weight loss by mutual reinforcement. Moreover, off-the-shelf approaches might be possible, given the existence of an established market for medications targeting cGMP pathways, with FDA- and EMA-approved drugs such as sildenafil and linaclotide. Sildenafil acts on adipocytes, possibly through cGMP-dependent protein kinase I and mechanistic/mammalian target of rapamycin (mTOR) signaling pathways, browning subcutaneous white fat, thus increasing energy expenditure (232).

 

Beloranib

 

Beloranib is an analogue of the natural chemical compound fumagillin and is a methionine aminopeptidase 2 (MetAP2) inhibitor acting to reduce production of new fatty acid molecules by the liver and converting stored fats into useful energy (233). It was first tested in 31 obese women, who were divided into four groups (0.1mg, 0.3mg, 0.9mg, or placebo twice weekly). A dose-dependent weight loss was shown after four weeks of 0.9mg Beloranib administration with mean 3.8kg loss vs 0.6kg in the placebo group. It also improved lipid metabolism and lowered C-reactive protein and adiponectin. A phase II double-blinded, randomized clinical trial examined the efficacy and safety of Beloranib administration (234).147 obese patients were divided into four groups: 0.6, 1.2, 2.4 mg subcutaneous injection or placebo. After twelve weeks of administration, a dose-dependent weight loss of -5.5, -6.9, -10kg, respectively, was reported, vs -0.4kg in the placebo group. The main adverse events were sleep disturbance and gastrointestinal abnormalities. Beloranib may also cause robust weight loss and hypophagia in rats with hypothalamic and genetic obesity (235). In 2015, however, a phase III clinical trial for Prader-Willi was stopped after a second patient death (236). (see Table 25)

 

Table 25. Beloranib

FDA approved/Phase

Phase III aborted in 2015 after second patient death in Prader-Willi trial

Mechanism of action

Fumagillin analogue with methionine aminopeptidase 2 inhibition that reduced fatty acid synthesis in the liver and converted stored fat into useful energy; originally designed as an angiogenesis inhibitor

Clinical Benefits

↑ weight loss, hypophagia,

↓ lipid metabolism, CRP, adiponectin, cardiovascular factors

Adverse events

Sleep disturbance, gastrointestinal abnormalities

 

Fibroblast Growth Factor (FGF21)

 

Fibroblast growth factor (FGF) 21, expressed primarily in the liver, but also found in adipose tissue, skeletal muscle, and pancreas, is a member of the FGF family and acts as a metabolic regulator of body weight, glucose metabolism, and lipid metabolism (237). In WAT, FGF21 induces glucose uptake and adiponectin secretion with browning of white adipose tissue. In brown adipose tissue, it stimulates glucose uptake and thermogenesis, thus increasing energy expenditure. In the liver, it blocks GH signaling, regulates fatty acid oxidation both in the fasted state and in mice consuming high-fat, low-carbohydrate ketogenic diet and it maintains lipid homeostasis (238). FGF21 is characterized by anti-inflammatory, anti-oxidative stress properties with its circulating concentration increasing during periods of muscle activity or critical stress (239). Although, it is an attractive anti-obesity and anti-diabetes target, FGF21 levels are increased in obese ob/ob and db/db mice and correlate positively with BMI in humans. Exogenous administration of FGF21 in DIO in mice show virtually no beneficial effects on glucose tolerance and lipid metabolism, suggesting that the obesity state is FGF21-resistant (240).

 

ALTERNATIVE AND COMPLEMENTARY TYPES OF TREATMENT OF OBESITY

 

Gut Microbiota

 

Recently, a major shift in research has occurred towards the investigation of gut microbiota effects on energy expenditure and metabolism. Gut microbiota are responsible for a significant amount of the interaction between the host and the nutritional environment. Soluble fiber such as galacto-oligosaccharides and fructo-oligosaccharides (FOS), are fermented by the gut microbiota into short-chain fatty acids (SCFAs) acetate, propionate and butyrate (241). This mechanism provides to host 10% of its daily energy requirement (242). These SCFAs are an energy source for colonic epithelium, liver, and peripheral tissues (243). By fermenting nondigestible dietary fibers, host metabolism is enhanced. In mice with DIO, SCFAs improved glucose metabolism, insulin resistance, and obesity. In other animal studies, butyrate-producing bacteria (F. prausnitzii) induced secretion of glucagon-like peptide 1 (GLP1) from colonic L cells through the fatty acid receptor FFAR2(244). Furthermore, butyrate and propionate activate intestinal gluconeogenesis. Butyrate, through a cAMP-dependent mechanism, promotes the gene expression involved in intestinal gluconeogenesis. Propionate, itself a substrate for intestinal gluconeogenesis, activates its expression viaa gut-brain neural circuit involving the fatty acid receptor FFAR3 (245).

 

Given the key role played by microbiota in host nutrient processing and metabolism, it is not surprising that data points to a strong relation between gut microbiota and obesity and diabetes in humans. A reduced gut microbial diversity and altered microbiota composition is observed in obese individuals. There is also a low rate of gut microbial richness and specific bacterial groups are enriched or decreased in obese patients in comparison with lean people (246). Moreover, chronic diseases, such as obesity, diabetes, and HIV are associated with chronic low-grade inflammation. Gut microbiota regulates this inflammation through several mechanisms. Lipopolysaccharides (LPSs) from the outer membrane of Gram-negative bacteria may translocate through the intestinal border and cause subsequent systemic inflammation (247). Indeed, the intestinal barrier of obese patients is more permeable compared with that of lean individuals. Bile acids are characterized by a strong relation with gut microbiota affecting host’s body-weight homeostasis. Bile acids are microbially altered metabolites that are first endogenously produced by the liver and further metabolized by the gut microbiota (248). FXR signaling is an important pathway connecting gut microbiota and bile acids.

 

Based on the above knowledge, several interventions involving manipulation of the microbiome have been proposed as anti-obesity treatment. A diet which contains soluble fiber, prebiotics and/or probiotics could enhance the growth of beneficial gut microbiota and boost host metabolism. Lately, there has been interest in berberine administration in T1D, T2D, gestational diabetes, and prediabetes. The early reports of interventions using probiotics appear successful (249). Fecal microbiota transplant (FMT), the transfer of fecal suspension from a healthy (lean) donor into the gastrointestinal tract of an individual with disease (obesity) in order to restore a healthy gut is a potentially novel option to treat obesity.  However, there is not enough data about the safety of this method, that is why it is only FDA approved for recurrent Clostridium difficile infection.

 

Anti-Obesity Vaccines (Ghrelin, Somatostatin, Ad36)

 

The idea of a vaccination against obesity is also intriguing. The main action of these vaccines would be based on suppressing appetite-stimulating hormones or blocking food absorption. Three vaccines have been tested so far:

 

  1. An anti-ghrelin vaccine was found not only to reduce appetite by decreasing hypothalamic orexigenic signals but also to increase energy expenditure in rodent and pigs (250). Despite the promising results in rodents, clinical trials in humans showed no weight loss despite the development of ghrelin autoantibodies after four injections of anti-ghrelin vaccine (251). Another study, however, showed that IgG anti-ghrelin autoantibodies could protect ghrelin from degradation, suggesting that an autoimmune response may be involved in the orexigenic effects of ghrelin (252).

 

  1. An anti-somatostatin vaccine. Somatostatin is a peptide hormone which is produced, mainly, in the hypothalamus as well as other tissues, such as the gastrointestinal system. Somatostatin has the ability to suppress GH and insulin-like growth factor 1 (IGF-1) secretion. Reduced GH is associated with obesity and increased adiposity. So, the somatostatin vaccine could increase the secretion of GH and IGF-1(253). However, clinical trials in mice failed to reduce food intake, though a 10% improvement of body weight was observed (254).

 

  1. A live adenovirus 36 (Ad36) vaccine. Adenovirus 36 increases the risk of obesity in humans, characterized by increased inflammation and adiposity (255). Mice were injected with live Ad36 vaccine and compared to the control group (unvaccinated) after 14 weeks. The control group had 17% greater body weight and 20% more epididymal fats versus the vaccinated group, which also had decreased inflammatory cytokines and macrophages in fat tissue (256). (see Table 26)

 

Table 26. Anti-Obesity Vaccines

Drug name

Mechanism of action

Weight loss vs placebo

Clinical Benefits

Anti-obesity vaccine: somatostatin vaccine

Increases the secretion of GH, IGF-1

10%

 

Adenovirus 36

Live adenovirus36

 

Decreases body weight, epididymal fat in mice, inflammatory cytokines and macrophages

 

Nanomedicine

 

The introduction of nanomedicine in the field of obesity treatment is highly novel (257). Nanoparticles can achieve targeted drug delivery along with minimized side effects. The poor water-solubility of anti-obesity drugs can be overcome via nano-encapsulation. More specifically, nanoemulsion of orlistat has been tried in order to overcome its high lipophilicity, to improve its dissolution and to avoid the pancreatic lipase inhibition caused by this pharmaceutical agent in vivo (258). Additionally, a conjugated polymer-nanocarrier was created in order to reduce the side effects of orlistat (259). In 2014, the ability of mesoporous silica particles to reduce body weight was investigated (260). They found that the silica particles embedded in food could sequestrate lipase in their small pores through a lipase-specific interaction, leading to decreased absorption of fat.

 

Appetite suppression is an alternative method to decrease food intake and impact energy homeostasis (261). As mentioned above, however, anti-ghrelin vaccine was formed using virus-like particles for obesity treatment. The passive delivery of anti-ghrelin antibodies did not lead to long-term inhibition of food intake. So, to solve this problem, investigators immunoconjugated ghrelin with virus proteins to create a vaccine that was able to trigger an immune response leading to generation of specific anti-ghrelin antibodies. This anti-ghrelin vaccine played an important role in maintaining energy homeostasis in a DIO murine model.

 

In other examples, nanomedicine has enhanced the action of antiangiogenic agents in the treatment of obesity. Detailed above, antiangiogenic therapy inhibits the progression of adipocyte hyperplasia and reduces weight gain. A targeting nanoparticle was created in order to enhance the accumulation of the antiangiogenic drug in WATs by delivering it to vascular endothelial cells. Unlike WAT, brown adipose tissue (BAT) is full of mitochondria and a robust vascular structure helps to induce thermogenesis, increasing energy expenditure, and decreasing body weight. Thus, two nanoparticle platforms delivering browning agents to adipose tissue vasculature were formed (262). PPARγ nuclear receptor agonists (including rosiglitazone) have been shown to be characterized by anti-inflammatory properties against obesity and atherosclerosis. However, they are associated with severe side effects that limit their therapeutic use (263). In another, a mitochondria-targeted nanoparticle delivers the proposed anti-obesity compound PLGA-bPEG-triphenylphosphonium (TPP) polymer (264). The PEG shell extends the circulation time of nanoparticles, and TPP could facilitate the internalization into the matrix space of mitochondria to achieve targeted drug delivery.

 

Instead of targeted delivery, a localized and sustained release of a browning agent is a promising alternative for facilitation of WAT browning. Two nanoparticles, one injectable (265) and one in a painless microneedle array patch (266) were introduced. In vivo studies revealed successful delivery of the model drug into the human adipose tissue followed by ~15% decrease of weight gain after a four-week treatment.

 

CONCLUSION

 

The field of anti-obesity molecular pharmacotherapy is expanding. The homeostasis of body weight and metabolism are tightly linked to the central nervous system. The latter is characterized by centers that send orexigenic and anorexigenic signals regulating starvation and satiety, reducing and increasing energy expenditure, respectively. Pharmaceutical multi-agents in single compounds containing active portions of two or more drugs may allow for simultaneous effects on several synergistic pathways affecting appetite control and energy expenditure. Such medications could achieve increased weight loss with fewer side effects. Furthermore, the possibility of improved formulations (e.g., injectable forms of anti-obesity drugs and or once weekly verses daily administration) serve to enhance compliance. Considering that obesity is a multifactorial disease, it needs multimodal treatment. In an era where a variety of different therapeutic options is the norm for the management of chronic diseases such as type 2 diabetes and hypertension, the hope is that this process will led to better personalized anti-obesity treatments, focusing on the special characteristics, needs, and comorbidities of each patient and the effectiveness and safety of the recommended therapy. Thus, before starting any therapy, it will be important to record the detailed medical profile of the patient. Hereditary or acquired diseases, lifestyle parameters, and psychiatric history have to be taken into account when anti-obesity treatment is tailored for each patient. Further on the therapeutic horizon and still in much need of research are the place for altering gut microbiota balance and development of anti-obesity vaccines, novel peptide-mediated delivery of nuclear hormones, single molecular multi-agonists, and nanotechnologies that improve drug delivery and hold promise in the future of molecular pharmacotherapy of obesity.

 

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Subcutaneous Adipose Tissue Diseases: Dercum Disease, Lipedema, Familial Multiple Lipomatosis and Madelung Disease

ABSTRACT

 

Subcutaneous adipose tissue diseases involving adipose tissue and its fascia, also known as adipofascial disorders, represent variations in the spectrum of obesity. The adipofascia diseases discussed in this chapter can be localized or generalized and include a common disorder primarily affecting women, lipedema, and four rare diseases, familial multiple lipomatosis, angiolipomatosis, Dercum disease, and multiple symmetric lipomatosis. The fat in adipofascial disorders is difficult to lose by standard weight loss approaches, including lifestyle (diet, exercise), pharmacologic therapy, and even bariatric surgery, due in part to tissue fibrosis. In the management of obesity, healthcare providers should be aware of this difficulty and be able to provide appropriate counseling and care of these conditions. Endocrinologists and primary care providers alike will encounter these conditions and should consider their occurrence during workup for bariatric surgery or hypothyroidism (lipedema) and in those that manifest, or are referred for, dyslipidemia or diabetes (Dercum disease). People with angiolipomas should be worked up for Cowden’s disease where a mutation in the gene PTEN increases their risk for thyroid and breast cancer. This chapter provides details on the pathophysiology, prevalence, genetics and treatments for these adipofascial disorders along with recommendations for the care of people with these diseases. 

 

INTRODUCTION

 

People with subcutaneous adipose tissue (SAT) diseases have fat within this compartment that grows abnormally in amount or structure, often causing pain and other discomfort.  Subcutaneous adipose tissue is loose connective tissue, or adipofascia, which is the most common type of connective tissue in vertebrates. The focus of this chapter is on abnormal SAT that has within it changes in blood vessels, lymphatic vessels, immune cells, mesenchymal stem cells, fascia, interstitial matrix organ, or other components that make up loose connective tissue. 

 

The SAT diseases discussed here include lipedema, which commonly occurs in women, and four rare adipose tissue diseases (RAD): familial multiple lipomatosis, angiolipomatosis, Dercum disease, and Madelung disease (1). Adipose tissue in SAT diseases is resistant to loss by usual measures including extreme dietary changes (both hypocaloric and in macronutrient content) and exercise. Because of this, it is often referred to persistent fat tissue. People with diabetes and/or obesity may have a mixture of normal and persistent fat, making the understanding of SAT diseases by clinicians important in the care of these patients. Persistent fat may also be found in conditions where adipose tissue proliferation occurs, such as during infection, in autoimmune diseases, in those with hypermobile joint disorders, or with exposure to environmental toxins. Information on subcutaneous adipose tissue diseases not discussed here can be found in recent reviews and other Endotext chapters, including those covering lipodystrophies (2-4), cellulite (5), obesity (6,7), and other fat depots such visceral fat (abdominal, perirenal, pericardial), and perivascular fat (8).

 

Along with the gut (9), subcutaneous adipose tissue is thought to be one of the largest endocrine organs in the body (10). Subcutaneous adipose tissue houses immune cells including monocytes/macrophages, mast cells, and lymphocytes, which produce some of the hormones secreted by fat tissue (11,12). Greater amounts of fat and immune cells result in an inflammatory process that can lead to insulin resistance and slow intrinsic pumping of lymphatic vessels, which, in turn, may prolong inflammation in this tissue (13).   

 

Patients, most often women with swelling, have slow blood flow in, and lymph flow out of, depots of increased fat on the abdomen (14,15) or the gynoid area (hips, thighs and buttocks) (16). Poor blood and lymph flow through fat tissue results in accumulation of fluid, cell waste material, proteins, cells and other metabolic products in the extracellular matrix (ECM) around adipocytes and other components of adipofascia, resulting in a hypoxic environment, especially in adipocytes furthest from their nutrient and oxygen sources. These adipocytes then send signals that recruit more immune cells, resulting in a state of sustained inflammation and tissue degradation. Connective tissue then replaces degraded tissue in a process called fibroplasia or fibrosis. When tissue ischemia occurs or ECM accumulation outpaces its degradation, fibrosis may become unchecked and lead to widespread pathological remodeling of the ECM culminating in permanent scar tissue that completely inhibits flow through the adipose tissue (17).

 

Obesity is a main cause of densification of fascia and fibrosis development in loose connective tissue (18). The result is a fibrotic mesh around adipocytes and fat lobules which has been well described (19). This sick fat, or adisopathy, increases the risk of metabolic disease (20). In addition, more fibrotic adipose tissue is less responsive to mobilization and reduction attempts through diet, exercise, use of weight loss medications, or bariatric surgery (19,21,22). All the SAT disorders in this chapter have a component of fibrosis in the tissue that prevents loss by usual measures. An important goal is to determine why the loose connective tissue in SAT diseases becomes fibrotic and adisopathic to prevent its occurrence and progression and treat when identified.

 

LIPEDEMA

 

Lipedema is a common SAT disease that was first described in 1940 at the Mayo clinic by Drs. Allen and Hines (23). A second seminal paper in 1951 provided a description of lipedema that is still commonly used for clinical diagnosis. Lipedema occurs almost exclusively in women but has been reported rarely in men (24-26).

 

Lipedema fat is located just under the skin on the limbs including upper arms, hips, buttocks, thighs, lower legs, generally sparing the trunk and feet. It feels nodular when palpated, may be painful to touch, and often has prominent superficial veins. Lipedema tissue can be found under the umbilicus and in some woman, a deeper nodular adipofascia is found in the lateral abdomen. This inner nodularity may reflect changes in the ECM that may be a precursor to lipedema fat if abdominal obesity develops. Disease defining key questions and physical examination characteristics can help to establish the diagnosis of lipedema (Figure 1).

Figure 1. Characteristics of lipedema that aid in establishment of diagnosis are listed, and many can be seen in the accompanying photo. This patient was diagnosed with Stage 3, type III and IV lipedema without lymphedema (see below). A quick and easy set of questions and exam findings are included to help in diagnosis of women with lipedema.

Classification of Lipedema

 

Lipedema is classified by stages and types (27). In Stage 1 (Figure 2) the skin surface is normal over an enlarged hypodermis that already has palpable pea-sized nodules in the fat. These pea-sized nodules represent enlargement of and fibrosis in the ECM and in the connective tissue surrounding the fat lobule. Stage 2 skin is uneven with indentations (like cellulite) representing thickening and contraction of underlying connective tissue fibers over increased fat with small to larger hypodermal masses. Lobular extrusions of skin, fat and fascia tissue in Stage 3 represent significant loss of elasticity in the adipofascia drastically inhibiting mobility; blood flow in and lymph flow out of the lobules is reduced resulting in inflammation followed by fibrosis; it is in this stage that fibrosis in the tissue becomes prominent and clearly palpable; fibrosis may or may not affect the skin and the skin may develop thinning and a looser connection to the underlying hypodermis (Figure 1). Modifications of diagnostic criteria for lipedema have been suggested (28).

 

Lymphedema can occur at any stage, but is more often found in women with Stage 3 lipedema when it is often called lipo-lymphedema or Stage 4 (26). Rather than use this combined term, however, it is preferable to identify the lipemia stage and state whether lymphedema is also present or not. Lymphedema can be identified in women with lipedema by visible swelling of the hands or feet, pitting edema, asymmetry between the size of one limb versus the other, and/or a positive Stemmer sign. A positive Stemmer sign occurs when edema in the limb makes it difficult to pinch skin on the great toe, top of the foot, finger, or dorsal hand. A negative Stemmer sign occurs when only skin is pinched. Other differences between lipedema and lymphedema include symmetry (lipedema tissue occurs symmetrically versus lymphedema, which is usually unilateral), sparing of the hands and feet in lipedema, and report by patients of pain in lipedematous but not lymphedematous tissue.

Figure 2. Three stages of legs of women with lipedema with subcategories of types. In Stage 1, the skin is smooth, and the legs can appear normal but there is pain, easy bruising and a nodular feel to the fat tissue. In Stage 2, the lipedema fat exhibits a mattress-like pattern indicating fibrosis under the skin that tethers on the skin that can be found on the upper legs (Type II) or extend down to the ankles (Type III). In lipedema Stage 3, there are folds of tissue and the lipedema fat usually extends down to the ankles. For description of the types of lipedema, see Figure 3.

The type of lipedema is defined by its anatomical location (29). Women with Type I lipedema have affected fat on the pelvis, buttocks and hips (saddle bag phenomenon). Women with Type II have affected fat from the buttocks to knees with formation of a tender deposits of fat around the inner side of and below the knee. Women with Type III lipedema have affected fat from the buttocks to ankles where a prominent “cuff,” or ledge, of fat tissue develops. Women with Type IV lipedema fat have affected arms and women with Type V lipedema, a rare type, have only affected lower legs. The most common phenotype of women with lipedema are combinations of II and IV or III and IV (Figure 3).

Figure 3. Types of lipedema fat. Lipedema fat may be located from the umbilicus down to the bottom of the hips (Type I), down to the medial knees usually including a pad of fat on the inner knee and below the knee (Type II), and down to the ankle (Type III) where a “cuff” of fat develops but spares the dorsal foot. Rarely only the lower legs are affected (Type V). Lipedema affecting the arms alone is rare (Type IV) and, instead, usually is found in combination with Type II or III lipedema. The arms can be variably affected with nodular lipedema fat around the cubit nodes, over the brachioradialis, down the medial arm to the wrist in line with the thumb or 5th digit, the entire lower arm, or the entire arm.

Prevalence of Lipedema

 

From one specialty lymphedema/obesity clinic in Germany, the prevalence of lipedema in women was estimated to be 11%. Estimates from similar clinics reported prevalence rates of lipedema ranging from 15 to 18.8% (30,31). The prevalence of lipedema in children in the United States in a vascular clinic was reported to be 6.5% (32). Examination of 62 women outside of clinics in Germany found a prevalence rate for all types of lipedema of 39% (33).  Using the lowest prevalence estimate in adults of 11%, over 16 million women in the US may be affected with lipedema.  

 

Genetics of Lipedema

 

The gene or genes underlying lipedema are not known, but another affected immediate family member has been reported in up to 60% of women (34-37). This is compatible with an autosomal dominant inheritance with incomplete penetrance (38) in which an affected parent has a 50% chance of passing lipedema to their child. Supportive of a genetic component, lipedema has been reported in children (32) and as early as infancy by some women. The phenotypic expression of lipedema may vary amongst affected females, especially if there is also obesity in the family. Males that carry the putative gene do not generally exhibit the phenotype, even the fathers of affected daughters. 

 

A mutation in the POU1F1/PIT-1 gene has been shown to cause multiple anterior pituitary deficiencies including thyroxine and growth hormone (GH) deficiency. A PIT-1 mutation was associated with the phenotypic presentation of lipedema in a short mother but not her short son who also carried the mutation, and not in her normal height daughter who did not carry the mutation (39). In mice with GH receptor (GHR) antagonism or lacking GH function, subcutaneous adipose tissue is increased more than other fat depots similar to lipedema in humans (40). No other cases of women with lipedema and a mutation in PIT-1 have been reported to date and women with lipedema are not known to be more likely to have short stature. Classic features of lipedema can be found in people with Williams syndrome caused by a chromosomal 7q11.23 microdeletion of ~1.6 million base pairs, which includes the elastin gene ELN (41). Loss of elasticity results in the reduction of the ability of tissue to contract back after being stretched. Changes in elasticity may therefore allow more fat to grow.  These reports suggest there may be more than one genetic mutation causing expression of the lipedema phenotype. Additional genes that may be important in the development of lipedema have been reviewed (42).

 

Pathophysiology of Lipedema

 

The cause of lipedema remains unknown. Given the predominance of occurrence in women, it is tempting to consider sex steroids, especially estrogen, as major triggers or contributors of this condition. That knee laxity in women peaks when estrogen levels decline between ovulation and post-ovulation is indictive of estrogen’s effect on connective tissue (43). Other observations that support this effect include reports that lipedema is often first noticed at the time of puberty in young girls and occasionally following pregnancy, when multiple hormone levels are high, including estrogen. Although data in men is sparse, those reported to have lipedema tend to have low testosterone or liver disease, both of which are associated with a relative increase in estrogen levels and therefore a higher estrogen to testosterone ratio (21). While higher levels of testosterone in women with polycystic ovarian syndrome are not protective against lipedema, the adipose tissue in women with this condition may be predisposed to lipedema due to abdominal obesity and inflammation associated with pre-diabetes or diabetes. A causative role for estrogen in the expression of lipedema remains speculative until well-controlled studies are conducted that quantify sex hormone levels, sex hormone receptors, tissue effects, metabolism or sex hormone driven pathways in men and women with lipedema. 

 

PROPOSED THEORIES TO EXPLAIN LIPEDEMA

 

Theory 1: Abnormal Blood Microvasculature

 

We and others (36,44,45) have advanced the theory that increased compliance from structural changes in connective tissue results in the ability to hold on to fluids, proteins and other constituents within the ECM and is causally important in the development of lipedema. As in lymphedema, changes in lipedema tissue may occur globally but are likely to also be found regionally in the same limb (46). Over 50% of women with lipedema are thought to have some kind of joint hypermobility consistent with a connective tissue disorder (25). Most women with lipedema and hypermobility fall into the Ehlers Danlos spectrum of disorders, the gene for which is not known (47,48). This hypothesis is consistent with loss of elasticity in tissue resulting in classic features of lipedema in Williams syndrome. Structures in loose connective tissue that contain elastin include blood vessels, lymph nodes, and connective tissue fascia that helps loose connective tissue hold its shape. Initial lymphatic vessels do not have elastic fibers, but elastic fibers support lymphatic vessels to open and close in response to pressure changes in the tissue; loss of elasticity could reduce the ability of lymphatic vessels to open with increased pressure in the ECM. Capillaries do not have elastic fibers but the loose connective tissue surrounding them does; as loose connective tissue enlarges due to growth of adipocytes, capillaries are at risk for dilation and distortion. Dilated and/or distorted capillaries may release their contents into tissues at a higher rate and/or amount in lipedema which initially, lymphatic vessels promptly pump out. Over time, due to compliance in fascia surrounding cells, malfunction of lymphatic vessels, and increased infiltrate leaving altered capillaries fill the ECM, with protein dense and salt-rich (49) deposits that accumulate in the interstitial space clogging flow through the tissue resulting in hypoxia. Hypoxia results in the secretion of hypoxia inducible factor (HIF)-1 by local adipocytes, which stimulates hypoxia response elements on a number of genes including the vascular endothelial growth factor (VEGF) gene and inducing proliferation of stem cells in the adipose tissue (50). Levels of VEGF have been shown to be elevated in women with lipedema (n=9) compared to women without lipedema (n=5) (51), supporting an underlying role for hypoxia in the pathogenesis of lipedema. 

 

Lymph fluid induces adipogenesis when added to adipocytes (52). Therefore, accumulation of pre-lymph fluid in the ECM may be a stimulating factor in adipogenesis. Mesenchymal stem cells isolated from lipedema stromal vascular fraction (SVF), a  heterogeneous collection of cells surrounding adipocytes within adipose tissue, contained the connective tissue cell marker CD90+ and endothelial/pericytic marker CD146+ (53). With ~50% of cells in the SVF expressing +CD146+, perivascular cells (pericytes) play a physiological role in vascular development and homeostasis (52). The presence of such high numbers of pericytes is consistent with a chronic capillary injury in lipedema leading to increased need for repair and increased protection of vessels.

 

As overworked lymphatic vessels eventually lose function, microaneurysms form in the lymphatic vessels in lipedemadous tissue, becoming high risk for breakage and leakage analogous to what happens in lymphedema (54,55). Adipokines, especially large adipokines such as leptin and monocyte chemoattractant protein (MCP)-1, become secreted primarily into the lymphatic system rather than blood capillaries (56). It would then follow that adipokine flow out of lipedema loose connective tissue would be diminished and systemic levels lower (e.g., leptin levels), leading to larger quantities of adipose tissue necessary to achieve weight homeostasis. 

