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Vitamin D: Production, Metabolism, and Mechanisms of Action

ABSTRACT

Vitamin D production in the skin under the influence of sunlight (UVB) is maximized at levels of sunlight exposure that do not burn the skin. Further metabolism of vitamin D to its major circulating form (25(OH)D) and hormonal form (1,25(OH)2D) takes place in the liver and kidney, respectively, but also in other tissues where the 1,25(OH)2D produced serves a paracrine/autocrine function: examples include the skin, cells of the immune system, parathyroid gland, intestinal epithelium, prostate, and breast. Parathyroid hormone, FGF23, calcium and phosphate are the major regulators of the renal 1-hydroxylase (CYP27B1, the enzyme producing 1,25(OH)2D); regulation of the extra renal 1-hydroxylase differs from that in the kidney and involves cytokines. The major enzyme that catabolizes 25(OH)D and 1,25(OH)2D is the 24-hydroxylase; like the 1-hydroxylase it is tightly controlled in the kidney in a manner opposite to that of the 1-hydroxylase, but like the 1-hydroxylase it is widespread in other tissues where its regulation is different from that of the kidney. Vitamin D and its metabolites are carried in the blood bound to vitamin D binding protein (DBP) and albumin--for most tissues it is the free (i.e., unbound) metabolite that enters the cell; however, DBP bound metabolites can enter some cells such as the kidney and parathyroid gland through a megalin/cubilin mechanism. Most but not all actions of 1,25(OH)2D are mediated by the vitamin D receptor (VDR). VDR is a transcription factor that partners with other transcription factors such as retinoid X receptor that when bound to 1,25(OH)2D regulates gene transcription either positively or negatively depending on other cofactors to which it binds or interacts.  The VDR is found in most cells, not just those involved with bone and mineral homeostasis (i.e., bone, gut, kidney) resulting in wide spread actions of 1,25(OH)2D on most physiologic and pathologic processes. Animal studies indicate that vitamin D has beneficial effects on various cancers, blood pressure, heart disease, immunologic disorders, but these non-skeletal effects have been difficult to prove in humans in randomized controlled trials. Analogs of 1,25(OH)2D are being developed to achieve specificity for non-skeletal target tissues such as the parathyroid gland and cancers to avoid the hypercalcemia resulting from 1,25(OH)2D itself. The level of vitamin D intake and achieved serum levels of 25(OH)D that are optimal and safe for skeletal health and the non-skeletal actions remain controversial, but are likely between an intake of 800-2000IU vitamin D in the diet and 20-50ng/ml 25(OH)D in the blood.

OVERVIEW

Rickets became a public health problem with the movement of the population from the farms to the cities during the Industrial Revolution. Various foods such as cod liver oil and irradiation of other foods including plants were found to prevent or cure this disease, leading eventually to the discovery of the active principle—vitamin D. Vitamin D comes in two forms (D2 and D3) which differ chemically in their side chains. These structural differences alter their binding to the carrier protein vitamin D binding protein (DBP) and their metabolism, but in general the biologic activity of their active metabolites is comparable. Vitamin D3 is produced in the skin from 7-dehydrocholesterol by UV irradiation, which breaks the B ring to form pre-D3. Pre-D3 isomerizes to D3 but with continued UV irradiation to tachysterol and lumisterol. D3 is preferentially removed from the skin, bound to DBP. The liver and other tissues metabolize vitamin D, whether from the skin or oral ingestion, to 25OHD, the principal circulating form of vitamin D. Several enzymes have 25-hydroxylase activity, but CYP2R1 is the most important. 25OHD is then further metabolized to 1,25(OH)2D principally in the kidney, by the enzyme CYP27B1, although other tissues including various epithelial cells, cells of the immune system, and the parathyroid gland contain this enzyme. 1,25(OH)2D is the principal hormonal form of vitamin D, responsible for most of its biologic actions. The production of 1,25(OH)2D in the kidney is tightly controlled, being stimulated by parathyroid hormone (PTH), and inhibited by calcium, phosphate and FGF23. Extrarenal production of 1,25(OH)2D as in keratinocytes and macrophages is under different control, being stimulated primarily by cytokines such as tumor necrosis factor alfa (TNFα) and interferon gamma (IFNg). 1,25(OH)2D reduces 1,25(OH)2D levels in cells primarily by stimulating its catabolism through the induction of CYP24A1, the 24-hydroxylase.  25OHD and 1,25(OH)2D are hydroxylated in the 24 position by this enzyme to form 24,25(OH)2D and 1,24,25(OH)3D, respectively.  This 24-hydroxylation is generally the first step in the catabolism of these active metabolites to the final end product of calcitroic acid, although 24,25(OH)2D and 1,24,25(OH)3D have their own biologic activities. CYP24A1 also has 23-hydroxylase activity that leads to a different end product. Different species differ in their ratio of 23-hydroxylase/24-hydroxyase activity in their CYP24A1 enzyme, but in humans the 24-hydroxyase activity predominates. Like CYP27B1, CYP24A1 is widely expressed. CYP24A1 is induced by 1,25(OH)2D in most tissues, which serves as an important feedback mechanism to avoid vitamin D toxicity. In the kidney, PTH inhibits CYP24A1, whereas FGF23, calcium and phosphate stimulates it, just the opposite of the actions of these hormones and minerals on CYP27B1. However, such regulation is not seen in other tissues.  In macrophages, CYP24A1 is either missing or defective, so in situations such as granulomatous diseases like sarcoidosis in which macrophage production of 1,25(OH)2D is increased, hypercalcemia and hypercalciuria due to elevated 1,25(OH)2D can occur without the counter regulation by CYP24A1.

The vitamin D metabolites are transported in blood bound to DBP and albumin. Very little circulates as the free form. The liver produces DBP and albumin, production that is decreased in liver disease, and these proteins may be lost in protein losing enteropathies or the nephrotic syndrome. Thus, individuals with liver, intestinal or renal diseases which result in low levels of these transport proteins may have low total levels of the vitamin D metabolites without necessarily being vitamin D deficient as their free concentrations may be normal.

The receptor for 1,25(OH)2D (VDR) is a transcription factor regulating the expression of genes which mediate its biologic activity. VDR is a member of a rather large family of nuclear hormone receptors which includes the receptors for glucocorticoids, mineralocorticoids, sex hormones, thyroid hormone, and vitamin A metabolites or retinoids. The VDR is widely distributed, and is not restricted to those tissues considered the classic target tissues of vitamin D. The VDR upon binding to 1,25(OH)2D heterodimerizes with other nuclear hormone receptors, in particular the family of retinoid X receptors. This complex then binds to special DNA sequences called vitamin D response elements (VDRE) generally within the genes it regulates, although these VDREs can be thousands of base pairs from the transcription start site. There are thousands of the VDREs in hundreds of genes, and the profile of active VDREs (and regulated genes) varies from cell to cell.  A variety of additional proteins called coregulators complex with the VDR to activate (coactivators) or inhibit (corepressors) VDR transcriptional activity. Coactivator factors involved in VDR mediated transcription include factors with histone acetylase activity, including steroid receptor coactivator (SRC) 1, SRC 2 and SRC 3, and CREB-binding protein p300, in addition to the SWI–SNF ATP dependent chromatin remodeling complex, methyltransferases and the Mediator complex (aka DRIP), which functions to recruit RNA polymerases. VDR binding sites are associated with sites for other transcription factors such as p63, C–EBPα, C–EBPβ, Runx2 and PU.1, which can cooperate with VDR and VDR coregulators to influence 1,25(OH)2D responses in target cells. Among other functions these coregulators reconfigure the chromatin structure to bring the VDR/VDRE to the transcription start site, explaining how such distant VDR/VDREs can regulate gene transcription.  In addition to coactivators there are a number of corepressors. One such corepressor of VDR action in the skin is called hairless, in that its loss or mutation, like that of the VDR, leads to altered hair follicle cycling resulting in baldness. Corepressors typically work by recruiting histone deacetylases (HDAC) or methyl transferases (MT) to the gene which reverses the actions of HAT, leading to a reduction in access to the gene by the transcription machinery. These coregulators can be specific for different genes, and different cells differentially express these coregulators, providing some specificity for the actions of 1,25(OH)2D and VDR.

In addition to regulating gene expression, 1,25(OH)2D has a number of non-genomic actions including the ability to stimulate calcium transport across the plasma membrane. The mechanisms mediating these non-genomic actions and their physiologic significance remain unclear. Similarly, it is not clear that all actions of the VDR require the ligand 1,25(OH)2D. The best example of this is the hair loss in animals and subjects with VDR mutations but not in animals and subjects with mutations in CYP27B1, the enzyme producing 1,25(OH)2D. As mentioned, the VDR is widely distributed, and the actions of 1,25(OH)2D are quite varied. The classic target tissues—bone, gut, and kidney—are involved with calcium homeostasis. The mechanisms by which 1,25(OH)2D regulates transcellular calcium transport are best understood in the intestine. Here 1,25(OH)2D stimulates calcium entry across the brush border membrane into the cell, transport of calcium through the cell, and removal of calcium from the cell at the basolateral membrane. Calcium entry at the brush border membrane occurs down a steep electrochemical gradient. It is controlled in large measure by a specific calcium channel called TRPV6 and in humans also by a homologous calcium channel TRPV5. Transport of calcium through the cell is regulated by a class of calcium binding proteins called calbindins. Much of the transport occurs within vesicles that form in the terminal web. Removal of calcium from the cell at the basolateral membrane requires energy and is mediated by the ATP requiring calcium pump or CaATPase (PMCA1b) as well as the sodium/calcium exchange protein (NCX1). 1,25(OH)2D induces TRPV6 and TRPV5, the calbindins, and the CaATPase, but not all aspects of transcellular calcium transport are a function of new protein synthesis. Animals null for calbindin 9k (the major calbindin in mammalian intestine) have little impairment of intestinal calcium transport. Animals null for TRPV6, on the other hand, have a reduction in intestinal calcium transport, but the deficit is not profound. Thus, it is likely that compensatory mechanisms for intestinal calcium transport exist that have yet to be discovered.  Similar mechanisms mediate 1,25(OH)2D regulated calcium reabsorption in the distal tubule of the kidney. The proteins involved are homologous but not identical (TRPV5 and Calbindin 28k, for example). The situation in bone, however, is less clear. VDR are found in osteoblasts, the bone forming cells. 1,25(OH)2D promotes the differentiation of osteoblasts and regulates the production of proteins such as collagen, alkaline phosphatase, and osteocalcin thought to be important in bone formation. 1,25(OH)2D also induces RANKL, a membrane bound protein in osteoblasts that enables osteoblasts to stimulate the formation and activity of osteoclasts. Thus 1,25(OH)2D regulates both bone formation and bone resorption. Some evidence suggests that the major effect of 1,25(OH)2D on bone is to provide adequate levels of calcium and phosphate from the intestine. The rickets of patients with a mutated VDR or of mice in which the VDR has been deleted can be prevented/corrected by normalizing serum calcium and phosphate levels by dietary means. On the other hand, normal bone formation is not restored, and with time the VDR null mice become osteoporotic despite the high calcium/phosphate diet. Moreover, the VDR in osteoblasts/osteocytes appears to control bone resorption especially when dietary calcium is limited. Whether subjects with VDR mutations also develop osteoporosis prematurely or fail to maintain serum calcium in times of calcium deficiency has not been reported.

The non-classic actions of 1,25(OH)2D include regulation of cellular proliferation and differentiation, regulation of hormone secretion, and regulation of immune function. The ability of 1,25(OH)2D to inhibit proliferation and stimulate differentiation has led to the development of a number of analogs in the hopes of treating hyperproliferative disorders such as psoriasis and cancer without raising serum calcium. Psoriasis is now successfully treated with several vitamin D analogs. Observational studies are promising with respect to adequate vitamin D nutrition and cancer prevention. However, supplementation with vitamin D of subjects with adequate vitamin D levels to start with has not been shown to decrease cancer incidence but may be beneficial for cancer mortality. 1,25(OH)2D inhibits parathyroid hormone secretion and stimulates insulin secretion. A number of analogs and 1,25(OH)2D itself are currently available for use in the treatment of secondary hyperparathyroidism accompanying renal failure. Epidemiologic evidence indicates that vitamin D deficiency is associated with increased risk of both type 1 and type 2 diabetes mellitus, but prospective clinical trials to demonstrate a role for vitamin D supplementation in preventing the conversion of prediabetes to diabetes has not shown benefit in vitamin D replete individuals. However, there may be benefit in vitamin D deficient patients.  The ability of 1,25(OH)2D to regulate immune function is likely part of its efficacy in the treatment of psoriasis. A number of other autoimmune diseases have been found in animal studies to respond favorably to vitamin D and 1,25(OH)2D or its analogs, and epidemiologic evidence linking vitamin D deficiency to increased incidence of these diseases has been reported. Similarly, epidemiologic evidence linking vitamin D deficiency to a number of respiratory illnesses is substantial, including increased risk of COVID-19 infections.

DISCOVERY

The first clear description of rickets was by Whistler (1) in 1645. However, it was not until the Industrial Revolution with the mass movement of the population from the farms to the smoke- filled cities that rickets became a public health problem, most notably in England where sunlight intensity was already marginal for much of the year. Mellanby (2) in Great Britain and McCollum (3) in the United States developed animal models for rickets and showed that rickets could be cured with cod liver oil. McCollum heated the cod liver oil to destroy its vitamin A content and found that it still had antirachitic properties; he named the antirachitic factor vitamin D. Steenbock and Black (4) then demonstrated that UV irradiation of food, in particular non saponifiable lipids, could treat rickets. Meanwhile, clinical investigations revealed that rickets could be prevented or cured in children with sunlight or artificial UV exposure (5,6) suggesting that what subsequently became known as vitamin D could be produced by irradiation of precursors in vivo. Ultimately, Askew et al. (7) isolated and determined the structure of vitamin D2 (ergocalciferol) from irradiated plant sterols (ergosterol), and Windaus et al. (8) determined the structures and pathway by which 7-dehydrocholesterol (7-DHC) in the skin is converted to vitamin D3 (cholecalciferol). The name vitamin D1 refers to what proved to be an error of an earlier identification, and is not used. The structures and pathways of production of vitamin D3 are shown in figure 1. The structures of vitamins D2 and D3 differ in the side chain where D2 contains a double bond (C22-23) and an additional methyl group attached to C24. In this chapter the designation of D will refer to both D3 and D2.

Figure 1. The production of vitamin D3 from 7-dehydrocholesterol in the epidermis. Sunlight (the ultraviolet B component) breaks the B ring of the cholesterol structure to form pre- D3. Pre-D3 then undergoes a thermal induced rearrangement to form D3. Continued irradiation of pre- D3 leads to the reversible formation of lumisterol3 and tachysterol3 which can revert back to pre-D3 in the dark.

Figure 2. The metabolism of vitamin D. The liver converts vitamin D to 25OHD. The kidney converts 25OHD to 1,25(OH)2D and 24,25(OH)2D. Other tissues contain these enzymes, but the liver is the main source for 25-hydroxylation, and the kidney is the main source for 1α-hydroxylation. Control of metabolism of vitamin D to its active metabolite, 1,25(OH)2D, is exerted primarily at the renal level where calcium, phosphorus, parathyroid hormone, FGF23, and 1,25(OH)2D regulate the levels of 1,25(OH)2D produced.

 METABOLISM

Vitamin D3 produced in the epidermis must be further metabolized to be active. The first step, 25-hydroxylation, takes place primarily in the liver, although other tissues have this enzymatic activity as well. As will be discussed below, there are several 25-hydroxylases. 25OHD is the major circulating form of vitamin D. However, in order for vitamin D metabolites to achieve maximum biologic activity they must be further hydroxylated in the 1α position by the enzyme CYP27B1; 1,25(OH)2D is the most potent metabolite of vitamin D and accounts for most of its biologic actions. The 1α hydroxylation occurs primarily in the kidney, although as for the 25-hydroxylase, other tissues have this enzyme. Vitamin D and its metabolites, 25OHD and 1,25(OH)2D, can also be hydroxylated in the 24 position. This may serve to activate the metabolite or analog as 1,25(OH)2D and 1,24(OH)2D have similar biologic potency, and 1,24,25(OH)3D has activity approximately 1/10 that of 1,25(OH)2D. However, 24-hydroxylation of metabolites with an existing 25OH group leads to further catabolism. The details of these reactions are described below.

Cutaneous Production of Vitamin D3

The precursor of vitamin D, 7-dehydrocholesterol (7-DHC) is on the Kandutsch-Russell cholesterol pathway. The final enzymatic reaction mediated by 7-dehyrocholesterol reductase converting 7-DHC to cholesterol is regulated by a number of factors including vitamin D and cholesterol which enhance its degradation thus enabling increased levels of 7-DHC for conversion to vitamin D (9). Although irradiation of 7-DHC was known to produce pre-D3 (which subsequently undergoes a temperature rearrangement of the triene structure to form D3), lumisterol, and tachysterol (figure 1), the physiologic regulation of this pathway was not well understood until the studies of Holick and his colleagues (10-12). They demonstrated that the formation of pre-D3 under the influence of solar or UV irradiation (maximal effective wavelength between 290-310) is relatively rapid and reaches a maximum within hours. UV irradiation further converts pre-D3 to lumisterol and tachysterol. Both the degree of epidermal pigmentation and the intensity of exposure correlate with the time required to achieve this maximal concentration of pre-D3, but do not alter the maximal level achieved. Although pre-D3 levels reach a maximum level, the biologically inactive lumisterol continues to accumulate with continued UV exposure. Tachysterol is also formed, but like pre-D3, does not accumulate with extended UV exposure. The formation of lumisterol is reversible and can be converted back to pre-D3 as pre-D3 levels fall. At 0oC, no D3 is formed; however, at 37oC pre-D3 is slowly converted to D3. Thus, short exposure to sunlight would be expected to lead to a prolonged production of D3 in the exposed skin because of the slow thermal conversion of pre-D3 to D3 and the conversion of lumisterol to pre-D3. Prolonged exposure to sunlight would not produce toxic amounts of D3 because of the photoconversion of pre-D3 to lumisterol and tachysterol as well as the photoconversion of D3 itself to suprasterols I and II and 5,6 transvitamin D3 (13).

Melanin in the epidermis, by absorbing UV irradiation, can reduce the effectiveness of sunlight in producing D3 in the skin. This may be one important reason for the lower 25OHD levels (a well-documented surrogate measure for vitamin D levels in the body) in Blacks and Hispanics living in temperate latitudes (14). Sunlight exposure increases melanin production, and so provides another mechanism by which excess D3 production can be prevented. The intensity of UV irradiation is also important for effective D3 production. The seasonal variation of 25OHD levels can be quite pronounced with higher levels during the summer months and lower levels during the winter. The extent of this seasonal variation depends on the latitude, and thus the intensity of the sunlight striking the exposed skin. In Edmonton, Canada (52oN) very little D3 is produced in exposed skin from mid-October to mid-April; Boston (42oN) has a somewhat longer period for effective D3 production; whereas in Los Angeles (34oN) and San Juan (18oN) the skin is able to produce D3 all year long (15). These findings apply to sea level. At higher elevations there is less atmospheric absorption of UVB, so that skiers can make vitamin D even in winter on sunny days. Peak D3 production occurs around noon, with a larger portion of the day being capable of producing D3 in the skin during the summer than other times of the year. Clothing (16) and sunscreens (17) effectively prevent D3 production in the covered areas. This is one likely explanation for the observation that the Bedouins in the Middle East, who totally cover their bodies with clothing, are more prone to develop rickets and osteomalacia than the Israeli Jews with comparable sunlight exposure.

Hepatic Production of 25OHD

The next step in the bioactivation of D2 and D3, hydroxylation to 25OHD, takes place primarily in the liver although a number of other tissues express this enzymatic activity. 25OHD is the major circulating form of vitamin D and provides a clinically useful marker for vitamin D status. DeLuca and colleagues were the first to identify 25OHD and demonstrate its production in the liver over 30 years ago, but ambiguity remains as to the actual enzyme(s) responsible for this activity. 25-hydroxylase activity has been found in both the liver mitochondria and endoplasmic reticulum, and the enzymatic activities appear to differ indicating different proteins. At this point most attention has been paid to the mitochondrial CYP27A1 and the microsomal CYP2R1. However, in mouse knockout studies and in humans with mutations in these enzymes, only CYP2R1 loss is associated with decreased 25OHD levels (18,19). However, deletion or mutation of CYP2R1 does not totally eliminate 25OHD production These are mixed function oxidases, but differ in apparent Kms and substrate specificities. 

The mitochondrial 25-hydroxylase is now well accepted as CYP27A1, an enzyme first identified as catalyzing a critical step in the bile acid synthesis pathway. This is a high capacity, low affinity enzyme consistent with the observation that 25-hydroxylation is not generally rate limiting in vitamin D metabolism. Although initial studies suggested that the vitamin D3-25-hydroxylase and cholestane triol 27-hydroxyase activities in liver mitochondria were due to distinct enzymes with differential regulation, the cloning of CYP27A1 and the demonstration that it contained both activities has put this issue to rest (20-22). CYP27A1 is widely distributed throughout different tissues with highest levels in liver and muscle, but also in kidney, intestine, lung, skin, and bone (20-23).  Mutations in CYP27A1 lead to cerebrotendinous xanthomatosis (24,25), and are associated with abnormal vitamin D and/or calcium metabolism in some but not all of these patients (25-27).  However, mice in which CYP27A1 is deleted actually have elevated 25OHD levels along with the disruption in bile acid synthesis (28). CYP27A1 can hydroxylate vitamin D and related compounds at the 24, 25, and 27 positions. However, D2 appears to be preferentially 24-hydroxylated, whereas D3 is preferentially 25-hydroxylated (29). The 1αOH derivatives of D are more rapidly hydroxylated than the parent compounds (30). These differences between D2 and D3 and their 1αOH derivatives may explain the differences in biologic activity between D2 and D3 or between 1αOHD2 and 1αOHD3.

The major microsomal 25-hydroxylase is CYP2R1, although other enzymes have been shown in in vitro studies to have 25-hydroxylase activity. This enzyme like that of CYP27A1 is widely distributed, although it is most abundantly expressed in liver, skin and testes (30). Unlike CYP27A1, CYP2R1 25-hydroxylates D2 and D3 equally (30). Several Nigerian families have been shown to have CYP2R1 mutations in family members with rickets (19,31).  These subjects respond to D therapy but suboptimally (19,31). Mice lacking CYP2R1 have reduced 25OHD levels, unlike mice lacking CYP27A1, but even the combined deletion of CYP2R1 and CYP27A1 does not reduce these levels more than about 70% (18). Thus, neither CYP27A1 nor CYP2R1 by themselves account for all 25-hydroxyase activity in the body, suggesting a role of other yet to be described 25-hydroxylases.

Studies of the regulation of 25-hydroxylation have not been completely consistent, most likely because of the initial failure to appreciate that at least two enzymatic activities were involved and because of species differences. In general, 25-hydroxylation in the liver is little affected by vitamin D status. However, CYP27A1 expression in the intestine (32) and kidney (33) is reduced by 1,25(OH)2D. Not surprisingly bile acids decrease CYP27A1 expression (34) as does insulin (35) through an unknown mechanism. Dexamethasone, on the other hand, increases CYP27A1 expression (36). CYP2R1 appears to be mediated by aspects of metabolism. Roizen et al. (37) found that the serum concentration of 25OHD, but not vitamin D, was decreased in mice fed a high fat diet to induce obesity compared with normal weight mice. Moreover, mRNA and protein levels of CYP2R1 were decreased in these obese mice.  The expression of other 25-hydroxylases (CYP27A1, CYP3A) or the catabolizing enzyme CYP24A1 was not altered. Aatsinki et al (38) examined the effect of high fat diet induced obesity, fasting, and type 2 diabetes as well as streptozotocin induced (type 1) diabetes on 25OHD levels in mice.  All these metabolic manipulations decreased the hepatic mRNA and protein concentration of CYP2R1. These authors then demonstrated that the decrease in CYP2R1 was mediated by PPARγ-coactivator-1α (PGC1α), a key metabolic regulator increased by fasting or diabetes. They then showed that the control of CYP2R1 gene expression by PGC1α involved another transcriptional regulator, estrogen-related receptor α (ERRα), which also binds to other nuclear receptors such as VDR and the glucocorticoid receptor (GR). Consistent with this is that dexamethasone, a ligand for GR, decreased hepatic CYP2R1 mRNA and protein concentrations by a mechanism mediated by increased PGC1α.

Renal Production of 1,25(OH)2D

1,25(OH)2D is the most potent metabolite of vitamin D, and mediates most of its hormonal actions. 1,25(OH)2D is produced from 25OHD by the enzyme 25OHD-1α hydroxylase (CYP27B1). The cloning of CYP27B1 by four independent groups (40-43) ended a long effort to determine the structure of this critical enzyme in vitamin D metabolism. Mutations in this gene are responsible for the rare autosomal disease of pseudovitamin D deficiency rickets (40,42,44,45). An animal model in which the gene is knocked out by homologous recombination reproduces the clinical features of this disease including retarded growth, rickets, hypocalcemia, hyperparathyroidism, and undetectable 1,25(OH)2D (46). Unlike Vdr null mice and VDR mutations in humans, alopecia is not part of this phenotype.

CYP27B1 is a mitochondrial mixed function oxidase with significant homology to other mitochondrial steroid hydroxylases including CYP27A1 (39%), CYP24A1 (30%), CYP11A1 (32%), and CYP11β (33%) (40). However, within the heme-binding domain the homology is much greater with 73% and 65% sequence identity with CYP27A1 and CYP24A1 (40). These mitochondrial P450 enzymes are located in the inner membrane of the mitochondrion, and serve as the terminal acceptor for electrons transferred from NADPH through ferrodoxin reductase and ferrodoxin. Expression of CYP27B1 is highest in epidermal keratinocytes (40), cells that previously had been shown to contain high levels of this enzymatic activity (47). However, the kidney also expresses this enzyme in the renal tubules as do the brain, placenta, testes, intestine, lung, breast, macrophages, lymphocytes, parathyroid gland, osteoblasts and chondrocytes (40,48-51). That said, the kidney is generally considered the major source of circulating levels of 1,25(OH)2D, with the extrarenal CYP27B1 activities providing for local needs under normal circumstances. However, extrarenal sources can lead to increased 1,25(OH)2D and calcium levels in some pathologic conditions to be discussed subsequently.

The principal regulators of CYP27B1 activity in the kidney are parathyroid hormone (PTH), FGF23, calcium, phosphate, and 1,25(OH)2D. Extrarenal production tends to be stimulated by cytokines such as IFN-gamma and TNF-α more effectively than PTH (52) and may be less inhibited by calcium, phosphate, and 1,25(OH)2D depending on the tissue. Administration of PTH in vivo (53) or in vitro (54,55) stimulates renal production of 1,25(OH)2D. This action of PTH can be mimicked by cAMP (53,55) and forskolin (56,57) indicating that at least part of the effect of PTH is mediated via its activation of adenylate cyclase. However, PTH activation of protein kinase C (PKC) also appears to be involved in that concentrations of PTH sufficient to stimulate PKC activation and 1,25(OH)2D production are below that required to increase cAMP levels (58). Furthermore, synthetic fragments of PTH lacking the ability to activate adenylate cyclase but which stimulate PKC activity were found to increase 1,25(OH)2D production (59). Direct activation of PKC with phorbol esters results in increased 1,25(OH)2D production. Although the promoter of CYP27B1 contains several AP-1 (PKC activated) and cAMP response elements, it is not yet clear how PTH regulates CYP27B1 gene expression (60). However, several mechanisms have been proposed. In one study the nuclear receptor 4A2 acting through a C/EBP consensus element appears to be involved (61). Another mechanism involves VDIR that is proposed to bind to a negative VDRE in the CYP27B1 promoter. When PKA is activated by PTH VDIR is phosphorylated and recruits the p300 complex with HAT activity, inducing gene transcription (62). Calcium modulates the ability of PTH to increase 1,25(OH)2D production. Calcium by itself can decrease CYP27B1activity (63,64) and block the stimulation by PTH (65). Given in vivo, calcium can exert its effect in part by reducing PTH secretion, but this does not explain its direct actions in vitro or its effects in parathyroidectomized or PTH infused animals. Phosphate deprivation can stimulate CYP27B1 activity in vivo (66,67) and in vitro (68). The in vivo actions of phosphate deprivation can be blocked by hypophysectomy (69,70) and partially restored by growth hormone (GH) (70,71) and insulin-like growth factor (IGF-I) (72). However, like PTH, the exact mechanism by which GH and/or IGF-I mediates the effects of phosphate on CYP27B1 expression remains unclear. More recently FGF23 has been shown to inhibit CYP27B1 activity in vivo and in vitro (73). FGF23 has been implicated as at least one of the factors responsible for impaired phosphate reabsorption and 1,25(OH)2D production in conditions such as X-linked and autosomal dominant hypophosphatemic rickets and oncogenic osteomalacia (74,75). FGF23 acts through FGF receptors 1 and 3 in conjunction with the coreceptor Klotho, but the mechanism by which FGF23 regulates CYP27B1 remains obscure. High phosphate stimulates FGF23 production from bone, and this is likely the major mechanism by which phosphate leads to decreased CYP27B1 activity (76).  1,25(OH)2D administration leads to reduction in CYP27B1 activity. In the kidney Meyer et al. (77) identified a region in the Cyp27b1 gene that when deleted blocked 1,25(OH)2D production. However, in other tissues no vitamin D response element has been identified in the promoter of the 1α-hydroxylase gene (60). In keratinocytes, 1,25(OH)2D has little or no effect on CYP27B1 mRNA and protein levels when given in vitro. When 24-hydroxylase activity is blocked, 1,25(OH)2D administration fails to reduce the levels of 1,25(OH)2D produced (78,79). Thus, the apparent feedback regulation of CYP27B1 activity by 1,25(OH)2D in most tissues, with the possible exception of the kidney, appears to be due to its stimulation of CYP24A1 and subsequent catabolism, not to a direct effect on CYP27B1 expression or activity. Moreover, 1,25(OH)2D stimulates FGF23 production and inhibits PTH production. Both actions will decrease, indirectly, the ability of 1,25(OH)2D to inhibit its own production (76).  Thus, renal and extrarenal regulation of CYP27B1 by 1,25(OH)2D may differ.

Renal Production of 24,25(OH)2D

The kidney is also the major producer of a second important metabolite of 25OHD, namely 24,25(OH)2D, and the enzyme responsible is 25OHD-24 hydroxylase (CYP24A1) [75]. CYP24A1 and CYP27B1 are homologous enzymes that coexist in the mitochondria of tissues where both are found, such as the kidney tubule. However, there genes are located on different chromosomes (chromosome 20q13 and chromosome 12q14 for CYP24A1 and CYP27B1, respectively, in humans). They share the same ferrodoxin and ferrodoxin reductase components. While CYP27B1 activates the parent molecule, 25OHD, CYP24A1 initiates a series of catabolic steps that lead to its inactivation. However, in some tissues 24,25(OH)2D has been shown to have biologic effects different from 1,25(OH)2D as will be described subsequently. CYP24A1 24-hydroxylates both 25OHD and 1,25(OH)2D. The 24-hydroxylation is then followed by oxidation of 24OH to a 24-keto group, 23-hydroxylation, cleavage between C23-24, and the eventual production of calcitroic acid, a metabolite with no biologic activity. CYP24A1 also has 23-hydroxylase activity, initiating steps that lead to 23/26 lactone formation. Different species have CYP24A1s that differ in their preference for the 24-hydroxylation vs 23-hydroxylation pathway. The human enzyme follows the 24-hydroxylation pathway. Analogs with differences in their side chain are also likely to differ in the pathway utilized. CYP24A1 catalyzes all the steps in this catabolic pathway (81) (82). Although CYP24A1 is highly expressed in the kidney tubule, its tissue distribution is quite broad. In general, CYP24A1 can be found wherever the VDR is found. The affinity for 1,25(OH)2D is higher than that for 25OHD, making this enzyme an efficient means for eliminating 1,25(OH)2D. Thus, CYP24A1 is likely to play the important role of protecting the body against excess 1,25(OH)2D. Indeed, inactivating mutations in CYP24A1 have been found to underlie the disease idiopathic infantile hypercalcemia (83), manifesting as the name suggests with elevated serum calcium and 1,25(OH)2D levels. These individuals may present for the first time as adults, often in the context of increased 1,25(OH)2D production as in pregnancy (84).  An animal model in which CYP24A1 has been knocked out likewise showed very high levels of 1,25(OH)2D when treated with vitamin D and impaired mineralization of intramembranous bone (85). The skeletal abnormalities could be corrected by crossing this mouse to one lacking the VDR suggesting that excess 1,25(OH)2D (which acts through the VDR) rather than deficient 24,25(OH)2D (which does not) is to blame (85).

The regulation of CYP24A1 in the kidney is almost the mirror image of that of CYP27B1. PTH and 1,25(OH)2D are the dominant regulators, but calcium, phosphate, insulin, FGF23, IGF-I, GH, and sex steroids may also play a role. 1,25(OH)2D induces CYP24A1. The promoter of CYP24A1 has two vitamin D response elements (VDREs) critical for this induction (86-88). Protein kinase C activation as by phorbol esters enhances this induction by 1,25(OH)2D (89). An AP-1 site is found adjacent to the proximal VDRE, but mutation of this site does not appear to block phorbol ester enhancement of CYP24A1 induction by 1,25(OH)2D (90). PTH, on the other hand, inhibits the expression of CYP24A1 in the kidney (91). This action can be reproduced with cAMP (92) and forskolin (56) indicating the role of PTH activated adenylate cyclase (93). PTH has no effect on intestinal CYP24A1, most likely because the intestine does not have PTH receptors. Surprisingly, however, PTH is synergistic with 1,25(OH)2D in stimulating CYP24A1 expression and activity in bone cells which do have PTH receptors, again through a cAMP mediated mechanism (94). This synergism is further potentiated by the addition of insulin (95) (96). FGF23 also induces CYP24A1 expression (97). Surprisingly this requires the VDR (97), since FGF23 also inhibits 1,25(OH)2D production and so would be expected to reduce CYP24A1 via a 1,25(OH)2D/VDR mechanism. Restriction in dietary phosphate reduces CYP24A1 expression consistent with a decrease in FGF23, but also in a manner blocked by hypophysectomy (98). GH and IGF-I can reduce CYP24A1 expression in hypophysectomized animals, suggesting that the phosphate effect on CYP24A1 like its opposing effect on CYP27B1, is mediated by GH and IGF-I (98) as well as FGF23. The region(s) of the CYP24A1 promoter mediating these actions of PTH and FGF23 as well as 1,25(OH)2D have recently been mapped (96). Similar to that for CYP27B1 this regulation differs in different cell types. Thus, although different regulators tend to have opposite effects on CYP24A1 and CYP27B1 expression the molecular mechanisms by which the regulation occurs also differ for each enzyme.

TRANSPORT IN BLOOD

The vitamin D metabolites are transported in blood bound primarily to vitamin D binding protein (DBP) (85-88%) and albumin (12-15%) (99-101). DBP concentrations are normally 4-8µM, well above the concentrations of the vitamin D metabolites, such that DBP is only about 2% saturated. DBP has high affinity for the vitamin D metabolites (Ka=5x108M-1 for 25OHD and 24,25(OH)2D, 4x107M-1 for 1,25(OH)2D and vitamin D), such that under normal circumstances only approximately 0.03% 25OHD and 24,25(OH)2D and 0.4% 1,25(OH)2D are free (100-102). Conditions such as liver disease and nephrotic syndrome resulting in reduced DBP and albumin levels will lead to a reduction in total 25OHD and 1,25(OH)2D levels without necessarily affecting the free concentrations (103) (figure 3). Similarly, DBP levels are reduced during acute illness, potentially obscuring the interpretation of total 25OHD levels (104). Earlier studies with a monoclonal antibody to measure DBP levels suggested a decreased level in African Americans consistent with their lower total 25OHD levels, but these results were not confirmed using polyvalent antibody-based assays (105). Vitamin D intoxication can increase the degree of saturation sufficiently to increase the free concentrations of 1,25(OH)2D and so cause hypercalcemia without necessarily raising the total concentrations (106).

The vitamin D metabolites bound to DBP are in general not available to most cells. Thus, the free or unbound concentration is that which is critical for cellular uptake as postulated by the free hormone hypothesis. Support for the concept that the role of DBP is to provide a reservoir for the vitamin D metabolites but that it is the free concentration that enters cells and exerts biologic function comes from studies in mice in which DBP has been deleted and in humans in which the gene is mutated. In DBP knockout mice the vitamin D metabolites are presumably all free and/or bioavailable. These mice do not show evidence of vitamin D deficiency unless placed on a vitamin D deficient diet despite having very low levels of serum 25OHD and 1,25(OH)2D (107). Tissue levels of 1,25(OH)2D were found to be normal in the DBP knockout mice as were markers of vitamin D action such as expression of intestinal TRPV6, calbindin 9k, PMCA1b, and renal TRPV5 (108). Recently a family in which a large deletion of the coding portion of the DBP gene (and adjacent NPFFR2 gene) has been reported (109). The proband had normal calcium, phosphate and PTH levels with vitamin D supplementation despite very low levels of 25OHD, 24,25(OH)2D, and 1,25(OH)2D that were not responsive to massive doses of vitamin D (oral or parenteral). The free 25OHD was nearly normal. The carrier sibling had vitamin D metabolite levels between those of the proband and the normal sibling. Thus, both the studies in DBP null mice and humans support the free hormone hypothesis while also supporting the role of DBP as a circulating reservoir for the vitamin D metabolites. Therefore, there is currently a debate as to whether the free concentration of 25OHD, for example, is a better indicator of vitamin D nutritional status than total 25OHD, given that DBP levels, and hence total 25OHD levels, can be influenced by liver disease, nephrotic syndrome, pregnancy, and inflammatory states (110,111). However, certain tissues such as the kidney, placenta, and parathyroid gland express the megalin/cubilin complex which is able to transport vitamin D metabolites bound to DBP into the cell. This is critical for preventing renal losses of the vitamin metabolites (112) and may be important for vitamin D metabolite transport into the fetus and regulation of PTH secretion. Indeed, mice lacking the megalin/cubilin complex have poor survival with evidence of osteomalacia indicating its role in vitamin D transport into critical cells involved with vitamin D signaling

Figure 3. Correlation of total 25OHD (A) and 1,25(OH)2D (C) levels to DBP; lack of correlation of free 25OHD (B) and 1,25(OH)2D (D) levels to DBP. Data from normal subjects (open triangles), subjects with liver disease (closed triangles, open circles), subjects on oral contraceptives (open triangles*), and pregnant women (open squares) are included. These data demonstrate the dependence of total 25OHD and 1,25(OH)2D concentrations on DBP levels which are reduced by liver disease. However, the free concentrations of 25OHD and 1,25(OH)2D are normal in most patients with liver disease. Reprinted with permission from the American Society for Clinical Investigation.

DBP was originally known as group specific component (Gc-globulin) before its properties as a vitamin D transport protein became known. It has three common polymorphisms which are useful in population genetics. These alleles have somewhat different affinities for the vitamin D metabolites (113), but which do not appear to alter its function. DBP is a 58kDa protein with 458 amino acids that is homologous to albumin and α-fetoprotein (αFP) (40% homology at the nucleotide level, 23% at the amino acid level) (114). These three genes cluster on chromosome 4q11-13 (115). DBP, like albumin and αFP, is made primarily but not exclusively in the liver-other sites include the kidney, testes, and fat.  DBP like other steroid hormone binding proteins is increased by oral (not transdermal) estrogens and pregnancy (100). In vitro, glucocorticoids and cytokines such as EGF, IL-6 and TGF-β have been shown to increase (glucocorticoids, EGF, IL-6) or decrease (TGF-β) DBP production (116).

Although transport of the vitamin D metabolites may be the major function for DBP, it has other properties. DBP has high affinity for actin, and may serve as a scavenger for actin released into the blood during cell death (117). DBP has also been shown to activate macrophages (118) and osteoclasts (119). However, in a mouse rendered deficient in DBP by homologous recombination (knock out) no obvious abnormality was observed except for increased turnover in vitamin D and increased susceptibility to osteomalacia on a vitamin D deficient diet (120). Evidence for osteopetrosis (indicating failure of osteoclast function) was not found.

MECHANISM OF ACTION

The hormonal form of vitamin D, 1,25(OH)2D, is the ligand for a transcription factor, the vitamin D receptor (VDR). Most if not all effects of 1,25(OH)2D are mediated by VDR acting primarily by regulating the expression of genes whose promoters contain specific DNA sequences known as vitamin D response elements (VDREs). There are thousands of VDREs throughout the gene, often thousands of base pairs away from the coding portion of the gene regulated.  However, some actions of 1,25(OH)2D are more immediate, and may be mediated by a membrane bound vitamin D receptor that has been less well characterized than the nuclear VDR or by the VDR acting outside of the nucleus. On the other hand, some actions of VDR do not require its ligand 1,25(OH)2D. Our understanding of the mechanism by which VDR regulates gene expression has increased enormously over the past few years.

VDR and Transcriptional Regulation

 The VDR was discovered in 1969 (121) (although only as a binding protein for an as yet unknown vitamin D metabolite subsequently identified as 1,25(OH)2D), and was eventually cloned and sequenced in 1987 (122,123). Inactivating mutations in the VDR result in hereditary vitamin D resistant rickets (HVDRR) (124). Animal models in which the VDR has been knocked out (125) (126) have the full phenotype of severe vitamin D deficiency indicating that the VDR is the major mediator of vitamin D action. The one major difference is the alopecia seen in HVDRR and VDR knockout animals, a feature not associated with vitamin D deficiency, suggesting that the VDR may have functions independent of 1,25(OH)2D at least in hair follicle cycling. The VDR is a member of a large family of proteins (over 150 members) that includes the receptors for the steroid hormones, thyroid hormone, vitamin A family of metabolites (retinoids), and a variety of cholesterol metabolites, bile acids, isoprenoids, fatty acids and eicosanoids. A large number of family members have no known ligands, and are called orphan receptors. VDR is widely, although not universally, distributed throughout the different tissues of the body (127). Many of these tissues were not originally considered target tissues for 1,25(OH)2D. The discovery of VDR in these tissues along with the demonstration that 1,25(OH)2D altered function of these tissues has markedly increased our appreciation of the protean effects of 1,25(OH)2D.

The VDR is a molecule of approximately 50-60kDa depending on species. The basic structure is shown in figure 4. The VDR is unusual in that it has a very short N-terminal domain before the DNA binding domain when compared to other nuclear hormone receptors. The human VDR has two potential start sites. A common polymorphism (Fok 1) alters the first ATG start site to ACG. Individuals with this polymorphism begin translation three codons downstream such that in these individuals the VDR is three amino acids shorter (424 aas vs 427 aas). This polymorphism has been correlated with reduced bone density suggesting it is of functional importance (128). The most conserved domain in VDR from different species and among the nuclear hormone receptors in general is the DNA binding domain. This domain is comprised of two zinc fingers. The name derives from the cysteines within this stretch of amino acids that form tetrahedral complexes with zinc in a manner which creates a loop or finger of amino acids with the zinc complex at its base. The proximal (N-terminal) zinc finger confers specificity for DNA binding to the VDREs while the second zinc finger and the region following provide at least one of the sites for heterodimerization of the VDR to the retinoid X receptor (RXR). The second half of the molecule is the ligand binding domain, the region responsible for binding 1,25(OH)2D, but which also contains regions necessary for heterodimerization to RXR. At the C-terminal end is the major activation domain, AF-2, which is critical for the binding to coactivators such as those in the steroid receptor coactivator (SRC) and vitamin D receptor interacting protein (DRIP) or Mediator families (129). In mutation studies of the homologous thyroid receptor, corepressors were found to bind in overlapping regions with coactivators in helices 3 and 5, a region blocked by helix 12 (the terminal portion of the AF2 domain) in the presence of ligand (130). Deletion of helix 12 promoted corepressor binding while preventing that of coactivators (130).

Figure 4. Model of the vitamin D receptor (VDR). The N terminal region is short relative to other steroid hormone receptors. This region is followed by two zinc fingers which constitute the principal DNA binding domain. Nuclear localization signals (NLS) are found within and just C-terminal to the DNA binding domain. The ligand binding domain makes up the bulk of the C-terminal half of the molecule, with the AF2 domain comprising the most C-terminal region. The AF2 domain is largely responsible for binding to co-activators such as the SRC family and DRIP (Mediator) in the presence of ligand. Regions on the second zinc finger and within the ligand binding domain facilitate heterodimerization with RXR. Corepressor binding is less well characterized but appears to overlap that of coactivators in helices 3 and 5, a region blocked by helix 12 in the presence of ligand. 

The ligand binding domain (LBD) for VDR has been crystallized and its structure solved (131). More recently the structure of the VDR/RXR heterodimer has been analyzed by high resolution cryoelectron microscopy (132).  These studies show that the VDR has a high degree of structural homology to other nuclear hormone receptors. It is comprised of 12 helices joined primarily by beta sheets. The 1,25(OH)2D is buried deep in the ligand binding pocket and covered by helix 12 (the terminal portion of the AF-2 domain). Assuming analogy with the unliganded LBD of RXRα and the ligand bound LBD of RARγ (133), the binding of 1,25(OH)2D to the VDR triggers a substantial movement of helix 12 from an open position to a closed position, covering the ligand binding pocket and putting helix 12 in position with critical residues from helices 3, 4, and 5 to bind coactivators. Coactivator complexes bridge the gap from the VDRE to the transcription machinery at the transcription start site (figure 5) (134).

Figure 5. 1,25(OH)2D-initiated gene transcription. 1,25(OH)2D enters the target cell and binds to its receptor, VDR. The VDR then heterodimerizes with the retinoid X receptor (RXR). This increases the affinity of the VDR/RXR complex for the vitamin D response element (VDRE), a specific sequence of nucleotides in the promoter region of the vitamin D responsive gene. Binding of the VDR/RXR complex to the VDRE attracts a complex of proteins termed coactivators to the VDR/RXR complex. The DRIP (Mediator) coactivator complex spans the gap between the VDRE and RNA polymerase II and other proteins in the initiation complex centered at or around the TATA box (or other transcription regulatory elements). SRC coactivators recruit histone acetyl transferases (HAT) to the gene promoting the opening up of its structure to enable the transcription machinery to work. Transcription of the gene is initiated to produce the corresponding mRNA, which leaves the nucleus to be translated to the corresponding protein.

Nuclear hormone receptors including the VDR are further regulated by protein complexes that can be activators or repressors (135). The role of corepressors in VDR function has been demonstrated (136) but is less well studied than the role of coactivators. One such corepressor, hairless, is found in the skin and may regulate 1,25(OH)2D mediated epidermal proliferation and differentiation as well as ligand independent VDR regulation of hair follicle cycling (137-139). The coactivators, which are essential for VDR function, form two distinct complexes, the interaction of which remains unclear (129). The SRC family has three members, SRC 1-3, all of which can bind to the VDR in the presence of ligand (1,25(OH)2D) (140). These coactivators recruit additional coactivators such as CBP/p300 and p/CAF that have histone acetyl transferase activity (HAT), an enzyme that by acetylation of lysines within specific histones appears to help unravel the chromatin allowing the transcriptional machinery to do its job. The domain in these molecules critical for binding to the VDR and other nuclear hormone receptors is called the NR box, and has as its central motif LxxLL where L stands for leucine and x for any amino acid. Each SRC family member contains three well conserved NR boxes in the region critical for nuclear hormone receptor binding. The DRIP (Mediator) complex is comprised of 15 or so proteins several of which contain LxxLL motifs (141). However, DRIP205 (Mediator 1) is the protein critical for binding the complex to VDR. It contains 2 NR boxes. Different NR boxes in these coactivators show specificity for different nuclear hormone receptors (142). Unlike the SRC complex, the DRIP complex does not have HAT activity (129). Rather the DRIP complex spans the gene from the VDRE to the transcription start site linking directly with RNA polymerase II and its associated transcription factors.  DRIP and SRC appear to compete for binding to the VDR. In keratinocytes DRIP binds preferentially to the VDR in undifferentiated cells, whereas SRC 2 and 3 bind in the more differentiated cells in which DRIP levels have declined (143). Thus in these cells DRIP appears to regulate the early stages of 1,25(OH)2D induced differentiation, whereas SRC may be more important in the later stages, although overlap in gene specificity is also observed (144,145) (146). These coregulators are not specific for VDR, but interact with a large number of other transcription factors. The DRIP (Mediator) complex can mark regions in the genome containing large numbers of sites for transcription factors including VDREs. These sites are known as super enhancers often regulating genes involved with cell fate determination (147).  Recently, SMAD 3, a transcription factor in the TGF-β pathway, has been found to complex with the SRC family members and the VDR, enhancing the coactivation process (148). Phosphorylation of the VDR may also control VDR function (149). Furthermore, VDR has been shown to suppress β-catenin transcriptional activity (150), whereas β-catenin enhances that of VDR (151).  Thus, control of VDR activity may involve crosstalk between signaling pathways originating in receptors at the plasma membrane as well as within the nucleus.

VDR acts in concert with other nuclear hormone receptors, in particular RXR (152). Unlike VDR, there are three forms of RXR--α, β, γ--and all three are capable of binding to VDR with no obvious differences in terms of functional effect. RXR and VDR form heterodimers that optimize their affinity for the vitamin D response elements (VDREs) in the genes being regulated. RXR appears to be responsible for keeping VDR in the nucleus in the absence of ligand (153). VDR may also partner with other receptors including the thyroid receptor (TR) and the retinoic acid receptor (RAR) (154,155), but these are the exceptions, whereas RXR is the rule. The VDR/RXR heterodimers bind to VDREs, which typically are comprised of two half sites each with six nucleotides separated by three nucleotides of nonspecific type; this type of VDRE is known as a DR3 (direct repeats with three nucleotide spacing). RXR binds to the upstream half site, while VDR binds to the downstream site (156). However, a wide range of VDRE configurations have been found at nearly any location within a gene (5’, 3’, introns) (157). Moreover, different tissues differ as to which VDREs actively bind VDR (158). 1,25(OH)2D is required for high affinity binding and activation, but the RXR ligand, 9-cis retinoic acid, may either inhibit (159) or activate (160) 1,25(OH)2D stimulation of gene transcription. A DR6 has been identified in the phospholipase C-γ1 gene that recognizes VDR/RAR heterodimers (154), and a DR4 has been found in the mouse calbindin 28k gene (161). Inverted palinodromes with 7 to 12 bases between half sites have also been found (151).  Furthermore, the half sites of the various known VDREs show remarkable degeneracy (table 1). The G in the second position of each site appears to be the only nearly invariant nucleotide. 1,25(OH)2D can also inhibit gene transcription through its VDR. This may occur by direct binding of the VDR to negative VDREs that in the PTH and PTHrP genes are remarkably similar in sequence to positive VDREs of other genes (162,163). However, inhibition may also be indirect. For example, 1,25(OH)2D inhibits IL-2 production by blocking the NFATp/AP-1 complex of transcription factors from activating this gene (164) through a mechanism not yet clear. Similarly, 1,25(OH)2D inhibits CYP27B1 in at least one renal cell line by an indirect mechanism involving VDR binding to VDIR (62,80). Thus, a variety of factors including the flanking sequences of the genes around the VDREs and tissue specific factors play a large role in dictating the ability of 1,25(OH)2D to regulate gene expression.

Non-Genomic Actions

A variety of hormones that serve as ligands for nuclear hormone receptors also exert biologic effects that do not appear to require gene regulation and may work through membrane receptors rather their cognate nuclear hormone receptors. Examples include estrogen (165), progesterone (166), testosterone (167), corticosteroids (168), and thyroid hormone (169). 1,25(OH)2D has also been shown to have rapid effects on selected cells that are not likely to involve gene regulation and that appear to be mediated by a different, probably membrane receptor. A model for such effects is shown in figure 6. Similar to other steroid hormones, 1,25(OH)2D has been shown to regulate calcium and chloride channel activity, protein kinase C activation and distribution, and phospholipase C activity in a number of cells including osteoblasts (170), liver (171), muscle (172), and intestine (173,174). These rapid effects of 1,25(OH)2D have been most extensively studied in the intestine. Norman's laboratory coined the term transcaltachia to describe the rapid onset of calcium flux across the intestine of a vitamin D replete chick perfused with 1,25(OH)2D (175). This increased flux could not be blocked with actinomycin D pretreatment (176), but was blocked by voltage gated L type channel inhibitors (177) and protein kinase C inhibitors (178). These animals had to be vitamin D replete and contain the VDR, indicating that the basic machinery for calcium transport was intact. On the other hand L type channel activators such as BAY K-8644 (179) and protein kinase C activators such as phorbol esters (177) could activate transcaltachia similar to 1,25(OH)2D.

Figure 6. Model for the non-genomic actions of 1,25(OH)2D. 1,25(OH)2D binds to a putative membrane receptor. This leads to activation of a G protein (GTP displacement of GDP and dissociation of the β and γ subunits from the now active α subunit). Gα -GTP activates phospholipase C (PLC) (β or γ) to hydrolyze phosphatidyl inositol bis phosphate (PIP2) to inositol tris phosphate (IP3) and diacyl glycerol (DG). IP3 releases calcium from intracellular stores via the IP3 receptor in the endoplasmic reticulum; DG activates protein kinase C (PKC). Both calcium and PKC may regulate the influx of calcium across the plasma membrane through various calcium channels including L-type calcium channels.  

A putative membrane receptor for 1,25(OH)2D (1,25(OH)2D membrane associated rapid response steroid binding protein (1,25D-MARRSBP) also known as ERp57) has been purified from the intestine (180) and subsequently cloned and sequenced (181). Its size is approximately 66kDa. Antibodies have been made against this putative receptor (182). These antibodies block the ability of 1,25(OH)2D to stimulate calcium uptake by isolated chick intestinal cells (183) and to stimulate protein kinase C activity in resting zone chondrocytes while inhibiting proliferation of both resting zone and proliferating zone chondrocytes (182). Analog studies also support the existence of a separate membrane receptor for 1,25(OH)2D. Because of the breaking of the B ring during vitamin D3 production from 7-dehydrocholesterol, the A ring can assume a conformation similar to the parent cholesterol molecule (6-s-cis) (shown as previtamin D3 in figure 1) or the more commonly depicted 6-s-trans form in which the A ring rotates away from the rest of the molecule (shown as vitamin D3 in figure 1). Analogs of 1,25(OH)2D can be produced which favor the 6-s-cis conformation or the 6-s-trans conformation. 1,25(OH)2-d5-previtamin D3 is one such analog locked into the 6-s-cis conformation. This analog has only weak activity with respect to VDR binding or transcriptional activation but is fully effective in terms of stimulating transcaltachia and calcium uptake by osteosarcoma cells when compared to 1,25(OH)2D (184). 6-s-trans analogs are not effective. However, some of these rapid actions of 1,25(OH)2D are not found in cells from VDR null mice suggesting that the VDR may be required for the expression and/or function of the membrane receptor or be the membrane receptor. In other cells both 1,25D-MARRSBP and VDR appear to be required for these rapid effects of 1,25(OH)2D (185,186).

The model (figure 6) emerging from these studies is that 1,25(OH)2D interacts with a membrane receptor to activate phospholipase C possibly through a G protein coupled process. Phospholipase C then hydrolyzes phosphatidyl inositol bis phosphate (PIP2) in the membrane releasing inositol tris phosphate (IP3) and diacyl glycerol (DG). These second messengers may then activate both the intracellular release of calcium from intracellular stores via the IP3 receptor and protein kinase C, either one or both of which could stimulate calcium channel activity leading to a further rise in intracellular calcium levels. In the intestine and kidney, the increased flux of calcium across the brush border membrane is then transported out of the cell at the basolateral membrane, completing transcellular transport. In other cells the increased calcium would need to be removed by other mechanisms after the signal conveyed by the rise in calcium is no longer required. Much work remains to prove this model including the physiologic requirement for a unique membrane receptor.

TARGET TISSUE RESPONSES: CALCIUM REGULATING ORGANS 

Intestine

Intestinal calcium absorption, in particular the active component of transcellular calcium absorption, is one of the oldest and best known actions of vitamin D having been first described in vitro by Schachter and Rosen (187) in 1959 and in vivo by Wasserman et al. (188) in 1961. Absorption of calcium from the luminal contents of the intestine involves both transcellular and paracellular pathways. The transcellular pathway dominates in the duodenum and cecum, and this is the pathway primarily regulated by 1,25 dihydroxyvitamin D (1,25(OH)2D) (189), although elements of the paracellular pathway such as the claudins 2 and 12 are likewise regulated by 1,25(OH)2D (reviews in (190,191). Figure 7 shows a model of our current understanding of how this process is regulated by 1,25(OH)2D. Calcium entry across the brush border membrane (BBM) occurs down a steep electrical-chemical gradient and requires no input of energy. Removal of calcium at the basolateral membrane must work against this gradient, and energy is required. This is achieved by the CaATPase (PMCA1b), an enzyme induced by 1,25(OH)2D in the intestine. Calcium movement through the cell occurs with minimal elevation of the intracellular free calcium concentration (192) by packaging the calcium in calbindin containing vesicles (193-195) that form in the terminal web following 1,25(OH)2D administration.

Figure 7. Model of intestinal calcium transport. Calcium enters the microvillus of the intestinal epithelial cell through TRPV6 (previously known as CaT1) calcium channel. Within the microvillus calcium is bound to calmodulin (CaM) which is itself bound to brush border myosin I (BBMI). BBMI may facilitate the movement of the calcium/CaM complex into the terminal web where the calcium is picked up by calbindin (CaBP) and transported through the cytoplasm in endocytic vesicles. At the basolateral membrane the calcium is pumped out of the cell by the Ca-ATPase (PMCA1b). 1,25(OH)2D enhances intestinal calcium transport by inducing TRPV6, CaBP, and PMCAb as well as increasing the amount of CaM bound to BBMI in the brush border.  

1,25(OH)2D regulates transcellular calcium transport using a combination of genomic and nongenomic actions. The first step, calcium entry across the BBM, is accompanied by changes in the lipid composition of the membrane including an increase in linoleic and arachidonic acid (196,197) and an increase in the phosphatidylcholine:phosphatidylethanolamine ratio (198). These changes are associated with increased membrane fluidity (197), which we have shown results in increased calcium flux (199). The changes in lipid composition occur within hours after 1,25(OH)2D administration and are not blocked by pretreatment with cycloheximide (198). In addition, an epithelial specific calcium channel, TRPV6, is expressed in the intestinal epithelium (200). This channel has a high degree of homology to TRPV5, a channel originally identified in the kidney (201,202). The tissue distributions of these channels are overlapping and can be found in other tissues, but TRPV6 appears to be the main form in the intestine (203,204). TRPV6 mRNA levels in the intestine of vitamin D deficient mice are markedly increased by 1,25(OH)2D, although similar changes are not found in the kidney (205).  Mice null for TRPV6 have decreased intestinal calcium transport (206).

Calcium entering the brush border must then be moved into and through the cytoplasm without disrupting the function of the cell. Electron microscopic observations indicate that in the vitamin D deficient animal, calcium accumulates along the inner surface of the plasma membrane of the microvilli (207,208). Following vitamin D or 1,25(OH)2D administration calcium leaves the microvilli and subsequently can be found in mitochondria and vesicles within the terminal web (193,194,207,208). The vesicles appear to shuttle the calcium to the lateral membrane where it is pumped out of the cell by the basolateral CaATPase, PMCA1b. These morphologic observations have been confirmed by direct measurements of calcium using x-ray microanalysis that demonstrate equivalent amounts of calcium within the microvilli of D deficient and 1,25(OH)2D treated animals but much higher amounts of calcium in the mitochondria and vesicles of the 1,25(OH)2D treated animals (194,209). Such data suggest that 1,25(OH)2D controls calcium entry into the cell primarily by regulating its removal from the microvillus and accumulation by subcellular organelles in the terminal web, although flux through calcium channels in the membrane such as TRPV6 also plays a major role.

The ability of 1,25(OH)2D to stimulate calcium entry into and transport from the microvillus does not require new protein synthesis (193,198,210). Cycloheximide does not block the ability of 1,25(OH)2D to increase the capacity of brush border membrane vesicles (BBMV) to accumulate calcium, although it does block the increase in alkaline phosphatase in the same BBMV [193]. Likewise, cycloheximide does not block the increase in mitochondrial calcium following 1,25(OH)2D administration, although it blocks the rise in calbindin and prevents the normal vesicular transport of calcium through the cytosol (193,211). Thus, nongenomic actions underlie at least some of these first steps in 1,25(OH)2D stimulated intestinal calcium transport within the microvillus, although the changes take hours, not minutes, to observe. The exact role for these nongenomic effects on calcium influx relative to the role of TRPV6 remains to be elucidated.

Calmodulin is the major calcium binding protein in the microvillus (212). Its concentration in the microvillus is increased by 1,25(OH)2D; no new calmodulin synthesis is required or observed after 1,25(OH)2D administration (213). Calmodulin is likely to play a major role in calcium transport within the microvillus, and inhibitors of calmodulin block 1,25(OH)2D stimulated calcium uptake by BBMV (214). Within the microvillus calmodulin is bound to a 110kD protein, myosin 1A (myo1A)) (previously referred to as brush border myosin 1). 1,25(OH)2D increases the binding of calmodulin to myo1A in brush border membrane preparations (213), although binding of calmodulin to the myo1A attached to the actin core following detergent extraction of the membrane appears to be reduced (215). The calmodulin/myo1A complex appears late in the development of the brush border, and is found in highest concentration in the same cells of the villus which have the highest capacity for calcium transport (216). Myo1A is located primarily in the microvillus of the mature intestinal epithelial cell, although small amounts have been detected associated with vesicles in the terminal web (217). Thus, the calmodulin/myo1A complex may be responsible for moving calcium out of the microvillus. Its exact role in calcium transport is unclear in that mice null for myo1A do not show reduced intestinal calcium transport(218)).  Calbindin is the dominant calcium binding protein in the cytoplasm (212,219), where it appears to play the major role in calcium transport from the terminal web to the basolateral membrane (190). The increase in calbindin levels in the cytosol following 1,25(OH)2D administration is blocked by protein synthesis inhibitors (210). Indeed, calbindin was the first protein discovered to be induced by vitamin D (219). Glenney and Glenney (212) observed that calbindin has a higher affinity for calcium than does calmodulin. The differential distribution of calmodulin and calbindin between microvillus and cytosol combined with the differences in affinity for calcium led Glenney and Glenney (212) to propose that in the course of calcium transport calcium flowed from calmodulin in the microvillus to calbindin in the cytosol with minimal change in the free calcium concentration in either location. However, the role of calbindin in intestinal calcium transport does not appear to be critical in that mice null for calbindin9k grow normally, have normal intestinal calcium transport, and their serum calcium levels and bone mineral content are equivalent to wildtype mice regardless of the calcium content of the diet (220). The CaATPase (PMCA1b) at the basolateral membrane and the sodium/calcium exchanger (NCX1) are responsible for removing calcium from the cell against the same steep electrochemical gradient as favored calcium entry at the brush border membrane (221). Related proteins are found in the renal distal tubule. As its name implies, the extrusion of calcium from the cell by the calcium pump requires ATP. This pump is a member of the PMCA family, and in the intestine the isoform PMCA1b is the major isoform found. This pump is induced by 1,25(OH)2D (222). Calmodulin activates the pump, but calbindin may do likewise (223). Deletion of Pmca1b reduces calcium absorption and blocks 1,25(OH)2D stimulation of such resulting in reduction in growth and bone mineralization (224)., Moreover, the deletion of protein 4.1R, which regulates PMCA1b expression in the intestine, results in decreased intestinal calcium transport (225). The role of NCX is not considered to be as important as PMCA1b for intestinal calcium transport (226).

The paracellular pathway has received less study, but accounts for the bulk of intestinal calcium transport in that the ileum accounts for around 80% of total calcium absorption essentially all by the paracellular pathway. Paracellular calcium absorption depends to a considerable extent on the gradient between the luminal calcium concentrations and the interstitial calcium concentrations. Thus, it is faster in the duodenum and upper jejunum than the ileum, but because the transit time in the ileum is so much longer than that of the upper GI tract, the ileum is where most of the calcium absorption takes place. Solvent drag plays a large part in moving calcium across the tight junctions between the epithelial cells (227) . Solvent flow follows the osmotic gradient which is maintained distal to the tight junction by the Na/K ATPase and sodium glucose cotransporter of the basolateral membrane which may be stimulated by 1,25(OH)2D (226,227). The tight junction itself provides both charge and size selectivity. The actomyosin ring around the tight junction contributes to the size selectivity (228). The claudins and occludins contribute to charge selectivity. Claudin 2, 12, 15 are negatively charged proteins enabling cations such as sodium and calcium to pass (229,230). 1,25(OH)2D stimulates the expression of claudins 2 and 12 (231). Prolactin stimulates claudin 15 expression, thought to contribute to the increased calcium absorption during pregnancy (232).

Although less studied, intestinal phosphate transport is also under the control of vitamin D. This was first demonstrated by Harrison and Harrison (233) in 1961. Active phosphate transport is greatest in the jejunum, in contrast to active calcium transport that is greatest in the duodenum. Cycloheximide blocks 1,25(OH)2D stimulated phosphate transport (234), indicating that protein synthesis is involved. Phosphate transport at both the brush border and basolateral membranes requires sodium. A sodium-phosphate transporter in the small intestine (NaPi-IIb), homologous to the type IIa sodium phosphate transporter in kidney, has been cloned and sequenced (235). Expression of NaPi-IIb is increased by 1,25(OH)2D (236). Transport of phosphate through the cytosol from one membrane to the other is poorly understood. However, cytochalasin B, a disrupter of microfilaments, has been shown to disrupt this process (237) suggesting that as for calcium, intracellular phosphate transport occurs in vesicles.