 

Other potential contributors to expression of lipedema include release of lipids from leaky lymphatic vessels in the gut and tissue level (57), which could mediate induction of adipogenesis and have an important role in the development of the loose connective tissue in lipedema (58). Finally, inflammation in response to hypoxia or injuries could facilitate the development of fibrosis within loose connective tissue, not only reducing flow through the tissue further, but also impeding fat loss during weight reduction (19,21,22).

 

Theory 2: Abnormal Lymphatic Vasculature

 

Another theory posits that fluid accumulation in the ECM results from a primary defect in lymphatic vessels. Such accumulation enhances permeability issues in surrounding blood microvessels (59). In support, of this hypothesis, one study of women with lipedema and obesity noted a mismatch in the number of lymphatic vessels and the increased numbers of blood vessels in affected tissue (60). Instead there was an increase in the size (area) and area/perimeter ratio of the lymphatic vessels. Increased angiogenesis but fewer numbers and dilated lymphatic vessels has also been reported in a diet-induced obesity model in mice (61).  In another supportive study of lipedemadous tissue free of lymphedema, an expansion in the size of lymphatic vessels but no significant changes in transport in of lymphatic fluid was reported (62). However, against this hypothesis of a primary defect in lymphatic tissue as the proximal cause of lipedema is that lymphatic vessel function as determined by lymphangioscintigraphy appears normal in many women with early stages of lipedema and only later can reductions in lymphatic flow rate or function be detected in many women with late stage lipedema (54,63,64).  

 

Markers of Obesity, Cardiometabolic Health, and Aortic Disease in Women with Lipedema

 

Hypertrophic adipocytes, a marker of an inflammatory environment at risk for insulin resistance and other metabolic dysfunction, are reported in loose connective tissue in lipedema from women regardless of whether they were obese or not (58,65). Adipogenesis has also been identified in lipedema loose connective tissue (58) as has hypertrophy and hyperplasia of adipocytes in people who developed obesity after lifestyle changes. Unhealthy hypertrophic adipocytes undergo necrosis and become surrounded by macrophages that phagocytize the dead adipocytes forming crown like structures on histological exams of tissue. Crown-like structures have also been found in the loose connective tissue of women with lipedema (53,58). 

 

Adipose tissue stem cells collected from subcutaneous adipose tissue from people with obesity have reduced adipogenic potential and proliferative ability (66). The same reduction in adipogenic potential including a reduced capacity to produce leptin by cells in culture was found for adipose tissue removed by tumescent liposuction from women with lipedema compared to women without lipedema (67). These data support the possibility that even in early stages of lipedema when BMI is in the non-obese range, lipedema fat tissue shares characteristics of adipose tissue taken from people with obesity. Thus, even though femoral adipose tissue is known to be cardioprotective (68), this association weakens in later stages of lipedema. The later the stage, the greater the obesity and metabolic risk, including lower high density lipoprotein (HDL) cholesterol levels, higher diastolic and systolic blood pressures, higher reported history of hypertension, and higher percentages of pre-diabetes (69).  As such women with advanced lipedema should be closely monitored for these conditions as part of their ongoing care.

 

Women with lipedema are thought to have a connective tissue disease along the spectrum of hypermobile Ehlers Danlos. Transthoracic 2D echocardiography (2DE) and Doppler imaging revealed that women with Stage 2 lipedema in their early 40s with BMI ~30 kg/m2 had impaired left ventricular apical rotation and left ventricular twist compared to people with lymphedema and those without either disease (70). Another paper by the same group used 2DE and Doppler imaging demonstrated enlarged ascending aortic systolic and diastolic diameters resulting in aortic stiffness in women with lipedema compared to controls (44). Individuals with Williams syndrome with loss of elasticity and features of lipedema also have aortic stiffness (71). These cardiac changes may reflect an underlying connective tissue disorder in women with lipedema and the possible need for cardiovascular screening even if lipid levels and other markers of metabolic syndrome are normal.

 

Imaging of Lipedema

There are currently no imaging exams that can be used to definitively differentiate lipedema fat from non-lipedemadous adipose tissue.  However, some imaging studies may be useful.  Nuclear medicine lymphangioscintigraphy (NM LAS) may be helpful in differentiating the presence of lymphedema in patients with lipedema. Flow of Technetium-99m-sulfur colloid injected dermally and taken up by the lymphatic vessels starting at the toe or finger webbing can be normal in lipedema (72), or the lymphatics can be tortuous especially below the knee (73). Other authors found slower lymphatic flow and a marked asymmetry of the lymphatic system in women with lipedema as compared with women without lipedema (74,75).          

 

Dual energy X-ray absorptiometry scans (DEXA) can be used for assessing whole body composition including regional fat mass and lean body mass in addition to bone mineral density; some scanners also estimate visceral fat mass. One study suggested that DEXA can be used to strengthen the confirmation of a diagnosis of lipedema in women, differentiating them from women without lipedema by a cutoff value of 0.46 for fat mass in the legs (kg) adjusted for BMI (76). Even though many women with lipedema also have obesity, the authors assert this cutoff value allows for a separation of lipedema of the legs from women without lipedema regardless of obesity.

 

Ultrasound of lipedema tissue compared to control tissue or tissue from women with lymphedema demonstrates thinner skin in agreement with previous data (77), and increased thickness and hypoechogenicity of the subcutaneous fat throughout the lower limb suggesting a diffuse increase in aqueous material (78). The hypoechogenicity was most significant in the distal extremity (medial calf) and may provide support to a clinical diagnosis when found.  Another group found no difference between ultrasonographic features of women with and without lipedema including compressibility and echogenicity (79). The control women were reported to have obesity or lipohypertrophy, an enlargement of the legs that is phenotypically similar to lipedema but painless. The definition of lipohypertrophy is unclear in the literature where authors have stated that symptoms of lipohypertrophy resolve with elevation suggesting a fluid component associated with the fat tissue (80). In personal communication with the authors, they state the relief of symptoms is due to lowering of pressure on the venous system suggesting that venous disease is important in the diagnosis of lipohypertrophy. Other authors state that lipohypertrophy is a precursor to lipedema which may explain why the latter ultrasound data showed no differences. Clearly, better means of distinguishing lipedema from those with larger legs but no lipedema is needed.

 

Finally, widening of lymphatic vessels up to 2 mm has been found by magnetic resonance imaging (MRI) of the legs of women with lipedema; women with lipedema and lymphedema had lymphatic vessel enlargement >3 mm (81). If this dilation of lymphatic vessels is consistent with lymphostatic decompensation (failure of lymphatic vessel function) in lipedema as the authors suggest, then salt should be found in the skin of women with lipedema as lymphatic vessels regulate Na+, Cl– and water in the skin, where reduced lymphatic vessel numbers are paralleled by increased blood pressure (82). Indeed Crescenzi et al. found increased salt in the skin and loose connective tissue of women with lipedema compared to women without lipedema, even in earlier Stage 1 lipedema where women tend to not have obesity (49).

 

Conditions Associated with Lipedema

 

OBESITY

 

Women with lipedema are often are often thought of as having common obesity whether or not they meet BMI criteria for this condition. The two striking differences between women with lipedema and women with obesity are that women with lipedema often have tenderness of the affected tissue and/or easy bruising of the skin overlying the lipedema fat, which is not found in women with common obesity (Table 1). However, some women with lipedema do not have pain in their tissues.  It is unclear if women with lipedema with pain have the same disease as women without pain. Of note, women with lipedema may have no pain in their lipedema fat tissue when they are well-controlled under treatment regimens, however, they should still be considered to have lipedema. For example, women with lipedema who eat low inflammatory foods, avoiding processed starch and sugar, who exercise most days of the week and wear compression garments on their legs can have minimal to no pain. Therefore, a good history is important to identify a history of pain in the tissue that would be indicate the presence of lipedema. As described above, helpful measures to differentiate women with lipedema from women with common obesity include the disproportionate distribution of their adipose tissue between the trunk and legs, any family history of lipedema, as well as a historical inability to lose much fat from the lipedema-affected areas. On the other hand, when women with obesity and lipedema lose more substantial weight through medical or surgical interventions, they can lose some fat from the areas with lipedema, which can then leave them with rolls of excess skin along in the areas of remaining lipedema fat tissue (Figure 4). 

 

Table 1. Clinical Similarities and Differences Between Lipedema and Obesity

Sign/Symptom

Lipedema

Obesity

Sex affected

Females

Females and Males

Onset

Puberty

Any age

Increased fat

Common

Common

Gynoid disproportion

Common

Possible

Influenced by lifestyle

No

Yes

Tenderness of the tissue

Common

Absent

Easy bruising

Common

Absent

Pitting edema

Uncommon

Uncommon

Stemmer sign

Negative

Negative

Able to lose fat from the legs/hips

Minimal

Common

 

Obesity (especially abdominal/visceral obesity) and/or polycystic ovary syndrome can worsen lipedema severity. This is thought to be mediated by increases in adipokines, tumor necrosis factor alpha (TNFa) and leptin that often accompany these conditions, which are associated with venous disease (83). Venous dysfunction that can lead to leakage of fluid back into tissue due to reflux may also be an important contributor to worsening of both lipedema and lymphedema, when present (84,85). 

Figure 4. Fat due to obesity and fat due to lipedema can be intermixed on the legs. With weight loss, the obesity fat can be lost resulting in excess skin and lipedema fat tissue remaining on the legs.

LYMPHEDEMA

 

Women with lipedema are at risk for developing lymphedema, which may happens in lipedema Stage 3 > Stage 2 > Stage 1 (26). The presence of lymphatic disease or lymphedema increases the risk of cellulitis and wounds, which can be difficult to manage and disfiguring. Women with heavy limbs and swelling should be considered for manual lymphatic drainage and deeper tissue therapies such as instrument assisted soft tissue therapies (e.g., Astym therapy, Graston technique) or manual therapies (e.g., myofascial therapies or other deep tissue therapies (86)), followed by compression plus reduction of obese adipofascia to reduce the risk of developing lymphedema.

 

PSYCHOSOCIAL

 

Psychosocial issues are prominent in women with lipedema including appearance-related distress and depression (87), which can result in eating disorders (88). This is not surprising in the United States where very thin women or photos of women that have been photoshopped to accentuate the appearance of leanness are posted on the internet and on television. Imagine how a woman in today’s society might feel who developed lipedema at puberty and is told to diet and exercise by friends, family and healthcare providers to lose weight, something that she has done for years to no avail. One author polled women in Germany and found a high rate of suicide attempts in women with lipedema (89). Lower mobility associated with lipedema and obesity were also found to affect quality of life in women living with lipedema (87) and may contribute to social isolation. Prevention and management of obesity in women with lipedema becomes paramount to maintain their quality of life. High anxiety is associated with hypermobile joint disorders (90), and because hypermobile joint disorders are associated with lipedema (25), anxiety should be assessed and treated to help women living with lipedema.

 

DERCUM DISEASE (PAINFUL LIPOMAS)

 

Lipedema can be present in the same individual who also has Dercum disease (see below), and in this instance, would be considered a mixed disorder. Authors have tried to differentiate women with lipedema from those with Dercum disease by examining populations and finding that people with Dercum disease tend to have other pain disorders including higher pain scores, fibromyalgia, abdominal pain, and migraines, and more often have lipomas, cognitive dysfunction and shortness of breath; whereas women with lipedema have more often fibrotic tissue, easy bruising, hypermobile joints, venous disease and edema of the feet (25).

 

Numerous other conditions apart from those described above have been associated with lipedema (Table 2). Of note, hypothyroidism is found in 27% of women with lipedema (25,26).  In one case, a woman with lipedema was described as having lymphedema and multiple symmetric lipomatosis (91).

 

Table 2. Co-Morbidities and Complications Associated with Lipedema (60, 92-94)

Musculoskeletal

Soft Tissue

Vascular

Other

Gait disturbance

Obesity; fat deposits

Lymphedema/Idiopathic Edema

Pain

Change in posture (e.g., lordosis)

Loss of skin elasticity

Dilated Capillaries Microangiopathy

Psychological distress/anxiety

Genu valgum and arthritis of the knees

Thinning of the skin

Bruising

Shortness of breath

Ankle pronation

Lipomas

Varicose veins

Venous insufficiency

Cellulitis

Hypermobile joints (Hypermobile Ehlers Danlos?)

Cellulite; fibrosis

Cherry angiomas

Slow metabolic rate

 

Clinical Care of Women with Lipedema

 

Depending on whether the astute clinician makes the diagnosis of lipedema during the course of taking a history and physical, affected patients may more typically seek care for an associated co-morbidity. For example, women with lipedemia who have thyroid disease and/or obesity may regularly be referred by their primary care provider to an Endocrinologist. Endocrinologists should be clued in to a possible diagnosis of lipedema in women who present with difficulty losing weight from their hips, buttocks and legs, and who are convinced they have a thyroid issue, but thyroid labs are normal. In desperation, patients with lipedema may ask for a “complete set of thyroid labs” including thyroid stimulating hormone (TSH), free T4, free T3, reverse T3 and thyroid peroxidase (TPO) antibodies to ensure that there is no thyroid issue, which may cause tension during a clinical visit when a provider chooses not to order all these laboratories. Other times, women with joint hypermobility are often cared for by rheumatologists and orthopedic surgeons, and those with lipedema and venous disease are often followed by vascular surgeons, physical therapists and lymphedema specialists.

 

DIAGNOSIS

 

Once the possibility of lipedema is considered, a good medical history will include an assessment of the food eaten, patterns of exercise, and a timeline of development of lipedema signs and symptoms with special attention to hormonal transitions in women including puberty, pregnancy, or menopause. Additionally, helpful findings on history include pain and easy bruising in affected areas and a family history of similar traits in other female members. The upper arms and legs should then be examined for physical manifestations of lipedema as described in more detail below. In the author’s experience, diagnosing lipedema in a woman who presents thinking they have another condition, such as thyroid disease but with normal thyroid function tests, and providing education and treatment recommendations can be transformative for the patient’s life and greatly enhance the patient-physician relationship. 

 

The physical exam to diagnose lipedema can be performed quickly if a woman can be seen in her underwear after donning a gown.  Visual inspection to establish disproportionality between the upper and lower body fat should be done initially and include a measure of the waist and hip ratio, which is also helpful in diagnosing central obesity.  Following this, examination (both visually and by palpation of fat tissues) should be performed with special attention to characteristics described in Table 3.

 

Table 3. Examination of Subcutaneous Fat for Lipedema, With or Without Obesity

No Obesity

Head

Normal

Neck

Normal

Arms

Normal (nodular fat tissue may be found around cubital nodes)

Wrist

Normal

Hands

Normal; Stemmer sign negative (no edema)

Abdomen

Normal (nodules may be found deep laterally or under the umbilicus)

Buttocks

Increased loose connective tissue; may be nodular and heavy

Hips

Increased loose connective tissue; may be nodular and heavy

Thighs

Increased loose connective tissue; may be nodular and heavy

Medial knee

Nodular or enlarged fat pad; usually tender

Under knee

Fat pad; may be nodular

Shin

May be covered in fat making the shin hard to palpate

Lateral malleolus

May have fat pad underneath

Ankle

Cuff may be very small but present in Type III lipedema

Feet

Normal; stemmer sign negative

Skin

Bruising; livedo reticularis; may see peau d’orange with long-standing disease

With Obesity

Head

May have hair loss and increased fat

Neck

May have filling of the supraclavicular fossae

Arms

Nodular fat tissue on upper and/or lower arms and around cubital nodes; hanging fat on upper arm that may be heavy

Wrist

A cuff of fat may be present; bend the hand back to easily see the cuff

Hands

Fat may be found at the base of the thumb, between the MCP joints or over the hand

Abdomen

Increased deposit of fat above and/or below umbilicus

Buttocks

Increased loose connective tissue; may be nodular and usually heavy

Hips

Increased loose connective tissue; may be nodular and heavy

Thighs

Increased loose connective tissue; may be nodular and usually heavy

Medial knee

Nodular fat pad; usually tender

Under knee

Fat pad; may be nodular

Shin

Usually covered in fat making the shin hard to palpate

Lateral malleolus

May have fat pad underneath

Ankle

Cuff present in Type III lipedema

Feet

May have increased fat; Stemmer sign negative (no edema) when no lymphedema

Skin

Bruising; livedo reticularis; may see peau d’orange with long-standing disease

There is a wide variation in the phenotype of lipedema (see Figure 2 Types) therefore lack of one or more physical exam finding does not negative the presence of lipedema.

 

At present, lipedema does not have an International Classification of Disease (ICD)-10 code but an ICD-11 code of EF02.2 has been proposed.  In the meantime, other ICD-10 codes useful when caring for patients with lipedema are listed in Table 4.

.

Table 4. ICD-10 Codes for Clinical Visits for Patients with Lipedema

Sign/Symptom

ICD-10 Code

Lymphedema/Swelling (may be non-pitting)

I89.0

Edema unspecified

R60.9

Lipomatosis not elsewhere classified

E88.2

Chronic pain

G89.4

Venous insufficiency

I87.2

Varicose veins

I83.10

Overweight

E66.3

Other Obesity

E66.8

Obesity (ICD-10 code varies by BMI)

Z68

 

Treatments for Lipedema

 

FOOD PLANS

 

Many women with lipedema bring along family members that can attest to their healthy or minimal eating and beneficial exercise patterns as they tend not to be initially believed by healthcare providers. There is very little data on the use of diets to reduce lipedema fat.  Although poorly studied, it is generally accepted that lipedema fat is resistant to weight loss mediated through lifestyle, which compounds patients’ frustrations when weight loss expectations are not met. In the absence of specific recommendations, dietary counseling can focus on establishing healthy eating patterns for overall health improvement and weight management.

 

Food plans are important in helping manage obesity that accompanies lipedema with a minimal goal of stabilizing weight and a maximal goal of losing obesity weight. The most successful food plans are those with low processed carbohydrates including added sugars that reduce insulin levels and inflammation and, therefore, reduce adipogenesis (95); fasting between meals (no snacking) has been suggested (96). One group used a 1200 calorie diet along with complete decongestive therapy to reduce volume in the legs of women with lipedema (97), but evidence for long-term weight loss maintenance by this approach is lacking.

 

EXERCISE

 

Exercise is important for women living with lipedema as the muscle action helps pump blood and lymph fluid through the limbs. However, women with lipedema have ~67% of the normative value for quadriceps muscle strength compared to women without this condition matched for age and BMI (98). One theory is that fibrosis from the fat tissue extends into and reduces muscle function. The Dutch guidelines for lipedema recommend graded exercise programs aimed at strength training and conditioning for women with unhealthy lifestyles or physical limitations, although they recognize that the body parts affected by lipedema tend to increase in tissue volume despite activity (80). These authors also state that exercise and heat can increase swelling and pain in the lipedematous areas. Anecdotally, women with lipedema appear to benefit greatly from water exercises, which may be in part due to the compressive effect of water on the body that helps mobilize fluid and soften fibrotic tissues, as well as from water jets that may also help reduce fluid in the adipofascia. Some women with lipedema have a concern about showing their bodies in public due to the commonality of public body shaming (99). Cropped pant, swim tights and other swimwear coverings have enabled more women with lipedema to feel comfortable during public swimming.  Garments with compression are generally recommended for women with lipedema to wear during land-based exercises especially, when using Nordic poles that improve the adipofasica of the arms and legs.

 

COMPRESSION GARMENTS

 

Compression garments are usually worn on the legs with a high waist (to treat fat on the abdomen) and on the arms as needed. Compression can be lower in millimeters of mercury (mm Hg) for lipedema than for lymphedema. For example, 15-25 mm Hg or Class I 20-30 mm Hg, compared to Class II 30-40 mm Hg or Class III 40-50 mm Hg. The type of knit for a lower pressure garment can be circular knit, which means it is seamless and is knitted on a round cylinder. Circular knit garments have more stretch and are best suited for women with lipedema that have less lymphedema or swelling. They can also stretch to fit any shape and size. Flat knit garments have less stretch and therefore provide better edema control. Flat knit is recommended especially for women with lipedema who have an ankle cuff or unusual shape requiring a custom fit and usually have a seam. A durable medical equipment (DME) order can be provided to patients to take to a medical supply store if they are able to get insurance coverage for compression garments. Therapists treating patients with lipedema can provide guidance on compression wear.

 

VENOUS DISEASE

 

Venous insufficiency has been documented in 25% of women with lipedema (92,100). When pitting edema is present, venous insufficiency should be investigated in women with lipedema by a venous duplex ultrasound of the legs. These studies are performed in a vascular lab and should specify to look at the greater and lesser veins of the legs to evaluate for venous insufficiency and not just thrombus. Care should be taken to treat venous disease conservatively first as there is no data showing correction of venous insufficiency by surgical means will improve lipedema. Anecdotally in reports from women with or without lipedema, lymphedema can occur after surgical treatment of venous insufficiency of the greater saphenous vein (101).

 

BARIATRIC SURGERY

 

Women with lipedema without some upper body obesity may respond poorly to bariatric surgery with regard to weight loss (102) and often feel like failures or are mistakenly told (directly or indirectly) by their providers that it was their fault, with devastating psychological impact.  Indeed, women that lose minimal weight from their lower abdomen, hips, and legs after bariatric surgery should be examined for the presence of lipedema. Even with less-than-expected weight loss, patients with lipedema should still be considered candidates for bariatric surgery as several procedures (e.g., laparoscopic gastric bypass and sleeve gastrectomy) have shown weight-independent benefits on glucometabolic outcomes, especially prediabetes and diabetes, and cardiovascular risk. When women do lose weight and it includes a portion of their lipedema-affected regions, it often results in accentuation of the “saddle bag” look (See Figure 4) and may worsen their body image anxieties.  Optimally, women with lipedema should be identified prior to bariatric surgery, counseled on their condition and how it might influence their overall weight-loss response, and be offered complete decongestive therapy and compression garments to reduce the risk of developing lymphedema after bariatric surgery and to improve weight loss success.  In addition, pre-surgery is a good time to initiate consultation with a plastic surgeon regarding options of removal of excess skin removal once weight stability is established post-operatively (usually between 1 and 3 years).

 

LIPOSUCTION

 

Women with lipedema typically have several medically necessary reasons for undergoing liposuction to remove lipedema fat, including:

  • Loss of mobility
  • Reduced quality of life
  • Joint damage or altered gait
  • Chronic pain
  • Failure to improve signs and symptoms associated with lipedema despite conservative therapy

 

Complete decongestive therapy including manual lymphatic drainage, compression garments, a healthy eating plan, and as much activity as allowed or possible are important before liposuction to improve outcomes. Due to the increased vascularity of the lipedematous tissue and blood loss with liposuction, post-procedure anemia is not uncommon. Therefore, labs prior to surgery should include a complete blood count (CBC) with platelet level as well as coagulation labs to include activated prothrombin time (aPTT), prothrombin time (PT), thrombin time (TT), andfibrinogen.  People with normal coagulation labs and easy bruising can have hereditary and acquired platelet defects, hereditary disorders of vascular and perivascular tissues including Ehlers Danlos Syndrome, and other disorders of blood clotting.  Any woman with lipedema and a personal or family history of bleeding or clotting should work with a healthcare provider to determine if additional testing is needed before liposuction surgery (103).

 

Removal of lipedema fat by liposuction that spares lymphatic vessels (wet, not dry, technique) has been performed primarily in Europe, especially in Germany, since the 1990s (104-107).  The fat is saturated with Klein solution which includes saline or lactated Ringers solution, an anesthetic such as lidocaine or prilocaine, epinephrine, sodium bicarbonate buffer(108), usually without steroid (109).  This tumescent technique provides turgor to the tissue allowing blunt microcannula to slide through the fat tissue avoiding creation of shearing forces and tissue damage. When power assisted, tiny, rapid vibrations of the microcannula break up fat which is then suctioned out of the tissue. Water jet assisted liposuction (WAL) uses jets of saline and Klein solution to release fat for suction with minimal damage to cells and vessels(106) without the waiting period required to tumesce the tissue. Laser assisted tumescent liposuction is another technique which some reserve for fibrotic areas such as the posterior thighs.

 

Most affected women undergo liposuction in stages, involving removal of an area of lipedema fat from the lower body and arms followed by a period of recovery and healing before returning to remove an adjacent region. The average number of surgeries for a women with Stage II lipedema ranged between two and three (110), but some had more than five (37). Patients are either awake during the liposuction procedure with or without conscious sedation (104,111,112), or general anesthesia, the former allowing for rapid recovery (111). Some medications used in general anesthesia reduce the pumping activity of lymphatic vessels (113-116). Prior to undergoing liposuction by a qualified surgeon, therefore, a patient should have a thorough understanding of the surgeon’s technique, whether the surgeon uses general anesthesia along with the type of analgesia, the number of surgeries performed by the surgeon and outcomes and their complication rate. After liposuction, the surgically treated areas may be quite tender and uncomfortable for days to weeks.

 

Most studies on liposuction are from surgeons performing the procedure, are not randomized or controlled, and do not include external oversight of data collection. Nevertheless, current data are compelling for benefit. Twenty-five women with lipedema had significant improvements in pain, tension in the legs, excessive warmth, muscle cramps, leg heaviness, tired legs, swelling, itching, general involvement of the skin, difficulty walking, quality of life, and appearance of the legs six months post-liposuction surgery (104). A larger study of 85 women from the same clinic demonstrated significant improvements six months after surgery for all complaints with the greatest improvement in quality of life (110). In a longer study from a different clinic, 21 women over an average of 3.7 years after their first liposuction procedure and 2.9 years after the second liposuction showed improvement in the parameters of body disproportion, swelling, edema and quality of life, except for bruising which improved in all but two of the women (105).  A retrospective study of women with Stage I or II lipedema from the same clinic, four, and eight years after liposuction, showed sustained improvements during follow-up for parameters including pain, sensitivity to pressure, edema, bruising, restriction of movement, cosmetic impairment, reduction of overall quality of life and overall impairment (117). The most interesting data was the reduced need for combined decongestive therapy four years after liposuction, which decreased further after eight years (37). 

 

Any surgery, including liposuction, requires that efficacy of the procedure and the medical necessity be demonstrated to the insurance company.  What are currently needed are well conducted randomized, controlled trials of sufficient numbers of patients with lipedema to determine which patients do and do not benefit from liposuction. In the meantime, documenting patient baseline characteristics and outcomes by surgeons in the United States will be important to understand the benefits of liposuction for lipedema in the US population compared to reports from other countries (e.g., Germany). It is notable that surgeons agree that quality of life is strongly and consistently improved by liposuction (104,110,117,118). 

 

COMPLETE DECONGESTIVE THERAPY

 

Complete decongestive therapy (CDT) is commonly recommended for the treatment of lymphedema and includes skin care, education on home exercise programs, manual lymphatic drainage (MLD) therapy, wrapping as needed to reduce fluid build-up, and skin care recommendations performed by physical and occupational therapists and licensed massage therapists that have undergone additional training. Many women with lipedema benefit from CDT with reduced pain, limb volume and capillary fragility (119-121). Near-infrared fluorescence lymphatic imaging (NIRFLI) has added additional techniques to MLD including the “Fill and Flush” method (122). Complete decongestive therapy also improves lymph flow in brain lymphatic vessels (123). Deeper tissue therapies to reduce fibrosis in the lipedema tissues may also be beneficial for patients with lipedema. 

 

PNEUMATIC COMPRESSION DEVICES

 

Studies have shown the benefit of advanced pneumatic compression devices (PCDs) in the treatment of lymphedema. There are also studies on the benefits of PCDs in the treatment of lipedema (119,124). Important for the distorted and dilated capillaries in lipedema (36,88), PCDs decrease capillary fragility (120), improving vessel quality. Along with manual therapy to improve flow of fluid through lipedema tissue, PCDs are also recommended in conjunction with liposuction surgery for lipedema (110). If a woman with lipedema responds well to manual therapy, or she tries a PCD and has a reduction in tissue volume, she should be offered PCD therapy to continue treatment at home when insurance will no longer cover CDT or when distance or commitments prevent regular professional visits. The PCD should ideally be an E0652 device with a segmented, multi-ported pump that allows for individual pressure calibration at each port. This allows the patient to alter pressure in areas of severe pain or for different shaped tissue. Pump garments should wrap around and treat the abdomen and pelvis when the legs are pumped, and the chest when the arms are pumped.  If basic compression pumps are prescribed (E0650; E0651), compression garments to protect the abdomen, pelvis, chest and/or head should be worn during pumping. Without these compression garments, fluid is pushed up the leg into the abdominal and pelvic area where it accumulates due to lymphatic dysfunction. As this fluid sits in the tissue with all its nutrients and protein, evidence suggests it may stimulate further adipogenesis (125). With an E0652 pump, the abdomen is treated along with the leg and the chest is treated along with the arm preventing pooling of lymph fluid. PCDs can be easily ordered by writing a prescription for durable medical equipment with multiple suppliers.