 Bone

Nutritional vitamin D deficiency, altered vitamin D responsiveness such as vitamin D receptor mutations (hereditary vitamin D resistant rickets), and deficient production of 1,25(OH)2D such as mutations in the CYP27B1 gene (pseudo vitamin D deficiency) all have rickets as their main phenotype. This would suggest that vitamin D, and in particular 1,25(OH)2D, is of critical importance to bone. Furthermore, VDR are found in bone cells (238,239), and vitamin D metabolites have been shown to regulate many processes in bone. However, the rickets resulting from vitamin D deficiency or VDR mutations (or knockouts) can be corrected by supplying adequate amounts of calcium and phosphate either by infusions or orally [214-217]. Moreover, deletion of VDR from bone cells does not result in rickets (240). This would suggest either that vitamin D metabolites do not directly impact bone, or that substantial redundancy has been built into the system.  However, arguing for a physiologically non-redundant direct action of vitamin D on bone is the development of osteoporosis and decreased bone formation in these VDR or CYP27B1 null mice not corrected by the high calcium/phosphate diet (241).  A further complicating factor in determining the role of vitamin D metabolites in bone is the multitude of effects these metabolites have on systemic calcium homeostatic mechanisms which themselves impact on bone. The lack of vitamin D results in hypocalcemia and hypophosphatemia that as implied above is sufficient to cause rickets. Moreover, part of the skeletal phenotype in vitamin D deficiency is also due to the hyperparathyroidism that develops in the vitamin D deficient state as PTH has its own actions on bone and cartilage. Furthermore, within bone the vitamin D metabolites can alter the expression and/or secretion of a large number of skeletally derived factors including insulin like growth factor-1 (IGF-I) (242), its receptor (243), and binding proteins (244,245), transforming growth factor β (TGFβ) (246), vascular endothelial growth factor (VEGF) (247), interleukin-6 (IL-6) (248), IL-4 (249), and endothelin receptors (250) all of which can exert effects on bone of their own as well as modulate the actions of the vitamin D metabolites on bone. Understanding the impact of vitamin D metabolites on bone is additionally complicated by species differences, differences in responsiveness of bone and cartilage cells according to their states of differentiation, and differences in responsiveness in terms of the vitamin D metabolite being examined. Thus, the study of vitamin D on bone has had a complex history, and uncertainty remains as to how critical the direct actions of the vitamin D metabolites on bone are for bone formation and resorption.

Bone develops intramembranously (e.g., skull) or from cartilage (endochondral bone formation, e.g., long bones with growth plates). Intramembranous bone formation occurs when osteoprogenitor cells proliferate and produce osteoid, a type I collagen rich matrix. The osteoprogenitor cells differentiate into osteoblasts which then deposit calcium phosphate crystals into the matrix to produce woven bone. This bone is remodeled into mature lamellar bone. Endochondral bone formation is initiated by the differentiation of mesenchymal stem cells into chondroblasts that produce the proteoglycan rich type II collagen matrix. These cells continue to differentiate into hypertrophic chondrocytes that shift from making type II collagen to producing type X collagen. These cells also initiate the degradation and calcification of the matrix by secreting matrix vesicles filled with degradative enzymes such as metalloproteinases and phospholipases, alkaline phosphatase (thought to be critical for the mineralization process), and calcium phosphate crystals. Vascular invasion and osteoclastic resorption are stimulated by the production of VEGF and other chemotactic factors from the degraded matrix. The hypertrophic chondrocytes also begin to produce markers of osteoblasts such as osteocalcin, osteopontin, and type I collagen resulting in the initial deposition of osteoid. Moreover, at least some of these chondrocytes further differentiate (or trans differentiate) into osteoblasts (251). Terminal differentiation of the hypertrophic chondrocytes and the subsequent calcification of the matrix are markedly impaired in vitamin D deficiency leading to the flaring of the ends of the long bones and the rachitic rosary along the costochondral junctions of the ribs, classic features of rickets. Although supply of adequate amounts of calcium and phosphate may correct most of these defects in terminal differentiation and calcification, the vitamin D metabolites, 1,25(OH)2D and 24,25(OH)2D, have been shown to exert distinct roles in the process of endochondral bone formation.

The VDR makes its first appearance in the fetal rat at day 13 of gestation in the condensing mesenchyme of the vertebral column then subsequently in osteoblasts and the proliferating and hypertrophic chondrocytes by day 17 (252). However, fetal development is quite normal in vitamin D deficient rats (253) and VDR knockout mice (126) suggesting that vitamin D and the VDR are not critical for skeletal formation. Rickets develops postnatally, becoming most manifest after weaning. The impairment of endochondral bone formation observed in vitamin D deficiency is associated with decreased alkaline phosphatase activity of the hypertrophic chondrocytes (254), alterations in the lipid composition of the matrix (255) perhaps secondary to reduced phospholipase activity (256), and altered proteoglycan degradation (257) due to changes in metalloproteinase activity (257,258). Both 1,25(OH)2D and 24,25(OH)2D appear to be required for optimal endochondral bone formation (259). However, in the CYP24A1 knockout mouse, that fails to produce any 24-hydroxylated metabolites of vitamin D, the skeletal lesion is defective mineralization of intramembranous (not endochondral) bone. Furthermore, the skeletal abnormality appears to be due to high circulating 1,25(OH)2D levels in that crossing this mouse with one lacking the VDR corrects the problem (85). Whether this reflects species differences between mice and other species (most studies demonstrating the role of 24,25(OH)2D in bone and cartilage have used rats and chicks) remains unknown. Chondrocytes from the resting zone of the growth plate of rats tend to be more responsive to 24,25(OH)2D than 1,25(OH)2D, whereas the reverse is true for chondrocytes from the growth zone with respect to stimulation of alkaline phosphatase activity (260), regulation of phospholipase A2 (stimulation by 1,25(OH)2D, inhibition by 24,25(OH)2D) (261), changes in membrane fluidity (increased by 1,25(OH)2D, decreased by 24,25(OH)2D) (262), and stimulation of protein kinase C activity (263). These actions of 1,25(OH)2D and 24,25(OH)2D do not require the VDR and are non-genomic in that they take place with isolated matrix vesicles and membrane preparations from these cells (260). As discussed earlier membrane receptors for these vitamin D metabolites have been found in chondrocytes that may mediate these non-genomic actions (264). Osteoblasts also differ in their response to 1,25(OH)2D depending on their degree of maturation (265). In the latter stages of differentiation, rat osteoblasts respond to 1,25(OH)2D with an increase in osteocalcin production (266), but do not respond to 1,25(OH)2D in the early stages. Mice, however, differ from rats in that 1,25(OH)2D inhibits osteocalcin expression (266). Similarly, the effects of 1,25(OH)2D on alkaline phosphatase (267) and type I collagen (268) are inhibitory in the early stages of osteoblast differentiation but stimulatory in the latter stages (265). Osteopontin is better stimulated by 1,25(OH)2D in the early stages than the late stages of differentiation (265,269). Osteocalcin and osteopontin in human and rat cells have well described VDREs in their promoters (270-272) (the mouse does not) (273). However, alkaline phosphatase and the COL1A1 and COL1A2 genes producing type I collagen do not have clearly defined VDREs, so it remains unclear how these genes are regulated by 1,25(OH)2D. These maturation dependent effects of 1,25(OH)2D on bone cell function may explain the surprising ability of excess 1,25(OH)2D to block mineralization leading to hyperosteoidosis (274,275) as such doses may prevent the normal maturation of osteoblasts.

In addition to its role in promoting bone formation, 1,25(OH)2D also promotes bone resorption by increasing the number and activity of osteoclasts (276). Whether mature osteoclasts contain the VDR and are regulated directly by 1,25(OH)2D remains controversial (277,278), but the VDR in osteoclast precursors is not required for osteoclastogenesis. Rather, the stimulation of osteoclastogenesis by 1,25(OH)2D is mediated by osteoblasts. Rodan and Martin (279) originally proposed the hypothesis that osteoblasts were required for osteoclastogenesis, and the mechanism has now been elucidated (280). Osteoblasts produce a membrane associated protein known as RANKL (receptor activator of nuclear factor (NF)-kB ligand) that activates RANK on osteoclasts and their hematopoietic precursors. This cell-to-cell contact in combination with m-CSF also produced by osteoblasts stimulates the differentiation of precursors to osteoclasts, and promotes their activity. 1,25(OH)2D regulates this process by inducing RANKL (281) as does PTH, PGE2, and IL-11, all of which stimulate osteoclastogenesis. 1,25(OH)2D requires the VDR in osteoblasts for this purpose, although the other hormones and cytokines do not. Osteoblasts from Vdr knockout mice fail to support 1,25(OH)2D induced osteoclastogenesis, whereas osteoclast precursors from Vdr knockout mice can be induced by 1,25(OH)2D to form osteoclasts in the presence of osteoblasts from wildtype animals (282). 

Kidney

The regulation of calcium and phosphate transport by vitamin D metabolites in the kidney has received less study than that in the intestine, but the two tissues have similar although not identical mechanisms. Eight grams of calcium are filtered by the glomerulus each day, and 98% of that is reabsorbed. Most is reabsorbed in the proximal tubule. This is a paracellular, sodium dependent process with little or no regulation by PTH and 1,25(OH)2D. Approximately 20% of calcium is reabsorbed in the thick ascending limb of the loop of Henle, 10-15% in the distal tubule, and 5% in the collecting duct (283). Regulation by vitamin D takes place in the distal tubule where calcium moves against an electrochemical gradient (presumably transcellular) in a sodium independent fashion (284). Phosphate, on the other hand, is approximately 80% reabsorbed in the proximal tubule, and this process is regulated by PTH (285). In parathyroidectomized (PTX) animals Puschett et al. (286-288)) demonstrated acute effects of 25OHD and 1,25(OH)2D on calcium and phosphate reabsorption. Subsequent studies indicated that PTH could enhance or was required for the stimulation of calcium and phosphate reabsorption by vitamin D metabolites (289,290).

The molecules critical for calcium reabsorption in the distal tubule appear to be the VDR, calbindin, TRPV5, and the BLM calcium pump (PMCA1b as in the intestine), a situation similar to the mechanism for calcium transport in the intestine. However, the calbindin in the kidney in most species is 28kDa, whereas the 9kDa form is found in the intestine in most species. The kidney has mostly TRPV5, whereas the intestine is primarily TRPV6. The calcium pump is the same isoform in both tissues (PMCA1b) although other forms of PMCA are also present. Calmodulin and a brush border myosin I like protein are also found in the kidney brush border, but their role in renal calcium transport has not been explored. VDR, calbindin, TRPV5, and PMCA1b colocalize in the distal tubule, but not all distal tubules contain this collection of proteins (201,202,291,292) suggesting that not all distal tubules are involved in calcium transport. 1,25(OH)2D upregulates the VDR (234), an action opposed by PTH (237). Calbindin is also induced by 1,25(OH)2D in the kidney(293,294). The activity of the calcium pump is increased by 1,25(OH)2D (295), but it is not clear that the protein itself is induced. The increased activity may be due to the induction of calbindin that increases its activity. The effect of 1,25(OH)2D on TRPV5 expression is stimulatory (205).

Phosphate reabsorption in the proximal tubule is mediated at the brush border by sodium dependent phosphate transporters (NaPi-2a and NaPi-2c) that rely on the baso-lateral membrane Na,K ATPase to maintain the sodium gradient that drives the transport process (296). It is not clear whether 1,25(OH)2D regulates the expression or activities of these transporters as it does in the intestine, although PTH clearly does. Like PTH, FGF23 blocks phosphate reabsorption, presumably by blocking NaPi-2a activity. Unlike PTH, FGF23 also blocks the renal production of 1,25(OH)2D, as discussed earlier.  The link between phosphate reabsorption and 1,25(OH)2D production remains unclear.

TARGET TISSUE RESPONSES: NON-CALCIUM TRANSPORTING TISSUES

In addition to the its effects on tissues directly responsible for calcium homeostasis, 1,25(OH)2D regulates the function of a wide number of other tissues. These all contain the VDR. Regulation of differentiation and proliferation is one common theme; regulation of hormone secretion is another; regulation of immune function is the third. In most cases 1,25(OH)2D acts in conjunction with calcium. Selected examples follow.

Regulation of Hormone Secretion

PARATHYROID GLAND (PTH SECRETION)

As previously mentioned, PTH stimulates the production of 1,25(OH)2D. In turn 1,25(OH)2D inhibits the production of PTH (297,298). The regulation occurs at the transcriptional level. Within the promoter of the PTH gene is a region that binds the VDR and mediates the suppression of the PTH promoter by 1,25(OH)2D (162,293,299-303). However, there is substantial controversy about whether this site is a single half site (299) or a more classic DR3 (292), whether one VDRE is involved or two (300), whether only VDR binds (299,303), whether VDR/RXR heterodimers bind (162,300), or whether VDR partners with a different protein (301). Some of the differences may reflect different species, but the nature of PTH gene suppression by 1,25(OH)2D remains incompletely understood. Calcium alters the ability of 1,25(OH)2D to regulate PTH gene expression. Calcium is a potent inhibitor of PTH production and secretion, acting through the calcium sensing receptor (CaSR) on the plasma membrane of the parathyroid cell. 1,25(OH)2D induces the CaSR in the parathyroid gland making it more sensitive to calcium (304). Animals placed on a low calcium diet have an increase in PTH and 1,25(OH)2D levels indicating that the low calcium overrides the inhibition by 1,25(OH)2D on PTH secretion (305,306). One possible explanation involves the protein calreticulin that binds to nuclear hormone receptors including VDR at KXGFFKR sequences, and inhibits their activity (307,308). Low dietary calcium has been shown to increase calreticulin levels in the parathyroid gland (309). The ability of 1,25(OH)2D to inhibit PTH production and secretion has been exploited clinically in that 1,25(OH)2D and several of its analogs are used to prevent and/or treat secondary hyperparathyroidism associated with renal failure. The parathyroid gland also expresses CYP27B1 and so can produce its own 1,25(OH)2D that may act in an autocrine or paracrine fashion to regulate PTH production (310). As noted earlier, the parathyroid gland is one of several tissues expressing the megalin/cubilin complex potentially enabling it to take up 25OHD and other D metabolites still bound to DBP.

PANCREATIC BETA CELLS (INSULIN SECRETION) 

1,25(OH)2D stimulates insulin secretion, although the mechanism is not well defined (311,312). VDR, CYP27B1 and calbindin-D28k are found in pancreatic beta cells (313-315), and  studies using calbindin-D28k null mice have suggested that calbindin-D28k, by regulating intracellular calcium, can modulate depolarization-stimulated insulin release (316).  Furthermore, calbindin-D28k, by buffering calcium, can protect against cytokine mediated destruction of beta cells (317).  A number of mostly case control and observational studies have suggested that vitamin D deficiency contributes to increased risk for type 2 diabetes mellitus (318). Moreover, several randomized clinical trials evaluating the ability of vitamin D supplementation to prevent the progression of prediabetes to diabetes indicate that vitamin D has a modest protective effect especially in vitamin D deficient subjects (319,320).

FIBROBLAST GROWTH FACTOR (FGF23)

 FGF23 is produced primarily by bone, and in particular by osteoblasts and osteocytes. 1,25(OH)2D3 stimulates this process, but the mechanism is not clear (322). Inasmuch as FGF23 inhibits 1,25(OH)2D production by the kidney, this feedback loop like that for PTH secretion maintains a balance in the levels of these important hormones. Mutations in the Phosphate regulating gene with Homologies to Endopeptidases on the X chromosome (PHEX) or FGF23 itself (which prevent its proteolysis) or conditions such as McCune-Albright disease and tumor induced osteomalacia in which FGF23 is overexpressed in the involved tissue led to hypophosphatemia and inappropriately low 1,25(OH)2D accompanied by osteomalacia. The role of PHEX, which was originally thought to cleave FGF23, in regulating FGF23 levels is not clear.  In contrast mutations in UDP-N-acetyl-α-D galactosamine:polypeptide N-acetylgalactosaminyltransferase (GALNT3), which glycosylates FGF23, or in FGF23 which blocks this glycosylation result in inhibited FGF23 secretion leading to hyperphosphatemia, increased 1,25(OH)2D, and tumoral calcinosis (323).

Regulation of Proliferation and Differentiation 

CANCER

 1,25(OH)2D has been evaluated for its potential anticancer activity in animal and cell studies for nearly 40 years (324). The list of malignant cells that express VDR is now quite extensive, and the list of those same cells that express CYP27B1 is growing. The accepted basis for the promise of 1,25(OH)2D in the prevention and treatment of malignancy includes its antiproliferative, pro-differentiating effects on most cell types. The list of mechanisms proposed for these actions is extensive, and to some extent cell specific (325). Among these mechanisms 1,25(OH)2D has been shown to stimulate the expression of cell cycle inhibitors p21 and p27 (326) and the expression of the cell adhesion molecule E-cadherin (150), while inhibiting the transcriptional activity of β-catenin (150,327,328). In keratinocytes, 1,25(OH)2D has been shown to promote the repair of DNA damage induced by ultraviolet radiation (UVR) (329) (330), reduce apoptosis while increasing survival after UVR (331), and increase p53 (332).  Epidemiologic evidence supporting the importance of adequate vitamin D nutrition (including sunlight exposure) for the prevention of a number of cancers (333-337) is extensive. Although numerous types of cancers show reduction (338), most attention has been paid to cancers of the breast, colon, and prostate. I (339) recently reviewed a number of meta-analyses of epidemiologic studies evaluating the association of vitamin D intake and/or 25OHD levels and the risk of developing these cancers.  The data supporting a reduction in risk for developing colorectal cancer and breast cancer in premenopausal females with higher vitamin D intake or higher serum 25OHD levels were considerably stronger than that for the prevention of prostate cancer. Prospective randomized controlled trial data are limited. In a prospective 4 yr. trial with 1100iu vitamin D and 1400-1500 mg calcium originally designed to look at osteoporosis the authors showed a 77% reduction in cancers after excluding the initial year of study (340), including a reduction in both breast and colon cancers. In this study, vitamin D supplementation raised the 25OHD levels from a mean of 28.8ng/ml to 38.4ng/ml with no changes in the placebo or calcium only arms of the study. However, this was a relatively small study in which cancer prevention was not the primary outcome variable. A substantially larger trial involving over 25,000 subjects treated in a two by two design with vitamin D and/or omega 3 fatty acid did not find a benefit of vitamin supplementation with respect to cancer incidence but appears to have shown a beneficial effect on mortality (341). Trials of 1,25(OH)2D and its analogs for the treatment of cancer have been disappointing. In a small study involving 7 subjects with prostate cancer treated with doses of 1,25(OH)2D up to 2.5µg for 6-15 months, 6/7 showed a decrease in the rise of prostate specific antigen (PSA), a marker of tumor progression (342), and one patient showed a decline. However, hypercalciuria was common and limiting. A preliminary report of a larger study involving 250 patients with prostate cancer using 45µg 1,25(OH)2D  weekly in combination with docetaxel demonstrated a non-significant decline in PSA, although survival was significantly improved (HR 0.67) (343). A larger follow-up study did not show increased survival (344).  The incidence of either hypercalcemia or hypercalciuria was not reported. Most likely until an analog of 1,25(OH)2D is developed which is both efficacious and truly non hypercalcemic, treatment of cancer with vitamin D metabolites will remain problematic.

SKIN 

 Epidermal keratinocytes are the only cells in the body with the entire vitamin D metabolic pathway. As described earlier, production of vitamin D3 from 7-dehydrocholesterol takes place in the epidermis. However, the epidermis also contains CYP27A1 (345), the mitochondrial enzyme that 25-hydroxylates vitamin D, and CYP27B1 (40,47), the enzyme that produces 1,25(OH)2D from 25OHD. The CYP27B1 in keratinocytes is differently regulated than CYP27B1 in renal cells. Although PTH stimulates CYP27B1 activity in the keratinocyte, the mechanism appears to be independent of cAMP (346). Cytokines such as tumor necrosis factor-α and interferon-γ stimulate CYP27B1 activity (347,348). 1,25(OH)2D does not exert a direct effect on CYP27B1 expression in keratinocytes, but regulates 1,25(OH)2D levels by inducing CYP24A1 thus initiating the catabolism of 1,25(OH)2D (79). CYP27B1 is expressed primarily in the basal cells of the epidermis (50); as the cells differentiate the mRNA and protein levels of CYP27B1and its activity decline (349).

1,25(OH)2D regulates keratinocyte differentiation in part by modulating the ability of calcium to do likewise (350). Therefore, it is important to understand the actions of calcium on this cell prior to examining the influence of 1,25(OH)2D (351-356)(357). If keratinocytes are grown at calcium concentrations below 0.07mM, they continue to proliferate but either fail or are slow to develop intercellular contacts, stratify little if at all, and fail or are slow to form cornified envelopes. Acutely increasing the extracellular calcium concentration (Cao) above 0.1mM (calcium switch) leads to the rapid redistribution of desmoplakin, cadherins, integrins, catenins, plakoglobulin, vinculin, and actinin from the cytosol to the membrane where they participate in the formation of intercellular contacts. Calcium also stimulates the redistribution to the membrane of protein kinase Cα (PKCα) (358,359) and the tyrosine-phosphorylated p62 associated protein of ras GAP (360,361) where they further the calcium signaling process. These early events are accompanied by a rearrangement of actin filaments from a perinuclear to a radial pattern which if disrupted blocks the redistribution of these proteins and blocks the differentiation process. Within hours of the calcium switch keratinocytes switch from making the basal keratins K5 and K14 and begin making keratins K1 and K10 (356) followed, subsequently, by increased levels of profilaggrin (the precursor of filaggrin, an intermediate filament associated protein), involucrin and loricrin (precursors for the cornified envelope) (362,363). Loricrin, involucrin and other proteins (364) are cross linked into the insoluble cornified envelope by the calcium sensitive, membrane bound form of transglutaminase (365,366), which like involucrin and loricrin increases within 24 hours after the calcium switch (367). Within 1-2 days of the calcium switch cornified envelope formation is apparent (355,368), paralleling transglutaminase activation (369). The induction of these proteins represents a genomic action (likely indirect) of calcium as indicated by a calcium induced increase in mRNA levels and transcription rates (356,363,369,370). The relevance of calcium induced differentiation in vitro to the in vivo situation is indicated by the steep gradient of calcium within the epidermis, with the highest levels in the uppermost (most differentiated) nucleated layers (371). Current evidence for the importance of calcium in epidermal function is that barrier disruption, which results in increased proliferation, is associated with loss of the calcium gradient, whereas increasing the calcium concentration in the epidermis with sonophoresis stimulates lamellar body secretion (372-376).

The keratinocyte senses calcium via a seven transmembrane domain, G protein coupled receptor (CaSR) (377) originally cloned from the parathyroid cell by Brown et al (378,379). Knocking out the CaSR blocks calcium induced differentiation in vitro (380,381) and in vivo (382). However, keratinocytes also produce an alternatively spliced variant of the CaSR as they differentiate (383). This variant CaSR lacks exon 5 and so would be missing residues 461-537 in the extracellular domain. A mouse model in which the full length CaSR has been knocked out continues to produce the alternatively spliced form of CaSR, but its epidermis contains lower levels of the terminal differentiation markers loricrin and profilaggrin, and keratinocytes from these mice fail to respond normally to calcium (383) consistent with the results when the full length calcium receptor was deleted in vitro (380,381). We have produced a conditional knockout of the CaSR allowing us to delete CaSR in the tissue of choice using cell specific cre recombinases that avoids the problem with the original global knockout (384). When the CaSR is deleted specifically in the keratinocyte, this mouse has a reduction in epidermal differentiation and barrier repair (382), but unlike the global knockout does not have abnormalities in overall calcium homeostasis, and rather than showing an increased calcium gradient in the epidermis has a blunted one. The conditional knockout mouse also lacks the alternatively spliced CaSR.

Inositol 1,4,5 tris phosphate (IP3) and diacylglycerol levels increase within seconds to minutes after the calcium switch implicating activation of the phospholipase C (PLC) pathway (385,386). Similar to intracellular calcium levels (Cai), the levels of inositol phosphates (IPs) remain elevated for hours after the calcium switch. The prolonged increase in IPs after the calcium switch may contribute to the plateau phase of Cai elevation and a prolonged elevation of diacylglycerol (DG) that would stimulate the protein kinase C (PKC) pathway. This prolonged increase in IPs appears to be due to calcium induction and activation of PLC (154,386,387), especially PLC-γ1.  Activation of PLC-γ1 by calcium involves a chain of events involving src kinase activation of phosphatidyl inositol 3 kinase and phosphatidyl inositol 4 phosphate 5  kinase 1α within the context of a membrane complex with E-cadherin leading to the formation of phosphatidyl inositol tris phosphate in the membrane which activates PLC-γ1 via its PH domain (388).  Phosphorylation of PLC-γ1 is not part of its activation by calcium unlike its activation by EGF (389). Knocking out Plcg1 blocks the ability of calcium to increase Cai and to induce involucrin and transglutaminase (387). Thus, like CaSR, PLC-γ1 is critical for the ability of calcium to regulate keratinocyte differentiation.

Phorbol esters, which bind to and activate PKC, are well known tumor promoters in skin However, the initial effects of phorbol esters in vitro are to promote differentiation in cells grown in low calcium (358,390,391), effects which are potentiated by calcium (383). Phorbol esters stimulate PKC, and PKC inhibitors block the ability of both calcium and phorbol esters to promote differentiation (391). Phorbol esters as well as calcium stimulate the expression of both keratin 1 and involucrin gene constructs each of which contains an AP-1 site within the calcium response element (CaRE) of the promoter for these genes (392,393). If the AP-1 site within the CaRE is mutated, neither calcium nor phorbol esters are effective (392,393). These CaREs also contain VDREs (DR3), which at least in the involucrin gene has been shown to mediate 1,25(OH)2D regulation of this gene (394). Phorbol esters do not reproduce all the actions of calcium on the keratinocyte, and vice versa, but cross talk between their signaling pathways is clearly present.

The observation that 1,25(OH)2D induces keratinocyte differentiation was first made by Hosomi et al. (395) and provided a rationale for the previous and unexpected finding of 1,25(OH)2D receptors in the epidermis (396). 1,25(OH)2D increases the mRNA and protein levels for involucrin and transglutaminase, and promotes CE formation at subnanomolar concentrations in preconfluent keratinocytes (370,397-399). Calcium affects the ability of 1,25(OH)2D to stimulate keratinocyte differentiation, and vice versa. Calcium in the absence of 1,25(OH)2D and 1,25(OH)2D at low (0.03mM) calcium raise the mRNA levels for involucrin and transglutaminase in a dose dependent fashion by stimulating gene expression. The stimulation of mRNA levels by calcium and 1,25(OH)2D is synergistic at early time points; however, longer periods of incubation lead to a paradoxical fall in the mRNA levels for these proteins. This is due to the fact that although transcription is increased by calcium and 1,25(OH)2D, stability of the mRNA is reduced in cells incubated with calcium and 1,25(OH)2D.

The transcriptional regulation by 1,25(OH)2D is both direct and indirect. Several genes contain VDREs (e.g. involucrin), but VDREs have not been found in all genes that are regulated by 1,25(OH)2D. Inhibition of PKC activity or mutation of the AP-1 site in the CaRE of the involucrin gene also blocks the ability of 1,25(OH)2D to regulate expression of involucrin (394). The ability of 1,25(OH)2D to increase intracellular calcium (Cai) (298) accounts for at least part of the ability of 1,25(OH)2D to induce differentiation. A rapid (presumably nongenomic) effect of 1,25(OH)2D on Cai has been described (400), although this response is controversial (398). Our studies indicate that the ability of 1,25(OH)2D to increase Cai requires time and gene transcription. 1,25(OH)2D increases CaSR mRNA levels and prevents their fall in cells grown in 0.03mM calcium (401). This results in an enhanced Cai response to extracellular calcium (Cao). 1,25(OH)2D also induces the family of PLCs (402). PLC-γ1 contains a VDRE in its promoter (154), which unlike the usual VDRE is a DR6 which binds VDR/RAR rather than VDR/RXR. Knocking out PLCG1 blocks 1,25(OH)2D induced differentiation (403) as well as calcium induced differentiation mentioned earlier. The other PLCs have not been studied as extensively, but are likely to show similar means of regulation by 1,25(OH)2D.

Our current working model for the mechanisms by which calcium and 1,25(OH)2D regulate keratinocyte differentiation is shown in figure 8. The keratinocyte expresses a CaSR that by coupling to and activating PLC controls the production of two important second messengers, IP3 and DG. PLC-β is likely to be activated acutely by CaSR via a G protein coupled mechanism, whereas PLC-γ1 is activated acutely by calcium stimulated non receptor tyrosine kinases and subsequently by PIP3 in the membrane. Both PLCs are induced by calcium and 1,25(OH)2D. IP3 stimulates the release of calcium from intracellular stores thus raising Cai. The initial release of calcium from these stores activates the Stim1/Orai1 channel in the membrane (404) that may stimulate proliferation of the basal keratinocytes and initiate their movement out of the basal layer. The increase in Cai and DG stimulates the activation of critical PKCs and their translocation to membrane receptors (RACK). PKC-α appears to be the most critical PKC for the subsequent events triggered by calcium in the keratinocyte, although PKCδ has also been implicated.  Activated PKC leads to the induction and activation of AP-1 transcription factors which regulate the transcription of a number of genes including keratin 1, transglutaminase, involucrin, loricrin, and profilaggrin required for the differentiation process. Activation of the CaSR also activates the RhoA kinase leading to activation of src kinases which by phosphorylating various catenins leads to the formation of the Ecadherin/catenin complex in the membrane (405). This complex recruits both PI3K and PIP5K1α required to maintain the PIP2 and PIP3 levels in the membrane (357). PIP3 activates PLC-γ, that is in turn activates the TRPC channels in the membrane to enable the prolonged increase in Cai required for differentiation (406). 1,25(OH)2D, which is produced by the keratinocyte in a highly regulated fashion, modulates calcium regulated differentiation at several steps. First, 1,25(OH)2D increases CaSR expression, thus making the cell more responsive to calcium. Secondly, 1,25(OH)2D induces all the PLCs again increasing the responsiveness of the cell to calcium. Finally, 1,25(OH)2D has a direct effect on the transcription of the genes such as involucrin. The net result is that both calcium and 1,25(OH)2D promote keratinocyte differentiation through interactive mechanisms.

Figure 8. A model of 1,25(OH)2D and calcium regulated keratinocyte differentiation. The G-protein coupled calcium receptor (CaSR) when activated by extracellular calcium activates Gα as described in the legend to figure 6. Gα stimulates PLC mediated hydrolysis of PIP2 to IP3 and DG. IP3 releases Cai from intracellular stores, and DG activates PKC. Depletion of intracellular calcium stores leads to influx of calcium across store operated calcium channels. PKC stimulation leads to activation of AP-1 transcription factors which along with calcium and 1,25(OH)2D activated transcription factors stimulate the expression of genes essential for the differentiation process. 1,25(OH)2D regulates this process by inducing CaSR and PLC as well as genes essential for cornified envelope formation such as involucrin and transglutaminase.

The VDR is also critical for hair follicle (HF) cycling. Unlike epidermal differentiation, hair follicle cycling is not dependent on 1,25(OH)2D. Alopecia is a well described characteristic of mice and humans lacking VDR (125,126,407) due to failure to regenerate the cycling lower portion of the HF after the initial developmental cycle is completed. Deletion of CYP27B1 (408) and CaSR (382) do not result in alopecia. Cianferotti et al. (409) attributed the loss of HF cycling in VDR null mice to a gradual loss of the proliferative potential in the stem cells of the HF bulge region. However, this conclusion has been challenged by Palmer et al. (410), who attributed the failure of HF cycling in the VDR null mouse in part to a failure of the progeny of these stem cells to migrate out of the bulge rather than their loss of proliferative potential suggesting a loss of activation. The role of VDR in the stem cells that regulate both HF cycling and epidermal regeneration is also important in the skin wound healing process. When the skin is wounded the progeny of stem cells from all regions of the HF and interfollicular epidermis (IFE) contribute at least initially (411,412), although the stem cells in the IFE make the most lasting contribution. Tian et al. (413) observed that topical 1,25(OH)2D enhanced wound healing, suggesting that unlike HF cycling, the wound repair required this VDR ligand. Luderer et al. (414) observed that in the global VDRKO, there was a reduction in TGFβ signaling in the dermis, and subsequently demonstrated that the VDR in macrophages but not in keratinocytes was responsible for macrophage recruitment during the inflammatory phase of cutaneous wound healing (415). Our studies have focused on the VDR in epidermal keratinocytes.  We have observed that re-epithelialization by the keratinocytes over the wound is impaired when the deletion of VDR from keratinocytes is accompanied by either a low calcium diet or a deletion of the CaSR (416). Thus like the role of calcium and CaSR in vitamin D regulated keratinocyte differentiation so a similar synergism is seen in wound healing. These results are consistent with the loss of E-cadherin/catenin complex formation in the VDRKO keratinocyte, a complex that maintains stem cells in their niches (417), regulates when stem cell division is symmetric (to maintain stem cell numbers) or asymmetric (initiating differentiation) (418), and is essential for the ability of keratinocytes to migrate as a sheet to re-epithelialize the wound (419). As noted previously calcium and the CaSR along with 1,25(OH)2D and VDR are required for E-cadherin/catenin complex formation during the differentiation process and so are involved in enabling its role in wound healing (420).

Immune System

The potential role for vitamin D and its active metabolite 1,25(OH)2D3 in modulating the immune response has long been recognized since the discovery of vitamin D receptors (VDR) in macrophages, dendritic cells (DC), and activated T and B lymphocytes, the ability of macrophages and DC as well as activated T and B cells to express CYP27B1, and the ability of 1,25(OH)2D3 to regulate the proliferation and function of these cells. While these are the key cells mediating the adaptive immune response, 1,25(OH)2D, VDR, and CYP27B1 are also expressed in a large number of epithelial cells which along with the aforementioned members of the adaptive immune response contribute to host defense by their innate immune response. The totality of the immune response involves both types of responses in complex interactions involving numerous cytokines. The regulation of these different responses and their interactions by 1,25(OH)2D3 is nuanced. In general, 1,25(OH)2D3 enhances the innate immune response primarily via its ability to stimulate cathelicidin, an antimicrobial peptide important in defense against invading organisms, whereas it inhibits the adaptive immune response primarily by inhibiting the maturation of dendritic cells (DC) important for antigen presentation, reducing T cell proliferation, and shifting the balance of T cell differentiation from the Th1 and Th17 pathways to Th2 and Treg pathways. Inflammatory autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis involve Th17 activation, a cell that expresses RANKL, and so can drive osteoclastogenesis leading to bone loss.     

ADAPTIVE IMMUNE RESPONSE

The adaptive immune response is initiated by cells specialized in antigen presentation, DC and macrophages in particular, activating the cells responsible for subsequent antigen recognition, T and B lymphocytes. These cells are capable of a wide repertoire of responses that ultimately determine the nature and duration of the immune response. Activation of T and B cells occurs after a priming period in tissues of the body, e.g., lymph nodes, distant from the site of the initial exposure to the antigenic substance, and is marked by proliferation of the activated T and B cells accompanied by post translational modifications of immunoglobulin production that enable the cellular response to adapt specifically to the antigen presented. Importantly, the type of T cell activated, CD4 or CD8, or within the helper T cell class Th1, Th2, Th17, Treg, and subtle variations of those, is dependent on the context of the antigen presented by which cell and in what environment. Systemic factors such as vitamin D influence this process. Vitamin D in general exerts an inhibitory action on the adaptive immune system. 1,25(OH)2D3 decreases the maturation of DC as marked by inhibited expression of the costimulatory molecules HLA-DR, CD40, CD80, and CD86, decreasing their ability to present antigen and so activate T cells (421). Furthermore, by suppressing IL-12 production, important for Th1 development, and IL-23 and IL-6 production important for Th17 development and function, 1,25(OH)2D3 inhibits the development of Th1 cells capable of producing IFN- and IL-2, and Th17 cells producing IL-17 (422). These actions prevent further antigen presentation to and recruitment of T lymphocytes (role of IFN-γ), and T lymphocyte proliferation (role of IL-2).  Suppression of IL-12 increases the development of Th2 cells leading to increased IL-4, IL-5, and IL-13 production, which further suppresses Th1 development shifting the balance to a Th2 cell phenotype. Treatment of DCs with 1,25(OH)2D3 can also induce CD4+/CD25+ regulatory T cells (Treg) cells (423) as shown by increased FoxP3 expression, critical for Treg development.  These cells produce IL-10, which suppresses the development of the other Th subclasses. Treg are critical for the induction of immune tolerance (424).  In addition, 1,25(OH)2D3 alters the homing of properties of T cells for example by inducing expression of CCR10, the receptor for CCL27, a keratinocyte specific cytokine, while suppressing that of CCR9, a gut homing receptor (425). The actions of 1,25(OH)2D3 on B cells have received less attention, but recent studies have demonstrated a reduction in proliferation, maturation to plasma cells and immunoglobulin production (426). 

 

1,25(OH)2D3 has both direct and indirect effects on regulation of a number of cytokines involved with the immune response (review in (427)). TNF has a VDRE in its promoter to which the VDR/RXR complex binds.  1,25(OH)2D3 both blocks the activation of NFκB via an increase in IκBα expression and impedes its binding to its response elements in the genes such as IL-8 and IL-12 that it regulates. 1,25(OH)2D3 has also been shown to bring an inhibitor complex containing histone deacetylase 3 (HDAC3) to the promoter of rel B, one of the members of the NFκB family, thus suppressing gene expression. Thus, TNF/NFkB activity is markedly impaired by 1,25(OH)2D3 at multiple levels. In VDR null fibroblasts, NFκB activity is enhanced. Furthermore, 1,25(OH)2D3 suppresses IFNγ, and a negative VDRE has been found in the IFNγ promoter. GM-CSF is regulated by VDR monomers binding to a repressive complex in the promoter of this gene, competing with nuclear factor of T cells 1(NFAT1) for binding to the promoter.

The ability of 1,25(OH)2D3 to suppress the adaptive immune system appears to be beneficial for a number of conditions in which the immune system is directed at self—i.e. autoimmunity (review in (428)). In a number of experimental models including inflammatory arthritis, psoriasis, autoimmune diabetes (e.g., NOD mice), systemic lupus erythematosis (SLE), experimental allergic encephalitis (EAE) (a model for multiple sclerosis), inflammatory bowel disease (IBD), prostatitis, and thyroiditis VDR agonist administration has prevented and/or treated the disease process. As will be discussed later, a number of these conditions are associated with bone loss either directly (e.g., inflammatory arthritis) or indirectly presumably via increased serum levels of inflammatory cytokines. These actions of 1,25(OH)2D3 were originally ascribed to inhibition of Th1 function, but Th17 cells have also been shown to play important roles in a number of these conditions including psoriasis (321),  experimental colitis (422), and rheumatoid arthritis (429), conditions that respond to 1,25(OH)2D3 and its analogs. Although few prospective, randomized, placebo-controlled trials in humans have been performed, epidemiologic and case control studies indicate that a number of these diseases in humans are favorably impacted by adequate vitamin D levels. For example, the incidence of multiple sclerosis correlates inversely with 25OHD levels and vitamin D intake, and early studies suggested benefit in the treatment of patients with rheumatoid arthritis and multiple sclerosis with VDR agonists (427,428). Similarly, IBD is associated with low vitamin D levels (430). Children who are vitamin D deficient have a higher risk of developing type 1 diabetes mellitus, and supplementation with vitamin D during early childhood reduces the risk of developing type 1 diabetes (review in (421)).  In VDR null mice myelopoiesis and the composition of lymphoid organs are normal, although a number of abnormalities in the immune response have been found.  Some of the abnormalities in macrophage function and T cell proliferation in response to anti-CD3 stimulation in these animals could be reversed by placing the animals on a high calcium diet to normalize serum calcium (431), indicating the important role of calcium in vitamin D regulated immune function as in skeletal development and maintenance, hormone regulation, and keratinocyte differentiation. Other studies have noted an increased number of mature DCs in the lymph nodes of VDR null mice, which would be expected to promote the adaptive immune response (432). Somewhat surprisingly, RANKL also increases the number and retention of DCs in lymph nodes (433) suggesting that at least this mechanism is not mediated via the RANKL/RANK system in VDR null mice, which I will discuss at length subsequently.  In contrast to these inhibitory actions of 1,25(OH)2D3, Th2 function as indicated by increased IgE stimulated histamine from mast cells is increased in VDR null mice (434). The IL-10 null mouse model of IBD shows an accelerated disease profile when bred with the VDR null mouse with increased expression of Th1 cytokines (435). Surprisingly, despite a reduction in natural killer T cells and Treg cells and a decreased number of mature DCs, VDR null mice bred with NOD mice do not show accelerated development of diabetes (436). Part of the difference in tissue response in VDR null mice may relate to differences in the ability of 1,25(OH)2D3 to alter the homing of T cells to the different tissues (425).  In allergic airway disease (asthma) Th2 cells, not Th1 cells, dominate the inflammatory response. 1,25(OH)2D3 administration to normal mice protected these mice from experimentally induced asthma in one study, blocking eosinophil infiltration, IL-4 production, and limiting histologic evidence of inflammation (437).  However, a study with VDR null mice using a comparable method of inducing asthma showed that lack of VDR also protected the mice from an inflammatory response in their lungs (438). In an extension of this study the investigators showed that wildtype (WT) splenocytes were only minimally successful at restoring experimental airway inflammation to VDR null mice, whereas splenocytes from these mice were able to transfer experimental airway inflammation to the unprimed WT host (439). Thus, the impact of vitamin D signaling on adaptive immunity depends on the specifics of the immune response being evaluated. 

Inhibition of the adaptive immune response may also have benefit in transplantation procedures (440).  In experimental allograft models of the aorta, bone, bone marrow, heart, kidney, liver, pancreatic islets, skin, and small bowel VDR agonists have shown benefit generally in combination with other immunosuppressive agents such as cyclosporine, tacrolimus, sirolimus, and glucocorticoids (440). Much of the effect could be attributed to a reduction in infiltration of Th1 cells, macrophages and DC into the grafted tissue associated with a reduction in chemokines such as CXCL10, CXCL9, CCL2, and CCL5.  CXCL10, the ligand for CXCR3, may be of particular importance for acute rejection in a number of tissues, whereas CXCL9 as well as CXCL10 (both CXCR3 ligands) may be more important for chronic rejection at least in the heart and kidney, respectively. Although there are no prospective trials of the use of VDR agonists in transplant patients, several retrospective studies in patients with renal transplants treated with 1,25(OH)2D3 have suggested benefit with respect to prolonged graft survival and reduced numbers of acute rejection episodes.

Suppression of the adaptive immune system may not be without a price. Several publications have demonstrated that for some infections including Leishmania major (441) and toxoplasmosis (442), 1,25(OH)2D3 promotes the infection (442), while the mouse null for VDR is protected (441). This may be due at least in part to loss of IFNγ stimulation of ROS and NO production required for macrophage antimicrobial activity (441). Furthermore, atopic dermatitis, a disease associated with increased Th2 activity (443), and allergic airway disease, likewise associated with increased Th2 activity, (437-439), may be aggravated by 1,25(OH)2D3 and less severe in animals null for VDR.

THE INNATE IMMUNE RESPONSE

The innate immune response involves the activation of toll-like receptors (TLRs) in polymorphonuclear cells (PMNs), monocytes and macrophages as well as in a number of epithelial cells including those of the epidermis, gingiva, intestine, vagina, bladder and lungs (review in (444)). There are 10 functional TLRs in human cells (of 11 known mammalian TLRs). TLRs are an extended family of host noncatalytic transmembrane pathogen-recognition receptors that interact with specific membrane patterns (PAMP) shed by infectious agents that trigger the innate immune response in the host. A number of these TLRs signal through adapter molecules such as myeloid differentiation factor-88 (MyD88) and the TIR-domain containing adapter inducing IFN-β (TRIF).  MyD88 signaling includes translocation of NFkB to the nucleus, leading to the production and secretion of a number of inflammatory cytokines. TRIF signaling leads to the activation of interferon regulatory factor-3 (IRF-3) and the induction of type 1 interferons such as IFNβ.  MyD88 mediates signaling from TLRs 2, 4, 5, 7 and 9, whereas TRIF mediates signaling from TLR 3 and 4. TLR1/2, TLR4, TLR5, TLR2/6 respond to bacterial ligands, whereas, TLR3, TLR7, and TLR 8 respond to viral ligands. The TLR response to fungi is less well defined. CD14 serves as a coreceptor for a number of these TLRs. Activation of TLRs leads to the induction of antimicrobial peptides (AMPs) and reactive oxygen species, which kill the organism. Among these AMPs is cathelicidin. Cathelicidin plays a number of roles in the innate immune response. The precursor protein, hCAP18, must be cleaved to its major peptide LL-37 to be active. In addition to its antimicrobial properties, LL-37 can stimulate the release of cytokines such as IL-6 and IL-10 through G protein coupled receptors, and IL-18 through ERK/P38 pathways, stimulate the EGF receptor leading to activation of STAT1 and 3, induce the chemotaxis of neutrophils, monocytes, macrophages, and T cells into the skin, and promote keratinocyte proliferation and migration (445). The expression of this antimicrobial peptide is induced by 1,25(OH)2D3 in both myeloid and epithelial cells (446,447).  In addition, 1,25(OH)2D3 induces the coreceptor CD14 in keratinocytes(448). Stimulation of TLR2 by infectious organisms like tuberculosis in macrophages (449) or stimulation of TLR2 in keratinocytes by wounding the epidermis (448) results in increased expression of CYP27B1, which in the presence of adequate substrate (25OHD) stimulates the expression of cathelicidin.  Lack of substrate (25OHD) or lack of CYP27B1 blunts the ability of these cells to respond to a challenge with respect to cathelicidin and/or CD14 production (447-449). In diseases such as atopic dermatitis, the production of cathelicidin and other antimicrobial peptides (AMPs) is reduced, predisposing these patients to microbial superinfections (450). Th2 cytokines such as IL-4 and 13 suppress the induction of AMPs(451). Since 1,25(OH)2D3 stimulates the differentiation of Th2 cells, in this disease 1,25(OH)2D3 administration may be harmful.  An important role of these AMPs besides their antimicrobial properties is to help link the innate and adaptive immune response. This interplay is well demonstrated in SARS-CoV-19 infections in which a dysfunctional and/or delayed innate immune response can lead to an unchecked adaptive immune response resulting in a massive release of proinflammatory cytokines, the “cytokine storm”, leading to destruction of the lungs and death (452). Patients with vitamin D deficiency appear to be more vulnerable to this infection (453).

Although many cells are capable of the innate immune response including bone cells, most studies have focused on the macrophage and the keratinocyte. Vitamin D regulation of the innate immune response in these two cell types is comparable, but differences exist.

Macrophages

The importance of adequate vitamin D nutrition for resistance to infection has long been appreciated but poorly understood. This has been especially true for tuberculosis. Indeed, prior to the development of specific drugs for the treatment of tuberculosis, getting out of the city into fresh air and sunlight was the treatment of choice. In a recent survey of patients with tuberculosis in London (454) 56% had undetectable 25OHD levels, and an additional 20% had detectable levels but below 9 ng/ml (22 nM).  In 1986 Rook et al. (455) demonstrated that 1,25(OH)2D3 could inhibit the growth of Mycobacterium tuberculosis.  The mechanism for this remained unclear until the publication by Liu et al. (449) of their results in macrophages. They observed that activation of the Toll-like receptor TLR2/1 by a lipoprotein extracted from M. tuberculosis reduced the viability of intracellular M. tuberculosis in human monocytes and macrophages concomitant with increased expression of the VDR and of CYP27B1 in these cells. Killing of M. tuberculosis occurred only when the serum in which the cells were cultured contained adequate levels of 25OHD, the substrate for CYP27B1. This provided clear evidence for the importance of vitamin D nutrition (as manifested by adequate serum levels of 25OHD) in preventing and treating this disease, and demonstrated the critical role for endogenous production of 1,25(OH)2D3 by the macrophage to enable its antimycobacterial capacity.  Activation of TLR2/1 or directly treating these cells with 1,25(OH)2D3 induced the antimicrobial peptide cathelicidin, which is toxic for M. tuberculosis. If induction of cathelicidin is blocked as with siRNA, the ability of 1,25(OH)2D3 to enhance the killing of M. tuberculosis is prevented (456). Furthermore, 1,25(OH)2D3 also induces the production of reactive oxygen species which if blocked likewise prevents the anti-mycobacterial activity of 1,25(OH)2D3 treated macrophages (457). The murine cathelicidin gene lacks a known VDR response element in its promoter, and so might not be expected to be induced by 1,25(OH)2D3 in mouse cells, yet 1,25(OH)2D3 stimulates antimycobacterial activity in murine macrophages. Murine macrophages, unlike human macrophages, utilize inducible nitric oxide synthase (iNOS) for their TLR and 1,25(OH)2D3 mediated killing of M. tuberculosis (457,458). Clinical trials attempting to treat tuberculosis patients with high levels of vitamin D have shown mixed results (459)(460).

Keratinocytes

Cathelicidiin and CD14 expression in epidermal keratinocytes is induced by 1,25(OH)2D3 (445,448).  In these cells butyrate, which by itself has little effect, potentiates the ability of 1,25(OH)2D3 to induce cathelicidin (461).  Keratinocytes treated with 1,25(OH)2D3 are substantially more effective in killing Staphyococcus aureus than are untreated keratinocytes. Wounding the epidermis induces the expression of TLR2 and that of its co-receptor CD14 and cathelicidin (448). This does not occur in mice lacking CYP27B1 (448). Unlike macrophages, 1,25(OH)2D3 stimulates TLR2 expression in keratinocytes as well as in the epidermis when applied topically (448) providing a feed forward loop to amplify the innate immune response. Wounding also increases the expression of CYP27B1.  This may occur as a result of increased levels of cytokines such as TNF-α and IFN-γ, both of which we have shown stimulate 1,25(OH)2D3 production, as well as by TGF-β and the TLR2 ligand Malp-2 (448). When the levels of VDR or one of its principal coactivators, SRC3, are reduced using siRNA technology, the ability of 1,25(OH)2D3 to induce cathelicidin and CD14 expression in human keratinocytes is markedly blunted (461).

Other Tissues

The VDR is widespread (127,462) (reviews). In some of these tissues the functional significance of the VDR and/or the effect of 1,25(OH)2D are unclear. Since several of the functions regulated by 1,25(OH)2D in some of these tissues may have clinical relevance, this section will focus on a select number of these tissues. 

HEART

A reduction in contractility has been observed in vitamin D deficient animals (463). This may be due to lack of vitamin D or the accompanying hypocalcemia and hypophosphatemia. However, in vitro 1,25(OH)2D stimulates calcium uptake by cardiac muscle cells (464,465). In addition, 1,25(OH)2D inhibits the expression of atrial naturetic factor, one of the few genes with a negative VDRE in its promoter (466).  Deletion of the VDR specifically in cardiac muscle leads to hypertrophy and fibrosis (467). Low circulating levels of 25OHD are associated with increased risk of myocardial infarction in men [436]. However, a large randomized clinical trial failed to show a protective effective of vitamin D supplementation to individuals with normal levels of 25OHD with respect to cardiovascular disease (341) 

SKELETAL MUSCLE 

Proximal muscle weakness is a hallmark of vitamin D deficiency, and reduced high energy substrates (ATP, creatinine phosphate) have been observed in that condition (468). Myoblasts contain VDR, although the expression of VDR in mature muscle cells is controversial. Muscle weakness may reflect the lower levels of calcium and phosphate rather than a reduction in 1,25(OH)2D. However, evidence for a direct role of 1,25(OH)2D and VDR in muscle function is increasing (469). Moreover, 1,25(OH)2D may have actions on muscle that do not require the VDR, at least the genomic functions of VDR. The Boland laboratory (470) has demonstrated acute effects of 1,25(OH)2D on calcium uptake, PLC, PLA2, PLD, PKC, and adenylate cyclase activities, all of which may alter muscle function.

PITUITARY

VDR have been found primarily in thyrotropes in vivo and in GH and prolactin secreting cell lines in vitro (471,472). 1,25(OH)2D increases TRH stimulated TSH secretion by a mechanism involving increased Cai and IP3 production (473,474), suggesting that induction of PLC by 1,25(OH)2D may be involved. 

BREAST

The breast contains VDR (475), and vitamin D plays a role in normal breast development (476). Moreover, breast cancer cells also contain VDR (477), and 1,25(OH)2D and its analogs reduce their proliferation in vivo and in vitro (478,479). This has obvious clinical implications for the treatment of breast cancer.

LIVER

Low levels of VDR have been found in the liver, particularly in stellate cells (480,481). Hepatic regeneration is impaired in vitamin D deficient animals, even when the serum calcium is normalized by a high calcium diet (482), suggesting a role for 1,25(OH)2D in hepatic cell growth and in the prevention of hepatic fibrosis (481).

LUNG 

VDR have been found in type II epithelial pneumocytes (483). 1,25(OH)2D stimulates their maturation including increased phospholipid production and surfactant release [437].These results are consistent with the abnormal alveolar development observed in pups born to vitamin D deficient mothers (484). In addition 1,25(OH)2D stimulates the innate immune response in bronchial epithelial cells and may provide protection in patients with cystic fibrosis with recurrent lung infections as well as in patient with Covid-19 infections (452,485) as discussed previously.

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Diabetes Mellitus in People with Cancer

ABSTRACT

 

There is increasing evidence of an association between cancer and diabetes mellitus. Patients with type II diabetes are at increased risk of malignancy due to shared risk factors between the two conditions, and people with a diagnosis of cancer may develop new onset diabetes or impaired glycemic control, partly as a result of the systemic anti-cancer treatments (SACT) they receive. Many newer targeted anti-cancer treatments can have off-target metabolic toxicities not seen with conventional chemotherapy agents. Early recognition of diabetes or hyperglycemia in people with cancer can improve outcomes. This chapter aims to summarize these associations, provide an overview of how different SACT modalities can impact on glycemic control, and highlight key recommendations for the management of this complex patient group.

 

INTRODUCTION

 

Diabetes mellitus (DM) is a rising global public health emergency, with recent estimates suggesting that over 780 million people globally will be affected by 2045 (1). DM is typically classified into broad categories including type 1 (T1DM), type 2 (T2DM), gestational, monogenic, pharmacologically-induced, endocrinopathy-driven and DM due to pancreatic disease/deficiency (sometimes referred to as type 3c) (2, 3). T2DM is regarded as the most common subtype and is reported to account for over 85% of cases (1). All types of DM can lead to multisystem microvascular (nephropathy, retinopathy, neuropathy) and macrovascular (ischemic heart disease, stroke and peripheral vascular disease) complications, with management of these complications placing a strain upon many health services.

 

People with a diagnosis of DM are also at higher risk for developing several cancers (4), with reasons for this in part due to shared risk factors between the two, including age, obesity, sedentary lifestyle, and diet (5,6). A recent umbrella review of meta-analyses found that risks of developing most cancers were higher in people with DM compared to those without, with the most convincing evidence seen in breast cancer, intrahepatic cholangiocarcinoma, colorectal cancer, and endometrial cancer. One exception in this study was prostate cancer, where the risk appeared lower in individuals with DM (4). In view of this increased cancer risk in people with DM, some groups even advocate that regular screening for underlying cancer should be part of routine DM assessments (7).

 

It is estimated that approximately 20% of people with cancer have concurrent diabetes (8). Individuals with cancer are also at an increased risk of developing new onset DM or hyperglycemia, independent of an underlying diagnosis of diabetes, whilst cancer patients with concurrent DM often experience worsening glycemic control (9). Reasons for poor glycemic control in these individuals include complications from systemic anticancer treatments (SACT) along with supportive medications to treat treatment side effects, and symptoms of the underlying malignancy. This chapter aims to summarize the complex relationship between malignancy and DM, particularly the effects of SACT on glycemic control and risk of DM, as well as outlining management guidelines for DM in people with cancer.

 

DIABETES/HYPERGLYCEMIA AND CANCER OUTCOMES                       

 

A number of observational studies have demonstrated that hyperglycemia is associated with poorer overall survival (OS) and increased risk of disease recurrence in a number of malignancies, solid and hematological (10-17), with a number of individual studies, and larger meta-analyses supporting this. One meta-analysis reviewed 12 studies comprising 9,872 people with a diagnosis of cancer without known diabetes. Individuals with hyperglycemia were found to have significantly worse disease-free survival (DFS) (hazard ratio (HR) 1.98, 95% confidence interval (CI) 1.20-3.27) compared to those without, as well as worse OS (HR 2.05, 95% CI 1.67-2.551) (18). A further meta-analysis of 4,241 patients with pancreatic cancer suggested that those individuals with concurrent DM (1,034) have poorer OS (HR 1.16, 95% CI 1.08-1.25) and a higher risk of on-treatment death than those without concurrent DM (19). Furthermore, in a meta-analysis of 8 studies in breast cancer, concurrent DM was found to confer a greater risk of death, and a later stage at presentation, as well as impact on the treatment given (20). People with DM also have a higher prevalence of oral cancers, as well as a higher mortality from these cancers (21).

 

In addition to this, a number of preclinical studies have suggested that hyperglycemia may specifically attenuate the efficacy of chemotherapy in people with cancer with or without diabetes, which could in part account for these observations (22). For example, hyperglycemia may attenuate chemotherapy-induced reactive oxygen species (ROS) production, which in-turn can diminish the efficacy of treatment (23). In vivo, there are some small series that have demonstrated an association between hyperglycemia and resistance to chemotherapy. A clinical study of 88 people with estrogen-receptor positive breast cancer demonstrated impaired glucose tolerance significantly correlated with disease progression in those patients receiving chemotherapy (24). Furthermore, high blood glucose levels irrespective of an underlying DM diagnosis, were shown to significantly enhance oxaliplatin resistance in individuals with stage III colorectal cancer receiving adjuvant chemotherapy (22). Studies such as these highlight the importance of adequate glycemic control during treatment for cancer to potentially improve outcomes, although these data are mainly from observational studies, with interventional studies lacking.

 

EFFECT OF DIABETES OR HYPERGLYCEMIA ON QUALITY OF LIFE IN PEOPLE WITH CANCER

 

Cancer-related symptoms and SACT side effects, such as fatigue, nausea, anorexia and pain can be debilitating to patients. When confounded by symptoms of hyperglycemia, the impact upon an individuals’ quality of life can be significant (25). Furthermore, the impact of a cancer diagnosis, as well as treatment and cancer-related symptoms can have major negative impacts on diabetes self-care (26), with data suggesting that adherence to glucose lowering drugs often decreases in individuals following a cancer diagnosis (27). A cancer diagnosis can also have financial and social impacts upon individuals, affecting access to healthy food and outpatient diabetes services, resulting in lower quality of life and a higher symptom burden (28). A systematic review of 10 studies, demonstrated poorer patient reported outcomes (PROs) in those diagnosed with both cancer and DM compared to having either one of these diseases alone (29).

 

 

People with DM are known to be at higher risk from infections, and undergoing SACT can exacerbate this, resulting in higher rates of infection and hospitalization observed in those with cancer and DM (30, 31). This in turn leads to higher rates of chemotherapy dose reductions and early treatment cessation (28, 32-34). A meta-analysis of 10 observational studies involving 8,688 cases found that the likelihood of developing chemotherapy-induced neutropenia was higher amongst individuals with DM/hyperglycemia than those without (odds ratio (OR) 1.32, 95% CI 1.06-1.64) (31). Chemotherapy-induced neutropenia poses a significant risk for infection and hospitalization in all people with cancer, with an associated rate of morbidity and mortality which is higher in those with raised blood glucose levels (30). In addition to severe hematological toxicity, more severe rates of non-hematological toxicity have also been associated with hyperglycemia during chemotherapy in people with prostate cancer and lymphoma (35). A single-center retrospective analysis found that individuals with cancer and DM who had good glycemic control had no increased risk of treatment-related complications compared with individuals without DM (36), suggesting that optimal glycemic control during SACT could improve tolerability, thereby reducing rates of admission and dose-limiting toxicity.

 

Conceivably, people with DM may be more prone to neuro- and nephrotoxic agents due to their underlying predisposition conferred by the DM. Indeed, a previous report suggested that taxane-based chemotherapy regimens resulted in a significantly higher rates of peripheral neuropathy in those with DM compared to those without (74.4% vs. 58.5%) (37). There are no convincing data to suggest that a concurrent cancer diagnosis accelerates the risk of diabetic nephropathy or retinopathy.

 

EFFECTS OF SYSTEMIC ANTICANCER THERAPIES ON GLYCEMIA  

 

Systemic anti-cancer therapies (SACT) encompass a wide range of treatments including cytotoxic chemotherapy, hormone therapy, targeted therapy, and immunotherapy, many of which can impact upon glycemic control directly or as a result of toxicity management or supportive medications which are given alongside treatment. Several anti-cancer agents have been demonstrated to increase the risk of hyperglycemia as summarized in Table 1, and many can do this even in those without a known diagnosis of DM. People receiving SACT are also at risk of developing a new diagnosis of diabetes. One study demonstrated that 11% of people (15/134) undergoing routine chemotherapy met the criteria for a new diagnosis of diabetes (using the diagnostic criteria as per guidelines from the UK National Institute for Clinical and Healthcare Excellence (NICE) and without a previous known diagnosis) based upon HbA1cmeasurements). The majority of these individuals (73%) had been receiving short course steroids with chemotherapy, and 40% were being treated in the curative/adjuvant setting (38). A second prospective cohort study in 90 people taking glucocorticoids as part of therapy protocols for primary brain tumor or metastases, lymphoma, or for bone marrow transplant, found non-DM range hyperglycemia in 58% and DM-range hyperglycemia in 18.9% (39). These individuals with hyperglycemia are also more likely to present with an emergency admission during cancer therapy than those with normoglycemia (40).

 

Table 1. SACT used in the Treatment of Cancer Demonstrated to be Associated with Worsening Glycemic Control

Type of SACT

Drug Examples

Risk of Diabetes/Hyperglycemia (Range of any grade)

Type of diabetes most likely to develop

Targeted therapy

 

mTOR inhibitors

Everolimus (41, 42)

12-50%

T2DM

Temsirolimus (42)

26%

PI3K inhibitors

Alpelisib (43)

37%

T2DM

Idelialisib (44)

28/30%

EGFR inhibitor

Osimertinib (45)

2%

T2DM

Panitumumab (46, 47)

1-10%

Multikinase inhibitor

Sunitinib (48-50)

0-8%

Risk of hypoglycemia

Reverses T1/T2DM, but also causes hyperglycemia

Pazopanib (50)

Tyrosine kinase inhibitor (TKI)

Nilotinib (51)

6%

T2DM

Ponatinib (52)

3%

ALK Inhibitor

Ceritinib (53)

49%

T2DM

FLT3 inhibitor

Midostaurin (54, 55)

7-20%

T2DM

Gilteritinib (56)

13%

Monoclonal antibody

Gemtuzumab (anti-CD33) *inpatient use (57)

10%

T2DM

Somatostatin Analogues

Octreotide, Lanreotide (58)

Up to 30%

T2DM, but risk of hypoglycemia

Chemotherapy

 

Anti-metabolite

5-fluorouracil (59, 60)

Up to 10%

T2DM

Pemetrexed (61, 62)

4%

Decitadine/Azacitidine (63)

6-33%

Alkylating agents

Busulfan (64)

66-67%

Platinum based

Oxaliplatin (65, 66)

4%

Anthracyclines

Doxorubicin (60, 67)

Up to 10%

Other

Arsenic trioxide (ATO) (68)

45%

 

Immune Checkpoint Inhibitors

 

PD-1

Nivolumab (69)

<1%

T1DM

Pembrolizumab (70)

1-2.2%

CTLA-4

Ipilumumab (69)

<1%

 

Combination ICP (71)

4%

Hormone Therapy

 

Hormone Treatment

ADT (44, 72)

Risk ratio 1.39 (95% CI 1.27-1.53) n=65,595 cases

T2DM

Tamoxifen (73)

Diabetes risk adj. odds ratio 1.24 (95% CI 1.08-1.42)

Abbreviations: ADT = androgen deprivation therapy; ALK – anaplastic lymphoma kinase; ATO – arsenic trioxide; CTLA-4 – cytotoxic T-lymphocyte protein-4; EGFR – epidermal growth factor receptor; FLT3 – FMS-like tyrosine kinase-3; ICP – immune checkpoint inhibitor; TKI – tyrosine kinase inhibitor; mTOR – mechanistic target of rapamycin; PI3K – phosphoinositide-3 kinase; PD-1 – programmed cell death protein-1; T1DM – type 1 diabetes mellitus; T2DM – type 2 diabetes mellitus

 

Cytotoxic Chemotherapy

 

Hyperglycemia occurs in between 10 and 30% of people undergoing cytotoxic chemotherapy for malignancy (74), and although often transient during treatment, can persist, or even lead to DM in some people. Poor glycemic control can increase the risk of infections and hospitalization (28, 34), as previously discussed, leading to treatment interruptions and dose reductions, as well as significant morbidity, and even mortality (33). A number of cytotoxic chemotherapy regimens are reported to cause hyperglycemia in people without diabetes, including commonly used drugs such as 5-fluorouracil (5-FU), platinum-based drugs (oxaliplatin, carboplatin, cisplatin) and anthracyclines (doxorubicin, epirubicin) (75). In one cohort study of 422 people receiving 5FU-based chemotherapy regimens for the treatment of early or advanced colorectal cancer, 11.6% (42 people) developed diabetes and a further 11.3% developed impaired fasting blood glucose (FBG) levels. Of the 42 people who developed diabetes, 7 required no treatment, 13 received diet control and physiotherapy only, and 22 received antidiabetic medication (75). In a second cohort of 185 people with head and neck cancer treated with platinum-containing regimens, 3.8% developed type 2 DM, with 3 presenting with hyperglycemic crises (DKA, HHS) (65). One possible contributing factor for developing impaired FBG levels and/or type 2 DM is the concurrent use of corticosteroids in highly emetogenic chemotherapy regimens, but an analysis of type 2 DM following anthracycline use in 3,147 lymphoma patients suggested that the use of these drugs independently increases the risk of T2DM, when data was adjusted for corticosteroid use, comorbidities, age, and gender. A threshold doxorubicin dose of 253mg was identified, below which there was no increased risk of developing T2DM (76). Risk of diabetes from cytotoxic chemotherapy may also increase with age, with one pediatric study suggesting that the risk was higher in acute lymphoblastic leukemia (ALL) patients aged > 10, compared with those < 10 years old (77).