 

DEEP TISSUE THERAPY

 

Women with lipedema treated with deep tissue manual therapy have reduced pain, fat tissue on the legs, tissue volume, tissue fibrosis and leaky or fibrotic vessels (86,126). This deep tissue therapy is in the spectrum of meridian massage shown to reduce body weight (127) and is thought to  improve lymphatic flow through lipedema fat tissue. Massage also reduced fat in preterm infants (128). Instrument-assisted soft tissue (IAST) therapy has cc in lipedema fat tissue with noted reduction in palpable fibrosis after treatment. Instrument-assisted soft tissue  techniques include Astym therapy, which increased fibroblast activation and number, production of fibronectin, movement, and decreased pain in patients with fibrosis (129) and Graston technique which reduces pain and improves movement (130), and are performed by physical therapists who can be located on websites for these techniques. Traditional Chinese gua sha tools or bian stones have been used to improve pain and function (131) as has cupping (132).  Pressure required to occlude lymphatic function in the upper limb was found to be 86 mm Hg, suggesting that deeper treatment into the tissue is safe, for example at pressures ranging from 15 to 25 mm Hg used to reduce scars (133), and will not damage lymphatic vessels (122).

 

PSYCHOLOGICAL SUPPORT

 

Women with lipedema have often spent years looking for answers and help for their condition.  Healthcare providers often hold strong negative attitudes and stereotypes about people with obesity, which may reduce the quality of care they provide to women with lipedema (134). Poor quality of life associated with mobility and appearance-related stress associated with lipedema can result in depression (87). For many patients, they experience a huge sense of relief when they finally get a diagnosis of lipedema after trying a myriad of diets and exercise programs, even bariatric surgery to lose the lipedema fat. In addition to treatment recommendations in this chapter, there are a number of things a healthcare provider can do to help improve the lives of people living with lipedema: 1) Reduce focus on body weight in lipedema and provide education on improving metabolism, reducing inflammation and improving quality of the lipedema fat tissue (reducing fibrosis); 2) Use motivational interviewing focusing on strides made to improve markers of health including healthy eating, activity, metabolic lab markers, and social interactions; 3) Ensure that the clinic environment is welcoming with tables and chairs that allow women with larger lower bodies to be comfortable; 4) Ensure that patients with lipedema have identity safety in clinic situations and encourage healthy social interactions at home and in on-line social groups that also provide safe affiliations known to improve satisfaction of life for women with lipedema (135); and 5) Ensure that the continuum of care includes adequate referral resources for counselling, physical therapy and message, and when indicated pain management specialists. Including providers that understand lipedema and the physical and the psychological burden this diagnosis carries for patients is especially important (134).

 

MEDICATIONS AND SUPPLEMENTS

 

There are no medications and supplements specifically for lipedema.  Instead, recommendations regarding use of medications and supplements for the treatment of lipedema should focus on reducing tissue inflammation, fibrosis, swelling, pain, and pharmacologic weight loss management for those who are overweight or have obesity.  Supplements used for lipedema are, in part, based on literature for lymphedema and venous disease, both complications of lipedema.  Some medications exacerbate symptoms in lipedema and should be avoided (Table 5). 

 

Sympathomimetic Amines

 

Sympathomimetic amines (SA) such as phentermine and amphetamine are approved by the food and drug administration (FDA) for the treatment of obesity. Sympathomimetic amines bind to adrenergic receptors (AR) located on adipocytes to induce lipolysis, reducing the storage of fat.  Adrenergic receptors are also located on blood vessels and lymphatic vessels. Activating AR on blood vessels induces vasoconstriction. Activating AR on lymphatic vessels improves the efficiency of lymphatic pumping by increasing the force of contraction (136); medications or supplements that improve lymphatic pumping are lymphagogues. Amphetamine and dextroamphetamine alone or in combination are also FDA-approved for the treatment of attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), and narcolepsy.  The use of SA for treatment of lipedema may be beneficial in reducing fat and improving lymphatic pumping. A retrospective questionnaire study found that low dose sympathomimetic amines improved quality of life, reduced weight, clothing size, pain and leg heaviness in women with lipedema (137). Contraindications of sympathomimetic amines include advanced arteriosclerosis, symptomatic cardiovascular disease, moderate to severe hypertension, hyperthyroidism, known hypersensitivity or idiosyncrasy to the sympathomimetic amines, and glaucoma.

 

Diosmin

 

Diosmin is a bioflavonoid found in the rind of citrus fruit and is traditionally prescribed for the treatment of inflammation associated with chronic venous insufficiency. Diosmin was shown to reduce oxidative stress markers in people with chronic venous insufficiency (138). Diosmin also functions as a lymphagogue, and in combination with its anti-inflammatory activity, reduces edema (138). Women with lipedema who have a feeling of heaviness in their legs, obvious edema, chronic venous insufficiency or Stage II and III lipedema report feeling less pain and improved swelling on diosmin based on the author’s experience. Diosmin can be found over the counter or ordered by prescription as a medical food.  Placing lemons, limes or other citrus in water to soak before drinking is a way to intake diosmin throughout the day.

 

Metformin

 

There are no current medications that can be used to reduce fibrosis already present in lipedema fat tissue, for which liposuction and deep tissue therapy are better modalities.  Metformin and resveratrol have been shown to reduce the development of hypoxia-inducible factor (HIF)-1 inflammation and fibrosis in mice fed a high fat diet (139). Metformin also prevented fibrosis and restored glucose uptake in fat after insulin stimulation, although it did not prevent side effects of doxorubicin that included tissue loss and inflammatory response (140).  Metformin should be considered early in women with obesity and lipedema Stages II and III where fibrosis in the fat tissue is prominent, as well as in women who have signs of metabolic syndrome (69).

 

Selenium

 

Selenium is a mineral found in the soil and in high concentration in Brazil nuts (Bertholletia excelsa). Selenium has been demonstrated to have anti-inflammatory effects on multiple levels of the inflammatory cascade (141-144).  Edema was significantly decreased after selenium intake in two placebo-controlled trials for people with lymphedema (145,146) and improved complete decongestive therapy while reducing the incidence of erysipelas infections in patients with chronic lymphedema (145). Each Brazil nut contains approximately 200 mcg of selenium with a no observed adverse effects for dietary intake of selenium up to 800 mcg daily (147).  Care must be taken to follow blood selenium levels as selenium deficiency and excess can both adversely affect glucose and lipid metabolism and potentiate the risk of development of type 2 diabetes in several animal studies, with less clear associations in human studies (148). One case report of a woman with lipedema showed reduced leg volume with a combination of selenium and Butcher’s broom (149).

 

Table 5: Medications and Supplements to Avoid When Treating People with Lipedema

Medication

Used for

Reason to avoid

Thiazolidinediones

Diabetes

Increases subcutaneous fat tissue; fluid retention

Calcium channel blockers

Hypertension

Fluid retention

Oral Corticosteroids*

Reduce inflammation

Weaken tissue; fluid retention; rebound inflammation

NSAIDs

Pain

Fluid retention

Sex hormones

Hormone replacement

Fluid retention; implicated to effect development of lipedema

Beta blockers

Cardiac health

Fluid retention

Clonidine

Hypertension

Fluid retention

Gabapentin

Pain

Fluid retention

Furosemide**

Edema

Concentrates protein in the interstitial organ eventually halting fluid flux

*Nasal or inhaled corticosteroids have less effect; oral corticosteroids should be used when medically necessary

**Aldactone and hydrochlorothiazide have less adverse effects in women with lipedema

NSAIDs: Non-steroidal anti-inflammatory drugs

 

Concluding Remarks on Lipedema

 

Lipedema is a common disease mostly in women resulting in an enlargement of the adipofascia on the limbs due to excess fibrosis in the tissue that typically defies expectations for loss by lifestyle, weight-loss medications, and bariatric/metabolic surgical interventions. The presence of fibrosis, especially in the interstitial spaces where it may serve to restrict blood and lymph out flow, is thought to contribute to the resistance of this tissue to weight loss. Women with lipedema should be recognized prior to weight loss efforts so that expectations can be discussed, and manual therapies and other treatments can be considered to improve outcomes.  Medications and supplements can be tried, but liposuction should be considered for women with lipedema who fail conservative measures and following weight loss with medications and/or bariatric surgery. There is a wide variety of presentations of lipedema in women due to co-morbidities and other genetic and environmental influences. Therefore, every affected woman should be considered on a spectrum and treatments personalized.

 

FAMILIAL MULTIPLE LIPOMATOSIS

 

Familial multiple lipomatosis (FML) is a rare adipose disorder (RAD) of multiple lipomas in subcutaneous fat (OMIM 151900).  Some members in an FML family may have only a few lipomas whereas others may have hundreds to thousands; it is not understood why there is unequal penetrance in families. Lipomas usually are not painful or tender to the touch except while growing; they may also cause a slight feeling of itching or burning when forming. Some lipomas can be tender if they develop in areas of pressure such as on the back of the legs, the lower back (pressure from a chair), or the lateral wrist due to repetitive stress such as comes from using a computer mouse (150,151). Another example of trauma-induced lipomatous growth includes movement of the xiphoid process (152).  

 

According to older FML literature (153), "pain may suddenly develop in one of the lipomas (called lipoma dolorosa), and will gradually extend to involve more and more of the discrete lipomas.” The authors state that lipoma dolorosa syndrome in families with FML is not the same as Dercum disease (see below) (154). This is confusing as individuals with painful lipomas in an FML family have been described as having Dercum disease. While painful lipomas in a person with FML may also be on the spectrum of Dercum disease, a more precise name is FML with painful lipomas, especially when a family history of FML is known.

 

It is interesting that by observation in some families with FML, the men will develop lipomas and the women often develop obesity in line with lipedema. This suggests an overlap between the development of one fat disorder (FML) and another (lipedema) and should prompt more detailed questions regarding other potentially affected family members.

 

Prevalence of FML

 

Familial multiple lipomatosis is considered to be a rare disease with an estimated prevalence of 1/50,000 (155).

 

Genetics of FML

 

Familial multiple lipomatosis is usually inherited in an autosomal dominant manner with males and females equally affected. The gene High Mobility Group AT-Hook 2 (HMGA2; 12q15) has been implicated in FML but is not thought to be causative. A mutation in partner and localizer of breast cancer (BRCA2, DNA-repair associated gene), called PALB2, was described in a family with FML (156). PALB2 is an intranuclear protein that anchors BRCA2 to nuclear structures.  PALB2 mutations are associated with a 2-fold increased risk of breast cancer, a Fanconi anemia subset, pancreatic cancer and ovarian cancer (156).

 

Conditions Associated with FML

 

In case reports, FML has been associated other rare or unusual disorders (Table 6).  Because multiple lipomas are often linked with mutations in tumor suppressor genes, FML can be considered clinically to be a marker for the presence of an underlying tumor suppression gene mutation and affected patients and their families should be appropriately screened.

 

For example, in MEN-1, lipomas have  been reported in association with a recessive mutation in a tumor suppressor gene (157).  In a family with retinoblastoma and multiple lipomas, the lipomas were present in people with a gene mutation in the RB1 gene who did not develop retinoblastomas (158). Multiple lipomas in Cowden's disease can be due to a germline inactivation of PTEN/MMAC1 that renders a person susceptible to thyroid and breast malignancies (159). Other genes including other tumor suppressor genes have been implicated in the growth of lipomas (160). For example, a mutation was found in the tumor suppression gene PALB2 in a family with multiple lipomas suggestive of a diagnosis of FML (156).  And finally, lipomatosis like that of FML has also been reported in two cases after chemotherapy (161,162), a treatment known to be associated with an increased risk of cancer development.  Because of these associations, people with multiple lipomas should be considered at increased risk for cancers and a referral to a geneticist considered.

 

Table 6. Disorders Found in Association with Multiple Lipomas

Atypical mole syndrome (163)

Gastroduodenal lipomatosis (no gastroduodenal lipomatosis in proband’s mother) (164)

Celiac disease (165)

Cowden’s disease (159)

Gastrointestinal stromal tumor (166)

Interhemispheric brain lipoma with corpus callosum hypoplasia and the malformation of cortical development in a young woman with refractory epilepsy (167)

Neurofibromatosis (NF1) (168)

Multiple endocrine neoplasia (MEN)-1 (169)

Retinoblastoma (Rb1) (158)

Legius Syndrome (SPRED 1); autosomal dominant; multiple café-au-lait macules and skin fold freckling, ± macrocephaly, a Noonan-like appearance, learning difficulties and/or attention deficit in children and lipomas in adults (170)

 

Pathophysiology of FML

 

The pathophysiology of lipoma growth in FML is not known. Single lipomas of subcutaneous fat tissue are the most common benign tumor growths in humans and may be induced by genetic changes, trauma, inflammation, or other causes.  As detailed above, multiple lipomas tend to be linked with tumor suppression genes. People with FML are known to be insulin sensitive, therefore an insulin-resistant metabolic cause of FML is unlikely (171). Additionally, the presence of the lipomas themselves do not confer insulin resistance.

 

Imaging of Lipomas in FML

 

Lipomas in FML are identified by palpation as connected to skin, surrounded by fat or connected to other structures such as muscle or solid fascial structures. Localized pain can assist in finding smaller lipomas. Silky or tight clothing can also assist in palpation. Sonographic evaluation is the best most inexpensive means to identify lipomas other than palpation, but the average sensitivity for three Radiologists when retrospectively reviewing sonographic exams of lipomas was only 48%, and average accuracy was 59% (172). Magnetic resonance imaging without contrast can be used to find lipomas (173), but small lipomas, lipomas without a capsule, and lipomas with minimal fibrosis or surrounding edema remain difficult to identify.  Radiologists were able to render the correct diagnosis for lipoma versus liposarcoma in 69% of cases (174). Computed tomography (CT) scans have been used to differentiate lipomas from liposarcomas (175) but should be used after sonography and MRI to avoid excess radiation exposure.

 

Evaluation of the Patient With FML

 

The initial workup for people with FML includes a family history of lipomas and cancer, and any associated conditions such as nevi or neuropathy. The exam incudes assessment for multiple lipomas usually located on the trunk, lower back, arms, and thighs; rarely on the upper back or calves. Skin should be examined for nevi and cherry angiomas, the latter seen commonly with multiple lipomatosis (176). Due to the associated cancer risk, the exam includes examination of the thyroid and breasts for nodules. Reflexes should be checked along with monofilament and/or vibration assessments for peripheral neuropathy. Cancer screening as appropriate for sex and age should be advised, and appropriate labs ordered (Table 7).  Although there is no definitive association of FML with dyslipidemia, statin therapy may be helpful in lowering lipoma size (177) and so a lipid panel is also appropriate.

 

Table 7. The FML Workup

Family history

Lipomas; cancer; nevi; celiac; neuropathy

Medical history

Lipomas; cancer; nevi; celiac; neuropathy

Physical Exam

Lipomas

Trunk, arms, low back, flanks, abdomen, thigh.

Attached to skin, muscle, other.

Skin

Nevi; cherry angiomas

Thyroid

Nodules

Nervous system

Reflexes; skin sensory exam (monofilament)

Laboratory Studies

Thyroid

TSH

Blood fats

Lipid panel; other per family history

Fibrin clot (found in angiolipoma)

D-dimer

Food/gluten intolerance

Celiac panel

 

Treatment of FML

 

The current management of FML includes screening for associated conditions such as cancer (Table 7) and consideration of a referral to genetics for tumor suppressor gene workups as needed. A healthy diet and an exercise plan to avoid or reduce obesity is important as obesity in families with FML can be associated with pain (154) and, anecdotally, triathletes notice a reduction in lipoma size during high intensity training. A statin has been shown to reduce a lipoma in a case report (177). Painful lipomas or those that interfere with activities of daily living can be excised as needed but these procedures can cause numerous scars (Figure 5).  Massive amounts of lipomas can occur on the arms, hips/flanks, buttocks and thighs. Therefore, this condition can be psychologically devastating and people with severe FML do not consider it benign.

 

Liposuction is an option to excision of lipomas in people with FML as it provides good results in terms of skin appearance, and there is reported lack of recurrence or growth or development of other lipomas in the same area for at least 12 months (178). Injections of collagenase have been shown to shrink or destroy lipomas with minimal pain and good cosmetic result in a published abstract (179). Similar data were found for the detergent, deoxycholic acid (180), but anecdotally care should be taken not to inject too much detergent that can remain in the tissue requiring excision to remove. Additional treatments such as cryotherapy have been suggested and reviewed (181). More data is needed for the efficacy of injections and other therapies for the lipomas in FML as they are preferable to more invasive and scarring surgical techniques.

Figure 5. Multiple scars after excision of lipomas in FML.

Concluding Remarks on FML

 

Familial multiple lipomatosis is a rare disease of multiple lipomas often associated with mutations in a tumor suppressor gene. Therefore, people identified with FML should be assessed for cancer. Liposuction should be considered to remove symptomatic lipomas and is preferable to surgical excision as multiple excisions leave many scars.

 

ANGIOLIPOMATOSIS

 

Angiolipomatosis also known as angiolipoma microthromboticum (OMIM 206550) is a rare disease of multiple angiolipomas and connective tissue that occurs commonly in men and usually begins after puberty; one case of a child with an angiolipoma in a family in which the father also had angiolipomatosis has been reported (182). Angiolipomas have been described as vascular malformations or vascular lipomas where blood vessels occupy between 10-90% of the angiolipoma. In families with familial angioliopomatosis, lipomas and angiolipomas can exist in the same person. Subcutaneous angiolipomas usually occur on the trunk and limbs, rarely on the head, hands, or feet (183). The angiolipomas can be the size of a rice grain, pea, a marble or much larger and are tender to the touch and can be associated with intense pain.  Angiolipomas may or may not be visible and may be palpable or non-palpable depending on their location and size. Numerous case reports describe epidural or extradural spinal angiolipomas, and rare cases report colonic (184), bronchial (185), joint (186), and testicular angiolipomas (187). Angiolipomas are known to be painful, although not always, and should be distinguished from other painful neoplasms (188). One case of angiolipomatosis was reported to occur after treatment with corticosteroids (189).

 

The loose connective tissue of angiolipomas contains adipose cells, fibrotic tissue, vessels with fibrin clots, and mast cells as salient features (Figure 6). Due to the large number of vessels, angiolipomas are bluish in color through the skin. Interestingly, the vessels in angiolipomas can grow from the dermis into the territory of the epidermis making the vessels palpable as small raised areas on the skin (Figure 7). A plethora of capillary “cherry” angiomas where a capillary grows and dilates through the epidermis may be found on the skin in areas of angiolipomas (Figure 8).

Figure 6. Angiolipoma with mast cell with enlarged multiple vessel lumens and degraded tissue. The black arrow points to a classic fried egg appearance of a mast cell stained with Alcian Blue in angiolipoma tissue. Red arrows point to small fat cell remnants likely non-functional as evidenced by the absence of nuclei. Blood vessels are numerous and large for location. The green arrow demonstrates the remnant of a capillary. Connective tissue is evident especially in the area surrounding the mast cell as bluish fibers. Magnification 100X.

Figure 7. Multiple cherry angiomas present on the legs and arms of a woman with angiolipomatosis.

Figure 8. Histological features of angiolipomas. A. Small area of hypervascularity in an angiolipoma (40X). B. Blood vessels in an angiolipoma grow up and through the epidermis and are palpable on the skin (40X). C. Empty and presumed dead and non-functional vessel on the left containing an eosinophil next to two functional blood vessel lumens containing red blood cells (100X). Microthrombi can be seen as pale areas especially between the right side of the dead vessel and the lumen of the active vessel. Dead vessels may result in hypoxia and ischemia causing pain. D. Non-functioning blood vessel to the right and smaller fat cells surrounded by an enlarged interstitial organ (40X).

Prevalence of Angiolipomatosis

The prevalence of angiolipomatosis is unknown but it is considered to be a rare disease (190,191). 

 

Genetics of Angiolipomatosis

 

Angiolipomatosis most often occurs sporadically, but a family history can be identified in a minority of cases as autosomal dominant (192) or autosomal recessive (193,194). There are no known genes identified to date for angiolipomatosis.

Pathophysiology of Angiolipomatosis

Angiolipomas likely arise from fascia and therefore may also be painful because fascia is highly innervated, and when inflamed, is a likely source of pain (195). Inflamed fascia has robust angiogenesis (196) and may be important in the initial development of angiolipomas as resident mesenchymal cells in fascia can develop into adipocytes (197). It is thought that microthrombi in angiolipomas leads to necrosis of blood vessels, adipocytes and other components of adipofascia. Other hypotheses regarding pain include nerve damage from limited blood flow and tethering by fibrotic tissue. 

 

Subcutaneous angiolipomas are assumed to be congenital in origin where pubertal hormones may induce differentiation of adult adipose-derived stromal adipogenic precursors that reside in adipofascia; these precursors develop into adipocytes in intimate association with blood vessels (197). Vascular proliferation is thought to occur after repeated trauma to the fascia resulting in the development of an angiolipoma. However, there is a question of whether angiolipomas can become autonomous as a cancer. Two of three cases of angiolipomas in one publication suggest a neoplastic nature for these tumors due to deletion of parts of chromosome 13, a region containing the retinoblastoma gene, a tumor suppressor gene (198). The neoplastic nature of angiolipomas should be considered in individuals with significant numbers of angiolipomas and anti-neoplastic treatments considered when other conservative therapies fail.

Imaging of Angiolipomatosis

Identification of angiolipomas in tissue by Ga-PSMA PET/CT (199), magnetic resonance imaging (200), and ultrasound (201) allows surgeons to identify superficial and deeper angiolipomas targeted for removal. 

 

Treatment of Angiolipomatosis

 

SURGICAL

 

The only definitive treatment of angiolipomas to date is individual resection by excision or liposuction (202). Angiolipomas are typically removed if they are painful or restrict movement. A surgical emergency may occur to prevent hemorrhage of angiolipomas which can compress the spinal cord (203,204). Karyotypes of DNA from the angiolipomas should be assessed to determine the neoplastic nature of the angiolipomas so as to prepare the patient for the potential of multiple resections throughout life (198).

 

A concern with resection of lipomas is that inflammation is often a sequela of the removal process. As fascia plays an important role in the pathophysiology of angiolipomas, generation of inflammation in the fascia by surgical techniques has been anecdotally noted to incite a pain crisis. Removal of angiolipomas must therefore be considered carefully and manual or IAST therapies for the fascia should be considered after any surgery to speed recovery and reduce pain.

 

PAIN MANAGEMENT

 

The necrosis of tissue in angiolipomas and inflammation of fascia with all of its nerve endings can cause severe pain in individuals with angiolipomatosis.  In a case report from Germany, the systemic administration of acetylsalicylic acid, diclofenac, ketotifen, ranitidine, tramadol, or tilidine combined with naloxone did not provide adequate pain relief. In contrast, the antidepressant doxepin, which also has antihistaminergic effects to control the release of mast cell mediators, demonstrated good therapeutic efficiency for the pain from angiolipomas (205).  Depending on the mast cell burden in angiolipomas and their systemic effects including flushing, itching, nausea, diarrhea, angioedema, pain and a cadre of other signs and symptoms (206,207), individuals with angiolipomatosis may be considered to have mast cell disease or mast cell activation disease. Treatments to reduce the burden of mast cells in angiolipomas such as histamine 1 and/or histamine 2 receptor blockers, montelukast, non-steroidal anti-inflammatory drugs, antihistaminergic bioflavonoids such as quercetin or pycnogenol, amphetamines, and possibly stronger immunosuppressants that have been used for mastocytosis such as sunitinib (208) or mast cell activation syndrome such as tofacitinib (209) or imatinib (210) may provide benefit for pain and growth of angiolipomas. Non-neoplastic therapy for mast cell activation disease should be considered prior to the use of antineoplastic agents, which have been described extensively (211). Patients with angiolipomatosis can have a poor quality of life due to extreme pain and fatigue and consider suicide.  In these individuals, use of anti-neoplastic agents should be considered early. 

 

Many individuals with angiolipomatosis require opioid pain management and should be under the care of pain management specialists. Unfortunately, opioids can activate mast cells requiring concurrent treatment of mast cell signs and symptoms (212). Opioids should not be withheld during a pain crisis, and in fact may need to be escalated before weaning back down after the pain crisis has resolved.

 

Concluding Remarks on Angiolipomatosis

 

People with angiolipomas have severe pain that can be out of proportion to the outward appearance of the individual. Treatment with mast cell stabilizers, pain medications, and surgical treatments of angiolipomas are all important in management.  More research is needed for this rare disease to enable individuals with angiolipomas to live a full and active life.

 

DERCUM DISEASE

 

Dercum disease (DD; OMIM 103200) is a term used to describe extremely painful adipofascial tissue that is resistant to loss by diet and exercise and poorly responsive to analgesics. Other names include adiposis dolorosa (a term that is also used to describe women with lipedema) and Morbus Dercum. While Dercum disease is defined as painful fatty masses accompanied by other signs and symptoms of a chronic healing cycle disorder (25) (213), there remains a lot of confusion in the literature as to what exactly Dercum disease is. One review article stated that people with Dercum disease have obesity and chronic pain (214), which can easily be confused with people who have obesity and chronic pain for a variety of reasons including fibromyalgia. The old classification of Dercum disease and a new classification remain inadequate to differentiate the overlapping disorders that are bundled together as Dercum disease (65,215,216) because they describe only the phenotype and not the history (Table 8).

 

Table 8. Comparison of Outdated Classifications of Dercum Disease.

Older Classification

Previous Recent Classification

Type I: Diffuse Type. Widespread occurrence of painful lipomas in a diffuse manner

Diffuse Type. Diffusely painful adipose tissue that may present as painful folds of fats containing fat nodules that feel like pearls located around lymph node beds. Mostly resembles lipedema but extends or start in the trunk which differentiates it from lipedema

Type II: Generalized Nodular

Nodular type. Intense pain in and around grape-like clustered lipomas of variable size most commonly on the arms, legs, lower back or thorax; can include angiolipomas. Most resembles familial multiple lipomatosis

Type III. Localized Nodular

Nodular type. Intense pain in and around grape-like clustered lipomas of variable size in defined areas; can include angiolipomas

Type IV: Juxta-articular

NA

NA

Mixed Type: Combination of diffuse and nodular

 

A better classification of painful adipofascia considers the history of the disease (Table 9). For example, women with lipedema who become obese and/or develop lymphedema can metabolically become toxic or ill leading to the growth of painful masses in fat tissue. The etiology of these masses is likely due to the presence of inflammation known to slow lymphatic pumping leaving more pre-lymph fluid in the ECM, inducing adipogenesis, as lymph (even pre-lymph) makes fat grow. Women with lipedema, obesity, and painful fatty masses have dominated some studies on Dercum disease leading the authors to describe women with Dercum disease as having obesity and chronic pain (214). The masses that develop in women with lipedema and metabolic syndrome are similar to those that develop on the abdomens of people who have obesity and do not have lipedema. These tender masses resolve with weight loss and have been called Ander’s disease or adiposis tuberosa simplex (217). A good history taken from a woman with lipedema and the label Dercum disease, may reveal the development of lipedema earlier in life and additional weight gain later in life with development of tender masses, allowing treatment to be focused on lipedema and obesity rather than on Dercum disease.

 

Table 9. Conditions with Painful Adipofascia which Have Been Labeled as Dercum Disease

Types

Comments

Obesity-associated

Non-painful lipomas resolve with weight loss (Ander’s disease)

Lipedema with obesity and/or lymphedema

Painful lipomas resolve with weight loss; lipedema fat tissue remains

Familial multiple lipomatosis (FML) with obesity

Lipomas may get smaller and pain reduce with weight loss

Angiolipomas with or without obesity

Weight loss does not affect angiolipomas but is important to reduce inflammation

Localized due to trauma (218)

Multiple lipomas in an area of trauma and not just a single lipoma; likely due to injury of the fascia as with angiolipomas

Toxic/infectious; present around lymph node beds or diffuse with or without obesity

Likely due to a healing cycle disorder, infection, methylation deficiency, high oxalate or other toxin overload.

 

Signs and symptoms of Dercum disease include chronic pain, fatigue, brain fog, insomnia, cardiac arrhythmia most often tachycardia (palpitations), gastrointestinal distress often similar to irritable bowel syndrome, muscle weakness, tremor or jerking of muscles (myoclonus), joint pains, insulin resistance and diabetes, hypothyroidism, and other autoimmune disorders (219).  The signs and symptoms of Dercum disease have been suggested to be in the spectrum of fibromyalgia (220).

 

Dr. Dercum’s first patient was a woman with obesity with fat similar to a woman with Stage 3 lipedema (221). Dr. Dercum and his medical resident described rapid changes in fat tissue shape and size in real time suggestive of edema or fluid shifts. Therefore, involvement of the lymphatic system is likely in Dercum disease. Irregular and thickened lymphatic vessels have been described in Dercum disease suggesting that an altered lymphatic system can contribute to changes in the adipofascial tissue that found by palpation (222). Once people with Dercum disease are more accurately described by phenotype, there will be a better chance of finding a gene or biomarkers.

 

Prevalence of Dercum Disease

 

Many women with lipedema have been miscategorized as having Dercum disease when actually they have lipedema and metabolic syndrome, making the prevalence estimate of 1/1000 for Dercum disease in Sweden too high (223). There are no other prevalence studies of Dercum disease although the angiolipomatosis type is considered rare and the obesity- or lipedema-associated types are likely common, but these individuals are better described with what they have, angiolipomas, obesity, or lipedema respectively, with metabolic disease rather than lumping them all together as Dercum disease simply due to the presence of pain in the tissue.

 

Genetics of Dercum Disease

 

There is(are) currently no known gene(s) for Dercum disease.  Continuing to include people with angiolipomas, FML, and lipedema under the same moniker of Dercum disease will make it very difficult to discover genes important for these diseases when examining populations. The study of genes for specific families may be more helpful to find gene mutations that can then be assessed in individuals with painful adipofascia.

 

One family with familial multiple lipomatosis was found to have members that developed pain in the lipomas consistent with Dercum disease (adiposis dolorosa). While many of the family members with FML and pain also had obesity, not all were. Therefore the authors concluded that “adiposis dolorosa may in fact be an expression of familial multiple lipomas” (154). It remains unclear, however, why some individuals would develop pain and some not in a family with FML. Fascia can become inflamed for a variety of reasons including surgery, trauma, infection, toxin or drug exposure, and development of obesity. Lipomas in people with FML are often connected by a tail of connective tissue to solid fascial structures in the body, and fascia is a source of preadipocytes (Figure 9).  It may be that lipomas in FML are a marker of fascial disease and that pain in and around the lipomas depends on the amount and extent of inflammation present.  Other genes may modify susceptibility to prolonged inflammation including those yet to be identified in fibromyalgia (224).

Figure 9. Lipomas with fascial component. A. Lipoma with obvious tail of connective tissue. Removal of the fascia is important along with the lipoma to reduce additional growth in the area of removal. B. Long piece of connective tissue weaving amongst multiple lipomas during resection.

Pathophysiology of Dercum Disease

 

The pathophysiology of Dercum disease needs to be determined by type, something that very few papers have done so accurately. In mostly women with lipedema type Dercum disease, substance P was lower in the spinal fluid compared to controls (225), confirming a strong pain component is present when women with lipedema develop obesity and metabolic syndrome. In another study, interleukin-6 levels were elevated in the fat from women with Dercum disease compared to women without lipedema supporting Dercum disease as an inflammatory disorder (226). Weight stabilization and when possible, weight loss in patients with obesity should be a focus for women who have developed metabolic disease, in addition to caring for their lipedema.

 

The juxta-articular type of Dercum disease where nodules in the adipofascial tissue are present around joints had been associated with rheumatoid arthritis (227). Lymph nodes are present around many joints including the elbow (cubit nodes), knees (popliteal nodes), and hips (femoral nodes), and in these locations adipofacial nodules have been found. Cases of juxta-articular Dercum disease suggest that inflammation in the adipofascia around joints may reduce lymphatic pumping in these areas resulting in a backup of fluid in the interstitial body leading to densification of fascia and eventually fibrosis around lobules of fat making them palpable as nodules. These nodules are tender due to inflammation of the fascia and nerves. As an example, a woman with rheumatoid arthritis was treated with tocilizumab, a humanized monoclonal antibody of class IgG1, targeting interleukin-6 receptors, and developed painful fatty masses of her knees documented by MRI (228). A similar pathophysiology would be likely for the trauma-induced Dercum disease. 

 

Familial multiple lipomatosis and angiolipomas have been previously discussed including why pain develops in angiolipomas. It is unclear why a person with FML would suddenly develop pain in and around lipomas qualifying for a diagnosis of Dercum disease (FML type with pain).  The presence of inflammation occurring in the body of a person with FML such as from obesity, trauma, hypermobile joint spectrum disorders, arthritis and any other inflammatory condition that includes the fascia in the inflammatory process likely accounts for the development of pain in FML. Resolving the inflammation in the fascia may reduce the pain and return the diagnosis back to FML alone.

 

One theory on the origin of Dercum Disease is based on the work of Robert Naviaux in which a failure of healing of inflammation occurs (213). According to Naviaux, a normal healing cycle includes normal wakefulness, restorative sleep, fitness and healthy aging. The cell “danger response” is an evolutionarily conserved cellular metabolic response activated when a cell encounters a threat that could injure or kill it, examples of which can be microbial, chemical, physical, or psychological in nature. Chronic disease occurs when cells fail to heal or contain inflammation, and a toxic repeating loop of incomplete recovery and re-injury occurs. Chronic pain disorders are included by Naviaux as a healing disorder and the author feels many people with Dercum disease fall into this category.

 

Imaging of Dercum Disease

 

The most inexpensive means to document lipomas in the adipofascia of people with Dercum disease is by ultrasound. Ultrasound findings include a hyperechogeneity (higher density) to the lipoma suggesting fibrotic tissue and no increased Doppler signal (minimal blood flow) (229).

 

Magnetic resonance imaging of the tissue of people with Dercum disease has found lymphedema in a woman and multiple lipomas in a man  (230). Nodular type lipomas have also been visualized by MRI in the tissue of people with Dercum disease (229). The lipomas were multiple, oblong, fatty lesions in the superficial subcutaneous adipose tissue, mostly < 2 cm in long axis diameter. A nodular ("blush-like") fluid signal was also found without the presence of contrast. According to Richard Semelka, MD, gadolinium contrast should not be used in people with Dercum disease unless absolutely necessary to avoid any risk for development of gadolinium deposition disease (231,232). MRI images demonstrate variability in the tissue of people with Dercum disease, from lymphedema to distinct lipomas, and exemplify the different phenotypes under the moniker of Dercum disease. To date there are no confirmed connections between multiple lipomas as in FML or trauma-induced Dercum disease and development of lymphedema.

 

Conditions Associated with Dercum Disease

 

Dercum disease has been associated with many conditions such as disrupted sleep cycle, headaches, cognitive difficulties, tachycardia, shortness of breath, and gastrointestinal symptoms (219). Many of these symptoms are consistent with mast cell activation disease (MCAD).  Therefore, MCAD is considered an associated condition in Dercum disease. Diabetes is common in Dercum disease (25,219) and cardiovascular disease should be evaluated for and treated in any person with Dercum disease especially if blood markers of inflammation are high, such as C-reactive protein. A woman with Dercum disease had a dysfunctional arteriolar venous reflex in her arm suggesting a blood vascular or nerve problem in Dercum disease (233).  Another case of a woman with the FML type of painful lipomas was described who had dizziness followed by left sided sensory-motor deficit suggestive of a vascular origin (234).  Anecdotally, some of the author’s patients with Dercum disease also have postural orthostatic tachycardia syndrome (POTS). Fibromyalgia is often an accompanying diagnosis in people with Dercum disease as are other pain syndromes such as migraines (25).

 

Many people with Dercum disease become concerned that the painful lipomas can spread throughout the body as with cancer.  Once case of a lipoma on the uterus of a woman with Dercum disease is known to the author, and two women with Dercum disease had invasive calcaneal lipomas that were resected.  Other lipomas in people with Dercum disease have been identified in the gastrointestinal system but these need to be verified. Lipomatous hypertrophy of the interatrial septum was found in one person with Dercum disease (235), but this type of fat can also be associated with obesity (236). Altered lymphatics were found in a few cases of Dercum disease (222). One family had Dercum disease with dysarthria, visual pursuit defect and progressive dystonia (237).

 

General swelling, a sensation of a “heaviness” in tissue, increased pain in one limb, or aching limbs all suggest that a lymphatic dysfunction may be present, which can be evaluated using lymphangioscintigraphy, which is performed in most nuclear medicine departments, or near infrared lymphatic imaging using indocyanine green, which is not yet to the point of being readily clinically available. Finding altered lymphatic function can change clinical management by steering practitioners towards prescribing manual lymphatic drainage therapy and compression garments to contain and support lymphatic flow.

 

Treatment of Dercum Disease

 

To maintain a healthy weight or lose excess adipofascial tissue, people with Dercum disease should be encouraged to eat a healthy diet such as Mediterranean, DASH, low processed sugar, plant-based low inflammatory foods, or foods that are low in histamine if mast cell activation disease is present or suspected; they should also undertake a graded exercise program.

 

PAIN MANAGEMENT OF DERCUM DISEASE

 

Signs and symptoms common in people with Dercum disease, including pain. should be treated symptomatically (Table 10). Opioids are often used for pain treatment for Dercum disease, but doses can escalate over time and care should be taken to try and find additional alternative treatments.

 

Table 10. Medications Used to Treat Pain and Other Symptoms in People with Dercum Disease

Medication

Comments

Deoxycholic acid (238)

Injection of deoxycholic acid reduced pain and size of lipomas in a man with FML type of Dercum disease

Doxepin (205)

Has antihistaminergic activity therefore useful for pain, depression and mast cell activation disease

Intravenous lidocaine (239)

Ketamine is often used in addition to or in place of lidocaine if not effective

Topical lidocaine (240)

Often combined with other medications in topical form such as EMLA (241)

Metformin (242)

Useful for metabolic disease and when inflammatory markers are high

Mexiletine and amitriptyline (243)

Mexilitine has been described as oral lidocaine and offers an alternative to opioids

Low dose naltrexone (244)

Effective for fibromyalgia pain, a condition often present in Dercum disease

Pregabalin (245)

Gabapentin has also been shown to reduce pain (246) but may increase edema

Sympathomimetic amines (137)

Phentermine, dextroamphetamine, amphetamine; sympathomimetic amines resolved lipomas and liver fat in two cases of Dercum disease.

 

NON-MEDICATION TREATMENT OF DERCUM DISEASE

 

People with Dercum disease should be offered manual or IAST therapies or pool/water therapy to reduce pain, improve mobility and impede progression of the disease. Manual lymphatic drainage combined with pregabalin improved weight and pain in a woman with Dercum disease  (245). It has also been reported that fascia improved, pain reduced, and fat was lost after women with lipedema and Dercum disease received deep tissue therapy (86,126).

 

Liposuction has been used as treatment for Dercum disease (247), reducing pain by one point on a visual analogue scale (248) and improving insulin sensitivity (249). Surgeons removing lipomas by liposuction must have experience in the removal of fibrotic tissue and manual or IAST therapies should be performed before and after any surgery to keep inflammation levels at a minimum.

 

Transcutaneous frequency rhythmic electrical modulation system (FREMS) reduced pain and the size of lipomas in one case of Dercum disease (250). Cycling hypobaric air around ten people with Dercum disease improved pain and mental quality of life after five days of therapy (251). Cycling air around the body by sequential pneumatic compression pump therapy is also useful for people with Dercum disease due to the presence of lymphatic dysfunction (222,229).

 

Concluding Remarks on Dercum Disease

 

People with Dercum disease have painful lipomas and other signs and symptoms of a healing disorder. The different types of Dercum disease need to be delineated before any gene or biomarker can be found. The pain and associated signs and symptoms of Dercum disease should be treated to improve quality of life. People with Dercum disease may be at high risk for cardiovascular disease and cardiovascular risk factors should be closely monitored and treated when appropriate.

 

MULTIPLE SYMMETRIC LIPOMATOSIS

 

Multiple symmetric lipomatosis (MSL; OMIM#151800) also known as Madelung disease, Launois-Bensaudesyndrome, cephalothoracic lipodystrophy, and benign symmetric lipomatosis is a rare disease first described by Brodie in 1846.This disorder is clearly not benign. Madelung reported data on 33 cases, but the classical description of the disease is attributed to Launois and Bensaude who published a detailed account of 65 cases in 1898. The literature on MSL was initially dominated by research on men with alcoholism; however, people who do not consume alcohol (252), women, and children are also affected (253).

 

There are different types of MSL described initially by two different groups and reclassified in 2018 based on a German cohort of 45 patients (Table 11 and Figure 10) (91). 

 

Table 11. Types of Multiple Symmetric Lipomatosis (Locations of Abnormal Fat Tissue)

Types

Old Classification (254)

Old Classification (255)

New German Classification (91)

I

Neck, shoulders, supraclavicular triangle, and proximal upper limbs

Neck, upper back, shoulder girdle,

and upper arms

Ia: Neck

Ib: Neck, shoulder girdle, upper arms

Ic: Neck, shoulder girdle, upper arms, chest, abdomen, upper and lower back

II

Abdomen and thighs

Shoulder girdle, deltoid region, upper arms, and thorax

Hips, bottom, and upper legs

III

Thigh or female type similar to lipedema

Gynecoid type: Thighs

and medial side of the knees

General distribution skipping head, forearms, and lower legs

IV

NA

Abdominal type: Abdomen

NA

Women tend to have Type II MSL and the authors state that it is difficult to differentiate women with lipedema from women with MSL type II. Criteria the authors used to distinguish the two are “the hips and bottom are affected [in Type II MSL] which are not affected in patients suffering from lipedema.”  Lipedema, however, does affect the hips and buttocks in Types I-III lipedema (Figure 3). Finding a gene or biomarker for lipedema and MSL will be ultimately be helpful in distinguishing these adipofascial disorders.

Figure 10. Two different presentations of MSL. The man has MSL with a Charcot Marie Tooth presentation with increased fat on the upper body even after multiple resections, and the woman on the right has increased fat on the arms and upper back consistent with the old classification of MSL Type II.

In rare cases, MSL SAT can invade the muscles of the tongue (256,257), vocal cords (258), and periorbital area (259). Tracheal or esophageal compression can occur resulting in superior vena cava syndrome (260).

 

Prevalence of MSL

 

Multiple symmetric lipomatosis is considered rare occurring 1:25,000 in a primarily male Italian population (261) and 1:25,000 in a German population where females outnumbered men 2.5:1 (91).

 

Genetics of MSL

 

Multiple deletions of mitochondrial DNA, and the myoclonus epilepsy and ragged red fibers (MERRF) tRNA(Lys) A>G(8344) mutation have been found in some cases of MSL (262,263) but not in others (264,265). Chalk et al. found no mitochondrial pathology or mutations in four siblings with MSL with a pattern favoring autosomal recessive (265). Another study examined individuals with mitochondrial mutations and found that MSL was a rare sign of mitochondrial disease with a strong association between multiple lipomas and lysine tRNA mutations (266). If triglyceride cannot be mobilized from fat, then along with adipogenesis (via microRNAs), fat would be expected to increase. Indeed, a mutation in the LIPE gene coding for hormone sensitive lipase was found to be mutated in a family with MSL and lipodystrophy (267).

 

Mutations in the MFN2 gene coding for mitofusin 2 have been found to cause MSL with Charcot Marie Tooth Disease (268). Mitofusin 2 helps to regulate the morphology of mitochondria by controlling the fusion process. Individuals with mutations in MFN2 have increased fat on the upper part of the body and a lipodystrophy or lack of fat on other aspects of the body. These data support pathophysiology of MSL hypothesis 1 above for the development of MSL but also support hypothesis 2 in that alcohol may cause widespread damage to mitochondria.

 

Pathophysiology of MSL

 

HYPOTHESIS 1: BROWN ADIPOSE TISSUE

 

The dorsocervical fat pad (buffalo hump) is thought to be a location of brown adipose tissue found both in MSL (264,269,270) and HIV-associated lipodystrophy (271-274) suggesting that the abnormal fat tissue in both of these conditions arises from brown adipocytes (275).  Uncoupling protein (UCP)-1 has been shown to be activated in HIV-associated lipodystrophy and in agreement, calcyphosine-like (CAPSL), important in early adipogenesis, was down-regulated and uncoupling protein (UCP)-1 upregulated in eleven individuals, one with familial MSL and ten with sporadic MSL disease (276). Stromal vascular cells grown out of MSL fat tissue resulted in multiloculated adipocytes consistent with brown adipocytes (277,278). These data suggest that altered pre-adipocyte mesenchymal stem cells, adipogenesis and energy metabolism are important in development of MSL fat. In support, microRNAs miR-125a-3p and miR-483-5p are significantly increased in the fat of patients with MSL. These microRNAs promote adipogenesis through regulating the RhoA/ROCK1/ERK1/2 pathway (279). Finally, stem cells from MSL tissue showed significantly higher proliferative activity (280) suggesting a defect in regulation of adipogenesis. That brown fat was not found by 18 F-fluorodeoxyglucose (18 F-FDG) uptake using PET/CT in areas of MSL tissue (281) does not rule out the brown fat hypothesis as there may be browning of MSL fat rather than as strict replication of brown adipocytes.

 

HYPOTHESIS 2: INFLAMMATION, ALCOHOL AND THE LYMPHATIC SYSTEM

 

Further research is needed to determine the exact pathophysiology involved in the development of MSL fat tissue, but the fact that alcohol is damaging to many tissues in the body suggests that inflammation may play a role. Interleukin-6 levels were elevated in MSL tissue compared to unaffected tissue (280), and ethanol intake increases CYP2E1 activity in adipose tissue, leading to apoptosis of adipocytes through activation of the pro-apoptotic Bcl-2 family protein Bid, resulting in activation of complement via C1q, and adipose tissue inflammation (282).

 

The liver produces over 50% of lymphatic fluid that enters the thoracic lymphatic ducts in the great veins in the neck (283).  When the liver is fatty or cirrhotic, the liver produces even more lymphatic fluid (284). That many men with men with MSL develop fat around the neck in the location of the thoracic ducts or abdomen where the digestive tract transports approximately 2/3 of lymphatic fluid, becomes intriguing and may suggest involvement of the lymphatic system.  Women have more developed vasculature, including lymphatics, in subcutaneous adipose tissue and therefore, if lymphatic vessels are important in the pathophysiology of MSL, they could be expected to have a different phenotype than men.  Rats provided acute alcohol intoxication were found to have mesenteric lymphatic hyperpermeability (thoracic duct was not examined), a peri-lymphatic adipose tissue inflammatory response, and an altered systemic adipokine profile (285). When lymphatic vessels leak, fat grows (52). Alcohol and other mediators of lymphatic vessel leakage may therefore play a role in MSL.

 

Disorders Associated with MSL

 

Associated disorders include liver disease, dyslipidemia, metabolic syndrome, hypertriglyceridemia, hypothyroidism, diabetes mellitus, and peripheral and autonomic neuropathy (Figure 11).

Figure 11. Disorders often associated with MSL (Madelung disease). Copyright © 2018 Szewc et al. (286). This work is published and licensed by Dove Medical Press Limited. Full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/).

Morbidity and mortality in MSL is thought to be high with sudden non-coronary death accounting for a large percentage of deaths in one series of primarily men (254). The neuropathology of MSL is a distal axonal demyelination different from that associated with alcohol intake and impairment of autonomic function has been suggested as a possible cause of sudden death; this impairment seems to prevalently involve the autonomic nervous system and not related to a high alcohol intake.

 

Treatment of MSL

 

Anyone with MSL should be encouraged to stop intake of alcohol. The only definitive treatment of MSL is liposuction or excision of the MSL tissue. The advantages of lipectomy is more complete removal of MSL tissue and better control of iatrogenic damage to nearby structures. Liposuction, however, achieves good cosmetic results and is simpler and less invasive than lipectomy (287). Multiple symmetric lipomatosis tissue tends to recur after liposuction and even excision. Therefore, other treatments are needed to slow down the progression of this disease to improve quality of life. Some believe that combining excision with liposuction can reduce recurrence (288).

 

Mesotherapy is a procedure that involves injections of multiple substances such as pharmaceuticals and/or vitamins into subcutaneous fat to reduce the fat tissue or cellulite. Such substances include phosphatidylcholine, multivitamins, pentoxifylline, aminophylline, hyaluronic acid, yohimbine, collagenase and others. Mesotherapy has been used to treat MSL but the injections can cause fibrosis which can make excision or liposuction difficult (289).

 

Concluding Remarks on MSL

 

Multiple symmetric lipomatosis is a rare adipofasial disorder associated with alcohol use, but not always. The pathophysiology is unknown but may involve early adipogenesis, mitochondrial dysfunction, and brown adipose tissue formation. Women with MSL may have lipedema and vice versa, therefore a gene or biomarker is needed to identify people with different types of MSL. Surgical treatment remains the only therapy for MSL.

 

OVERALL CONCLUSIONS ON ADIPOFASCIAL DISEASES

 

Aipofascial diseases occur when there is an increase in adipofascial tissue on the body that becomes fibrotic and is resistant to loss by lifestyle change. Until such time that better understanding of the pathophysiology of these disorder hints at other treatment modalities, these disorders often require removal by surgical means. Many of the diseases overlap, making identification difficult and will remain so until additional genes or biomarkers are clinically available. 

 

A comparison table of the five adipofascial disorders presented in this chapter can be helpful (Table 12).

 

Table 12.  Comparison of Adipofascial Diseases

Characteristic

Lipedema

DD

MSL

FML

Angiolipomas

Fat Location

Limbs

Global

Upper body

Trunk, arms, thighs

Global

Diet-resistant fat

Yes

Yes

Yes

Yes

Yes

Lipomas

+

+++

+++

+++

+++

Time SAT change

Puberty

Adult

Adult

Child, adult

Young adult

Painful SAT

Yes

Yes

Not usually

Not usually

Yes

Sex

Female

Female

Male

Male, female

Male, female

Lymphatic dysfunction

Yes

Yes

Yes

Possible

Unknown

Prevalence

Common

Rare

Rare

Rare

Rare

Associated conditions

Lymphedema

Autoimmune; diabetes

Neuropathy

Moles; neuropathy

Unknown

Inheritance Pattern

Autosomal dominant; incomplete penetrance

Autosomal dominant; sex-specific influence

Autosomal dominant or recessive

Autosomal dominant

Autosomal dominant; spontaneous

Gene

None

None

LIPE (267)

MFN2 (268)

tRNALys(266)

PALB2(156)

None

Biomarkers

None

None

miRNA (279)

None

None

Abbreviations: miRNA: microRNA; PALB2; Partner and localizer of BRCA2

 

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Normal Physiology of Growth Hormone in Adults

ABSTRACT

Growth hormone (GH) is an ancestral hormone secreted episodically from somatotroph cells in the anterior pituitary. Since the recognition of its multiple and complex effects in the early 1960s, the physiology and regulation of GH has become a major area of research interest in the field of endocrinology. In adulthood, its main role is to regulate the metabolism. Pituitary synthesis and secretion of GH is stimulated by episodic hypothalamic secretion of GH releasing factor and inhibited by somatostatin. Insulin-like Growth Factor I (IGF-I) inhibits GH secretion by a negative loop at both hypothalamic and pituitary levels. In addition, age, gender, pubertal status, food, exercise, fasting, sleep and body composition play important regulatory roles. GH acts both directly through its own receptors and indirectly through the induced production of IGF-I. Their effects may be synergistic (stimulate growth) or antagonistic, as for the effect on glucose metabolism: GH stimulates lipolysis and promotes insulin resistance, whereas IGF-I acts as an insulin agonist. The bioactivity of IGF-I is tightly controlled by several IGF-I binding proteins. The mechanisms underlying the insulin antagonist effect of GH in humans are causally linked to lipolysis and the ensuing elevated levels of circulating free fatty acids. The nitrogen retaining properties of GH predominantly involve stimulation of protein synthesis, which could be either direct or mediated through IGF-I, insulin or lipid intermediates. In the present chapter, the normal physiology of GH secretion and the effects of GH on intermediary metabolism throughout adulthood, focusing on human studies, are presented.

INTRODUCTION

Harvey Cushing proposed in 1912 in his monograph "The Pituitary Gland" the existence of a "hormone of growth", and was thereby among the first to indicate that the primary action of growth hormone (GH) was to control and promote skeletal growth. In clinical medicine GH (also called (somatotrophin) was previously known for its role on promoting growth of hypopituitary children, and for its adverse effects in connection with hypersecretion as observed in acromegaly. The multiple and complex actions of human GH were, however, acknowledged shortly after the advent of a pituitary-derived preparation of the hormone in the late fifties - as reviewed by Raben in 1962 (1).

In the present chapter we will briefly review the normal physiology of GH secretion and the effects of GH on intermediary metabolism throughout adulthood. Other important physiological effects of GH are presented in the review on GH replacement in adults.

GROWTH HORMONE

GH is a single chain protein with 191 amino-acids and two disulfide bonds. The human GH gene is located on chromosome 17q22 as part of a locus that comprises five genes. In addition to two GH related genes (GH1 that codes for the main adult growth hormone, produced in the somatotrophic cells found in the anterior pituitary gland and, to a minor extent, in lymphocytes, and GH2 that codes for placental GH), there are three genes coding for chorionic somatomammotropin (CSH1, CSH2 and CSHL) (also known as placental lactogen) genes (2,3). The GH1 gene encodes two distinct GH isoforms (22 kDa and 20 kDa). The principal and most abundant GH form in the pituitary and blood is the monomeric 22K-GH isoform, representing also the recombinant GH available for therapeutic use (and subsequently for doping purposes) (3). Administration of recombinant 22K-GH exogenously leads to a decrease in the 20K-GH isoform, and thus testing both isoforms is used to detect GH doping in sports (4).

As already mentioned, GH is secreted by the somatotroph cells located primarily in the lateral wings of the anterior pituitary. A recent single cell RNA sequencing study performed in mice showed that GH-expressing cells, representing the somatotrophs, are the most abundant cell population in the adult pituitary gland (5). The differentiation of somatotroph cell is governed by the pituitary transcription factor 1 (Pit-1). Data in mice suggest that the pituitary holds regenerative competence, the GH-producing cells being regenerated form the pituitary’s stem cells in young animals after a period of 5 months (6).

Physiological Regulation of GH Secretion

The morphological characteristics and number of somatotrophs are remarkably constant throughout life, while their secretion pattern changes. GH secretion occurs in a pulsatile fashion, and in a circadian rhythm with a maximal release in the second half of the night. So, sleep is an important physiological factor that increases the GH release. Interestingly, the maximum GH levels occur within minutes of the onset of slow wave sleep and there is marked sexual dimorphism of the nocturnal GH increase in humans, constituting only a fraction of the total daily GH release in women, but the bulk of GH output in men (7).

GH secretion is also gender-, pubertal status- and age- dependent (Figure 1 and Figure 4) (8). Integrated 24h GH concentration is significantly greater in women than in men and greater in the young than in older adults. The serum concentration of free estradiol, but not free testosterone, correlates with GH, and when correcting for the effects of estradiol, neither gender nor age influence GH concentration. This suggests that estrogens play a crucial role in modulating GH secretion (8). During puberty, a 3-fold increase in pulsatile GH secretion occurs that peaks around the age of 15 years in girls and 1 year later in boys (9).

Figure 1 The secretory pattern of GH in young and old female and male. In young individuals the GH pulses are larger and more frequent and that female secrete more GH than men (modified from (8)).

Pituitary synthesis and secretion of GH is stimulated by episodic hypothalamic hormones. Growth hormone releasing hormone (GHRH) stimulates while somatostatin (SST) inhibits GH production and release. GH stimulates IGF-I production which in turn inhibits GH secretion at both hypothalamic and pituitary levels. The gastric peptide ghrelin is also a potent GH secretagogue, which acts to amplify hypothalamic GHRH secretion and synergize with its pituitary GH-stimulating effects (Figure 2) (10). Interestingly, recently germline or somatic duplication of GPR101 has been shown to constitutively activate the cAMP pathway in the absence of a ligand, leading to GH release. Although the precise physiology of GPR101 is unclear, it is worth mentioning it since it clearly has an effect on GH pathophysiology (11).

In addition, a multitude of other factors may impact the GH axis, most probably due to interaction with GRHR, somatostatin, and ghrelin. Estrogens stimulate the secretion of GH, but inhibit the action of GH on the liver by suppressing GH receptor (GHR) signaling. In contrast, androgens enhance the peripheral actions of GH (12). Exogenous estrogens potentiate pituitary GH responses to submaximal effective pulses of exogenous GHRH (13) and mute inhibition by exogenous SST (14). Also, exogenous estrogen potentiates ghrelin’s action (15).