 

Exact mechanisms of how and why some cytotoxic chemotherapies can lead to hyperglycemia or T2DM remain unclear. Proposed mechanisms include the induction of an inflammatory state which predisposes to hyperglycemia (78) or direct metabolic effects on tissues vital to glucose homeostasis such as skeletal muscles (79).

 

Oral Targeted Anticancer Agents

 

Many new targeted cancer therapies inhibit various points in the insulin receptor signaling pathway including the commonly used class of tyrosine kinase inhibitors (TKIs) (80). Reported effects of targeted TKIs on blood glucose metabolism range from the development of metabolic syndrome and diabetes via the blocking of insulin signaling (80), as well as erratic glycemic control and even hypoglycemia in those with pre-existing type 1 or type 2 DM49, (81, 82). In contrast some TKIs may improve glycemic control suggesting that management of these individuals needs to be individualized with no one-size-fits-all management algorithm. Reversibility of these effects is also unclear, with reported improvements in glycemic control and HbA1c levels following dose reductions or treatment termination (83).

 

Inhibitors of mTOR (everolimus, temsirolimus or ridaforolimus) have also been shown to impact glycemic control since mTOR is a protein kinase that plays a key role in regulating cell growth as well as lipid and glucose metabolism (84, 85). Meta-analyses looking into these effects have demonstrated significantly higher rates of hyperglycemia, hypercholesterolemia, and hypertriglyceridemia compared with controls (86, 87) In isolated cases, the effects have been severe enough to precipitate DKA (88). To date, studies have not demonstrated either positive or negative associations between treatment response rates and incidence of metabolic complications (89).

 

As novel targeted agents continue to be introduced to manage a range of cancers, it is expected that metabolic toxicities continue to be reported given the homeostatic function of many of these druggable targets. Whilst some of these agents will provide meaningful benefit in terms of survival for people with advanced cancers, such as the PI3Kainhibitor alpelisib for PI3KA-mutated metastatic breast cancer (43), glycemic control needs to be at the forefront of the prescriber’s mind at initiation, to ensure adequate management of toxicities.

 

Hormone Therapy

 

ANDROGEN DEPRIVATION THERAPY

 

Androgen deprivation therapy (ADT) is recognized as a risk factor for development of diabetes, metabolic syndrome, and cardiovascular disease (72, 90, 91). In a large observational study of over 35,000 men treated for prostate cancer, ADT in the form of gonadotropin-releasing hormone (GnRH) agonists, oral antiandrogens, a combination of the two, or orchiectomy was associated with a significantly increased risk of diabetes, coronary heart disease, myocardial infarction, and sudden cardiac death (90). These findings are supported by other studies, including a meta-analysis of over 150,000 men with prostate cancer receiving ADT (72), with association observed with all forms of ADT, with the weakest association with anti-androgen therapy alone.

 

ESTROGEN TARGETED THERAPY

 

Studies examining the effect of estrogen-targeted therapies on the development of diabetes in women with breast cancer are less clear cut. Whilst one retrospective cohort analysis failed to demonstrate a link between tamoxifen use and the development of DM (92), two large population-based studies demonstrated a significant association between tamoxifen use and the development of diabetes in women diagnosed with breast cancer (73, 93); The first of these studies included almost 15,000 Canadian women aged 65 years or older diagnosed with early breast cancer, whilst the second included over 22,000 women in Taiwan aged 20 years and over. Whilst tamoxifen appears to increase the risk of developing DM, aromatase inhibitor therapy does not, with no link found in any of these three studies.

 

Immune Checkpoint Inhibitors

 

Immune checkpoint inhibitors (ICPi), including cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PDL-1) inhibitors are a sub-class of monoclonal antibody treatments that have revolutionized cancer treatment over the last decade. First approved for use in the treatment of melanoma, ICPi are now recognized as providing a survival benefit across a number of cancers, and are increasingly used in early-stage cancers in the adjuvant setting and also in combination with chemotherapy (94). Whilst clinically effective, ICPi can lead to a spectrum of immune-related adverse events (IRAEs). Endocrine IRAEs include hypophysitis, thyroiditis, adrenalitis and de novo diabetes. The risk of developing de novo diabetes is low, occurring in 0.2-4% of ICPi treated individuals depending on the immunotherapy given (69). The immune checkpoint PD-1 and its ligand PD-L1 have been shown to have an important immune homeostatic function in the pancreas by promoting beta cell maturation and preventing immune-mediated beta cell destruction (95). To date, there is no convincing evidence for a physiological role for CTLA-4 within the pancreas. PD-1 inhibitors, PD-L1 inhibitors, and combination CTLA-4/PD-1 therapy have been demonstrated to precipitate diabetes more commonly than CTLA-4 inhibitors alone. The underlying clinical presentation is akin to type 1 diabetes (70) and believed to be precipitated by inappropriate activation of self-reactive T-cells and destruction of insulin-producing pancreatic islet β-cells. ICP-induced insulin deficiency may lead to new-onset insulin-dependent diabetes or worsening pre-existing type 2 diabetes. Up to 75% of people who develop ICP-induced hyperglycemia/diabetes present with diabetic ketoacidosis (DKA) (96-98). Presentations are frequently acute with a precipitous increase in blood glucose (99). Therefore ICP-induced diabetes can be discriminated from ‘standard’ type 1 diabetes mellitus, by its tendency towards a faster onset, apparently fulminant course, and high degree of antibody negativity (99). The nomenclature of the condition in the published literature varies mainly between ‘type 1 like’ to ‘fulminant’ with there being differences between the presentation of ICPi-induced diabetes and type 1 and fulminant diabetes. Kyriacou and colleagues compared the characteristics of 75 published cases and concluded that there is some overlap with type 1 DM and fulminant DM. However, this was felt to be insufficient overlap for ICPi diabetes to be wholly classified as either type 1 like or fulminant (100). Nevertheless, the recognition that these agents can precipitate rapid beta cell destruction which results in an unusually high number of emergency presentations is key. Treatment of non-endocrine IRAEs is typically with high dose steroids, often for prolonged periods of time. At present, steroids are used in up to a third of people receiving ICPs, further increasing the risk of hyperglycemia, and steroid induced T2DM.

 

An analysis of the World Health Organizations (WHO) pharmacovigilance database over a 4-year period detected 283 cases of ICP-induced diabetes mellitus, 50.2% of which presented with DKA, and 6% of whom were on concurrent steroids at diagnosis (101). There was a wide variability in duration of ICP treatment, and timing of DM onset, occurring even up to 8 months after cessation of ICP treatment. A systematic review of 90 cases, demonstrated a diagnosis of DM on average after 4.5 cycles of ICP (102). C-peptide levels were usually low or undetectable at diagnosis, islet autoantibodies were positive in 53%, with a predominance of glutamic acid decarboxylase antibodies, and susceptible HLA genotypes present in 65% (102). HbA1c levels were relatively low, consistent with the observed rapid onset of beta cell inflammation. Importantly, an elegant albeit small single-center study, used radiological and biochemical phenotyping to demonstrate that ICPi DM is irreversible (103). This has important clinical implications such that any individual diagnosed with ICPi-induced DM should be counselled around an expected life-long requirement of insulin.

 

Glucocorticoid (Steroid) Treatment

 

Glucocorticoids (GC) increase insulin resistance and glucose production and inhibit the production and secretion of insulin by pancreatic beta cells, as well as acting centrally to counteract the appetite-reducing effects of insulin (104). As such they are commonly associated with the development of hyperglycemia and diabetes. GCs have a direct hyperglycemic effect which starts very early after ingestion (105, 106). They typically cause an increase in blood glucose levels 4-8 hours after ingestion leading to a peak blood glucose level between midday meal and evening meal (106, 107). One in ten people not known to have diabetes develop GC-induced diabetes (108) an effect which is dose dependent (109). The incidence of glucocorticoid-induced hyperglycemia has been shown to occur in up to 30% of individuals without diabetes (110), but could be as high as 50%. The consequences of missing it can lead to significant harm, including the development of Hyperosmolar Hyperglycemic State (HHS), hospitalization, and in extreme circumstances, death. In a single center UK prevalence study 12.8% (120/940) of inpatients were found to be on glucocorticoids, however only 20.5% of these individuals (25/120) had their blood glucose levels measured during admission, demonstrating how infrequently glucose is measured in hospital (111). It is important to ensure that if glucocorticoid (steroid) induced hyperglycemia does occur, it is picked up early.

 

The use of GCs, is common in advanced cancer, to reduce peri-lesional edema, relieve pain, control nausea, combat fatigue, or boost appetite. For oncological emergencies such as cerebral metastases, superior vena-cava obstruction (SVCO), or metastatic spinal cord compression (MSCC), high dose GC treatment is integral to patient management. Furthermore, GC treatments are the backbone of many hematological cancer treatment regimens, and are often used as supportive anti-emetic medications, or to prevent allergic reactions, in many solid tumor regimens (105), and, as discussed above, the main first-line treatment for the management of ICP toxicity. In one study, the incidence of glucocorticoid-induced diabetes was 20% in those with newly diagnosed gastrointestinal cancer following at least 3 cycles of highly or moderately emetogenic chemotherapy, including dexamethasone as a supportive medication. Furthermore, almost 60% of people in the study exhibited signs of insulin resistance and multivariate analysis showed a significant association between the cumulative dose of dexamethasone and the incidence of corticosteroid-induced diabetes (112). In a separate smaller study of 16 women without diabetes with ovarian or endometrial cancer receiving carboplatin/paclitaxel chemotherapy with dexamethasone as supportive care, almost all experienced elevated interstitial glucose levels with diurnal variation during the first five days of treatment (113). For those who receive prednisolone as part of a treatment regimen for hematological malignancies, rates of steroid-induced diabetes and hyperglycemia have been reported to be as high as 32.5% and 47% respectively, highlighting the scale of this issue (114, 115).

 

Supra-physiological doses of glucocorticoids approximate to a dose of prednisolone greater than 5mg per day – or an equivalent dose of the alternative synthetic GC (Table 2). With increasing dose of GC, the risk of potential hyperglycemia increases, and in people without pre-existing diabetes, a glucocorticoid dose equivalent of >12mg dexamethasone and longer acting steroids are associated with a greater degree of hyperglycemia (116). As duration of GC treatment increases, it becomes increasingly likely that hyperglycemia may not resolve once the GCs are withdrawn, with those groups at particular risk of developing glucocorticoid induced diabetes, shown in Table 3.

 

Table 2. Glucocorticoid Dose Equivalent

Glucocorticoid (steroid)

Potency (equivalent doses)

Duration of action (half-life, in hours)

Hydrocortisone

20 mg

8

Prednisolone

5 mg

16-36

Methylprednisolone

4 mg

18-40

Dexamethasone

0.8 mg

36-54

Betamethasone

0.8 mg

26-54

 

Table 3. Risk Factors for Glucocorticoid-Inducted Diabetes

Pre-existing type 1 or type 2 diabetes

Family history of diabetes

Increasing age

Obesity

Ethnic minorities

Impaired fasting glucose or impaired glucose tolerance

Polycystic ovarian syndrome

Previous gestational diabetes

Previous development of hyperglycemia on glucocorticoid therapy

Concurrent cytotoxic therapy known to cause hyperglycemia

 

HYPOGYCEMIA IN PEOPLE ON SACT

 

Although anti-cancer therapies and glucocorticoid use lead predominantly to hyperglycemia, there are risks of hypoglycemia that require consideration. People at risk of hypoglycemia should be counselled on the signs and symptoms to be aware of, and of the requirement to inform the driver and vehicle licensing agency should they experience any episodes of hypoglycemia requiring third party assistance.

 

Poor oral intake and nausea/vomiting from the underlying cancer or treatments put individuals at increased risk of hypoglycemia. Poor glycemic control can cause weight loss and precipitate nutrition impact symptoms (NIS) such as nausea, poor appetite, and altered bowel movements, further increasing the risks of hypoglycemia, particularly when dietary intake has been poor for some time. People with diabetes on an insulin secretagogue (sulfonylureas or meglitinides), or those on insulin, are also at higher risk of hypoglycemia.

 

In patients with end-stage metastatic disease, and shortened life expectancy, tight glucose control is not indicated, potentially placing individuals at unnecessary risk for hypoglycemia, particularly in those with a poor performance status >2. Individual risk for hypoglycemia and prognosis should be considered and recommended glycemic measurement targets are between 6.0 mmol/L – 15 mmol/L (108 – 225 mg/dl) (117).

 

People with new onset ICPi-induced insulin deficiency often have labile glucose control (99). More relaxed glucose targets may be required to avoid hypoglycemia wherever possible. Immune checkpoint inhibitors can also induce hypopituitarism leading to secondary adrenal insufficiency. This may lead to hypoglycemia (together with any of the following - hyponatremia, hyperkalemia and hypotension). Adrenalitis leading to primary adrenal insufficiency is very rare. Presentation of adrenal insufficiency ranges from asymptomatic laboratory alterations to the acutely unwell, with management depending on the severity (118). Other causes of adrenal or pituitary deficiency leading to hypoglycemia include metastases at these sites, surgery, irradiation, azole class of anti-fungal medication, and inappropriate abrupt cessation of glucocorticoid medication.

 

In oncology patients being weaned from long-term steroids, glucose monitoring will need to be continued after glucocorticoid cessation, with doses of anti-diabetic treatments adjusted accordingly, and individuals advised on risks of hypoglycemia. Caution is also required whilst using certain hematological anti-cancer therapies, including lenalidomide (119) and bortezomib (120), which can precipitate hypoglycemia, particularly in people with an underlying diagnosis of diabetes.

 

All cancer patients at risk from hypoglycemia should receive advice regarding appropriate treatment with 15–20 g of fast-acting carbohydrate, taken immediately (121). Comprehensive guidelines from the Joint British Diabetes Societies for Inpatient Care on the management of hypoglycemia can be found at this reference (122).

 

MANAGEMENT RECOMMENDATIONS  

 

Despite the effects of hyper- and hypoglycemia in people with diabetes (PWD) and those without known diabetes in cancer, there is a sparsity of guidance on the specific management considerations of these individuals. To address this, collaborative guidelines have recently been produced by the UK Chemotherapy Board (UKCB) and Joint British Diabetes Society for Inpatient Care (JBDS) (123, 124). The scope of these guidelines are to provide advice for the oncology/hemato-oncology and diabetes multidisciplinary teams to manage people with diabetes, commencing anti-cancer/ steroid therapy, as well as identifying individuals without a known diagnosis of diabetes who are at risk of developing hyperglycemia and new onset diabetes. These guidelines are intended for the outpatient management of people with cancer, particularly in the setting of the oncology/hemato-oncology clinic, and provision of advice for individuals at home, but where necessary, may be applied to inpatients as well. Whilst covering these guidelines in detail is beyond the scope of this chapter, key management considerations are summarized in tables 4-9.

 

Table 4. At Baseline

·       HbA1c and venous plasma glucose should be checked in all people with cancer at baseline clinic visit

·       Provide high risk individuals with capillary blood glucose (CBG) meter and glucose testing strips, or if baseline plasma glucose is ≥12 mmol/L (216 mg/dl)

·       Individuals with raised baseline HbA1c (>47 mmol/mol [6.5%]) should be referred to primary care for management of hyperglycemia prior to next follow up visit

·       When initiating SACT/glucocorticoids individuals must be informed of the risk of developing hyperglycemia/diabetes and potential symptoms to expect

·       The recommended glucose target level is 6.0-10.0 mmol/L (108 – 180 mg/dl), allowing a range of 6.0-12.0 mmol/L (108 – 216 mg/dl)

·       There are differences in opinion at where the threshold for intervention should be drawn - 12.0 mmol/L (216 mg/dl) is a pragmatic threshold

 

Table 5. Commencing Glucocorticoids (GC) /Systemic Anti-Cancer Therapy

·       Check baseline HbA1c and random venous plasma glucose before starting therapy

·       Monitor random plasma glucose at each treatment visit

·       Educate patients in symptoms of hyperglycemia

·       Consider commencing gliclazide 40mg if raised blood glucose ≥12mmol/L (216 mg/dl) on two occasions

·       Gliclazide may require frequent and significant increases in dose to reduce glucose levels, particularly on high dose steroids

·       Inform diabetes care provider if persistently raised blood glucose

·       If blood glucose is ≥20mmol/L (360 mg/dl), rule out DKA/HHS

 

Table 6. Commencing Immune Checkpoint Inhibitors (ICP)

·       Educate patients to be aware of symptoms of hyperglycemia

·       Rule out DKA or HHS which often occurs precipitously

·       Withhold ICP if evidence of ICP-induced diabetes emergency. Once patient has been regulated with insulin substitution, consider restarting ICP

·       Almost all patients require insulin therapy – refer urgently to diabetes team

 

Table 7. Managing Nausea and Vomiting

·       People with diabetes should be made aware of likely exacerbation of hyperglycemia whilst on anti-emetic therapy

·       People with diabetes receiving emetogenic chemotherapy should be offered an NK1 antagonist (e.g., aprepitant) with a long acting 5HT3 inhibitor (e.g., ondansetron)

·       Consider the use of a GC in the first cycle and reduce doses or withdraw completely based on the PWD’s emetic control and on blood glucose management

 

Table 8. For Non-Insulin-Treated Individuals with Type 2 Diabetes

·       Check baseline HbA1c and random venous plasma glucose before starting therapy

·       Monitor random plasma glucose at each treatment visit

·       Educate patients in symptoms of hyperglycemia

·       If plasma glucose is ≥12 mmol/L (216 mg/dl) on two occasions, screen for symptoms of hyperglycemia and ketonuria/ketonemia

·       In individuals already on a sulphonyurea such as gliclazide or meglitinides, up-titrate morning dose of gliclazide to a maximum doses of 240 mg. Evening dose of gliclazide may be initiated to achieve a maximum daily dose of 320 mg

·       Insulin therapy may be required

·       In individuals on a diet-controlled regimen, or on other non-sulfonylurea treatments (e.g., metformin, DPP4 inhibitors, pioglitazone, SGLT2 inhibitors) commence gliclazide 40 mg, and up-titrate

 

Table 9. For Insulin-Treated Individuals with Type 2 Diabetes

·       Check baseline HbA1c and random venous plasma glucose before starting therapy

·       Monitor random plasma glucose at each treatment visit

·       If plasma glucose is ≥12 mmol/L (216 mg/dl) on two occasions, screen for symptoms of hyperglycemia and ketonuria/ketonemia

·       Contact usual diabetes team for support in titrating insulin

·       Consider titrating insulin by 10-20% of the original dose daily

·       Individuals should be made aware of ‘sick day rules’ with insulin administration

 

Full management guidelines can be found at the UK Chemotherapy Board (UKCB) and Joint British Diabetes Society for Inpatient Care (JBDS) websites.

 

ADDITIONAL MANAGEMENT CONSIDERATIONS: CHOICE OF DIABETES THERAPEUTIC AGENT

 

Special consideration should also be given to the non-glycemic effects of hypoglycemic agents, including specific side effects and the impact on weight. Although weight reduction is associated with improvement in glycemic and metabolic profile in people with type 2 diabetes and is a key consideration in the choice of therapy, significant weight loss would usually be an unwanted effect in the oncology population. Indeed, weight gain is often used as a metric of improving nutritional state, especially in cancer related cachexia. This also has implications when counselling people with cancer about dietary choices when there is an additional cancer diagnosis. It is imperative that personalized advice is offered by healthcare professionals considering the global impact on the individual of any dietary or even lifestyle advice. SGLT2 inhibitors and GLP-1 agonists, for their potential weight reduction effects, are therefore less attractive options in the oncology setting. Insulin and sulfonylureas, on the other hand, offer an anabolic effect and therefore may be more desirable. Gastrointestinal side effects are common among hypoglycemic agents including metformin, DPP4 inhibitors, and GLP-1 agonists, and have the potential to complicate issues with nausea, vomiting, and oral intake from the underlying cancer and its treatment. Similarly, poor oral intake and nephrotoxic effects of certain SACT, added to a potential osmotic diuretic effect of SGLT2

inhibitors, could also increase the risk of acute kidney injury. The associated risk of genital tract infections with SGLT2 inhibitors would also be an additional consideration especially within an immunocompromised population (125). The impact and significance of these non-glycemic effects in the oncology population clearly differ to that of the general population, therefore highlighting the importance of a personalized approach with regular review of patients’ diabetes treatment through their oncology journey. 

 

CONCLUSIONS

 

It is common practice in oncology to initiate systemic anti-cancer therapy (including chemotherapy, targeted treatment, immunotherapy and steroids) in people with pre-existing diabetes. Diabetes, or risk of developing diabetes are by no means a contraindication to treatment but treating clinicians should be aware of the risks to patients, and counsel them appropriately. As more sophisticated anti-cancer treatments become licensed for use, the metabolic effects of these treatments will become better understood, and oncology teams should utilize and collaborate with endocrinology and primary care services to minimize the risks to individuals from poor glycemic control and diabetes. The recent publication of specific guidelines should act as a reference aid for clinicians and wider healthcare professionals to aid in risk recognition, diagnostic and screening for treatment induced diabetes, and provide the tools to appropriately manage these individuals and reduce the risks of complications.

 

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Monogenic Disorders Causing Hypobetalipoproteinemia – copy

ABSTRACT

 

Monogenic mutations leading to hypobetalipoproteinemia are rare. The monogenic causes of hypobetalipoproteinemia include familial hypobetalipoproteinemia, abetalipoproteinemia, chylomicron retention disease, loss of function mutations in PCSK9, and loss of function mutations in angiopoietin-like protein 3 (ANGPTL3) (Familiar Combined Hypolipidemia). This chapter describes the etiology, pathogenesis, clinical and laboratory findings, and the treatment of these rare monogenic disorders.

 

INTRODUCTION

 

Monogenic mutations leading to hypobetalipoproteinemia are rare. The monogenic causes of hypobetalipoproteinemia include familial hypobetalipoproteinemia (FHBL), abetalipoproteinemia (ABL), chylomicron retention disease (CMRD), loss of function mutations in PCSK9, and loss of function mutations in angiopoietin-like protein 3 (ANGPTL3) (Familial Combined Hypolipidemia, FCH). Increased understanding of the genetic and the molecular underpinnings of these disorders has allowed a focused prioritization of therapeutic targets for drug development. Table 1 summarizes genetic, lipid, and clinical features of the major hypobetalipoproteinemia syndromes. Of note the parental lipid profile is normal in abetalipoproteinemia and chylomicron retention disease.

 

It should be recognized that secondary, non-familial, forms of hypobetalipoproteinemia occur and include strict vegan diet, malnutrition, hyperthyroidism, malignancy, and chronic liver disease. In addition, hypobetalipoproteinemia can also be due to polymorphisms in multiple genes that together result in hypobetalipoproteinemia (polygenic etiology) (1-3). In a study of 111 patients with LDL-C levels below the fifth percentile 36% had monogenic hypobetalipoproteinemia, 34% had polygenic hypobetalipoproteinemia, and 30% had hypobetalipoproteinemia from an unknown cause (1). In a study of women with an LDL-C ≤1st percentile (≤50 mg/dL) 15.7% carried mutations causing monogenic hypocholesterolemia and 49.6% were genetically predisposed to a low LDL-C on the basis of an extremely low weighted polygenetic risk score (3).

 

Table 1. Characteristics of the Hypobetalipoproteinemia Syndromes

 

Inheritance

Effected gene

Prevalence

Lipids

Clinical features

FHBL

ACD

Truncation mutations in Apo B

1:1000 – 1:3000

Apo B <5th percentile,

LDL-C 20- 50 mg/dL

Hepatic steatosis

Mild elevation of transaminases. Lower prevalence of ASCVD

ABL

AR

MTTP

<1:1,000,000

Triglycerides < 30 mg/dl,

Cholesterol < 30 mg/dl),

LDL and Apo B undetectable

Hepatic steatosis

Malabsorption, steatorrhea, diarrhea, and failure to thrive.

Deficiency of fat-soluble vitamins.

PCSK9

ACD

Loss of function mutations in PCSK9

 

Heterozygous – mild to moderate reduction in LDL-C

Homozygous – LDL-C ~15 mg/dl

Normal health; significantly lower prevalence of ASCVD

FCH

ACD

Loss of function mutations in ANGPTL3

Very rare

Panhypolipidemia

Normal health; significantly lower prevalence of ASCVD

CMRD

AR

SAR1B

Very rare

LDL-C and HDL-C -decreased by 50%,

Triglycerides - normal

hypocholesterolemia associated with failure to thrive, diarrhea, steatorrhea, and abdominal distension

ACD- autosomal co-dominant; AR- autosomal recessive; FHBL- familial hypobetalipoproteinemia; ABL- abetalipoproteinemia; FCH- Familiar Combined Hypolipidemia; CMRD- chylomicron retention disease, MTTP- microsomal triglyceride transfer protein; ANGPTL3- angiopoietin-like protein 3; ASCVD- atherosclerotic cardiovascular disease

 

FAMILIAL HYPOBETALIPOPROTEINEMIA  

 

Familial Hypobetalipoproteinemia (FHBL) is most commonly due to truncation mutations in the gene coding for Apo B (4-6). Variants that lead to truncated proteins that are 30% in length or shorter have more severe signs and symptoms than those with longer truncated proteins (4,5). The truncated forms of Apo B found in FHBL are generally non-functional (truncation decreases lipidation and secretion) and are catabolized quickly, resulting in markedly reduced levels in the plasma (Apo B <5th percentile and LDL-C typically between 20- 50 mg/dL) (5,6). Although there is one normal allele in heterozygous FHBL, plasma Apo B levels are approximately 25% of normal rather than the predicted 50% (6). These lower than expected levels result from a lower secretion rate of VLDL Apo B from the liver, decreased production of LDL Apo B, increased catabolism of VLDL, and extremely low secretion of the truncated Apo B (4-6). Given the reduced substrate (Apo B) for lipid (predominantly triglyceride) loading, fatty liver develops in these patients (4,7). Hepatic steatosis and mild elevation of liver enzymes are common in heterozygous FHBL (4,7). Interestingly, individuals with monogenic hypobetalipoproteinemia had a much greater prevalence of hepatic dysfunction than individuals with polygenic hypobetalipoproteinemia (1). In contrast to non-alcoholic fatty liver disease, FHBL is not associated with hepatic or peripheral insulin resistance (7). This observation, however, does not imply that hepatic steatosis associated with FHBL is benign. There are several reports of steatohepatitis, cirrhosis, and hepatocellular carcinoma in patients with FHBL and it is estimated that 5-10% of individuals with FHBL develop relatively more severe nonalcoholic steatohepatitis (4). Because of the risk of developing liver disease liver function tests should be checked every 1-2 years and a hepatic ultrasound in those with elevated liver transaminases (4). While hepatic fat accumulation is the rule, there is generally sufficient chylomicron production to handle dietary fat. However, oral fat intolerance and intestinal fat malabsorption have been reported (4). On the positive side the decrease in proatherogenic lipoproteins has been associated with a reduced risk of cardiovascular disease (8).

 

Given the association of FHBL and low LDL-C, Apo B has been an attractive target for drug development. Indeed, unraveling the genetic and molecular mechanisms of FHBL provided the motivation to pharmacologically antagonize Apo B synthesis for therapeutic gains. This culminated in the production of mipomersen, a synthetic single strand anti-sense oligonucleotide to Apo B (9,10). Essentially, anti-sense oligonucleotides contain approximately ~20 deoxyribonucleic acid (DNA) base pairs complementary to a unique messenger ribonucleic acid (mRNA) sequence. The hybridization of the anti-sense oligonucleotide to the mRNA of interest leads to its catabolism via RNase H1, with markedly reduced mRNA levels and ultimately reduced target protein levels. In this case, mipomersen binds to Apo B mRNA leading to reduced production of the protein, and mimicking (albeit to a lesser extent) FHBL. Mipomersen is the first anti-sense oligonucleotide approved by the United States Food and Drug Administration (FDA) and was commercialized in 2013 with a limited indication for adjunctive LDL-C lowering in patients with homozygous familial hypercholesterolemia (HoFH) (10). It is an injectable agent administered subcutaneously once a week. In the clinical trials, mipomersen was associated with a reduction of LDL-C by 21% in subjects with HoFH and 33% in subjects with heterozygous familial hypercholesterolemia (HeFH) (10). Interestingly, it was also found to lower Lp(a) by 21- 23% (10). While it is highly efficacious in LDL-C lowering, it has side effects, many of which can be predicted based on the experience with FHBL (e.g., hepatic steatosis, elevated liver enzymes) (10). It is also associated with injection site reactions in a considerable number of subjects (10). In May 2018 sales were discontinued due to safety concerns related to increased liver transaminases and fatty liver.

 

Homozygous hypobetalipoproteinemia (HHBL) is extremely rare (4). These patients are homozygous or compound heterozygous for mutations in the Apo B gene. The clinical manifestations mimic ABL (see below) (4).

 

ABETALIPOPROTEINEMIA  

 

Abetalipoproteinemia (ABL) is a rare disorder characterized by very low plasma concentrations of triglyceride and cholesterol (under 30 mg/dl) and undetectable levels of LDL and Apo B (5,11,12). HDL-C levels are usually normal or modestly reduced. It is due to mutations in the gene that codes for microsomal triglyceride transfer protein (MTTP) (5,11-13). MTTP lipidates nascent Apo B in the endoplasmic reticulum to produce VLDL and chylomicrons in the liver and small intestine, respectively (13,14). Unlipidated Apo B is targeted for proteasomal degradation leading to the absence of Apo B containing lipoproteins in the plasma (and thus markedly reduced levels of LDL-C and triglycerides) (13,14). Similar to FHBL, VLDL production is inhibited (12). The reduced triglyceride export from the liver leads to hepatic steatosis, which rarely may progress to steatohepatitis, fibrosis, and cirrhosis (7,11). Additionally, lack of MTTP facilitated lipidation of chylomicrons in the small intestine results in lipid accumulation in enterocytes with associated malabsorption, steatorrhea, and diarrhea (5,11). The malabsorption and diarrhea lead to failure to thrive during infancy (5,11). Acanthocytosis may encompass 50% of circulating red blood cells (red blood cells with spiked cell membranes, due to thorny projections) (11,12). An additional issue of importance related to ABL is deficiency of fat-soluble vitamins (11). Early diagnosis of ABL and homozygous hypobetalipoproteinemia is extremely important as vitamin E deficiency culminates in atypical retinitis pigmentosa, spinocerebellar degeneration with ataxia, and vitamin K deficiency can lead to a significant bleeding diathesis (11). High dose supplementation with fat soluble vitamins early in life can prevent these devastating complications (5,11). Additional treatment measures include a low-fat diet and supplementation with essential fatty acids (5,11).

 

Given the very low level of atherogenic lipoproteins and lipids associated with ABL, there was interest in inhibiting MTTP therapeutically. Lomitapide is an oral MTP inhibitor that has been developed over the course of many years (10,15). In early trials, it was tested at a relatively high dose and the side effect profile was prohibitive (nausea, flatulence, and diarrhea). The more recent clinical trial program tested lower doses with drug titration in subjects with HoFH (10,15). On an intention to treat basis, LDL-C was decreased by 40% and apolipoprotein B by 39% (10). In patients who were actually taking lomitapide, LDL-C levels were reduced by 50% (10). In addition to decreasing LDL-C levels, non-HDL-C levels were decreased by 50%, Lp(a) by 15%, and triglycerides by 45% (10). Lomitapide received the same limited indication as mipomersen for adjunctive treatment of patients with HoFH (10). Besides the gastrointestinal issues already alluded to, its side effect profile includes hepatic steatosis (10). Its long-term safety has not been established.

 

PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9 (PCSK9)

 

Proprotein convertase subtilisin/ kexin type 9 (PCSK9) belongs to the proprotein convertase class of serine proteases (16-18). After synthesis, PCSK9 undergoes autocatalytic cleavage. This step is required for secretion, most likely because the prodomain functions as a chaperone and facilitates folding (16,17). PCSK9 is associated with LDL particles and the LDL-receptor (LDLR) (18). In 2003, Abifadel reported the seminal work that mapped PCSK9 as the third locus for autosomal dominant hypercholesterolemia (Familial Hypercholesterolemia- FH) (19). This finding revealed a previously unknown actor involved in cholesterol homeostasis and served to launch a series of investigations into PCSK9 biology. As it turns out, PCSK9 functions as a central regulator of plasma LDL-C concentration (16-18). It binds to the LDLR and targets it for destruction in the lysosome (16-18). Overactivity of PCSK9 results in a decrease in LDLR and an increase in LDL-C levels while decreased activity of PCSK9 results in an increase in LDLR and a decrease in LDL-C.

 

Since the discovery of gain-of-function mutations in PCSK9 as a cause of FH, investigators have also uncovered loss of function mutations of PCSK9. Loss-of-function mutations in PCSK9 are associated with low LDL-C levels and markedly reduced ASCVD (16,17). In African Americans 2.6 percent had nonsense mutations in PCSK9 that resulted in a 28 percent reduction in LDL-C and an 88 percent reduction in the risk of coronary heart disease (20). The hypolipidemia is not associated with liver abnormalities or other disorders. Interestingly, rare individuals homozygous or compound heterozygotes for loss of function mutations in PCSK9 have been reported with extremely low levels of LDL-C (~15 mg/dl), normal health and reproductive capacity, and no evidence of neurologic or cognitive dysfunction (18,21,22). Collectively, these observations served as further motivation to pursue antagonism of PCSK9 as a therapeutic target. Antagonizing PCSK9 would prolong the lifespan of LDLR, leading to significant reductions in plasma LDL-C levels.

 

There are numerous approaches to inhibiting PCSK9 including humanized monoclonal antibodies (mAbs), gene silencing, and use of small inhibitory peptides (18). Thus far, approaches utilizing mAbs are FDA approved (10). The two fully human monoclonal antibodies (alirocumab and evolocumab) targeting PCSK9 became commercially available in 2015. Clinical trials of mAbs targeted to PCSK9 have demonstrated remarkable efficacy in LDL-C reduction (~50% reduction in LDL-C as monotherapy and ~65% reduction in LDL-C in combination with a statin) with an excellent short-term safety and tolerability profile (10). Moreover, a large randomized controlled trial (FOURIER) demonstrated incremental improvement with a 15% reduction in the composite primary endpoint of major adverse cardiovascular outcome with addition of evolocumab on top of standard of care in patients with stable vascular disease (23). Additionally, the ODYSSEY OUTCOMES trial also demonstrated a similar reduction in major adverse cardiovascular events with alirocumab vs. placebo in patients with recent acute coronary syndromes (24). Finally, inclisiran, a small interfering RNA that inhibits translation of PCSK9, is approved in Europe but not yet in the US (10).

 

FAMILIAL COMBINED HYPOLIPIDEMIA

 

Familial combined hypolipidemia (FCH) is due to loss of function mutations in the gene encoding angiopoietin-like protein 3 (ANGPTL3) (25,26). ANGPTL3 inhibits various lipases, such as lipoprotein lipase and endothelial lipase (25,26). Therefore, loss of function mutations in ANGPTL3 relinquishes this inhibition resulting in more efficient metabolism of VLDL and HDL particles (25,26). In addition, to increasing VLDL clearance the secretion of VLDL is also decreased due to a decrease in free fatty acid flux to the liver (25). LDL clearance is increased but the mechanism remains to be fully elucidated (25). Studies have suggested that ANGPTL3 inhibition lowers LDL-C by limiting LDL particle production due to ANGPTL3 inhibition and increased endothelial lipase activity reducing VLDL-lipid content and size, generating remnant particles that are efficiently removed from the circulation rather than being further metabolized to LDL (27). Clinically, FCH manifests as panhypolipidemia (decreased triglycerides, LDL-C, and HDL-C) (25,26). Interestingly, heterozygotes for certain nonsense mutations in the first exon of ANGPTL3 have moderately reduced LDL-C and triglyceride levels while compound heterozygotes have significant reductions in HDL-C as well (25,26).  Homozygosity or compound heterozygosity for other loss-of-function mutations in exon 1 of ANGPTL3 have no detectable ANGPTL3 in plasma and striking reductions of atherogenic lipoproteins with HDL particles containing only apo A-I and preß-HDL. Individuals who are heterozygous for the loss of function mutations in ANGPTL3 have normal HDL-C levels and significantly reduced LDL-C (<25th percentile) (25,26).

 

A pooled analysis of cases of familial combined hypolipidemia was published 2013 (28). One hundred fifteen individuals carrying 13 different mutations in the ANGPTL3 gene (14 homozygotes, 8 compound heterozygotes, and 93 heterozygotes) and 402 controls were evaluated. Homozygotes and compound heterozygotes (two mutant alleles) had no measurable ANGPTL3 protein. In heterozygotes, ANGPTL3 was reduced by 34-88%, according to genotype. All cases (homozygotes and heterozygotes) demonstrated significantly lower concentrations of all plasma lipoproteins (except for Lp(a)) as compared to controls. Familial combined hypolipidemia is not associated with any comorbidity. In fact, the prevalence of fatty liver was the same as controls. However, ANGPTL3 deficiency is associated with a reduced risk of cardiovascular disease (25,29).

 

Recently, evinacumab, a human monoclonal antibody against ANGPTL3, was approved for the treatment of Homozygous Familial Hypercholesterolemia (10). Evinacumab decreases LDL-C levels by mechanisms independent of LDL receptor activity (10).

 

CHYLOMICRON RETENTION DISEASE

 

Chylomicron retention disease (CMRD), known also as Anderson’s disease for the individual who first described the condition in 1961, is a rare inherited lipid malabsorption syndrome (30,31). It is due to mutations in the SAR1B gene which codes for the protein SAR1b, a small GTPase, involved in intracellular protein trafficking (30). Mutations in SAR1b result in the failure of pre-chylomicrons to move from the endoplasmic reticulum to the golgi (30). This disorder usually presents in young infants with diarrhea, steatorrhea, abdominal distention, and failure to thrive (30,31). Patients with CMRD demonstrate a specific autosomal recessive hypocholesterolemia that differs from other familial hypocholesterolemias. CMRD is associated with a 50% reduction in both plasma LDL-C and HDL-C with normal fasting triglyceride levels (30,31). Mutations in SAR1B do not affect VLDL secretion by the liver. The decrease in HDL-C is postulated to be due to a decrease in Apo A-I secretion and cholesterol efflux by the small intestine (30). The mechanism accounting for the decrease in LDL-C is not clear. The usual increase in triglycerides and chylomicron levels following a fat meal is blocked (30). The duodenal mucosa is white on endoscopy and intestinal biopsy reveals cytosolic lipid droplets and lipoprotein-sized particles in enterocytes (30). As one would expect the absorption of fat-soluble vitamins (A, D, K, and E) and essential fatty acids is impaired (30,31).

 

Treatment for individuals with CMRD is similar to that described above for individuals with ABL (31).

 

REFERENCES

 

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Hypophysitis

ABSTRACT

 

Hypophysitis is an inflammation of the pituitary gland and is a rare cause of hypopituitarism. It can be primary (idiopathic) or secondary to sella and parasellar lesions, systemic diseases, or drugs (mainly immune checkpoint inhibitors). Primary hypophysitis has five histologic variants: lymphocytic, granulomatous, xanthomatous, IgG4-related, and necrotizing. Lymphocytic hypophysitis is the most common form; it is likely an autoimmune disease and is more frequently observed in females during pregnancy or postpartum. Granulomatous hypophysitis is the second most common variant and possible secondary causes of granulomatous infiltration of the pituitary should be excluded before concluding that a case of granulomatous hypophysitis is idiopathic. Xanthomatous, necrotizing, and IgG4-related hypophysitis are very rare and the latter is often the manifestation of a systemic disease with multi-organ involvement (IgG4-related disease). Immune checkpoint inhibitors are monoclonal antibodies increasingly used for the treatment of solid and hematological malignancies. They cause a T-lymphocyte activation and proliferation that lead to the anti-tumor response, and may cause autoimmune manifestations known as part of what is called “immune-related adverse events”. A significant number of patients treated with immune checkpoint inhibitors develop immune-related hypophysitis and require prompt diagnosis and treatment. Regardless of the etiology, patients with hypophysitis present with various signs and symptoms caused by the pituitary inflammation that can lead to hypopituitarism and compression of sella and parasellar structures. Contrary to other causes of hypopituitarism, adrenocorticotropic hormone and thyroid-stimulating hormone deficiencies are very frequent in the early stages of hypophysitis and must be identified immediately. The diagnosis of hypophysitis is based on clinical, laboratory, and radiological data; while pituitary biopsy is the gold standard test for diagnosing primary hypophysitis, it should be reserved only for selected cases. Magnetic resonance imaging is the technique of choice for suspected hypophysitis, and the main differential diagnoses are pituitary adenomas in adults, germinomas, and Langerhans cell histiocytosis in adolescents, and metastases in those receiving immune checkpoint inhibitors. The mainstay of treatment of patients with hypophysitis is pituitary hormone replacement. Those with severe signs and symptoms of sella compression should be treated with high-dose glucocorticoids, which usually cause an excellent initial response, although relapse of the pituitary inflammation is common. Pituitary surgery should be considered in patients who do not respond to glucocorticoids and have progressive and debilitating symptoms. Pituitary fibrosis and atrophy often develop in the late stage of the disease, with persistent hypopituitarism.

 

 

INTRODUCTION

 

Hypophysitis is a generic term that includes a variety of conditions that cause inflammation of the pituitary gland. It is an infiltrative cause of hypopituitarism and can cause symptoms related to sella compression and pituitary hormone deficiencies.

 

Hypophysitis can be classified according to the anatomic location of pituitary involvement (adenohypophysitis, infundibulo-neurohypophysitis, or panhypophysitis) and the cause (primary or secondary forms) (Table 1) (1-4). The primary forms are characterized by an idiopathic inflammatory process confined to the pituitary gland, while the secondary forms are triggered by a definite etiology (drugs and intracranial or systemic diseases). Five histologic variants of primary hypophysitis have been described: lymphocytic, granulomatous, xanthomatous, IgG4-related, and necrotizing (Table 1). Lymphocytic hypophysitis is the most common form of hypophysitis and occurs most commonly in women during late pregnancy and the postpartum period. However, thanks to the increasing use over the last two decades of monoclonal antibodies inhibiting immune checkpoints for the treatment of several solid and hematological malignancies, new immune-related adverse events have emerged, with hypophysitis being a relatively common occurrence.

 

Table 1. Classification of Hypophysitis

CAUSE: PRIMARY AND SECONDARY HYPOPHYSITIS

· Primary hypophysitis:

o Isolated

o Associated with autoimmune diseases:

•  Polyglandular autoimmune syndromes

•  Autoimmune thyroiditis (Hashimoto thyroiditis)

•  Autoimmune adrenalitis

•  Type 1 diabetes mellitus

•  Lymphocytic parathyroiditis

•  Idiopathic inflammatory myopathy

•  Systemic lupus erythematosus

•  Sjogren’s syndrome

•  Rheumatoid arthritis

•  Primary biliary cirrhosis

•  Atrophic gastritis

•  Optic neuritis

•  Myocarditis

•  Temporal arteritis

•  Bechet’s disease

•  Retroperitoneal fibrosis

•  Erythema nodosum

•  Idiopathic thrombocytopenic purpura

•  Dacryoadenitis

•  Autoimmune thrombocytopenia

•  Autoimmune encephalitis

· Secondary hypophysitis:

o Drugs:

•  Immune checkpoint inhibitors

•  Interferon-α

•  Ribavirin

•  Ustekinumab

o Sella and parasellar diseases*:

•  Germinoma

•  Rathke’s cleft cyst

•  Craniopharyngioma

•  Pituitary adenoma

•  Primary pituitary lymphoma

o Systemic diseases:

•  IgG4-related disease**

•  Sarcoidosis

•  Granulomatosis with polyangiitis (Wegener’s granulomatosis)

•  Langerhans cell histiocytosis

•  Erdheim-Chester’s disease

•  Rosai-Dorfman disease

•  Inflammatory pseudotumor

•  Tolosa-Hunt syndrome

•  Takayasu’s arteritis

•  Cogan’s syndrome

•  Crohn’s disease

o Thymoma and other malignancies (anti-Pit-1 antibody syndrome)

o Infections:

•  Bacteria (Mycobacterium tuberculosis; Treponema pallidum; Tropheryma whipplei; Borrelia; Brucella)

•  Viruses (Cytomegalovirus; Herpes simplex; Varicella-zoster virus; Influenza viruses; Coronavirus; Enterovirus; Coxsackie; Tick-Borne encephalitis virus; Hantavirus)

•  Mycoses (Aspergillus; Nocardia; Candida albicans; Pneumocystis jirovecii)

•  Parasites (Toxoplasma gondii)

ANATOMIC LOCATION OF PITUITARY INVOLVEMENT

· Adenohypophysitis: the inflammation involves the anterior pituitary. It accounts for ~65% of cases of primary hypophysitis

· Infundibulo-neurohypophysitis: the inflammation involves the posterior pituitary and the stalk. It accounts for ~10% of cases of primary hypophysitis

· Panhypophysitis: the inflammation involves the entire gland. It accounts for ~25% of cases of primary hypophysitis

HISTOPATHOLOGY FORMS OF PRIMARY HYPOPHYSITIS

· Lymphocytic hypophysitis (68%)

· Granulomatous hypophysitis (19%)

· IgG4-related (plasmocytic) hypophysitis (8%)**

· Xanthomatous hypophysitis (4%)

· Necrotizing hypophysitis (<1%)

· Mixed forms (lymphogranulomatous; xanthogranulomatous)

* The infiltrate focuses around the lesion rather than diffuse in the entire gland. This secondary form of pituitary infiltration is generally a histopathological finding and patient’s signs and symptoms are otherwise related to the primary sella and parasellar mass.

** IgG4-related hypophysitis can be isolated, but is often a manifestation of systemic disease with the involvement of multiple organs.

 

PRIMARY HYPOPHYSITIS

 

Primary hypophysitis is a rare disease, with just over 1300 published cases so far (5). The incidence is estimated to be ~1 in 9 million/year (4,6), and hypophysitis accounts for ~0.4% of pituitary surgery cases (2). Five histologic variants of primary hypophysitis have been described, and there are mixed forms as well. Table 2 summarizes the epidemiological and histopathological features of these variants (2,5,7-9). Primary hypophysitis, apart from the rare IgG4-related and necrotizing variants, occurs more frequently in young females. The clinical manifestations of all forms of primary hypophysitis are similar and are linked to the degree of pituitary involvement and the associated hormonal deficiencies.

 

Table 2. Characteristics of the Various Forms of Primary Hypophysitis

 

Lymphocytic

Granulomatous

IgG4-related

Xanthomatous

Necrotizing

Prevalence

The most common subtype (68%*).

The second most common subtype (19%*).

Rare (8%*). Higher prevalence in Japan and Korea.

Very rare (4%*).

Extremely rare (<1%).

Gender predominance

Female, ~3:1

Female, ~3:1

Male, ~2:1

Female, ~3:1

Male, ~2:1

Association with pregnancy

Yes. ~70% of patients present during pregnancy or postpartum.

No

No

No

No

Mean age at presentation

4th decade (women).

5th decade (men).

5th decade

7th decade (men).

2nd-3rd decade (women).

4th decade

Six cases reported (aged 12, 20, 33, 39, 40, and 52).

Histopathology

Diffuse lymphocyte infiltration (primarily T cells) of the pituitary gland. Lymphoid follicles can be observed and occasional plasma cells, eosinophils, and fibroblasts may also be present. Pituitary fibrosis and atrophy may occur in later stages of the disease.

Large numbers of multinucleated giant cells and histiocytes with granuloma formation.

Extensive gland infiltration by plasma cells with a high degree of IgG4 positivity. Storiform fibrosis is observed**. Pituitary fibrosis and atrophy occur in later stages of the disease, if not treated.

Foamy histiocytes (lipid-rich macrophages) without the presence of granulomas. Plasma cells and small round mature lymphocytes are also observed. Pituitary fibrosis may be seen in later stages of the disease.

Diffuse non-hemorrhagic necrosis with surrounding lymphocytes, plasma cells and eosinophils.

* Prevalence derived by published cases after excluding those where the pathologic variant is unknown. Forty-one cases with mixed histology findings have been published.

** Storiform fibrosis: dense, wire-like strands of fibrotic collagen deposition radiating outward from a central point.

 

Lymphocytic Hypophysitis

 

Lymphocytic hypophysitis is the most common histologic variant of primary hypophysitis (4,5,8). It shows a striking temporal association with pregnancy, with ~70% of cases in women presenting during pregnancy or postpartum. Most patients present in the last month of pregnancy or in the first 2 months after delivery (4). Lymphocytic hypophysitis is believed to have an autoimmune etiology. This is supported by the lymphocytic infiltration of the pituitary, the link with pregnancy, the frequent association with other autoimmune diseases (Table 1), the frequent finding of pituitary antibodies in these patients (see below), the association with particular human leukocyte antigen alleles (1), the improvement of symptoms in response to immunosuppressive drugs, and animal models of primary hypophysitis (10).

 

Granulomatous Hypophysitis

 

Granulomatous hypophysitis is the second most common subtype of primary hypophysitis and its etiology is unknown. Before concluding that a case of granulomatous hypophysitis is “primary” (i.e., idiopathic), known possible causes of granulomatous infiltration of the pituitary should be excluded. Possible secondary causes of granulomatous hypophysitis include tuberculosis, sarcoidosis, syphilis, Langerhans’ histiocytosis, granulomatosis with polyangiitis (formerly known as Wegener’s granulomatosis), and Rathke’s cleft cyst rupture (see “Hypophysitis secondary to sella and parasellar disease” and “Hypophysitis secondary to systemic disease” below). (11).

 

 

IgG4-related hypophysitis can be isolated (primary hypophysitis) but is often a manifestation of systemic disease with involvement of multiple organs (14,15). Some authors include IgG4-related hypophysitis among the histologic variants of primary hypophysitis, while others report this among the secondary forms of hypophysitis. Considering that the diagnosis and management does not change according to the classification used, we will discuss the features of IgG4-related hypophysitis in this section.

 

The etiology of this disease is poorly understood and may involve autoimmunity and/or an abnormal tolerance to unspecified allergens and infectious agents (16,17). IgG4-related disease is diagnosed more frequently in older males and is characterized by a dense lymphoplasmacytic infiltration with a predominance of IgG4-positive plasma cells in the affected tissue and storiform fibrosis in the more advanced stages of the disease (Table 2). One or (more frequently) multiple organs can be affected including lymph nodes, pancreas, liver, salivary and lacrimal glands, retroperitoneum, aorta, pericardium, thyroid, lungs, kidneys, skin, stomach, prostate, ovaries, and the pituitary gland (17-19). Overall, the prevalence of pituitary involvement in IgG4-related disease is believed to be low (2-8%) (20). Nonetheless, a recent cohort study from Japan screened 27 patients with IgG4-related pancreatitis via pituitary MRI and found 1 case of hypophysitis with hypopituitarism and 4 cases of empty sella (21). Patients with pituitary abnormalities were more likely to have multi-organ disease. If confirmed by large-scale studies, these findings would advocate for screening for hypophysitis especially in patients with multiple IgG4-related organ involvement.

 

IgG4-related disease is considered a rare cause of hypophysitis, although a Japanese group reported a strikingly high prevalence of IgG4-related hypophysitis in 170 consecutive patients with hypopituitarism/central diabetes insipidus and a clinical diagnosis of hypophysitis (4% and 30% respectively) (22). Moreover, Bernreuther et al. reviewed retrospectively 29 cases of biopsy-proven primary hypophysitis previously diagnosed as “lymphocytic” or “not otherwise specified, non-granulomatous” and found that 41.4% of cases fulfilled the criteria for IgG4-related hypophysitis, suggesting that this entity might be more frequent than previously thought (23). Two recent reviews of the literature found that the epidemiology of IgG4-related hypophysitis may differ according to sex: affected men were older, more likely to have systemic disease and higher IgG4 serum levels; women were younger and often presenting with isolated pituitary disease, lower IgG4 serum levels, and a concomitant diagnosis of other autoimmune diseases (24,25).

 

The diagnosis of IgG4-related hypophysitis is confirmed by characteristic histopathologic findings at pituitary biopsy. However, pituitary biopsy is an invasive procedure and other criteria can be used to establish the diagnosis (Table 3)(26).

 

Table 3. Diagnostic Criteria for IgG4-related Hypophysitis

Criteria

Established diagnosis

Criterion 1

PITUITARY HISTOPATHOLOGY: Mononuclear infiltration of the pituitary gland, rich in lymphocytes and plasma cells, with >10 IgG4-positive cells/high-power field. *

CRITERION 1

 

or

 

CRITERIA 2 + 3

 

or

 

CRITERIA 2 + 4 + 5

Criterion 2

PITUITARY MRI: Sella mass or thickened pituitary stalk.

Criterion 3

OTHER INVOLVEMENT: Biopsy-proven involvement in other organs.

Criterion 4

SEROLOGY: Serum IgG4 level >140 mg/dL (1.4 g/L).

Criterion 5

RESPONSE TO TREATMENT: Shrinkage of the pituitary mass and symptom improvement with corticosteroids.

* Low level of infiltration may be seen if the patient is receiving treatment with glucocorticoids (27)

 

It should be considered that patients with IgG4-related hypophysitis have multi-organ involvement in 60-90% of cases. Therefore, they should receive an extensive evaluation for establishing the extent of the disease after the initial diagnosis. The diagnostic work-up should include physical examination, laboratory evaluation, and whole-body imaging (19).

 

Xanthomatous Hypophysitis

 

The pituitary shows cystic-like areas of liquefaction infiltrated by lipid-rich macrophages. It has been suggested that many cases of xanthomatous hypophysitis may represent an inflammatory response to components of a ruptured Rathke’s cleft cyst (see “Hypophysitis secondary to sella and parasellar disease” below) (12,13).

 

Necrotizing Hypophysitis

 

Necrotizing hypophysitis has been reported in six patients (of which only five histology-proven) (28-30). Five patients presented with diabetes insipidus and some degree of anterior pituitary dysfunction was described in all reported cases. Frontal headache at presentation was reported in three patients (28,29). One patient presented with photophobia (29). Five patients were treated surgically and all but one had persistent postoperative panhypopituitarism and central diabetes insipidus (28-31).

 

Clinical Presentation of Primary Hypophysitis

 

The signs and symptoms at diagnosis, as well as the pituitary hormone abnormalities depend on the degree of pituitary involvement (Table 4) (4,5,8).

 

Primary hypophysitis more frequently involves the anterior pituitary and patients typically present with severe headaches, visual disturbances due to chiasmal compression, and symptoms of adrenal insufficiency. Contrary to other causes of hypopituitarism, impaired adrenocorticotropic hormone (ACTH) and thyroid-stimulating hormone (TSH) secretion is very frequent in the early stages of primary hypophysitis, putting these patients at increased risk of life-threatening adrenal insufficiency. A large case series from Germany has highlighted that secretion of gonadotropins is also impaired very frequently in these patients (32). Growth hormone (GH) deficiency and hyperprolactinemia can also occur.

 

Less frequently, the inflammation can involve primarily the posterior pituitary and the stalk. Patients with infundibulo-neurohypophysitis typically present with diabetes insipidus and other pituitary hormone deficiencies are less common. As expected, signs of both anterior and posterior pituitary involvement coexist in panhypophysitis (that is, inflammation of the entire gland).

 

Table 4. Clinical Presentation and Prevalence of Pituitary Hormone Abnormalities at Diagnosis in Patients with Primary Hypophysitis According to the Degree of Pituitary Involvement

SIGNS AND SYMPTOMS AT DIAGNOSIS

Adenohypophysitis

(~65% of cases)

Infundibulo-neurohypophysitis

(~10% of cases)

Panhypophysitis

(~25% of cases)

All forms *

· Headache: 53%

· Visual disturbances: 43%

· Adrenal insufficiency: 42%

· Hyperprolactinemia: 23%

· Hypothyroidism: 18%

· Hypogonadism: 12%

· Lactation failure: 11%

· Polydipsia/polyuria: 1%

· Polydipsia/polyuria: 98%

· Headache: 13%

· Adrenal insufficiency: 8%

· Hyperprolactinemia: 5%

· Hypogonadism: 3%

· Visual disturbances: 3%

· Hypothyroidism: 0%

· Lactation failure: 0%

· Polydipsia/polyuria: 83%

· Headache: 41%

· Adrenal insufficiency: 19%

· Visual disturbances: 18%

· Hypothyroidism: 17%

· Hyperprolactinemia: 17%

· Hypogonadism: 14%

· Lactation failure: 5%

· Headache: 48%

· Adrenal insufficiency: 38%

· Polydipsia/polyuria: 34%

· Visual disturbances: 32%

· Hypogonadism: 21%

· Hyperprolactinemia: 20%

· Hypothyroidism: 16%

· Lactation failure: 8%

PITUITARY HORMONE ABNORMALITIES AT DIAGNOSIS

Adenohypophysitis

(~65% of cases)

Infundibulo-neurohypophysitis

(~10% of cases)

Panhypophysitis

(~25% of cases)

All forms

· ACTH deficiency: 56%

· TSH deficiency: 44%

· FSH/LH deficiency: 42%

· GH decreased: 26%

· Hyperprolactinemia: 25%

· Hyperprolactinemia: 23% ***

· ADH deficiency: 0%

· ADH deficiency: 98%

· FSH/LH deficiency: 8% **

· Hyperprolactinemia: 5% ***

· Hyperprolactinemia: 0%

· ACTH deficiency: 0%

· TSH deficiency: 0%

· GH decreased: 0% **

· ADH deficiency: 95%

· GH decreased: 51%

· FSH/LH deficiency: 47%

· ACTH deficiency: 46%

· Hyperprolactinemia: 40% ***

· TSH deficiency: 39%

· Hyperprolactinemia: 16%

· ADH deficiency: 63%

· ACTH deficiency: 60%

· FSH/LH deficiency: 55%

· TSH deficiency: 50%

· Hyperprolactinemia: 39%

· GH decreased: 37%

Abbreviations: ACTH, adrenocorticotropic hormone; ADH, antidiuretic hormone; FSH, follicle-stimulating hormone; GH, growth hormone; LH, luteinizing hormone; TSH, thyroid-stimulating hormone.

* Other possible symptoms at diagnosis include weight gain (~20%) and temperature dysregulation (rare) (32,33).

** Some case series have reported a high prevalence of GH and FSH/LH deficiency in patients with infundibulo-neurohypophysitis (34).

*** Hyperprolactinemia may be related to stalk compression (disconnection hyperprolactinemia) or to the immune-mediated destruction of prolactin-secreting cells.

 

Granulomatous hypophysitis can be associated with more severe symptoms than lymphocytic hypophysitis, with two case series documenting more frequent occurrence of headache, chiasmal compression, and hypopituitarism (32,35). A review of the literature found that the most common symptoms of granulomatous hypophysitis at presentation were headache (61%), visual changes (40%), polyuria/polydipsia (27%) and cranial nerve palsies (27%); panhypopituitarism and diabetes insipidus were found in 49% and 27% of cases, respectively (11). Cases of compression of the cavernous part of the internal carotid artery have also been described (36).

 

Clinical data regarding xanthomatous and IgG4-related hypophysitis are less robust due to the rarity of these variants. Gutenberg et al. found that xanthomatous hypophysitis did not cause chiasmal compression and was associated with a low risk of diabetes insipidus and a less severe anterior pituitary hormone impairment than lymphocytic or granulomatous hypophysitis (FSH/LH and GH deficiencies are more common than TSH and ACTH deficiencies) (35). IgG4-related hypophysitis involves frequently both the pituitary and the stalk (~65%) and causes panhypopituitarism, anterior hypopituitarism and central diabetes insipidus in ~50%, ~25% and ~18% of cases, respectively (37). Cases of intrachiasmal abscess and spreading to the cavernous sinus have also been reported (38,39).

 

Primary hypophysitis is rare in children, with less than 100 cases reported in the literature of which only a few were biopsy-proven (40-42). The clinical presentation, however, seems to differ from adults. A review of the literature showed that the most common presenting symptoms in children are caused by antidiuretic hormone (ADH) deficiency (85%) (42). GH deficiency is found in 76% of cases, while FSH/LH, TSH and ACTH deficiencies were less common than in adults (32%, 29% and 20%, respectively). Headaches and visual disturbances were also rarely reported (17% and 8% of cases, respectively) (42). As central diabetes insipidus and growth retardation are the most common presenting symptoms in children with primary hypophysitis, the more frequent intracranial germinomas and Langerhans cell histiocytosis, as well as craniopharyngiomas, have to be considered in the differential diagnosis (43). Moreover, children with a presumptive diagnosis of hypophysitis are at risk of developing germinomas later in life (up to 3 years after the initial diagnosis) and require extended follow-up (42,44). Germinomas are also a documented cause of secondary hypophysitis (see “Hypophysitis secondary to sella and parasellar disease” below).

 

Imaging and Differential Diagnosis of Primary Hypophysitis

 

Magnetic resonance imaging (MRI) of the sella region typically shows an enlarged pituitary. In order to avoid unnecessary surgery, primary hypophysitis needs to be differentiated from other sella and parasellar masses (Table 5)(45), with pituitary adenomas being the most frequent differential diagnosis in adults.

 

Table 5. Differential Diagnosis of Hypophysitis

SELLA AND PARASELLAR MASSES

·   Pituitary adenomas (including pituitary apoplexy);

·   Pituitary metastases: the differential diagnosis is particularly important in patients with suspected hypophysitis and malignant tumors receiving immune checkpoint inhibitors;

·   Other sella and parasellar tumors (e.g., craniopharyngiomas, germinomas, gliomas, lymphomas, meningiomas, pituicytomas, chordomas, teratomas, dermoids and epidermoids);

·   Rathke’s cleft cyst;

·   Abscesses.

OTHER

·   Physiological hypertrophy of the pituitary in children and adolescents (especially pubertal females) and perimenopausal women;

·   Pituitary hyperplasia associated with pregnancy;

·   Sheehan’s syndrome at onset;

·   Thyrotropic hyperplasia associated with severe, untreated primary hypothyroidism.

 

Primary hypophysitis typically presents as a homogeneous pituitary enlargement with intense and homogeneous enhancement post-gadolinium and no deviation of the stalk (Figure 1); these and other features can help differentiate between primary hypophysitis and pituitary adenomas at MRI (Table 6) (1,4,46,47). Gutenberg et al. developed a score using variables such as age, association with pregnancy, and MRI findings to distinguish hypophysitis from pituitary adenomas with high accuracy (47). Further differential diagnoses, especially for lymphocytic hypophysitis, are the physiologic pituitary enlargement associated with pregnancy and Sheehan’s syndrome, although these patients have no history of obstetric hemorrhage (48,49). A cautious balance between radiological, clinical, and laboratory findings is necessary to reach the correct diagnosis and avoid inappropriate treatment (50).

 

 Table 6. Differential Imaging Characteristics of Primary Hypophysitis and Pituitary Adenomas

MRI

Primary hypophysitis

Pituitary adenoma

Pre-gadolinium

ACUTE / SUB-ACUTE PHASE:

·   Homogeneous pituitary enlargement with symmetrical suprasellar expansion;

·   Suprasellar extension with compression and displacement of chiasm;

·   Stalk thickened but not deviated; *

·   Loss of bright spot of the neurohypophysis in case of involvement of the posterior pituitary. **

 

CHRONIC PHASE:

·   Pituitary atrophy;

·   Empty sella.

·   Microadenoma (<1cm): unilateral, asymmetric endosellar mass;

·   Macroadenoma (>1cm): expanding, not homogeneous pituitary mass with asymmetrical suprasellar expansion;

·   Compression and displacement of chiasm (macroadenoma);

·   Contralateral deviation of the stalk;

·   The bright spot of the neurohypophysis can be usually seen. **

Post-gadolinium

·   Intense and homogeneous enhancement of the pituitary mass. Cystic areas have been described, especially in the xanthomatous variant;

·   Dural tail sign can be present (thickening of the enhanced dura that resembles a tail extending from a mass). ***

·   Slight, delayed and not homogeneous enhancement. Cystic and necrotic areas are frequently observed in macroadenomas;

·   Dural tail usually absent. ***

Abbreviations: MRI, magnetic resonance imaging.

* An enlarged pituitary stalk can also be found in other intracranial pathologies (e.g., sarcoidosis, metastases, Langerhans cell histiocytosis, germinoma, craniopharyngioma, astrocytoma, pituitary adenoma, lymphoma, tuberculosis, Erdheim-Chester’s disease) (51).