GH release correlates inversely with intraabdominal visceral adiposity via mechanisms that may depend on increased free fatty acids (FFA) flux, elevated insulin, or free IGF-I.

Figure 2. Factors that stimulate and suppress GH secretion under physiological conditions.

GROWTH HORMONE RELEASING HORMONE

GHRH is a 44 amino-acid polypeptide produced in the arcuate nucleus of the hypothalamus. These neuronal terminals secrete GHRH to reach the anterior pituitary somatotrophs via the portal venous system, which leads to GH transcription and secretion. Moreover, animal studies have demonstrated that GHRH plays a vital role in the proliferation of somatotrophs in the anterior pituitary, whereas the absence of GHRH leads to anterior pituitary hypoplasia (16). In addition, GHRH up-regulates GH gene expression and stimulates GH release (17). The secretion of GHRH is stimulated by several factors including depolarization, α2-adrenergic stimulation, hypophysectomy, thyroidectomy and hypoglycemia, and it is inhibited by SST, IGF-I, and activation of GABAergic neurons.

GHRH acts on the somatotrophs via a seven trans-membrane G protein-coupled stimulatory cell-surface receptor. This receptor has been extensively studied over the last decade leading to the identification of several important mutations. Point mutations in the GHRH receptors, as illustrated by studies done on the lit/lit dwarf mice, showed a profound impact on subsequent somatotroph proliferation leading to anterior pituitary hypoplasia (18). Unlike the mutations in the Pit-1 and PROP-1 genes, which lead to multiple pituitary hormone deficiencies and anterior pituitary hypoplasia, mutations in the GHRH receptor lead to profound GH deficiency with anterior pituitary hypoplasia. Subsequent to the first GHRH receptor mutation described in 1996 (19), an array of familial GHRH receptor mutations have been recognized over the last decade. These mutations account for almost 10% of familial isolated GH deficiencies. An affected individual will present with short stature and a hypoplastic anterior pituitary. However, they lack certain typical features of GH deficiency such as midfacial hypoplasia, microphallus, and neonatal hypoglycemia (20).

SOMATOSTATIN (SST)

SST is a cyclic peptide, encoded by a single gene in humans, which mostly exerts inhibitory effects on endocrine and exocrine secretions. Many cells in the body, including specialized cells in the anterior paraventricular nucleus and arcuate nucleus, produce SST. These neurons secrete SST into the adenohypophyseal portal venous system, via the median eminence, to exert effects on the anterior pituitary. SST has a short half-life of approximately 2 minutes as it is rapidly inactivated by tissue peptidase in humans.

SST acts via a seven trans-membrane, G protein coupled receptor and, thus far, five subtypes of the receptor have been identified in humans (SSTR1-5). Although all five receptor subtypes are expressed in the human fetal pituitary, the adult pituitary only expresses 4 subtypes (SSTR1, SSTR2, SSTR3, SSTR5). Of these four subtypes, somatotrophs exhibit more sensitivity to SSTR2 and SSTR5 ligands in inhibiting the secretion of GH in a synergistic manner (21). Somatostatin inhibits GH release but not GH synthesis.

GHRELIN

Ghrelin is a 28 amino-acid peptide that is the natural ligand for the GH secretagogue receptor. In fact, ghrelin and GHRH have a synergistic effect in increasing circulating GH levels (7). Ghrelin is primarily secreted by the stomach and may be involved in the GH response to fasting and food intake.

Clinical Implications

GH levels – influence of body composition, physical fitness and age

With the introduction of dependable radioimmunological assays, it was recognized that circulating GH is blunted in obese subjects, and that normal aging is accompanied by a gradual decline in GH levels (22,23). It has been hypothesized that many of the senescent changes in body composition and organ function are related to or caused by decreased GH (24), also known as "the somatopause".

Studies carried out in the late 90s have uniformly documented that adults with severe GH deficiency are characterized by increased fat mass and reduced lean body mass (LBM) (25). It is also known that normal GH levels can be restored in obese subjects following massive weight loss (26), and that GH substitution in GH-deficient adults normalizes body composition. What remains unknown is the cause-effect relationship between decreased GH levels and senescent changes in body composition. Is the propensity for gaining fat and losing lean mass initiated or preceded by a primary age-dependent decline in GH secretion and action? Alternatively, accumulation of fat mass secondary to non-GH dependent factors (e.g. life style, dietary habits) results in a feedback inhibition of GH secretion. Moreover, little is known about possible age-associated changes in GH pharmacokinetics and bioactivity.

Cross-sectional studies performed to assess the association between body composition and stimulated GH release in healthy subjects show that adult people (mean age 50 yr) have a lower peak GH response to secretagogues (clonidine and arginine), while females had a higher response to arginine when compared to males. Multiple regression analysis, however, reveal that intra-abdominal fat mass is the most important and negative predictor of peak GH levels, as previously mentioned (27). In the same population, 24-h spontaneous GH levels also predominantly correlated inversely with intra-abdominal fat mass (Figure 3) (28).

Figure 3. Correlation between intra-abdominal fat mass and 24-hour GH secretion.

A detailed analysis of GH secretion in relation to body composition in elderly subjects has, to our knowledge, not been performed. Instead, serum IGF-I has been used as a surrogate or proxy for GH status in several studies of elderly men (29-31). These studies comprise large populations of ambulatory, community-dwelling males aged between 50-90 yr. As expected, the serum IGF-I declined with age (Figure 4), but IGF-I failed to show any significant association with body composition or physical performance.

Figure 4. Changes in serum IGF-I with age; modified from (32).

GH action: Influence of age, sex and body composition

Considering the great interest in the actions of GH in adults, surprisingly few studies have addressed possible age-associated differences in the responsiveness or sensitivity to GH. In normal adults the senescent decline in GH levels is paralleled by a decline in serum IGF-I, suggesting a down-regulation of the GH-IGF-I axis. Administration of GH to elderly healthy adults has generally been associated with predictable, albeit modest, effects on body composition and side effects in terms of fluid retention and modest insulin resistance (33). Whether this reflects an unfavorable balance between effects and side effects in older people or the employment of excessive doses of GH is uncertain, but it is evident that older subjects are not resistant to GH. Short-term dose-response studies clearly demonstrate that older patients require a lower GH dose to maintain a given serum IGF-I level (34,35), and it has been observed that serum IGF-I increases in individual patients on long-term therapy if the GH dosage remains constant. Moreover, patients with GH deficiency older than 60 years are highly responsive to even a small dose of GH (36). Interestingly, there is a gender difference response to GH treatment with men being more responsive in terms of IGF-I generation and fat loss during therapy, most probably due to lower estrogen levels that negatively impact the GH effect on IGF-I generation in the liver (37).

The pharmacokinetics and short-term metabolic effects of a near physiological intravenous GH bolus (200μg) were compared in a group of young (30 year) and older (50 year) healthy adults (38). The area under the GH curve was significantly lower in older subjects, whereas the elimination half-life was similar in the two groups, suggesting both an increased metabolic clearance rate and apparent distribution volume of GH in older subjects. Both parameters showed a strong positive correlation with fat mass, although multiple regression analysis revealed age to be an independent positive predictor. The short-term lipolytic response to the GH bolus was higher in young as compared to older subjects. Interestingly, the same study showed that the GH binding proteins correlated strongly and positively with abdominal fat mass (39).

A prospective long-term study of normal adults with serial concomitant estimations of GH status and adiposity would provide useful information about the cause-effect relationship between GH status and body composition as a function of age. In the meantime, the following hypothesis is proposed (Figure 5): 1. Changes in life-style and genetic predispositions promote accumulation of body fat with aging; 2. The increased fat mass, leads to increased FFA availability, and induces insulin resistance and hyperinsulinemia; 3. High insulin levels suppress IGF binding protein (IGFBP)-1 resulting in a relative increase in free IGF-I levels; 4. Systemic elevations of FFA, insulin and free IGF-I suppress pituitary GH release, which further increases fat mass; 5. Endogenous GH is cleared more rapidly in subjects with a high amount of fat tissue.

At present it is not justified to treat the age-associated deterioration in body composition and physical performance with GH especially due to concern that the ensuing elevation of IGF-I levels may increase the risk for the development of neoplastic disease (For an extensive discussion of GH in the elderly see the chapter on this topic in the Endocrinology of Aging section of Endotext).

Figure 5. Hypothetical model for the association between low GH levels and increased visceral fat in adults.

Life-long GH deficiency

A real-life model for GH effects in human physiology is represented by patients with life-long severe reduction in GH signaling due to GHRH or GHRH receptor mutations, combined deficiency of GH, prolactin, and TSH, or global deletion of GHR. They show short stature, doll facies, high-pitched voices, and central obesity, and are fertile (40). Despite central obesity and increased liver fat, they are insulin sensitive, partially protected from cancer and present a major reduction in pro-aging signaling and perhaps increased longevity (41). The decrease of cancer risk in life-long GH deficiency together with reports on the permissive role of GH for neoplastic colon growth (42), pre-neoplastic mammary lesions (43), and progression of prostate cancer (44) demands, at least, a careful tailoring of GH substitution dosage in the GH deficient patients.

GH and the immune system

Although the majority of data on the relation between GH and the immune system are from animal studies, it seems that GH may possess immunomodulatory actions. Immune cells, including several lymphocyte subpopulations, express receptors for GH, and respond to its stimulation (45). GH stimulates in vitro T and B-cell proliferation and immunoglobulin synthesis, enhances human myeloid progenitor cell maturation, and modulates in vivo Th1/Th2 (8) and humoral immune responses (46). It has been shown that GH can induce de novo T cell production and enhance CD4 recovery in HIV+ patients. Another study with possible clinical relevance showed that sustained GH expression reduced prodromal disease symptoms and eliminated progression to overt diabetes in mouse model of type 1 diabetes, a T-cell–mediated autoimmune disease. GH altered the cytokine environment, triggered anti-inflammatory macrophage (M2) polarization, maintained activity of the suppressor T-cell population, and limited Th17 cell plasticity (46). JAK/STAT signaling, the principal mediator of GHR activation, is well-known to be involved in the modulation of the immune system, so is tempting to assume that GH may have a role too, but clear data in humans are needed.

Growth Hormone Signaling in Humans

Growth hormone RECEPTOR (GHR) activation

GHR signaling is a separate and prolific research field by itself (47), so this section will focus on recent data obtained in human models.

GHRs have been identified in many tissues including fat, lymphocytes, liver, muscle, heart, kidney, brain and pancreas (48,49). Activation of receptor-associated Janus kinase (JAK)-2 is the critical step in initiating GH signaling. One GH molecule binds to two GHR molecules that exist as preformed homodimers. Following GH binding, the intracellular domains of the GHR dimer undergo rotation, which brings together the two intracellular domains each of them binding one JAK2 molecule. This, in turn, induces cross-phosphorylation of tyrosine residues in the kinase domain of each JAK2 molecule followed by tyrosine phosphorylation of the GHR (48,50). Phosphorylated residues on GHR and JAK2 form docking sites for different signaling molecules including signal transducers and activators of transcription (STAT) 1, 3, 5a and 5b. STATs bound to the activated GHR-JAK2 complex are subsequently phosphorylated on a single tyrosine by JAK2 allowing dimerization and translocation to the nucleus, where they bind to DNA and activate gene transcription. A STAT5b binding site has been characterized in the IGF-I gene promoter region (51). Attenuation of JAK2-associated GH signaling is mediated by a family of cytokine-inducible suppressors of cytokine signaling (SOCS) (52). SOCS proteins bind to phosphotyrosine residues on the GHR or JAK2 and suppress GH signaling by inhibiting JAK2 activity and competing with STATs. For example, it has been reported that the inhibitory effect of estrogen on hepatic IGF-I production seems to be mediated via up regulation of SOCS-2 (53).

Data on GHR signaling derive mainly from rodent models and experimental cell lines, although GH-induced activation of the JAK2/STAT5b and the mitogen activated protein kinase (MAPK) pathways have been recorded in cultured human fibroblasts from healthy human subjects (54). STAT5b in human subjects is critical for GH-induced IGF-I expression and growth promotion as demonstrated by the identification of mutations in the STAT5b gene of patients presenting with severe GH insensitivity in the presence of a normal GHR (55). Activation of GHR signaling in vivo has been reported in healthy young male subjects exposed to an intravenous GH bolus vs. saline (56). Significant tyrosine phosphorylation of STAT5b was recorded after GH exposure at 30-60 minutes in muscle and fat biopsies, but there was no evidence of GH-induced activation of PI 3-kinase, Akt/PKB, or MAPK (56).

GH and insulin signaling

GH impairs the insulin mechanism but the exact mechanisms in humans are still a matter of debate. There is no evidence of a negative effect of GH on insulin binding to the receptor (57,58), which obviously implies post-receptor metabolic effects.

There is animal and in vitro evidence to suggest that insulin and GH share post-receptor signaling pathways (59). Convergence has been reported at the levels of STAT5 and SOCS3 (60) as well as on the major insulin signaling pathway: insulin receptor substrates (IRS) 1 and 2, PI 3-kinase (PI3K), Akt, and extracellular regulated kinases (ERK) 1 and 2 (61-63). Studies in rodent models suggest that the insulin-antagonistic effects of GH in adipose involve suppression of insulin-stimulated PI3-kinase activity (59,64). In 2001 it was demonstrated that GH induces cellular insulin resistance by uncoupling PI3K and its downstream signals in 3T3-L1 adipocytes (65)]. A follow up study has shown that GH increased p85α expression and decreased PI3K activity in adipose tissue of mice, supporting the previous report of a direct inhibitory effect of GH on PI3K activity (64). However, a study performed in healthy human skeletal muscle showed, as expected, that the infusion of GH induced a sustained increase in FFA levels and subsequently insulin resistance as assessed by the euglycemic clamp technique, but was not associated with any change in the insulin-stimulated increase in either IRS-1/PI3K or PKB/Akt activity (66). It was subsequently showed that insulin had no impact on GH-induced STAT5b activation or SOCS3 mRNA expression (67).

Because GH and insulin share some common intracellular substrates, a hypothesis arose claiming that competition for intracellular substrates explains the negative effect of GH on insulin signaling (59). Furthermore, studies have shown that SOCS proteins negatively regulate the insulin signaling pathway (68). Therefore, another possible mechanism by which GH alters the action of insulin is by increasing the expression of SOCS genes.

INSULIN-LIKE GROWTH FACTOR-I

Physiology of IGF-I

GH acts both directly through its own receptor and indirectly through the induced production of IGF-I. GH stimulates synthesis of IGF-I in the liver and many other target tissues (Figure 6); about 75% of circulating IGF-I is liver-derived. IGF-I is a 70 amino-acid peptide, found in the circulation, 99% bound to transport proteins (IGFBP) in the circulation.

Following the initial discovery of IGF-I, it was thought that GH governs somatic growth only by IGF-I produced by the liver (69). However, in the 1980s this hypothesis was challenged by the identification of IGF-I production in numerous tissues. IGF-I is known as a global and tissue-specific growth factor as well as an endocrine factor. In some tissues IGF-I acts as a potent inhibitor of cellular apoptosis.

Figure 6. GH is produced in the pituitary gland. In the periphery, GH acts directly and indirectly through stimulation of IGF-I production. In the circulation, the liver is the most important source of IGF-I (75%) but other tissues (e.g. brain, adipose tissue, kidney, bone, and muscles) may contribute. Under GH stimulation the muscle, adipose tissue, and bone have been shown to secrete IGF-I that has a paracrine/autocrine effect.

Interestingly, insulin and IGF-I share many structural and functional similarities, implying that they originated from the same ancestral molecule. Both molecules could have been part of the cycle of food intake and consequent tissue growth. The IGF-I gene is a member of the insulin gene family and the IGF-I receptor is structurally similar to the insulin receptor in its tetrameric structure, with 2 alpha and 2 beta subunits (70). The alpha subunit binds IGF-I, IGF-II, and insulin; however, the subunit has a higher affinity towards IGF-I compared to IGF-II and insulin. Although insulin and IGF-I share many similarities, during evolution the functionality of the two molecules has become more divergent, where insulin plays a more metabolic role and IGF-I is more involved in cell growth.

The IGF-I receptor is expressed in many tissues in the body. However, the receptor number on each cell is strictly regulated by several systemic and tissue factors including circulating GH, iodothyronines, platelet-derived growth factor, and fibroblast growth factor. Following the binding of the IGF-I molecule, the receptor undergoes a conformational change which activates tyrosine kinase, leading to auto-phosphorylation of tyrosine. The activated receptor phosphorylates IRS-2, which in-turn activates the RAS activating protein SOS. This complex activates the MAPK pathway leading to the stimulation of cell growth (71,72).

The IGFBP family comprises six binding proteins (IGFBP 1-6) with a high affinity towards IGF-I and II. Apart from regulating the free plasma IGF fraction, IGFBPs also play an important role in the transport of IGF into different tissues and extravascular space. IGFBP-3 and IGFBP-2 are the most abundant forms seen in plasma and are saturated with IGF-I due to their high affinity: 75% of IGF-I is bound to IGFBP-3. Interestingly, similar to IGF-I, IGFBP-3 production is also regulated by GH. In the plasma, IGFBP-3 is bound to a protein called acid labile subunit (ALS), which stabilizes the “IGFBP3-IGF-I” complex, prolonging its half-life to approximately 16 hours (73). IGFBP-1, on the other hand, is present in lower concentration in plasma than IGFBP-2 and 3. However, due to lower affinity for IGF-I, IGFBP-1 is usually in an unsaturated state and changing plasma concentrations of IGFBP-1 become important in determining the unbound fraction of IGF-I. A recently new discovered player in the regulation of IGF-I bioavailability is the pregnancy-associated plasma protein-A2 (PAPP-A2) that cleaves IGFBP3 and 5 and releases IGF-I. Homozygous mutations in PAPP-A2 result in growth failure with elevated total but low free IGF-I (74). Low IGF-I bioavailability impairs growth and glucose metabolism in a mouse model of human PAPP-A2 deficiency and treatment with recombinant human IGF-I in PAPP-A2 deficient patients improves growth and bone mass and ameliorates glucose metabolism (74,75).

Effects of IGF-I

Studies on hypophysectomized animals overexpressing IGF-I demonstrate the independent anabolic effects of IGF-I (76). IGF-I plays a key role in growth, where it acts not only as a determinant of postnatal growth, but also as an intra-uterine growth promoter. Total inactivation of the IGF-I gene in mice produce a perinatal mortality of 80% with the surviving animal showing significant growth retardation compared to controls (77). Human IGF-I deficiency can be either due to GH deficiency, GHR inactivation, or IGF-I gene mutation. Interestingly, infants with congenital GH deficiency and GHR mutations present with only minor growth retardation, whereas the rare patient with IGF-I deficiency, secondary to a homozygous partial deletion of the IGF-I gene, presents with severe pre- and postnatal growth failure, mental retardation, sensorineural deafness and microcephaly (78-80). The differences in the clinical presentation are most likely due to the fact that some degree of IGF-I production is present in patients with GH deficiency, and GHR and GHRH defects. The important growth promoting role of IGF-I is further demonstrated by studies on transgenic mice. Only 6-8% postnatal growth retardation is presented in mice with liver-selective deletion of IGF-I gene showing low serum IGF-I concentrations, whereas animals with total IGF-I deletion or those with only peripherally produced IGF-I deletion showed marked growth retardation (81).

Both elevated and reduced levels of serum IGF-I are associated with excess mortality in human adults (82). In addition, it is well recognized in many species including worms, flies, rodents and primates that a reciprocal relationship exists between longevity and activation of the insulin/IGF axis (82). In this regard, it is noteworthy that calorie restriction is associated with increased longevity and reduced insulin/IGF activity in many species (83), albeit GH levels being increased by calorie restriction and fasting (84).

In the context of GH and IGF-I physiology it can be concluded that 1) during childhood and adolescence the combined actions of GH and IGF-I in the presence of sufficient nutrition promote longitudinal growth and somatic maturation, 2) continued excess IGF-I activity in adulthood increases the risk for cardiovascular and neoplastic diseases and hence reduces longevity, and 3) calorie restriction, which suppresses IGF-I activity and stimulates GH secretion, may promote longevity also in human adults (84).

METABOLIC EFFECTS OF GROWTH HORMONE

The nutritional status dictates the effects of GH. In the state of ‘feast’ and sufficient nutrient intake where insulin is increased in the liver and IGF-I production is stimulated, GH promotes protein anabolism. Whereas, in a state with decreased nutrient intake and during the sleep and exercise, the direct effects of GH are more predominant and this is mainly characterized by stimulation of lipolysis.

Glucose Homeostasis and Lipid Metabolism

The involvement of the pituitary gland in the regulation of substrate metabolism was originally detailed in the classic dog studies by Houssay (85). Fasting hypoglycemia and pronounced sensitivity to insulin were distinct features of hypophysectomized animals. These symptoms were readily corrected by administration of anterior pituitary extracts. It was also noted that pancreatic diabetes was alleviated by hypophysectomy. Finally, excess of anterior pituitary lobe extracts aggravated or induced diabetes in hypophysectomized dogs. Furthermore, glycemic control deteriorated following exposure to a single supraphysiological dose of human GH in hypophysectomized adults with type 1 diabetes mellitus (86). Somewhat surprisingly, only modest effects of GH on glucose metabolism were recorded in the first metabolic balance studies involving adult hypopituitary patients (87,88).

More recent studies on glucose homeostasis in GH deficient adults have generated results which at first glance may appear contradictory. Insulin resistance may be more prevalent in untreated GH deficient adults, whereas the impact of GH replacement on this feature seems to depend on the duration and the dose (89).

Below, some of the metabolic effects of GH in human subjects, with special reference to the interaction between glucose and lipid metabolism, will be reviewed.

Studies in Normal Adults

More than fifty years ago, it was shown that infusion of high-dose GH into the brachial artery of healthy adults reduced forearm glucose uptake in both muscle and adipose tissue, which was paralleled by increased uptake and oxidation of FFA (90). This pattern was opposite to that of insulin, and GH in the same model abrogated the metabolic actions of insulin.

Administration of a GH bolus in the post-absorptive state stimulates lipolysis following a lag time of 2-3 hours (91). Plasma levels of glucose and insulin, on the other hand, change very little. This is associated with small reductions in muscular glucose uptake and oxidation, which could reflect substrate competition between glucose and fatty acids (i.e. the glucose/fatty acid cycle) (Figure 7). In line with this, sustained exposure to high GH levels induces both hepatic and peripheral (muscular) resistance to the actions of insulin on glucose metabolism together with increased (or inadequately suppressed) lipid oxidation. Apart from enhanced glucose/fatty acid cycling, it has been shown that GH-induced insulin resistance is accompanied by reduced muscle glycogen synthase activity (57) and diminished glucose dependent glucose disposal (92). However, insulin binding and insulin receptor kinase activity from muscle biopsies is not affected by GH (57).

Lessons from Acromegaly

Active acromegaly clearly unmasks the diabetogenic properties of GH. In the basal state plasma glucose is elevated despite compensatory hyperinsulinemia. In the basal and insulin-stimulated state (euglycemic glucose clamp) hepatic and peripheral insulin resistance is associated with enhanced lipid oxidation and energy expenditure (93). There is evidence to suggest that this hyper-metabolic state ultimately leads to beta cell exhaustion and overt diabetes mellitus (94), but it is also shown that the abnormalities are completely reversed after successful surgery (93). Conversely, it has been shown that administration of GH in supraphysiological doses for only two weeks induces comparable acromegaloid - and reversible - abnormalities in substrate metabolism and insulin sensitivity (95).

Interaction of Glucose and Lipid Metabolism

The effect of FFA on the partitioning of intracellular glucose fluxes was originally described by Randle et al. (96). According to this hypothesis (the glucose/fatty acid cycle), oxidation of FFA initiates an upstream, chain-reaction-like, inhibition of glycolytic enzymes, which ultimately inhibits glucose uptake (Figure 7).

Figure 7. The glucose fatty-acid cycle. A. Randle proposed in 1963 that increased FFA compete with and displace glucose utilization leading to a decreased glucose uptake. The hypothesis stated that an increase in fatty acid oxidation in muscle and fat results in higher acetyl CoA in mitochondria leading to inactivation of two rate-limiting enzymes of glycolysis (i.e., phosphofructokinase (PFK) and pyruvate dehydrogenase (PDH) complex). A subsequent increase in intracellular glucose-6-phosphate (glucose 6-P) results in high intracellular glucose concentrations and decreased glucose uptake by muscle and fat. B. However, in contrast to the proposed hypothesis by Randle, studies using MR spectroscopy have shown reductions in intramyocellular glucose 6-P and glucose concentrations and have led to an alternative hypothesis. The new hypothesis proposes that a transient increase of intracellular diacylglycerol (DAG) activates the theta isoform of protein kinase C (PKCθ) that causes increased serine phosphorylation of IRS-1/2 and consecutively decrease PI3K activation and glucose-transport activity leading to decrease intracellular glucose concentrations.

The Randle hypothesis remains an appealing model to explain the insulin-antagonistic effects of GH when considering its pronounced lipolytic effects. To support this, experiments have shown that co-administration of anti-lipolytic agents and GH reverses GH-induced insulin resistance (97). Moreover it has been shown that GH-induced insulin resistance is associated with suppressed pyruvate dehydrogenase activity in skeletal muscle (98). However, according to the Randle hypothesis, the fatty acid-induced insulin resistance will result in elevated intracellular levels of both glucose and glucose-6-phosphate (Figure 7), whereas the muscle biopsies from GH deficient adults after GH treatment have revealed increased glucose but low-normal glucose-6-phosphate levels (99).

Implications for GH Replacement

Regardless of the exact mechanisms, the insulin antagonistic effects may cause concern when replacing adult GH deficient patients with GH, since some of these patients are insulin resistant in the untreated state. There is evidence to suggest that the direct metabolic effects on GH may be balanced by long-term beneficial effects on body composition and physical fitness, but some studies report impaired insulin sensitivity in spite of favorable changes in body composition. There is little doubt that these effects of GH are dose-dependent and may be minimized or avoided if an appropriately low replacement dose is used. Still, the pharmacokinetics of subcutaneous (s.c.) GH administration is unable to mimic the endogenous GH pattern with suppressed levels after meals and elevations only during post absorptive periods, such as during the night. This may be considered the natural domain of GH action, which coincides with minimal beta-cell challenge. This reciprocal association between insulin and GH and its potential implications for normal substrate metabolism was initially described by Rabinowitz & Zierler (100). The problem arises when GH levels are elevated during repeated prandial periods. The classic example is active acromegaly, but prolonged high dose s.c. GH administration may cause similar effects. Administration of GH in the evening probably remains the best compromise between effects and side effects (101), but it is far from physiological.

We know and understand that hypoglycemia is a serious and challenging side effect of insulin therapy as a consequence of inappropriately high insulin levels (during fasting). As a corollary, we must realize that hyperglycemia may result from GH therapy. It is therefore important to carefully monitor glucose metabolism and to use the lowest effective dose when replacing adults with GH.

Effects of GH on Muscle Mass and Function

The anabolic nature of GH is clearly evident in patients with acromegaly and vice versa in patients with GH deficiency. A large number of in vitro and animal studies throughout several decades have documented stimulating effects of GH on skeletal muscle growth. The methods employed to document in vivo effects of GH on muscle mass in humans have been exhaustive, including whole body retention of nitrogen and potassium, total and regional muscle protein metabolism using labeled amino-acids, estimation of LBM by total body potassium or dual x-ray absorptiometry, and direct calculation of muscle area or volume by computerized tomography and magnetic resonance imaging.

Effects of GH on Skeletal Muscle Metabolism in Vitro and in Vivo

The clinical picture of acromegaly and gigantism includes increased LBM of which skeletal muscle mass accounts for approximately 50%. Moreover, retention of nitrogen was one of the earliest observed and most reproducible effects of GH administration in humans (1). Thoroughly conducted studies with GH administration in GH deficient children, using a variety of classic anthropometric techniques, strongly suggested that skeletal muscle mass increased significantly during treatment (102,103). Indirect evidence of an increase in muscle cell number following GH treatment was also presented (103).

These early clinical studies were paralleled by experimental studies in rodent models. GH administration in hypophysectomized rats increased not only muscle mass, but also muscle cell number (i.e. muscle DNA content) (103). Interestingly, the same series of experiments revealed that work-induced muscle hypertrophy could occur in the absence of GH. The ability of GH to stimulate RNA synthesis and amino-acid incorporation into protein of isolated rat diaphragm suggested direct mechanisms of actions, whereas direct effects of GH on protein synthesis could not be induced in liver cell cultures (104). Another important observation of that period was that GH directly increases the synthesis of both sarcoplasmic and myofibrillar protein without affecting proteolysis in a rat model (105).