** The bright spot may be absent in up to 20% of healthy subjects (especially the elderly).

*** The dural tail sign is not specific to hypophysitis. It can be observed in meningioma (most frequently) and other intracranial pathologies (e.g. lymphoma, chloroma, metastasis, multiple myeloma, glioblastoma multiforme, aspergillosis, chordoma, schwannoma, pleomorphic xanthoastrocytoma, hemangiopericytoma, granulomatosis with polyangiitis, sarcoidosis, medulloblastoma, eosinophilic granuloma, pituitary adenoma, pituitary apoplexy, Erdheim-Chester’s disease) (52)

Figure 1. Magnetic resonance imaging findings in a case of primary hypophysitis. Panel A) T1-weighted image, sagittal section. Panel B) T1-weighted image, coronal section. Panel C) T1-weighted image post-gadolinium, sagittal section. Panel D) T1-weighted image post-gadolinium, coronal section. A homogeneous enlargement of the pituitary with thickening of the stalk can be seen. The mass shows intense and homogeneous enhancement post-gadolinium.

Autoantibodies in Primary Hypophysitis

 

Several authors have assessed the presence and utility of serum autoantibodies (pituitary and/or hypothalamic antibodies) in patients with primary hypophysitis:

 

  • An autoimmune etiology for lymphocytic hypophysitis was suggested by the presence of pituitary antibodies that may recognize α-enolase, GH, the pituitary gland-specific factors 1a and 2 (PGSF1a and PGSF2), regulatory prohormone-processing enzymes commonly produced in the pituitary gland (PC1/3, PC2, CPE and 7B2), secretogranin II, chromosome 14 open reading frame 166 (C14orf166), the corticotroph-specific transcription factor TPIT, and chorionic somatomammotrophin (HCS) (53-61). Several techniques have been used to detect pituitary antibodies in primary hypophysitis (ELISA, radioligand assay, immunoblotting, and immunofluorescence) and the prevalence of antibody-positive hypophysitis is 11-73% depending from the antigen(s) tested and the technique used (7,62). However, the pathogenic role of these autoantibodies is unclear and they are not specific to hypophysitis. For example, pituitary antibodies were identified by indirect immunofluorescence in ~45% of patients with biopsy-proven hypophysitis, but were also found in the serum of patients with isolated central diabetes insipidus (35%), germinomas (33%), isolated anterior hormone deficiencies (29%), prolactinomas (27%), Rathke’s cleft cysts (25%), craniopharyngiomas (17%), non-functioning pituitary tumors (13%), GH-secreting pituitary tumors (12%), and healthy subjects (5%) (62-65). They can also be found in patients with autoimmune endocrine disorders, especially Hashimoto thyroiditis (63). However, indirect immunofluorescence using human pituitary gland as a substrate and showing a granular cytosolic staining pattern was most commonly found in patients with hypophysitis and isolated hormone deficiencies (62); therefore, the finding of this staining pattern can be useful to clinicians in establishing a diagnosis of hypophysitis;

 

  • The detection of hypothalamic antibodies targeting corticotropin-releasing hormone (CRH)-secreting cells in some patients with GH/ACTH deficiency but with pituitary antibodies targeting only GH-secreting cells indicates that an autoimmune aggression to the hypothalamus can be responsible for some cases of lymphocytic hypophysitis (66). Consequently, not only pituitary but also hypothalamic autoimmunity may contribute to anterior pituitary dysfunction in a subset of patients with primary hypophysitis;

 

  • A search for ADH antibodies in patients with primary hypophysitis may help identifying patients who are prone to developing autoimmune central diabetes insipidus (67). These antibodies alone are not a good diagnostic marker for posterior pituitary involvement, but may serve as a predictive marker of gestational or post-partum diabetes insipidus (68,69);

 

  • Anti-Rabphilin antibodies have been proposed to be a biomarker for lymphocytic infundibulo-neurohypophysitis (70). Rabphilin is involved in the release of hormones or neurotransmitters and is expressed mainly in the brain, including the posterior pituitary and hypothalamus where ADH is present. Whether anti-Rabphilin antibodies are a cause of central diabetes insipidus or a result of infundibulo-neurohypophysitis is unknown. However, anti-Rabphilin antibodies are detected in 76% of patients with infundibulo-neurohypophysitis and 11% of patients with lymphocytic hypophysitis. In contrast, these antibodies are absent in patients with sella/suprasellar masses without lymphocytic hypophysitis, suggesting that this antibody may serve as a biomarker for the diagnosis of infundibulo-neurohypophysitis and may be useful for the differential diagnosis in patients with central diabetes insipidus (45);

 

  • Primary hypophysitis can eventually evolve in pituitary fibrosis and atrophy, documented at imaging as an “empty sella”. Lupi et al. have found pituitary antibodies in 6% of patients with an empty sella not linked to previous head trauma. In this cohort, the presence of pituitary antibodies also correlated with the presence of hypopituitarism (71);

 

  • Antibodies recognizing GH and one peptide from proopiomelanocortin (POMC) have been described in a patient with IgG4-related hypophysitis (72).

 

Natural History of Primary Hypophysitis

 

Primary hypophysitis can be self-limiting and spontaneous remission may occur (Figure 2). Considering the low prevalence of the disease, however, robust data regarding the natural history of primary hypophysitis are lacking (54). Moreover, most of the literature regards lymphocytic hypophysitis, while data from other histology subtypes are less robust. A review of 76 cases of primary hypophysitis from Germany has shown that patients not receiving any active treatment had improvement, stability or progression of the pituitary involvement at MRI in 46%, 27% and 27% of cases, respectively; pituitary deficiencies improved, remained stable or worsened in 27%, 55% and 18% of patients, respectively (73). A previous study by Khare et al. showed that spontaneous resolution of sella compression symptoms occurred in all patients managed conservatively and that 33% had complete or partial recovery of pituitary function (74). Park et al. also reported similar findings (75).

 

Primary hypophysitis frequently evolves to fibrosis and pituitary atrophy in the chronic stages of the disease, which often impair pituitary function (Figure 2). The evolution to empty sella has also been shown in a mouse model of autoimmune hypophysitis (76). Caturegli et al. reported that only 4% of patients had spontaneous remission with recovery of pituitary function, while most patients will require long-term replacement of one or more pituitary axes (4,54). Whether medical treatment with glucocorticoids has a positive impact on the natural history of primary hypophysitis is still a matter of debate.

Figure 2. Natural History of Primary Hypophysitis.

Most of the published case series mainly focus on the more frequent lymphocytic hypophysitis. Granulomatous hypophysitis can cause more severe signs and symptoms (headache, chiasmal compression and anterior/posterior hypopituitarism). Xanthomatous hypophysitis seems to cause sella compression and pituitary dysfunction less frequently. IgG4-related hypophysitis can cause various degree of involvement of the anterior pituitary, posterior pituitary and the stalk. Necrotizing hypophysitis is extremely rare and is associated with a high risk of panhypopituitarism and diabetes insipidus. The chronic stage of the disease is most likely related to the extent of damage of the pituitary. Some authors have suggested that some cases of lymphocytic hypophysitis may evolve to the granulomatous form, as mixed forms can rarely be observed. A death rate of 7% has been described in large case series of patients with primary hypophysitis and is probably related to unrecognized acute adrenal insufficiency.

 

Diagnosis and Treatment of Primary Hypophysitis

 

Pituitary biopsy is the gold standard to confirm the diagnosis of primary hypophysitis. This procedure, however, should be considered only in equivocal cases and when the outcome of the biopsy is expected to change the therapeutic management, and should be performed by a neurosurgeon with extensive expertise in pituitary surgery.

 

Due to the rarity of the disease, the management of hypophysitis is controversial. An algorithm in line with the more recent literature is reported in Figure 3. Initial evaluation of patients with suspected hypophysitis involves clinical and laboratory assessment. Patients with a suspicion of hypophysitis based on biochemical results should undergo a pituitary MRI, as well as visual assessment to check visual fields and acuity. Secondary causes of hypophysitis and other sella/parasellar masses should be considered in the differential diagnosis.

 

The mainstay of treatment of primary hypophysitis is pituitary hormone replacement (77,78). As outlined above, ACTH production is frequently impaired at presentation, and most patients will require glucocorticoid replacement. This should be started before thyroxine replacement (if TSH deficiency is present as well) to avoid precipitating acute adrenal insufficiency.

 

Conservative management is recommended for primary hypophysitis unless symptoms are severe and progressive. The only exception to this rule is IgG4-related hypophysitis that – like other manifestations of the disease – should be promptly treated to revert symptoms and prevent irreversible fibrosis (79,80). The mainstay of treatment are glucocorticoids, which often cause remission of symptoms within a few weeks. A typical starting dose is prednisone 30-40 mg/day (or equivalent), which should be continued for 2-4 weeks, and then tapered gradually over 2-6 months (19). However, some patients may benefit from long-term maintenance glucocorticoid therapy (with or without a steroid-sparing agent), especially in case of extensive multi-organ involvement. Relapse is possible and multiple courses of high-dose glucocorticoids are often necessary. Rituximab has also been used in patients with poor response to glucocorticoids (19,81,82). A case of IgG4-related hypophysitis successfully treated with azathioprine has also been reported (83).

 

High-dose glucocorticoids are the first-line treatment to improve the swelling of the pituitary and improve the symptoms related to significant sella compression. Anterior pituitary function can recover after pulse corticosteroid therapy, although >70% of patients will require long-term replacement with one or more hormones (4); central diabetes insipidus rarely recovers. Honegger et al. documented excellent initial responses to high-dose glucocorticoids, with radiological improvement, stability and progression in 65%, 31% and 4% of cases, respectively (73). However, these patients carried a higher risk of side effects (weight gain, psychiatric symptoms, peripheral edema, diabetes mellitus and glaucoma) and relapse of the pituitary inflammation was documented in 38% of cases. Relapses occurred 2-17 months after starting pulse steroids and the risk or relapse did not correlate with either initial glucocorticoid dose or treatment duration (73). Hormone deficiencies improved with glucocorticoids only in 15% of patients, while they remained stable or worsened in 70% and 15% of cases, respectively (73). Lupi et al. performed a review of the literature and found somewhat better outcomes with medical therapy, reporting pituitary mass reduction in 84% of cases, improving anterior pituitary function in 45%, and restored posterior pituitary function in 41% after high-dose glucocorticoids and/or azathioprine, with a relatively low risk of relapse (14%) (84). Recently, Chiloiro et al. found in a small prospective double-arm study that high-dose glucocorticoid treatment – compared with simple observation – was associated with higher rates of hypophysitis resolution and pituitary function recovery (85). The authors also showed that positive pituitary antibodies, a diagnosis of diabetes insipidus and secondary hypogonadism at the time of presentation, and specific MRI findings (a thicker pituitary stalk, a smaller pituitary volume, and the evidence of posterior pituitary involvement at MRI including absent bright spot) predicted better clinical outcomes following glucocorticoid therapy. These findings should be confirmed in a larger prospective cohort.

 

Whether central diabetes insipidus is an unfavorable prognostic factor for response to glucocorticoids is unclear. The abovementioned study by Chiloiro et al. suggests better outcomes in patients with central diabetes insipidus at the time of hypophysitis diagnosis (85); however, Lupi et al. found that patients with concomitant anterior and posterior pituitary dysfunction responded poorly to glucocorticoids, which were unable to revert the hypopituitarism (86). Glucocorticoid therapy was also found to be less effective in granulomatous or xanthomatous hypophysitis (35). In glucocorticoid-resistant cases and when high-dose glucocorticoids cause unacceptable side effects, immunosuppressive drugs such as azathioprine, methotrexate, and cyclosporin A have been used successfully. However, the long-term effects are unclear (1). Rituximab has also been employed to treat steroid-refractory hypophysitis (36,87-89).

 

Surgery should be considered only in cases with serious and progressive deficits of the visual field, visual acuity, or nerve paralysis not responsive to medical treatment. Surgery generally improves sella compression in the short term; however, Honegger et al. observed progression/relapse of the disease in 25% of patients after a mean follow-up of 3 years (73). Pituitary function improved only in 8% of patients after surgery, and the rates of resolution of chiasmal compression were also low (73). Further supporting the limited role of surgery in the management of hypophysitis, two small observational studies found that surgery did not impact significantly on the resolution of neurological symptoms or hormonal deficits during follow-up (90,91).

 

Stereotactic radiotherapy (radiosurgery) has been effectively employed in selected patients who have failed medical treatment or suffer from repeated recurrence of lymphocytic hypophysitis (92,93).

 

Figure 3. Diagnosis and management of primary hypophysitis. 1 Check random ACTH and cortisol if acute adrenal insufficiency is suspected. Consider confirmatory testing (e.g., Synacthen) if equivocal or borderline results. The Synacthen test can give false-positive results in the early stages of central adrenal insufficiency. During pregnancy and in patients receiving oral estrogens, the rise of corticosteroid-binding globulin (CBG) leads to falsely elevated cortisol levels and the normal reference ranges and stimulated cortisol cut-offs do not apply. 2 Pituitary surgery can also provide histology for definitive diagnosis.

DRUG-INDUCED HYPOPHYSITIS: IMMUNE CHECKPOINT INHIBITORS

 

Immune checkpoint inhibitors are monoclonal antibodies increasingly used for solid and hematological malignancies (94). They block several regulators of the immune activation (immune checkpoints), enhancing the host’s immune response to tumor cells (Figure 4). These drugs have shown a favorable toxicity profile and significant anti-tumor activity but, because of their mechanism of action, new typical side-effects have emerged (immune-related adverse events, irAEs) (Figure 4) (95,96).

 

Figure 4. Mechanism of Action of Immune Checkpoint Inhibitors. Tumor antigens are presented to T-cells by antigen-presenting-cells (APCs) via the interaction of the major histocompatibility complex (MHC) and the T-cell receptors, representing the primary signal for activating T-cells. Another costimulatory signal involving interaction between B7 on APCs and CD28 on T-cells is needed to complete T-cell activation and expansion (Panel A). Several co-receptors act as negative modulators of immune response at different molecular checkpoints. The CTLA-4 is induced in T-cells at the time of their initial response to antigen. CTLA-4 is transported to the cell surface proportionally to the antigen stimulation; it binds to B7 with greater affinity than CD28, resulting in specific T-cell inactivation (Panel B). The PD-1/PD1-L1 pathway is not involved in initial T-cell activation: it regulates inflammatory responses in peripheral tissues sustained by already activated effector T-cells. Activated T-cells up-regulate PD-1 and inflammatory signals in the tissue induce the expression of PD1-L1s, which downregulate the activity of T-cells, protecting normal tissues from collateral destruction; this mechanism is also exploited by tumor cells to evade the immune system response (Panel B). Monoclonal antibodies that block either CTLA-4 or PD1/PD1-L1 increase cytotoxic T-cell activity by expanding T-cell activation and proliferation (Panel C). The eventual T-cell reactivation is responsible for the both anti-tumor response and the immune-related adverse events associated with these drugs.

irAES mirror the immune response reactivation induced by immune checkpoint inhibitors and may predict better survival and response to the treatment of the underlying malignancy (97-100). irAEs can affect multiple organs and systems, including the pituitary and other endocrine glands (Table 7) (101).

 

Table 7. Immune-Related Adverse Events Associated with Immune Checkpoint Inhibitors

ENDOCRINOPATHIES

OTHER SYSTEMS AND ORGANS

PITUITARY: Hypophysitis.*

 

THYROID: Thyroiditis (both hypo- and hyperthyroidism); Euthyroid Graves’ ophthalmopathy.

 

ADRENAL GLANDS: Adrenalitis.*

 

PANCREAS: Insulinopenic diabetes mellitus.

SKIN: Rash/inflammatory dermatitis; Bullous dermatoses; Stevens-Johnson syndrome; Toxic epidermal necrolysis; Drug rash with eosinophilia and systemic symptoms syndrome; Drug-induced hypersensitivity syndrome; Acute generalized exanthematous pustulosis; Alopecia areata; Vitiligo; Psoriasis.

 

GASTROINTESTINAL SYSTEM: Colitis; Hepatitis; Pancreatitis.

 

LUNGS: Pneumonitis.

 

MUSCOSKELETAL SYSTEM: Arthritis; Polymyalgia-like syndrome; Myositis; Vasculitis.

 

KIDNEY: Nephritis.

 

CARDIOVASCULAR SYSTEM: Myocarditis; Pericarditis; Arrhythmias; Heart failure; Vasculitis; Venous thromboembolism.

 

NERVOUS SYSTEM: Guillain-Barré syndrome; Myasthenia gravis; Peripheral neuropathy; Autonomic neuropathy; Aseptic meningitis; Encephalitis; Transverse myelitis.

 

HEMATOLOGY: Autoimmune hemolytic anemia; Acquired thrombotic thrombocytopenic purpura; Hemolytic uremic syndrome; Aplastic anemia; Lymphopenia; Immune thrombocytopenia; Acquired hemophilia.

 

EYE: Uveitis; Iritis; Episcleritis; Blepharitis.

* Immune checkpoint inhibitors can cause both primary adrenal insufficiency (rarer) and secondary adrenal insufficiency (more frequent).

 

Epidemiology

 

Hypophysitis may occur as a complication during treatment with immune checkpoint inhibitors. Ipilimumab, a monoclonal antibody against the cytotoxic T lymphocyte antigen-4 (CTLA-4) is the drug that has been more strongly associated with this immune-related adverse event (Table 8) (5,102-112). The overall incidence of hypophysitis is 12% in patients treated with anti-CTLA-4 antibodies and 0.5% in patients treated with anti-programmed death 1 (PD1) antibodies (113,114).

 

Table 8. Immune Checkpoint Inhibitors and the Risk of Hypophysitis

Category

Drug

Approved and off-label indications

Incidence of reported hypophysitis in clinical studies

Anti-CTLA-4

(70% of published hypophysitis cases)

Ipilimumab

Unresectable or metastatic melanoma; Adjuvant treatment in melanoma; Relapsed hematologic cancer.

Up to 17.4% (G3-G4: 0.3-17.4%)

Tremelimumab

Malignant mesothelioma; Hepatocellular carcinoma. This drug is not FDA approved.

0-2.6% (G3-G4: 1%)

Anti-PD1 (24% of published hypophysitis cases)

Nivolumab

Metastatic colorectal cancer; Recurrent or metastatic squamous cell head and neck cancer; Hepatocellular carcinoma; Classical Hodgkin’s lymphoma; Unresectable or metastatic melanoma; Adjuvant treatment in melanoma; Progressive, metastatic non-small cell lung cancer; Progressive small cell lung cancer; Advanced renal cell cancer; Urothelial carcinoma; Platinum-resistant ovarian cancer.

0-3% (G3-G4: 0.5%)

Pembrolizumab

Metastatic or recurrent locally advanced gastric cancer; Recurrent or metastatic squamous cell head and neck cancer; Relapsed or refractory classical Hodgkin’s lymphoma; Relapsed chronic lymphocytic leukemia; Unresectable or metastatic melanoma; Unresectable or metastatic microsatellite instability-high cancer; Metastatic non-small cell lung cancer; Metastatic, non-squamous Non-small cell lung cancer (in combination with Pemetrexed and Carboplatin); Locally advanced or metastatic urothelial carcinoma; Advanced Merkel cell carcinoma.

0-4.8% (G3-G4: 0-2.4%)

Dostarlimab

Mismatch repair deficient recurrent or advanced endometrial cancer; Mismatch repair deficient recurrent or advanced solid tumors.

No cases published. Hypophysitis is listed as a possible adverse reaction in <10% of treated patients in the product information.

Cemiplimab

Cutaneous squamous cell carcinoma.

1 case reported

Toripalimab

Melanoma; several solid malignancies (development stage).

1 case reported

Geptanolimab (still in development stage)

Peripheral T-cell lymphoma; Alveolar soft part sarcoma; Cervical cancer; Non-Hodgkin's lymphoma; Liver cancer; Colorectal cancer; Non-small cell lung cancer.

1 case reported

Anti-PD1-L1 (2% of published hypophysitis cases)

Atezolizumab

Metastatic non-small cell lung cancer; Locally advanced or metastatic urothelial carcinoma.

1% (G3-G4: 1%)

Avelumab

Metastatic Merkel cell carcinoma; Locally advanced or metastatic urothelial carcinoma; Advanced non-small cell lung cancer.

1 case reported

Durvalumab

Advanced non-small cell lung cancer; Locally advanced or metastatic urothelial carcinoma.

1 case reported

Combination therapy

(4% of published hypophysitis cases)

Ipilimumab + Nivolumab

Unresectable or metastatic melanoma; Progressive small cell lung cancer; Non-small cell lung cancer; Advanced renal cell cancer; Malignant mesothelioma; Recurrent glioblastoma.

Up to 12.8% (G3-G4: 1.5-8.7%)

Ipilimumab + Pembrolizumab

Advanced melanoma; Advanced renal cell carcinoma.

0-9.1% (G3-G4: 0-6%)

Durvalumab + Tremelimumab

Advanced non-small cell lung cancer.

0%

Abbreviations: CTLA-4, cytotoxic T lymphocyte antigen-4; FDA, Food and Drug Administration; G3, grade 3 immune checkpoint inhibitor-induced hypophysitis (see Table 11); G4, grade 4 immune checkpoint inhibitor-induced hypophysitis (see Table 11); PD1, programmed death 1; PD1-L1, programmed death 1 Ligand 1.

 

Pathogenesis

 

The pathogenesis of anti-CTLA-4 antibody-induced hypophysitis involves type II and IV hypersensitivity, as well as the humoral immune response (Figure 5). This has been suggested by histopathological findings of patients with hypophysitis following treatment with Ipilimumab (alone or in combination with Nivolumab or Pembrolizumab), evidence of pituitary antibodies in the serum of these patients, association with specific human leucocyte antigens, and animal models of anti-CTLA-4-induced hypophysitis (8,15,115-118).

 

Evidence regarding the pathophysiology of anti-PD1/PD1-L1 antibody-induced hypophysitis is scant, but immune response reactivation most likely targets ACTH-secreting cells because of the very frequent isolated ACTH deficiency (5). Kanie et al. recently postulated that ectopic expression of ACTH in the tumor may contribute to some cases of anti-PD1/PD1-L1 antibody-induced hypophysitis, as a form of paraneoplastic syndrome (119). Furthermore, Bellastella et al. identified a higher prevalence of anti-pituitary and anti-hypothalamus antibodies in patients with cancer treated with anti-PD1/PD1-L1 agents (120). In a small longitudinal study, the same group also found that more than half of patients who start anti-PD1/PD1-L1 treatment developed anti-pituitary or anti-hypothalamus antibodies after 9 weeks of treatment, with a concomitant increase prolactin and a reduction in ACTH and IGF-1 levels compared to baseline (120). These preliminary results need to be validated in a larger cohort, but the presence of anti-hypothalamus antibodies would suggest that – at least in some patients – hypothalamic autoimmunity might contribute to the development of anti-PD1/PD1-L1 antibody-induced pituitary dysfunction.

 

Figure 5. Proposed pathogenesis of anti-CTLA-4 antibody-induced hypophysitis. Anti-CTLA-4 antibody-induced hypophysitis accounts for ~70% of immune-checkpoint induced hypophysitis cases. The CTLA-4 antibody binds to pituitary CTLA-4 antigen, inducing complement activation and infiltration with macrophages and other inflammatory cells, leading to phagocytosis and enhanced antigen presentation. Subsequently, autoimmune type IV hypersensitivity reactions start, with infiltration of the anterior pituitary by autoreactive T lymphocytes that eventually leads to pituitary cytotoxicity and inflammation. Moreover, patients with anti-CTLA-4 antibody-induced hypophysitis develop pituitary antibodies that predominantly recognize TSH- FSH- and ACTH-secreting cells. Pituitary cytotoxicity in anti-PD1/PD1-L1 antibody-induced hypophysitis presumably affects mostly ACTH-secreting cells, as isolated ACTH deficiency is the most common occurrence in these patients.

Clinical Characteristics

 

There are important differences between primary hypophysitis and immune checkpoint-induced hypophysitis (Table 9)(5,8,103). The latter does not have a female predominance (8,121) and seems to present more frequently with hypopituitarism at diagnosis. Both forms of hypophysitis are more commonly associated with an initial deficit of ACTH, FSH/LH and TSH, but symptoms of adrenal insufficiency and confirmed ACTH deficiency are much more common in patients with immune checkpoint-induced hypophysitis (8,113,114). Central diabetes insipidus can occur in a substantial share of primary hypophysitis cases (i.e., the infundibulo-neurohypophysitis and panhypophysitis variants), while it is extremely rare in immune-checkpoint induced hypophysitis. Pituitary enlargement and visual impairment are much more common in primary hypophysitis, while the size of the pituitary may appear normal in immune checkpoint inhibitor-induced hypophysitis (in absence of a baseline pituitary MRI) and optic chiasm involvement is rare (5,8).

 

Table 9. Comparison Between Primary and Immune Checkpoint Inhibitor-Induced Hypophysitis

Characteristics

Primary hypophysitis

Immune checkpoint inhibitor-induced hypophysitis

Etiology

Autoimmune.

Immune response reactivation.

Epidemiology

·   More prevalent in young females (female:male ratio ~3:1), apart from the rare IgG4-related form that is more common in older males.

·   The onset of the lymphocytic subtype is strongly associated with late pregnancy and the post-partum period.

·  The epidemiology is most likely influenced by the underlying malignancy.

·  0.5-12% of treated patients develop hypophysitis, depending on the drug used.

·  The female:male ratio is ~1:4 and the mean age at onset is ~60 years (older male patients appear to be the group carrying the higher risk).

·  No prior cancer therapy is associated with higher risk of developing hypophysitis.

Time after the initiating event

Unknown. The median duration of symptoms before clinical presentation

is varies according to the anatomic location of the pituitary involvement:

· Adenohypophysitis (during pregnancy): 4 months;

· Adenohypophysitis (outside of pregnancy): 12 months;

· Infundibulo-neurohypophysitis: 3 months;

· Panhypophysitis: 4 months.

· Ipilimumab: median time to onset 9-11 weeks (range 1-35); *

· Pembrolizumab: median time to onset 16 weeks (range 1-52); *

· Nivolumab: median time to onset 21-22 weeks (range 6-48); *

· Ipilimumab + Nivolumab: median time to onset 11-12 weeks (range 3-32). *

Symptoms at presentation

· Headache: 48%

· Adrenal insufficiency: 38%

· Polydipsia/polyuria: 34%

· Visual disturbances: 32%

· Hypogonadism: 21%

· Hypothyroidism: 16%

·Adrenal insufficiency: 81% **

·Headache: 45%

·Hypothyroidism: 18%

·Hypogonadism: 11%

·Visual disturbances: 6%

·Polydipsia/polyuria: 2%

Pituitary hormone abnormalities

· ADH deficiency: 63%

· ACTH deficiency: 60%

· FSH/LH deficiency: 55%

· TSH deficiency: 50%

· GH decreased: 37%

· Hyperprolactinemia: 39%

·   ACTH deficiency: 96% **

·   TSH deficiency: 63%

·   FSH/LH deficiency: 59%

·   GH decreased: 19%

·   Hyperprolactinemia: 11%

·   ADH deficiency: 4%

Abnormal MRI at presentation

97% of cases

64% of cases ***

Histopathology

Marked infiltration of lymphocytes of the pituitary gland, typically accompanied by scattered plasma cells, eosinophils and fibroblasts, and in later disease stages by fibrosis.

T-cell infiltration and IgG-dependent complement fixation and phagocytosis.

Treatment

Usually good response to glucocorticoids.

Good response to glucocorticoids of the symptoms related to sella compression.

Outcome

Variable: from complete recovery to persistent hypopituitarism.

Pituitary enlargement (if present) eventually resolves. TSH and FSH/LH deficiencies often recover, while central adrenal insufficiency persists almost invariably.

Abbreviations: ACTH, adrenocorticotropic hormone; ADH, antidiuretic hormone; CTLA-4, cytotoxic T lymphocyte antigen-4; FSH, follicle-stimulating hormone; LH, luteinizing hormone; GH, growth hormone; MRI, magnetic resonance imaging; TSH, thyroid-stimulating hormone.

* Data from the prescribing information of Ipilimumab, Pembrolizumab and Nivolumab.

** Anti-PD-1/PD1-L1 antibody-induced hypophysitis typically presents with isolated ACTH deficiency, while CTLA-4 antibody-induced hypophysitis more frequently leads to multiple hormone deficiencies (Table 10).

*** MRI abnormalities are transient, can be subtle and precede clinical symptoms in ~50% of cases. Anti-PD-1/PD1-L1 antibody-induced hypophysitis typically lacks MRI changes and causes no mass effect symptoms (Table 10).

 

The onset of immune checkpoint-induced hypophysitis varies according to the drug used (Table 9); early onset has been reported and it can appear also several months after the initiation of the immunotherapy (122,123). The risk of hypophysitis with Ipilimumab appears to be dose-dependent, with a higher prevalence in those receiving 10 mg/kg vs. 3 mg/kg (124-126). Conversely, patients receiving concomitant cytotoxic chemotherapy or with brain radiotherapy-pretreated metastases might be protected from the risk of developing hypophysitis, presumably through immune cell depletion (95,127).

 

Patients with immune checkpoint-induced hypophysitis typically present with nonspecific symptoms of adrenal insufficiency like fatigue, headache, myalgia, nausea, vomiting, reduced appetite, light-headedness, and dizziness, whilst symptoms of other anterior pituitary hormone deficiencies are less common at the time of diagnosis (Table 9) (113,114). Manifestations of adrenal insufficiency often overlap with those of the underlying malignancy but must not be overlooked because of the risk of developing life-threatening adrenal crisis. Visual disturbances are very rare (the pituitary enlargement, if present, is often minor and transient) and central diabetes insipidus is extremely uncommon (95,113,114,128,129). Other less frequent symptoms include confusion, hallucinations, memory loss, labile moods and depression (including suicidal ideation), insomnia, temperature intolerance, and chills (130,131). Importantly, up to 45% of patients can be asymptomatic and are diagnosed only at laboratory evaluation, highlighting the importance of regular monitoring (123,132).

 

Associated irAEs have been reported in about half of patients with immune checkpoint inhibitor-induced hypophysitis (133). By far, the most common associated irAE was thyroiditis (~30%), followed by colitis (~20%), skin reactions (~15%), pneumonitis (~5%), and hepatitis (~5%) (133).

 

Patients with anti-CTLA-4 antibody-induced hypophysitis tend to have a more diverse clinical presentation than those with anti-PD-1/PD1-L1 antibody-induced hypophysitis. The latter typically presenting later during treatment, with severe isolated ACTH deficiency (which frequently leads to hyponatremia at the time of diagnosis), and no significant pituitary enlargement both clinically and radiologically. Also, treatment discontinuation is less frequently required in patients with anti-PD-1/PD1-L1 antibody-induced hypophysitis (Table 10) (5,81,114,134-136).

 

Table 10.  Comparison Between Anti-CTLA-4 and Anti-PD1/PD1-L1 Antibody-Induced Hypophysitis

Characteristics

Anti-CTLA-4 antibody-induced hypophysitis

Anti-PD1/PD1-L1 antibody-induced hypophysitis

Number of cases reported

192 (74% males)

69 (72% males)

Mean time to onset (95% CI)

10.5 weeks (9.8-11.2)

Anti-PD1: 27.0 weeks (20.9-33.1)

Anti-PD1-L1: 27.8 weeks (0-58.0)

Mean doses to onset

3.4 doses

10.3 doses

Symptoms at presentation

· Adrenal insufficiency: 75%

· Headache: 60%

· Hypothyroidism: 21%

· Hypogonadism: 16%

· Visual disturbances: 8%

· Polydipsia/polyuria: <1%

·Adrenal insufficiency: 91%

·Hypothyroidism: 7%

·Headache: 4%

·Polydipsia/polyuria: 3%

·Hypogonadism: 0%

·Visual disturbances: 0%

Pituitary hormone abnormalities at presentation *

· ACTH deficiency: 95%

· TSH deficiency: 85%

· FSH/LH deficiency: 75%

· GH decreased: 27%

· Hyperprolactinemia: 7%

· ADH deficiency: 2%

·   ACTH deficiency: 97%

·   Hyperprolactinemia: 20%

·   FSH/LH deficiency: 13%

·   TSH deficiency: 4%

·   GH decreased: 3%

·   ADH deficiency: 3%

Prevalence of hyponatremia at presentation **

39% of cases

62% of cases

Abnormal MRI at presentation

81% of cases

18% of cases

Discontinuation of the immune checkpoint inhibitor

· No: 56%

· Yes, temporarily: 3%

· Yes, permanently: 41%

· No: 70%

· Yes, temporarily: 20%

· Yes, permanently: 10%

Outcome

· Long-term hypopituitarism: 89%

· Pituitary function recovery after treatment: 5%

· Spontaneous resolution: 1%

· Death: 5%

· Recurrence after treatment: 0%

· Long-term hypopituitarism: 90%

· Pituitary function recovery after treatment: 6%

· Spontaneous resolution: 0%

· Death: 4%

· Recurrence after treatment: 0%

* Pituitary hormone deficiencies can be isolated or combined (especially in the case of anti-CTLA-4 antibody-induced hypophysitis). ACTH + TSH deficiency is the most frequent combination observed in these patients.

** Most likely related to cortisol deficiency. It can be a clue to the diagnosis.

Abbreviations: ACTH, adrenocorticotropic hormone; ADH, antidiuretic hormone; CTLA-4, cytotoxic T lymphocyte antigen-4; FSH, follicle-stimulating hormone; LH, luteinizing hormone; GH, growth hormone; MRI, magnetic resonance imaging; PD1, programmed death 1; PD1-L1, programmed death 1 Ligand 1; TSH, thyroid-stimulating hormone.

 

According to the degree of symptoms and of the severity of the disease, immune checkpoint-induced hypophysitis is graded 1 to 4 (Table 11) (78). Grade 3 toxicity or worse (including death) has been described in 2-10% of reported hypophysitis cases (137,138).

Abbreviations: ADL, activities of daily living.

Table 11. Grading of Immune Checkpoint Inhibitor-Induced Hypophysitis

Grade

Description

Grade 1

Asymptomatic or mild symptoms.

Grade 2

Moderate symptoms, able to perform ADL.

Grade 3

Severe symptoms, medically significant consequences, unable to perform ADL.

Grade 4

Severe symptoms, life-threatening consequences, unable to perform ADL.

Grade 5

Death.

 

Diagnosis and Management

 

An algorithm for diagnosis and management of immune checkpoint-induced hypophysitis in line with the more recent literature is shown in Figure 6. Patients should be regularly monitored with clinical assessment and hormonal tests during treatment with immune checkpoint inhibitors. Almost invariably, patients who develop hypophysitis have ACTH deficiency and cases of fatal acute adrenal insufficiency have been reported (139); this highlights the importance of pituitary function assessment at baseline and during treatment, also in asymptomatic patients (140). If there is a strong suspicion of adrenal insufficiency on clinical grounds (e.g. G3-G4 symptoms), glucocorticoid replacement should be started without delay (141). TSH deficiency is also very common (>60% of patients). Indeed, a fall in serum TSH and free T4 have been suggested to be early signs of immune checkpoint inhibitor-induced hypophysitis and can be a clue to the diagnosis (141-144). A recent paper has identified antibodies against two autoantigens (anti-GNAL and anti-ITM2B) that may aid in the diagnosis of and predict the risk of developing immune checkpoint-induced hypophysitis (145). Moreover, Kobayashi et al. evaluated the usefulness of pituitary antibodies and human leukocyte antigen alleles in predicting immune checkpoint-induced pituitary dysfunction. The authors showed distinct and overlapped patterns of pituitary antibodies and human leukocyte antigen alleles between patients who developed hypophysitis (n=5) or isolated ACTH deficiency (n=17) (117). The usefulness of anti-GNAL and anti-ITM2B antibodies, pituitary antibodies, and human leukocyte antigen alleles as biomarkers in the clinical setting needs to be validated in larger cohorts of patients.

 

Patients with suspected drug-induce hypophysitis should undergo a pituitary MRI and visual assessment (141,146). The importance of obtaining pituitary imaging was recently highlighted in a retrospective study by Nguyen et al., where 33% of hypophysitis cases would have been missed if no MRI were carried out (143). Also, pituitary MRI is important for the differential diagnosis of other pituitary lesions, in particular metastases (Table 12). Faje et al. reported that ~50% of patients with immune checkpoint-induced hypophysitis presented with diffuse pituitary enlargement at MRI before the onset of clinical symptoms (98). 18F-FDG PET performed as part of the staging of the underlying malignancy can show intense radiotracer uptake and may precede clinical symptoms and biochemical abnormalities (147,148); however, its routine use for the diagnosis of hypophysitis is not recommended.

 

Current guidelines on the management of immune-checkpoint induced hypophysitis suggest clinicians to consider with holding treatment in G1-G2 hypophysitis until the patient is stabilized on hormone replacement (101). We believe that patients with immune checkpoint inhibitor-induced hypophysitis should not stop treatment unless they develop severe and progressive symptoms (G3-G4 hypophysitis). In fact, this type of hypophysitis if often self-limiting and most of patients do not show progression of sella compression. Therefore, the decision whether to withhold a treatment that can have a significant impact on the progression-free survival of the underlying malignancy should be balanced carefully. When G3-G4 hypophysitis is suspected, a course of high-dose corticosteroids given during the acute phase may result in inflammation reversal and ameliorate the compression of sella and parasellar structures. Whether high-dose glucocorticoids have an impact on the anti-tumor effect of immune checkpoint inhibitors is uncertain. Earlier evidence suggested a neutral effect on survival (99,149,150); however, a study from Faje et al. questioned this, showing reduced survival among patients with melanoma treated with high-doses glucocorticoids for Ipilimumab-induced hypophysitis (100,126). Nonetheless, treatment should not be delayed in patients with severe symptoms of sella compression.

 

The resolution of the neuroradiological abnormalities is usually observed within 2 months (128,130). Treatment with high-dose glucocorticoids, however, does not restore ACTH deficiency and most patients will require long-term replacement (Table 10) (5,123). On the other hand, thyroid and gonadal deficiencies often recover and the need for hormone replacement needs to be reassessed in the long term (123,124,143,151,152). In addition, patients developing irAEs can be severely ill and can present with a “euthyroid sick syndrome” and/or a “sick eugonadal syndrome” that can affect the interpretation of the laboratory results (130).

 

Figure 6. Diagnosis and Management of Immune Checkpoint Inhibitor-Induced Hypophysitis. 1 Some authors suggest laboratory evaluation before the first infusion, then at 8 weeks for patients receiving Ipilimumab (i.e., prior to cycle 3) and then at week 16 if there are no interim signs/symptoms suggestive of hypophysitis. Other authors recommend laboratory evaluation for hypophysitis prior to each infusion of immune checkpoint inhibitors in the first 12-16 weeks of treatment, in order to pick up early or late onset of the disease. 2 Check random ACTH and cortisol if acute adrenal insufficiency is suspected. Exclude recent glucocorticoid use and concomitant treatment that may alter serum cortisol measurement (e.g., oral estrogens). As a guide, in patients that are unwell serum cortisol >450 nmol/L makes the diagnosis of adrenal insufficiency unlikely. Adrenal insufficiency is possible if morning cortisol 200-450 nmol/L or random cortisol 100-450 nmol/L; consider confirmatory testing with Synacthen, although this can give false-positive results in the early stages of central adrenal insufficiency. Adrenal insufficiency is likely if morning cortisol <200 nmol/L or random cortisol <100 nmol/L and patients should be started on hormone replacement. These cut-offs should be seen only as a guide and need to be adapted to local laboratory assays and reference ranges. Patients receiving immune checkpoint inhibitors can also develop adrenalitis and primary adrenal insufficiency. These patients have high ACTH and renin/aldosterone should be measured to investigate mineralocorticoid deficiency. 3 IGF-1 is valuable to confirm changes from baseline that may suggest new-onset hypophysitis. However, further tests to prove GH deficiency are not required because these patients would not be treated (active malignancy). 4 Pituitary MRI is normal in ~20% and ~80% of hypophysitis cases associated with anti-CTLA-4 and anti-PD1/PD1-L1 antibodies, respectively. Therefore, normal imaging does not exclude hypophysitis. MRI changes can be very subtle (Table 11). 5 We believe that patients with immune checkpoint inhibitor-induced hypophysitis should not stop treatment unless they develop severe and progressive symptoms (G3-G4 hypophysitis). Once the acute symptoms of hypophysitis have resolved, restarting treatment with immune checkpoint inhibitors is not contraindicated. Adequately treated, long-term hypopituitarism is not a contraindication to restarting immune checkpoint inhibitors.

An important differential diagnosis in patients with suspected drug-induced hypophysitis and a sella mass are pituitary metastases (Table 12) (95,141,153-155). The early studies on immune checkpoint inhibitors mainly assessed their efficacy in patients with advanced melanoma. Pituitary metastases are rare in melanoma (~2.5% of pituitary metastasis cases reported in the literature); however, these drugs are increasingly used for other malignancies including lung cancer, which accounts for ~25% of pituitary metastases (153). Central diabetes insipidus in immune checkpoint inhibitor-induced hypophysitis is extremely rare; therefore, a sella mass associated with diabetes insipidus is strongly suggestive of a metastasis.

 

Table 12. Differential Diagnosis of Immune Checkpoint Inhibitor-Induced Hypophysitis and Pituitary Metastases

Characteristics

Immune checkpoint inhibitor-induced hypophysitis

Pituitary Metastases

Clinical presentation

·   Central diabetes insipidus is extremely rare;

·   Anterior pituitary insufficiency is very common (chiefly ACTH, FSH/LH and TSH deficiency).

·   Headache is a frequent presenting symptom.

·   Central diabetes insipidus is the most common hormonal abnormality (~45%);

·   Cranial nerve deficits due to involvement of the chiasm and the cavernous sinus are common (22-28%);

·   Anterior pituitary insufficiency has been described in ~24% of patients;

·   Headache and retro-orbital pain have been reported in ~16% of patients;

Imaging

MRI:

·   Mild-to-moderate diffuse enlargement of the pituitary (up to 60-100% of the baseline size). Pituitary height typically does not exceed 2 cm. Pituitary enlargement resolves in most cases over the course of weeks/months. Empty sella can develop in the long term.

·   Extension into the cavernous sinus or above the sellar diaphragm is uncommon.

·   Homogeneous (more frequent) or heterogeneous enhancement (less frequent) post-gadolinium;

·   Suprasellar extension with compression and displacement of chiasm is uncommon;

·   The pituitary stalk may be thickened but not deviated;

·   The posterior pituitary is preserved in most of cases.

MRI:

·   Sella or suprasellar mass;

·   Isointense or hypointense mass on T1WI, with a usually high-intensity sign on T2WI;

·   Homogeneous enhancement post-gadolinium, although hemorrhage, necrosis and areas of cystic degeneration can be observed;

·   Thickened and enhancing pituitary stalk is possible, but it is typically less common than immune checkpoint inhibitor-induced hypophysitis;

·   Presence of other brain metastases (~15%);

·   Invasion of the cavernous sinus, chiasm, or hypothalamus (~14%)

·   Loss of bright spot of the neurohypophysis (~13%);

·   Dumbbell-shaped mass (~11%);

·   Sphenoid sinus invasion (~9%);

 

CT: may show bony destruction.

Abbreviations: CT, computed tomography; MRI, magnetic resonance imaging; T1WI, T1 weighted images; T2WI, T2 weighted images.

 

DRUG-INDUCED HYPOPHYSITIS: OTHER DRUGS

 

Reversible or irreversible hypopituitarism may be a rare side effect following treatment with interferon-α, and interferon-α/ribavirin combination therapy has been associated with cases of granulomatous hypophysitis with anterior pituitary dysfunction (156-160). The anti-interleukin-12 and -23 monoclonal antibody ustekinumab (used in the treatment of psoriasis) has been associated with a case of hypophysitis with panhypopituitarism (161).

 

HYPOPHYSITIS SECONDARY TO SELLA AND PARASELLAR DISEASE

 

Pituitary inflammation can be triggered by sella and parasellar disease. The infiltrate is mainly lymphocytic or xanthogranulomatous and focuses around the lesion rather than diffusing to the entire gland (4).

 

Germinoma

 

Germinomas are rare brain tumors predominantly affecting prepubertal children. They are highly immunogenic tumors and can induce a strong immune response that can involve the pituitary leading to secondary hypophysitis (162-169). Histologically, lymphocytic or granulomatous hypophysitis is seen in ~80% and ~20% of cases linked to germinomas, respectively (169).

 

Germinomas arising in the sella and parasellar region are difficult to differentiate from hypophysitis in children because of similar clinical features (diabetes insipidus + GH deficiency + visual disturbances). This differentiation, nevertheless, is critical for patient care due to different treatments of the two diseases. Biopsy-proven cases of primary hypophysitis are extremely rare in children and adolescents (41); therefore, in children below 10 years a germinoma should be considered the most likely diagnosis.

 

Tumor markers such as α-fetoprotein, β-human chorionic gonadotropin, or placental alkaline phosphatase in the cerebrospinal fluid may be useful for diagnosing germinoma. However, a pituitary biopsy is the gold standard for differentiating the two conditions, although germinomas can have a marked lymphocytic infiltrate that can outnumber the neoplastic cells making differential diagnosis difficult (168). If germinoma is part of the histologic differential diagnosis, markers for germinomas such as Oct3/4, PLAP and NANOG may be useful.

 

Finally, it should be noted that the hypopituitarism caused by sella germinomas can precede for years a visible pituitary mass, so that prolonged symptomatic periods prior to diagnosis are common (168).

 

Rathke’s Cleft Cyst

 

The rupture of Rathke’s cleft cyst can cause hypophysitis associated with visual disturbances, headache and hypopituitarism including – very frequently – central diabetes insipidus (170-175). Histopathology can show lymphocytic, granulomatous, xanthomatous or mixed forms of hypophysitis (174). Some authors have suggested that many cases of xanthomatous hypophysitis may actually be related to rupture of Rathke’s cleft cysts (12,13).

 

Other Sella and Parasellar Masses

 

Cases of secondary hypophysitis have been described in association with craniopharyngiomas (176), pituitary adenomas (177-182) and primary pituitary lymphomas (177,183).

 

HYPOPHYSITIS SECONDARY TO SYSTEMIC DISEASE

 

Sarcoidosis

 

Sarcoidosis is a multisystem inflammatory disease of unknown origin characterized by the formation of non-caseating granulomas that can involve all organ systems. The central nervous system can be affected in 5-15% of patients (neurosarcoidosis) and may be the presenting feature of the disease (184). Granulomas can involve the pituitary, hypothalamus and the stalk in ~0.5% of patients with sarcoidosis, resulting in varying grade of hypopituitarism (185,186). ~60% of the cases reported in the literature are males presenting in the 3rd and 4th decade. Central diabetes insipidus, FSH/LH deficiency and hyperprolactinemia are among the most frequent hormone abnormalities (187). Patients with sarcoidosis and hypothalamic-pituitary involvement tend to have more frequent multi-organ involvement, as well as neurosarcoidosis and sinonasal involvement (187).

 

Granulomatosis with Polyangiitis

 

Granulomatosis with polyangiitis (previously known as Wegener’s Granulomatosis) is an antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis of unknown etiology with multisystem involvement and formation of necrotizing granulomas and vasculitis in small- and medium-sized blood vessels. Pituitary involvement is a rare and usually late manifestation of the disease (186,188,189), but it can also be the presenting complaint (190,191). Secondary hypogonadism and central diabetes insipidus are the most common endocrine abnormalities; diabetes insipidus can recover after adequate treatment of the underlying vasculitis, while anterior pituitary dysfunction is permanent in the majority of patients (192).

 

Langerhans Cell Histiocytosis

 

Langerhans cell histiocytosis is a rare disease mainly occurring in childhood, involving clonal proliferation of myeloid Langerhans cells that can infiltrate multiple organs (bones, skin, lymph nodes, lungs, thymus, liver, spleen, bone marrow, and central nervous system including the pituitary). Patients often carry the BRAF V600E mutation in the clonal myeloid cells (193).

 

The most common endocrine abnormality in patients with Langerhans cell histiocytosis is hypothalamic-pituitary infiltration causing central diabetes insipidus. These patients usually have multi-organ and cranio-facial involvement, although localized disease of the hypothalamic-pituitary region has been reported (194,195). Up to 40% of patients develop symptoms consistent with diabetes insipidus within the first four years, particularly if there is multisystem involvement and proptosis (196-198). Anterior pituitary hormone deficiency is also possible at diagnosis and during follow up (194,199).

 

Langerhans cell histiocytosis and germinoma are the most common cause of central diabetes insipidus in children and adolescents; therefore, germinoma should always been considered in the differential diagnosis (200).

 

The definitive diagnosis of Langerhans cell histiocytosis is the biopsy-proven infiltration of the pituitary with Langerhans cells with eosinophils, neutrophils, small lymphocytes, and histiocytes. However, pituitary biopsy is invasive and the diagnosis can be suggested by the presence of the characteristic histopathologic features in other tissues when a multisystem disease is present. For patients with suspected disease isolated to the pituitary, identification of BRAF-V600E in the peripheral blood or cerebrospinal fluid can support the diagnosis and rule out germinoma, although it does not distinguish Langerhans cell histiocytosis from Erdheim-Chester disease (see below) (201).

 

When hypophysitis secondary to Langerhans cell histiocytosis is suspected but pituitary biopsy is not available, it is reasonable to initiate therapy empirically with a plan to follow disease response with MRI. Treatment options include prednisone, alone or in combination with vinblastine, cladribine and vemurafenib, alongside desmopressin and other pituitary hormone replacements to treat hypopituitarism.

 

Erdheim-Chester Disease

 

Erdheim-Chester’s disease is a rare multisystem histiocytic disorder, most often seen in adults, which may be confused with Langerhans cell histiocytosis. Histiocytic infiltration leads to xanthogranulomatous infiltrates of multiple tissues (bones, skin, lungs, facial, orbital and retro-orbital tissue, retroperitoneum, cardiovascular system and cerebral nervous system including the pituitary). Long bone pain and symmetric osteosclerotic lesions suggest this diagnosis, which is confirmed by tissue biopsies showing histiocytes with non-Langerhans features. Patients often carry the BRAF V600E mutation in the clonal myeloid progenitor cells (193).

 

Pituitary involvement may manifest as central diabetes insipidus and anterior hypopituitarism, which typically persist even with radiographic regression of the disease. As for Langerhans cell histiocytosis, the definitive diagnosis of Erdheim-Chester’s disease is the finding of the typical histologic features at pituitary biopsy, which can be supported by the finding of the BRAF V600E mutation. Treatment options include vemurafenib, interferon-α, dabrafenib, trametinib, cobimetinib, cladribine, cyclophosphamide and glucocorticoids.

 

Rosai-Dorfman Disease

 

Pituitary involvement has been described in Rosai-Dorfman disease, a rare histiocytic disorder. Patients may have both anterior pituitary dysfunction, central diabetes insipidus and visual disturbances (202,203).

 

Inflammatory Pseudotumor

 

The inflammatory pseudotumor is a rare inflammatory disorder commonly involving the lung and orbit. It can be isolated or associated with the IgG4-related disease (204). Pituitary infiltration is a rare manifestation and patients can present with anterior and posterior hypopituitarism. The inflammatory pseudotumor can also spread to the sphenoid sinus, the cavernous sinus and the optic chiasm (205-207).

 

Tolosa-Hunt Syndrome

 

Tolosa-Hunt syndrome is a painful ophthalmoplegia caused by idiopathic retro-orbital inflammation involving the cavernous sinus or the superior orbital fissure. Histology shows nonspecific granulomatous or nongranulomatous inflammation. Patients with pituitary involvement present with anterior and posterior hypopituitarism, diplopia and retro-orbital pain (often unilateral) (208-212).

 

Other Systemic Diseases

 

Cases of secondary hypophysitis have been described in association with Takayasu’s arteritis (granulomatous hypophysitis) (213), Cogan’s syndrome (214) and Crohn’s disease (215,216). A case of isolated ACTH deficiency in a patient with Crohn’s disease has also been published (217).

 

OTHER CAUSES OF SECONDARY HYPOPHYSITIS

 

Thymoma and Other Malignancies (Anti-Pit-1 Antibody Syndrome)

 

Pit-1 is essential for the differentiation, proliferation, and maintenance of somatotrophs, lactotrophs, and thyrotrophs in the pituitary (218). Yamamoto et al. described three cases of acquired combined TSH, GH, and PRL deficiency, with circulating anti-Pit-1 antibodies (219). Cytotoxic T-cells that react against Pit-1 are likely the cause of anti-Pit-1 antibody syndrome (220-222). All these patients later developed thymomas that express Pit-1. Removal of the thymoma resulted in a decline in antibody titer, suggesting that aberrant expression of Pit-1 in the thymoma plays a causal role in the development of this syndrome (223). A handful of cases of anti-Pit-1 antibody syndrome not associated with thymoma have since been published. The malignancies causing this paraneoplastic syndrome included diffuse large B-cell lymphoma of the bladder and a metastatic cancer of unknown origin (222,224). Based on these cases, Yamamoto et al. have proposed diagnostic criteria for anti-PIT-1 hypophysitis (Table 13).

 

Table 13. Diagnostic Criteria for Anti-PIT-1 Hypophysitis

Criteria

Probable diagnosis

Established diagnosis

Criterion 1

Acquired specific GH, PRL, and TSH deficiency. *

CRITERION 1

CRITERION 1

 

and

 

CRITERION 2

Criterion 2

Presence of anti-PIT-1 antibody or PIT-1-reactive T cells in the circulation.

Criterion 3

Coexistence of thymoma or malignant neoplasm. **

* The secretion of other pituitary hormones is not impaired. The MRI of the pituitary is typically normal, but a slight atrophy of the anterior pituitary can be observed.

** Criterion 3 may help the diagnosis and clarify pathogenesis but may not be necessarily obvious at the time of diagnosis.

Infections

 

Infections of the pituitary are a rare cause of hypophysitis and hypopituitarism (225). They can affect either exclusively the pituitary area or as a part of disseminated infections. Risk factors are diabetes mellitus, organ transplantation, human immunodeficiency virus infection, non-Hodgkin lymphoma, chemotherapy, and Cushing’s syndrome. They can occur by (186):

 

  • Hematogenous spread in immuno-compromised hosts;
  • Contiguous extension from adjacent anatomical sites (meninges, sphenoid sinus, cavernous sinus and skull base);
  • Previous infectious diseases of the CNS of different etiologies;
  • Iatrogenic inoculation during trans-sphenoidal surgery.

 

However, in the majority of cases of pituitary abscess an obvious cause cannot be identified.

Tuberculosis can cause granulomatous involvement of the hypothalamus, the pituitary or the stalk. Tubercular meningitis and hypothalamic-pituitary involvement seem to affect mostly anterior pituitary function (226).

 

Several viruses can cause meningitis, meningoencephalitis and encephalitis that can involve the hypothalamic-pituitary region. Partial or complete hypopituitarism may develop as a result (186). A study by Leow et al. has shown that ~40% of patients with severe acute respiratory syndrome (SARS)-associated with Coronavirus infection can develop reversible central adrenal insufficiency, suggesting a possible inflammation of the pituitary in these patients (227). Hantavirus can also cause viral hypophysitis with pituitary ischemia and hemorrhage as part of the hemorrhagic fever with renal syndrome (HFRS), leading to partial or complete hypopituitarism, including diabetes insipidus (186,228,229).

 

Mycoses with hypothalamic-pituitary involvement are extremely rare. Patients frequently present with central diabetes insipidus and anterior pituitary dysfunction (mainly FSH/LH deficiency and hyperprolactinemia) (186).

 

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Benign Prostate Disorders

ABSTRACT

 

Benign prostatic hyperplasia (BPH) is among the commonest urological abnormalities affecting the aging male. The cause of the increase in prostatic volume is multifactorial, but current research has implicated hormonal aberrations. Clinical assessment of the patient is integral to determining the optimal treatment strategy. Exclusion of prostatic cancer and complications of BPH are critical prior to the commencement of conservative and non-invasive strategies. Recently, the introduction of pharmaceutical agents has changed the landscape of management of BPH. Alpha-blockers, 5-alpha reductase inhibitors, and phosphodiesterase-5 inhibitors provide significant symptomatic improvement for BPH, particularly when used in combination. Invasive surgical therapies remain the gold standard for refractory and complicated BPH disease. Advances in technology have provided new methods to perform prostatectomy including: bipolar resection, laser resection, ablation, enucleation or vaporization. Newer, minimally invasive measures have been introduced in an attempt to limit patient morbidity, specifically operative complications, sexual and urinary function. While results are promising, these emerging therapies have limited long-term data. The purpose of the current chapter is to provide an overview of the current knowledge of benign prostatic hyperplasia.

 

INTRODUCTION

 

The prostate is an organ linked inextricably to the endocrine system. During the development of the prostate, the epithelium and mesenchyme are under the control of testicular androgens, and interact to form an organized secretory organ. Furthermore, the endocrine system plays a key mechanistic role in many prostate diseases, and many therapies for prostatic diseases are aimed at the manipulation of the endocrine system. The gland resides in the true anatomical pelvis and forms the most proximal aspect of the urethra. It has been stated that the prostate gland is the male organ most commonly afflicted with either benign or malignant neoplasms (1). Therefore, it is an organ with which every physician and surgeon needs to be familiar. We will focus on BPH, the most prevalent of benign disorders affecting the prostate.

 

EMBRYOLOGY

 

The development, growth and cytodifferentiation of the prostate are androgen-dependent and occur via embryonic cell-to-cell interactions between the mesenchyme (undifferentiated connective tissue) that induce epithelial development while the epithelium induces mesenchymal differentiation (2).

 

In the developing prostate, urogenital sinus mesenchyme acting under the influence of testicular androgens induces ductal morphogenesis, the expression of epithelial androgen receptors, regulates epithelial proliferation and specifies the expression of prostatic-lobe specific secretory proteins. The developing prostatic epithelium reciprocally induces the differentiation and morphological patterning of smooth muscle in the urogenital sinus mesenchyme (2). In the prostate, it is traditional to consider androgens as promoters of growth, while activin and tumor growth factor-beta1 (TFG-β1) are regarded as potent growth inhibitors. These factors do not act independently, however, and cross-talk occurs between the signaling pathways at a sub-cellular level (3).

 

The first step in development of the prostate begins with the urogenital sinus mesenchyme signaling to the epithelium, causing it to form epithelial buds. Androgens then induce bud elongation, branching and epithelial differentiation (3). Prenatally, the androgen receptor (AR) is expressed only in the mesenchyme, not in the epithelium. Initial epithelial development is thus controlled via paracrine interactions where activation of stromal androgen receptors stimulates growth factors and induces growth in adjacent prostatic epithelial cells (4).

 

At the 5th week, the mesonephric (Wolffian) duct opens onto the lateral surface of the urogenital sinus and gives rise to the ureteric bud (Figure 1). By the 7th week, the growth of the urogenital sinus involves the progressive incorporation of the terminal part of the mesonephric duct into the wall of the urogenital sinus. They eventually open into the Mullerian tubercle that is the future verumontanum of the prostate. At their termination the paramesonephric (Mullerian) ducts fuse and are surrounded by the mesonephric ducts. At 10 weeks, prostatic epithelial buds begin to arise from the circumference of the urethra, around the orifice of the paramesonephric ducts. They develop predominantly on the posterior surface of the junction of the mesonephric ducts, forming two concentrations, above and below them (5).

Figure 1. The embryological origin and development of the prostatic urethra and the prostate, adapted from Delmas (5).

During the fetal period at about 6 months, multiple outgrowths arise from the prostatic portion of the urethra, particularly the posterior surface of the urethra, and grow into the surrounding mesenchyme. Glandular epithelium of the prostate differentiates from the endodermal cells of the urethra, and outgrowths of glandular epithelium protrude into the associated mesenchyme differentiate into the dense stroma and smooth muscle fibers of the prostate. In contrast, the prostatic glandular epithelium outgrowths situated on the anterior surface regress and are replaced by fibromuscular tissue. This region becomes the future anterior commissure of the prostate 5,6).

 

ANATOMY

 

According McNeal’s model of the prostate (7), four different anatomical zones may be distinguished that have anatomo-clinical correlation (Figure 2):

 

  • The peripheral zone: is the area forming the postero-inferior aspect of the gland and represents 70% of the prostatic volume. It is the zone where the majority (60-70%) of prostate cancers form.
  • The central zone: represents 25% of the prostate volume and contains the ejaculatory ducts. It is the zone which usually gives rise to inflammatory processes (e.g., prostatitis).
  • The transitional zone: this represents only 5% of the total prostatic volume. This is the zone where benign prostatic hypertrophy occurs and consists of two lateral lobes together with periurethral glands. Approximately 25% of prostatic adenocarcinomas also occur it this zone.
  • The anterior zone: predominantly fibromuscular with no glandular structures.

 

The prostate weighs approximately 20g by the age of 20 and has the shape of an inverted cone, with the base at the bladder neck and the apex at the urogenital diaphragm (8). The prostatic urethra does not follow a straight line as it runs through the center of the prostate gland but it is actually bent anteriorly approximately 35 degrees at the verumontanum (where the ejaculatory ducts join the prostate) (9).

Figure 2. 1= Peripheral Zone, 2= Central Zone, 3= Transitional Zone, 4= Anterior Fibromuscular Zone. B= Bladder, U= Urethra, SV= Seminal Vesicle (adapted from Algaba (10)).

HISTOLOGY

 

The prostate consists of stromal and epithelial elements. Smooth muscle cells, fibroblasts and endothelial cells are in the stroma and the epithelial cells are secretory cells, basal cells and neuroendocrine cells (Figure 3).

Figure 3. Histology of a prostate gland affected by benign prostatic hyperplasia.

The columnar secretory cells are tall with pale to clear cytoplasm. These cells stain positively with prostate-specific antigen (PSA) (11). Basal cells are less differentiated than secretory cells and are devoid of secretory products such as PSA (12). Finally, neuroendocrine cells are irregularly distributed throughout ducts and acini, with a greater proportion in the ducts. The prostate has the greatest number of neuroendocrine cells of any of the genitourinary organs (13).  Glands are structured with open and closed cell types with the open type facing the inside of the duct having a monitoring role over its contents. Most cells contain serotonin, but other peptides that are present include somatostatin, calcitonin, gene-related peptides and katacalcin (11). The cells co-express PSA and prostatic acid phosphatase. Their function is unclear, but it is speculated that these cells are involved with local regulation by paracrine release of peptides (11). Prostatic ducts and acini are distinguished by architectural pattern at low power magnification. The prostate becomes more complex with ducts and branching glands arranged in lobules and surrounded by stroma with advancing age.

 

Figure 4. Diagram outlining the structure of the prostate gland with regard to ducts, glandular cells and their relationship to blood vessels.

PHYSIOLOGY

 

At present, there is only limited knowledge of all of the secretory products of the prostate and how these relate to reproduction and infertility. However, the main role of the prostate as a male reproductive organ is to produce prostatic fluid that accounts for up to 30 per cent of the semen volume. Prostatic fluid promotes sperm motility, and it is a milky, alkaline fluid containing PSA, citric acid, calcium, zinc, acid phosphatase and fibrinolysin among its many constituents (Table 1) (14).  During ejaculation, alpha-adrenergic stimulation of prostatic smooth muscle expresses seminal fluid containing sperm from the ampulla of the vas deferens into the posterior urethra (15). Interestingly, abnormal growth of the prostate is only experienced by humans and dogs, and why other mammals are spared is a mystery (16).

 

Table 1.  The Composition of Human Semen (adapted from Ganong (17))

Color

White, opalescent

Specific Gravity

1.028

pH

7.35-7.50

Volume

3ml

SPECIFIC COMPONENTS OF SEMEN

Gland/Site

Volume in ejaculate

Features

Testis/Epididymis

0.15ml (5%)

Average approximately spermatozoa 80 million/ml

Seminal Vesicle

1.5-2ml (50-65%)

Fructose (1.5-6.5 mg/ml) phosphorylcholine ergothioneine, ascorbic acid, flavins prostaglandins, bicarbonate

Prostate

0.6-0.9ml (20-30%)

Spermine, citric acid, cholesterol, phospholipids, fibrinolysin, fibrinogenase, zinc, acid phosphatase, prostate-specific antigen

Bulbourethral Glands

< 0.15ml (<5%)

Clear mucus

 

ENDOCRINE CONTROL OF PROSTATIC GROWTH

 

Intraprostatic signaling systems are important for the regulation of cell proliferation and extracellular matrix production in the prostatic stroma. Central to this premise is the balance between factors such as TGF-β1, that induces extracellular matrix production, suppresses collagen breakdown and cell proliferation and factors such as fibroblast growth factor 2 and insulin-like growth factors that are mitogenic in the stromal compartment (18). Other endocrine pathways are being investigated, and there is a growing body of evidence suggesting an abnormality in the insulin-like growth factor axis is playing a role in the pathogenesis of BPH (19).

 

Testosterone

 

Prostatic epithelial cells express the androgen receptor (20). From the beginning of embryonic differentiation to pubertal maturation and beyond, androgens are a prerequisite for the normal development and physiological control of the prostate (21). Androgens help maintain the normal metabolic and secretory functions of the prostate, and they are also implicated in the development of BPH and prostate cancer. Androgens do not act in isolation, and other hormones and growth factors are being investigated (22).

 

Androgens also interact with prostate stromal cells which release soluble paracrine factors that induce growth and development of the prostatatic epithelium (4). These paracrine pathways might be critical in regulating the balance between proliferation and apoptosis of prostate epithelial cells in the adult (22).