In a human study, the in vivo effects of systemic and local GH and IGF-I administration on total and regional protein metabolism revealed that GH administration for 7 days in normal adults increased whole body protein synthesis without affecting proteolysis (106), and comparable results have been obtained in other human studies (107-110).

Based on these studies it seems that the nitrogen-retaining properties of GH predominantly involve stimulation of protein synthesis without affecting (lowering) proteolysis. Theoretically, the protein anabolic effects of GH could be either direct or mediated through IGF-I, insulin, or lipid intermediates. GHR are present in skeletal muscle (49), which allows for direct GH effects; alternatively, GH may stimulate local muscle IGF-I release, which subsequently acts in an autocrine/paracrine manner. The effects of systemic IGF-I administration on whole body protein metabolism seem to depend on ambient amino-acid levels in the sense that IGF-I administered alone suppresses proteolysis (111) whereas IGF-I in combination with an amino-acid infusion increase protein synthesis (112). It is therefore likely that the muscle anabolic effects of GH, at least to some extent, are mediated by IGF-I. By contrast, it is repeatedly shown that insulin predominantly acts through suppression of proteolysis and this effect(s) appears to be blunted by co-administration of GH (113). The degree to which mobilization of lipids contributes to the muscle anabolic actions of GH has so far not been specifically investigated.

An interesting discovery has been that infusion of GH and IGF-I into the brachial artery increases forearm blood flow several fold (110,114). This effect appears to be mediated through stimulation of endothelial nitric oxide release leading to local vasodilatation (115,116). Thus, it appears that an IGF-I mediated increase in muscle nitric oxide release accounts for some of the effects of GH on skeletal muscle protein synthesis. This increase in muscle blood flow may also contribute to the GH-induced increase in resting energy expenditure, since skeletal muscle metabolism is a major determinant of resting energy expenditure (23). Moreover, it is plausible that the reduction in total peripheral resistance seen after GH administration in adult growth hormone deficiency is mediated by nitric oxide (116).

Effects of GH Administration on Muscle Mass and Function in Adults without GH-Deficiency

As previously mentioned, the ability of acute and more prolonged GH administration to retain nitrogen in healthy adults has been known for decades, and more recent studies have documented a stimulatory effect on whole body and forearm protein synthesis.

Rudman et al. were the first to suggest that the senescent changes in body composition were causally linked to the concomitant decline in circulating GH and IGF-I levels (23). This concept has been recently reviewed (117), and a number of studies with GH and other anabolic agents for treating the sarcopenia of ageing are currently in progress.

Placebo-controlled GH administration in young healthy adults undergoing a resistance exercise program for 12 weeks showed a GH induced increase in LBM, whole body protein balance, and whole body protein synthesis, whereas quadriceps muscle protein synthesis rate and muscle strength increased to the same degree in both groups during training (118). In a similar study in older men, GH also increased LBM and whole body protein synthesis, without significantly amplifying the effects of exercise on muscle protein synthesis or muscle strength (119). An increase in LBM but unaltered muscle strength following 10 weeks of GH administration plus resistance exercise training was also recorded (120). A more recent study in older men observed a significant increase (4.4 %) in LBM with GH, but no significant effects on muscle strength (121). Finally, a meta-analysis of studies administering GH to healthy adult subjects showed that it increases LBM and reduces fat mass without improving muscle strength or aerobic exercise capacity (122).

Numerous studies have evaluated the effects of GH administration in chronic and acute catabolic illness. A comprehensive survey of the prolific literature within this field is beyond the scope of this review, but it is noteworthy that HIV-associated body wasting is a licensed indication for GH treatment in the USA. In these patients, GH treatment for 12 weeks has been associated with significant increments in LBM and physical fitness (123,124).

CONCLUSIONS

The GH/IGF-I axis is specifically regulated and is involved in a multitude of processes during all the aspects of life from intrauterine growth, to childhood and puberty, adulthood and lastly elderly stages of life. GH acts directly or via its principal metabolite, IGF-I, and has a wide range of physiological roles being a metabolic active hormone in adulthood. The nutritional status of an organism dictates the effects of GH, either an impairment of insulin action (fasting state) or promoting protein anabolism (fed state). As our knowledge of GH normal physiology increases, our ability to understand and specifically target the GH/IGF-I pathway for a diverse range of therapeutic purposes should also increase. Normal aging is associated with a gradual decline in serum IGF-I levels that run in parallel with reductions in muscle mass and function and other senescent changes in organ function. The cause-effect relationship is uncertain, but GH administration to elderly people without pituitary disease has not proven beneficial and sustained supra-physiological IGF levels and actions are likely to be harmful. On the other hand, a stimulation of endogenous GH secretion induced by exercise and calorie restriction may contribute to healthy aging.  

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Social and Environmental Factors Influencing Obesity

ABSTRACT

 

The evidence for social and environmental factors that contribute to obesity are often underappreciated. Obesity prevalence is significantly associated with sex, racial ethnic identity, and socioeconomic status, which creates complex relationships between each of these characteristics. Food availability remains an important factor associated with obesity that relates to differences in prevalence seen across geographical areas and higher rates of obesity within low socioeconomic status individuals. Proliferation of high calorie, energy dense food options that are or perceived as more affordable combined with reductions in occupational and transportation related physical activity can contribute to a sustained positive energy balance.  Additionally, environments experiencing deprivation, disorder, or high crime have been shown to be associated with higher odds of obesity, which may appear more frequently in low social status individuals. Both objective and subjective measures of social status and inequality are associated with increased energy intake and decreased energy expenditure, which could place individuals of low social status at greater risk for obesity development. Given the complexity of this multifactorial disease, effective obesity care requires knowledge of these complex relationships and an integration between the health systems and surrounding community. Resources for practicing clinicians regarding methods of screening for social and environmental factors in clinical care are provided in addition to information on a program that has been widely dispersed and made accessible to those who may be the most at risk. 

 

INTRODUCTION

 

Many medical providers appreciate the significant social and environmental determinants of obesity but are unsure how to address them. Others consider these factors outside of their control and scope of practice, and are thus hesitant to even broach the topic with their patients. Finally, many medical providers still attribute obesity to causes within a person’s control, such as dietary choices, amount of exercise, or willpower, (1, 2) which perpetuates a stigma that accompanies this disease.  Specifically, the prevailing stigma is that those who suffer from obesity represent a population who lack the willingness to change their poor lifestyle habits or harbor a character flaw that, at its extreme, infers immoral behaviors (e.g., gluttony). In reality, obesity is a multifactorial disease (3) that is caused by a combination of biological, genetic, social, environmental, and behavioral determinants. In order to address this gap in the understanding of the social and environmental determinants of obesity and improve the care of patients with obesity, this chapter will review the evidence for the social and environmental determinants of obesity development. The specific areas to be covered include social identity, social status, societal trends, and influences of the built, industrial, and social environments, all factors that are closely associated with the prevalence or incidence of obesity or that impact efforts to prevent and treat this disease.  Resources for the busy clinician that will support implemental changes in one’s practice to improve the care and management of patients with obesity, as well as evidenced-based opportunities for advocacy in the community, will be included in the final section.

 

This chapter is divided into three primary sections based on the progression of thought and evidence surrounding the social and environmental determinants of obesity: individual characteristics, environmental characteristics, and social hierarchy influences. Individual characteristics are those that are attributed to the individual with obesity such as their sex, age, race, ethnicity, and socioeconomic status (SES). Environmental characteristics surround the individual, including the physical spaces where people live, work, and play, as well as sociocultural norms. The social hierarchy refers to social status or social rank of individuals within larger society or a local community.

 

INDIVIDUAL CHARACTERISTICS

 

The prevalence of obesity varies according to key individual characteristics such as age, sex, race and ethnicity, and SES. The prevalence of obesity increases cross-sectionally across the lifespan: from 13.9%, in early childhood (2-5 years old)  to 18.4% in childhood (6-11 years old),  20.6% in adolescence (12-19 years old), 35.7%, in young adulthood (20-39 years old),  42.8% in adulthood (40-59 years old), and 41.0% in older adulthood (≥60 years old) (4).  As of 2016, the prevalence of adult obesity in women in the United States was 41.1% and in men was 37.9% (4).  In the decade between 2007-2008 and 2015-2016, obesity significantly increased only in women (4), suggesting a sex-specific vulnerability to expression of this disease. Additionally, when race and ethnicity are considered, significant interactions between race and sex emerge. Non-Hispanic black, non-Hispanic Asian, and Hispanic women all have significantly higher prevalence of obesity than men with the same racial ethnic identity (5). In men and women, non-Hispanic Asians have significantly lower prevalence of obesity compared to all other major races and ethnicities in the United States (Note: not adjusted for ethnic specific cut points for Asians), and Non-Hispanic blacks and Hispanics have significantly higher prevalence of obesity compared to Non-Hispanic whites (5). It is not fully clear why differences in obesity prevalence by race and ethnicity are present, but some evidence points to differences in genetic backgrounds that affect body composition and fat distribution (6, 7), and to differences in cultural body image standards (8). Additionally, in the United States, race and ethnicity are confounded with SES, which is one of the most potent indicators of overall health in the United States (9).

 

A significantly greater proportion of underrepresented racial ethnic minorities are considered low SES compared to non-Hispanic Asians and non-Hispanic whites in the United States. Socioeconomic status is a composite measure that can be represented by measures of income, educational attainment, or occupational status. In the 2017 Census, 21.2% of non-Hispanic blacks and 18.3% of Hispanics lived below the poverty level compared to 8.7% of non-Hispanic whites and 10% of non-Hispanic Asians (10). Non-Hispanic Asians (53.9%) and non-Hispanic whites (36.2%) are more likely to earn a bachelor’s degree than non-Hispanic blacks (22.5%) and Hispanics (15.5%) (11). In terms of health, low SES in childhood is associated with adult development of cardiovascular risk factors and a 20% increase in the odds of having central obesity (as defined by a waist circumference >102 cm for men or > 88 cm for women) (12). In adult women, obesity prevalence increases with decreasing income and educational attainment; however, in non-Hispanic black women, obesity prevalence differs by education gradients but not by income gradients (13). Conversely, non-Hispanic black men have a higher prevalence of obesity in the highest income group, but all the men’s racial ethnic groups showed similar relationships between obesity rates and education gradients as women (13). Higher SES is also associated with healthy lifestyle behaviors that are often the first line of prevention or treatment for obesity. On the other hand, low SES is associated with less leisure time physical activity (14) and consumption of energy-dense diets that are nutrient poor (15); however, SES is not the only factor that influences these behaviors. Further exploration of how SES affects resources and the ability to practice healthy behaviors is expounded upon in the next section.

 

ENVIRONMENTAL CHARACTERISTICS   

 

Geography

 

Obesity prevalence differs by geographical region in the United States with the South and the Midwest having the highest level of obesity among adults (16). The Midwest and South also have high rates of diabetes and metabolic syndrome, which frequently accompany obesity (16).  Approximately 55% of global increases in BMI can be attributed to rising BMI in rural areas, and this may be as high as 80% in low- and middle-income countries (17). Rural areas are associated with 1.36 higher odds of obesity compared to urban areas; however, mediation analysis shows that individual educational attainment, neighborhood median household income, and neighborhood-built environment features reduce these odds by 94% and render the relationship statistically insignificant (18). Rural areas tend to have farther distances between residences and supermarkets, clinical settings, and recreational opportunities, which may be impacting the ability to practice healthy behaviors that prevent obesity. This is one example of the “built environment”, which alludes to the infrastructure of a geographic area that influences proximity to and types of resources, transportation methods, and neighborhood quality.

 

Food Availability

 

The frequency and type of food vendors in a neighborhood determines the types of foods that residents can purchase. Historically, evidence has suggested that fast food restaurant density is associated with obesity prevalence. A state-level analysis of fast food restaurant density and the number of residents per restaurant accounted for 6% of the variance in state obesity prevalence (19). Individual-level factors can interact with built environmental factors (like fast food restaurant density) to increase the odds of obesity. For example, one study in older adults showed that residents who ate 1-2 times per week at a fast food restaurant (odds ratio [OR]: 1.878), did not meet current physical activity guidelines (OR: 1.792), had low self-efficacy for eating healthy food (OR: 1.212), or identified as non-Hispanic black (OR: 8.057) and lived in a high density fast food neighborhood were more likely to have obesity than older adults who lived in a low density fast food neighborhood (20). On the other hand, recent research suggests that fast food restaurant density is not associated with obesity prevalence and the food consumed in these establishments’ accounts for less than 20% of the total energy intake (21). This could reflect the widespread availability of fast food nationally, which weakens the ability to dissect links between its presence and increased consumption specific to obesity.

 

The term “food desert” is often used to describe areas with limited access to affordable and nutritious food (e.g. supermarkets) and these vary significantly according to neighborhood socioeconomic and racial/ethnic composition (22, 23). Food desert designation has been positively linked to obesity in the United States and simply switching from a non-food desert census tract to a food desert census tract can increase the odds of obesity by 30%, when all other relevant factors are held constant (24). Conversely, access to supermarkets does not automatically result in healthier eating behavior and weight status. A systematic review showed that five out of six studies looking at supermarket access did not find increased fruit and vegetable consumption with greater accessibility; however, four out of five studies looking at changes in weight status found lower BMI and prevalence of obesity in areas with high access to supermarkets compared to low access areas (25). A large natural experiment found that the opening of a new supermarket improved overall diet quality in the neighborhood, but did not affect fruit and vegetable intake or BMI (26). Interestingly, the only positive outcome directly associated with regular use of the new supermarket was higher perceived access to healthy food (26). Although these findings are mixed, it is important to acknowledge that changes in food choices at a neighborhood level might occur too slowly to be captured in these studies.

 

In addition to food availability and quality, the shift in food type, amount, and pricing is also relevant to the obesity epidemic. For example, available evidence strongly supports a greater risk of weight gain and type 2 diabetes with increased consumption of sugar-sweetened beverages (27). North America still has the highest per capita sales of calorie sugar-sweetened beverages, but is slowly starting to shift to low-calorie sugar sweetened beverages, though sports and energy drink consumption continue to increase (28). Portion sizes in the most popular fast-food, take-out, and family style restaurants exceed current USDA and FDA standard-recommended portion amounts as well as what had been historically served in past decades (29). Increased portion sizes have been robustly linked to increases in energy intake in both adults and children; however, evidence is limited that decreasing portion size results in decreased energy intake (30). In addition, fast foods, snack foods, and foods available through convenience stores are typically ultra-processed (high in processed grains and added sugars; low in fiber and unsaturated fats).  A recent study found that keeping macronutrient content the same, meals that were ultra-processed resulted in greater food intake and weight gain over a two-week follow-up compared to consumption of non-processed foods (31). Contributing to increased intake of fast-foods and ultra-processed foods is the marketing techniques implemented by food industries across multiple mediums. Though adults have shown to be less susceptible to the effects of food advertising, experimental studies with children produce a moderate effect size for increased food consumption after food advertising exposure (32). Food advertising targeted at children is focused on brand building and emotive messages may not be discerned as such by this vulnerable population (33). Another common misconception confronting consumers is that healthy foods are more expensive, but research suggests this perception is based on misleading price metrics as well as changes in fruit and vegetable convenience and level of preparedness (34). Price per calorie metrics show fruits and vegetables to be more expensive than less healthy foods; however, price per average portion and price per edible 100 grams actually shows that fruits and vegetables are less expensive (34). In times of financial constraint, socioeconomically disadvantaged groups maximize energy value for money resulting in energy-dense, nutrient poor diets that contribute to obesity (35).

 

Transportation

 

Infrastructure can dictate means of transportation and neighborhood walkability, which is associated with weight status. High neighborhood walkability has been found to be associated with decreased prevalence of overweight and obesity (36), which can link back to structural differences discussed earlier between urban and rural areas (urban areas having higher walkability). Transport-related physical activity decreased by 17.8% between 1965 and 2009 in the United States, which could be due to growing ubiquity of car ownership and supportive infrastructure for automotive transport in the United States (37). Proximity to recreational facilities, recreational facility density, access to sidewalks and paths that remove pedestrians from traffic hazards, and access to parks, have all been reported to be facilitators of physical activity in qualitative and quantitative research (38, 39).

 

The quality of infrastructure in a neighborhood and the perceived aesthetics of homes, shops, and recreational facilities can impact the use of these facilities. A study in a high-income neighborhood and a low-income neighborhood showed that even though the number of recreational facilities was equitable in the neighborhoods, the residents of the low-income neighborhood perceived that they had less access to recreational facilities (40).

 

Additional neighborhood descriptors that are associated with obesity include neighborhood deprivation, disorder, and crime. Neighborhood deprivation, a composite score of socioeconomic position of individuals in a neighborhood that is used to assign a rank to that neighborhood, shows that high levels of deprivation are associated with a 20% increased odds of overweight (41). Neighborhood physical disorder refers to the presence of vandalism, abandoned lots or vehicles, garbage, and quality of building conditions. Women in an urban area with high neighborhood physical disorder have a 1.43 greater odds of obesity (42). Persons living in areas of high crime have a 28% reduced odds of achieving higher levels of physical activity and, conversely, perceived safety increases the odds of achieving higher levels of physical activity by 27% (43). Living in a neighborhood with high crime has been found to be associated with increased weekly snack consumption in women (42). The relevance of the neighborhood environment to obesity is further exemplified in the Moving to Opportunities Study (44). The Department of Housing and Urban Development randomly assigned just under 5000 families in Chicago, Baltimore, Boston, Los Angeles, and New York public housing to 3 possible conditions: receive a housing voucher to move to a low-poverty census track with moving counseling, receive a standard unrestricted housing voucher and no moving counseling, or receive nothing. Despite the fact that this study was not focused on weight or diabetes outcomes, participants that received the voucher to move to a low-poverty census track had 4.61 percentage points lower prevalence of BMI > 35, BMI > 40, and glycated hemoglobin ≥ 6.5% than participants who received nothing (44), showing that a mere change in environment from high- to low-poverty rates was enough to have a significant impact.

 

Work Environment and Advances in Communication Technology

 

As the built environment and food environment have changed in the United States, so has the work environment. From 1960 to 2010, jobs in the U.S. private industry shifted from 50% requiring at least moderate to vigorous physical activity to less than 20% requiring this level of activity intensity (45). National Health and Nutrition Examination Survey data has documented an association between decreases in work-related energy expenditure and weight gain over the same time period (45). These changes in occupation related physical activity could be due to improvements in labor-saving technology. Technology advances are not confined to the work environment and have spread into many facets of daily life, such as improvements in smart personal communication devices, internet media platforms, marketing techniques, and enhanced audio-visual media.  Studies show that marketing for unhealthy foods is often targeted at more vulnerable populations such as Non-Hispanic blacks (46) and Hispanics (47). Additionally, the availability of information about healthy weight-loss behaviors on the internet is poor when searched for in Spanish (48).  “Screen time” or the time spent using technology that utilizes a screen interface has been found to be associated with increased risk for obesity (49-51); however, many app companies and academic researchers are now using that same technology to help with obesity prevention and treatment (52-54). 

 

SOCIAL HIERARCHY     

 

Animal research consistently shows that animals of subordinate status experience adverse physiological and behavioral changes compared to their high status counterparts: higher levels of cortisol (primates) (55), elevated blood pressure (rats, rabbits, baboons, macaques) (56), elevated heart rate (primates) (56), accumulation of visceral fat (rats) (57), increased ad-libitum energy-dense food consumption (macaques, rats) (57, 58), cardiovascular disease (mice) (59), and shortened lifespan (mice) (59). This implies that social standing, regardless of species, has physiological implications and could be contributing to obesity development and poor health. The findings from animal models thus serve as the basis for parallel outcomes reported in humans of low social status.

 

Social status can be measured objectively or subjectively. Objective measures typically include socioeconomic status (SES) variables, such as income, education, or occupation, which were discussed as individual level factors at the beginning of this chapter. Social status can also be represented by manifestations of status differentials, including inequality between groups or measurable differences in the ability for someone to obtain basic life necessities, such as food security. High levels of absolute income/wealth may be related to health not only through better material conditions, but also through social position.  However, in an analysis of two nationally representative British panel studies, ranked position of income/wealth, not absolute income/wealth, predicted adverse health outcomes such as obesity, presence of chronic disease, and poor ratings of physical functioning and pain (60). In a worldwide study of physical activity, countries with large activity inequality predicted obesity better than the total volume of physical activity within the country (61). Activity inequality is identified by calculating a Gini coefficient for population step count data from each country, 0 = complete equality, 1= complete inequality. Individuals in the top five countries for physical activity inequality (Saudi Arabia, USA, Egypt, Canada, Australia) were 196% more likely to have obesity than individuals from more equal societies that did not have large disparities in step counts across the population. Gender differences account for 43% of the inequality observed, however, this effect was mitigated in societies that rated higher in walkability (61). Inequality can also drive calorie consumption. Individuals who are experimentally induced to view themselves as poor in reference to others exhibited increased calorie intake (62). Additionally, individuals who believed they were poorer or wealthier than an interaction partner exhibited higher levels of anxiety in regards to that difference in status that, in turn, led to increased calorie consumption (62).

 

Food insecurity affects approximately 11.8 percent of families in the United States and has been linked to obesity and diabetes. Food insecurity occurs when “the intake of one or more members of a household is reduced and eating patterns are disrupted (sometimes resulting in hunger) because of insufficient money and other resources for food” (63). In women, food insecurity status predicts overweight/obese status differentially across racial ethnic groups. Non-Hispanic white women who are food insecure are 41% more likely to have overweight or obesity whereas Hispanic women who are food insecure are 29% more likely to have overweight and obesity (64). Among non-Hispanic black women and men, food insecurity did not predict overweight or obesity status (64). A population-based study in Canada revealed that persons in food insecure households had double the risk of developing type 2 diabetes compared to persons in food secure households, even after controlling for age, gender, income, race, physical activity, smoking status, alcohol consumption, diet quality, and BMI (65). Reduced food availability is theorized to initiate compensatory biological mechanisms that boost caloric intake, decrease resting metabolic rate, and increase storage of adipose tissue as a protective mechanism for survival (66). Research in youth has provided evidence for a moderating effect of food insecurity on the relationship between income and subjective social status (67). This means that low income is more strongly associated with low subjective social status when the household is also food insecure.

 

Subjective measures of social status (SSS) are typically measured by asking individuals to place themselves on 10-rung ladders based on where they perceive their rank within society and the community. Experimental evidence demonstrates a relationship between feelings of low social status and increased calorie intake. Cornil and Chandon showed that hometowns of National Football League teams consumed more calories after a team loss than hometowns of winning teams or of hometowns where teams didn’t play (68). Manipulations of social status in an experimental setting show that acute eating behavior post experimental manipulation consists of higher calorie food choices and higher total calorie intake in the low status group (69). Additionally, individuals randomized to a low social status condition, had increased levels of ghrelin, a hormone that stimulates appetite, as compared to the high social status condition, suggesting a physiological hunger response to low perceived social status (70). Studies of physical activity and SSS show that low SSS is associated with significantly lower levels of moderate to vigorous physical activity (71, 72), which could contribute to a lower overall energy expenditure. Closely related to SSS are other perceptive representations of status differentials, such as perceived discrimination, which is associated with increased weight and BMI in women (73) and increased abdominal adiposity in non-Hispanic whites (74).

 

Researchers have integrated individual and environmental factors into design and development of interventions to improve weight outcomes or weight-related behaviors (healthy eating, physical activity); however, not all of them are successful. For example, a study among low-income women with children in rural Mexico randomly assigned families to cash or in-kind transfers (food baskets) and found that women in the food basket and cash groups actually gained weight compared to women in the control group (75). This study and others that show weight gain occurring in spite of access to resources or poverty relief imply accounting for individual and environmental factors alone may not paint a complete picture of obesity development. Granted, it is important to consider that systemic environmental changes, such as placement of sidewalks or fruits and vegetables in a corner store, may not be adequately captured in a short time frame typical of academic studies. However, the small or nonexistent changes observed when resources are supplied warrants further investigation into deeper realms of social hierarchical constructs, as well as continued study of individual and environmental factors to improve treatment and prevention of obesity.

 

CLINICAL IMPLICATIONS AND CONCLUSIONS

 

Given the extent of the information on individual, environmental, and social hierarchy constraints on obesity development, it is important to understand how these can merge with clinical care. It is evident that there is no one simple solution and effective care requires knowledge of these complex relationships and an integration between the health system and the surrounding community. For example, based on the knowledge that the social determinants of health can influence diabetes and its comorbidities, the American Diabetes Association recommends in its clinical guidelines that providers “assess the social context… and apply that information to treatment decisions” (76). In conjunction with recognition of the impact of social and environmental determinants on multiple chronic diseases, some researchers propose that “community vital signs” be integrated into the electronic health record (EHR) (77) and some community health centers have begun pilot testing a social determinants questionnaire in their HER (78). Knowledge provided by these “vital signs” and social determinants could help providers make appropriate lifestyle-tailored recommendations for the patient.

 

Discussing context surrounding food in a patient’s life can provide insight into the realistic expectations for a patient’s diet.  Food insecurity can be identified with a short two question screener (79) and implementation in clinics has shown that screening improves clinician awareness of food insecurity, helping to better understand the lengths to which it affects patient treatment (80). Positive responses from physicians after pilot testing that incorporates screening into clinical practice mitigates concerns that discussions about food security would be stigmatizing to the patient (80). Patients who identify as food insecure can be referred to local food banks or community programs that will connect patients with resources at a federal and community level.

 

Patients that are finding it difficult to follow lifestyle modification recommendations to lose weight to prevent diabetes development may benefit from the Diabetes Prevention Program. The Diabetes Prevention Program is a lifestyle program focused on weight loss through dietary change and increased physical activity. While the overall weight loss was modest (~4% after 4 years), participants lowered their chances of developing diabetes by 58% during long-term follow-up (81). This program has been adapted for implementation and dissemination purposes and now the CDC’s National Diabetes Prevention (National DPP) program is available at almost 2,000 sites across the United States including many YMCAs, with a mix of online and in-person options.  This program is covered for eligible individuals by Medicare and many private insurers and cost for non-covered patients is variable and often income-based or free.  Initial evaluation of the real-world evidence for implementation of the National DPP have been promising with 35% achieving 5% weight loss and 42% meeting the activity goal of 150 minutes per week (82). Locations with the best participant retention and attendance share the following qualities: referrals from healthcare providers or health systems, provision of non-monetary incentives for participation, and use of cultural adaptations to address participant needs (83). The National DPP provides an affordable, easy and local referral source so that the provider can be assured their patients are receiving evidence-based lifestyle management in an ongoing program.  

 

RESOURCES

 

Figure 1 below shows the age-adjusted prevalence of obesity in adults by race and ethnicity, and sex from the Centers for Disease Control 2017 National Center for Health Statistics Data Brief (5).

Figure 1. Prevalence of Obesity by Race/Ethnicity and Sex

Questions to Incorporate into Your EHR About Food Insecurity

  1. “We worried whether (my/our) food would run out before (I/we) got money to buy more” Was that often true, sometimes true, or never true for (you/your household) in the last 12 months?
  2. “The food that (I/we) bought just didn't last and (I/we) didn't have money to get more” Was that often true, sometimes true, or never true for (you/your household) in the last 12 months?

Information on the Diabetes Prevention Program

https://nccd.cdc.gov/DDT_DPRP/Registry.aspx

 

Opportunities for Advocacy 

The Obesity Action Coalition: https://www.obesityaction.org/

The Obesity Society:  https://www.obesity.org/

STOP Obesity Alliance: http://stop.publichealth.gwu.edu/

Rudd Center for Food Policy and Obesity: http://www.uconnruddcenter.org/weight-bias-stigma

 

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Growth Hormone in Aging

ABSTRACT

 

Growth hormone (GH) serves important roles in adult life, including maintenance of lean body mass and bone mass, promoting lipolysis, thereby limiting visceral adiposity, and regulating carbohydrate metabolism, cardiovascular system function, aerobic exercise capacity, and cognitive function. Younger adults with growth hormone deficiency (AGHD) exhibit abnormalities in body composition, physical and cognitive function, and quality of life which are reversed by GH replacement therapy. With advancing age GH production declines, paralleled by physical and functional alterations similar to those of AGHD; however, the degree to which the decrease in GH contributes to these age-related changes is unknown. Seemingly in opposition to the theory that the diminished GH secretion of older age is a net detriment are observations that animal models of congenital GH deficiency have remarkably increased life span and humans with congenital GH deficiency may have decreased rates of age-related diseases such as diabetes and cancer. Several short-term studies aiming to increase GH in older adults by a variety of interventions including exercise, administration of GH, or treatment with GH secretagogues have demonstrated consistent effects to improve body composition, yet inconsistent effects on physical and cognitive function. While side effects of GH administration in older adults include edema, arthralgias, and elevated blood glucose, data regarding the possible long-term effects on “hard end points” such as risk of fractures, cancer, cardiovascular disease, life expectancy, and mortality are lacking.