 

The appropriate balance between testosterone and its 5-alpha reduced metabolites is key to normal prostate physiology (note the metabolic pathways for androgen metabolism are described in Endotext, Endocrinology of Male Reproduction, Androgen Physiology, Pharmacology and Abuse, D Handelsman). The metabolism of testosterone to dihydrotestosterone (DHT) and its aromatization to estradiol are recognized as the key events in prostatic steroid response. 

Figure 5. Conversion of testosterone to dihydrotestosterone (DHT) by 5alpha reductase

Testosterone, to be maximally active in the prostate, must be converted to DHT by the enzyme 5-alpha reductase (Figure 5) (23). DHT has a much greater affinity for the androgen receptor than does testosterone, and DHT accumulates in the prostate even when circulating concentrations of testosterone are low (24,25). Based on rat studies, DHT is about twice as potent as testosterone at equivalent androgen concentrations (26). Therefore, prostatic DHT concentrations may remain similar to those in young and elderly men, despite the fact that serum testosterone concentrations generally decline with age (23). In the prostate, the total level of testosterone is 0.4 ng/g and the total of DHT is 4.5 ng/g (27). The total serum concentration of testosterone in the blood is approximately 10 times higher than DHT (17). Circulating DHT, by virtue of its low serum plasma concentration and tight binding to plasma proteins, is of diminished importance as a circulating androgen affecting prostate growth (16). Intra-prostatic androgens are remarkably independent of serum concentrations (28), and circulating androgen concentrations often do not correlate with intraprostatic concentrations (29).

 

Estrogen

 

A role for estrogens in the prostate pathology of the ageing male appears likely with accumulating evidence that estrogens, alone or in combination with androgens, are involved in inducing aberrant growth and/or malignant change. Animal models have supported this hypothesis in the canine model, where estrogens “sensitize” the ageing dog prostate to the effects of androgen (30). The evidence is less clear in humans. Estrogens in the male are predominantly the products of peripheral aromatization of testicular and adrenal androgens (31). While the testicular and adrenal production of androgens declines with ageing, concentrations of total plasma estradiol do not decline. This has been ascribed to the increase in fat mass with ageing (the primary site of peripheral aromatization) and to an increased aromatase activity with ageing. However, free or bioavailable estrogens may decline due to an increase in sex hormone binding globulin, which could translate to lower intraprostatic concentrations of the hormone. The potentially adverse effects of estrogens on the prostate might be due to a shift in the intra-prostatic estrogen: androgen ratio with ageing. 

 

Estrogen, which acts through estrogen receptors (ER) alpha and beta, has been implicated in the pathogenesis of benign and malignant human prostatic tumors (32-34). As stated above, BPH is thought to originate in the transitional zone (TZ) and prostate cancer the peripheral zone (PZ) of the prostate. Receptor studies have found ER-alpha and ER-beta types distributed in human normal and hyperplastic prostate tissues, using in situ hybridization and immunohistochemistry. ER-alpha expression was restricted to stromal cells of the PZ. In contrast, ER-beta was expressed in the stromal and epithelial cells of PZ as well as TZ. These findings suggest that estrogen might play a crucial role in the pathogenesis of BPH through ER-beta (33). Investigations are ongoing and could result in a new range of therapies directed against BPH and prostate cancer. Dietary phytoestrogens (in soy and other vegetables) or selective estrogen receptor modulators are currently being investigated with regard to their role in the development of BPH and prostate cancer (31). Such ER modifiers might oppose some of the effects of natural estrogen by modulating ER receptors, thus reducing the local impact of androgens that need active ER receptors, effectively making them anti-androgenic compounds, but this hypothesis requires more investigation (35).

 

BENIGN PROSTATIC HYPERPLASIA (BPH)

 

BPH is an age-related and progressive neoplastic condition of the prostate gland (36). BPH can only be diagnosed definitively by histology. BPH in the clinical setting is characterized by lower urinary tract symptoms (LUTS, see below and Table 2). There is no causal relationship between BPH and prostate cancer (37). Clinically apparent BPH has a significant effect on quality of life, particularly its effects on nocturia and bladder dysfunction. The overall prevalence of BPH is 10.3%, with an overall annual incidence rate of 15 per 1000 man-years, increasing with age (3 per 1000 at age 45-49 years, to 38 per 1000 at 75-79 years). For a symptom-free man at age 46, the 30-year risk of clinical BPH is 45% (38). The true prevalence and incidence of clinical BPH will vary according to the criteria used to describe the condition; however, it has been estimated that the prevalence of BPH is rising due to increases in modifiable risk factors such as obesity (39). It is crucial to acknowledge that LUTS can exist without signs of BPH – as the symptoms can be caused by variations in the sympathetic nervous stimulation of prostatic smooth muscle, variability of prostatic anatomy (viz., enlarged median lobe of the prostate), and the variable effects of bladder physiology from the obstruction and aging.

 

There have been several studies demonstrating the fact that clinical BPH is a progressive disease. The Olmsted county study (40) showed that with each year there were deteriorations in symptom scores, peak flow rates, and increases in prostate volumes based on transrectal ultrasound scanning (TRUS). The risk of acute urinary retention (AUR) increased with flow rates below 12 ml/sec and with glands greater than 30ml. Studies have also demonstrated that those with larger prostates (>40 ml) and with serum PSA greater than 1.4 ng/ml were more likely to develop acute urinary retention (41). Treatment, however, has changed with the advent of effective non-surgical therapies. Between 1992-1998 there has been a significant lengthening of the period between first diagnosis of LUTS secondary to clinical BPH and surgery, associated with the earlier and increased use of specific medical treatments (42). From the patients perspective, the goals of therapy are to improve quality of life, reduce symptoms, and avoid surgery while ensuring safety from the complications of BPH (43).

 

Risk Factors for BPH

 

The only clearly defined risk factors for BPH are age and the presence of circulating androgens. BPH does not develop in men castrated before the age of forty (44),  but other factors may influence the prevalence of clinical disease. These include the following:

 

GENETICS

 

Clinical BPH appears to run in families. If one or more first degree relatives are affected, an individual is at greater risk of being afflicted by the disorder (45). In a study by Sanda et al (46), the hazard-function ratio for surgically treated BPH amongst first degree relatives of the BPH patients as compared to controls was 4.2 (95% CI, 1.7 to 10.2). The incidence of BPH is highest and starts earliest in blacks than Caucasians and is lowest in Asians (37). Interestingly, despite having larger prostate glands, the age-adjusted risk of BPH was the same for blacks as for whites (RR = 1.0, 95% Cl 0.8-1.2) (47). Furthermore, in an Asian population, men presenting with BPH are likely to have higher symptom scores than blacks or Caucasians (48).

 

DIET

 

Diet has been reported as a risk factor for the development of BPH. Large amounts of vegetables and soy products in the diet may explain the lower rate of BPH in Asia when compared to countries with Western, non-Asian diets. In particular, certain vegetables and soy are said to be high in phytoestrogens, such as genestin, that have anti-androgenic effects by an undetermined mechanism on the prostate in vitro (49).

 

Studying migrant populations with their heterogeneous environmental exposures increases the probabilities of identifying potential risk factors for BPH. Therefore, the association of alcohol, diet, and other lifestyle factors with obstructive uropathy was investigated in a cohort of 6581 Japanese-American men, examined and interviewed from 1971 to 1975 in Hawaii. After 17 years of follow-up, 846 incident cases of surgically treated obstructive uropathy were diagnosed with BPH. Total alcohol intake was inversely associated with obstructive uropathy (p < 0.0001). The relative risk was 0.64 (95% confidence interval: 0.52-0.78) for men drinking at least 25 grams of alcohol per month compared with nondrinkers. Among the 4 sources of alcohol, a significant inverse association was present for beer, wine, and sake, but not for spirits. No association was found with education, number of marriages, or cigarette smoking. Increased beef intake was weakly related to an increased risk (p = 0.047), while no association was found with the consumption of 32 other food items in the study (50).

 

METABOLIC SYNDROME

 

There is a growing body of evidence supporting the association between obesity or metabolic syndrome and the development of BPH. The risk of BPH appears to be independently associated with the individual components of BPH including central obesity, hyperinsulinemia, insulin resistance and dyslipidemia. Despite this, the precise causation of this association has not been clearly identified. Recent studies have suggested that in this setting, BPH is a consequence of the metabolic syndrome-associated metabolic derangements, altered sex hormone concentrations and lowered sex-hormone binding globulin concentrations (51). One study found in a cohort of 415 men, that indicators of metabolic syndrome (abnormal concentrations of insulin resistance, subclinical inflammatory state, and sex hormone globulin changes) were significantly associated with increased risk of BPH (52).  Mechanisms associated with metabolic syndrome have been discussed as possible targets for future therapies for BPH (53).

 

CHRONIC INFLAMMATION

 

There is strong evidence to suggest that inflammation and inflammatory markers are involved in the pathogenesis of BPH. Inflammation within the prostate can be caused by several factors including bacteria, virus, autoimmune disease, diet, metabolic syndrome, and hormone imbalances (53). This leads to the activation of inflammatory cells, release of cytokines, expression of growth factors, and ultimately abnormal proliferation of epithelial and stromal cells of the prostate. Proliferation induces a cycle of hypoxia and recruitment of more growth factors resulting in increased prostate volume and BPH (54). The REDUCE study of 8224 prostate biopsy samples of men with BPH found that 77.6% had cells of chronic inflammation on histology (55).

 

Anti-inflammatory medications have been studied in combination with BPH medications. A meta-analysis of three randomized controlled trials (n=183) in this area found that non-steroidal anti-inflammatory drugs improved IPSS scores by a mean of 2.89 points and increase peak urine flow by a mean of 0.89m/s (56).

 

OTHER RISK FACTORS

 

It has not been possible to delineate any other risk factors for BPH such as coronary artery disease, liver cirrhosis, or diabetes mellitus. Traditionally it has been believed that there is no causal relationship between malignant and benign prostatic hypertrophy (37) and recent data from large trials continue to support this premise (57).  Alternative theories have emerged but more data directly linking association with causality are required (58).

 

PATHOPHYSIOLOGY OF BPH

 

Natural History

 

BPH is a histological diagnosis, but its clinical manifestations occur after growth has occurred to such a degree and in such a strategic location within the gland, namely the transitional zone, that it impairs bladder emptying and results in LUTS. One can consider the natural history of BPH as involving two phases:

 

(i) The pathological or first phase of BPH is asymptomatic and involves a progression from microscopic to macroscopic BPH. Microscopic BPH will develop in almost all men if they live long enough but in only about half will progress to macroscopic BPH. This would suggest that additional factors are necessary to cause microscopic to progress to macroscopic BPH (59). The pathological phase involves development of hyperplastic changes in the transitional zone of the prostate (60). While there is wide variability in prostate growth rates at an individual level, prostate volume appears to increase steadily at about 1.6% per year in randomly selected community men (61).

 

(ii) The clinical or second phase of BPH involves the progression from pathological to ‘clinical BPH’ that is synonymous with the development of LUTS. Only about one half of patients with macroscopic BPH progress to develop clinical BPH (59). BPH consists of mechanical and dynamic components and it is these components that are responsible for the progression from pathological to clinical BPH (62). In clinical BPH, the ratio of stroma to epithelium is 5:1 whereas in the case of asymptomatic hyperplasia the ratio is 2.7:1. A significant contribution is therefore made by stroma to the infravesical obstruction of BPH (63).

 

DISEASE MANIFESTATIONS OF BPH

 

Lower Urinary Tract Symptoms (LUTS)

 

Lower urinary tract symptoms (LUTS) are highly prevalent and the majority of LUTS in men is produced by BPH, but may be contributed to by a variety of conditions (Figure 6). LUTS are traditionally divided into voiding or obstructive and storage or irritative symptoms (Table 2). Voiding symptoms are more common, however it is storage symptoms that are most bothersome and have a greater impact on a patient's life (64,65). The prevalence of clinical BPH rises with age and approximately 25% of men age 40 or over will suffer from LUTS (66).

 

Figure 6. Interaction of the many factors involved in the pathogenesis of LUTS. Other causes of LUTS (top right) include all of the differential diagnoses included in Table 5 (see below).

Hesitancy

Poor stream

Intermittent stream

Straining to pass urine 
Prolonged micturition 
Sense of incomplete bladder emptying 
Terminal dribbling

Urinary frequency

Urgency

Urge incontinence

Nocturia

 

In the past, LUTS suggestive of bladder outflow obstruction (BOO) secondary to BPH were referred to as ‘prostatism’, once other causes such as a urinary tract infection or prostate cancer were excluded. The pathology behind the symptoms was thought to be obstruction due to prostatic gland enlargement alone. However, it is now recognized that voiding/obstructive symptoms result from direct urinary flow obstruction whilst storage/irritative symptoms appear to be due to secondary bladder dysfunction (67). Thus, LUTs occurs after the prostate enlargement causes obstruction, and the bladder voiding is secondarily affected leading irritable symptoms (Figure 7).

 

Figure 7. Diagrammatic representation of BPH with the enlarged prostate transition zone causing obstruction of the prostatic urethra and the secondary changes in the bladder leading to hypertrophy of the detrusor muscle (copyright Nathan Lawrentschuk 2012).

 

This concept has been further refined in that obstructive symptoms are thought to result not only from mechanical obstruction due to glandular enlargement, but also dynamic obstruction secondary to contraction of the smooth muscle of the prostate, urethra and bladder neck. This dynamic obstruction is a result of sympathetic nervous system mediated stimulation of alpha-1 adrenoceptors. Storage symptoms appear to be caused by detrusor instability related to detrusor muscle changes in response to obstruction, such as bladder wall hypertrophy and collagen deposition in the bladder (68,69). Adrenoceptors may be further sub-divided into alpha1A and alpha1D subtypes, with alpha1A predominant in the prostate and alpha 1D in the bladder. Thus, blockade of alpha1A may be necessary for reduction of obstruction whereas the blockade of alpha1D may be required to relieve storage symptoms (70) (see below).

 

It has been suggested that the etiology of LUTS related to BPH is even more complex than outlined above, with extra-prostatic mechanisms such as bladder wall ischemia and changes in the central nervous system being implicated (71). Normal lower urinary tract function is complex, and theoretically any disruption of the pathway for micturition (Figure 8 below) may lead to LUTS (72).

 

It is worth noting the relationship between LUTS and sexual dysfunction, with sexual dysfunction being highly prevalent in men with LUTS

By sexual dysfunction, we refer to decreased libido, erectile dysfunction, decreased ejaculation and other ejaculation disorders. Kassabian

expands on the relationship and agrees with Leilefeld et al

in suggesting that the relationship is coincidental and both are common in the ageing male.

 

Figure 8- Normal micturition pathways (reproduced with permission from Physiology and pathophysiology of lower urinary tract symptoms, Drugs of Today, Vol 37, p. 7, Michel MC(72)).

 

COMPLICATIONS OF BPH

 

The complications of BPH are summarized in Table 3.

 

Table 3. Common Complications of BPH

·       Urinary retention

·       Recurrent Urinary Tract Infections

·       Bladder Calculi

·       Hematuria

·       Secondary bladder instability

·       Renal Impairment

 

Urinary Retention (Acute and Chronic)

 

As the prostate volume increases with age, the likelihood of acute urinary retention (AUR) and symptom severity both increase while urinary flow rates fall. In one study of more than 2000 men, those with a maximum urinary flow rate (Qmax) <12 ml/s had a 4 times greater risk for AUR than did men with a Qmax >12ml/s (76). AUR is usually painful and necessitates the insertion of a per urethral indwelling or suprapubic urinary catheter.

 

If the urinary retention is not dealt with in a timely fashion, the detrusor muscle becomes distended and damaged, contributing to poor detrusor function and an inability to adequately empty the bladder. The retention of urine becomes painless over time, and the sequelae of retained urine such as recurrent UTI, calculi, and renal impairment may develop.

Furthermore, a situation of overflow incontinence may develop whereby the bladder automatically empties once the volume reached exceeds its new, larger capacity. The passage of urine is typically uncontrolled, and this may often be the first presentation for someone with advanced BPH. The bladder remains full despite the emptying, which is only partial.

 

In situations of chronic urinary retention, relieving the bladder outflow obstruction might not restore normal detrusor function. These patients often need to use intermittent self-catheterization or have permanent drainage to keep their bladder empty and to reduce damage to the upper urinary tract.

 

Recurrent Lower Urinary Tract Infection (UTI)

 

The best host defense against infection in the lower urinary tract might be normal flow of urine and bladder emptying. In BPH, bladder outflow obstruction results in disruption of this mechanism with retention and pooling of urine in the bladder, giving organisms the opportunity to multiply rather than be flushed out. Despite this logical assumption, there is little evidence in the literature to support this theory. Nevertheless, men with significant clinical BPH are probably at risk of UTI, and men with UTI should be assessed for signs of BPH.

 

Bladder Calculi

 

In developed countries, the most prevalent cause of bladder calculi is bladder outlet obstruction owing to BPH (77). Of those who undergo prostate surgery for BPH, approximately 2% of all patients are found to have bladder stones (78). Stones occur in this situation due to urinary stasis combined with high urinary solute concentrations, which leads to crystal precipitation (79). Chronic infection with urease-producing organisms may predispose to the development of stones and rarely stones pass from the upper tract to act as a nidus in the bladder (79) . Bladder calculi associated with BPH remains an absolute indication for transurethral resection of the prostate (TURP) (80,81) because of the risk or recurrence of stone formation. However, the necessity of surgery is being challenged by the expanding use of medical management in treating BPH (81).

 

Hematuria

 

The incidence of hematuria with BPH is uncertain, however; in a retrospective review of almost 4000 patients undergoing TURP, Mebust et al (80) noted that hematuria was an indication for surgery in 12% of patients. It is hypothesized that BPH, with its increased acinar and stromal cell proliferation, stimulates increased vascularity via angiogenesis. These new and prolific vessels may be easily disrupted leading to recurrent bleeding (82). This is supported by Foley et al (83) who found the microvessel density to be higher in those patients with BPH having hematuria after histological studies. It is also hypothesized that 5-alpha reductase inhibitors might reduce angiogenesis and theoretically reduce the risk prostate bleeding. Finasteride has been suggested as an option in treating the problem of hematuria (84-86).

 

Detrusor (Bladder) Instability

 

The definition of detrusor instability is the development of a detrusor contraction which exceeds 15cm H2O at a bladder volume of less than 300ml (87). Detrusor instability is not a specific term related to BPH, but implies LUTS secondary to detrusor pathology. These symptoms are normally storage related and consist of urgency, frequency, urge incontinence, and nocturia. In BPH, the normal dynamics of the bladder are altered due to detrusor muscle stretching due in turn to retention of urine and contraction against an obstructed outlet. Although not completely understood, some of the detrusor instability may be related to changes at the adrenoceptors level, rather than just from obstruction and its consequences alone. In normal bladder physiology, beta-adrenoceptors are believed to be involved in the relaxation of the bladder during storage of urine (71). In some patients, however, the administration of noradrenaline leads to contraction of the detrusor muscle which may be blocked by an alpha-1 adrenoceptor antagonist (88). This implies the presence of alpha-adrenoceptors in the detrusor muscle in at least some patients. Furthermore, alpha-adrenoceptor antagonists have been shown to relieve storage and voiding symptoms in men without obstruction and storage symptoms in women (71,89-92). Alpha adrenoceptor subtypes in the human bladder are predominantly of the alpha1D and alpha1A type. In animal models, the alpha1D receptors become more abundant with bladder obstruction (93), and it may be speculated that this is the case in humans and that these receptors, once up-regulated, play a role in storage symptoms (71).

 

Renal Insufficiency

 

Renal insufficiency results from obstructive uropathy secondary to the bladder outlet obstruction of BPH. In an analysis of patients receiving treatment for BPH, 13.6% (range 0.3-30%) had renal insufficiency (78). Certainly, an abnormal creatinine is an indication to further investigate the upper urinary tract with imaging. Obviously, other concurrent causes of renal insufficiency need to be excluded. Those patients with renal insufficiency undergoing surgery are at increased risk (25%) of postoperative complications such as acute renal failure and urosepsis compared to patients without (17%) insufficiency (80).

 

HISTORY

 

A comprehensive medical history must be evaluated and should include the use of a voiding diary, the International Prostate Symptom Score (IPSS) and a discussion of the role of PSA testing (94).  An outline of the evaluation and treatment options for LUTS is shown in Table 4 and is discussed in greater depth below (95,96). Previous urological disease should be documented including previous urological surgery, UTI, bladder or renal calculi, renal disease and penoscrotal pathology. Any risk factors for surgery such as diabetes mellitus, immunosuppression, ischemic heart disease, respiratory problems, smoking as well as a comprehensive list of medications should be noted. Medications with anti-cholinergic properties should be noted, as these may contribute to the patient’s symptoms. The use of antihypertensives must be noted as any alpha-blocker treatment initiated could potentially cause severe hypotension.

 

As discussed in the section on differential diagnosis, consideration needs to be given to neurologic causes of voiding dysfunction such as stroke or Parkinson’s disease.

 

Table 4. A Summary of Diagnosis and Treatment Options in BPH

EVALUATION of LUTS

ESSENTIAL

1. History

2. Digital Rectal Exam (DRE)

3. Urinalysis 
4. Serum creatinine 
5. PSA, if > 10-year life expectancy 
6. International Prostate Symptom Score (IPSS) or AUA symptom index

SELECTED 
1. Uroflowmetry 
2. Imaging - especially if hematuria, UTI, urolithiasis 
3. Post Void Residual (PVR) estimation 
4. +/-Pressure flow studies 
5. +/-Cystoscopy

TREATMENT OPTIONS

MEDICAL THERAPY

1. Phytotherapy

2. Monotherapy:

       a. Alpha blockers

       b. 5-alpha reductase inhibitors

       c. PDE5 Inhibitors

3. Combination therapy:

       a. Alpha blocker + 5-alpha reductase inhibitor

       b. PDE5 inhibitor + alpha blocker (experimental)

SURGERY 
1
. Invasive surgery

       a. Transurethral resection of the prostate (TURP) 
       b. Laser prostatectomy/treatment 

       c. Open prostatectomy 
2. Minimally invasive measures

       a. Transurethral Incision of the prostate (TUIP) 

       b. Thermo ablative strategies (TUMT, TUNA)

       c. Chemical ablative (PRX-302, NX-1207, TEAP)

       d. Mechanical (Urolift, prostatic stent)

       e. Others (prostatic artery embolization, histotripsy, Rezum, aquablation)

 

International Prostate Symptom Score (IPSS)

 

The American Urologic Association (AUA) Symptom Index was developed as a standardized instrument to assess the degree of bladder outlet obstruction in men (89). It is widely used and consists of seven questions that assess emptying, frequency, intermittency, urgency, weak stream and straining with each graded with a score of 0-5. Total score ranges 0-35. The index categorizes patients as:

  1. Mild (score £7)
  2. Moderate (score 8-19)
  3. Severe (score 20-35).

 

The International Prostate Symptom Score (IPSS) is a modification of the AUA Symptom Index adding a single question assessing the quality of life or bother score based on the patient’s perception of the problem (Figure 9) (97). Both the AUA and IPSS questionnaires, although not specific for BPH, prostate volume, urinary flow rate, post-void residual volume, or bladder outlet obstruction, have been validated and are sensitive enough to be to be used in the evaluation of symptoms and selection of treatment (98-100). Many would argue that the score is the primary determinant of whether or not a patient proceeds to further treatment. Further, these questionnaires are a valuable objective measure when determining the response to treatments for BPH.

 

Figure 9. International Prostate Symptom Score (IPSS) Sheet (101,102)

 

EXAMINATION

 

General appearance is of importance, especially in identifying those with neurological disease (e.g., past stroke, Parkinson’s disease) or other major co-morbidities (obesity, severe osteoarthritis, diabetes) that may impact on treatment or further investigation. An abdominal examination should identify those in marked urinary retention, any abnormal masses, and previous surgical scars. A careful assessment of the scrotum and its contents as well as the penis is also warranted to exclude any other pathology. The digital rectal examination (DRE) is important in identifying prostatic abnormalities, including clinically apparent prostate carcinoma (103). Prostate size, texture, and tenderness should all be assessed, as should anal tone. Any nodules should be carefully noted. Constipation may also be a contributing factor to urinary retention and anal tone should also be recorded.

 

DIFFERENTIAL DIAGNOSIS OF BPH

 

It is important to acknowledge that the diagnosis of BPH often relies on surrogate measures until a histological diagnosis is confirmed. These range from clinical (symptom scores), physiological (uroflowmetry), anatomical (prostatic volume on DRE or TRUS) and biochemical (PSA values) measurement. Although all of these measurements capture some component of BPH, none of them is specific for BPH (104). Surrogate measures are likely to represent a continuum of disease severity without the existence of a threshold. Thus, differential diagnoses need to always be considered and where appropriate, excluded. In table 5 below, some of the more obvious differential diagnoses are listed, but will not be examined in detail.

 

Table 5. Differential Diagnoses for LUTS

Inflammatory Conditions

 

1. Urinary Tract Infection

2. Prostatitis

3. Bladder Calculi 
4. Interstitial Cystitis 
5. Tuberculous Cystitis

Neoplastic Conditions

 

1. Prostate cancer

2. Bladder transitional cell carcinoma (usually CIS)

3. Urethral cancer

Neurological Conditions

1. Parkinson's disease

2. Stroke

3. Multiple Sclerosis 
4. Cerebral Atrophy 
5. Shy-Drager Syndrome

Other Causes of Urinary Obstruction

1. Urethral stricture

2. Severe phimosis

3. Bladder neck dyssynergia 
4. External sphincter dyssynergia

 

PROSTATITIS

 

Prostatitis is a common condition that must be excluded from other causes of LUTS and is a common cause of visits to primary care physicians and urologists. It may present as an acute bacterial infection or may be chronic, occasionally progressing to a debilitating illness. In practice, the clinical diagnosis of prostatitis depends on the history and physical examination, but there is no characteristic physical finding or diagnostic laboratory test. Patients with prostatitis experience considerable morbidity and may remain symptomatic for many years. Unfortunately, there is limited understanding of the pathophysiology and optimal treatment for most patients. Prostatitis has been sub-classified and an abbreviated version is shown in Table 6.

 

·       Table 6. The National Institute of Health (USA) Consensus Classification of Prostatitis Syndromes

·       Acute bacterial prostatitis

·       Chronic bacterial prostatitis

·       Chronic prostatitis/chronic pelvic pain syndrome

·       Inflammatory

·       Non-inflammatory

·       Asymptomatic inflammatory prostatitis

 

Acute Prostatitis

 

Clinical features suggestive of acute prostatitis (Type 1, in Table 6 above) include dysuria and urinary frequency as well as perineal pain (Table 7). Systemic symptoms such as fever, rigors, myalgia and sweats are often a feature. On examination, the patient is normally febrile, and may be overtly septic depending on the infection severity. A digital rectal exam finds an extremely tender prostate, which is often intolerable to the patient. An abscess is occasionally palpated.

 

Table 7. Clinical Symptoms in Prostatitis (adapted from Lobel (105))

Genital symptoms

1. Dribbling

2. Inguinal pain

3. Testicular pain

4. Retropubic pain 
5. Perineal pain 
6. Urethral Burning

General Symptoms

1. Backache

2. Sweating

3. Tiredness

4. Cold feet

 

Investigations should include a mid-stream urine sample for microscopy, culture for bacteria, and antibiotic sensitivity. The most common organisms are typical uropathogenic bacteria such as Escherichia coli (E. coli). Blood cultures for bacteria and antibiotic sensitivity should also be considered. Prostatic massage is usually contraindicated in patients with acute prostatitis due to pain and the risk of precipitating sepsis. A treatment regime is highlighted in Table 8.  If there is failure to respond to therapy, evaluation for a prostatic abscess using a transrectal ultrasound scan or computed tomography scan may be required. If necessary, perineal or transurethral drainage of an abscess may be undertaken. At least 4 weeks of antibiotic therapy is recommended in all patients to try to prevent chronic bacterial prostatitis. Following resolution of acute prostatitis, the urinary tract should be investigated for any structural problems (106,107).

 

Table 8. Treatment of Acute Prostatitis

1. Hydration

2. Rest and hospitalization if severe

3. Empirical therapy with antibiotic until urine culture and sensitivities available

4. For patients requiring parenteral therapy antibiotics covering the likely organisms: broad spectrum cephalosporins, for example, cefuroxime, cefotaxime, or ceftriaxone plus gentamicin

5 Oral treatment according to sensitivities.: quinolones, such as ciprofloxacin or norfloxacin.  For patients intolerant of, or allergic to, quinolones: trimethoprim or co-trimoxazole;

6. Analgesics, such as non-steroidal anti-inflammatory drugs Suprapubic catheterization if catheterization needed - per urethral catheters may precipitate abscess formation

 

Chronic Prostatitis

 

As the presentation may be localized to the genital region or non-specific (see Table 7) a careful history and examination along with specialized diagnostic tests are needed to identify this condition. Investigations may involve prostatic massage to express organisms and/or white blood cells for analysis. Urine sample collection is often done in phases to aid in the localization process: first void urethral urine; mid-stream bladder urine; post-prostatic massage sample. Urine microscopy and quantitative culture is then undertaken. Semen analysis for excessive white blood cell numbers may also be indicative of chronic prostatitis. Serum PSA concentrations are often elevated in acute prostatitis or in an active phase of chronic prostatitis. Trans-rectal ultrasound might be considered but not recommended to differentiate the different forms of chronic prostatitis. Urinary tract localization procedures (culture of first void urethral urine; mid-stream bladder urine; post-prostatic massage samples of urine correlating to urethra, bladder and prostate) although theoretically correct, are often not used in clinical practice (106,107).

 

The various classifications of chronic prostatitis are listed in Table 6. Patients with chronic bacterial prostatitis (type II prostatitis) experience recurrent episodes of bacterial urinary tract infection caused by the same organism, usually E. coli, another Gram-negative organism, or enterococcus. Between symptomatic episodes of bacteriuria, lower urinary tract cultures can be used to document an infected prostate gland as the focus of these recurrent infections. Acute and chronic bacterial prostatitis represent the best understood, but least common, prostatitis syndromes (106,107).

 

Unfortunately, more than 90% of symptomatic patients have chronic prostatitis/chronic pelvic pain syndrome (type III). This term recognizes the limited understanding of the causes of this syndrome for most patients and the possibility that organs other than the prostate gland may contribute to this syndrome. Urological pain (normally in the perineum or associated with voiding or intercourse) is now recognized as a primary component of this syndrome. Active urethritis, urogenital cancer, urinary tract disease, functionally significant urethral stricture, or neurological disease affecting the bladder must be excluded. Patients with the inflammatory subtype (type IIIA) of chronic prostatitis/chronic pelvic pain syndrome have leukocytes in their expressed prostatic secretions post prostate massage urine or in semen.

 

In contrast, patients with the non-inflammatory subtype of chronic prostatitis (type III B) have no evidence of inflammation. In essence, they have no evidence of active infection nor of inflammation on available investigative techniques taken at a particular point in time. Repeat investigations are therefore done to be sure adequate sampling has been undertaken. This condition may be difficult to treat and requires intensive counselling, information and reassurance to the patient to be successfully managed (107).

 

Finally, asymptomatic inflammatory prostatitis (type IV) is diagnosed in patients who have no history of genitourinary tract pain complaints. It is often an incidental finding on prostatic biopsy done for other reasons (e.g., a raised PSA). Treatment is usually not required.

 

Treatment of Chronic Prostatitis

 

All patients should have investigations as outlined above. A summary of treatment options is shown in Table 9. Those patients with chronic prostatitis secondary to bacterial infection (type II) require a prolonged course of antibiotics (often up to three months) and should then be re-cultured to ensure eradication of the organism. Some urologists argue that these patients should also have investigation of their urinary tract by way of cystoscopy and at minimum, an ultrasound to ensure no anatomical abnormality that may be responsible.

 

Patients with asymptomatic prostatitis (IV) require no treatment but those with the inflammatory (IIIA) and non-inflammatory (IIIB) are more difficult.  Patients with type IIIA disease have excessive leukocytosis in their specimens but no bacteria. However, because their symptoms may be due to a pathogen that is difficult to isolate, a further course of antibiotics (6-12 weeks) with coverage of chlamydia and ureaplasma should be given (105). If this antibiotic course is not therapeutic, then a focus should be on anti-inflammatory medications (which may be used in conjunction with the course of antibiotics). If anti-inflammatory treatment fails, then patients should be treated as below, for type IIIB.

 

Current treatment for Type IIIB patients requires multiple therapies. Triple-therapy involves high dose alpha-blocker (3 month minimum), analgesia, and muscle relaxant (benzodiazepines). Initially, a narcotic analgesic should be changed to a non-steroidal anti-inflammatory (NSAID) if a response occurs after 2 weeks. The NSAID should be continued for at least 6 weeks, but stopped if there is no response at 2 weeks. If the triple treatment fails, other avenues must be explored, including biofeedback, relaxation exercises, psychotherapy, and lifestyle changes (soft cushions, cease bike-riding). The focus is on improving quality of life and minimizing symptoms, not curing the disease (105).

 

·       Table 9. Management and Treatment of Chronic Prostatitis

·       Oral and written patient education

·       Pharmacological treatment for chronic bacterial prostatitis chosen according to antimicrobial sensitivities include quinolones such as ciprofloxacin; ofloxacin; norfloxacin. For those allergic to quinolones: minocycline; doxycycline; trimethoprim-sulfamethoxazole; co-trimoxazole; in many regions, trimethoprim sulfamethoxazole is first line therapy because of better safety profile than quinolones.

·       Other treatments for chronic bacterial prostatitis: radical transurethral prostatectomy or total prostatectomy in carefully selected patients.

·       Empirical treatments for chronic abacterial prostatitis

·       Treat as for chronic bacterial prostatitis with a quinolone or tetracycline

·       Alpha blockers: terazosin, doxazosin, alfuzosin, tamsulosin, silodosin

·       Non-steroidal anti-inflammatory drugs

·       Stress management. Referral for psychological assessment as appropriate; diazepam. Note: benzodiazepines are considered but not recommended in clinical practice because of dependency

·       Adequate follow-up and counselling, often with professional support

·       Cernilton (pollen extract)

·       Bioflavonoid quercetin

·       Transurethral microwave thermotherapy

 

INVESTIGATIONS OF LUTS

 

As outlined by Tubarro et al (94), the aim of investigations for LUTS should be threefold: (1) to evaluate the possible relationship between prostatic enlargement, lower urinary tract symptoms and signs of bladder outlet obstruction; (2) to quantify the severity of benign prostatic enlargement-related symptoms and signs and (3) to rule out the presence of a prostate cancer.

 

Urinalysis

 

Urinalysis is used to screen for urinary tract infection as a cause of LUTS in order to identify those with microscopic or macroscopic hematuria. A formal urine culture may be undertaken if the analysis was suspicious for infection.

 

Post-Void Residual Urine Volume (PVRU)

 

Although there is a high degree of intra-individual variation in the PVRU, it may still provide valuable information with regard to bladder emptying. Although it does not distinguish adequately between bladder outlet obstruction or poor detrusor function, it can identify a bladder emptying problem and be used as a marker for improvement.  Due to its inability to differentiate between causes, the United States guidelines on BPH suggest it is an optional investigation (78). Greater than 300ml is considered a potential risk factor for upper urinary tract dilatation and renal impairment (108).  The PVRU does have the advantage of being used as a monitoring investigation in those opting for non-surgical therapy for BPH. It is readily and quickly performed in the office or hospital setting using portable ultrasound equipment.

 

Laboratory Investigations

 

Serum creatinine is recommended by most guidelines for the investigation of BPH and an elevated serum creatinine would be an indication to evaluate the upper urinary tract (96).

Serum PSA has several implications in the diagnosis and management of BPH, including (1) providing a prediction of the prostate volume (2) providing the prediction of disease course, and (3) providing a risk assessment for prostate cancer. Indeed, in multiple placebo arms of large double-blind clinical trials, the serum PSA is an independent predictor of the risk of acute urinary retention and progression to BPH-related surgery (109). While the PSA provides useful information in the aforementioned domains, in clinical practice the main utility of PSA testing in the setting of LUTS is to exclude prostate malignancy. In patients presenting with isolated LUTS, current guidelines suggest its use only if a diagnosis of prostate cancer will change management or if the PSA can assist in decision-making in patients at high risk of BPH progression. 

 

Upper Urinary Tract Imaging

 

Urinary tract ultrasound or computerized tomography are appropriate modalities. Most would consider upper tract imaging as mandatory if hematuria is present and recommend it if there was a history of urolithiasis, urinary tract infection, or renal insufficiency. Intravenous pyelography still has a role in certain cases, as other modalities do not outline the anatomy of the collecting system with such definition (94).

 

Urodynamics

 

Urodynamics is a general term for a collection of investigations useful in quantifying the activity of the lower urinary tract during micturition (110). Complete pressure-flow urodynamics are complex and usually involve fluoroscopy, video recording, bladder and rectal pressure measurement, as well as an assessment of urine flow. The simplest urodynamics are pressure-flow studies, requiring only voiding into a measuring device to obtain flow rates, and may easily be done in the office setting.

 

With regard to the investigation and diagnosis of conditions underlying LUTS, when considering inexpensive, safe, and completely reversible treatments, one may opt to avoid urodynamics studies initially. However, when considering irreversible, expensive, or potentially morbid therapy, such studies are considered mandatory. Many patients will not have urodynamics studies based on the first premise above (110). However, in reality, many surgeons and physicians will have simple pressure-flow studies readily available and will perform these as part of an initial consultation. More complex studies require time and are costly, and so should be reserved for particular situations as discussed below.

 

Urinary Flow Rate (Uroflowmetry)

 

Uroflowmetry is considered by some as the single most useful urodynamic technique for the assessment of obstructive uropathy. The purpose of the uroflow examination is to record one or more micturitions that are representative of the patient’s usual voiding pattern. Therefore, more than one micturition is often required and it is necessary to confirm with the patient if the flow was better, worse or about the same as their normal pattern, otherwise intra-individual variability may lead to false assumptions (111). The study may be performed in the office or as part of other urodynamic studies in the laboratory or operating suite.

 

Figure 10 indicates the most common urinary flow parameters measured. Of these, the peak flow rate is the most closely correlated with the extent of outflow obstruction (Table 10). Total voiding time is prolonged in obstruction and has a reduced Qmax. Poor detrusor contractility is impossible to distinguish from bladder outflow obstruction on uroflowmetry so other urodynamics investigations such as a cytometry are indicated.

 

Figure 10. Uroflowmetry in a normal individual- diagram above and actual reading below (Table 10).

 

Table 10. Interpretation of Uroflowmetry Results.

Flow rate- Qmax

Interpretation

>15ml/sec

Unlikely to be significant obstruction

<10ml/sec

Likely to be significant obstruction or weak detrusor activity

10-15ml/sec

Equivocal

 

Urodynamics- Pressure-Flow Studies

 

Various measurements may be used to define detrusor pressures and urethral sphincter pressures as an aid to diagnosis in specific circumstances. This is relevant in patients with LUTS who have had a stroke (or other neurologic disease) where bladder function may have sensory deficits or unstable detrusor contractions that may need alternate management. Nevertheless, detrusor instability is not considered a negative factor with respect to the outcome of BPH surgery (94), provided it is adequately managed. Some have even suggested that the detection of detrusor instability in patients with LUTS is only of minor diagnostic importance (112).

 

Urethrocystoscopy

 

The performance of this investigation depends on patient history and proposed surgical intervention. It is necessary where there is a history of microscopic or macroscopic hematuria to exclude bladder tumors or stones. A history or suspicion of urethral strictures, bladder tumors, or prior lower urinary tract surgery should also prompt this investigation. Surgeons may also use urethroscystoscopy when planning different surgical treatments or invasive therapies.

 

Transrectal Ultrasound Scanning (TRUS)

 

Compared to TRUS, methods of determining prostate size such as DRE, urethrocystoscopy, and retrograde urethrography are poor (113). It is often conducted in unison with biopsies of the prostate for suspected carcinoma, but is also a useful tool for assessing the size of an enlarged prostate so that the best mode of management may be undertaken, such as open versus endoscopic surgery.

 

OVERVIEW OF TREATMENT OF BPH

 

The primary aim of any treatment for BPH in the vast majority of men is to relieve bothersome obstructive and irritative symptoms (114) (Table 2). Treatment is often undertaken on an elective basis for such patients. Those in whom complications of BPH occur have treatment done urgently as a matter of course. A range of treatment options are available and may be tailored to the needs of every individual, taking into account their disease manifestations, success rates of treatment, possible complications, and patient preference.

 

WATCH AND WAIT/LIFESTYLE CHANGE

 

Many men who present with LUTS are often seeking a full assessment of their prostatic health rather than immediate treatment of symptoms that may not be exceptionally bothersome. People with mild symptoms may wish to pursue lifestyle changes as a way of improving their quality of life but with the option of review if such measures fail or symptoms worsen. Furthermore, when an adequate history is taken, hidden agendas such as fear of prostate cancer may even be revealed and fears allayed.

 

Often drinking habits may be responsible for symptoms such as nocturia, where considerable fluid volumes are consumed in the evening. Reducing fluid intake may diminish nocturia and evening urgency. Furthermore, caffeine and alcohol acting as diuretics can further exacerbate LUTS. Simple shifts in daily fluid intake may fulfil patient expectations and result in satisfactory outcomes. Voiding diaries are useful for making patients aware of drinking habits and may be the catalyst for initiating and monitoring changes. Bladder retraining (by using timed voiding, strengthening pelvic floor exercises, and monitoring oral intake) is also an option in some individuals, once a voiding diary has been examined.

 

Medications may also play a role with LUTS. Measures such as diuretic restriction in evenings often prevents nocturia and frequency, provided the diuretic can be taken earlier in the afternoon.

 

It is important to discuss options with the patient and that they he be made aware that the possibility of damage to their upper urinary tract or to the detrusor muscle may result if their symptoms deteriorate and they do not seek medical attention.

 

PHYTOTHERAPY FOR BPH

 

Phytotherapy, or the use of plant extracts, is becoming widely used in the management of many medical conditions including BPH (Table 11) (115). Often these agents are promoted to aid “prostatic health” and a significant proportion of men try them. Factors also contributing to their widespread use include the perception that they are supposedly ''natural'' products; the presumption of their safety (although this is not adequately proven); their alleged potential to assist in avoiding surgery, and even the unproven claim that they may prevent prostate cancer. The widespread availability of these products (without prescription) in vitamin shops, supermarkets, pharmacies, and over the internet has contributed to their usage and reflects the demand for these phytotherapeutic agents. The mechanisms of action are poorly understood but have been proposed to be (1) anti-inflammatory, (2) inhibitors of 5-alpha reductase, and more recently (3) through alteration in growth factors (116).

 

Phytotherapy, although promising, lacks long-term, good quality clinical data (117). Nevertheless, because there is a large placebo effect associated with treatment of voiding symptoms, the use of herbal products that have few or no side effects may be a reasonable first-line approach for many patients (118). However, patients should be counselled that the efficacy, mechanisms of action and long-term effects of these agents are not known and they must be aware of the limitations before proceeding (119).

 

The most popular phytotherapeutic agents are extracted from the seeds, barks and fruits of plants. Products may contain extracts from one or more plants and different extraction procedures are often used by manufacturers. Thus, the composition and purity of products may differ even if they originated from the same plant. Basic research on one product may not be easily transferred to another making the gathering of data and giving of advice difficult (120).

 

Table 11. Phytotherapy Used in the Treatment of Benign Prostatic Hyperplasia

Phytotherapeutic plant extract

Proposed Mechanism of action

Saw palmetto- fruit

(Serenoa repens)

Antiandrogenic, Anti-inflammatory

African plum- bark

(Pygeum africanum)

Antiandrogenic, potential growth factor manipulation, anti-inflammation actions

Pumpkin- seed

(Cucurbita pepo)

Phytosterols are thought to be amongst the active compounds

Cernilton- pollen

(Secale cereal, Rye)

Inhibition of alpha-adrenergic receptors

South African star grass- root

(Hypoxis rooperi)

Antiandrogenic, alteration in detrusor function

Stinging nettle- root

Steroid hormone manipulation reducing prostate growth

Opuntia- flower

(Cactus)

Unknown

Pinus- flower

(Pine)

Unknown

 

Saw Palmetto Berry (Serenoa repens)

 

Extracts from the berries of the American dwarf palm (saw palmetto) are the most popular and widely available plant extracts used to treat symptomatic BPH today (121,122). At least eight possible mechanisms of action for saw palmetto have been advocated including anti-androgenic properties, anti-inflammatory properties, induction of apoptosis to name a few (120). Several studies have found that saw palmetto suppresses growth and induces apoptosis of prostate epithelial cells by inhibition of various signal transduction pathways (123). However, it is most commonly believed that saw palmetto works as a naturally occurring weak 5-alpha reductase inhibitor, blocking the conversion of testosterone to DHT, as demonstrated in several in vitro studies (118, 124-127). Thus, saw palmetto may be expected to reduce prostate size. While demonstrated in animal models (128), this is not the case in several trials using saw palmetto in men with BPH (129,130). The only trial to show in vivo effects of saw palmetto involved needle biopsies of the prostate gland, before and after treatment with saw palmetto or placebo. Although the mechanism is unclear, there was a significant increase in prostatic epithelial contraction in the saw palmetto group (131).

 

Clinical evidence reporting the use of saw palmetto is conflicting. In a meta-analysis of 18 randomized studies relating to saw palmetto extracts, almost 3000 men with BPH were studied and the authors concluded that “the evidence suggests that saw palmetto improves urologic symptoms and flow rates but that further research is needed using standardized preparations to determine long term effectiveness” (115). When analyzing flow rate and symptom score alone from this meta-analysis, the effect of Seronoa repens (the scientific name of saw palmetto was to increase the flow rate by a further 2.28 ml/sec (standard error, SE, 0.29) over placebo which gave an increase of 1.09 ml/sec (SE 0.45). Serenoa repens also reduced the IPSS by 4.7 (SE 0.41), which is comparable to that found with finasteride and tamsulosin monotherapy (132).

 

Conversely, a recently published Cochrane review concluded Serenoa repens was no more effective than placebo for treatment of urinary symptoms consistent with BPH (133). This update of a prior review, nine new trials involving 2053 additional men (a 65% increase) were included. The main comparison was again Serenoa repens versus placebo where three trials were added with 419 subjects and three endpoints (IPSS, peak urine flow, prostate size). Overall, 5222 subjects from 30 randomized trials ranging from four to 60 weeks were assessed. The vast majority were double blinded and treatment allocation concealment was adequate in just over half the studies.

 

In summary, some saw palmetto studies have shown improved symptom scores compared to placebo but generally no change in flow rates (134). However, large reviews cast doubt on its efficacy. In general, there is a real paucity of well performed, adequately powered, and placebo-controlled trials in the use of phytotherapy in clinical BPH. It is generally well tolerated at a dose of 320mg/day, but its efficacy has not been compared with alpha-blockers regarding efficacy, and has not been shown to reduce complications of BPH with long term use. Finally, the product quality and purity cannot always be assured.

 

African Plum Tree (Pygeum africanum)

 

Extracts come from the bark of the African plum tree. It is hypothesized, based on in vitro observation, that it acts on the prostate through inhibition of fibroblast growth factors, has anti-estrogenic effects, and inhibits chemotactic leukotrienes. No strong clinical data exists of its efficacy although trials are in progress (116,119).

 

Pumpkin Seed (Cucurbita pepo)

 

Dried or fresh seeds have been taken to relieve symptoms. Phytosterols are thought to be amongst the active compounds. Side effects have not been reported but evidence is lacking with no current clinical trials (135).

 

Rye Pollen (Secale cereale)

 

This is prepared from rye grass pollen extract. In a systematic review summarizing evidence from randomized and clinically controlled trials (114), rye pollen was found to be well tolerated but only achieved modest improvement in symptom outcomes and did not significantly improve objective measures such as peak and mean urinary flow rates. Again, several mechanisms of action have been proposed including an improvement in detrusor activity, a reduction in prostatic urethral resistance, inhibition of 5-alpha reductase activity, and an influence on androgen metabolism in the prostate (119).

 

Other Extracts

 

South African Star Grass (Hypoxis rooperi), Opuntia (Cactus flower), stinging nettle, and Pinus (Pine flower) have also been studied and used, however the data numbers are small and the types of trials do not allow conclusions to be drawn at this stage (116).

 

MEDICAL THERAPY FOR BPH – MONOTHERAPY AGENTS

 

In 1986, Caine (62) proposed that infravesical obstruction in men with symptomatic BPH comprised both static and dynamic components. The static component of obstruction is related primarily to the mechanical obstruction caused by the enlarging prostatic adenoma whereas the dynamic component is principally determined by the tone of the prostatic smooth muscle. Two avenues for pharmacotherapy have therefore evolved, namely shrinking the prostate tissue or relaxing the smooth muscle of the prostate. Prostatic smooth muscle tone is under the influence of the autonomic nervous system. Thus, any pharmacologic agent that may interfere with the functioning of this system could alter resistance in smooth muscle tone and resulting symptoms.

 

Medical therapy is now first-line treatment for most men with symptomatic BPH. They are non-invasive, reversible, cause minimal side effects, and significantly improve symptoms (81,136). With these recommendations, the rates of prescriptions for the medical management for BPH have increased drastically over the past decade (137,138). This increased interest has further led to the development of safer, more efficacious agents.

 

Alpha-Blockers

 

There are 3 main components to clinically significant BPH: static, dynamic and detrusor muscle components as outlined above. The dynamic component is associated with an increase in smooth muscle tone of the prostate. These smooth muscle cells contract under the influence of noradrenergic sympathetic nerves, thereby constricting the urethra (139). Prostatic tissue contains high concentrations of both alpha1 and alpha2 adrenoceptors – 98% of the alpha1 adrenoceptors are associated with stromal elements of the prostate (140). Thus alpha1-receptor blockers relax smooth muscle, resulting in relief of bladder outlet obstruction that enhances urine flow (87). Different subtypes of alpha1 receptors have been identified, with alpha1A predominating. Two alpha1A-adrenoceptors generated by genetic polymorphism have been identified with different ethnic distributions but similar pharmacologic properties (36).

 

It was demonstrated in 1978 that phenoxybenzamine, a non-selective alpha1/alpha2 blocker, was effective in relieving the symptoms of BPH (141).  Side effects were significant and included dizziness and palpitations. Many of the side effects of the alpha-blockers were mediated by alpha2-receptors (142).Thus, alpha1 selective antagonists such as terazosin, doxazosin, and  prazosin and were developed that had fewer side effects than phenoxybenzamine (67). Doxazosin, alfuzosisn, and terazosin have gained favor in clinical practice because they are longer acting than prazosin. Due to side effects, many alpha1 selective antagonists need to be titrated and are often started at the lowest dose and built up over time to the maximal dose or a dose where clinical effects are satisfactory.

 

More recently, highly uro-selective alpha1A selective agents have been introduced including tamsulosin and silodosin. Due to the uro-selective nature, there is significant reduction in risk of systemic side-effects when compared to the less selective agents. However, the increased potency of these agents results in an increased compromise to bladder neck function and as a result, increases the risk of ejaculatory dysfunction.

 

PRAZOSIN

 

Prazosin (titrated up to 5mg day) has been shown to significantly increase flow rates by 36-59% compared to placebo 6-28% but 17% of men discontinued the drug due to side effects such as dizziness (21%), headache (14%), syncope (3.4%) and retrograde ejaculation (13%).

 

ALFUZOSIN  

 

Alfuzosin (5 mg bid or 10 mg daily) has shown symptom score reduction of 31-65% (compared to placebo 18-39%) and flow rate increases of 22-54% (compared to placebo 10-30%). Hence the results were similar to those of prazosin but with only 3-7% discontinuations due to dizziness (3-7%), headache (1-6%) and syncope (<1 %) (143,144).

 

TERAZOSIN  

 

Terazosin (2-10mg) had a symptom score reduction of 40-70% (compared to placebo 16-58%) and improved flow rates 19-40% (placebo 5-46%). Between 9-15 % of men discontinued the drug, related to dizziness (10-20%), headache (1-7%), asthenia (7-10%), syncope (0.5-1.0%), and postural hypotension (3-9%). Thus, terazosin was effective and superior to placebo in reducing symptoms and increasing the peak urinary flow rate. The effect of terazosin on the peak urinary flow rate was apparent in studies as soon as 8 weeks of therapy. Most importantly, the effect of terazosin on symptoms and peak urinary flow rate was independent of the baseline prostate size for the range of prostate volumes reported (145).

 

DOXAZOSIN  

 

Doxazosin (4-12mg/day) is a selective alpha1-adrenoceptor antagonist, and produced a significant increase in maximum urinary flow rate (2.3 to 3.6 ml. per sec) at doses of 4 mg, 8 mg and 12 mg, and in average flow rate compared with placebo. The increase in maximum flow rate was significantly greater than placebo within 1 week of initiating therapy and the drug significantly decreased patient-assessed total, obstructive, and irritative BPH symptoms. Blood pressure was significantly lower with all doxazosin doses compared with placebo. Adverse events, primarily mild to moderate in severity, were reported in 48% of patients on doxazosin compared to 35% on placebo, with only 11% discontinuing treatment (a similar number to placebo). The main side effects were dizziness (15-24%), headache (12%) and hypotension (5-8%), and abnormal ejaculation (0.4%) (146,147).

 

TAMSULOSIN  

 

Tamsulosin (0.4 mg once or twice daily dose) is a selective alpha blocker for the alpha1A subtype which predominates in the human prostate, having 12 times more affinity for the receptors in the prostate than in the aorta thereby reducing side effects mediated through blood vessels receptors. Symptom scores were reduced by 20-50% (placebo 18-30%), flow rates improved 20-45% (placebo 5-15%) but only 3-7% of men discontinued drug because of dizziness (3-20%), headache (3-20%), syncope (0.3%), and retrograde ejaculation (5-10%). The rate of retrograde ejaculation was much higher than alfuzosin but the blood pressure lowering side effects are less with tamsulosin(148). There are different formulations including extended release with lower pharmacological peaks and troughs which may offer fewer side effects.

 

SILODOSIN  

 

Silodosin (8mg daily) is a highly selective blocker for the alpha1A receptor subtype. It has the highest affinity for alpha1A receptors of the medications discussed here. Symptoms scores were reduced by 40-50% (placebo 20-30%), flow rates improved by 17-30% (placebo 5-14%). Despite these favorable urinary outcomes, a significant proportion of patients experienced ejaculatory dysfunction (13-23%). These rates are higher compared to tamsulosin, however discontinuation rates secondary to ejaculatory dysfunction remains at 1-2%. Typical side effects include thirst (10%), loose stools (9%) and dizziness (5%) (149,150). Similar results were found in a recent meta-analysis of silodosin. Compared to tamsulosin, the combination of 13 studies found silodosin showed little to no difference in urological symptom scores and quality of life whilst increasing sexual adverse events. The same results were reported when silodosin was compared to naftopidil and alfuzosin (151).

 

Several meta-analyses have demonstrated that all non-selective alpha1-adrenoceptor antagonists seem to have similar efficacy in improving symptoms and flow rates (152). The difference between non-selective alpha 1-adrenoceptor antagonists is related to their side effect profile. Overall, alfuzosin appear to be better tolerated than doxazosin, terazosin and prazosin(153). More recent analyses suggest that the highly uro-selective alpha1A blockers are more efficacious compared to non-selective alpha blockers with regards to urinary symptoms and urine flow improvement (154-156). Further, these highly selective agents appear to have a favorable systemic side-effect profile at a cost of ejaculatory function when compared to non-selective alpha blockers.

 

Table 12. Commonly Used Alpha-Blockers

Group

Drug

Nonselective alpha blockers

·       Phenoxybenzamine

·       Nicergoline

·       Thymoxamine

Selective alpha1 blockers

·       Prazosin

·       Alfuzosin

Super-selective alpha1A blockers

·       Tamsulosin

·       Silodosin

Long-acting alpha1 blockers

·       Terazosin

·       Doxazosin

 

5-Alpha Reductase Inhibitors

 

The enzyme 5-alpha reductase is crucial in the amplification of androgen action in the prostate by modulating the conversion of testosterone to DHT (Figure 5). Within the prostate, 90% of testosterone is converted to DHT (78,157). There are 2 isoforms of the enzyme 5-alpha reductase which are encoded by separate genes (158). Type 1 isoenzyme is expressed highly in the skin, liver, hair follicles, sebaceous glands, and prostate whereas type 2 is responsible for male virilization of the male fetus, and in adulthood resides in prostate, genital skin, facial and scalp follicles (159,160). Inhibitors of these enzymes potentially decrease serum and intra-prostatic DHT concentrations, thus reducing prostatic tissue growth.

 

FINASTERIDE

 

Finasteride was the first of these to be studied in humans and shown to decrease DHT concentrations (161). It acts predominantly on the type 2 isoenzyme of 5-alpha reductase. There is some evidence that patients on finasteride experience fewer serious complications associated with the progression of BPH compared with those prescribed an alpha blocker, such as acute urinary retention or undergoing BPH-related surgery, but more prospective data is needed (162). Finasteride reduces serum DHT concentrations by 65-70% and prostatic concentrations by 85-90%, although the intraprostatic concentrations of testosterone are reciprocally elevated as the testosterone is not being converted to DHT.

 

Because 5-alpha reductase inhibitors work by reducing prostatic tissue volume, baseline prostate size has a significant impact on its efficacy with larger glands (>50ml) being likely to respond (163,164). After treatment for one year with finasteride, there was a significant decrease (17-30%) in total gland size with the greatest size reduction in the periurethral component of the prostate, which has the greatest impact on obstructive symptoms (78,165,166). There was a 60-70% decrease in serum DHT concentration, a 25% decrease in prostate volume, and a symptom score reduction of 13-30% (vs placebo 4-20%). Urinary flow rate improved 7-20% (vs placebo 3-15%) and was more pronounced with prostates > 40ml. The side effect profile included a decreased libido in 10%, ejaculatory dysfunction in 7.7%, and impotence in 15.8%. But adverse events resulted in only 4% of patients discontinuing treatment (117,167). There was a 50% reduction in the risk of AUR and in the need for surgery (30%).  Finasteride has also found a role in the treatment of BPH-related hematuria although its role in reduction of perioperative bleeding is not well defined (84,117).

 

More recently, as part of the Prostate Cancer Prevention Trial involving over 18,000 men, it was concluded that finasteride delays the appearance of prostate cancer whilst reducing the risk of urinary problems. However, there was a reported increased risk of high-grade prostate cancer leading to the discontinuation of this study. This point remains controversial as some believe due to the gland shrinking that sampling was altered and by virtue of a smaller area the likelihood of finding an aggressive tumor was increased (168). In any case, the benefits in terms of improved LUTS needs to be weighed against the potential sexual side effects and potential small but significant increased risk of high-grade prostate carcinoma (169,170) and compared to the option of using adrenoreceptor blockers. Despite the findings, more evidence is needed before advising patients to cease finasteride. However, they do need to be counselled on the small, but significant risks of developing aggressive prostate cancer (171).

 

DUTASTERIDE  

 

Dutasteride unlike finasteride blocks both the type I and Type II 5-alpha reductase isomers showing a 60-fold greater inhibition of the type 1 isoenzyme than finasteride plus activity against the type 2 isoform (23,117). In terms of monotherapy, a one year randomized, double-blinded comparison of finasteride and dutasteride in men with BPH (EPICS: Enlarged Prostate International Comparator Study) found a trend for dutasteride improvement over finasteride in IPSS (International Prostate Symptom Score) that did not reach statistical significance (abstract)  (172). Another non-randomized comparative trial with 240 patients, published only in abstract form, showed a small improvement in AUASI and Qmax for dutasteride (173). However, dutasteride and finasteride have never been compared in long-term therapy, either as monotherapy or in combination with an alpha-blocker. These medications appear to exert continued effects beyond 1 year so comparison after only 1 year is likely to be premature.

 

The tolerability of 5-alpha reductase inhibitors in most studies has been excellent with the most relevant adverse effects being related to sexual function. They include reduced libido, erectile dysfunction, and, less frequently, abnormal ejaculation (74,174). Specifically for dutasteride in the Combat study (175), in the monotherapy arm of 1623 patients the side effect were: erectile dysfunction (6.0%) ; retrograde ejaculation (0.6%); altered (decreased) libido (2.8%); ejaculation failure (0.5%); semen volume decreased (0.3%); loss of libido (1.3%); breast enlargement (1.8%); nipple pain (0.6%); breast tenderness (1.0%), and dizziness (0.7%).

 

As with finasteride, the REduction by DUtasteride of prostate cancer Events (REDUCE) trial now fully reported has demonstrated similar results to the PCPT trial in reducing prostate cancer (176). Again, a higher risk of developing more aggressive cancer was demonstrated- but in this study it was not statistically significant. Indeed, some organizations such as the Canadian Urological Association have been dismissive of this point in recent guidelines (171). Needless to say, careful counselling of men regarding this issue is again required, particularly for younger men who will be on dutasteride for many years.

 

5-Alpha Reductase Inhibitors to Reduce Hematuria and Intraoperative Hemorrhage for Prostate Surgery

 

While considered an off-label use, there is some evidence that suggests that 5-alpha reductase inhibitors may be useful in the setting of (177):

  • Recurrent hematuria secondary to BPH
  • To reduce gland size and/or impact on angiogenesis to reduce intraoperative bleeding for prostate surgery.

 

No large randomized trials exist but an extensive summary of the literature is available (177).

 

Phosphodiesterase 5 Inhibitors

 

Phosphodiesterase 5 (PDE5) inhibitors (e.g., sildenafil, tadalafil and vardenafil) have been used predominantly to treat erectile dysfunction in men. However, recent data suggest they are effective for the treatment of LUTS secondary to BPH. Specifically, the cyclic nucleotide monophosphate cyclic GMP represents an important mediator in the control of the lower urinary tract outflow region (bladder, urethra). PDE5 inhibitors exert effects by several mechanisms including: calcium-dependent relaxation of endothelial smooth muscle, alteration of the spinal micturition reflex pathways, and increased blood flow to the lower urinary tract. PDE inhibitors are regarded as efficacious, have a rapid onset of action, and favorable effect-to-side-effect ratio (178).

 

The rationale for using tadalafil for BPH stems from the following three observations: first, the prevalence of LUTS, BPH, and erectile dysfunction (ED) increases with age; second, phosphodiesterase-5 inhibition mediates smooth muscle relaxation in the lower urinary tract; and third, early evidence demonstrates that PDE5 inhibitors such as tadalafil are successful in treating LUTS and ED (179). Results of several randomized controlled trials have demonstrated reproducible reductions in IPSS, symptoms, and improved quality of life compared to placebo. Data suggests tadalafil 5mg improves IPSS by 22-37% and the improvement occurs within one week of commencement, with a duration of 52 weeks (180). The adverse event profile was acceptable and consistent with that previously reported in men with ED (blurred vision, headache, back ache, nausea, etc.), with discontinuation rates of 2%. Not unexpectedly, in the same study tadalafil significantly improved the International Index of Erectile Function-Erectile Function score in sexually active men with erectile dysfunction at twelve weeks. Meta-analytical data confirms these findings suggesting that PDE5 inhibitors improve IPPS and erectile function, with no significant effect on maximal urinary flow rate (181). Other PDE5 inhibitors are being studied including sildenafil and vardenafil (178). The theoretical advantage is treating BPH and erectile dysfunction with one agent (182). To date, tadalafil is the only PDE5 inhibitor that is FDA approved for use for the treatment of BPH. Data on the long-term effects on symptoms and disease progression is not available at present.

 

Anticholinergic Medications

 

High-level evidence suggests that for selected patients with bladder outlet obstruction due to BPH and concomitant detrusor overactivity, combination therapy with an alpha-receptor antagonist and anticholinergic can be helpful (183).Such agents help particularly with the irritative urinary symptoms of frequency and urgency. Caution is recommended, however, when considering these agents in men with an elevated residual urine volume or a history of spontaneous urinary retention (171).

 

Botulinum Toxin A Injection

 

Injection of botulinum toxin A into the prostate is a novel treatment for LUTS secondary to BPH. First reported in 2003 (184), trans-perineal injection of 100 units of botulinum toxin into each lobe of the prostate under trans-rectal guidance is required. In this randomized controlled trial, thirty patients demonstrated significant improvement in IPSS (65% decrease) and serum PSA (51% decrease) compared to controls, who had injections of saline without botulinum toxin A, at a median follow-up of 20 months. Subsequent long-term follow-up of 77 patients up to 30 months has shown similar results – significant reduction in IPSS (approximately 50% lower), significant improvement in maximum flow rate (approximately 70% higher), and significant reduction in serum PSA values (approximately 50% lower).Importantly, no adverse events were noted (185).

 

Summary of Monotherapy Medical Treatment

 

The first line of medical treatment is an alpha-blocker, as the majority of patients treated have a prostate volume of less than 40ml. In men with larger prostates (greater than 40cc), a 5-alpha reductase inhibitor (e.g., finasteride or duasteride) alone or in combination with an alpha-blocker would be appropriate. Patients who are likely to respond to 5-alpha reductase inhibition will do so at the same relative magnitude as an alpha-blocker, but it will take a longer period of time (months as opposed to weeks). There is likely to be a 20-30 reduction in symptoms and a 1-2ml per second increase in urinary flow (167). Side-effect profiles of medical treatments are also important, as discussed above. For example, with regard to sexual function, tamsulosin and silodosin have an increased risk of retrograde ejaculation and finasteride increases sexual dysfunction (74). These may be important factors in choosing therapies. Finally, the emergence of PDE5 inhibitors for the treatment of men with LUTS secondary to BPH alters the landscape with an ability to treat men with BPH and ED with one agent. Multiple randomized trials and associated meta-analyses demonstrate the reproducible benefits of PDE5 inhibitors on urinary and erectile function.

 

MAJOR STUDIES OF MEDICAL TREATMENT OF BPH

 

The medical treatment of clinical BPH has come under increasing scrutiny through larger trials that have become imperative for their introduction into clinical practice. Some of these larger trials have been selected and are discussed below.

 

Veterans Affairs Study

 

In the Veterans Affairs Cooperative Studies Benign Prostatic Hyperplasia Study Group (186), a total of 1,229 subjects with clinical BPH were randomized to 1 year of placebo, finasteride, terazosin or drug combination. The primary outcome measures were the AUA symptom score and the peak urinary flow rate. The percentage of subjects who rated improvement as marked or moderate with placebo, finasteride, terazosin and combination was 39, 44, 61 and 65%, respectively, only the latter two were superior to placebo. There was no significant relationship between baseline prostate volume and treatment response to finasteride or with the other treatments (terazosin or combination). There was a significant but weak relationship between change in AUA symptom score and peak flow rate in the finasteride and combination groups. The symptom responses with terazosin were not related to peak flow rate or baseline prostate volume. In men with clinical BPH, finasteride and placebo are equally effective, while terazosin and combination are significantly more effective. In men with clinical BPH and large prostates, the advantage of finasteride over placebo in terms of symptom reduction, impact on bother due to symptoms and quality of life was small at best, while the advantage of terazosin (alone or in combination with finasteride) over finasteride alone and placebo was highly significant. The authors concluded that alpha1 blockers, such as terazosin, should be first line medical treatment for BPH(186). Another arm of this study observing surgical treatment versus watchful waiting is discussed below.

 

PLESS Study

 

The Proscar Long-term Efficacy and Safety Study (PLESS) was a 4-year, randomized, double-blind, placebo-controlled trial assessing the efficacy and safety of finasteride 5mg (Proscarä) in 3040 men, aged 45 to 78 years, with symptomatic BPH, enlarged prostates on TRUS volume criteria, and no evidence of prostate cancer (187,188). Finasteride use reduced the risk of developing acute urinary retention by 57% and the need for BPH-related surgery by 55% (189) in comparison to placebo. A modified AUA symptom score was used (because trial was undertaken prior to formal AUA being developed) and showed a statistically significant reduction in mean score of 3 for finasteride and 1.2 for placebo, starting at a level of 15 for both groups (188). Compared with placebo, men treated with finasteride experienced an increased incidence of new drug-related sexual adverse events (erectile dysfunction, decreased libido, ejaculation disorder) only during the first year of therapy with 4% of men discontinuing because of such events (187).

 

PREDICT Trial

 

The Prospective European Doxazosin and Combination Therapy (PREDICT) Trial was constructed to evaluate the efficacy and tolerability of the selective alpha1-adrenergic antagonist doxazosin and the 5-alpha reductase inhibitor finasteride, alone and in combination, for the symptomatic treatment of benign prostatic hyperplasia. It was a prospective, double-blind, placebo-controlled trial involving 1,095 men aged 50 to 80 years. The dose of finasteride was 5 mg/day. Doxazosin was initiated at 1 mg/day, and titrated up to a maximum of 8 mg/day over approximately 10 weeks according to the response of the maximal urinary flow rate (Qmax) and IPSS. An intent-to-treat analysis of 1,007 men showed doxazosin and doxazosin plus finasteride combination therapy produced statistically significant improvements in total IPSS and Qmax compared with placebo and finasteride alone. Finasteride alone was not significantly different statistically from placebo with respect to Qmax or total IPSS. The treatments were generally well tolerated. They concluded that doxazosin was effective in improving urinary symptoms and urinary flow rate in men with benign prostatic hyperplasia, and was more effective than finasteride alone or placebo. The addition of finasteride did not provide further benefit to that achieved with doxazosin alone (146).

 

MTOPS Study and Predictors of Clinical Progression

 

The Medical Therapy of Prostatic Symptoms (MTOPS) study is a double-masked, placebo-controlled, multi-center, randomized clinical trial with 4 study arms 1) placebo; 2) doxazosin (4 to 8 mg); 3) finasteride (5 mg) and 4) combination of both doxazosin and finasteride. 3,047 men were randomized equally to the 4 groups (190). Baseline parameters analyzed included age at randomization, transrectal ultrasound (TRUS) volume, AUA symptom score, Qmax, PVRU, and PSA. Reduction in the risk of BPH progression was analyzed by one covariate at a time regression models of absolute risk of BPH progression versus baseline covariates. Groups compared were combination versus doxazosin, combination versus finasteride and finasteride versus doxazosin (190).

 

In the main finding, disease progression, defined as an increase in AUASS (American Urology Association Symptom Score- similar to IPSS to score LUTS) of 4, AUR, renal insufficiency, recurrent UTIs and urinary incontinence, was prevented equally by doxazosin and finasteride with an even greater effect when both medications were combined. In conflict with the Veterans Affairs and PREDICT studies, finasteride alone did improve overall symptoms and peak urine flow compared to placebo at 4 years and with even more so when combined with doxazosin (190). This finding coincides with the long-term open label ARIA dutasteride study described above showing a cumulative symptom benefit of treatment up to 4 years (191)

 

A small part of the MTOPS study focused on the routinely available measure of serum PSA, in an attempt to predict a patient’s future risk of BPH clinical progression, acute urinary retention and BPH-related invasive therapy, permitting an informed decision concerning the value of medical therapy over watchful waiting (192). In MTOPS, 737 patients were assigned to placebo and followed for an average of 4.5 years. Clinical progression of BPH was pre-defined as either a 4-point increase in AUA symptom score, acute urinary retention, incontinence, renal insufficiency, or recurrent UTI. The need for BPH-related invasive therapy was a secondary outcome. These data are summarized in Table 13, where those having a lower PSA, had a lower rise in symptom score, and a reduced risk of acute urinary retention or invasive treatment compared to those with a higher PSA. The sub-group with the highest baseline PSA was also likely to have larger prostate glands, making the findings intuitive. However, as with many such findings, translating an individual PSA to a population study is difficult, as other factors will determine progression or regression of symptoms, not just PSA.

 

Table 13. Progression of BPH Symptomatically of Placebo Group only, to AUR or Further Intervention Based on Baseline PSA (adapted from Kaplan et al).

Baseline PSA tertiles (ng/ml)

Progression of symptom score (points)

Acute urinary retention Risk over study period

Invasive Treatment Risk

<1.2

3.10

0.18

0.6

1.2-2.5

3.47

0.35

1.33

>2.5

7.21

1.46

2.13

 

CombAT Trial

 

Combination therapy with a 5-alpha reductase (dutasteride) and the alpha blocker, tamsulosin, in men with moderate-to-severe benign prostatic hyperplasia and prostate enlargement was also further studied in the Combination of Avodart™ and Tamsulosin™ (CombAT) trial. The rationale was the same as those outlined for the MTOPS trial. In summary, it is a 4-year, global, multicenter, randomized, double-blind, parallel-group study designed to investigate the benefits of combination therapy with the dual 5-ARI dutasteride and the alpha-blocker tamsulosin compared with each monotherapy in improving symptoms and long-term outcomes in men with moderate-to-severe symptoms of BPH and prostate enlargement. Symptoms and long-term outcomes (AUR and surgery) were assessed as separate primary endpoints at two and four years, respectively. Eligible patients were at least 50 years old with prostate volume ≥30 cm3 and PSA level ≥1.5 ng/mL. Almost 5,000 men were enrolled (193). Perhaps the only criticism is the lack of placebo control arm in the study. 

 

The results at four years (194) demonstrated that combination therapy was superior to tamsulosin monotherapy but not dutasteride monotherapy at reducing the relative risk of AUR or BPH-related surgery. Combination therapy was also significantly superior to both monotherapies at reducing the relative risk of BPH clinical progression. Combination therapy provided significantly greater symptom benefit than either monotherapy. Safety and tolerability were reasonable and in line with expectations for both medications.  Certainly, at four years the CombAT data supports the long-term use of dutasteride and tamsulosin combination therapy in men with moderate-to-severe LUTS due to BPH and prostatic enlargement.

 

EMERGING COMBINATION THERAPY REGIMES

 

With the increasing body of evidence supporting the use of PDE5 inhibitors in the setting of BPH, a number of trials support its use in combination therapy. To date, there are smalls studies of alfuzosin and tadalafil, tamsulosin and sildenafil, and tamsulosin and vardenafil. These early studies suggest that combination therapy is more effective than monotherapy for urinary and erectile function with a good safety profile (195,196). A meta-analysis of 11 randomized controlled trials (n=855) looked at alpha blockers with or without PDE5 inhibitors. This analysis found men receiving PDE5 inhibitors had a mean improvement of 1.66 points on IPSS, mean increase of 0.94 ml/s maximum urinary flow rate, and improved erectile function (197). Larger series with longer-term follow up is required to definitively define the role of these combination therapies in current practice.

 

Summary of Combination Therapy for Men with BPH

 

In the larger studies where the standard endpoint of prostate symptom score was measured, a greater impact of dutasteride over tamsulosin was observed.  Considering urinary flow rate (Qmax), combination therapy outperformed dutasteride in those with PSA and prostate volumes above the 75 percentile. Clearly, those with larger prostates and higher PSAs derive a greater benefit with dutasteride coinciding with the size reduction impact of this drug.

 

In summary, the results of the MTOPS and CombAT trials both suggest combination therapy is better than 5-alpha reductase monotherapy at the 4-year mark. The higher incidence of adverse effects, the increased cost of combination therapy, and the need for prolonged therapy argue for a reductionist medical approach to this condition. One recent small study investigated the discontinuation of 5-alpha reductase inhibitors in patients on combination therapy and found prostate regrowth and worsening of symptoms after 1 year of cessation, emphasizing the importance of 5-alpha reductase inhibitors in prolonged therapy (198). In an opposing design, the SMART trial (Symptom Management After Reducing Therapy) observed the effect of removing the alpha blocker (tamsulosin) after 6 months of combined therapy with dutasteride (199). With I-PSS as the primary outcome, the investigators found that 77% of patients had symptoms that were the same or better after only 3 months of alpha blocker removal. In reference to the CombAT study, the effects of dutasteride continue past two years suggesting that removal of the alpha blocker at later time points may be even less noticeable. However, the CombAT study is quite powerful as it does demonstrate that the natural history of BPH is not altered by taking alpha blocker alone. The rate of AUR and need for surgery was unaltered at about 18%, and thus while tamsulosin helps LUTS, it does not alter disease progression. Combination therapy did lead to reductions in prostate volume, and changed natural history to reduce rates of AUR and surgery. The largest benefit was in the men with the largest glands.

 

The emergence of newer agents including PDE5 inhibitors gives rise to an increasing number of combination-therapies under investigation. Long-term follow-up is required on these newer combinations. As such, combination treatment will continue to shape the management of BPH for years to come.

 

SURGICAL TREATMENT OF BPH

 

Invasive Surgical Therapies

 

Traditionally prostatectomy by an open approach or TURP has been considered the gold-standard for refractory or complicated BPH (indications listed in Table 14). At present approximately 90% of prostatectomies are done by TURP. Open prostatectomy should be considered when a gland is estimated to weigh more that 75g, where large bladder calculi exist that may not be dealt with endoscopically, where large bladder diverticula requiring repair exist, if complex urethral conditions or when orthopedic abnormalities prevent positioning in lithotomy for TURP. Contraindications to open prostatectomy include a small fibrous gland, prostate adenocarcinoma, previous prostatectomy or other surgery of the pelvis preventing access (200).

 

Table 14. Indications for Prostatectomy

·       Acute Urinary Retention

·       Recurrent or persistent urinary tract infections

·       Significant bother from LUTS secondary to bladder outflow obstruction not responding to medical therapy

·       Recurrent hematuria known to be of prostatic origin

·       Bladder Calculi

 

TRANSURETHRAL RESECTION OF THE PROSTATE (TURP)

 

TURP remains the most common surgical treatment for BPH (201) and remains the ‘gold standard’ by which other surgical (and even medical) treatments are measured (74). TURP involves either regional or general anesthesia, with most patients spending a minimum of one night in the hospital. TURP involves surgically debulking the periurethral and transitional zones of the prostate to relieve obstruction. Debulking is done by electrocautery in the standard TURP through endoscopic instruments introduced into the urethra and bladder. Tissue is resected in small pieces until the hyperplastic tissue is removed and a new channel for passage in the prostatic urethra created in the capsule left behind, much like fashioning a pumpkin for a Halloween jack o’lantern. Despite using electrocautery, there are mild to severe degrees of hemorrhage, depending on the gland size. However, transfusions are rarely needed and the procedure is relatively free of life-threatening complications and most patients experience satisfactory resolution of their micturition symptoms. Studies on urinary peak flow rates and invasive pressure flow have demonstrated the superiority of TURP over minimally invasive therapies (202). Complications of TURP include failure to void (6%), hemorrhage requiring transfusion (1-4%), clot retention (3%), infection (2%), bladder neck contracture or urethral stricture (6%), transurethral resection syndrome (2%), and rarely incontinence (80,203,204). 

 

TURP is plagued by the potential for morbidity, specifically: retrograde ejaculation, erectile dysfunction, and urinary incontinence. Retrograde ejaculation is reported to occur in almost all patients undergoing TURP as the normal bladder neck mechanism which contracts to allow antegrade ejaculation is surgically resected. Counselling prior to surgery must include a discussion of the impact on sexual performance and also fertility. Erectile dysfunction (ED) may be associated with TURP either via thermal nerve injury or emotional stress and was reported in early studies at a rate of 4-40%. This has now been shown to be an overestimation (74,206). The rate of ED in the AUA Cooperative study was found to be 13% in 1,000 men (80), however this must be compared to increases of around 20% of ED in untreated groups with BPH. Although ED is often quoted as a side effect of TURP, Kassabian concluded that TURP (or even any other surgical therapy) did not appear to have a long-term effect on erectile function or libido (74). Incontinence is infrequent and typically is a result of intra-operative damage to the external urinary sphincter. Large pooled analysis revealed rates of incontinence following TURP of around 1% (207).

 

There has been one randomized controlled trial (Veterans Affairs Cooperative Study Group, see above) comparing TURP to “watchful waiting” or reassurance (203). This demonstrated that TURP showed greater benefit with 66% of patients having a decrease in symptoms post TURP compared to 28% who were undergoing ‘watchful waiting”. 

 

One significant modification to the standard TURP using monopolar cautery with glycine as an irrigant has been the use of bipolar cautery using normal saline as the irrigant. The latter has been termed bipolar transurethral resection in saline (TURIS). Glycine alters serum osmolality when absorbed through venous channels in the prostate as the system is under pressure which potentially leading to hyponatremia and also glycine directly itself has an impact on the nervous system. This syndrome is termed “TURP syndrome” and dictates that monopolar resections should be abandoned at around the one-hour mark or when significant venous breach occurs. The TURIS or bipolar technologies thus have the advantage of the ability to carry out resections for a longer time due to very few issues with absorption of saline systemically as opposed to glycine. Large series meta-analysis illustrated comparable efficacy and morbidity profiles when compared to monopolar TURP (207,208). A further modification of the bipolar technology is so called plasmakinetic vaporization where some data is emerging (209,210). This vaporizes rather than resects the prostate tissue. Compared to TURP, plasmakinetic energies results in similar improvements to IPSS up to 12-month follow up (211).

 

LASER THERAPY FOR BPH – ENUCLEATION OR VAPORIZATION OF THE PROSTATE  

 

There are several evolving therapies for BPH involving various lasers including Nd-YAG, Holmium, and now Thulium lasers. These laser energies may be utilized in various methods to resect, enucleate, or vaporize the prostate. Laser as an energy source has an advantage over standard electrocautery by being relatively bloodless and does not carry the risk of hyponatremia, which may rarely occur via absorption of irrigation fluid in a standard TURP (212).

 

Photoselective Vaporization of the Prostate (PVP)

 

The characteristic 532-nm wavelength laser is selectively absorbed by hemoglobin within prostatic tissue (213,214). Introducing this energy to the prostate results in selective vaporization of prostatic tissue, with effective hemostasis and relatively little tissue coagulation (1.5 – 0.3mm margin). Initially launched as a 60W prototype, the laser was ultimately introduced to the urology community as an 80W system that has been the predominant device used in clinical trials. This first generation used an Nd:YAG laser beam passed through a potassium-titanyl-phosphate (KTP) crystal, halving the wavelength (to 532nm), doubling the laser's frequency, and resulting in a green light. In 2006, the 120W lithium triborate laser (LBO) laser was introduced using a diode pumped Nd:YAG laser light that is emitted through an LBO instead of aKTP crystal, resulting in a higher-powered 532 nm wavelength while still using the same 70-degree deflecting, sidefiring, silica fiber delivery system. More recently, a 180W version has been released (215). This increase in energy corresponds with reduced lasering and operating time (216). Two-year data from the GOLIATH trial illustrates that the 180W version provides durable symptom improvement that is comparable to traditional TURP (217).

 

Compared to TURP, PVP has been shown to have an improved side-effect profile, time of catheterization, hospital stay, and improvement in urinary flow rate (218,219). Clinically, the advantage of PVP is that the length of stay in hospital is usually under 24 hours and it can be performed on anticoagulated patients. Outcomes have demonstrated a reduced frequency and severity of clinical complications, however it was limited to smaller prostate sizes (215).

 

In summary, several laser wavelengths (Potassium titanyl phosphate [KTP], Holmium:Yttrium aluminum garnet [Ho:YAG], Thulium), and delivery systems (end-firing; side-firing; interstitial) are available for PVP, and each has particular characteristics and potential advantages (171,219). In current practice, the use of 532nm 180W PVP (Greenlight) lasers is becoming increasingly more common due to significantly reduced operative times.  

 

Holmium:YAG Laser for Enucleation (HoLEP) or Resection of the Prostate (HoLRP)

 

This laser may be used to enucleate the prostate and remove the tissue in pieces (HoLEP) or to vaporise the tissue (HoLRP). HoLRP is an operation involving laser resection of the prostate tissue via an endoscope, similar to a standard TURP using electrocautery as outlined above. The fragments of prostate tissue are made small enough to irrigate out prior to detachment from the prostate (220). HoLEP again uses a Holmium laser but the laser acts like a finger would at an open prostatectomy, shelling out tissue until it floats in the bladder. The tissue is then morcellated and extracted. This technique may be safely used in large prostate glands (those weighing >100g) as an alternative to open prostatectomy as discussed below (212). Initial studies have demonstrated that HoLEP improved flow rates by 56-119% and by TURP 96-127%, and symptom scores reduced in both groups by 60%. Further, these studies reported a reduced length of hospital stay, clot retention rates, the occurrence of hyponatremia, strictures but had a slightly higher risk of reoperation (221-223). Pooled data of recent randomized trials suggest HoLEP results in significantly improved maximal flow rate, IPSS, transfusion rate at a cost to operative time (224). Patients are usually kept in hospital a little longer with the Holmium:YAG compared to the PVP technique. However, the Holmium:YAG laser has a longer track record. The disadvantage is that treatment with the Holmium:YAG is quite a complex procedure to learn as it widely resects all the prostatic tissue. HoLRP with its inherent wavelength and laser properties is not photoselective for prostate tissue and as such causes more coagulation and necrosis and has not been popular as a therapeutic intervention.

 

Thulium Laser

 

A two-micron continuous-wave is produced with a wavelength of 2013nm. This wavelength is close to the water absorption peak in tissue. This provides several advantages including excellent hemostasis with minimal thermal injury to surrounding tissue. Tissue may be incised accurately or vaporized depending on the settings utilized. Initial reports in 2005 reported the use of a 50-Watt Thulium: YAG (Tm:YAG) laser (225). More recently an improved 120 W laser has been produced, allowing for up to 1.08g of vaporization per minute (226). With the high degree of accuracy of focal ablation, various resection techniques have been reported including: Tm laser resection of the prostate-tangerine technique (TmLRP-TT), Tm vaporization (ThuVaP), Tm vaporesection (ThuVaRP), Tm vapoenucleation (ThuVEP), Tm enucleation (ThuLEP). Combinations of the available techniques allow prostate removal rates to be increase to 2-3 grams per minute (227).

 

When compared to TURP, TmVaRP offered similar urinary symptom improvement however TURP was superior in improving max voiding velocity post operatively (228). Furthermore, no improvements in reduced blood loss or decreased length of hospital stay were observed (228). Similarly, a meta-analysis of four clinical trials comparing ThuVEP and HoLEP showed both lasers were effective in reducing BPH symptoms but found ThuVEP to have slightly reduced blood loss and shorter lasting urinary incontinence post procedure (229). This evidence largely suggests that the choice between TURP, HoLEP, and thulium laser is based on availability and surgeon experience.

 

Visual Laser Thermoablation of the Prostate (VLAP)

 

Alternate minimally invasive laser therapies such as VLAP rely on deep thermal coagulation of the prostate by Nd:YAG laser with later necrosis and sloughing of the prostate tissue (230). They are not photoselective for prostate tissue and do not vaporize the tissue as PVP lasers do. They require prolonged catheterization and have a failure rate of around 10% as reported by Chacko et al in a randomized trial in 2001 (231). Such therapies differ from debulking surgery and require a post-procedure period for resolution of symptoms with the advantages being lack of general or regional anesthesia. Durability and tolerability remain issues for such therapies with re-treatment rates between 10 and 49% (202). Certainly, further studies, using randomization, larger sample sizes, and comprehensive measures of outcomes and adverse events, are still needed to better define the role of laser techniques for treating benign prostatic obstruction (221).

 

OPEN SIMPLE PROSTATECTOMY

 

This is the oldest, most invasive therapy for BPH (232). This form of surgery was the standard for men with BPH for over a century however was often associated with complications and prolonged hospital stays. The number of simple prostatectomies being performed has declined since the introduction of TURP and laser energies.

 

It is commonly done through a transvesical approach, but may be done retropubically. Early complications of this operation include hemorrhage, blood transfusion, sepsis, and urinary retention with the most common late complication being bladder neck stricture (2-3%) (200). TURP has lower perioperative morbidity but open prostatectomy produces equivalent, if not superior improvement with a similar or lower re-operation rate (233). Sexual dysfunction is not likely to be altered by the surgery (74,234) however ED is still quoted at 3-5% risk (200). Retrograde ejaculation occurs in 90% patients. Other complications of surgery such as deep vein thrombosis, myocardial infarction, and stroke are less than 1% (200).

 

SUMMARY OF INVASIVE SURGICAL TECHNIQUES

 

Over the past decade, significant advances have been made regarding the invasive management for BPH. Traditionally, TURP has been reserved for refractory or complicated BPH. However, recent advances in laser technologies have resulted in a marked uptake in the use of laser prostatectomy. Novel approaches utilizing laser energy allows for the enucleation, resection, or ablation of prostatic tissue. Multiple meta-analyses demonstrate equivocal efficacy when comparing TURP and laser prostatectomy. In light of this information, in patients where prostatectomy is indicated it is reasonable to proceed with either of the energy sources discussed above based on surgeon and patient preferences.

 

Minimally Invasive Surgical Therapies (MIST)

 

Minimally invasive therapies for BPH have evolved in the past decade with the goal being to achieve symptomatic improvement that is durable, without the morbidity associated with surgery or the long-term side effects or compliance issues associated with medical therapies (202). The aim of such treatments is to achieve results similar to TURP but with minimal anesthesia, hospitalization, and morbidity. An overview of earlier randomized controlled trials in 2000 by Tubaro et al (235) comparing minimally invasive and invasive modalities of treatment found re-treatment rates to be higher in the minimally invasive group. They concluded that at the time, none of the minimally invasive treatments were superior to TURP from a cost and benefit standpoint and that TURP remains the gold standard of treatment.

 

More recently, an increasing number of therapeutic options have been developed to improve durability without limitation to the minimally-invasive approach. Multiple ablative (thermo or chemical) or mechanical options have been introduced with early data available. Accordingly, current practice suggests a markedly increasing use of MIST, particularly in the younger patients (236). While the precise role of MIST is not clear, some view such treatments as in-between medical and TURP and we await long term data on all proposed therapies.

 

TRANSURETHRAL INCISION OF THE PROSTATE (TUIP)

 

A similar approach to a TURP is used except that no surgical debulking is undertaken. Between one and three incisions are made into the prostate at the level of the bladder neck back almost to the insertion of the ejaculatory ducts. This releases the “ring” of BPH tissue at the bladder neck, creating a larger opening. There is a reduced risk of morbidity such as hemorrhage. In some instances, ejaculation may be preserved in younger men, especially if one incision is made. The procedure only works if the tissue in the periurethral area is not too bulky, otherwise a “ball-valve” mechanism of adenoma may develop. Therefore, TUIP should be recommended to men with smaller prostates (237). Laser may be used for incisions of the prostate, as well as standard electrocautery (212). Some studies have shown TUIP to have similar IPSS outcomes to TURP but lower urine peak flow rate. Understandably, TUIP has also been shown to give better outcomes in terms of ejaculatory function (238).

 

THERMO-ABLATIVE THERAPIES

 

Thermoablation is the principle underlying the several minimally invasive available treatments that have been introduced thus far (239) and these include transurethral microwave thermotherapy (TUMT), transurethral electrovaporization of the prostate (TUVP), and transurethral needle ablation (TUNA). Collectively, these therapies have been shown to have similar or decreased efficacy when compared to TURP but have a slightly better morbidity profile at this stage. Longer follow up data will determine the true efficacy and risk profiles for these thermo-ablative therapies.

 

Transurethral Microwave Therapy (TUMT)

 

An intraurethral antenna emits microwave radiation and delivers heat to a targeted region of the prostate. Histologically, this results in well-controlled coagulative necrosis. A number of series have been published reporting outcomes following TUMT. Multiple studies have compared TUMT versus TURP, which have demonstrated the sustained effect of mild symptom improvement when compared to TURP. A recent review reported a reduced efficacy when compared to TURP with regards to IPSS improvement at 12 months (65% decrease compared to 77% with TURP) and urinary flow rate (70% increase compared to 119% with TURP) (240,241). Retreatment rates are high, ranging between 10-22% compared to 4-8% following TURP. Despite this limitation to efficacy, TUMT provides significant benefits when compared to TURP including improved sexual function, hospitalization, hematuria, transfusions rates (242). Because of lower effectiveness compared to TURP, TUMT is considered a second line option at this stage (243).

 

Transurethral Electrovaporization of the Prostate (TUVP)

 

TUVP uses heat from a monopolar or bipolar high voltage electrical current to vaporize tissue (237). Theoretically this technique could have an ablative as well as coagulative effect. To date, a meta-analysis of randomized controlled trials comparing TUVP and TURP have shown no significant differences in IPSS, quality of life or post void residual volumes. Similar rates of complications have also been found however this is limited by short follow up durations (244,245). Furthermore, TUVP did not lead to a reduction in postoperative morbidity or shorter hospital stays (246).

 

Transurethral Needle Ablation (TUNA)

 

Radiofrequency ablation between two electrodes results in thermal ablation and resulting coagulative necrosis of tissue. Several randomized trials have been performed with only short-to-midterm follow up available. As with other forms of MIST, concerns regarding durability are present. A 5 year follow up demonstrated that 58% of patients had maintained symptom control, however 21% needed re-treatment (247). Meta-analytical data confirms an improved IPSS and urinary flow rate at one-year, however to a significantly lower magnitude when compared to TURP (248). Similar to TUMT and TUVP, TUNA has a favorable morbidity profile when compared to TURP.

 

MECHANICAL THERAPIES

 

Urolift

 

Prostatic urethral lift (PUL) is a novel procedure that is characterized by the placement of non-absorbable implants within the prostatic urethra. When placed correctly, these implants provide anterolateral traction to the lateral lobes of the prostate without necessitating tissue ablation. Advantages of PUL are that it is a short, simple procedure that can be done under local anesthesia and has low complication rates. However, the presence of an obstructing median lobe poses a hurdle for procedure due to the inability to place an implant to the median lobe safely. This exclusion criteria prevents a large portion of men with BPH from undergoing this procedure. The BPH6 was a randomized controlled trail that prospectively compared the PUL with TURP. This study reported that PUL improves IPSS to 52% compared to 72% following TURP. Maximal urinary flow rates improved to a modest degree (41% compared to 144% following TURP). Interestingly the preservation of native prostatic tissue results in preserved erectile and ejaculatory function (249). Pooled analysis of available studies confirm these modest improvements in urinary and sexual function (250,251). Further, this procedure is well-tolerated and is performed in the outpatient setting under local anesthetic in a vast majority of cases. Morbidity is representative of typical MIST procedures with small proportions of patients reporting dysuria, urinary tract infection, and hematuria. Durability is among the main concern surrounding this procedure. Only three-year data has been published at present, reporting a modest IPSS improvement (252). Further comparative robust studies are required to determine the role of the PUL in current practice.

 

Intra-Prostatic Stents

 

In keeping with the principles of minimal invasion, a stent or coil is placed into the urethra at the point of maximal obstruction under local anesthesia, endoscopic and radiographic guidance. Stents may be temporary/biodegradable or permanent. Although effective in the short term, they do have a significant complication rate raising concerns over safety and large randomized controlled trials are needed to establish their long-term efficacy and their true role in the management of BPH (204,253,254).

 

Transurethral Ethanol Ablation of the Prostate (TEAP)

 

Deep intra-prostatic injection of pure ethanol results in chemical ablation of the prostate. Of the limited studies available, 4 year follow up suggests sustained response in 73% of patient, with 23% requiring retreatment. More robust comparative data is required prior to more formal recommendations for the use of TEAP.  

 

Fexapotide Triflutate – NX-1207

 

NX-1207 is injected into the transition zone of the prostate to ablate the tissue, but the precise mechanism by which NX-1207 acts has not been published to date. Trials of NX-1207 have shown a mean improvement of 5.7 points on IPSS score for men receiving one injection compared to placebo at mean 43 months follow up. When compared to men taking oral BPH medications, fewer in the NX-1207 group (8% vs 27%) required additional BPH intervention at 3 years (255). Long-term follow up of men receiving this chemical show durable reductions in symptom scores to 6.5-year follow-up (256). NX-1207 is well tolerated, with low rates of mild hematuria, dysuria and infection. No sexual dysfunction or incontinence has been reported for either agent.

 

Topsalysin - PRX-302

 

PRX-302 is a genetically modified bacterial pro-toxin that is activated by PSA within the prostatic tissue and forms transmembrane cellular pores that lead to apoptosis. Like TEAP and NX-1207, PRX-302 is injected into the transition zone of the prostate. PRX-302 results in a transient reduction in symptoms score that do not appear to be maintained at 12-month follow-up (257). Similar to NX-1207, it is well tolerated, with low rates of mild hematuria, dysuria and infection. No sexual dysfunction or incontinence has been reported.

 

Botulinum Toxin subtype A (botox)

 

Botox is a toxin produced by the bacterium Clostridium Botulinum. Its mechanism of action for intraprostatic injections is poorly understood however theories include glandular necrosis and the blockage of alpha-adrenergic receptors resulting in smooth muscle relaxation (258). Phase 2 single arm studies have shown that intraprostatic botox has minimal side effects but has a re-treatment rate as high as 29% (185)

 

OTHER THERAPIES

 

Aquablation

 

Aquablation involves a transrectal ultrasound guided, robot-assisted, high velocity saline stream. This results in the ability to ablate glandular tissue without the requirement of heat. Real-time monitoring is available and allows the surgeon to ensure sparing of the prostatic capsule. Early studies have demonstrated its safety and feasibility. The WATER trial showed that aquablation was not inferior to TURP for improving IPSS scores at 6 months follow up with slightly improved rates of anejaculation (259). Longer follow up data from this study is needed to prove long term efficacy and assess long term complication rates.  

 

Prostatic Artery Embolization (PAE)

 

PAE is performed by a trained interventional radiologist. Unilateral or bilateral prostatic arteries are injected with an embolic agent - which is typically ethanol-based. With increasing experience, technical success has increased to greater than 90%. A metanalysis of 13 studies including 1,254 men found that PAE demonstrated a mean 16.2 increase in IPSS score and improved quality of life that remained statistically significant after 3 years follow up. Transient dysuria and urinary frequency were reported in 10% and 16% of men, respectively. Post embolization syndrome was reported in 3.6% of men and only three cases of major post-operative complications were recorded (260).

 

More recently, two-year follow up data from a randomized controlled trial of PAE vs TURP was published. Reduction in IPSS score was similar in both arms however TURP men showed better urinary flow, post void residual volume, reduced prostate volume but more erectile dysfunction. 21% of men who had PAE required subsequent TURP within the 2-year period. PAE adverse events were less frequent than TURP but distribution within the severity classes were similar (261).

 

Water Vapor Therapy - Rezum

 

Rezum uses radiofrequency to create thermal energy in the form of water vapor. This vapor is delivered transurethrally under cystoscopy to the prostate and causes instant cell necrosis through cell membrane disruption. It is frequently performed under local anesthetic in the outpatient setting. Two retrospective studies showed improvement in IPSS, urinary flow and post void residual volume at 6 and 12 months follow up (262,263). A randomized trial of Rezum vs sham procedure showed a mean improvement of 7 points on IPSS score and an improvement in quality of life in the Rezum group. Peak urinary flow rate was improved by 6.2ml/s in the rezum group and was sustained at 12 months (264). Morbidity is minimal and is in-line with those experienced following alternate MISTs (265).

 

Histotripsy

 

Histotripsy is the use of extracorporeal ultrasound energy that produces extreme pressure changes within the prostatic tissue. This pressure changes result in localized clusters of microbubbles which cause mechanical fractionation. Collapse of these microbubbles leads to cellular destruction and prostatic cavitation (266). The method of prostate injury allows the procedure to be monitored through ultrasound in a real-time setting. One safety and feasibility trial has been published to date reporting three cases of transient urinary retention, 1 case of minor anal abrasion, and one case of microscopic hematuria out of 25 men. No serious intraoperative complications occurred (267).

 

SUMMARY OF MINIMALLY INVASIVE THERAPIES

 

A myriad of minimally invasive therapies (MIST) has been developed to reduce the morbidity of surgical BPH management. Current evidence in MIST is characterized by improvements in symptom and urinary flow rates similar or slightly less than TURP with high rates of retreatment. Despite this, these procedures are very well tolerated and may be performed as an outpatient. Further, these therapies are highlighted by the significant reduced risk of sexual dysfunction. Some consider that MIST might be suitable in younger patients that are willing to accept less urinary improvement to preserve sexual function. Elderly or co-morbid men might also benefit given many of these procedures can be performed under local anesthetic or in the outpatient setting. However, the precise role for MIST has not become clear to date. It is clear that MISTs are emerging and will likely become a prevalent treatment option in the management of BPH.  

 

MEASURING OUTCOMES AND EFFECTIVENESS OF TREATMENT

 

When considering the effectiveness of any treatment for BPH, one must consider the efficacy and tolerability of invasive or medical therapies (i.e., the effect on both subjective symptoms and urinary flow and incidence of adverse effects), the long-term effectiveness, the impact on daily life activities (quality of life) and the costs (268). Large scale randomized controlled trials provide information on the tolerability and efficacy of treatment options and evidence-based databases such as Cochrane reviews, may further analyze evidence-based data from multiple trials.

 

CONCLUSION

 

In conclusion, BPH is a common urological condition that is increasing in incidence in conjunction with the aging male population. If left untreated, BPH can lead to lower urinary tract obstructive symptoms that can significantly affect the quality of life of men.

 

As outlined in this chapter, the diagnosis of BPH begins with a detailed history of presenting complaint and interrogation of any lower urinary tract signs or symptoms. Questionnaires such as the IPSS score can help quantify the severity of these symptoms along with a uroflowmetry and PVR scan. A urinalysis, serum creatinine, and serum PSA should be ordered to investigate for prostate cancer, UTI, and renal failure. Imaging with USS or CT is not indicated unless there is concurrent hematuria, UTI, or urolithiasis.

 

Several options are now available for the treatment of BPH. Non-surgical management consists of medications such as alpha-blockers, 5-alpha reductase inhibitors, and PDE5 inhibitors which are available as monotherapy or in combination. BPH refractory to medical management is treated with surgical management which includes invasive and minimally invasive procedures. TURP remains the most widely used procedure for surgical BPH management and simple prostatectomies are reserved for larger prostates, complicated BPH or if TURP cannot be performed. These two procedures form the gold standard of treatment. Laser treatments when available offer good patient outcomes and have potential benefits when compared to TURP. The decision for either of these modalities of treatment is still largely dependent on availability and surgeon experience. The majority of minimally invasive treatment options are still experimental however may have a potential benefit for carefully selected men.

 

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Obesity In The Elderly

ABSTRACT

 

As the proportion of population above age 65 grows, so too increases the prevalence of those individuals who are obese. This phenomenon of an elderly population with obesity is the source of much research and debate with regards to treatment recommendations. It appears that older individuals on the extreme ends of the BMI spectrum, those who are underweight and those who are morbidly obese, have an increased risk of mortality. One major concern in the treatment of obese, elderly individuals is that many may have sarcopenic obesity which can be worsened with weight loss where some degree of lean body mass loss is inevitable. While various methods of weight loss may be recommended in some elderly who are obese, it is clear that any chosen method should be accompanied by a resistance training program in order to preserve muscle mass.

 

INTRODUCTION

 

The aging population in the U.S. is expected to more than double by 2050, increasing from 40.2 million to 88.5 million people (1). In tandem with this increase in elderly individuals is the high prevalence of those who are both elderly and obese. The significance of the increasing number of elderly individuals with obesity in terms of appropriate care and associated healthcare costs is the source of much debate.

 

PREVALENCE

 

Approximately 35% of adults in the U.S. aged 65 and over between 2007-2010 were obese as defined by body mass index (BMI, weight in kilograms over height in meters squared). In crude numbers this represents over 8 million adults aged 64-74 years and almost 5 million adults aged 75 and over (1). For individuals aged 75 and over there is a lower prevalence of obesity (27.8%) compared to those aged 65-74 years (40.8%) (1). A growing number of elderly are residing in nursing home (NH) facilities, and in line with this trend, researchers are examining the prevalence of obesity in NH facilities and its impact on healthcare utilization. Between 2000 and 2010, the prevalence of moderate to severe obesity in NHs increased from 14.7% to 23.9% (2). The rapid growth of the elderly population, which can largely be attributed to the aging baby boomers, will mark a change in the population’s composition in terms of sex ratios and ethnic diversity. Sex ratios of the population are projected to shift to include a larger share of elderly men (3). Moreover, the racial and ethnic make-up of this elderly cohort of patients is expected to develop to include more Hispanic individuals and a larger proportion of racial groups other than white. Between 2010 and 2050, the number of Hispanic people 65 years and older will increase from 2.9 to 17.5 million and the number of non-Hispanic individuals 65 years and older will increase from 37.4 to 71 million (3). These numbers of elderly individuals with obesity are also expected to increase as the population ages. Paradoxically, increased longevity does not necessarily translate to extra years spent in healthy living but may in fact result in more years spent in chronic poor health.

 

PATHOPHYSIOLOGY

 

Aging is accompanied by alterations in body composition. Fat free mass composed mostly of skeletal muscle declines by 40% between ages 20 and 70 years (4). Following age 70, both fat free mass and fat mass decrease together. With aging, there is also a redistribution of fat mass mainly in the visceral component but deposits are also observed in skeletal muscle and liver. The balance between energy intake and energy expenditure determines body fat mass. In the elderly, energy intake does not appear to increase significantly or may even decrease over time; therefore, decreased energy expenditure plays an important role in increasing fat mass with aging (4). After the age of 20, resting metabolic rate decreases by 2-3% per decade mainly due to a loss of fat free mass (4).  In addition to a decrease in resting metabolic rate, physical activity declines and there is an increase in sedentary time, which accounts for approximately half the loss in total energy expenditure with aging (4).

 

The redistribution of body fat centrally leads to the production of pro-inflammatory cytokines (5). Pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) lead to muscle loss and sarcopenia due to their catabolic effects (6). This loss of muscle mass leads to adverse outcomes such as decreased mobility and increased frailty.

 

Endocrinologic changes that occur with aging also play a role in the pathophysiology of obesity including a decrease in growth hormone, testosterone, and DHEA in addition to resistance to leptin and insulin.

 

HEALTHCARE OUTCOMES: THE POSITIVE AND THE NEGATIVE

 

Limitations To BMI Measurements

 

The American College of Cardiology and the American Heart Association define adults as overweight if BMI ≥ 25 kg/m2 and obese as BMI ≥ 30 kg/m2 regardless of age range. Accurately assessing obesity outcomes in the elderly can be a challenge given the drawbacks of defining obesity by BMI. Other methods have been utilized including hydrostatic densitometry (underwater weighing), dual-energy x-ray absorptiometry (DXA), and waist circumference. Given that BMI can either underestimate or overestimate body fat mass in the elderly and the fact fat deposition in the elderly tends to be accumulated intraabdominally, measurement of waist circumstance may be a better way of assessment. Despite its drawbacks, most studies analyzing healthcare outcomes in the obese elderly have utilized BMI as an assessment tool.

 

The Obesity Paradox

 

According to existing studies and meta-analyses, a higher BMI can be protective in the elderly. In an analysis of 13 observational studies from 1966 to 1999 examining cardiovascular mortality in non-hospitalized subjects aged 65 and above, a U-shaped curve was observed with an increase in right curve only when BMI was above 31-32 kg/m2 (7). A subsequent meta-analysis showed that BMI in overweight range did not confer an increased risk of mortality and a BMI in moderately obese range was only associated with a modest increase in mortality risk by 10% independent of gender, disease and smoking status (8). In a large, multi-ethnic study of community dwelling men and women aged 65 and above, the lowest hazard ratios (HRs) for mortality were seen in individuals with BMI 25 to less than 30 and BMI 30 to less than 35. HRs for mortality were increased when BMI was below 25 or higher than 35 (9). Similarly, in a large study of mortality in over 10,000 patients with type 2 diabetes mellitus and a median age of 63 years followed for a median of 10.6 years, a lower mortality risk was observed in overweight (BMI ≥ 25 kg/m2) and a higher mortality risk in those who were underweight (BMI ≤ 18.5 kg/m2) or obese (BMI ≥ 30 kg/m2) (10). A subsequent systemic review and meta-analysis evaluating the association of BMI with all-cause and cardiovascular mortality in subjects with type 2 diabetes mellitus, showed a strong non-linear relationship between BMI and all-cause mortality in both men and women. The lowest risk was seen in those with BMI 31-35 kg/m2 and 28-31 kg/m2. Lower BMI values were associated with higher mortality in both sexes (11). Combining available data suggests that BMI < 25 and > 35 kg/m2 is associated with higher mortality (41) (Figure 1).

Figure 1. BMI and Mortality in Elderly

While there are positive effects of obesity including increased energy reserve and prevention of malnutrition, protection from bone mineral density loss and osteoporosis, and delay in cognitive decline, there are also potential biases which may account for the obesity paradox seen in the elderly. The survival effect is one such bias which postulates that the remaining living elderly with obesity are more resistant to the complications of obesity compared to those who were perhaps more susceptible and therefore died earlier. Many studies are epidemiologic in design with the limitation of reverse causation where an overestimation of mortality risk can occur if unintentional weight loss due to an underlying disease occurs prior to BMI measurements and are then compared to the BMI of healthy group. Finally, cohort effects can be seen as subjects in different environments practicing different lifestyles are compared to one another (12).

 

One of the most significant complications of obesity in the elderly is the metabolic syndrome. This clustering of risk factors including increased waist circumference, hypertension, dyslipidemia, and glucose intolerance increases the likelihood of diabetes and cardiovascular disease. Obesity can stress the joints leading to joint dysfunction and mobility impairment as well as lead to pulmonary dysfunction and obstructive sleep apnea. Certain cancers are associated with higher BMIs including breast, uterine, colon and leukemia.

 

Weight Loss

 

Numerous population-based studies have found that weight loss in older persons is associated with increased mortality (13, 42, 43, 44). This is also true in diabetes (14). Obviously, a part of this may be due to the disease itself causing weight loss, but a number of studies have used different approaches to control for this. The negative effects of weight loss are muscle loss (sarcopenia), the protective effect of fat (on hip fracture for example), lipolysis leading to accelerated atherosclerosis, and fat loss leading to release of fat-soluble toxins into circulation (15). Fat and protein loss can also lead to drug toxicity due to the alteration of the pharmacokinetics of medications that are either fat-soluble or protein-bound (15). The benefits of weight loss need to be weighed against the risks in older persons (Figure 2).

 

Figure 2. Risk and Benefits of Weight Loss in the Elderly

 

Sarcopenic Obesity

 

Diet-induced weight loss in both younger and elderly adults consist of 75% fat tissue loss and 25% is fat free mass loss (16, 17). Hypothetically, in the elderly with obesity the loss of lean body mass is buffered by the already increased muscle mass. This proved to be a falsely reassuring concept when sarcopenic obesity was first described in the early 2000s. Sarcopenia is defined as the loss of skeletal mass and function and leads to frailty, disability, and loss of independence in the elderly. Elderly individuals with obesity have the unique difficulty in that although weight gain causes increased lean body mass and fat mass, the increased muscle mass is of poor quality. In a study by Villareal and colleagues, 52 obese elderly adults, 52 nonobese frail adults and 52 nonobese, nonfrail subjects matched for age and sex were compared. Elderly adults with obesity showed lower muscle quality compared with the other two groups in addition to reduced functional performance, aerobic capacity, strength, balance, and walking speed (18). In essence, the elderly with obesity cohort were sarcopenic and their increased adiposity proved deleterious. Subsequent studies have continued to demonstrate that sarcopenic obesity is associated with and precedes the onset of instrumental activities of daily living (IADLs) disability in community dwelling elderly (19). However, elderly subjects who are obese with increased muscle mass have better outcomes compared to those with low muscle mass. Determining which individuals who are elderly and obese have sarcopenia is important clinically and can be accomplished inexpensively and easily by measuring muscle strength via handgrip dynamometry or gait speed. The brief SARC-F questionnaire (Table 1) can also be used to identify obese individuals with poor muscle function (20). Another method for measuring and monitoring skeletal muscle mass is the use of creatine (methyl-d3) creatine dilution. In this noninvasive test, an oral tracer dose of D3-creatine is given and then subsequently measured in a fasting morning urine sample. Creatine dilution is a better measure of functional muscle mass than DXA (21).

 

Table 1. SARC-F Questionnaire

Component

Question

Scoring

Strength

How much difficulty do you have in lifting and carrying 10 pounds?

None = 0

Some = 1

A lot of unable = 2

Assistance in walking

How much difficulty do you have walking across a room?

None = 0

Some = 1

A lot, use of aids, or unable = 2

Rise from a chair

How much difficulty do you have transferring from a chair or bed?

None = 0

Some = 1

A lot or unable without help = 2

Climb stairs

How much difficult do you have climbing a flight of 10 stairs?

None = 0

Some = 1

A lot or unable = 2

Falls

How many times have you fallen in the past year?

None = 0

1-3 falls = 1

4 or more falls = 2

Score: ≥ 4 predictive of sarcopenia

 

TREATMENT

 

 

Select elderly individuals with obesity and BMI ≥ 30 kg/m2 who either have metabolic derangements or functional impairment may be recommended for weight loss provided that muscle and bone loss can be avoided (22).

 

Lifestyle Changes: Dietary Changes & Physical Exercise

 

Weight loss can be achieved alone by a moderate caloric deficit of 500-1000 kcal/day which leads to 1-2 pounds lost per week and 8-10% over 6 months (4).  However, dietary changes should be prescribed in conjunction with an exercise program consisting of aerobic, resistance and balance training to promote functionality and improve frailty (23). In a study of 107 frail elderly subjects with obesity randomized to control, diet group with 500-750 kcal deficit with 1 gm protein/kg/day, and a multi-component exercise and diet group, the combined exercise and diet group was more effective. The combined group had better physical performance scores, functional status, and aerobic capacity. Subjects also lost less lean body mass and bone mineral density compared to the diet group (24). Additionally, lifestyle interventions can reduce disease burden. In the Diabetes Prevention Program, men and women ≥ 65 years with obesity were more likely to achieve 7% weight loss compared to their younger (age ≤ 45 years) counterparts with obesity, at 3 years, 63% and 27% respectively. For every kilogram lost through diet and physical activity, the incidence of T2DM was decreased by 16% over a 3-year period (25).

 

In order to prevent muscle catabolism, elderly individuals with obesity with or at risk for sarcopenic obesity should be counseled on a less restrictive caloric deficit of 200-500 kcal/day combined with a recommended protein intake of 1.0-1.5 gm/kg assuming normal renal function.

 

Pharmacotherapy

 

There is limited data on safety and efficacy of weight loss medications in the elderly as they have largely been excluded from clinical trials. The FDA has approved five medications for chronic weight management: Semaglutide, Liraglutide, Naltrexone/Bupropion, Phentermine/Topiramate, and Orlistat. Additionally, metformin has been studied as a weight loss medication in obese, non-diabetic subjects. There is also a study in progress of elderly Japanese patients with type 2 diabetes assessing the efficacy and safety of empagliflozin, a sodium-glucose cotransporter-2 inhibitor (SGLT2i), known to cause weight loss (EMPA-ELDERLY). In this population, the effects on skeletal muscle mass, muscle strength, and physical performance will be assessed in subjects age 65 and older with type 2 diabetes on Empagliflozin (26). Overall, drug interactions, affordability, efficacy, and safety are all potential drawbacks to pharmacotherapy for weight loss in the elderly. However, there are no studies of outcomes of anorectic drugs used with exercise to protect muscle and bone.

 

SEMAGLUTIDE  

 

The weekly injectable glucagon-like peptide (GLP-1) receptor agonist was approved in 2021 for chronic weight management in adult patients with BMI of 30 kg/m2 or greater or 27 kg/m2 or greater plus a weight-related comorbid condition (hypertension, type 2 diabetes or dyslipidemia) as an adjunct to reduced calorie diet and increased physical activity. In the clinical trials, 233 (8.8%) of patients were between 65 and 75 years and 23 (0.9%) were 75 years or older and no differences in safety or efficacy were observed (27).

 

LIRAGLUTIDE

 

Liraglutide, a daily injectable GLP-1 receptor agonist was approved at doses of 3mg daily for weight loss by the FDA in 2014 for chronic weight management. This incretin-based therapy appears to have a short-term effect on decreasing gastric emptying but a long lasting central anorectic effect leading to a mean weight loss of 5.8kg in clinical studies (28, 29). The concern surrounding any weight loss in the elderly is the loss of skeletal muscle mass and sarcopenia. In a small study of elderly subjects who were either overweight or obese with type 2 diabetes mellitus treated with liraglutide 3mg daily in addition to metformin, reductions in fat mass and android fat were observed with the beneficial effect of preserved muscle tropism (30). A multicenter randomized, double-blind, parallel-group study of subjects with type 2 diabetes mellitus aged 18-80 years evaluated the effects of Liraglutide (as monotherapy or in combination with metformin) at various doses approved for treatment of diabetes mellitus (0.6mg, 1.2mg, 1.8mg daily) compared to individuals treated with Glimepiride or placebo. Mean body weight was reduced from baseline in all liraglutide treatment arms (up to 3.2 kg) and reduced fat tissue mass (1.0-2.4 kg) more than lean mass (1.5 kg) while glimepiride increased the mass of one or both tissue types (31). CT assessment also confirmed that reductions in fat tissue mass occurred in both abdominal subcutaneous and visceral fat compartments (31).

 

CONTRAVE

 

Contrave, the combination of naltrexone, an opioid antagonist, and bupropion, an aminoketone antidepressant, was FDA approved in 2014 for chronic weight management. Only 2% (62 of 3,239 subjects) in the Contrave clinical trials were over age 65 years and none older than 75 years (32). Data is lacking in terms of safety in older individuals, but given potential for neuropsychiatric disturbances, seizures, increased blood pressure and heart rate; extreme caution should be observed with this medication in the elderly.

 

QSYMIA

 

The combination of phentermine, a sympathomimetic amine anorectic, and topiramate extended release, an antiepileptic rug was FDA approved for chronic weight management in 2012. A small proportion of the subjects (254 total, 7%) studied in Qsymia clinical trials were aged 65 and older (33). While no differences in safety or effectiveness were observed, the adequate study numbers are also lacking. Given the side effect profile including risk of increased heart rate, acute myopia and secondary angle closure glaucoma, cognitive impairment and elevated creatinine, caution should be taken with starting this medication in elderly. Lower doses should be chosen and potential drug-drug interactions evaluated.

 

ORLISTAT

 

Orlistat acts as a pancreatic and gastric lipase inhibitor and leads to a 6.5-7.5 lb loss at one year. Its major side effects include steatorrhea, flatulence, fecal incontinence and malabsorption of fat-soluble vitamins. It appears to be equally efficacious with similar tolerance in a both the younger and elderly population (34).

 

METFORMIN

 

Metformin, a biguanide antidiabetic medication developed in the 1950s, may be a safe option to achieve modest weight loss even in nondiabetic individuals. In a small study of middle-aged nondiabetic subjects with obesity, metformin 2500mg daily without further caloric restriction or increased physical activity requirement resulted in a mean weight loss of 5.8 +/- 7kg (5.6+/-6.5%) compared to untreated controls (35). It may therefore be an efficacious and cost-effective strategy in elderly persons pending further studies.

 

Bariatric Surgery 

 

According to the NIH, bariatric surgery procedures including sleeve gastrectomy, laparoscopic adjustable gastric banding (LAGB), Roux-en-Y gastric bypass (RYGB), and biliopancreatic diversion with or without duodenal switch are potential options for individuals with obesity between ages 18 and 64 with BMI ≥ 40 kg/m2 or BMI ≥ 35 kg/m2 with additional co-morbidities. The American Diabetes Association has recommended lower BMI cutoffs of ≥ 30 kg/m2 for select individuals with uncontrolled hyperglycemia despite medical therapy (36). A retrospective review at a major surgical center in the U.S., found that of the 393 older patients (age > 65 years) who underwent bariatric surgery, older subjects had a higher comorbid burden compared to younger patients but exhibited comparable complication rates to patients under the age of 65 (37).  In a systematic review of over 8,000 patients aged 60 years and older who underwent bariatric surgery, outcomes (resolution of hypertension, diabetes, lipid disorders) and complication rates were similar to a younger population, independent of type of procedure (38).  While age should not necessarily be a barrier to recommending bariatric, this must be balanced against the limited existing data from pooled results of mostly small studies. Furthermore, bariatric surgery in the young and the elderly should always be coupled with resistance exercise.

 

Cryolipolysis

 

Cryolipolysis is FDA approved for treatment of focal fat deposits in the flanks, abdomen and thighs. In this procedure, fat cells are destroyed through a process of thermal reduction by which temperatures below normal but above freezing induce apoptosis-mediated cell death (39). Damaged adipocytes are then removed via an inflammatory response (39). This procedure has the advantage of being less invasive, does not require anesthesia with no downtime. In a retrospective review of a single surgery center with 528 subjects with age ranging from 18-79 years, the procedure was well tolerated with no adverse events and only 3 cases of mild or moderate pain reported to resolve in 4 or fewer days (40). However, there are limitations regarding the evaluation of the literature on this procedure thus far, including short follow-up time (typically 2-3 months), variability in cooling intensity factor (CIF) applied, differences in the evaluation of efficacy, and differences in the duration of procedure.

 

CONCLUSION

 

The landscape of the population is certainly changing and is marked by two significant trends: an increasingly elderly population and an ongoing obesity epidemic. This will undoubtedly impact families, social structures, and healthcare costs. How to appropriately care for these individuals will be the subject of much debate and further research. Physicians will need to balance the potential danger of weight loss in older persons against the complications of obesity to decide on the best patient centered approach. One clear recommendation is that all weight loss regimens in the elderly need to be coupled with a comprehensive resistance exercise program.

 

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Pathogenesis of Type 2 Diabetes Mellitus

ABSTRACT

 

Numerous distinct pathophysiologic abnormalities have been associated with type 2 diabetes mellitus (T2DM).  It is well established that decreased peripheral glucose uptake (mainly muscle) combined with augmented endogenous glucose production are characteristic features of insulin resistance. Increased lipolysis, elevated free fatty acid levels, along with accumulation of intermediary lipid metabolites contributes to further increase glucose output, reduce peripheral glucose utilization, and impair beta-cell function.  Adipocyte insulin resistance and inflammation have been identified as important contributors to the development of T2DM. The presence of non-alcoholic fatty liver disease [NAFLD] is now considered an integral part of the insulin resistant state.  The traditional concepts of “glucotoxicity” and lipotoxicity, which covers the process of beta cell deterioration in response to chronic elevations of glucose and lipids, has been expanded to encompass all nutrients [‘nutri-toxicity”].  The delayed transport of insulin across the microvascular system is also partially responsible for the development of tissue insulin resistance.   Compensatory insulin secretion by the pancreatic beta cells may initially maintain normal plasma glucose levels, but beta cell function is already abnormal at this stage, and progressively worsens over time.  Concomitantly, there is inappropriate release of glucagon from the pancreatic alpha-cells, particularly in the post-prandial period. It has been postulated that both impaired insulin and excessive glucagon secretion in T2DM are secondary to an “incretin defect”, defined primarily as inadequate release or response to the gastrointestinal incretin hormones upon meal ingestion.  To a certain extent, the gut microbiome appears to play a role in the hormonal and metabolic disturbances seen in T2DM.  Moreover, hypothalamic insulin resistance (central nervous system) also impairs the ability of circulating insulin to suppress glucose production, and renal tubular glucose reabsorption capacity may be enhanced, despite hyperglycemia.   These pathophysiologic abnormalities should be considered for the treatment of hyperglycemia in patients with T2DM. 

 

NORMAL GLUCOSE HOMEOSTASIS

 

In the post-absorptive state (10-12 hour overnight fast), the majority of total body glucose disposal takes place in insulin independent tissues (1).  Under basal conditions, approximately 50% of all glucose utilization occurs in the brain, which is insulin independent and becomes saturated at a plasma glucose concentration of about 40 mg/dl (2). Another 25% of glucose uptake occurs in the splanchnic area (liver plus gastrointestinal tissues) and is also insulin independent (3). The remaining 25% of glucose metabolism in the post-absorptive state takes place in insulin-dependent tissues, primarily muscle (4,5). Basal glucose utilization averages ~2.0 mg/kg.min and is precisely matched by the rate of endogenous glucose production (1,3-7). Approximately 85% of endogenous glucose production is derived from the liver, and the remaining amount is produced by the kidney (1,8,9). Approximately one-half of basal hepatic glucose production is derived from glycogenolysis and one-half from gluconeogenesis (9,10).

 

Following glucose ingestion, the balance between endogenous glucose production and tissue glucose uptake is disrupted. The increase in plasma glucose concentration stimulates insulin release from the pancreatic beta cells, and the resultant hyperinsulinemia and hyperglycemia serve (i) to stimulate glucose uptake by splanchnic (liver and gut) and peripheral (primarily muscle) tissues and (ii) to suppress endogenous glucose production (1,3-7,11-14).

 

Hyperglycemia, in the absence of hyperinsulinemia, exerts its own independent effect to stimulate muscle glucose uptake and to suppress endogenous glucose production in a dose dependent fashion (14-16). The majority (~80-85%) of glucose that is taken up by peripheral tissues is disposed of in muscle (1,3-7,11-14), with only a small amount (~4-5%) being metabolized by adipocytes (17). Although fat tissue is responsible for only a fraction of total body glucose disposal, it plays a very important role in the maintenance of total body glucose homeostasis (see below). Insulin is a potent inhibitor of lipolysis and even small increments in the plasma insulin concentration exerts a potent anti-lipolytic effect, leading to a marked reduction in the plasma free fatty acid level (18). The decline in plasma FFA concentration results in increased glucose uptake in muscle (19) and contributes to the inhibition of endogenous glucose production (16,20). Thus, changes in the plasma FFA concentration in response to increased plasma levels of insulin and glucose play an important role in the maintenance of normal glucose homeostasis (21,22).

SITE OF INSULIN RESISTANCE IN TYPE 2 DIABETES (T2DM)

The maintenance of whole-body glucose homeostasis is dependent upon a normal insulin secretory response by the pancreatic beta cells and normal tissue sensitivity to the independent effects of hyperinsulinemia and hyperglycemia (i.e., the mass-action effect of glucose) to augment glucose uptake. In turn, the combined effects of insulin and hyperglycemia to promote glucose disposal are dependent on three tightly coupled mechanisms: (i) suppression of endogenous (primarily hepatic) glucose production; (ii) stimulation of glucose uptake by the splanchnic (hepatic plus gastrointestinal) tissues; and (iii) stimulation of glucose uptake by peripheral tissues, primarily muscle (1,4,14). Muscle glucose uptake is regulated by flux through two major metabolic pathways: glycolysis (of which ~90% represents glucose oxidation) and glycogen synthesis.

 

Hepatic Glucose Production

 

In the overnight fasted state, the liver of healthy subjects produces glucose at the rate of ~1.8-2.0 mg.kg-1.min-1 (1,3,4,6,18,54). This glucose flux is essential to meet the needs of the brain and other neural tissues, which utilize glucose at a constant rate of ~1-1.2 mg.kg-1.min-1 (2,169).  Brain glucose uptake accounts for ~50-60% of glucose disposal during the post-absorptive state and this uptake is insulin independent. Therefore, brain glucose uptake occurs at the same rate during absorptive and post-absorptive periods and is not altered in T2DM (214).  Following glucose ingestion, insulin is secreted into the portal vein and glucagon release is inhibited, and this new hormonal ratio is carried to the liver, where it suppresses hepatic glucose output. If the liver does not perceive this insulin signal and continues to produce glucose, there will be two superimposed inputs of glucose into the body, one from the liver and another from the gastrointestinal tract, and marked hyperglycemia will ensue.

 

In subjects with T2DM and mild to moderate fasting hyperglycemia (140-200 mg/dl, 7.8-11.1 mmol/L) basal endogenous glucose production [EGP] is increased by ~0.5 mg/kg.min. Consequently, during the overnight sleeping hours (i.e., 2200 h to 0800 h), the liver of an 80-kg individual with diabetes and modest fasting hyperglycemia adds an additional 35 g of glucose to the systemic circulation. The increase in basal EGP is closely correlated with the severity of fasting hyperglycemia (1,3,4,6,18,54,157-159,162). Thus, in T2DM with overt fasting hyperglycemia (>140 mg/dl, 7.8 mmol/l), an excessive rate of EGP and glucose output is the major abnormality responsible for the elevated fasting plasma glucose concentration. The close relationship between fasting plasma glucose concentration and EGP has been demonstrated in numerous studies (164-166,170-174).

 

In the post-absorptive state, the fasting plasma insulin concentration in subjects with T2DM is 2-4-fold greater than in subjects without diabetes. Because hyperinsulinemia is a potent inhibitor of EGP (1,3,4-6,16,18,164,165,175), hepatic resistance to the action of insulin must be present in the post-absorptive state to explain the excessive output of glucose. Hyperglycemia per se also exerts a powerful suppressive action on EGP (15,167,175-177). Therefore, the liver, primarily, also must be glucose resistant with respect to the inhibitory effect of hyperglycemia to suppress glucose output, and this has been well documented (15,167,178,179).

 

Using the euglycemic insulin clamp technique in combination with tritiated glucose, the dose response relationship between endogenous glucose production and the plasma glucose concentration has been defined by Groop, DeFronzo, et al (18). The following points should be emphasized: (i) first, the dose-response curve relating inhibition of EGP to the plasma insulin concentration is quite steep, with an effective dose for half-maximal insulin concentration (ED50) of ~30-40 µU/ml; (ii) in individuals with T2D the dose response curve is shifted to the right, indicating the presence of hepatic resistance to the inhibitory effect of insulin on hepatic glucose production. However, at plasma insulin concentrations within the high physiologic range (~100 µU/ml), the hepatic insulin resistance can be largely overcome and a near normal suppression of EGP can be achieved; (iii) the severity of the hepatic insulin resistance is related to the severity of the diabetic state. In T2DM with mild fasting hyperglycemia, an increment in plasma insulin concentration of 100 µU/ml causes a complete suppression of EGP. However, in diabetic subjects with more severe fasting hyperglycemia, the ability of the same plasma insulin concentration to suppress EGP is impaired (18). These results suggest that there is an acquired component of hepatic insulin resistance and that this defect becomes progressively worse as the diabetic state decompensates over time.

 

The glucose released by the liver in the post-absorptive state can be derived from either glycogenolysis or gluconeogenesis (6,16,176). Studies employing the hepatic vein catheter technique have shown that the uptake of gluconeogenic precursors, especially lactate, is increased in subjects with T2DM (180). Consistent with this observation, radioisotope turnover studies, using lactate, alanine, and glycerol have shown that ~90% of the increase in HGP above baseline can be accounted for by accelerated gluconeogenesis (181,182). More recent studies employing 13C-magnetic resonance imaging (183) and D2O (184,185) have confirmed the important contribution of accelerated gluconeogenesis to the increase in HGP. An increased rate of glutamine conversion to glucose also has been shown to contribute to the elevated rate of gluconeogenesis in subjects with T2DM (186), which may be, in part, derived from renal gluconeogenesis (8). The mechanisms responsible for the increase in hepatic gluconeogenesis include hyperglucagonemia (187), increased circulating levels of gluconeogenic precursors (lactate, alanine, glycerol) (181,188), increased FFA oxidation (18,162,189), enhanced sensitivity to glucagon (190) and decreased sensitivity to insulin (1,4.18,164,165). Although the majority of evidence indicates that increased gluconeogenesis is the major cause of the increase in EGP in subjects with T2DM (181- 186), it is likely that accelerated glycogenolysis also contributes to it (181,191). 