 

INTRODUCTION

 

The decline in growth hormone secretion observed with aging is associated with changes in body composition and physical and psychological function that are similar to those seen in younger adult patients with growth hormone deficiency. These changes include reductions in lean body mass and muscle strength and an increase in body fat, particularly in the visceral compartment. Memory and cognitive function gradually deteriorate with age. Deep (slow-wave) sleep also decreases markedly with age, together with a decrease in nighttime growth hormone secretion, and sleep disorders become a significant clinical problem in old age. Although these changes only show an association, and it is still unknown whether there is any causal link between them, they have led to speculation that replacing or stimulating growth hormone may reverse some of the detrimental features of the aging process (1).

 

 

The trophic hormones which rise at puberty, including sex steroids and GH, have dramatic effects on body composition and strength. Their levels plateau in young adult life and then decline progressively, accompanied by a loss of muscle mass and aerobic capacity, and an increase in abdominal fat. These changes resemble some features of hypogonadism and adult GH deficiency (2).

 

After the third decade of life, there is a progressive decline of GH secretion by approximately 15% for every decade of adult life. Integrated measurements of daily GH secretion demonstrate that secretion peaks at puberty at about 150 µg/kg day, then decreases to approximately 25 µg/kg/day by age 55 (3). The reduction in GH secretion results from a marked reduction in GH pulse amplitude, with only very little change in pulse frequency (4). This process is characterized by lack of day-night GH rhythm resulting from loss of nocturnal sleep-related GH pulses (Figure 1) (4). Growth Hormone Binding Protein decreases from 60 years of age, theoretically increasing the amount of bioavailable growth hormone (5). This decrease is thought to parallel the decrease in growth hormone receptors with age. Though slow-wave sleep (SWS) decreases with age most studies administering GH or GHRH to seniors did not improve SWS. This finding suggests that the age-related decline in GH does not cause reduced SWS, although the reverse may be true.

FIGURE 1. Patterns of GH secretion in younger and older women and men. There is a marked age-related decline in GH secretion in both sexes and a loss of the nighttime enhancement of GH secretion seen during deep (slow-wave) sleep. This decrease is primarily due to a reduction in GH pulse amplitude, with little change in pulse frequency. L = large GH pulses, S = small GH pulses. From Ho et al. 1987 (4).

Circulating levels of IGF-I, the main mediator for the trophic effects of growth hormone, also decline with age (Figure 2). The majority of circulating IGF-I is produced in the liver under the control of GH. It appears that the age-related decline in IGF-I production is a direct result of decreases in GH and there is no evidence to suggest increased “GH resistance”. In fact, studies of GH replacement therapy in patients with pituitary disorders and dose-response studies demonstrate a reduction in GH dose necessary to maintain normal IGF-I concentrations in older subjects, although this is due at least in part to the higher susceptibility to side effects from GH and also that their target IGF-1 is lower (6-7).

FIGURE 2. Changes in serum IGF-I with increasing age. Modified from Juul A et al,1994 (8).

POTENTIAL MECHANISMS UNDERLYING THE DECLINE IN GH SECRETION WITH AGE

 

Three hypothalamic factors regulate GH secretion: somatostatin (SRIF), growth-hormone releasing hormone (GHRH) and ghrelin (9) (Figure 3). Somatostatin is a noncompetitive inhibitor of GH secretion as well as of other hormones. It modulates the GH response to GHRH. GHRH is the principal stimulator for GH synthesis and release. Ghrelin is the endogenous ligand to the growth hormone secretagogue receptor-1a (GHSR-1a). Ghrelin is secreted primarily by the stomach and has appetite-stimulating activity separate from its effect on GH secretion. Although recent preclinical data suggest that not all the effects of ghrelin are mediated through GHSR-1a (10), its orexigenic and GH secretagogue effects require the presence of the GHSR-1a (11).Acylated Ghrelin levels decrease with age (12) Growth hormone secretagogues (GHS) are synthetic molecules that stimulate the GHSR-1a exhibiting strong growth hormone-releasing activity.

 

A variety of stimuli and inhibitors, such as exercise, sleep, food intake, stress and body composition have effects on the hypothalamic factors that regulate GH production (13). All of these factors interact to generate the physiological patterns of pulsatile GH secretion.

FIGURE 3. Major components of the GH neuroregulatory system (3).

There are several mechanisms that could explain the age-related decrease in GH secretion. Possibilities include decreased secretion of GHRH or ghrelin, increase in inhibition by somatostatin, increased sensitivity of somatotrophs to negative feedback inhibition by IGF-I, decline in pituitary responsiveness to GHRH, and pituitary and/or hypothalamic responsiveness to ghrelin.

 

Whether the aging pituitary responds normally to GHRH and ghrelin is still a matter of debate. Although earlier studies suggest no age–related decline in GH responsiveness to GHRH (13), more recent reports suggest a gender-independent, age-related decline in the GH responsiveness to GHRH and ghrelin (14) (Figure 4).There is no age-related increase in GH sensitivity to IGF-1 (15); however, there may be relative deficiency in GHRH and ghrelin secretion, and an increase in SRIF secretion, in older individuals (16). The density of GHRS-1a receptors in the hypothalamus decreases with aging and this is thought to be responsible for the age-related decreased response to some GHS (17). The aging pituitary is also less responsive to exercise, sleep, and other physiologic stimuli. Based on these observations it is most likely that the age-related change in GH secretion is multifactorial in etiology and is caused by changes above the level of the pituitary.

FIGURE 4. Approximate peak GH relative change in response to I.V. bolus of Ghrelin and GHRH is diminished in older adults compared to young males. Adapted from Broglio F et al. (14).

DECREASE IN GH IN NORMAL AGING: SIMILARITIES WITH AND DIFFERENCES FROM ADULT GROWTH HORMONE DEFICIENCY

 

Although not a perfect parallel with aging, adult growth hormone deficiency (AGHD) is the best documented source of information on signs and symptoms of reduced GH secretion, effects of treatment, dosing strategies appropriate for adults, and side effects and safety of GH replacement.

 

Several aspects of normal aging resemble features of the AGHD syndrome, including decrease in muscle and bone mass, increased visceral fat, diminishing exercise and cardiac capacity, atherogenic alterations in lipid profile, thinning of skin, and many psychological and cognitive problems (18-19) (Table 1). Although these changes and the GH deficit of aging are milder than seen in AGHD, they remain clinically significant (20).

 

Table 1. Features of Adult Growth Hormone Deficiency (3)

Increased fat mass especially abdominal fat

Decreased lean body mass

Decreased muscle strength

Decreased cardiac capacity

Decreased exercise performance

Decreased bone mass

Decreased RBC volume

Atherogenic lipid profile

Thin dry skin

Impaired sweating

Poor venous access

Psychosocial problems-

Ø  low self-esteem

Ø  depression

Ø  anxiety

Ø  fatigue and listlessness

Ø  sleep disturbances

Ø  emotional lability and poor self-control

Ø  social isolation

Ø  poor marital and social-economic performance

 

It is important to distinguish the normal decrease in GH secretion associated with aging from true AGHD. Although aging is a state of relative physiologic GH deficiency, it is not a disease in itself and is clearly a separate entity from AGHD. This is demonstrated by higher GH secretion and physiological responses seen in older adults when compared with AGHD patients of similar age (2, 20). Moreover, aging per-se is not an indication for AGHD diagnosis testing or administration (21, 22).

 

Biochemical tests for AGHD diagnosis are imperfect, and their accuracy is strongly affected by the pre-test probability of the condition. Therefore, the most important indicator of the likelihood of GHD is the clinical context (21). In the majority of cases this is due to tumors arising in the region of the sella turcica or the treatment for these tumors including surgery and radiation, but there are other etiologies. Traumatic brain injury is an increasingly recognized cause of AGHD and may occur without coexisting deficiencies in other anterior pituitary hormones (22). IGF-1 levels alone are generally not enough for AGHD diagnosis, hence the need for provocative testing with the insulin tolerance test, glucagon stimulation test, or when available the combined GHRH-arginine test (23).

 

The GHS and ghrelin mimetic, macimorelin, has recently been approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for AGHD diagnosis., Macimorelin provides a simple, orally-available, well-tolerated, reproducible, and safe diagnostic test for AGHD with comparable accuracy to the insulin tolerance test in adults (24). Since studies of macimorelin excluded individuals over the age of 65 or with a BMI >40, the safety and efficacy of this test in these populations has not been established.

 

 

The age-related increase in body mass index, changes in body composition, and diminished functional capacity parallel the age-associated decline in growth hormone secretion (18, 19). The alterations in body composition that are most pronounced in normal aging include a reduction in bone density and in muscle mass and strength, an increase in body fat, and adverse changes in lipoprotein profiles (2, 16). This decline in GH production is initially clinically silent, but over time may contribute to sarcopenic obesity and frailty.

 

The decline in GH may also play a role in cognitive changes observed with aging. One of the many systems for classifying different cognitive domains is grouping them as either “crystallized” or “fluid” intelligence. Crystallized intelligence generally refers to vocabulary and long-term memory; whereas the fluid intelligence includes short term memory and active problem-solving and demonstrates a more marked age-related decline. Several studies have shown a correlation between plasma IGF-I concentrations and performance on tests of fluid intelligence (25), suggesting that GH may play a role in maintenance of fluid intelligence.

 

Mechanistic insights into the role of growth hormone and IGF-I in age-related alterations in cognitive function were assessed in several studies by Sonntag and colleagues demonstrating somatotrophic effects on rodent brain aging (26). These studies suggest that deficiencies in GH and IGF-I contribute to the functional decline in senescent rats whereas augmentation of GH or IGF-I improved cognitive function, increased glucose utilization throughout the brain, increased cortical vascularity, and ameliorated age-related decline in hippocampal neurogenesis. Although some small studies suggest a positive effect of GHRH or GH replacement on cognition (27), there is insufficient data to recommend GH deficiency testing or GH treatment solely for this purpose (22).

 

GROWTH HORMONE IN AGING: CAVEATS REGARDING LONGEVITY

 

Animal studies have called into question the hypothesis that interventions aiming to increase growth hormone secretion and IGF-I should be considered a net benefit (28). In numerous species from nematodes to rodents, caloric restriction, which lowers IGF-1, has been associated with an increased life span. Mice with growth hormone resistance and profoundly reduced IGF-I levels appear healthy and have increased life expectancy, along with reduced fasting glucose and insulin levels (29, 30). In mice with mutations in the gene necessary for the differentiation of pituitary cells to produce GH, life span increases by 42% and tumor development is delayed (30). These findings are accentuated when combined with caloric restriction, while treatment with GH actually reduces lifespan. Mice treated with a metalloproteinase that cleaves an IGF-binding protein to decrease the bioavailability of IGF-1 have a 38% longer lifespan and a lower incidence of tumors. In experiments conducted with a mouse strain prone to developing age-related cognitive decline and decreased life expectancy, treatment with a GHRH-receptor antagonist resulted in increased telomerase activity, improvements in some markers of oxidative stress, improved cognition, and increased mean life expectancy (31) On the other hand, mice treated with a growth hormone antagonist made by a single amino acid substitution in GH do not show increased lifespan.

 

The mechanism of increased longevity seen in these mice populations is complicated. Some mouse models with increased lifespan have insulin resistance, while those that develop overt diabetes have a shortened lifespan. Long living mice are either leaner than normal or have increased subcutaneous adipose tissue, both of which may have protective aging effects. Caloric restriction and decreased GH/IGF-1 signaling improves resistance to cellular stress, inhibits mTOR by rapamycin, leading to longevity and may be responsible for enhanced tumor resistance.

 

The parallel between these results and human senescence is not clear. This last assertion is underscored by the recent report that longevity is not increased, but rather reduced in women in a Brazilian population with a GHRH receptor mutation (32) and in a Swiss cohort of patients with isolated GH deficiency from a homozygous mutation spanning the GH1 gene (33). In an Ecuadorian-kindred with GH receptor deficiency and very low levels of IGF-I, however, rates of cancer and diabetes were markedly reduced compared to unaffected people in the same communities (34). A group of Croatian patients with dwarfism and deficiencies in GH, TSH, prolactin, FSH, and LH as a result of a homozygous PROP1 mutation do not die prematurely, do not develop diabetes mellitus, and have delayed appearance of grey hair (30). Ultimately, although size and lifespan appear to have an inverse relationship in some animal studies, no formal longevity studies have been performed in humans with dwarfism.

 

GROWTH HORMONE THERAPY IN NORMAL AGING

 

A large literature of over 2000 published papers has led to a general consensus that GH replacement can reverse many abnormalities in AGHD patients. Recent reviews of this literature report reduced fat mass, increased lean body mass, improved exercise capacity and cardiac function, improved bone mineral density, and enhanced quality of life by subjective and objective measures (35-37).

 

The similarities between aging and adult GH deficiency, while not exact, have led to interest in administering GH directly or stimulating GH secretion in aging individuals. However, the starting point and the target are not the same in the two conditions, and we cannot assume safety and efficacy will be the same.

 

To date, studies of interventions to increase GH effect in elderly subjects include administration of GH, IGF-I, GHRH, and ghrelin mimetic (GHS) either alone or in combination with each other, sex steroids, or exercise. The first studies of GH treatment in non-GHD older adults were performed not long after its effects in AGHD were demonstrated. In 1990 Rudman and colleagues reported that healthy men above the age of 60 who were treated with GH for 6 months responded with an 8.8% increase in lean body mass, a 14.4% decrease in adipose tissue mass, and a 1.6% increase bone mineral density (BMD) only at the vertebral spine (38). Although the change in BMD was quite small it was especially remarkable considering that most studies of AGHD have required one year or more of therapy to show an improvement in bone density. The changes in body composition persisted after one year of growth hormone treatment (39).

 

Although the Rudman study did not include any functional measures, given these results, it was postulated that GH treatment might also improve muscle strength and functional performance. Despite an absence of demonstrated functional efficacy, some clinics began to prescribe GH treatment to healthy older persons. Acknowledging this growing practice and the lack of information on the subject, the NIH National Institute on Aging issued a call for applications in 1991 to study trophic factors in aging. Several studies of GH, either alone or in combination with sex steroids, IGF-I, or exercise conditioning, and one study of GHRH were funded and have since been completed. These reports generally demonstrate that GH replacement in normal seniors can increase levels of IGF-I to the young adult normal range. However, lower doses of GH were used in subsequent studies to maintain IGF-I levels in the normal range for healthy young adults, since attempts to reproduce the doses of the initial study of Rudman and colleagues led to severe side effects. Following is a summary of the findings of these studies.

 

Effects of GH Treatment on Strength and Functional Performance

 

Though GH treatment of otherwise healthy seniors has been shown to have potentially beneficial effects on body composition, studies of physical functional effects have been generally disappointing and inconsistent.

 

In a relatively large study, comparing the effects of 6 months of growth hormone treatment with placebo in men aged 70 to 85, Papadakis and colleagues reported a 13% reduction in fat mass and a 4% increase in lean body mass in the treatment group, effects consistent with earlier studies; however, there was no effect of growth hormone on knee or handgrip strength or endurance (40). It is important to point out that this seemingly negative result is likely undercut and confounded by the excellent baseline functional status of the study subjects, who were close to the top of the range on many of the tests used, even before treatment. It is very likely that such a "ceiling effect" led to difficulties in demonstrating further improvements due to treatment with growth hormone.

 

In a separate study, Taaffe and co-workers showed that exercise training improved strength and exercise capacity, but growth hormone treatment did not further augment this effect (41). Several other similar studies have since been completed (42). These studies were conducted for 6–12 months, each at a single site; therefore, only short-term outcomes and side effects, not long-term risks, could be observed. Their results do not provide guidance on the effects of GH on long-term clinical outcomes or “hard” endpoints such as falls or fractures, maintenance of functional status, or effects on cardiovascular morbidity and mortality – outcomes that could establish more definitively the rationale for GH treatment in normal aging. Though few long-term risks have been observed, this is mainly indicative of an absence of information rather than a demonstration of safety. In 2004 a review of various interventions for sarcopenia and muscle weakness in the elderly concluded that GH therapy produces a high incidence of side-effects, does not increase strength, and that resistance training is the most effective intervention for increasing muscle mass and strength in the elderly (43).

 

In 2007 Liu and colleagues published a systematic review of the safety and efficacy of growth hormone in the healthy elderly (42). After a mean treatment duration of 27 weeks, GH treated individuals had decrease in fat mass of 2.1 kg and an equal increase in lean body mass of 2.1 kg, with no change in weight overall. Total cholesterol levels trended downward (0.29 mmol/L), though not significantly after adjustment for the change in body composition. Other outcomes, including bone density and other serum lipid levels, did not change. Despite higher doses of GH per kilogram of body weight, women treated with GH did not increase lean body mass and achieved only borderline significant decreases in fat mass, indicating a difference in response to GH therapy between genders. Persons treated with GH were significantly more likely to experience soft tissue edema, arthralgias, carpal tunnel syndrome, and gynecomastia and were somewhat more likely to experience the onset of diabetes mellitus and impaired fasting glucose (Figure 5).

Figure 5. Adverse events in participants treated with growth hormone versus those not treated. From Liu H et al. (42)

Effects of GH Treatment on Cognition, Sleep, and Mood

 

As noted above, rodent studies have shown that GH administration increases brain vascularity and improves performance on some cognitive tests, but systematic tests of cognitive effects of GH treatment in humans are lacking (44). A seemingly contradictory finding was reported in 2017 by Basu and colleagues who demonstrated that spatial learning and memory were improved in 12-month-old GH receptor antagonist transgenic mice when compared to their wild type controls, proposing that GH antagonism as well may have cognitive benefits in aging rodents (45). A trial of GH therapy in patients with Down syndrome showed an increase in head circumference but no effect on cognitive performance (46). Early reports suggested that GH increased deep sleep (SWS), but subsequent studies have failed to confirm this and indeed have found increased sleep fragmentation and reduced total deep sleep (27). While GH treatment of adult GH deficiency improves self-rated quality of life (QoL) scores using any of a number of questionnaires, there are no solid comparable data for normal aging.

 

Combination Interventions with GH and Sex Steroids

 

In a 6-month study of healthy men and women over the age of 65 using GH alone or in combination with estrogen/progestin in women and testosterone in men, GH administration increased lean body mass (LBM) in women with or without estrogen/progestin (47). In men, GH and testosterone increased LBM when given alone and had an additive effect in combination. In men, total fat mass decreased with either testosterone or GH alone, but the decrease was greatest with the combination, whereas in women GH decreased fat mass while sex steroids did not change fat mass. Body strength did not improve in women and slightly increased in men only in the GH + testosterone arm of the trial. There was no evidence that co-administration of sex steroids altered the frequency or severity of GH-related side effects.

 

A 2006 British study randomized healthy older men to 6 months treatment with GH, testosterone patch (Te), or combination of both GH and testosterone (GHTe) and compared results to placebo (48). Both GH treated groups experienced similar increases in lean body mass, while this parameter was unchanged by testosterone treatment alone. Fat mass decreased only in the GH/testosterone combination group. Similarly, mid-thigh muscle cross-sectional area and exercise capacity (VO2 max) was increased only in GHTe and not in the GH or Te groups. There was no difference among the groups in 5 of 6 muscle strength measures except for strength of knee flexion that was found to be increased in the GHTe group. Both GH treated groups reported improvement in a quality of life questionnaire. Overall GH treatment was well tolerated in this study, with most GH-related side effects resolving with dose adjustment.

 

A study published in 2009 randomized men over the age of 65 having IGF-I levels in the bottom tertile of the reference range, and who were treated with testosterone after a “Leydig cell clamp”, to three groups of daily doses of GH either 0, 5 mcg/kg or 10 mcg/kg (49). After 16 weeks the investigators were able to demonstrate significant synergistic effects of GH treatment, when added to testosterone replacement, on all parameters studied including decrease in total fat mass and truncal fat as well as increase in lean body mass and maximal voluntary muscle strength and aerobic endurance. A slight increase in systolic blood pressure was noted in the study, but did not appear to be related to GH therapy.

 

Growth Hormone and Exercise

 

Regular exercise has been shown to increase lean body mass, muscle strength, and aerobic capacity in older men (50). Vigorous exercise acutely stimulates growth hormone secretion, a physiologic response that has been utilized as a screening test for growth hormone deficiency in children.

 

The growth hormone response to exercise decreases with aging (51). This finding led to speculation that some of the effects of exercise might be mediated via effects on growth hormone and IGF-I. Although exercise stimulates an acute rise in growth hormone secretion, subsequent overnight growth hormone secretion is inhibited (52). In older adults, even intensive exercise does not elevate serum IGF-I level (53). Therefore, the effects of exercise on muscle mass and function seem to be separate from those of growth hormone.

 

Studies assessing the effects of adding GH to progressive resistance training regimens in older adults have found little to no additional benefit of GH therapy on measures of muscle strength or other measures of muscle composition, but did find that GH therapy led to greater reductions in fat mass than resistance training alone (41, 54, 55).

 

Adverse Effects of GH Treatment

 

Side effects observed during clinical trials of growth hormone treatment in normal aging must be taken into consideration in a different way from those in patients treated for adult growth hormone deficiency. The possibility that some of the hormonal changes observed with aging could represent adaptive responses must be considered. Whether increasing growth hormone above the age-appropriate normal range may have as many risks, both acute and delayed, as benefits is a worthwhile hypothesis to examine. The acute side effects of growth hormone are largely hormonal. The most worrisome long-term potential side effect, of special importance in the older population where baseline risk is elevated, is the risk of cancer. Though there is no definitive evidence that GH replacement in AGHD increases the risk of de novo or recurrent malignancy, but several case reports note development of cancer after treatment with GH and because it is a mitogen, the use of GH is contraindicated in active malignancy (35, 36). Nevertheless, GH replacement is not associated with tumor regrowth in AGHD patients with pituitary tumors (56).

 

Older persons are more sensitive to replacement with GH and more susceptible to the side effects of therapy. The acute side effects are due to the hormonal effects of over-replacement, which can be avoided or relieved with careful dose titration. Patients who are older, heavier, or female are more prone to develop complications (22). Common side effects of GH replacement include fluid retention, with peripheral edema, arthralgia, and carpal tunnel syndrome (Figure 5) Although glucose levels often increase with initiation of GH, these levels generally return toward normal with the improvement in body composition and reduced insulin resistance. However, some studies report persistent elevations in fasting glucose and insulin with chronic GH treatment. Other less frequently reported side effects include headache, tinnitus, and benign intracranial hypertension (22). Hypothyroidism is common in the elderly. GH can accelerate both the clearance of thyroxine and promote its conversion to triiodothyronine, and therefore may have variable effects in hypothyroid patients who are on thyroid hormone replacement.

 

GROWTH HORMONE RELEASING HORMONE (GHRH) AND GROWTH HORMONE SECRETAGOGUES (GHS)

 

GHRH and GHS stimulate the secretion of GH. Since most AGHD is caused by pituitary lesions, and these patients, unlike healthy seniors, are unresponsive to GHRH or GHS, there are few studies of treatment with these agents.

 

Theoretically, treatment with GHRH or GHS should lead to more physiologic GH replacement, leading to a pulsatile rather than prolonged elevation in GH and preserving the ability for negative feedback inhibition of GH by increasing IGF-I. GHRH and GHS effects are influenced by the same factors which modulate endogenous GHRH secretion, such as negative feedback by somatostatin. This normal negative feedback regulation would be expected to result in buffering against overdose. The side effects of GHRH treatment are similar in character to GH treatment but are milder and less frequent. Since the GHS are smaller molecules than GH, and generally resistant to digestive enzymes, they can be administered via the oral, transdermal or nasal routes.

 

Growth Hormone Releasing Hormone (GHRH)

 

There are several published trials of GHRH treatment in normal aging (57, 58). Once daily GHRH injections can stimulate increases in GH and IGF-I at least to the lower part of the young adult normal range (57). In a study of 6 months treatment with daily bedtime subcutaneous injections of GHRH(1–29)NH2, alone or in combination with formal exercise conditioning, IGF-I levels increased by 35% (56). Participants had an increase in lean body mass and decrease in body fat (mainly abdominal visceral fat). However, there was no improvement in strength or aerobic fitness with GHRH injections. This study confirmed the benefits of exercise but showed no effect upon IGF-I levels; thus, it appears that GH/GHRH and exercise work through different mechanisms. Subjects receiving GHRH also showed no change in scores on an integrated physical functional performance test of activities of daily living, but there was a significant decline in physical function in the placebo group. This finding, suggesting that GHRH can stabilize if not improve physical function, needs confirmation.

 

Sleep and cognition were also studied in this GHRH trial, with unexpected results. GHRH failed to improve and may even have impaired deep sleep, despite the rise in IGF-I and pulsatile GH. However, GHRH treatment was associated with improved scores in several domains of fluid (but not crystallized) intelligence – those measures previously found to be correlated with circulating IGF-I levels (25).

 

A 2006 study of the effects of 6-months daily treatment with sermorelin acetate, a GHRH analogue, on cognitive function of 89 elderly adults found significant improvement on several cognitive assessments, particularly those involving problem solving, psychomotor processing speed, and working memory, but no change on tests reflecting crystallized intelligence (27). Higher GH levels were associated with higher Wechsler Adult Intelligence Scale performance IQ scores, and greater increases in IGF-1 were associated with higher verbal fluency test scores, while gender, estrogen status, and initial cognitive function did not interact with the GHRH effect on cognition.

 

A 2013 pilot study of 30 elderly adults given a stabilized analogue of GHRH, tesamorelin, versus placebo, used magnetic resonance spectroscopy to examine the effects of inhibitory and excitatory neurotransmitters (60). After 20 weeks GABA levels were increased in all brain regions, N-acetylaspartylglutamate levels were increased in the dorsolateral frontal cortex, and myo-inositol (an osmolyte linked to Alzheimer disease) levels were decreased in the posterior cingulate, with similar results across adults with mild cognitive impairment (MCI) and those with normal cognitive function. Treatment related changes in serum IGF-1 were positively correlated with changes in GABA and negatively correlated with myo-inositol. There was a favorable treatment effect on cognition (p = .03), but no significant associations were observed between treatment-related changes in neurochemical and cognitive outcomes.

 

The follow-up study of 152 elderly patients on tesamorelin versus placebo included those with amnestic MCI (early stage Alzheimer’s disease) and analyzed executive function, episodic memory, mood, sleep, insulin sensitivity, glucose tolerance, body composition, and IGF-1 levels (61). GHRH had a favorable effect on cognition (P = .002) in both groups. Treatment related increases in IGF-I were associated with higher composite change scores in executive function (p = .03). Visual memory, mood, sleep, hemoglobin A1c, and 2-hour OGTT glucose and insulin responses were not affected in either population, though GHRH treatment was associated with increased fasting plasma insulin levels in adults with MCI. Treatment with GHRH reduced body fat by 7.4% (p < .001) and increased lean muscle mass by 3.7% (p < .001), across both populations. Ultimately though, the clinical significance of these results cannot be assessed as no data was collected regarding functional status.

 

In a non-controlled 3-month trial of GHRH(1-44)amide in 10 postmenopausal women, increases in both GH and IGF-I levels as well as decreased visceral fat were demonstrated (57). This study also reported improvements from baseline in selected measures of functional performance including timed walking and stair climbing.

 

Thus, as is the case with GH, studies of treatment of healthy seniors with GHRH have arrived at a consensus on hormonal and body composition effects but inconsistent functional effects. There is a very encouraging but still unconfirmed positive effect on some domains of fluid intelligence.