 

The presence of both direct and indirect effects of insulin in suppressing EGP and release into the circulation were recently demonstrated in animals using intra-portal and systemic insulin infusions (430). The results provided evidence that, in addition to a direct action of insulin on hepatic enzymes, the inhibition of adipose tissue lipolysis represents an important mechanism by which insulin regulates the rate of gluconeogenesis.  This is therefore, accomplished indirectly, by controlling the supply of free fatty acids, which are essential to the process of glucose synthesis de novo.  The rate-limiting step in achieving fast and complete inhibition of adipose tissue lipolysis is the transendothelial transport of insulin across tissue capillaries.  Additional data obtained during systemic infusions of free fatty acids and in experiments where adipocyte lipolytic factors were manipulated, together with observation in mice lacking hepatic Foxo1 & Akt1/2 signaling have confirmed this indirect action of insulin on gluconeogenesis (430-432).  These findings have generated the hypothesis that in patients with T2DM, insulin may be transported slowly across tissue capillaries, which delays the inhibition of lipolysis with subsequent impairment of the suppression of EGP.

 

On the other hand, animal studies (431), where insulin was infused directly into the portal vein, mimicking normal insulin secretory pattern, showed that there is complete and swift inhibition of EGP.  These observations were confirmed when plasma glucagon and fatty acid levels were clamped at basal values, and in conditions where brain insulin action was blocked.  Authors conclude that the direct hepatic effect of insulin in the regulation of EGP is more relevant and that, the indirect effect is redundant in physiological conditions.  Acute insulin suppression of endogenous gluconeogenesis is largely an indirect effect mediated by the inhibition of adipose tissue lipolysis, which reduces delivery of non-esterified fatty acids and glycerol to the liver.  The major direct effect of insulin on hepatic glucose metabolism is the regulation of glycogen metabolism.  Hyperglycemia and hyperinsulinemia are required to maximally stimulate net hepatic glycogenesis.  In T2DM, lipid-induced hepatic insulin resistance, high rates of adipose tissue lipolysis and hyperglucagonemia impair glucose metabolism in the liver (432).

 

Because of the inaccessibility of the liver in man, it has been difficult to assess the role of key enzymes involved in the regulation of gluconeogenesis (pyruvate carboxylase, phosphoenol- pyruvate carboxykinase), glycogenolysis (glycogen phosphorylase), and net hepatic glucose output (glucokinase, glucose-6-phosphatase). However, considerable evidence from animal models of T2DM and some evidence in humans have implicated increased activity of PEPCK and G-6-Pase in the accelerated rate of hepatic glucose production (192-194).

 

Recently, changes in hypothalamic insulin signaling have been shown to affect endogenous glucose production. The activation of the insulin receptor in the third cerebral ventricle is capable of suppressing glucose production, independent of plasma insulin or other counter-regulatory hormones.  Conversely, central antagonism to insulin signaling impairs the ability of circulating insulin to inhibit glucose production (6A).  These observations have raised the possibility that hypothalamic insulin resistance contributes to hyperglycemia in T2DM.

The Role of the Kidney

The kidney also has been shown to produce glucose and estimates of the renal contribution to total endogenous glucose production have varied from 5% to 20% (8,9,195). These varying estimates of the contribution of renal gluconeogenesis to total glucose production are largely related to the methodology employed to measure glucose production by the kidney (196). One unconfirmed study suggests that the rate of renal gluconeogenesis is increased in T2DM with fasting hyperglycemia (197). Arguing against this possibility are studies employing the hepatic vein catheter technique which have shown that all of the increase in total body EGP (measured with 3-3H-glucose) in T2DM can be accounted for by increased hepatic glucose output (measured by the hepatic vein catheter technique) (3). A more relevant aspect on the role of the kidney in the dysregulation of glucose homeostasis in diabetes is the maintenance of hyperglycemia, which results from a maladaptive enhancement of the tubular glucose transport threshold (9A, 9B). It has been hypothesized that in response to an elevated glucose load presented to the proximal tubular lumen, the sodium glucose co-transporter system increases its reabsorptive capacity by upregulating the SGLT-2 expression and kinetics (9C).  However, more recent studies conducted in humans who underwent unilateral nephrectomy were not able to confirm the over-expression of either SGLT-2 or SGLT-1 proteins in proximal renal tubules of patients with T2DM compared to non-diabetic controls (433, 434).  The augmented tubular glucose transport described in patients with type 1 and type 2 diabetes may result from a functional enhancement of the activity of these co-transporters.  The elevated renal threshold to plasma values between 220-250 mg/dl for the excretion of glucose into the urine in these patients, thus may be secondary to a sustained hyperglycemia. If this is confirmed, the maladaptive process of recycling a substantial amount of glucose back into the peripheral circulation may be attenuated with near-normoglycemia, possibly reversible. In any case, this contribution of the kidney to hyperglycemia in diabetic patients represents one additional pathogenic mechanism that has been underappreciated.

Peripheral (Muscle) Glucose Uptake

Muscle is the major site of glucose disposal in man (1,3-5,14). Under euglycemic hyperinsulinemic conditions, approximately 80% of total body glucose uptake occurs in skeletal muscle (1,3-5). Studies employing the euglycemic insulin clamp in combination with femoral artery/vein catheterization have examined the effect of insulin on leg glucose uptake in subjects with T2DM and control subjects (3). Since bone is metabolically inert with regards to carbohydrate metabolism and adipose tissue takes up less than 5% of an infused glucose load (17,198,199), muscle represents the major tissue responsible for leg glucose uptake.

 

In response to a physiologic increase in plasma insulin concentration (~80-100 μU/ml), leg (muscle) glucose uptake increases linearly, reaching a plateau value of 10 mg/kg leg wt per minute (3). In contrast, in lean subjects with T2DM, the onset of insulin action is delayed for ~40 min and the ability of the hormone to stimulate leg glucose uptake is markedly blunted, even though the study is carried out for an additional 60 min in the group with T2DMto allow insulin to more fully express its biological effects (3). During the last hour of the insulin clamp study, the rate of glucose uptake was reduced by 50% in the group with T2DM (3). These results provide conclusive evidence that the primary site of insulin resistance during euglycemic insulin clamp studies performed in subjects with T2DM resides in muscle tissue. Using the forearm and leg catheterization techniques (13,153,200,202), a number of investigators have demonstrated a decreased rate of insulin-mediated glucose uptake by peripheral tissues. The use of positron emission tomography (PET) scanning to quantitate leg glucose uptake in subjects with T2DM has provided additional support for the presence of severe muscle resistance to insulin in diabetic subjects (203).   

 

Vascular and Myocardial Insulin Resistance

 

The first and rate-limiting step in insulin-mediated glucose disposal is the transit of insulin from the plasma to the muscle.  Crossing of insulin from the circulation into the muscle interstitium is governed by vascular endothelium.  The transendothelial transport depends on the insulin receptor binding to the endothelial cell membrane and requires the activation of the nitric oxide synthase.  The transport of insulin across the endothelial cell layer appears to involve a complex vesicular trafficking process, which is saturable.  Insulin is known to promote capillary vasodilation particularly in the postprandial period to facilitate entry and distribution of fuel substrates, including glucose.  Several studies sampling lymph and interstitial glucose, using dialysis techniques, have suggested that a delay in insulin transfer from the plasma to the tissue may play an important role in the development of insulin resistance (427-429).  Thus, impairment of insulin action may be secondary to a decrease in capillary density [chronic situations] or to a defective increase in blood flow or micro-capillary recruitment [acute conditions] (429). These abnormalities have been described in obese insulin-resistant and in the skin flow response of patients with diabetes. 

 

Myocardial insulin resistance translates to abnormal intracellular signaling and reduced glucose oxidation rates in animal models of obesity (435).  It adversely affects myocardial mechanical function and tolerance to ischemia and reperfusion.  The heart is a dynamic organ that requires continuous energy in the form of ATP in order to meet contractile demands.  This is achieved via a constant supply of blood-borne oxidizable substrates.  The majority of ATP is derived from fatty acid oxidation [60-70%].  Glucose and lactate extracted from the circulation account for the remainder 30-40%.  However, when blood glucose and insulin levels are elevated, such as immediately after a meal, glucose becomes the major fuel for myocardial oxidation and, it may represent up to 70% of the total substrate oxidation by cardio-myocytes.  Long–chain fatty acids are taken up by the heart proportionately to circulating levels, via a passive facilitated transport.  Once inside the cytosol, they are degraded into acetyl-CoA moieties that enter the mitochondrial oxidative phosphorylation process.  The excess fatty acids are re-esterified to form diacyl- and triacyl-glycerides and, these lipid intermediates are stored in the form the myo-cellular lipid pool.  Glucose enters the myocardial cells both via GLUT-1 passive and insulin-stimulated GLUT-4 active transport.  These are dictated by myocardial contraction demands and circulating insulin levels.  Intracellular glucose is phosphorylated and, either stored as muscle glycogen or anaerobically oxidized to pyruvate. Under normal oxygen delivery, pyruvate is converted to acetyl-CoA, which enters mitochondrial oxidation.  In conditions of ischemia, low oxygen forces the conversion of pyruvate into lactate (435).

 

It is believed that myocardial insulin resistance with typical defects in glucose transport and oxidation develops, in part, because of an excess supply of fatty acids.  In addition to a direct competition with glucose utilization, there is evidence that the accumulation of intracellular lipid intermediates interferes with insulin signaling.  The molecular defects responsible for the insulin resistance in the cardio-myocytes are analogous to the skeletal muscle.  The local generation of reactive oxygen species and other elements also participate in obstructing insulin action. Although the cellular and metabolic manifestations may be similar, the consequences of insulin resistance in the heart muscle tends to express with lower tolerance for ischemia and poor mechanical function.  Consequently, patients with insulin resistance are susceptible to earlier and more severe cardiovascular complications.   

 

Splanchnic (Hepatic) Glucose Uptake

 

In humans, it is difficult to catheterize the portal vein, and glucose disposal by the liver has not been examined directly. Using the hepatic vein catheterization technique in combination with the euglycemic insulin clamp, the contribution of the splanchnic (liver plus gastrointestinal) tissues to overall glucose homeostasis has been examined in lean subjects with T2DM with mild to moderate fasting hyperglycemia (3). In the post-absorptive state, there is a net release of glucose from the splanchnic area (i.e., negative balance) in both control and subjects with T2DM, reflecting glucose production by the liver. In response to insulin, splanchnic glucose output is promptly suppressed (reflecting the inhibition of HGP) and, by 20 min, the net glucose balance across the splanchnic region declines to zero (i.e., there was no net uptake or release) (3). After 2 h of sustained hyperinsulinemia, there is a small net uptake of glucose (~0.5 mg.kg- 1.min-1) by the splanchnic area (i.e., positive balance). This uptake is virtually identical to the rate of splanchnic glucose uptake observed in the basal state, indicating that the splanchnic tissues, like the brain, are insensitive to insulin at least with respect to the stimulation of glucose uptake (3,5,6,175). There was no difference between diabetic and control subjects for glucose taken up by the splanchnic tissues at any time during the insulin clamp study (3).

 

The results of these studies illustrate another important point: namely, that under conditions of euglycemic hyperinsulinemia, very little of the infused glucose is taken up by the splanchnic (and therefore hepatic) tissues (3,5,6,175). During the insulin clamp, the rate of whole-body glucose uptake averaged 7 mg.kg-1.min-1, and of this, only 0.5 mg.kg-1.min-1 or 7%, was disposed of by the splanchnic region. Because the difference in insulin-mediated total body glucose uptake between the T2DM and control groups during the euglycemic insulin clamp study was 2.5 mg.kg-1.min-1, from a purely quantitative standpoint it is obvious that a defect in splanchnic (hepatic) glucose removal never could account for the magnitude of impairment in total body glucose uptake following intravenous glucose/insulin administration. However, after glucose ingestion, the oral route of administration and the resultant hyperglycemia conspire to enhance splanchnic (hepatic) glucose uptake (6,7,11,12,16,26,175) and, under these conditions, diminished hepatic glucose uptake has been shown to contribute to the impairment in glucose tolerance in T2DM (see discussion below) (6,204,205).

 

Summary: Whole Body Glucose Utilization

 

Insulin-mediated whole body glucose utilization during the euglycemic insulin clamp represents essentially skeletal muscle glucose utilization. There is a noticeable decrease for glucose taken up in the body in T2DM patients compared with non-diabetic subjects. On the other hand, net splanchnic glucose uptake, quantitated by the hepatic venous catheterization technique, is similar in both groups and averaged 0.5 mg.kg-1.min-1. Adipose tissue glucose uptake accounts for less than 5% of total glucose disposal (17,198,199). Brain glucose uptake, estimated to be 1.0-1.2 mg.kg-1.min-1 in the post-absorptive state (2,169,206), is unaffected by hyperinsulinemia (169). Muscle glucose uptake (extrapolated from leg catheterization data) in control subjects accounts for ~75-80% of the total glucose uptake (1,3,4). In subjects with T2DM, the largest part of the impairment in insulin-mediated glucose uptake is accounted for by a defect in muscle glucose disposal. Even if adipose tissue of subjects with T2DM took up absolutely no glucose, it could, at best, explain only a small fraction of the defect in whole body glucose metabolism.

 

Glucose Disposal during OGTT

 

In everyday life, the gastrointestinal tract represents the normal route of glucose entry into the body. However, the assessment of tissue glucose disposal following glucose ingestion presents a challenge because of the difficulties in quantitating the rate of glucose absorption, suppression of hepatic glucose production, and organ (liver and muscle) glucose uptake. Moreover, because the plasma glucose and insulin concentrations are changing simultaneously, it is difficult to draw conclusions about insulin secretion or insulin sensitivity.

 

To address these issues, Ferrannini, DeFronzo, and colleagues (7,11,12,205) administered oral glucose to healthy control subjects in combination with hepatic vein catheterization to examine splanchnic glucose metabolism. The oral glucose load and endogenous glucose pool were labeled with [1-14C] glucose and [3-3H] glucose, respectively, to quantitate total body glucose disposal (from tritiated glucose turnover) and endogenous HGP (difference between the total rate of glucose appearance, as measured with tritiated glucose, and the rate of oral glucose appearance, as measured with [1-14C] glucose).

 

During the 3.5 h after glucose (68 g) ingestion: (i) 19 g, or 28%, or the oral load was taken up by splanchnic tissues; (ii) 48 g, or 72%, was disposed of by peripheral (non-splanchnic) tissues; (iii) of the 48 g taken up by peripheral tissues, the brain (an insulin-independent tissue) accounted for ~15 g (~1 mg.kg-1.min-1), or 22%, of the total glucose load (12); (iv) basal HGP declined by 53%. Similar percentages for splanchnic glucose uptake (24%-29%) and suppression of HGP (50%-60%) in normal subjects have been reported by other investigators (13,204,207-209). The contribution of skeletal muscle to the disposal of an oral glucose load has been reported to vary from a low of 26% (207) to a high of 56% (208), with a mean of 45% (11,13,207-209). These results emphasize several important differences between oral and intravenous glucose administration. After glucose ingestion: (i) EGP is less completely suppressed, most likely due to activation of local sympathetic nerves that innervate the liver (210); (ii) peripheral tissue (primarily muscle) glucose uptake is quantitatively less important; (3) splanchnic glucose uptake is quantitatively much more important.

 

In individuals with T2DM (12,204,205,211,212) the disposition of an oral glucose load is significantly altered. The disturbance in glucose metabolism is accounted for by two factors: (i) decreased tissue glucose uptake and (ii) impaired EGP suppression. Splanchnic glucose uptake is similar in diabetic and control groups. Inappropriate suppression of EGP accounted for nearly one-third of the defect in total-body glucose homeostasis, while reduced peripheral (muscle) glucose uptake accounted for the remaining two-thirds. Since hyperglycemia per se enhances splanchnic (hepatic) glucose uptake in proportion to the increase in plasma glucose concentration (24,175), the splanchnic glucose clearance (SGU/plasma glucose concentration) is markedly reduced in all subjects with T2DM following glucose ingestion. Using a combined insulin clamp/OGTT technique, impairment in glucose uptake by the splanchnic tissues in subjects with T2DM has been demonstrated directly (213).

 

The gastrointestinal incretin hormones, which are produced in response to nutrient intake and potentiate the stimulus to insulin secretion in the postprandial period have been implicated as additional factors in the pathogenesis of T2DM (4A,28-30). The combined actions of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) can account for most of the incretin effect in normal subjects (4B). Recent demonstration that in T2DM the incretin effect is impaired, diminished or absent (4B) has rekindled interest in the potential role of these gastrointestinal peptides in the abnormal handling of glucose by splanchnic tissues and perhaps, in the decline in beta-cell insulin secretion.

 

When viewed in absolute terms, most studies have demonstrated that the total amount of glucose taken up by all tissues of body over the 4-hour period following the ingestion of an oral glucose load is normal (13) or slightly decreased (204,205,211). However, this occurs at the expense of postprandial hyperglycemia. Thus, the efficiency of glucose disposal, i.e., the glucose clearance (tissue glucose uptake/plasma glucose concentration), is severely reduced. It should be emphasized that it is not the absolute glucose disposal rate, but rather the increment in glucose disposal above baseline that determines the rise in plasma glucose concentration above the fasting value. Every published study (13,204,205,211) has demonstrated that the incremental response in whole-body glucose uptake is moderately to severely reduced in individuals with T2DM. Similar results have been reported for forearm muscle glucose uptake (13,201,202,208,209), pointing out the important contribution of diminished muscle glucose disposal to impaired oral glucose tolerance in T2DM.

 

In summary, results of the OGTT indicate that both impaired suppression of EGP and decreased tissue (muscle) glucose uptake contribute approximately equally to the glucose intolerance of T2DM. The efficiency of the splanchnic (hepatic) tissues to take up glucose (as reflected by the splanchnic glucose clearance) also is impaired in individuals with T2DM.

 

Summary of Insulin Resistance in T2DM

 

Insulin resistance involving both muscle and liver are characteristic features of the glucose intolerance in individuals with T2DM. In the basal state, the liver represents a major site of insulin resistance, and this is reflected by overproduction of glucose despite the presence of both fasting hyperinsulinemia and hyperglycemia. This accelerated rate of hepatic glucose output is the primary determinant of the elevated fasting plasma glucose concentration in T2DM. Although tissue (muscle) glucose uptake in the post-absorptive state is increased when viewed in absolute terms, the efficiency with which glucose is taken up (i.e., the glucose clearance) is diminished. After glucose infusion or ingestion (i.e., in the insulin stimulated state) both decreased muscle glucose uptake and impaired suppression of HGP contribute to the insulin resistance. Following glucose ingestion, the defects in insulin-mediated glucose uptake by muscle and the suppression of glucose production by insulin contribute approximately equally to the disturbance in whole-body glucose homeostasis in T2DM. However, under euglycemic hyperinsulinemic conditions, EPG is largely suppressed and impaired muscle glucose uptake is primarily responsible for the insulin resistance.

DYNAMIC INTERACTION BETWEEN INSULIN SENSITIVITY AND INSULIN SECRETION IN T2DM

 

Subjects with T2DM manifest abnormalities both in tissue (muscle, fat, and liver) sensitivity to insulin and in pancreatic insulin secretion. To understand how these two metabolic disturbances interact to produce the full-blown diabetic condition, it is necessary to quantitate insulin action and insulin secretion in the same individual over a wide range of insulin sensitivity. This dynamic interaction is demonstrated graphically by results obtained in healthy, lean, young normal glucose tolerant women who received a euglycemic insulin clamp (1 mU.kg-1.min-1) and were stratified into quartiles based upon the rate of insulin-mediated glucose disposal (49).

 

Insulin secretion was measured independently on a separate day with a +125 mg/dl hyperglycemic clamp. Insulin resistance and insulin secretion were strongly and positively correlated (r=0.79, p<0.001).  Women who were the most insulin resistant (quartile 1) had the highest fasting plasma insulin concentrations and highest early and late phase plasma insulin responses. Similar results relating the plasma insulin response and the severity of insulin resistance have been reported in normal glucose tolerant subjects with the minimal model technique (46,47) and the insulin suppression test/oral glucose tolerance test (214).

 

A number of groups have examined the dynamic interaction between insulin secretion and insulin sensitivity in subjects with T2DM (1,4,34,35,38,39,42,46-48,58-61,150,162).

DeFronzo (4) studied lean (ideal body weight < 120%) and obese (ideal body weight > 125%) subjects with varying degrees of glucose tolerance as follows: Group I-obese subjects (n=24) with normal glucose tolerance; Group II-obese subjects (n=23) with impaired glucose tolerance; Group III-obese subjects (n=35) with overt diabetes, subdivided into those with a hyperinsulinemic response and those with a hypoinsulinemic response during a 100-gram OGTT; Group IV-normal weight subjects with T2DM (n=26); Group V-normal weight subjects (n=25) with normal glucose tolerance. All subjects ingested 100 g of glucose to provide a measure of glucose tolerance and insulin secretion. Whole-body insulin sensitivity was quantitated with the euglycemic insulin (~100 µU/ml) clamp technique, which was performed with indirect calorimetry to quantitate rates of glucose oxidation and non-oxidative glucose disposal. The later primarily reflects glycogen synthesis (215).

 

In normal weight subjects with T2DM, insulin-mediated whole-body glucose uptake was reduced by 40-50% and the impairment in insulin action resulted from defects in both oxidative and non-oxidative glucose metabolism (4). Obese individuals without T2DM were as insulin resistant as the normal-weight subjects with T2DM (4). Defects in both glucose oxidation and glucose storage contributed to the insulin resistance in the obese nondiabetic group. From the metabolic standpoint, therefore, obesity and T2DM closely resemble each other.

 

Similar results concerning reduced whole-body insulin sensitivity in individuals with obesity and T2DM have been reported by other investigators (160,161,166,216-218). Despite nearly identical degrees of insulin resistance, normal-weight subjects with T2DM manifested fasting hyperglycemia and marked glucose intolerance, whereas the obese individuals without diabetes had normal or only minimally impaired oral glucose tolerance (4). This apparent paradox is explained by the plasma insulin response during the OGTT. Compared with control subjects, the obese group without diabetes secreted more than twice as much insulin, and this was sufficient to offset the insulin resistance. In contrast, in normal-weight subjects with T2DM, the pancreas, when faced with the same challenge, was unable to augment its secretion of insulin sufficiently to compensate for the insulin resistance. This imbalance between insulin supply by the beta-cells and the insulin requirement by tissues resulted in a frankly diabetic state, with fasting hyperglycemia and marked glucose intolerance.

 

The fact that plasma insulin response to the development of insulin resistance typically is increased during the natural history of T2DM does not mean that the beta cell is functioning normally. To the contrary, recent studies (4C) have demonstrated that the onset of beta-cell failure occurs much earlier and is more severe than previously appreciated.

 

Recognizing that simply measuring plasma insulin response to a glucose challenge does not provide a valid index of beta cell function, a series of studies were conducted in subjects with normal glucose tolerance (NGT), impaired glucose tolerance (IGT) and T2DM, using an oral glucose tolerance test to evaluate the increment in insulin secretion in response to an increment in plasma glucose. A euglycemic insulin clamp to measure insulin sensitivity was also performed to address the adjustment of the beta cell to the body’s sensitivity to insulin.

 

Thus, the results yielded a better measure of beta-cell function expressed per increment of plasma glucose and corrected for the degree of insulin resistance, the so-called disposition index [ΔI/ΔG ÷IR]. These data revealed a substantial decrease in beta-cell function, most evident in individuals with IGT who had lost anywhere from 60 to 85% of the total insulin secretory capacity.

 

When obesity and diabetes coexist in the same individual, the severity of insulin resistance is only slightly greater than that in either the normal-weight diabetic or nondiabetic obese groups (4), and the magnitude of the defects in glucose oxidation and non-oxidative glucose disposal are similar in all obese and diabetic groups. Although hyperinsulinemic and hypoinsulinemic obese diabetic subjects were equally insulin resistant, the severity of glucose intolerance is worse in the hypoinsulinemic group, and this was related entirely to the presence of severe insulin deficiency.

 

In the obese nondiabetic subjects, tissue sensitivity to insulin is markedly reduced, but glucose tolerance remains perfectly normal because the beta cells are able to augment their insulin secretory capacity appropriately to offset the defect in insulin action. As the obese individual develops impaired intolerance, there is a further reduction in insulin-mediated glucose disposal, which is due primarily to a decrease in glycogen synthesis. However, there is only a small additional impairment in glucose tolerance, because the beta cells are able to further augment their secretion of insulin to counteract the deterioration in insulin sensitivity. The progression of the obese, glucose intolerant person to overt diabetes is heralded by a decline in insulin secretion without any worsening of insulin resistance. The obese diabetic has tipped over the top of Starling's curve of the pancreas and is now on the descending portion. Even though the plasma insulin response is increased compared to nondiabetic control subjects, it is not elevated appropriately for the degree of insulin resistance and there is evidence that there is ~80% of beta-cell functional loss by the time of diagnosis in diabetic subjects. The beta cell insulin response during the OGTT is best represented by the change in plasma insulin over the change in plasma glucose concentration, taking into consideration the degree of insulin resistance for each individual, the so-called Insulin Secretion /Insulin Resistance Index or Disposition Index as shown in Figure 1 below.

Figure 1- Log normalization of the relationship between 2-hour plasma glucose and Insulin Secretion/ Insulin Resistance in subjects with normal glucose tolerance (NGT), impaired glucose tolerance (IGT) and patients with type 2 diabetes (T2DM). There is a linear decline in the insulin secretory capacity with the development of the disease, such that by the time clinical diabetes with hyperglycemia become evident, the loss of beta-cell secretion of insulin is below 5% of NGT controls.

 

The natural history of T2DM described above is consistent with results in humans and monkeys published by other investigators (33-39,42,43,59-61,98,150). In lean subjects with a wide range of glucose tolerance, Reaven et al (42) demonstrated that the progression from normal to impaired glucose tolerance was marked by the development of severe insulin resistance, which was counterbalanced by a compensatory increase in insulin secretion. The onset of T2DM was associated with no (or only slight) further deterioration in tissue sensitivity to insulin.  Rather, insulin secretion declined and the impairment in beta cell function was paralleled by a decrease in glucose tolerance.  A similar sequence of events has been documented prospectively in Pima Indians (34-39,58,60), in Caucasians (1,4,41,42,44,47,59, 162, 219), Pima Indians (34-39,58,60,219), and Pacific Islanders (33,62,220) and, is consistent with the development of T2DM in the rhesus monkeys (48).  As monkeys grow older, they become obese and develop a diabetic condition closely resembling human T2DM. The earliest detectable abnormality in this primate model is a decrease in tissue sensitivity to insulin. Because of a compensatory increase in insulin secretion, the fasting plasma glucose concentration and glucose tolerance remain normal.

 

The studies detailed above indicate that insulin resistance is an early and characteristic feature of the natural history of T2DM in high-risk populations. Overt diabetes develops only in those individuals whose beta cells are unable to appropriately augment their secretion of insulin to compensate for the defect in insulin action. It should be recognized, however, that there are well-described populations with T2DM in whom insulin sensitivity is normal at the onset of diabetes, whereas insulin secretion is severely impaired (81-83). How frequently this occurs in a typical patient with T2DM remains to be determined. This insulinopenic variety of diabetes appears to be more common in African-Americans, elderly subjects, and in lean Caucasians.  In this later group, it is important to exclude type 1 diabetes, since ~10% of Caucasians with older onset diabetes are islet cell antibody and/or GAD positive (220).

 

Primary Hypersecretion of insulin

 

An alternative view to explaining the “state of insulin resistance” is the notion that primary beta cell overstimulation results in insulin hypersecretion.  This leads to the development of obesity and insulin resistance, and then, to beta cell exhaustion (436).  In a model that presupposes beta cell hypersecretion as the initial manifestation of beta cell dysfunction, insulin sensitivity is modulated by insulin secretion.  When beta cell hypersecretion occurs, the responsiveness of insulin-sensitive tissues to insulin is downregulated and, these tissues become insulin resistant.  The latter becomes necessary to maintain normal glucose tolerance, without the adverse outcome of hypoglycemia.  However, considering that beta cell hypersecretion is primary and ‘fixed’, when insulin sensitivity is acutely improved, hypoglycemia would be expected to ensue.  In either case, the demonstration of the existence of a feedback loop that regulates glucose metabolism has made it clear that assessment of the adequacy of beta cell function requires knowledge of both the degree of insulin sensitivity and the magnitude of the insulin response. 

 

When considering the feedback loop governing glucose metabolism, in the face of increased insulin secretion, insulin resistance should develop as a protective measure to maintain normal glucose concentrations without hypoglycemia. This is supported by observations in patients with insulinomas, in whom the risk of hypoglycemia is reduced by the downregulation of insulin action with the development of insulin resistance (437).  Further support for this downregulation of insulin action comes from studies in healthy individuals with normal glucose tolerance in whom insulin resistance developed during 3–5 days of chronic physiologic hyperinsulinemia, achieved by insulin infusion balanced by glucose infusion to prevent hypoglycemia (438).  Higher basal insulin levels have been documented in individuals with obesity and impaired glucose tolerance before the development of T2DM and, identified as a risk factor for diabetes.  However, in these studies, OGTT glucose levels were already higher in those who progressed and could be a confounder. Thus, although studies provide some support for the concept of a potential independent pathogenic role of primary hyperinsulinemia in dysglycemia a stronger, more definitive proof is still missing. 

 

Therefore, while it is clear that T2DM is a heterogeneous condition characterized by beta cell failure, whether beta cell dysfunction or primary hyperinsulinemia is the early event in the pathogenesis of dysglycemia is now up for debate. Although there is sufficient evidence in humans (and animal models) to support the principal defect as being early beta cell dysfunction associated with reduced insulin secretion, it is incumbent on the proponents of the primary hyperinsulinemia hypothesis to undertake further studies to make their case more forcefully. Improved understanding of whichever mechanism underlies beta cell dysfunction should allow us to provide better preventative and therapeutic interventions for T2DM.

 

Delayed Insulin Clearance in Diabetes

 

The role of delayed (or decreased) insulin clearance as a contributor to insulin resistance and to the development of T2DM has been studied.  Insulin availability in the systemic circulation is determined by the rate of beta cell secretion and its rate of hepatic/peripheral/renal clearance.  Insulin levels modulate expression and activity of the insulin receptors in target tissues, which ultimately determines insulin action. The main site of insulin clearance is the liver that removes approximately 50% of endogenous insulin with the remainder being cleared by the kidneys and the skeletal muscle.  Receptor-mediated insulin endocytosis is the primary mechanism by which insulin is removed from the circulation and inactivated.  Upon binding to its receptor, the insulin-receptor complex is internalized through the formation of clathrin-coated vesicles, and is delivered to the endosomes; the acidification of the endosomes then allows the dissociation of the hormone from its receptor and their sorting in different directions. Most of the internalized insulin is next targeted to lysosomes where it is degraded, whereas a smaller fraction remains intact. Both degradation products and intact insulin are segregated in recycling vesicles and released from cell.  Defects in the intracellular processing of insulin have been reported in cells from insulin resistant individuals and reduced insulin clearance has been observed in individuals with IGT.  More recently, it has also been demonstrated that reduced insulin clearance predicts the development of T2DM independently of confounding factors.  There is evidence in animal model of fat-induced insulin resistance supporting the idea that decreased insulin clearance may serve as a compensatory mechanism to alleviate b-cell stress from excessive demand in these conditions of insulin resistance (439).  The extent to which delayed insulin clearance is responsible for the advancement of insulin resistance and its role in the pathogenesis of T2DM remains unknown.    

 

Nutrient-induced Stress on Insulin Secretion

 

There is growing support to the theory that an excess of calorigenic nutrients ingested over time presents the pancreatic islet beta-cells with an overwhelming burden, which might lead to toxic hormonal and metabolic adaptations.  It is well recognized that the short-term effects of glucose, lipids and amino acids perfusing the beta cells in the endocrine pancreas include the stimulation of insulin biosynthesis and secretion.  Excessive exposure to these nutrients is believed to over-stimulate the beta cells with a constant and uninterrupted demand for insulin release and, possibly induce changes in tissue insulin sensitivity.  Chronically, abundant nutritional intake will trigger augmented insulin secretion and insulin resistance, both of which have been shown to contribute to the pathogenesis of T2DM.  Eventually, there is altered glucose sensing and depletion of insulin stores.  The de-differentiation, with beta cell death that follows is likely to play a role in the progression of the disease.  Thus, the traditional concepts of “glucotoxicity” and lipotoxicity”, which defines the process of beta cell deterioration in response to chronic elevation of glucose and lipids in the pericellular milieu, has now been expanded to encompass all nutrients [‘nutri-toxicity”].

 

The biochemical mechanisms underlying beta cell adaptation and failure associated with “nutri-toxicity” are not entirely clear, but appear to be related to oxidative stress.  Various pathways in the cytosol, endoplasmic reticulum [ER] and mitochondria are involved, which tend to affect the insulin secretory capacity of the beta cell.  In conditions of mild-to-moderate “nutri-stress”, such as in overweight/obesity, there is exaggerated basal and nutrient stimulated insulin secretion.  Slightly elevated blood glucose concentration, hyperinsulinemia and insulin resistance become progressively more evident.  Obesity with beta cell failure and T2DM result when there is more advanced and prolonged nutri-stress”.  The metabolic machinery of the beta cell is overwhelmed and, there is mitochondrial and ER dysfunction, which result in severe oxidative stress.  As a consequence, insulin synthesis and secretion become impaired and there is intra- cellular accumulation of toxic metabolites with beta cell de-differentiation and death (440).

 

 

ROLE OF THE ADIPOCYTE IN THE PATHOGENESIS OF T2DM

 

The majority (>80%) of persons with T2DM in the US are overweight (221). Both lean and especially obese persons with T2DM are characterized by day-long elevations in the plasma free fatty acid concentration, which fail to suppress normally following ingestion of a mixed meal or oral glucose load (222). Free fatty acids (FFA) are stored as triglycerides in adipocytes and serve as an important energy source during conditions of fasting. Insulin is a potent inhibitor of lipolysis, and restrains the release of FFA from the adipocyte by inhibiting the enzyme hormone sensitive lipase. In patients with T2DM the ability of insulin to inhibit lipolysis (as reflected by impaired suppression of radioactive palmitate turnover) and reduce the plasma FFA concentration is markedly reduced (17). It is now recognized that chronically elevated plasma FFA concentrations can lead to insulin resistance in muscle and liver (1,4,19,21,22,51,162,223,224) and impair insulin secretion (22,225,226). Thus, elevated plasma FFA levels can cause/aggravate three major pathogenic disturbances that are responsible for impaired glucose homeostasis in individuals with T2DM and the "triumvirate" (muscle, liver, beta cell) was joined by the "fourth musketeer" (227) to form the "disharmonious quartet". In addition to FFA that circulate in plasma in increased amounts, individuals with T2DM and obese individuals without T2DM have increased stores of triglycerides in muscle (228,229) and liver (230,231) and the increased fat content correlates closely with the presence of insulin resistance in these tissues. Triglycerides in liver and muscle are in a state of constant turnover and the metabolites (i.e., fatty acyl CoAs) of intracellular FFAs have been shown to impair insulin action in both liver and muscle (1,4,92). This sequences of events has been referred to as "lipotoxicity" (1,4,22,93). Evidence also has accumulated to implicate "lipotoxicity" as an important cause of beta cell dysfunction (22,93) (see earlier discussion).

 

Adipocyte Inflammation and Insulin Resistance

 

Increased risk of developing T2DM is found in patients who have chronic, low-grade adipocyte inflammation and who are also insulin resistant (441).  The mechanisms of adipose tissue inflammation and the related insulin-resistant state are complex.  Visceral adiposity is known to be highly active in releasing numerous inflammatory cytokines [Adipokines] that are strongly implicated in the genesis of tissue insulin resistance and T2DM.  Adipokines provide an important link between obesity and insulin resistance IR.  Adiponectin is a unique adipokine that is inversely related to the metabolic syndrome, T2DM, and atherosclerosis.  Adiponectin increases fatty acid oxidation while reducing glucose production in liver, and ablation of the adiponectin gene in mice induces insulin resistance and T2DM.  Adiponectin is also anti-inflammatory; it suppresses tumor necrosis factor (TNF) actions in nonalcoholic fatty liver disease and inhibits nuclear factor kappa-beta [NFκB and monocyte adhesion to endothelial cells.  Human resistin is an adipokine secreted by infiltrating inflammatory cells in human adiposity and can stimulate synthesis and secretion of other cytokines in adipocytes and endothelial cells.  Leptin, a well-known adipokine, normally functions centrally to suppress appetite, but most obese patients are leptin resistant and have increased circulating leptin.  In obesity, hyperleptinemia contributes to inflammation through modulation of T-cell and monocyte functions.  A role for retinol-binding protein 4 [RBP-4], a more recently described adipokine has been proposed to be linked to inflammation.

 

Visfatin is a novel adipokine that is increased in obesity, is pro-inflammatory, and has an insulin-mimetic effect via binding to the insulin receptor.  A member of the lipocalin family, lipocalin-2, also known as neutrophil gelatinase–associated lipocalin, modulates inflammation and is another adipokine that is elevated in the adipose tissue of obese mouse models and in the plasma of obese and insulin-resistant humans.  In vitro studies suggest that lipocalin-2 induces insulin resistance in adipocytes and hepatocytes. The plasma level of another member of the lipocalin family, lipocalin-type prostaglandin D synthase, serves as a biomarker of coronary atherosclerosis.  Thus, multiple adipose-secreted factors that are capable of impairing the cellular action of insulin have been suggested to be involved in the development of insulin resistance and facilitate the development of T2DM.

 

It should be recognized that nutritional fatty acids can modulate the inflammatory response, particularly via NFκB activity, and promote insulin resistance.  Further-more, inflammatory modulation of adipocyte differentiation increases free fatty acid release. The mechanisms of free fatty acid-associated insulin resistance include protein kinase C (PKC) activation, endoplasmic reticulum stress, and increased oxidative burden.  Free fatty acids also inhibit insulin receptor substrates [IRSs] and induce insulin resistance in skeletal muscle and liver.  Increased fatty acid flux from adipose tissue to liver causes hepatic insulin resistance by increasing gluconeogenesis, glycogenolysis, and glucose-6-phosphatase expression and activity, and by enhancing lipogenesis and triglyceride synthesis attributable to activation of the transcription factor sterol-CoA regulatory element binding protein.  Finally, free fatty acids cause endothelial insulin resistance and damage by impairing insulin and nitric oxide–dependent signaling, thus contributing to the vascular injury observed in adiposity.

 

The initial insult in obese individuals that triggers inflammation and systemic insulin resistance may occur through recruitment of macrophages and innate immune antigen activation of inflammatory receptors in the membrane.  This can be perpetuated with secretion of chemokines, retention of macrophages in adipose, and secretion of adipokines.  The inflammatory milieu induces adipocyte inflammatory cascades, such as the NFκB pathway, via activation of various kinases, and this modulates adipocyte transcription factors, attenuates insulin signaling, and increases the release of pro-inflammatory adipokines and free fatty acids. Inflammatory attenuation of adipocyte differentiation further exacerbates adipose dysfunction. These paracrine and endocrine adipose inflammatory events induce a systemic inflammatory and insulin-resistant state, favoring the development of T2DM.

 

FFA and Muscle Glucose Metabolism

 

Four decades ago, Randle (232) proposed that increased FFA oxidation restrains glucose oxidation in muscle by altering the redox potential of the cell and by inhibiting key glycolytic enzymes. The excessive FFA oxidation: (i) leads to the intracellular accumulation of acetyl CoA, a potent inhibitor of pyruvate dehydrogenase (PDH), (ii) increases the NADH/NAD ratio, causing a slowing of the Krebs cycle, and (iii) results in the accumulation of citrate, a powerful inhibitor of phosphofructokinase (PFK). Inhibition of PFK leads to the accumulation of glucose-6-phosphate (G-6-P) which in turn inhibits hexokinase II. The block in glucose phosphorylation causes a buildup of intracellular free glucose which restrains glucose transport into the cell via the GLUT4 transporter. The resultant decrease in glucose transport was postulated to account for the impairment in glycogen synthesis, although a direct inhibitory effect of fatty acyl Co-As on glycogen synthase also has been demonstrated (233). This sequence of events via which accelerated plasma FFA oxidation inhibits muscle glucose transport, glucose oxidation, and glycogen synthesis is referred to as the "Randle Cycle" (232). It should be noted that the same scenario would ensue if the FFA were derived from triglycerides stored in muscle (228,229) or from plasma (222).

 

Felber and coworkers (59,159,162,234,235) were amongst the first to demonstrate that in obese non-diabetic and diabetic humans, basal plasma FFA levels and lipid oxidation (measured by indirect calorimetry) are increased and fail to suppress normally after glucose ingestion. The elevated basal rate of lipid oxidation was strongly correlated with a decreased basal rate of glucose oxidation, as well as with reduced rates of glucose oxidation and non-oxidative glucose disposal (glycogen synthesis) following ingestion of a glucose load. Further validation of the Randle Cycle in man has come from studies employing the euglycemic insulin clamp. In normal subjects, physiologic hyperinsulinemia (80-100 μU/ml) causes a 60-70% decline in plasma FFA concentration and a parallel decline in plasma FFA and total body lipid oxidation (18). When Intralipid is infused concomitantly with insulin to maintain or increase the plasma FFA concentration/oxidation, both glucose oxidation and non-oxidative glucose disposal are inhibited in a dose dependent fashion (223). Using magnetic resonance imaging, it has been shown that the FFA-induced inhibition of non-oxidative glucose disposal reflects impaired glycogen synthesis (236). The inhibitory effect of elevated plasma FFA levels can be observed at all plasma insulin concentrations, spawning the physiologic and pharmacologic range (223).

The inhibitory effect of an acute elevation in plasma FFA concentration on muscle glucose metabolism is time dependent. Thus, the earliest (within 2 hours) observed abnormality is a defect in glucose oxidation (237), as would be predicted by operation of the Randle cycle (232). This is followed (between 2-3 hours) by defects in glucose transport and phosphorylation and eventually (after 3-4 hours) by impaired glycogen synthesis.

 

Biochemical/Molecular Basis of FFA-Induced Insulin Resistance

 

The original description of the Randle cycle was formulated based upon experiments performed in rat diaphragm and heart muscle (232). More recent studies performed in human skeletal muscle suggest that mechanisms in addition to those originally proposed by Randle are involved in the FFA-induced insulin resistance. Thus, several groups (236,238,239) have failed to observe a rise in muscle G-6-P and citrate concentrations when insulin-stimulated glucose metabolism was inhibited by an increase in the plasma FFA concentration. Elevated plasma FFA levels also failed to inhibit muscle phosphofructokinase activity. Thus, while increased FFA/lipid oxidation and decreased glucose oxidation are closely coupled, as originally demonstrated by Randle, mechanisms other than product (i.e., elevated intracellular G-6-P and free glucose concentrations) inhibition of the early steps of glucose metabolism must be invoked to explain the defects in glucose transport, glucose phosphorylation and glycogen synthesis.

 

Studies in humans and animals have shown a strong inverse correlation between insulin- stimulated glucose metabolism and increased intramuscular lipid pools, including triglyceride (240-242), diacyl-glycerol (DAG) (243,244), and long chain fatty acyl CoAs (FA-CoA) (245). An acute elevation in plasma FFA concentration leads to an increase in muscle fatty acyl CoA and DAG concentrations. Both long chain fatty acyl CoAs and DAG activate PKC theta (243), which increases serine phosphorylation with subsequent inhibition of IRS-1 tyrosine phosphorylation (246,247). Consistent with this observation, two groups have shown that in human muscle elevated plasma FFA levels inhibit insulin-stimulated tyrosine phosphorylation of IRS-1, the association of the p85 subunit of PI-3 kinase with IRS-1, and activation of PI-3-kinase (248,249). Direct effects of long chain fatty acyl CoAs on glucose transport (250), glucose phosphorylation (251), and glycogen synthase (233) also have been demonstrated in muscle. Lastly, increased muscle ceramide levels (secondary to increased long chain fatty acyl CoAs) have been shown to interfere with glucose transport and to inhibit glycogen synthase in muscle via activation of PKB (252). In summary, elevated plasma FFA concentrations can induce insulin resistance in muscle via multiple mechanisms involving alterations in a variety of intracellular lipid signaling molecules which exert their inhibitory effects on multiple steps (insulin signal transduction system, glucose transport, glucose phosphorylation, glycogen synthase, pyruvate dehydrogenase, Krebs cycle) involved in glucose metabolism.

 

Fatty Liver Disease in T2DM

 

As the epidemics of obesity increases worldwide in conjunction with T2DM, there is a parallel and proportionate increase in the prevalence of nonalcoholic fatty liver disease (NAFLD).  A subtype of NAFLD, which can be characterized as nonalcoholic steato-hepatitis (NASH) is a potentially progressive liver disease that can lead to cirrhosis, hepatocellular carcinoma, liver transplantation, and death.  NAFLD is also associated with extrahepatic manifestations such as chronic kidney disease, cardiovascular disease and sleep apnea.  Despite this important burden, we are only beginning to understand its pathogenesis and the contribution of environmental and genetic factors to the risk of developing the progressive course of fatty liver disease.  Of interest, however, despite the fact that the risk of liver-related mortality and the advancement to liver fibrosis are increased in patients with NAFLD, the leading cause of death is cardiovascular disease (442-443).

 

NAFLD and NASH are stages of fatty liver disease that are associated with obesity, insulin resistance, T2DM, hypertension, hyperlipidemia, and metabolic syndrome.  In these individuals, a net retention of lipids within hepatocytes, mostly in the form of triglycerides, is a prerequisite for the development of fatty liver disease.  The primary metabolic abnormality leading to lipid accumulation (steatosis), however, is not well understood, but it could potentially result from insulin resistance and alterations in the uptake, synthesis, degradation or secretory pathways of hepatic lipid metabolism. Insulin resistance represents the most reproducible factor in the development of fatty liver disease.  There is also some evidence that lipids synthesis de novo”, a process derived from excess non-utilized carbohydrates accumulated in hepatocytes contributes to the intracellular lipid pool.  Once an excessive amount of lipids accumulate inside the hepatocytes, a steatotic liver develops.  This makes the cellular architecture of the liver vulnerable to further injury, when challenged by additional insults. There is a presumption that progression from simple, uncomplicated steatosis to steato-hepatitis to advanced fibrosis results from two operating “hits” due to: i) insulin resistance with further accumulation of fat within hepatocytes, and ii) generation of reactive oxygen species due to lipid peroxidation with cytokine production and Fas ligand induction.  The oxidative stress and lipid peroxidation are key factors in the development and progression from steatosis to more advanced stages of liver damage.  In addition, this sequence of events reflects similar systemic processes, which worsen tissue insulin resistance with impairment of insulin secretion and accelerated atherogenesis, related primarily to the pro-inflammatory state (442).

 

FFA and Blood Flow

 

Insulin is a vaso-dilatory hormone and the stimulatory effect of insulin on muscle glucose metabolism has been shown to result from: (i) a direct action of insulin to augment muscle glucose metabolism, and (ii) increased blood flow to muscle (253,254). The vaso-dilatory effect of insulin is mediated via the release of nitric oxide from the vascular endothelium (255). In insulin resistant conditions, such as obesity and T2DM, some investigators have suggested that as much as half of the impairment in insulin-mediated whole body and leg muscle glucose uptake is related to a defect in insulin's vaso-dilatory action (253,254), although the link between insulin-mediated vasodilation and increased blood flow, as well as the underlying mechanisms have been challenged by others (256, 256A). More recent studies employed contrast-enhanced ultrasonography using 1-methyl-xantine to demonstrate that insulin infusion promotes capillary recruitment in healthy individuals. These data have suggested that there is a time-dependent effect of insulin on regional blood flow redistribution with capillary pre-sphincter relaxation preceding vasodilation and consequent increase in skeletal muscle glucose metabolism (256B). These observations also provided a partial explanation for the discrepant findings reported on the topic of insulin, fatty acids and vasodilatation.

 

Because T2DM and obesity are insulin resistant states characterized by day-long elevation in the plasma FFA concentration (222) and impaired endothelium dependent vasodilation (253), investigators have examined the effect of increased plasma FFA levels on limb blood flow and muscle glucose uptake (257,258). In healthy, non-diabetic subjects an acute physiologic increase in plasma FFA concentration inhibited metha-choline (endothelium dependent) but not nitroprusside (endothelium independent) stimulated blood flow in association with an impairment in insulin-stimulated muscle glucose disposal. In subsequent studies, the inhibitory effect of FFA on insulin-stimulated leg blood flow was shown to be associated with decreased nitric oxide availability (259). FFA elevation also inhibits nitric oxide production in endothelial cell cultures by decreasing nitric oxide synthase activity (259). Since the IRS-1/PI-3 kinase signal transduction pathway is involved in the regulation of nitric oxide synthase activity (260), one could hypothesize that FFA-induced inhibition of the insulin signal transduction pathway is responsible for the blunted vaso-dilatory response to the hormone.

 

FFA and Hepatic Glucose Metabolism

 

The liver plays a pivotal role in the regulation of glucose metabolism (1,4,6,11,16,205). Following carbohydrate ingestion, the liver suppresses its basal rate of glucose production and takes up approximately one-third of the glucose in the ingested meal (12,24,25,205).

Collectively, suppression of glucose production and augmentation of hepatic glucose uptake account for the maintenance of nearly one-half of the rise in plasma glucose concentration following ingestion of a carbohydrate meal.  Hepatic glucose production is regulated by a number of factors, of which insulin (inhibits) and glucagon and FFA (stimulate) are the most important. In vitro studies have demonstrated that plasma FFA are potent stimulators of endogenous glucose production and do so by increasing the activity of pyruvate carboxylase and phosphoenolpyruvate carboxy-kinase, the rate limiting enzymes for gluconeogenesis (261,262).  FFA also enhances the activity of glucose-6- phosphatase, the enzyme that ultimately controls the release of glucose by the liver (263).

 

In normal subjects, increase plasma FFA levels stimulate gluconeogenesis (264,265), while a decrease in plasma FFA concentration reduces gluconeogenesis (264). It has been shown that a significant portion of the suppressive effect of insulin on hepatic glucose production is mediated via inhibition of lipolysis and a reduction in circulating plasma FFA concentrations (16,266,267).  Moreover, FFA infusion in normal humans under conditions that simulate the diabetic state (268) and in obese insulin-resistant subjects (269) enhances hepatic glucose production, most likely secondarily to stimulation of gluconeogenesis.  In subjects with T2DM, the fasting plasma FFA concentration and lipid oxidation rate are increased and are strongly correlated with both the elevated fasting plasma glucose concentration and basal rate of hepatic glucose production (18,51,59,162,190,270). The relationship between elevated plasma FFA concentration, FFA oxidation, and hepatic glucose production in obesity and T2DM is explained as follows: (i) increased plasma FFA levels, by mass action, augment FFA uptake by hepatocytes, leading to accelerated lipid oxidation and accumulation of acetyl CoA. The increased concentration of acetyl CoA stimulates pyruvate carboxylase, the rate limiting enzyme in gluconeogenesis (261,262), as well as glucose-6-phosphatase, the rate-controlling enzyme for glucose release from the hepatocyte (263); (ii) the increased rate of FFA oxidation provides a continuing source of energy (in the  form of ATP) and reduced nucleotides (NADH) to drive gluconeogenesis; (iii) elevated plasma FFA induce hepatic insulin resistance by inhibiting the insulin signal transduction system (244- 248). In patients with T2DMthese deleterious effects of elevated plasma FFA concentrations occur in concert with increased plasma glucagon levels (181,190,271), increased hepatic sensitivity to glucagon, and increased hepatic uptake of circulating gluconeogenic precursors.

 

The Role of Gut Microbiome

 

Recently the potential role of the gut microbiome in metabolic disorders such as obesity and T2DM has been identified (444).  Obesity is associated with changes in the composition of the intestinal microbiota, and the obese microbiome seems to be more efficient in harvesting energy from the diet.  Lean male donor fecal microbiota transplantation (FMT) in males with the metabolic syndrome resulted in a significant improvement in insulin sensitivity in conjunction with an increased intestinal microbial diversity, including a distinct increase in butyrate-producing bacterial strains.  Such differences in gut microbiota composition might function as early diagnostic markers for the development of T2DM in high-risk patients.  Products of intestinal microbes such as butyrate may induce beneficial metabolic effects through enhancement of mitochondrial activity, prevention of metabolic endotoxemia, and activation of intestinal gluconeogenesis via different routes of gene expression and hormone regulation. There is currently an enormous effort in trying to better understand, amongst other things, whether bacterial products (like butyrate) have the same effects as the intestinal bacteria that produce it, in order to ultimately pave the way for more successful interventions for obesity and T2DM.   Rapid development of the currently available techniques, including the use of fecal transplantations, has already shown promising results, so there is hope for novel therapies based on the microbiota in the future.

 

Summary: FFA and the Pathogenesis of Obesity and T2DM

 

n obese individuals and in the majority (>80%) of subjects with T2DM, there is an expanded fat cell mass and the adipocytes are resistant to the anti-lipolytic effects of insulin (18). Most individuals with obesity or T2DM are characterized by visceral adiposity (272) and visceral fat cells have a high lipolytic rate, which is especially refractory to insulin (273). Not surprisingly, both T2DM and obesity are characterized by an elevation in the mean day-long plasma FFA concentration. Elevated plasma FFA levels, as well as increased triglyceride/fatty acyl CoA content in muscle, liver, and beta cell, lead to the development of muscle/hepatic insulin resistance and impaired insulin secretion.

 

THE OMNIOUS OCTET

Figure 2. Summary of the Eight Principal Mechanisms Contributing to Hyperglycemia in Patients with Type 2 Diabetes

 

The eight principle known causes leading to hyperglycemia through the pathogenesis of T2DM are summarized in Figure 2. It is already established that decreased peripheral glucose uptake combined with augmented endogenous (hepatic) glucose production are characteristic features of insulin resistance. Increased lipolysis with accumulation of intermediary lipid metabolites contributes to further enhance glucose output while reducing peripheral utilization. Compensatory insulin secretion by the pancreatic beta-cells eventually reaches a maximum and, then it progressively deteriorates. Concomitantly, there is inappropriate release of glucagon from the pancreatic alpha-cells, particularly in the post- prandial period. It has been postulated that both impaired insulin and excessive glucagon secretion in T2DM are facilitated by the “incretin defect”, defined primarily as inadequate response of the gastrointestinal “incretin” hormones to meal ingestion in addition to islet-cell resistance to the potentiating action on insulin-secretion by these gastrointestinal peptides. Moreover, considering that hypothalamic insulin resistance (central nervous system) with an elevated sympathetic drive, typically seen in patients with T2DM also impair the ability of circulating insulin to suppress glucose production. The fact that renal tubular glucose reabsorption capacity is enhanced in diabetic patients also contributes to the development and maintenance of chronic hyperglycemia.  Thus, the time has arrived to advance the concept from the “triumvirate” to the “omnious octet” (4A). Further, recent observations have recognized that a chronic low-grade inflammation with activation of the immune system are involved in the pathogenesis of obesity-related insulin resistance and T2DM (4D). Adipose tissue, liver, muscle and pancreas are themselves sites of inflammation in presence of obesity. Infiltration of macrophages and other immune cells as well as the presence of pro-inflammatory cytokines in these tissues has been associated with insulin resistance and beta-cell impairment. The possibility that endothelial dysfunction and changes in vascular capillary permeability affect peripheral insulin action has also been raised (4E). These pathogenic mechanisms must be taken into account when deciding for the treatment of hyperglycemia in patients with T2DM.

 

CELLULAR MECHANISMS OF INSULIN RESISTANCE

 

The stimulation of glucose metabolism by insulin requires that the hormone must first bind to specific receptors that are present on the cell surface of all insulin target tissues (1,274-277). After insulin has bound to and activated its receptor, "second messengers" are generated and these second messengers initiate a series of events involving a cascade of phosphorylation- de-phosphorylation reactions (1,274-280) that eventually result in the stimulation of intracellular glucose metabolism. The initial step in glucose metabolism involves activation of the glucose transport system, leading to influx of glucose into insulin target tissues, primarily muscle (1,281,282). The free glucose, which has entered the cell, subsequently is metabolized by a series of enzymatic steps that are under the control of insulin. Of these, the most important are glucose phosphorylation (catalyzed by hexokinase), glycogen synthase (which controls glycogen synthesis), and phosphofructokinase (PFK) and PDH (which regulate glycolysis and glucose oxidation, respectively).

 

Insulin Receptor/Insulin Receptor Tyrosine Kinase

 

The insulin receptor is a glycoprotein consisting of two alpha subunits and two beta subunits linked by disulfide bonds (1,274-277). The alpha subunit of the insulin receptor is entirely extracellular and contains the insulin-binding domain. The beta subunit has an extracellular domain, a transmembrane domain, and an intracellular domain that expresses insulin- stimulated kinase activity directed towards its own tyrosine residues (1,274-277). Insulin receptor phosphorylation of the beta subunit, with subsequent activation of insulin receptor tyrosine kinase, represents the first step in the action of insulin on glucose metabolism (274- 277). Mutagenesis experiments have shown that insulin receptors devoid of tyrosine kinase activity are completely ineffective in mediating insulin stimulation of cellular metabolism (283,284). Similarly, mutagenesis of any of the three major phosphorylation sites (at residues 1158, 1163, and 1162) impairs insulin receptor kinase activity, resulting in a decrease in the acute metabolic and growth promoting effects of insulin (283,285).

 

Insulin Receptor Signal Transduction

 

Following activation, insulin receptor tyrosine kinase phosphorylates specific intracellular proteins, of which at least nine have been identified (282). Four of these belong to the family of insulin-receptor substrate proteins: IRS-1, IRS-2, IRS-3, IRS-4 (the others include Shc, Cbl, Gab-1, p60dok, and APS). In muscle IRS-1 serves as the major docking protein that interacts with the insulin receptor tyrosine kinase and undergoes tyrosine phosphorylation in regions containing amino acid sequence motifs (YXXM or YMXM).  When phosphorylated, these serve as recognition sites for proteins containing src-homology 2 (SH2) domains (where y = tyrosine, M = methionine, and x - any amino acid) (274,275).  Mutation of these specific tyrosines severely impairs the ability of insulin to stimulate glycogen synthesis and DNA synthesis, establishing the important role of IRS-1 in insulin signal transduction (282). In liver, IRS-2 serves as the primary docking protein that undergoes tyrosine phosphorylation and mediates the effect of insulin on hepatic glucose production, gluconeogenesis and glycogen formation (287). In adipocytes, Cbl represents another substrate which is phosphorylated following its interaction with the insulin receptor tyrosine kinase, which is required for stimulation of GLUT 4 translocation.

 

Phosphorylation of Cbl occurs when the CAP/Cbl complex associates with flotillin in caveolae, or lipid rafts, containing insulin receptors (288,289).

 

In muscle, the phosphorylated tyrosine residues on IRS-1 mediate an association between the two SH2 domains of the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase), leading to activation of the enzyme (274-284,290,291). PI3-kinase is a heterodimeric enzyme comprised of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit. The latter catalyzes the 3-prime phosphorylation of phosphatidylinositol (PI), PI-4-phosphate, and PI-4,5- diphosphate, resulting in the stimulation of glucose transport (274-277). Activation of PI3-kinase by phosphorylated IRS-1 also leads to activation of glycogen synthase (274,275), via a process that involves activation of PKB/Akt and subsequent inhibition of kinases such as GSK-3 (292) and activation of protein phosphatase 1 (PP1) (293). Inhibitors of PI3-kinase impair glucose transport (274-277,294) by interfering with the translocation of GLUT 4 transporters from their intracellular location (281,282) and block the activation of glycogen synthase (295) and hexokinase (HK)-II expression (296). The action of insulin to increase protein synthesis and inhibit protein degradation also is mediated by PI-3 kinase and involves the activation of mTOR (297,298). mTOR controls translation machinery by phosphorylation and activation of p70 ribosomal S6 kinase (p70rsk) (297) and phosphorylation of initiation factors (299). Insulin also promotes hepatic triglyceride synthesis via increasing the transcription factor steroid regulatory element-binding protein (SREBP)-1c (300), and this lipogenic effect of insulin also appears to be mediated via the PI3-kinase pathway (274).

 

Other proteins with SH2 domains, including the adapter protein Grb2 and Shc, also interact with IRS-1 and become phosphorylated following exposure to insulin (274-276,301). Grb2 and Shc serve to link IRS-1/IRS-2 to the mitogen-activated protein (MAP) signaling pathway, which plays an important role in the generation of transcription factors (274,275). Following the interaction between IRS-1/IRS-2 and Grb2 and Shc, Ras is activated, leading to the stepwise activation of Raf, MEK, and ERK.  Activated ERK then translocates into the nucleus of the cell, where it catalyzes the phosphorylation of transcription factors.  These promote cell growth, proliferation, and differentiation (274-276,301-303).  Blockade of MAP kinase pathway prevents stimulation of cell growth by insulin but has no effect on the metabolic actions of the hormone (304-306).

 

Under anabolic conditions insulin stimulates glycogen synthesis by simultaneously activating glycogen synthase and inhibiting glycogen phosphorylase (307-309). The effect of insulin is mediated via the PI3 kinase pathway which inactivates kinases, such as glycogen synthase kinase-3 and activates phosphatases, particularly protein phosphatase 1 (PP1). It is believed that PP1 is the primary regulator of glycogen metabolism (307-310). In skeletal muscle, PP1 associates with a specific glycogen-binding regulatory subunit, causing the activation [de-phosphorylation] of glycogen synthase; PP1 also inactivates [phosphorylates] glycogen phosphorylase.  The precise steps that link insulin receptor tyrosine kinase/PI 3-kinase activation to the stimulation of PP1 have yet to be defined. Some evidence suggests that p90 ribosomal S6- kinase may be involved in the activation of glycogen synthase (274). Akt also has been shown to phosphorylate and thus inactivate GSK-3 (292). This decreases glycogen synthase phosphorylation, leading to the enzyme activation (292). A number of studies have convincingly demonstrated that inhibitors of PI3-kinase also inhibit glycogen synthase activity and abolish glycogen synthesis (274,293,310). From the physiological standpoint, it makes sense that activation of glucose transport and glycogen synthase should be linked to the same signaling mechanism to provide a coordinated stimulation of intracellular glucose metabolism.

 

Insulin Signal Transduction Defects in T2DM

 

Both receptor and post-receptor defects have been shown to contribute to insulin resistance in individuals with T2DM. Some, but not all studies have demonstrated a modest 20-30% reduction in insulin binding to monocytes and adipocytes from patients with T2DM (1,311- 316). This reduction is due to a decreased number of insulin receptors without change in insulin receptor affinity. In addition to the decreased number of cell-surface receptors, a variety of defects in insulin receptor internalization and processing have been described (314,315).

 

However, some caution should be employed in interpreting these studies. Muscle and liver, not adipocytes, represent the major tissues responsible for the regulation of glucose homeostasis in vivo and insulin binding to solubilized receptors obtained from skeletal muscle biopsies and liver has been shown to be normal in obese and lean diabetic individuals when expressed per milligram of protein (312,313,316-318). Moreover, a decrease in insulin receptor number cannot be demonstrated in over half of subjects with T2DM (319,320), and it has been difficult to demonstrate a correlation between reduced insulin binding and the severity of insulin resistance (321,322). The insulin receptor gene has been sequenced in a large number of patients with T2DM from diverse ethnic populations using denaturing-gradient gel electrophoresis or single- stranded conformational polymorphism analysis, and, with very rare exceptions (323), physiologically significant mutations in the insulin receptor gene have not been observed (324,325). This excludes a structural gene abnormality in the insulin receptor as a cause of common T2DM.

 

Insulin receptor tyrosine kinase activity has been examined in a variety of cell types (skeletal muscle, adipocytes, hepatocytes, and erythrocytes) from normal-weight and obese diabetic subjects. Most (278,301,312,313,320,326-328), but not all (317,329) investigators have found reduced tyrosine kinase activity that cannot be explained by alterations in insulin receptor number or insulin receptor binding. However, near-normalization of the fasting plasma glucose concentration, (by weight loss) has been reported to correct the defect in insulin receptor tyrosine kinase activity (330). This observation suggests that the defect in tyrosine kinase is acquired and results from some combination of hyperglycemia, defective intracellular glucose metabolism, hyperinsulinemia, and insulin resistance - all of which improved after weight loss. A glucose-induced reduction in insulin receptor tyrosine kinase activity has been demonstrated in rat fibroblast culture in vitro (331). Insulin receptor tyrosine kinase activity assays are performed in vitro, and the results of these assays could provide misleading information with regard to insulin receptor function in vivo. To circumvent this problem, investigators have employed the euglycemic hyperinsulinemic clamp in combination with muscle biopsies and anti- phospho-tyrosine immunoblot analysis (301). Such analysis yields a "snap shot" of the insulin- stimulated tyrosine phosphorylation state of the receptor in vivo. The results of these studies have demonstrated a substantial decrease in insulin receptor tyrosine phosphorylation in both obese nondiabetic and subjects with T2DM (301,328). When insulin-stimulated insulin receptor tyrosine phosphorylation was examined in normal-glucose-tolerant or impaired- glucose-tolerant individuals at high risk of developing T2DM, a normal increase in tyrosine phosphorylation of the insulin receptor has been observed (332). These observations are consistent with the concept that impaired insulin receptor tyrosine kinase activity in patients with T2DM is acquired secondarily to hyperglycemia or some other metabolic disturbance.