 

Ghrelin Mimetics and Growth Hormone Secretagogues (GHS)

 

Ghrelin, a 28 amino-acid octanoylated peptide, is produced in the stomach and increases before meals and during overnight fasting. Ghrelin acts at both hypothalamic and pituitary levels via mechanisms distinct from GHRH. Ghrelin therefore has different effects from GHRH or GH; subjects often gain body weight, lean and fat mass via a number of GH dependent and independent mechanisms (62). The effects of ghrelin on GH secretion depend in part on the presence of GHRH. If GHRH secretion declines with aging, as is thought to be the case, ghrelin’s effects may be blunted. While the effects of these two GHS differ clinically, they have synergistic effects on GH release, and therefore supplementation of both substances may be more effective than either alone. Nevertheless, ghrelin is more potent than GHRH at eliciting GH secretion (14). Additionally, there are other substances which can enhance GH response to GHS by suppressing somatostatin secretion, including arginine and beta-adrenergic antagonists, which could potentially enhance treatment effects (59).

 

Several studies have shown short-term effects of GHS on GH secretion, but few studies of their chronic effects in normal aging have been reported. Bowers and colleagues showed that chronic repeated injections or subcutaneous infusions of GH-releasing peptide-2 (GHRP-2) could stimulate and maintain increases in episodic GH secretion and raise IGF-I levels (63).

Results of a one-year double-blind, randomized, placebo-controlled, modified-crossover clinical trial of the Merck orally active ghrelin mimetic MK-677 in healthy high functioning older adults were published in 2008 (64). Daily administration of MK-677 significantly increased growth hormone and IGF-I levels to those of healthy young adults without serious adverse effects. Mean fat-free mass decreased in the placebo group but increased in the MK-677 group. No significant differences were observed in abdominal visceral fat or total fat mass. Body weight increased 0.8 kg in the placebo group and 2.7 kg in the MK-677 group (P = 0.003). Fasting blood glucose level increased an average of 0.3 mmol/L (5 mg/dL) in the MK-677 group (P = 0.015), and insulin sensitivity decreased. The most frequent side effects were an increase in appetite that subsided in a few months and transient, mild lower-extremity edema and muscle pain. Low-density lipoprotein cholesterol levels decreased in the MK-677 group relative to baseline values (change, -0.14 mmol/L) (-5.4 mg/dL) P = 0.026); no differences between groups were observed in total or high-density lipoprotein cholesterol levels. Changes in bone mineral density consistent with increased bone remodeling occurred in MK-677 recipients. Increased fat-free mass did not result in changes in strength or function.

 

A multicenter trial of the Pfizer investigational oral GHS, capromorelin, in pre-frail older men and women recruited over 300 subjects and was initially planned as a two-year intervention (65). The study was stopped, however, after all subjects had been treated for 6 months and many for 12 months, due to failure to see an increase in percent lean body mass, which was a pre-set non-efficacy termination criterion. Absolute lean body mass did increase significantly, but due to the appetite-stimulating and lipogenic/anti-lipolytic effect of ghrelin mimetics – unforeseen in early 1999 when the study was designed and ghrelin was still unknown – subjects also gained weight (about 1.5 Kg) and this washed out the effect on percent lean body mass. However, even this truncated study is currently the largest clinical trial of chronic GHS treatment in aging. It showed the expected increases in IGF-I levels and (as noted) total lean body mass. There were also encouraging effects on physical functional performance. Of seven functional tests, one improved significantly after 6 months of treatment, and another after 12 months. Two other measures showed non-significant trends toward improvement, and the three remaining measures showed no effect. Effects on clinical endpoints such as falls could not be assessed with this relatively brief duration of treatment. Side effects were generally mild, including increases in fasting blood sugar within the normal range. Interestingly, there was a self-reported deterioration of sleep quality, though formal sleep testing was not performed. Cognition was not studied in this trial. The reasons for the difference in functional outcomes between the two trials are not clear, but it is speculated that this may reflect differences in the populations studied. The MK-677 study recruited a robustly healthy population of seniors in whom further improvement in physical function might be difficult to achieve, while the capromorelin trial was limited to participants already manifesting a decline in function.

 

Thus, as with GH and GHRH, reports of the hormonal and body composition effects of ghrelin mimetic GHS in normal aging are relatively consistent, but there is no consensus on functional effects among these very few studies, and of course none could assess long-term clinical outcomes or risks.

 

The novel GHS, anamorelin, is currently under clinical development for cancer anorexia and cachexia syndrome (CACS), a syndrome overrepresented in the elderly.  In a phase II randomized, double-blind, placebo-controlled study, 3 days of treatment increased body weight and appetite in these patients when compared to placebo (66). Over 3 months of treatment, anamorelin increased body weight, LBM, hand grip strength, and quality of life (QOL). Anamorelin also increased IGF-1 and IGF binding protein (IGFBP)-3. It was well-tolerated, but it induced a small increase in glucose and insulin concentrations (67). Unfortunately, two large, international, randomized, double-blind, placebo-controlled phase III studies in patients with advanced non-small cell lung cancer and CACS (ROMANA 1 and 2) did not show improvements in handgrip strength with anamorelin, in spite of increased LBM, fat mass, body weight and appetite-related QoL compared to placebo (68). Although these studies were not restricted to the elderly, the mean age of the population was above 60 years of age in all studies.

 

CONCLUSIONS

 

While aging is not a disease, it results in alterations in body composition and functional decline with subsequent frailty and loss of independence. Interventions that slow this decline could potentially prolong the capacity for independent living and improve quality of life, but this has not yet been demonstrated. It is unknown whether the decrease in trophic hormones including sex steroids and growth hormone that occur with aging represents an adaptive or pathological process. Aging may represent a milder form of adult GHD, and since GH replacement in frank AGHD has met with success, it may be logical to reason that GH replacement or stimulation by GHRH or GHS might be beneficial in aging. However, older persons are more sensitive to GH, and thus more susceptible to the side effects of replacement. To date, definitive conclusions regarding functional effects of treatments in normal aging aimed at increasing GH levels to those of young healthy persons have been elusive. Until more studies are undertaken to determine the long-term effects of GH and GHS supplementation, conclusive statements about the merits of treatment cannot be made. Long term studies on hard clinical endpoints, such as falls and fracture rates, function measures, quality of life, and decreased morbidity and mortality from vascular disease need to be performed in order to establish the role, if any, for GH and GHS treatment in normal aging. In the meantime, GH use for anti-aging purposes is currently prohibited by US federal law (69, 70).

 

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Endocrine Hypertension

CLINICAL RECOGNITION

 

Hypertension is defined differently by various societies with a blood pressure exceeding 139/89 mm Hg for adults aged 18 years or older generally considered being elevated, based on the mean of 2 or more properly measured seated BP readings on each of 2 or more office visits. Hypertension affects approximately 31% of Americans when using the above cutoff level. Blood pressure control is suboptimal and is achieved in less than 1 in 3. For children, hypertension is defined as an average systolic BP and/or diastolic BP that is greater than the 95th percentile for age, gender, and height on more than 3 occasions. Normal BP in children is defined as a SBP and DBP less than the 90th percentile for age, gender, and height. Figure 1 provides an overview of classification of BP for adults 18 years and older.

Figure 1. Classification of Hypertension. AHA, American Heart Association; ACC, American College of Cardiology; ESC, European Society of Cardiology; ESH, European Society of Hypertension; DHL, German Hypertension League; NICE, National Institute for Health and Care Excellence of the United Kingdom. DBP, diastolic blood pressure; SBP, systolic blood pressure. Modified from: Jordan J, Kurschat C, Reuter H. Arterial hypertension. Dtsch Arztebl Int. 2018 Aug 20;115(33-34):557-568

 

Less than 5% of hypertension is endocrine related, the vast majority being “essential”. Endocrine hypertension is suggested by finding physical or historical clues suggesting a specific endocrine disease or patient’s failure to respond to conventional therapy. The first step when evaluating a patient with suspected endocrine-related hypertension is to exclude other causes of secondary hypertension. A detailed medical history and review of systems should be obtained. The onset of hypertension and the response to previous anti-hypertensive treatment should be determined. A history of target organ damage (i.e. retinopathy, nephropathy, claudication, heart disease, abdominal or carotid artery disease) and the overall cardiovascular risk status should also be explored in detail. Moreover, a detailed family history may provide valuable insights into familial forms of endocrine hypertension.

 

A secondary cause of hypertension should be suspected with the following:

  • Young age
  • Resistant hypertension
  • Need for more than 3 antihypertensives to control blood pressure
  • Very high blood pressure >180/110 mm Hg
  • Family history of kidney disease
  • Hypokalemia
  • Plethora with features of Cushing’s syndrome
  • Spells with variable blood pressure spikes
  • Features of growth hormone excess
  • Features of hypothyroidism, i.e. swollen eyes, dry skin
  • Signs and symptoms of hyperthyroidism, i.e. palpitations, weight loss
  • Retinal angiomas (?von Hippel Lindau disease)

 

Table 1 provides a specific description of the clinical presentation of endocrine conditions related to hypertension.

 

Table 1. Clinical Findings in Patients with Endocrine Hypertension

Condition

Clinical presentation

Primary hyperaldosteronism

Diastolic hypertension, headache, muscle weakness, hypokalemia, metabolic alkalosis

Cushing’s syndrome

Fatigue, weight gain, round face, proximal myopathy, plethora, hirsutism, buffalo hump, central obesity

Pheochromocytoma

Headache, palpitation, sweating, pallor, paroxysmal BP

Hyperthyroidism

 

Tremor, tachycardia, atrial fibrillation, weight loss, goiter, ophthalmopathy, pretibial myxedema

Hypothyroidism

 

Fatigue, cold intolerance, weight gain, nonpitting edema, periorbital puffiness

CAH: 11beta-hydroxylase

deficiency

Virilization, tall stature, hirsutism, advanced bone age, amenorrhea

CAH: 17alpha-hydroxylase

deficiency

Pseudohermaphroditism (male), sexual infantilism (female), hypokalemia

Liddle syndrome

Severe hypertension, hypokalemia, and metabolic alkalosis

Apparent mineralocorticoid

excess

Growth retardation/short stature, hypertension, hypokalemia, diabetes insipidus,

Pseudohypoaldosteronism

type 2

Short stature, hyperkalemic metabolic acidosis, normal aldosterone

Glucocorticoid Resistance

 

Ambiguous genitalia, precocious puberty, hirsutism, oligo/anovulation

Hyperparathyroidism

Bones, stones, abdominal groans, and psychic moans

Acromegaly

 

Headache, jaw enlargement, macroglossia, amenorrhea, impotence, diabetes mellitus, hypertension, heart failure

Insulin Resistance

 

Hypertension, abdominal/visceral obesity, dyslipidemia, and insulin resistance

 

It is also important to identify correctly patients with hypertensive emergencies (increased BP and acute target-organ damage) and provide the necessary urgent treatment. A focused exam must be undertaken quickly with the purpose of rapid identification of the acute target-organ damage. Hypertensive urgency is defined as a SBP > 180 mm Hg or DBP >120 mm Hg with minimal or no target-organ damage. The following tables shows the common hypertensive emergencies and the possible types of acute end-organ injury. Approx. 1% of Americans with hypertension will present with a hypertensive emergency.

 

Table 2. Common Causes of Hypertensive Emergencies

Medication noncompliance

Renovascular and renoparenchymal disease

Pre-eclampsia/eclampsia

Malignant hypertension

Acute increase in sympathetic activity (Pheochromocytoma crisis)

Autonomic dysfunction (Guillain-Barré syndrome, post-spinal cord injury) and

Central nervous system disorders (head injury, cerebral infarction / hemorrhage)

Drugs

   Sympathomimetics (cocaine, amphetamines incl. crystal meth, phencyclidine, etc)

   MAO inhibitors and the ingestion of tyramine-containing foods

   Withdrawal from clonidine and other central alpha2 adrenergic receptor agonists

 

Table 3. Hypertensive Emergency Acute End-Organ Injury

Cerebrovascular

     Subarachnoid or intracerebral hemorrhage

     Ischemic stroke

     Encephalopathy

 Renal damage

     Acute renal failure, scleroderma renal crisis, microangiopathic hemolytic anemia

 Cardiac

     Heart failure

     Acute coronary syndromes

     Acute aortic dissection

Eye

     Hemorrhage

     Exudate

     Papilledema

 

DIAGNOSIS AND DIFFERENTIAL DIAGNOSIS

 

Idiopathic (primary or essential) hypertension accounts for approximately 95% of diagnosed cases. It is estimated that approximately 5% of hypertensive patients have identifiable conditions that result in blood pressure elevation (secondary hypertension). Endocrine hypertension accounts for approximately 3% of the secondary forms of hypertension and is a term assigned to states in which hormonal derangements result in clinically significant hypertension. The major causes of secondary hypertension are summarized in table 4.

 

Table 4. Classification of Hypertension

Essential (95%)

Secondary causes (5%)

Endocrine Hypertension

Adults

    Cushing’s Syndrome

    Primary aldosteronism

    Pheochromocytoma

    Hyperthyroidism

    Hypothyroidism

    Hyperparathyroidism

    Acromegaly

    Insulin Resistance

Children

    CAH: 11beta-hydroxylase deficiency

    CAH: 17alpha-hydroxylase deficiency

    Apparent mineralocorticoid excess

    Liddle syndrome

    Pseudohypoaldosteronism type 2

    Glucocorticoid Resistance

    Insulin resistance

    Constitutive activation of the MR (Geller syndrome)

Non-Endocrine Hypertension

    Polycystic kidney disease

    Glomerular disease

    Renovascular

·           Atherosclerosis (older individuals)

·           Fibromuscular dysplasia (women)

·           Other: Scleroderma, vasculitis (PAN)

    Medications (Contraceptive drugs, NSAIDs, nasal decongestants with     adrenergic effects, MAOIs, steroids, methamphetamine, cocaine)

     Obstructive sleep apnea

     Coarctation of aorta

     Pre-eclampsia, eclampsia

     Polycythemia vera

 

PATHOPHYSIOLOGY


Cushing’s Syndrome

Hypercortisolemia is associated with hypertension in approximately 80% of adult cases and half of children. In Cushing’s syndrome there is increased hepatic production of angiotensinogen and cardiac output, reduced production of prostaglandins via inhibition of phospholipase A, increased insulin resistance, and oversaturation of 11beta-Hydroxysteroid Dehydrogenase activity with increased mineralocorticoid effect through stimulation of the mineralocorticoid receptor.

 

Primary Aldosteronism (PA)

 

PA can be a sporadic or familial condition. Most cases of PA are caused by bilateral adrenal hyperplasia and less commonly by an aldosterone-producing adrenal adenoma. Very rarely, PA can be caused by an adrenal carcinoma or unilateral adrenal cortex hyperplasia (also called primary adrenal hyperplasia). Familial aldosteronism is estimated to affect at least 2% of all patients with primary hyperaldosteronism and is classified as type 1, 2, 3, and 4. In familial hyperaldosteronism type 1, an autosomal dominantly inherited chimeric gene defect in CYP11B1/CYPB2 (coding for 11beta-hydroxylase/aldosterone synthase) causes ectopic expression of aldosterone synthase activity in the cortisol-producing zona fasciculata, making mineralocorticoid production regulated by corticotropin. The hybrid gene has been identified on chromosome 8. Familial hyperaldosteronism type 2 is not glucocorticoid-remediable. During the last years, other forms of familial aldosteronism were identified with 18-oxoF 10-1,000 higher (in type 3) than seen in familial hyperaldosteronism type 1 and/or type 2. Familial hyperaldosteronism type 3 is caused by germline mutations in the potassium channel subunit KCNJ5 and familial hyperaldosteronism type 4 is caused by germline mutations in the CACNA1H gene, which encodes the alpha subunit of an L-type voltage-gated calcium channel (Cav3.2).

 

Pheochromocytoma

 

These rare neuroendocrine tumors are composed of chromaffin tissue containing neurosecretory granules. Adrenal pheochromocytomas and most paragangliomas located in the abdomen produce and secrete catecholamines which can cause paroxysmal or sustained hypertension with hypertensive crisis.

 

Hyperthyroidism

 

Hyperthyroidism increases systolic blood pressure by increasing heart rate, decreasing systemic vascular resistance, and raising cardiac output. In thyrotoxicosis, patients usually are tachycardic and have high cardiac output with an increased stroke volume and elevated systolic blood pressure.

 

Hypothyroidism

 

Hypothyroid patients have impaired endothelial function, increased systemic vascular resistance, extracellular volume expansion, and an increased diastolic blood pressure. Hypothyroid patients have higher mean 24-h systolic BP and BP variability on 24-h ambulatory BP monitoring.

 

Congenital Adrenal Hyperplasia: 11beta-hydroxylase deficiency (5% of CAH)

 

11beta-hydroxylase is responsible for the conversion of deoxycorticosterone (DOC) to corticosterone (precursor of aldosterone) and 11-deoxycortisol to cortisol. In approximately 2/3 of individuals affected by a deficiency of this enzyme, monogenic low renin hypertension with low aldosterone levels ensues caused by accumulation of 11-deoxycortisol and DOC.

 

Congenital Adrenal Hyperplasia: 17alpha-hydroxylase deficiency

 

This enzyme deficiency is rare and leads to diminished production of cortisol and sex steroids. Chronic elevation of ACTH causes an increased production of DOC and corticosterone with subsequent hypertension, hypokalemia, low aldosterone concentrations with suppressed renin.

 

Apparent Mineralocorticoid Excess

 

Low-renin hypertension can present in various forms; one of them is apparent mineralocorticoid excess (AME), an autosomal recessive disorder caused by deficiency of the 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) enzyme. This enzyme converts cortisol to the inactive cortisone in renal tubular cells. The lack of this enzyme results in high levels of cortisol in renal tubule cells, which activates the mineralocorticoid receptor.

 

Liddle Syndrome

 

Liddle described patients with severe hypertension, hypokalemia, and metabolic alkalosis, who had low plasma aldosterone levels and plasma renin activity. “Gain of function” mutations in the genes coding for the beta- or gamma-subunit of the renal epithelial sodium channel, located at chromosome 16p13, lead to constitutive activation of renal sodium resorption and subsequent volume expansion.

 

Pseudohypoaldosteronism Type 2

 

This condition is transmitted in an autosomal dominant fashion, and can cause low renin hypertension. Hypertension in these patients may develop as a consequence of increased renal salt reabsorption, and hyperkalemia ensues as a result of reduced renal K excretion despite normal glomerular filtration and aldosterone secretion. Abnormalities such as activating mutations in the amiloride-sensitive sodium channel of the distal renal tubule are responsible for the clinical phenotype.

 

Glucocorticoid Resistance or Chrousos Syndrome

 

This autosomal recessive or dominant inherited disorder is rare and caused by inactivating mutations of the glucocorticoid receptor gene. Permanent elevation of ACTH can lead to stimulation of adrenal compounds with mineralocorticoid activity (corticosterone, DOC), and elevation of cortisol may lead to stimulation of the mineralocorticoid receptor, resulting in hypertension. In women, hirsutism and oligomenorrhea may develop through stimulation of androgens.

 

Constitutive Activation of the Mineralocorticoid Receptor (MC receptor)

 

The MC receptor can be mutated leading to the onset of hypertension before age 20. “Gain of function” mutations in the MC gene on chromosome 4q31 were identified. The inheritance pattern is autosomal-dominant.

 

DIAGNOSTIC TESTS NEEDED AND SUGGESTED

The presence of clinical signs and symptoms suggestive of endocrine hypertension (see table 1) should lead to a general screening for the most common forms of endocrine hypertension (Table 5).

 

Table 5. Screening Tests for Endocrine Causes of Hypertension

Cushing’s Syndrome

24-hour urinary cortisol, overnight dexamethasone suppression test, midnight salivary cortisol

Primary Hyperaldosteronism

Plasma aldosterone: renin ratio

Pheochromocytoma

Urinary or plasma metanephrines, urinary catecholamines

Thyroid Dysfunction

TSH, FT4, T3

 

In patients with a positive screening test, subsequent confirmation by various testing modalities is necessary (Table 6). These steps may involve supplementary laboratory tests and localization imaging tests (CT, MRI).

 

Table 6. Tests for Diagnosing the Most Prevalent Forms of Endocrine Hypertension

Cushing’s Syndrome
ACTH-dependent (5-10%) (ACTH > 20 ng/L)

    High-dose Dexamethason suppression test or CRH test

         If positive, then pituitary MRI and/or bilateral inferior petrosal sinus sampling

         If negative, then chest/abdomen MRI and/or 68Ga-DOTATATE PET/CT scan or

         Octreoscan

ACTH-independent (90-95%) (ACTH <10 ng/L)

          Adrenal CT or MRI

Hyperaldosteronism
Salt suppression test

    positive if aldosterone excretion > 12 to 14 µg/d while urine Na > 200 mEq/day

or other suppression tests: fludrocortisone suppression and captopril challenge

Adrenal CT or MRI

Adrenal vein sampling

Pheochromocytoma
Anatomic imaging (CT/MRI):

    abd/pelvis if negative then chest/head and neck

Functional imaging

    [123/131] Iodine-Metaiodobenzylguanidine scan

    specific PET ([18F] Fluorodopamine, [18F]Fluorodopa) scan

    non-specific PET ([18F] Fluorodeoxyglucose)

Genetic testing

 

If the above conditions have been ruled out but the suspicion of an endocrine cause of hypertension is still high, we should move to the next step and test for rare causes of hypertension. The diagnostic strategy is described in table 7.

 

Table 7. Testing for Rare Causes of Endocrine Hypertension

CAH: 11beta-hydroxylase deficiency
↑11-deoxycortisol, ↑DOC, ↑ 19-nor-DOC

↓renin, ↓↓ aldosterone,

↑urinary 100*THS/(THE+THF+5αTHF) and 100*THDOC/(THE+THF+5αTHF) ratios

Genetic testing

CAH: 17alpha-hydroxylase deficiency

↑DOC, ↓11-deoxycortisol, ↓↓ aldosterone

↓renin, ↓plasma 17-hydroxyprogesterone,
↑urinary 100*THDOC/(THE+THF+5αTHF) and (THA+THB+5αTHB)/(THE+THF+5αTHF) ratios

Genetic testing

Apparent mineralocorticoid excess

↓renin, ↓K, low aldosterone

↑ 24 h urinary free cortisol / cortisone
↑urinary (THF+5αTHF)/THE

Genetic testing

Liddle Syndrome
↓renin, ↓ aldosterone, ↓urinary THALDO
Genetic testing (ENaC gene)

Pseudohypoaldosteronism type 2
↑K, hyperchloremic metabolic acidosis,
↓aldosterone, ↓renin, ↓serum HCO3,

↓urinary THALDO

Genetic testing (ENaC gene)

Glucocorticoid Resistance Syndrome
↑cortisol, ↑ACTH, ↑androgens

Genetic testing

Constitutive Activation of the Mineralocorticoid Receptor

↑K, ↓aldosterone, ↓renin

↓urinary THALDO

Genetic testing

THE-tetrahydrocortisone; THF- tetrahydrocortisol; THA-tetrahydro 11-dehydro-corticosterone; THB-tetrahydrocorticosterone; DOC-deoxycorticosterone; THALDO-tetrahydro aldosterone

 

THERAPY

In the face of a hypertensive crisis, rapid action is important and the underlying disorder and the individual patient’s comorbidities determine the treatment approach. Aortic dissection will require rapid lowering of blood pressure, whereas blood pressure in an ischemic cerebrovascular event should be lowered modestly considering the cerebral perfusion and intracranial pressures. Among 1000 participants with intracerebral hemorrhage and a mean systolic blood pressure of 201 mm Hg at baseline lowering the SBP to 110 to 139 mm Hg did not result in a lower rate of death or disability than standard reduction to a target of 140 to 179 mm Hg (Qureshi AI et al. NEJM 2016).  For acute hypertension following stroke, labetalol, nicardipine, and nitroprusside are commonly administered with labetalol being considered first line therapy. For cocaine intoxication, phentolamine and nitroprusside are recommended. For an adrenergic crisis due to pheochromocytoma, phentolamine, nitroprusside and urapidil are preferred. For the management of a hypertensive emergency in pregnant and postpartal women, intravenous labetalol next to magnesium sulfate, ketanserine, hydralazine, and nicardipine are considered first line medications. Immediate release oral nifedipine can also be given, especially when no intravenous access is available.  

 

In general, in the first hour of treatment the mean arterial blood pressure should be reduced by 15% to 20% from baseline and then another 10%-15% over the following 2 to 6 h with a further gradual reduction over the next 24 h to reach normal blood pressure levels.

 

The most common used intravenous drugs and their dose and duration of action are listed in the table 8.

 

Table 8. Commonly Used Intravenous Drugs

Agent

Dose

Onset/

duration of action

Vasodilators

 

 

Nitroprusside

0.25-10 mcg/kg/min

0.5-1 min/ 1-10 minutes

Nitroglycerine

5-200 mcg/kg/min

1-2 min/ 3-5 minutes

Nicardipine

5-15 mg/h, increase every 15 min

5-10 min/ 1-4h

Fenoldopam

Initial dose:0.1 mg/kg/min followed by  0.05 to 0.1 mcg/kg/min q 15-20min till normal BP

10 min/ 30 minutes

Hydralazine

10-20 mg q 20-30min

10-20 min/3-8h

Beta-blockers

 

 

Labetalol

20-80 mg as bolus every 10-20 min. or

0.5-2 mg/kg/min

5-10 min/2-6h

Esmolol

0.5-1 mg/kg bolus; 50-300 mcg/kg/min

1-2 min / 10-30 min

Alpha-blocker

 

 

Phentolamine

1-5 mg bolus q 5-15min; 0.5-1 mg/h infusion

1-2 min/ 3-10 min

Urapidil

12.5-25 mg bolus; 5-40 mg/h infusion

3-5 min / 4-6 h

Antagonist of 5-HT2 (hydroxytryptamine) receptors

Ketanserin

5 mg bolus, repeat; 2-6 mg/h infusion

1-2 min / 30-60 min

 

Once the diagnosis of a specific cause of endocrine hypertension has been established, treatment oriented toward the endocrine diseases should be instituted (see specific chapters in Endotext that discuss the treatment of these disorders in depth).

 

Table 9. Treatment for Endocrine Causes of Hypertension

Cushing’s Syndrome

Adrenolytic Therapy

    Metyrapone 250-6000 mg/day in 3-4 doses daily (oral)

    Ketoconazole 200-1200 mg/day in up to 4 daily doses (oral)

    Mitotane up to 4-12 g/day (oral)

    Etomidate intravenously at 0.3 mg/kg/h based on the serum cortisol levels

Somatostatin analogues

    Pasireotide 600-900 µg twice daily s.c.

Dopamine agonists

    Cabergoline initially 0.5 mg/week, titrated to 4.5 mg/week (oral)

Alkylating drugs

    Temozolomide (experimental, oral)

Glucocorticoid receptor antagonists

    Mifepristone, CORT112716, 113083 (oral)

Primary aldosteronism

Mineralocorticoid receptor antagonist

    Eplerenone 50 - 300 mg / day (oral)

    Spironolactone 50-225 mg/day (oral)

Glucocorticoids (GRA)

    Dexamethasone (low dose i.e. 0.5 mg)

Pheochromocytoma

a-adrenoceptor blocker± Β-blockers

    Phenoxybenzamine at 10-20 mg (titrated up based on SBP) twice daily for 2 weeks before surgery

    Propranolol or other beta-blocker for reflex tachycardia

Hypertensive crisis

    Phentolamine i.v. bolus of 2.5 mg-5 mg at 1 mg/min

    Sodium nitroprusside as an alternative at 0.25-10 mcg/kg/min

Hyperthyroidism
Thyroid storm

    Aggressive hydration of up to 3-4 L/d of crystalloid

    Antithyroid drugs

    Methimazole 20-30 mg q 6-12h, then 5-40 mg/d

    Propylthiouracil (second line) 200 mg q 4-6hr initially then 100-150 mg/day BID

    Dexamethasone (up to 2 mg q6h)

    β-blocker

    Propranolol 40 mg q6h titrated to SBP

    Iodide i.e. Lugol’s solution 1-2 drops

Hypothyroidism

Levothyroxine

    (1.6 mcg/kg/day)-lower dose for patients at risk for ischemic heart disease

Myxedema coma

    Loading dose 5-10 mcg/kg T4 iv then 50-100 mcg iv qd and steroid replacement (i.e.hydrocortisone  5-10 mg/hr) until normalization of  adrenal  function

GRA- Glucocorticoid-remediable aldosteronism

 

FOLLOW-UP


The long-term management of patients with the respective underlying endocrine disorder is discussed in depth in other sections of ENDOTEXT, for instance, the adrenal and pituitary sections.

 

REFERENCES

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          Endocrinology Series, Springer, New York, 2013, ISBN: 978-1-60761-547-7 (Print), ISBN-10: 978-1-60761-548-4 (online)