 

A physiologic increase in the plasma insulin concentration stimulates tyrosine phosphorylation of the insulin receptor and IRS-1 in lean healthy subjects to 150-200% of basal values (280,301,328,332,333). In obese subjects without T2DM, the ability of insulin to activate these two early insulin receptor signaling events in muscle is reduced, while in subjects with T2DM insulin has no significant stimulatory effect on either insulin receptor or IRS-1 tyrosine phosphorylation (301). The association of p85 protein and PI3-kinase activity with IRS-1 also is greatly reduced in obese non-diabetic and subjects with T2DM compared to lean healthy subjects (301,328- 334). Insulin also failed to increase the association of the p85 subunit of PI3-kinase with IRS-2 in muscle, indicating that T2DM is characterized by a combined defect in IRS-1 and IRS-2 function (301,328). The decrease in insulin stimulation of the association of the p85 regulatory subunit of PI3-kinase with IRS-1 is closely correlated with the impairment in muscle glycogen synthase activity and in vivo insulin-stimulated glucose disposal (301). Defective regulation of PI3-kinase gene expression by insulin also has been demonstrated in skeletal muscle and adipose tissue of subjects with T2DM (335). In animal models of diabetes, an 80% decrease in IRS-1 phosphorylation and a greater than 90% reduction in insulin-stimulated PI3-kinase activity have been reported (336).

 

In the insulin resistant, normal glucose tolerant offspring of two parents with T2DM, IRS-1 tyrosine phosphorylation and the association of p85 protein/PI3-kinase activity with IRS-1 are markedly decreased despite normal tyrosine phosphorylation of the insulin receptor; these insulin signaling defects are correlated closely with the severity of insulin resistance, measured with the euglycemic insulin clamp technique (332). In summary, a defect in the association of PI3-kinase with IRS-1 and its subsequent activation appears to be a characteristic abnormality in T2DM, is closely correlated with in vivo muscle insulin resistance, and is unrelated to a disturbance in insulin receptor tyrosine phosphorylation. Several groups (337,338) have reported that a common mutation in the IRS-1 gene (Gly 972 Arg) is associated with T2DM, insulin resistance, and obesity, but the physiologic significance of this mutation remains to be established (339).

 

The profound insulin resistance of the PI3-kinase signaling pathway contrasts markedly with the ability of insulin to stimulate MAP kinase pathway activity in insulin-resistant individuals with T2DM and in individuals with obesity without T2DM (301,328). Hyperinsulinemia increases MEK1 activity and ERK1/2 phosphorylation and activity to the same extent in lean healthy individuals as in patients with insulin resistance and obesity without T2DM and patients with T2DM (301,328). This finding of selective insulin resistance is similar to that recently observed in vasculature of Zucker fatty rats (340). Two possible reasons for this difference are alternate insulin signaling pathways and differential signal amplification. With regard to the former, the MAP kinase pathway can be activated either through Grb2/Sos interaction with IRS-1/IRS-2 or with Shc. Because IRS-1 tyrosine phosphorylation is dramatically reduced in the diabetics, it is possible that insulin activation of the MAP kinase pathway in vivo primarily occurs through Shc activation. There is evidence from in vitro studies to support this concept (341). Like ERK and MEK activity, insulin increased Shc phosphorylation to the same extent in lean and obese nondiabetic and subjects with T2DM (301). These results indicate that, in T2DM, insulin induces sufficient activation of the insulin receptor tyrosine kinase to increase Shc phosphorylation normally. It also is possible that differential signal amplification in the PI3-kinase and MAP kinase pathways can explain their differing susceptibilities to the effects of insulin resistance.

 

Maintenance of insulin stimulation of the MAP kinase pathway in the presence of insulin resistance in the PI3-kinase pathway may be important in the development of insulin resistance. ERKs can phosphorylate IRS-1 on serine residues (342), and serine phosphorylation of IRS-1 and the insulin receptor itself has been implicated in de-sensitization insulin receptor signaling (343). Continued ERK activity, when IRS-1 function already is impaired, could lead to a worsening of insulin resistance. Thus, subjects with T2DM or obesity have inappropriately high MAP kinase activity. One also could postulate that insulin resistance in the metabolic (PI3- kinase) pathway, with its compensatory increase in beta cell function and hyperinsulinemia, leads to excessive stimulation of the MAP kinase pathway in vascular tissue (301,302). This would result in the proliferation of vascular smooth muscle cells, increased collagen formation, and increased production of growth factors and inflammatory cytokines, possibly explaining the accelerated rate of atherosclerosis in individuals with T2DM (340A, 340B).

 

Glucose Transport

 

Activation of the insulin signal transduction system in insulin target tissues leads to the stimulation of glucose transport. The effect of insulin is brought about by the translocation of a large intracellular pool of glucose transporters (associated with low-density microsomes) to the plasma membrane (281,282,344). There are five major, different facilitative glucose transporters with distinctive tissue distributions (281,282,345,346) (Table 1). GLUT4, the transporter regulated by insulin is found in insulin-sensitive tissues (muscle and adipocytes), has a Km of ~5 mmol/l, which is close to that of the plasma glucose concentration, and is associated with HK-II (347- 349).  In adipocytes and muscle, its concentration in the plasma membrane increases markedly after exposure to insulin, and this increase is associated with a reciprocal decline in the intracellular GLUT4 pool.  GLUT1 represents the predominant glucose transporter in the insulin- independent tissues (brain and erythrocytes), but also is found in muscle and adipocytes. It is located primarily in the plasma membrane, where its concentration changes little after the addition of insulin. It has a low Km (~1 mmol/l) and is well suited for its function, which is to mediate basal glucose uptake. It is found in association with HKI (347-349).  GLUT2 predominates in the liver and pancreatic beta-cells, where it is found in association with a specific hexokinase, HKIV (347-350).  In the beta-cell, HKIV is referred to as gluco-kinase (350,351). GLUT2 has a high Km, (~15-20 mmol/l) and, as a consequence, the glucose concentration in cells expressing this transporter rises in direct proportion to the increase in plasma glucose concentration. This characteristic allows these cells to respond as glucose sensors.  In summary, each tissue has a specific glucose transporter and associated hexokinase, which allows it uniquely to carry out its specialized function to maintain whole-body glucose economy.

 

Table 1. Classification of Glucose Transport and HK Activity According to their Tissue Distribution and Functional Regulation

Organ

Glucose transporter

HK computer

Classification

Brain

GLUT1

HK-I

Glucose dependent

Erythrocyte

GLUT1

HK-I

Glucose dependent

Adipocyte

GLUT4

HK-II

Insulin dependent

Muscle

GLUT4

HK-II

Insulin dependent

Liver

GLUT2

HK-IVL

Glucose sensor

GK beta-cell

GLUT2

HK-IVB (glucokinase)

Glucose sensor

Gut

GLUT3-symporter

-

Sodium dependent

Kidney

GLUT3-symporter

-

Sodium dependent

 

Glucose transport activity in patients with T2DM uniformly has been found to be decreased in adipocytes (281,282,320,351,352) and muscle (281,282,354-356). In adipocytes from humans with T2DM and rodent models of diabetes, there is a severe reduction in GLUT4 mRNA and protein, and the ability of insulin to elicit a normal translocation response and to activate the GLUT4 transporter after its insertion into the cell membrane is impaired (281,282,320,353,357). In contrast, muscle tissue obtained from lean and obese subjects with T2DM exhibits normal or increased levels of GLUT4 mRNA expression and normal levels of GLUT4 protein (358-361). Moreover, acute (2- 4-h) physiological hyperinsulinemia does not increase the number of GLUT4 transporters in muscle in either healthy subjects or subjects with T2DM (358-361). Several studies have demonstrated an increase in muscle GLUT4 mRNA levels in response to insulin in control subjects (333,360), but not in subjects with T2DM (360), suggesting insulin resistance at the level of gene transcription. However, the physiological significance of the blunted increase in muscle GLUT4 mRNA levels in subjects with T2DM is unclear, since both basal and insulin- stimulated GLUT4 protein levels are normal. Large populations of subjects with T2DM have been screened for mutations in the GLUT4 gene (362,363). Such mutations are very uncommon and, when detected, have been of questionable physiologic significance.

 

The results summarized above indicate that the gene (GLUT4) encoding the major insulin- responsive glucose transporter and its transcriptional/translational regulation are not impaired in T2DM. However, in contrast to the normal expression of GLUT4 protein and mRNA in muscle of subjects with T2DM, every study that has examined adipose tissue has reported reduced basal and insulin-stimulated GLUT4 mRNA levels, decreased GLUT4 transporter number in all subcellular fractions, diminished GLUT4 translocation, and impaired intrinsic activity of GLUT4 (281,282,353,361,364). These observations demonstrate that GLUT4 expression in humans is subject to tissue-specific regulation. Although insulin does not increase GLUT4 expression in muscle, it stimulates the translocation of GLUT4 transporters from their intracellular location to the cell membrane (354,365,366). In humans with T2DM, the ability of insulin to stimulate GLUT4 translocation in muscle is impaired (354,367). Using a novel triple- tracer technique, the in vivo dose-response curve for the action of insulin on glucose transport in forearm skeletal muscle has been examined in nondiabetic and subjects with T2DM (368-370). Insulin-stimulated inward muscle glucose transport is severely impaired in subjects with T2DM who are studied under euglycemic conditions. The defect in glucose transport cannot be overcome by repeating the insulin clamp at each subject's normal fasting glucose (hyperglycemia) level. Since the number of GLUT4 transporters in the muscle of subjects with T2DM is normal (358-361), impaired GLUT4 translocation (281,354,367) and decreased intrinsic activity of the glucose transporter (366,371) must be responsible for the defect in muscle glucose transport. Impaired in vivo muscle glucose transport in T2DM also has been demonstrated using MRI (372) and PET (373).

 

Glucose Phosphorylation

 

Glucose phosphorylation and glucose transport are tightly coupled phenomena (374). Isozymes of hexokinase (HKI-HKIV) catalyze the first committed intracellular step of glucose metabolism, the conversion of glucose to glucose-6-phosphate (G-6-P) (347-350,375) (Table 1). HKI, HKII, and HKIII are single-chain peptides that have a number of properties in common, including a very high affinity for glucose and product inhibition by G-6-P. HKIV, also called gluco-kinase, has a lower affinity for glucose and is not inhibited by G-6-P. Gluco-kinase (HKIVB) is believed to be the glucose sensor in the beta-cell, while HKIVL plays an important role in the regulation of hepatic glucose metabolism.

 

In both rat (375-377) and human (333,348,378-380) skeletal muscle, HKII transcription is regulated by insulin. HKI also is present in human skeletal muscle, but it is not regulated by insulin (378). In response to physiological euglycemic hyperinsulinemia, HKII cytosolic activity, protein content, and mRNA levels increase by 50-200% in healthy non-diabetic subjects (378,380) and this is associated with the translocation of hexokinase II from the cytosol to the mitochondria (381). In contrast, insulin has no effect on HK-I activity, protein content, or mRNA levels (378).

 

In forearm muscle, insulin-stimulated glucose transport (measured with the triple tracer technique) has been shown to be markedly impaired in lean subjects with T2DM (370). However, since the rate of intracellular glucose phosphorylation was impaired to an even greater extent, insulin caused an increase in the intracellular free glucose concentration. By performing the insulin clamp at each subject’s normal level of fasting hyperglycemia, normal rates of whole- body glucose disposal and a normal rate of glucose influx into muscle was elicited. However, the rate of intracellular glucose phosphorylation increased only modestly; consequently, there was a dramatic rise in the free glucose concentration within the intracellular space that is accessible to glucose. These observations indicate that in individuals with T2DM, while both glucose transport and glucose phosphorylation are severely resistant to the action of insulin, impaired glucose phosphorylation (HKII) appears to be the rate-limiting step for insulin action. A similar pattern of impaired muscle glucose phosphorylation and transport is present in the insulin-resistant, normal glucose-tolerant offspring of two diabetic parents (382). These results are consistent with dose-response studies using PET to evaluated glucose phosphorylation and transport in skeletal muscle of subjects with T2DM (373). They also are consistent with 31P-NMR studies (383) which demonstrate that, during hyperinsulinemia, muscle G-6-P concentrations decline in subjects with T2DM versus control subjects. However, subsequent studies using 31P-NMR in combination with 1-14C-glucose suggest that the defect in insulin-stimulated muscle glucose transport exceeds the defect in glucose phosphorylation and is responsible for the decline in muscle glucose-6-P concentration (372). Because of methodologic differences, the results of the triple tracer (370) and MRI (372) studies cannot be reconciled at present. Nonetheless, observations from these studies are consistent in demonstrating that the defects in glucose phosphorylation and glucose transport in muscle are established early in the natural history of T2DM and cannot be explained by glucose toxicity (91). Clear evidence that HKII activity is crucial for glucose uptake derives from studies in transgenic mice who overexpress HKII. In this model, HKII over-expression increased both insulin- and exercise-stimulated muscle glucose uptake (384).

 

In healthy nondiabetic subjects, physiologic hyperinsulinemia for as little as 2-4 hours increases muscle HKII activity, gene transcription, and translation (333,378). In lean subjects with T2DM insulin-stimulated HKII activity and mRNA levels are markedly reduced compared to controls (383,385). Decreased basal muscle HKII activity and mRNA levels (385) and impaired insulin-stimulated HKII activity (379,380,386,387) in subjects with T2DM have been reported by other investigators. A decrease in insulin-stimulated muscle HKII activity also has been described in individuals with IGT (388). Because of its central role in insulin-mediated muscle glucose metabolism, several groups have looked for point mutations in the HKII gene in individuals with T2DM (388-390). Although several nucleotide substitutions have been found, none have been located close to the glucose and ATP binding sites and none have been associated with insulin resistance. Thus, an abnormality in the HKII gene is unlikely to explain the inherited insulin resistance in common variety T2DM.

 

Glycogen Synthesis

 

After glucose is phosphorylated by hexo-kinase II, it either can be converted to glycogen or enter the glycolytic pathway. Of the glucose that enters the glycolytic pathway, ~90% is oxidized. At low physiologic plasma insulin concentrations, glycogen synthesis and glucose oxidation are of approximately equal quantitative importance. With increasing plasma insulin concentrations, glycogen synthesis predominates (18,391). If the rate of glucose oxidation (determined by indirect calorimetry) is subtracted from the rate of whole-body insulin-mediated glucose disposal (determined from the insulin clamp), the difference represents non-oxidative glucose disposal (or glucose storage) (17,360), which primarily reflects glycogen synthesis (1,4,162,216,392).  Glucose conversion to lipid accounts for <5% of total body glucose disposal (18,198,199) and, less than 5-10% of the glucose taken up by muscle is released as lactate (5,393,394).  

 

Reduced insulin-stimulated glycogen synthesis is a characteristic finding in all insulin-resistant states, including obesity, diabetes, and the combination of obesity plus diabetes (1,4,18,43,59,159,162,218,219,377,393-395). Impaired glycogen synthesis also represents the major cause of insulin resistance in obese subjects with normal or only slightly impaired glucose tolerance (1,4,162,218,393,395,396).  Thus, the inability of insulin to promote glycogen synthesis is a characteristic and early defect in the development of insulin resistance in both obesity and T2DM. The emergence of overt diabetes with fasting hyperglycemia is associated with a major reduction in insulin-mediated non-oxidative glucose disposal (glycogen synthesis) in all ethnic groups (1,4,18,162,377,396).  Impaired glycogen synthesis also has been demonstrated in the normal-glucose-tolerant offspring of two diabetic parents (43,397), in the first-degree relatives of people with T2DM (41,398,399), and in a normoglycemic twin of a monozygotic twin pair in which the other has T2DM (101).

 

Using NMR imaging spectroscopy, a decrease in insulin-stimulated incorporation of [1H, 13C]- glucose into muscle glycogen of subjects with T2DM has been demonstrated directly (215). In T2DM, there was a marked lag in the onset of insulin-stimulated glycogen synthesis that was similar to the delay in insulin-mediated leg muscle glucose uptake. The rate of glycogen synthesis in subjects with T2DM was decreased by ~50%, paralleling the decrease in total glucose uptake by leg muscle (3).  Also, impaired muscle glycogen synthesis accounted for essentially all of the defect in whole body glucose disposal.

 

In summary, an abundance of convincing evidence demonstrates that impaired glycogen synthesis is the major metabolic defect in normal glucose tolerant subjects with obesity, in individuals with IGT, and in patients with overt diabetes. Moreover, numerous studies have documented that the earliest detectable metabolic abnormality responsible for the insulin resistance in normal glucose tolerant individuals who are destined to develop T2DM is impaired glycogen synthesis (4,41,43,101,382,392,399,400).

 

Glycogen synthase is the key insulin-regulated enzyme which controls the rate of muscle glycogen synthesis (307,308,310,379,401,402). Insulin enhances glycogen synthase activity by stimulating a cascade of phosphorylation/de-phosphorylation reactions (307,308,361-363,403) (see above discussion of insulin receptor signal transduction), which ultimately lead to activate PP1 (also called glycogen synthase phosphatase) (307,308,310,402). The regulatory subunit (G) of PP1 has two serine phosphorylation sites, called site 1 and site 2.  Phosphorylation of site 2 by cAMP-dependent kinase (PKA) inactivates PP1, while phosphorylation of site 1 by insulin activates PP1, leading to the stimulation of glycogen synthase (307,308,402,404). Phosphorylation of site 1 of PP1 by insulin in muscle is catalyzed by insulin-stimulated protein kinase 1 (ISPK-1) (309,405), which is part of a family of serine/threonine protein kinases termed ribosomal S6-kinases.  Because of their central role in muscle glycogen formation, considerable attention has focused on the three enzymes glycogen synthase, PP1, and ISPK-1 in the pathogenesis of insulin resistance in T2DM.

 

Glycogen synthase exists in an active (dephosphorylated) and an inactive (phosphorylated) form (307-310). Under fasting conditions, total glycogen synthase activity in subjects with T2DM is reduced and the ability of insulin to activate glycogen synthase is severely impaired (301,384,406-410). An impaired ability of insulin to activate glycogen synthase also has been demonstrated in the normal glucose tolerant relatives of individuals with T2DM (400).

Insulin-mediated activation of glycogen synthase and insulin-stimulated glycogen synthase gene expression has been shown to be impaired in cultured myocytes and fibroblasts from subjects with T2DM (411,412). Studies in insulin-resistant nondiabetic and diabetic Pima Indians have documented that the ability of insulin to activate muscle PP1 (glycogen synthase phosphatase) is severely reduced (413). PP1 dephosphorylates glycogen synthase, leading to its activation. Therefore, a defect in PP1 appears to play an important role in muscle insulin resistance (309).

 

The effect of insulin on glycogen synthase gene transcription and translation in vivo has been studied extensively. Most studies (378,414,415) have shown that insulin does not increase glycogen synthase mRNA or protein expression in human muscle studied in vivo. However, glycogen synthase mRNA expression is decreased in muscle of patients with T2DM (415,416), explaining in part the decreased glycogen synthase activity in this disease. However, the major abnormality in glycogen synthase regulation in T2DM and other insulin resistant conditions is its lack of de-phosphorylation and activation by insulin as a result of insulin receptor signaling abnormalities (see previous discussion). The glycogen synthase gene (417) has been the subject of intensive investigation. An association between glycogen synthase gene markers and T2DM has been demonstrated in Japanese, French, Finnish, and Pima Indian populations. However, DNA sequencing has revealed either no mutations (418) or rare nucleotide substitutions (419,420) that cannot explain the defect in insulin-stimulated glycogen synthase. Nonetheless, the association between the glycogen synthase gene and T2DM (418) suggests that another gene close to the glycogen synthase gene may be involved in the development of T2DM. The genes encoding the catalytic subunits of PP1 (421) and ISPK-1 (422) have been examined in insulin-resistant Pima Indians and Danes with T2DM. Several silent nucleotide substitutions were found in the PP1 and ISPK-1 genes in the Danish population; the mRNA levels of both genes were normal in skeletal muscle (422). No structural gene abnormalities in the catalytic subunit of PP1 were detected in Pima Indians (422). Thus, neither abnormalities in the PP1 and ISPK-1 genes nor abnormalities in their translation can explain the impaired enzymatic activities of glycogen synthase and PP1 that have been observed in vivo. Similarly, there is no evidence that an alteration in glycogen phosphorylase plays any role in the abnormality in glycogen formation in T2DM (423). In summary, glycogen synthase activity is severely impaired in patients with T2DM and in insulin-resistant normal glucose tolerant individuals who are predisposed to develop T2DM. However, the defect cannot be explained by an abnormality in the genes encoding glycogen synthase or is promoter or by other key genes - PP1 or ISPK-1 - involved in the regulation of glycogen synthase activity.

 

Glycolysis/Glucose Oxidation

 

Glucose oxidation accounts for ~90% of total glycolytic flux, while anaerobic glycolysis accounts for the other 10% (393,394). Two enzymes, phosphofructokinase (PFK) and pyruvate dehydrogenase (PDH) play pivotal roles in the regulation of glycolysis and glucose oxidation, respectively. In individuals with T2DM the glycolytic/glucose oxidative pathway has been shown to be impaired in many individuals with T2DM (393,394). Although one study suggested that the activity of PFK is modestly reduced in muscle biopsies from subjects with T2DM (424), the majority of evidence indicates that the activity of PFK is normal (407,412,417). Insulin has no effect on muscle PFK activity, mRNA levels, or protein content in either nondiabetic or diabetic individuals (417). PDH is a key insulin-regulated enzyme whose activity in muscle is acutely stimulated by a physiological increment in the plasma insulin concentration (415). Three previous studies have examined PDH activity in patients with T2DM. Insulin-stimulated PDH activity is decreased in isolated subcutaneous human adipocytes from patients with T2DM (425) and in skeletal muscle from subjects with T2DM undergoing euglycemic hyperinsulinemic clamps (426). However, when patients with T2DM had muscle biopsies during hyperglycemic hyperinsulinemic clamps, activation of PDH by insulin was normal (409), in concert with normalized rates of muscle glucose uptake. These results suggest that insulin stimulation of PDH activity is influenced by glycolytic flux.

 

Both obesity and T2DM are associated with accelerated FFA turnover and oxidation (1,4,18,162), which would be expected, according to the Randle cycle (232), to inhibit PDH activity and consequently glucose oxidation (see prior discussion). Thus, any observed defect in glucose oxidation or PDH activity could be acquired secondarily to increased FFA oxidation and feedback inhibition of PDH by elevated intracellular levels of acetyl-CoA and reduced availability of NAD. Consistent with this observation, the rates of basal and insulin- stimulated glucose oxidation have been shown to be normal in the normal glucose tolerant offspring of two parents with T2DM (43) and in the first-degree relatives of subjects with T2DM (41,423), while it is decreased in subjects with overt T2DM (1,4,393,394,427). Studies examining PHD activity in muscle tissue from lean diabetic subjects with mild fasting hyperglycemia are needed before the role of this enzyme in the development of insulin resistance in T2DM can be established or excluded.

 

In summary, post-binding defects in insulin action primarily are responsible for the insulin resistance in T2DM. Diminished insulin binding, when present, is small, occurs in individuals with IGT or very mild diabetes, and results secondarily from downregulation of the insulin receptor by chronic sustained hyperinsulinemia. In patients with T2DM and overt fasting hyperglycemia, post-binding defects are responsible for the insulin resistance. A number of post-binding defects have been documented, including diminished insulin receptor tyrosine kinase activity, insulin signal transduction abnormalities, decreased glucose transport, reduced glucose phosphorylation, and impaired glycogen synthase activity. The glycolytic/glucose oxidative pathway appears to be largely intact and, when defects are observed, they appear to be acquired secondarily to enhanced FFA/lipid oxidation. From the quantitative standpoint, impaired glycogen synthesis represents the major pathway responsible for the insulin resistance in T2DM, and family studies suggests that a defect in the glycogen synthetic pathway represents the earliest detectable abnormality in T2DM. Recent studies link the impairment in glycogen synthase activation to a defect in the ability of insulin to phosphorylate IRS-1, causing a reduced association of the p85 subunit of PI 3-kinase with IRS-1 and decreased activation of the enzyme (PI 3-kinase).

 

Mitochondrial Dysfunction

 

In obesity and T2DM, impaired oxidation, reduced mitochondrial contents, lowered rates of oxidative phosphorylation and the production and release of excessive reactive oxygen species (ROS) have been reported. Mitochondrial biogenesis is regulated by various transcription factors such as peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), peroxisome proliferator-activated receptors (PPARs), estrogen-related receptors (ERRs), and nuclear respiratory factors (NRFs).  Mitochondrial fusion is promoted by mitofusin 1 (MFN1), mitofusin 2 (MFN2) and optic atrophy 1 (OPA1), while fission is governed by the recruitment of dynamin-related protein 1 (DRP1) by adaptor proteins, such as mitochondrial fission factor (MFF), mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51), and fission 1 (FIS1).  Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and PARKIN promote DRP1-dependent mitochondrial fission, and the outer mitochondrial adaptor MiD51 is required in DRP1 recruitment and PARKIN-dependent mitophagy.  Several molecular abnormalities affecting these critical aspects of mitochondrial dynamics have been identified in obese individuals and in patients with T2DM (446). 

 

The generation of new mitochondria, mitochondrial biogenesis, is presumed to be defective in patients with T2DM because the expressions of PGC-1α and its targeted genes are reduced.  They are associated with an impaired ability to produce mitochondrial ATP and increased ROS production from the electron transport chain.  There is some preliminary evidence that stimulation of mitochondrial biogenesis by pharmacological activation targeting these molecules is beneficial in the treatment of T2DM and obesity.  The accumulation of damaged or depolarized mitochondria in pancreatic β cells is associated with oxidative stress and favors subsequent development of diabetes.  Mitochondria in pancreatic β cells are continuously recruited in the fusion and fission processes.  In a cultured pancreatic β cell line (INS-1), high levels of glucose- and palmitate-induced mitochondrial fusion arrested and reduced respiratory function.  In INS1 cells, mitochondria with fission demonstrated reduced Δψ and decreased levels of the fusion protein OPA1. The inhibition of fission machinery proteins using DRP1 and FIS1 RNAi resulted in decreased mitochondrial autophagy, the accumulation of oxidized mitochondrial proteins, reduced respiration, and impaired insulin secretion.  All of these suggest that selective fission of damaged mitochondria is followed by their removal by autophagy.  In another study, INS-1 cells were treated with palmitate and high glucose, and the fragmentation of mitochondria with reduced fusion activities was observed. The application of FIS1 RNAi that shifts the dynamic balance to favor fusion is able to prevent mitochondrial fragmentation, maintain mitochondrial dynamics, and prevent apoptosis.  Thus, although not entirely elucidated, abnormal mitochondrial fusion and fission dynamics in the pancreatic β cells may play an important role in beta cell dysfunction and the progression of T2DM.

 

Obesity and T2DM are associated with impaired skeletal muscle oxidation, reduced mitochondrial contents, and lowered rates of TCA cycle enzymes and OXPHOS.  Patients with T2DM and obesity also demonstrated reduced expression of MFN2, which may be related to the reduced function of mitochondria in skeletal muscle.  In 17 subjects with obesity, 12 weeks of exercise improved insulin sensitivity and fat oxidation.  Skeletal muscle biopsy in these patients revealed that decreased phosphorylation and reduction of DRP1 at serine 616 were negatively correlated with increases in fat oxidation and insulin sensitivity (447).  In this same study, there was a trend towards an increase in the expression of both MFN1 and MFN2.  Studies in hepatocytes have recently demonstrated that the role of MAMs in calcium, lipid, and metabolite exchange is altered in obesity and T2DM.  Although the ER and mitochondria play distinct cellular roles in the process of intermediary metabolism, obesity is known to lead to a marked reorganization of MAMs, which results in mitochondrial calcium overload, reduced respiratory function, and augmented oxidative stress (448).  In contrast, disrupting the integrity of MAMs by knocking out cyclophilin D leads to hepatic insulin resistance through the disruption of inter-organelle Ca2 transfer, ER stress, mitochondrial dysfunction, lipid accumulation, the activation of c-Jun N-terminal kinase and PKCε.  In addition to the beta-cell and skeletal muscle defects described earlier, these altered molecular pathways in the liver represent potential targets for new pharmaceutical intervention to be explored in future studies including individuals with obesity and patients with T2DM.

 

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340A. Cersosimo E, XiaoJing X, Musi N. Role of insulin signaling in vascular smooth muscle cell migration, proliferation and inflammation. Am J Physiol: Cell Physiol. C652-C657, Feb 15th, 2012

 

340B. Cersosimo E, XiaoJing X, Upala S, Triplitt C, Musi N.  Acute Insulin Resistance Stimulates and Insulin Sensitization Attenuates Vascular Smooth Muscle Cell Migration and Proliferation.  Physiological Reports Vol. 2, Iss. 8; e12123, August, 2014

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Update On Pancreatic Transplantation In The Management Of Diabetes

ABSTRACT

Pancreas transplantation is the most effective therapeutic option that can restore insulin independence in beta-cell penic recipients with diabetes. Because of life-long immunosuppression and the initial surgical risk, pancreas transplantation is a therapeutic option only in selected patients with diabetes. Based on renal function, candidates for pancreas transplantation can be classified into three categories: uremic patients, post-uremic patients (following a successful kidney transplantation), and non-uremic patients. Uremic patients are best treated by a simultaneous kidney-pancreas transplantation. Post-uremic patients can receive a pancreas after kidney transplantation. Non-uremic patients can receive a pancreas transplant alone, if diabetes is poorly controlled resulting in hypoglycemia unawareness, and in the presence of evolving chronic complications of diabetes. Results of pancreas transplantation have improved over time and are currently non-inferior to those of renal transplantation alone in recipients without diabetes. A functioning pancreatic graft can prolong patient survival, dramatically improves quality of life of recipients, and may ameliorate the course of chronic complications of diabetes. Unfortunately, because of ageing of the donor population and lack of timely referral of potential recipients, the annual volume of pancreas transplants is declining. Considering that the results of pancreas transplantation depend on center volume, and that adequate center volume is required also for training of newer generations of transplant surgeons, centralization of pancreas transplantation activity should be considered. The recent world consensus conference on pancreas transplantation provides an independent appraisal of the impact of pancreas transplantation on modern management of diabetes as well as expert guidelines for the practice of pancreas transplantation.

INTRODUCTION

Transplantation of an immediately vascularized pancreas allograft (PTx) is currently the most effective therapy to consistently restore insulin-independence in beta-cell depleted recipients with diabetes (1-3). Islet cell transplantation may achieve the same result, especially in patients who require fewer insulin units (4-5). As compared with PTx, islet cell transplantation is associated with lower procedure-related morbidity but requires the same immunosuppression, may necessitate multiple donors, and insulin-independence, when achieved, is not often maintained long-term (1-5). However, results reported very recently from centers of excellence show, that in properly selected patients, islet cell transplantation may achieve insulin-independence rates similar to those of PTx (6).

Unfortunately, PTx is not indicated in all insulin-dependent patients with diabetes because of the initial risk associated with surgery (7) and the need for life-long immunosuppression (8). In the appropriate recipient, however, PTx prolongs survival, especially when associated with kidney transplantation (9,10), restores near-normal metabolic control (11-14), improves the course of secondary complications of diabetes (11,12,15-26) and dramatically improves quality of life (27).

PTx includes several approaches. In the most common scenario a pancreas allograft is transplanted simultaneously with a kidney in patients with insulin-dependent diabetes and end stage diabetic nephropathy (simultaneous pancreas-kidney transplantation; SPK). Grafts are typically obtained from a single deceased donor. Alternatively, a cadaver pancreas can be transplanted simultaneously with a living donor kidney (SCPLK) (28), or a segmental pancreas graft and a kidney graft can be donated from the same live donor (SLPK) (29). The pancreas can also be transplanted alone (PTA), in pre-uremic recipients, or after a successful kidney transplant (PAK), in post-uremic recipients. When the pancreas is transplanted without a kidney from the same donor, the graft is considered to be solitary because renal function cannot be used to anticipate rejection in the pancreas (so called “sentinel kidney” function) (30). In rare circumstances the pancreas is transplanted in the setting of multivisceral organ transplantation (31). This type of PTx is not considered in this review, since it is not performed in the typical recipient with diabetes to primarily reverse diabetes, but rather for technical reasons in the context of a multiorgan graft required to address specific, and rare, conditions requiring this extreme type of transplantation.

THE BURDEN OF DIABETES

Thanks to the availability of exogenous insulin therapy, Type 1 diabetes has changed from an immediately fatal disease to a chronic disease. Sub-optimal metabolic control, coupled with genetic predisposition (32-34), can lead to the development of severe secondary complications many years after the diagnosis of diabetes. These complications are associated with significant morbidity and reduce life expectancy of affected individuals. Patients with diabetes who have poor metabolic control despite intensive insulin therapy and/or who develop progressive secondary complications can benefit from PTx as near-physiologic metabolism is re-established. These complications include: retinopathy, nephropathy, neuropathy, and cardiovascular disease. Diabetic nephropathy is the leading indication to PTx, as either SPK or PAK.

Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both (35). Diabetes mellitus can be classified into four types: type 1 (resulting from autoimmune destruction of beta-cells, and accounting for 5-10% of all cases), type 2 (caused by relative insulin deficiency in the setting of insulin resistance, typically associated with obesity, and representing some 90% of the cases), gestational diabetes (first diagnosed during pregnancy), and a heterogeneous group identified as “other specific types” (35).

In nearly all countries diabetes has a high, and continuously growing, prevalence (36,37). In Western countries, these figures are mainly due to changes in life style, including diet high in saturated fats and decreased physical activity, eventually leading to obesity. Regarding type 1 diabetes, which accounts for most potential recipients of PTx, the prevalence of the disease in the United States is estimated to be 1,250,000 persons, with an annual incidence of 35,000 new cases (38).

Diabetes causes significant morbidity and increases mortality in affected individuals (35,39). The risk of heart disease and stroke is increased 3 to 5-fold, and 50-70% of patients with diabetes die of these events. Fifteen years after the onset of diabetes, diabetic retinopathy is present in the majority of patients. Eventually, 20-30% of patients with diabetes will develop severe visual impairment over the years. Reduction in the incidence of diabetic nephropathy among patients with type 1 diabetes, by approximately 10%, was overcompensated by a 20% increase in the incidence of this complication in patients with type 2 diabetes, leading to a net increase of the prevalence of diabetic nephropathy among dialyzed patients and confirming diabetic nephropathy as the leading cause of end-stage renal failure (39). Incidence of end-stage renal disease in patients with diabetes is higher compared to the patients without diabetes, with a relative risk of 6.2 in the white population and 62.0 among Native Americans. Diabetic neuropathy, in its several forms, affects up to 50% of people with diabetes. In combination with reduced blood flow, neuropathy in the feet increases up to 25-fold the chance of foot ulcers and of several fold eventual limb amputation (40).

TREATMENT GOALS IN DIABETES

There is a large amount of evidence recommending that glycated hemoglobin (HbA1c) should be maintained below 7.0% to reduce the incidence of microvascular disease (35,41). However, the effects of intensive diabetes management on the occurrence of macrovascular complications remains somewhat elusive, tending to be more evident in type 1 diabetes (42), as compared with type 2 diabetes (43,44). More stringent metabolic control (e.g., HbA1c 6.0–6.5%), when achieved without significant hypoglycemia or other adverse effects of treatment, can be preferred in patients with short disease duration, long life expectancy, and without significant cerebrovascular disease (41). On the other hand, less tight metabolic control (e.g., HbA1c 7.5–8.0%) can be accepted in patients at risk of severe hypoglycemia and/or with limited life expectancy, advanced vascular complications, or extensive comorbid conditions (41).

INDICATIONS FOR PANCREAS TRANSPLANTATION AND CANDIDATE SELECTION

PTx is performed to restore an endogenous source of servoregulated insulin production in beta-cell penic patients with diabetes. In technically successful PTx, restoration of beta-cell mass is consistently and reproducibly expected to induce insulin-independence, although at the price of significant surgical morbidity and life-long immunosuppression (2,45). In most patients with diabetes there is a clear advantage in receiving a pancreas graft, when also a kidney graft is needed to reverse end-stage renal failure. Moreover, PTx is indicated in selected patients with complicated and/or labile diabetes, when the risk of surgery and immunosuppression is surpassed by the ongoing risk of ineffective insulin therapy (2,45,46).

Based on these principles, the prototype recipient for PTx is a patient with type 1 diabetes without detectable c-peptide, poor metabolic control and/or progressive secondary complications of diabetes. However, selected patients with type 2 diabetes with high insulin needs, low to mild insulin resistance, and non- or mildly obese, may achieve insulin-independence after PTx and enjoy results similar to those of patients with type 1 diabetes (2,45,46).

Since failure of conventional, insulin-based, therapy is required to become eligible for PTx, most recipients have a 20- to 25-year history of diabetes. By this time, most recipients have developed end-stage nephropathy and also require a kidney transplant. Ideally, these patients should receive an SPK transplant because diabetic nephropathy is associated with high mortality rate, and 75% of insulin-dependent patients with diabetes do not survive longer than 5 years with dialysis (47-49). SPK improves patient survival versus either dialysis or deceased donor kidney transplantation (9,10,50).

In fragile recipients deemed not suitable for SPK, renal transplantation from a live donor is an attractive possibility either as definitive treatment or as a bridge to PAK. Actually, live donor renal transplantation may be worthily pursed also in patients otherwise eligible for SPK because of organ shortage (2,45,46). SCPLK provides an additional transplant opportunity, since it still exploits the benefits of live donation for the kidney but does not require the sequential PAK to correct the diabetes. The main disadvantages of SCPLK are the fact that the pancreas is a solitary graft, and that live renal donation cannot be programmed as it has to be performed when the deceased donor pancreas graft becomes available. To do so, three surgical teams have to work simultaneously (one for the deceased donor, one for the live donor, and one for the transplant) making organization and coordination quite complex (28). Considering that correction of uremia is key in these patients (10), but that ideal donors suitable for SPK are becoming extremely rare (51), when a deceased donor is available a kidney alone transplantation (KTA) should be considered as a valid alternative to leaving the patient with end-stage renal disease while waiting for a SPK donor, who may never become actually available. After KTA, PAK could allow correction of diabetes, thus preventing recurrence of diabetic nephropathy in the renal graft in the long-term period. Paradoxically, surgical complications associated with PAK could jeopardize renal function in the short-term period making the indication for PAK a matter of debate especially in terms of baseline renal function. Although there is no agreed cut-off of renal function to safely proceed with PAK, a stable renal function with a creatinine clearance of at least 60 ml/min/1.73 m2, and a negative urine analysis are all considered important criteria (2,46,52).

According to the American Diabetes Association, PTA may be an option in selected patients with diabetes who have recurrent hypoglycemia unawareness, and/or have medical or psychological problems with insulin therapy (52). Normal or near-normal renal function is also required because the anticipated long-term beneficial effects of sustained insulin-independence on diabetic nephropathy may be surpassed by accelerated deterioration of renal function caused mostly by the nephrotoxic effects of immunosuppressants (22,50,53). Additional evidence shows that also patients with progressive complications (i.e., reversible nephropathy, progressive retinopathy, and severe neuropathy) may improve significantly with PTA (13,20). Although the impact of PTA on patient survival is still debated (54,55), in suitable recipients, PTA improves the course of diabetic retinopathy (18), diabetic neuropathy (13), and diabetic nephropathy (22,50,53), and reduces the level of cardiovascular risk (13,15).

Each patient eligible for PTx is, by definition, at high risk for cardiovascular disease, making cardiac and vascular work up key in this transplant population. In recipients of solitary pancreas grafts (PAK and PTA) accurate estimate of renal function is also mandatory, as the risk of renal dysfunction/failure is reduced when the GFR is ≥ 60-70 mL/min (56). The decision to pursue a solitary PTx should hence be well balanced against the inherent risks of PTx. On the contrary, insulin-dependent patients with diabetes have in SPK their ideal treatment modality. The evaluation process in these patients should explore all possible venues to permit transplantation because continued dialysis is associated with short survival. Unfortunately, many patients are already too sick when they are first referred for transplantation and cannot be offered the chance of SPK.

Although type-2 diabetes is often characterized by obesity and peripheral insulin resistance, recent studies have demonstrated that the old paradigm is no longer generally applicable. Several studies showed improved glycemic control after pancreas transplantation in subsets of patients with type 2 diabetes, especially if body mass index is less than 35 kg/m2 (57).

CURRENT PANCREAS TRANSPLANTATION ACTIVITY

According to the International Pancreas Transplant Registry (IPTR) and the US Organ Procurement and Transplantation Network (OPTN) approximately 51,000 PTx have been performed worldwide (> 31,000 from the United States and >20,000 from other countries) (51,56). Considering that reporting to these registries is mandatory only for US Centers, the real number of PTx performed worldwide exceeds reported registry figures.

According to IPTR data, the total number of PTx steadily increased in the United States until 2004 (peaking at a total of 1484) but has since declined substantially with fewer than 1000 procedures performed in 2014 and in 2015. The overall amount of pancreas transplants decreased slightly, from 1027 in 2018 to 1015 in 2019(56). This remains considerably higher than the nadir of 947 reported in 2015, with a slight decrease attributed to declining in PTAs (124 to 99) and PAKs (68 to 44) from 2018 to 2019. In fact, SPKs continued to increase, from 835 to 872, the highest annual number of SPKs performed in the last decade.

The reasons for the decline in PTx activity are not immediately understood. In the history of solid organ transplantation good results, such as those currently achieved by PTx, typically portend higher volumes. Decline in PTx volumes coincided with a reduction in the number of active PTx centers with only 11 Institutions performing ≥ 20 PTxs per year and most centers performing < 5 PTxs annually (51). The outcome of PTx is known to be influenced by center volume (58). Additionally, lower PTx volumes per center are expected to reduce the opportunities for training of younger generations of transplant physicians and surgeons, thus potentially worsening future outcomes of PTx and further reducing the volumes of PTx, in a vicious circle.

The reason for the current decline in PTx activity is multifactorial. Some factors are historical, such as limited referral of potential recipients (51), and incomplete procurement of pancreas grafts from otherwise suitable donors (59). Other factors, however, are newer and less correctable with educational or training programs for healthcare professionals (60). These factors include the progressive ageing of donor population (61), the increasing number of obese donors (62), and the growing proportion of cerebrovascular accidents as a cause of brain death (61). The combination of these epidemiologic factors makes the “ideal” pancreas donor (age ≤ 40 years, low BMI, death due to trauma, short stay in the intensive care unit, and hemodynamic stability without, or with low dose, vasoactive amines) extremely rare (63). These factors, along with the duration of cold graft storage, are summarized in the Pancreas Donor Risk Index (63). This index, conceived to optimize the use of all grafts suitable for PTx, has instead promoted additional donor selection and further reduced the number of PTx (64). Although it is known that PTx can be pursued using marginal donors with good results (65,66), most centers are not willing to accept this type of donor, as their use may be associated with higher rates of early graft failure.

IMPACT OF COVID-19 PANDEMIC ON PANCREAS TRANSPLANTATION

The global coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 virus reduced the worldwide transplant activity due to the overload of the health system and concern for patient safety. Since the first few months of the pandemic, the transplant community worked on characterizing infection, morbidity, and mortality from COVID-19 in the transplanted or waitlisted patient comparing outcomes to the general population. According to a worldwide survey, pancreas transplant activity declined shortly after the beginning of the COVID-19 pandemic because of both a reduction in patient referrals and utilization of deceased donors (67). There are limited clinical data on COVID-19 in PTx recipients, including a few case reports (68,69) and small series (70-73). As detailed in a recent review, COVID-19 in PTX recipients was mostly managed by reduction of immunosuppression with withdrawal of antimetabolites. Despite lower immunosuppression, the risk of rejection and graft loss does not appear to be clearly increased (74).

PANCREAS TRANSPLANTATION FROM DONORS AFTER CARDIAC DEATH

Shortage of suitable brain-dead donors (DBD), has forced the transplant community to explore the venue of donation after cardiac death (DCD). Based on Maastricht criteria (64) there are four categories of DCD donors. PTx is pursued in type 3 DCD donors, also known as controlled DCD donors. In this category of donors, cardiac arrest is awaited following withdrawal of ventilatory support in patients with fatal brain injuries who are not expected to progress to brain death (64). The use of this type of donors is associated with high organizational needs and may be influenced by national attitudes and regulations (65), but the results of PTx are quite encouraging making this source of grafts worth of further exploration (75-78).

In a recent systematic review and meta-analysis, Shahrestani and Co-workers identified 18 studies on PTx from DCD donors. No difference was noted in allograft survival (hazard ratio, 0.98; 95% confidence interval [95% CI], 0.74-1.31; p= 0.92), and recipient survival up to 10 years after PTx between DBD and DCD donors (hazard ratio, 1.31; 95% CI, 0.62-2.78; p= 0.47). The odds ratio for vascular thrombosis was 1.67 times higher in PTx from DCD organs (95% CI, 1.04-2.67; p= 0.006), but this difference was not evident in PTx from a subgroup of DCD who were treated with heparin (78).

GRAFT PROCUREMENT, PRESERVATION, AND TRANSPLANTATION TECHNIQUES

The history of pancreas transplantation has been shaped by developments in surgical techniques (7) and advancements in immunosuppressive regimens (79). It is now accepted that pancreas grafts are composed by the entire gland with an attached duodenal segment and that the organs are procured with minimal dissection in the donor during the heart beating period. A single arterial conduit is prepared at the back-table, usually by anastomosing the peripheral branches of a Y-shaped donor iliac graft to the cut ends of the superior mesenteric and splenic arteries (80). In rare circumstances, a segmental pancreas graft made of the body and tail of the gland, can be transplanted. This type of graft is used when there are concerns on perfusion of the pancreatic head/duodenum to allow PTx in otherwise “difficult to transplant” recipients, such as patients with high immunization titers. A segmental pancreas graft is also used from live donors (29). Pancreas grafts are highly sensitive to ischemia-reperfusion injury (63). Despite the incidence of surgical complications not significantly increasing until 20 hours of preservation (81), most centers now prefer to maintain the period of cold storage to ≤ 12 hours (82).

At the moment, the gold standard for pancreas graft preservation is static cold storage using the University of Wisconsin solution (83). When the period of cold storage is not exceedingly long also Celsior (84) and histidine-tryptophan-ketoglutarate (85) can be accepted. The use of histidine-tryptophan-ketoglutarate has been associated with higher rates of graft pancreatitis (86). Reduction of perfusion volumes are thought to prevent these complications. IGL-1 in a newer preservation solution, but data on PTx are yet scarce (87). As with other organs, machine perfusion is being explored also for pancreas allografts. The potential of this innovative preservation strategy in PTx remains to be established (88).

Regarding transplantation techniques, it is quite surprising that none was clearly shown to be superior over the other procedures (89). Despite this, some surgical techniques have become very popular and are currently considered standard procedures for PTx. The main variations in PTx technique regard the site for venous drainage (either systemic or portal) and the site for exocrine drainage (either urinary or enteric). In enterically drained grafts other major variations are the use of a Roux-en-Y isolated loop or the creation of a direct anastomosis between the donor duodenum and the recipient small bowel (90), duodenum (91-94), or stomach (95).

The combination of systemic venous effluent and enteric exocrine drainage is currently prevalent (7) as the alleged metabolic and immunologic advantages of portal venous drainage have not been unambiguously proven (96). Bladder drainage along with the inclusion in the graft of a duodenal segment (97 PTx is not employed very frequently at the present time because of frequent urologic and metabolic complications.

The greatest innovation in surgical technique is the description of laparoscopic, robotic-assisted, PTx. The initial experience by Boggi et al (98,99) was recently duplicated at the University of Illinois at Chicago (100). This makes PTx a minimally invasive procedure and is associated with obvious advantages but has high organizational needs, and requires surgeon and team training in advanced robotic procedures.

IMMUNOSUPPRESSIVE PROTOCOLS

Current state-of-the art immunosuppression in PTx was recently reported in a review article (101) and practice recommendations were provided by the proceedings of the first world consensus conference on pancreas transplantation (WCCPTx) (102-103).

Although the immunologic outcome of PTx has improved over the years, rejection still occurs quite frequently (from 20-30% in SPK to around 40% in PTA) (104). Accordingly, the use of T-cell depleting antibody induction is still preferred in some 90% of recipients, while an anti-interleukin-2 receptor antibody alone is used in the remaining 10%. In last two decades, maintenance immunosuppression regimens have employed tacrolimus and mycophenolate in over 80% of the patients (105-106). The use of cyclosporine and/or mammalian target of rapamycin has been mostly considered in the setting of switching in case of documented side effects related to the standard regimen (107) Steroids may be withdrawn or minimized to avoid their side effects, including the risk of glucose intolerance (108-109). The recent evidence that development of donor specific antibodies occurs in PTx and is associated with worse immunologic outcome, further compounds the field and could require the adoption of newer protocols for the treatment of antibody-mediated rejection such as a combination of anti CD20, intravenous immunoglobulins, and protease inhibitors (110). Early experiences suggest that switch from calcineurin inhibitors to belatacept, a T-cell co-stimulation blocker used to prevent acute rejection in adult renal transplant recipients, may reduce nephrotoxicity without evidence of increased risk of kidney or pancreas rejection (111,112). Belatacept may represent an important strategy for preservation of renal and pancreatic function after SPK transplantation, either as first-line or rescue therapy. A trial in primary SPK transplantation (NCT01790594), using belatacept for induction and for maintenance, in combination with mycophenolate mofetil and low dose calcineurin inhibitors, with early steroid withdrawal, was recently completed.

According to a recent review no major improvement in immunosuppressive regimens used for PTx was achieved during the last 20 years. Most PTx patients receive induction with depleting antibodies and maintenance with a combination of a calcineurin inhibitor (with tacrolimus being more prevalent than cyclosporine) plus mycophenolate and steroid maintenance. Newer drug combinations and well-designed prospective studies are needed to further improve the outcome of PTx (101).

POST-TRANSPLANT COMPLICATIONS

PTx carries the highest risk of post-transplant complications among all solid organ transplants, as a consequence of the medical complexity of recipients with diabetes and the susceptibility of pancreas allografts to develop vascular thrombosis and pancreatitis. Occurrence of post-operative complications reduces the rate of graft survival, with allograft pancreatectomy being required in some 5% of PTx recipients, but does not affect patient survival (113). Life-threatening complications still occur in approximately 3% of recipients, mostly because of development of an arterial pseudoaneurysm or an arteroenteric fistula (114).

In the long–term, malignancies as well as bacterial, viral, and fungal infections remain a significant cause of mortality and morbidity (114). Among a cohort of 360 SPK transplants, overall 5-year patient survival was 84%, but 25 recipients (6.9%) developed malignant tumors. Almost one-fourth of the cancers were skin tumors and 5 patients developed post-transplant lymphoproliferative disorders (PTLD) (106). According to the SRTR/Annual Data Report the cumulative incidence of PTLD at 4 years is 2.3% after PTA, 0.9% after SPK, and 1.1% after PAK. The higher frequency of PTLD in PTA patients is likely related to their increased immunosuppression and higher rates of acute rejection (104,116,117). The incidence of other cancers is 3- to 4-fold higher compared with the background population (115).

PATIENT AND GRAFT SURVIVAL

According to the International Pancreas Transplant Registry, 5- and 10-year graft function rates in 21,383 PTx, performed from 1984 to 2009, are 73 and 56%, respectively, for SPK; 64 and 38%, respectively, for PAK; and 53 and 36%, respectively, for PTA (1).

Cardiovascular and/or cerebrovascular events are the leading cause of recipient death either short- (<3 months post-transplant) and long-term (>1-year post-transplant) (118). In patients with type 1 diabetes, SPK has been shown in several studies to increase the observed versus expected lifespan, as compared with a kidney transplant alone (119,120). According to a large study of 13,467 patients, using data from the US Scientific Renal Transplant Registry and the US Renal Data System, the patient survival rate at 10 years post-transplant was significantly higher in recipients of a SPK than of a KTA from a deceased donor. In fact, recipients of a SPK had the greatest longevity (23.4 years), as compared with 20.9 years for recipients of a KTA from a living donor and 12.8 years for recipients of a KTA from a deceased donor (10,121).

In recipients of PAK, evidence shows that the PTx improves long-term patient and kidney graft survival rates. Also, glomerular filtration rates are significantly higher after PAK than after KTA (122). In recipients of PTA who have brittle diabetes mellitus, the mortality rate at 4 years is lower than that in the waiting list candidates (123). Earlier reports stating a survival disadvantage for recipients of solitary pancreas transplants (PTA and PAK) compared with patients on the waiting list for a transplant now seem to be unsubstantiated (54).

Pancreas graft survival rate is based on insulin independence. In the past decade, unadjusted graft survival rates at 1 year were 89% for SPK, 86% for PAK and 82% for PTA. Equivalent figures at 5 years were 71%, 65%, and 58%, respectively (118). More recently, 10-year actual insulin independence rates have been reported to exceed 80% in SPK and 60% in PTA (12,13).

The greatest improvements are seen in the gains over time in the estimated half-life (50% function) of pancreas grafts. The estimated half-life is now 14 years for SPK, and 7 years for both PAK and PTA. Moreover, the estimated half-life has increased to 10 years in recipients of PAK or PTA with a functioning pancreas graft at 1-year post-transplant. The longest pancreas graft survival time, by category, has been 26 years (SPK), 24 years (PAK) and 23 years (PTA) (124).

The leading cause of pancreas loss is rejection (125,126). Autoimmunity is also increasingly recognized as a cause of graft failure (127,128). The diagnosis of pancreatic rejection is based on laboratory markers and imaging techniques, but core biopsy remains the final diagnostic tool. In SPK, a rise in serum creatinine can be a surrogate for pancreas rejection suspicion; however, discordant kidney and pancreas rejection have been described (129). An increase in serum amylase and lipase, although not specific, can be an initial sign of pancreatic immune-activation. Hyperglycemia occurs only in cases of severe beta-cell dysfunction or destruction, and therefore it is a late marker of rejection. Guidelines for the diagnosis of PTx rejection have been recently updated with major implementation for the identification of antibody mediated rejection (130). Pancreatic antibody mediated rejection is a combination of serological and immunohistological findings consisting of donor specific antibody detection, morphological evidence of microvascular injury, and C4d staining in interacinar capillaries. The newest Banff schema recognizes different patterns of immunoactivation, including the recurrence of autoimmune diabetes that is characterized by insulitis and/or selective beta-cell destruction. Among the different causes of graft loss, recent studies have proven that despite immunosuppression, the recurrence of autoimmune disease is not a rare event (129). Historical experience with segmental PTx in identical twins showed that, without immunosuppression, autoimmune destruction of beta cells occurs early after PTx (131). Immunosuppression prevents such recurrence in most, but not in all, patients (127).

Graft failure of any organ has a negative impact on patient survival. In recipients of SPK, kidney graft loss increases the relative risk of death by a factor of 17.6 and pancreas graft loss by a factor of 3.1. In recipients of PAK, kidney graft loss increases the relative risk of death by a factor of 4.3 and pancreas graft loss by a factor of 4.1. In recipients of PTA, pancreas graft loss increases the relative risk of death by a factor of 4.1 (132).

EFFECTS OF PANCREAS TRANSPLANTATION ON ACUTE DIABETES COMPLICATIONS

The excess mortality seen in type 1 diabetes is largely related to diabetes and its comorbidities. Acute complications are represented by hyperglycemic syndromes (most commonly ketoacidosis, less frequently the hyperosmolar syndrome) and hypoglycemia induced by exogenous insulin therapy. They contribute to 80% of all early (<10-year diabetes duration) deaths, and for a 15% of deaths thereafter. Most early acute deaths result from diabetic ketoacidosis (often at diabetes onset or after an acute illness), whereas later acute deaths tend to result from hypoglycemic episodes (133,134). Successful PTx restores a regulated endogenous insulin production and eliminates the need for exogenous insulin administration. As such, no acute diabetic complication is seen in patients with fully functioning pancreatic graft. In addition, PTx improves hypoglycemia counter-regulation, by improving catecholamine and glucagon responses to glucose lowering. These improvements are stable and long-lasting, and have been shown up to 19 years from the grafting (135). Recently, the use of beta cell replacement therapy has been discussed for patient with problematic hypoglycemia, defined as two or more episodes per year of severe hypoglycemia or as one episode associated with impaired awareness of hypoglycemia (136). In such cases, if appropriate educational and technological interventions are not sufficient to improve the condition, PTx is indicated (136). It is therefore reasonable to consider PTx in patients with type 1 diabetes who are at proven risk for serious episodes of insulin-induced hypoglycemia and who demonstrate refractoriness to conventional medical management (135,136).

EFFECTS OF PANCREAS TRANSPLANTATION ON CHRONIC DIABETES COMPLICATIONS

Chronic diabetes complications are a major burden of the disease, dramatically contributing to deterioration of quality of life and reduced survival in the population with type 1 diabetes (137). They can be broadly separated into two categories: microvascular and macrovascular. The first ones are due to damage of small vessels involving eyes, kidneys and nerves, while the others are related to damage in larger blood vessels.

Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common, highly specific microvascular complication of diabetes, with prevalence strongly related to duration of diabetes and the levels of glycemic control. Numerous studies have been performed to elucidate the role of PTx on the clinical course of this complication. Initial work (138,139) found that SPK with subsequent normalization of blood glucose concentrations did not play a role in preventing or reversing retinal damage, but more recent studies support the view that PTx has beneficial effects. In a study conducted on 48 successful SPK, a careful eye examination was performed before and up to 60 months after grafting, with standardized classification of DR (19). The results showed, compared with a group of non-transplanted, matched patients with type 1 diabetes, that SPK recipients had a significantly higher rate of improvement or stabilization of the retinal lesions, depending on the severity of retinopathy at the time of transplantation. A report describing 112 patients with functioning SPK showed an improvement and/or stabilization in 73.5% patients with non-proliferative retinopathy, with an important decrease in the number or ophthalmologic procedures after a period of 4 years (140). Regarding the role of PTA, the course of DR was studied prospectively in PTA recipients and in non-transplanted patients with type 1 diabetes, with a follow-up of almost 3 years (18). The PTA and non-PTA groups consisted respectively of 33 (follow-up: 30 +/- 11 months) and 35 patients (follow-up: 28 +/- 10 months). Best corrected visual acuity, slit lamp examination, intraocular pressure measurement, ophthalmoscopy, retinal photographs, and in selected cases angiography were performed by the authors. At baseline, 9% of PTA and 6% of non-PTA patients had no diabetic retinopathy, 24 and 29% had non-proliferative diabetic retinopathy (NPDR), whereas 67 and 66% had laser-treated and/or proliferative diabetic retinopathy (LT/PDR), respectively. No new case of diabetic retinopathy occurred in either group during follow-up. In the NPDR PTA group, 50% of patients improved by one grading, and 50% showed no change. In the LT/PDR PTA, stabilization was observed in 86% of cases, whereas worsening of retinopathy occurred in 14% of patients. In the NPDR non-PTA group, diabetic retinopathy improved in 20% of patients, remained unchanged in 10%, and worsened in the remaining 70%. In the LT/PDR non-PTA group, retinopathy did not change in 43% and deteriorated in 57% of patients. Overall, the percentage of patients with improved or stabilized diabetic retinopathy was significantly higher in the PTA group (18). Therefore, although cases of early deterioration of diabetic retinopathy have been reported after pancreas transplantation (141), current evidence indicates delay of development and/or increased rate of stabilization of this complication following functioning pancreatic graft (142,143).

Diabetic Kidney Disease

Type 1 diabetes mellitus patients present a high risk of developing renal complications. Diabetic kidney disease, or CKD attributed to diabetes, occurs in 20 – 40% of patients with diabetes and is the leading cause of end-stage renal disease (ESRD) (144). Progression to ESRD in this patient population has important prognostic implications (48,145) and proves to be resistant to most nephroprotective therapeutic measures (146). As discussed above, simultaneous pancreas-kidney transplantation (SPK) in T1D patients is associated with improved patient survival compared to solitary cadaveric renal transplantation (10,121,147,148). Regarding the survival of the grafted kidney, the SPK approach generally guarantees better results compared with the cadaveric donor kidney only transplant. In long-term results (>10 years), the kidney graft survival rate in SPK is equal or better compared to that observed with a living donor solitary renal transplantation (149). Successful long-term normoglycemia as obtained by a functioning pancreas can also prevent recurrence of diabetic glomerulopathy in the kidney graft, as shown histologically by comparing renal biopsies from SPK or PAK versus kidney transplant alone (follow-up 1 to 6 years, approximately). In addition, SPK has been reported to be associated with better creatinine levels and reduced urinary albumin excretion in SPK patients, compared to kidney alone grafted individuals (150). Along similar lines, in patients with type 1 diabetes and long-term normoglycemia after successful SPK transplantation, kidney graft ultrastructure and function were better preserved compared with LDK transplantation alone (151). Altogether, the available information indicates that pancreas transplantation plays a role in protecting the grafted kidney and preventing the recurrence of diabetic nephropathy in renal allografts.

In the case of PTA, the effects on the native kidneys are not fully established yet. Currently available immunosuppressive drugs are nephrotoxic, and this places pancreas transplantation recipients, like other solid organ recipients (152), at risk for post-transplant nephropathy (153,154). Gruessner et al. (155) showed that a serum creatinine level above 1.5 mg/dL, recipient age below 30 years and or tacrolimus levels > 12 mg/dl at 6 months were significantly associated with the development of overt renal failure after PTA. However, in another study (156) no significant deterioration of renal function was observed at 1 year after PTA in patients with glomerular filtration rate (GFR) of about 50 ml/min. Initial work from our group showed no significant change in creatinine concentration and clearance and an improvement in proteinuria at 1 year after PTA (22). More recently, we reported the results achieved in 71 PTA recipients 5 years after transplantation (13,20). In this series proteinuria improved significantly, and only one patient developed ESRD. In the 51 patients with sustained pancreas graft function, kidney function (serum creatinine and glomerular filtration rate) decreased over time with a slower decline in recipients with pretransplant eGFR less than 90 ml/min in comparison to those with pretransplant eGFR greater than 90 ml/min; this finding is possibly due to the correction of hyperfiltration following normalization of glucose metabolism. However, another study (157) reported an accelerated decline in renal function after PTA in the patient population with lower pretransplant GFR. Important information on this issue has been provided by a study conducted with 1135 adult recipient of first PTA (55). The authors have subdivided their series of recipients into three groups, depending on the eGFR (ml/min/1.73 m2): ≥ 90 (n: 528), 60-89 (n: 338) and < 60 (n: 269). The patients were followed up to 10 years and the outcome was ESRD, according to the need for maintenance dialysis or kidney transplantation. The results indicated that at 10 years the cumulative probability of ESRD was 21.8%, 29.9% and 52.2% in recipients with pre-transplant eGFR ≥ 90, 60-89 and < 60 ml/min/1.73 m2, respectively (55). Overall, data available indicates the renal function before PTA as a major factor affecting post-transplantation evolution of the function of the native kidneys. The course of diabetic nephropathy after pancreas transplantation has also been characterized histologically (158-160). Fioretto et al. (161) performed protocol biopsies in patients who had received a successful PTA and found that, whereas 5 years after transplant the histologic lesions of diabetic nephropathy were unaffected, at 10 years reversal of diabetic glomerular and tubular lesions was evident. The histologic reversibility of diabetic nephropathy was previously shown in the case of transplantation of human cadaveric kidneys into recipients without diabetes (162,163) and is supported by the current favorable outcome of deceased diabetic donor kidneys (164). Of interest, a recent study has shown that mortality in PTA recipients who develop ESRD is similar to that found in type 1 diabetic patients on dialysis (165). Therefore, current evidence indicates that normoglycemia ensuing after successful pancreas transplantation prevents and may even reverse diabetic nephropathy lesions in native kidneys and kidney grafts. This has to be balanced with the potential nephrotoxic effects of immunosuppression.

Diabetic Neuropathy

Diabetic neuropathy affects approximately 50% of T1D patients and is associated with reduced survival (166,167). All types of pancreas transplantation may have beneficial effects on diabetic neuropathy (sensory, motor, and autonomic) (168-172). Navarro et al. (171) compared the course of diabetic neuropathy in 115 patients with a functioning pancreas transplantation (31 SPK, 31 PAK, 43 PTA without and 10 PTA with subsequent kidney transplantation) and 92 control patients over 10 years of follow-up. Using clinical examination, nerve conduction studies, and autonomic function tests, the authors found significant improvements in the transplanted groups (similar across the different subgroups) (171). Allen et al. demonstrated a gradual, sustained, and late improvement in nerve action potential amplitudes, consistent with axonal regeneration and partial reversal of diabetic neuropathy, in SPK recipients. Two distinct patterns of neurological recovery were analyzed: conduction velocity improved in a biphasic pattern, with a rapid initial recovery followed by subsequent stabilization. In contrast, the recovery of nerve monophasic amplitude continued to improve for up to 8 years (170). Similarly, we found a significant improvement in Michigan Neuropathy Screening Instrument scores (173), vibration perception thresholds, nerve conduction studies, and autonomic function tests in a series of PTA patients with long-term follow-up (13,20). The beneficial effects of pancreas transplantation on cardiac autonomic neuropathy were also reported by Cashion et al. (174) using 24 h heart rate variability monitoring. However, spectral analysis of heart rate variation was performed by Boucek et al. (175), but without significant findings. Interestingly, Martinenghi et al. (172) monitored nerve conduction velocities in five patients who underwent SPK, reporting a significant improvement which was strictly dependent on pancreas graft function. Nerve regeneration is defective in patients with diabetes (166). In a case report, Beggs et al. (176) performed sequential sural nerve biopsies after PTA and found histologic evidence of nerve regeneration. Quantification of nerve fiber density in skin biopsies (177-179) or in gastric mucosal biopsies obtained during endoscopy (180) is an interesting tool to assess diabetic neuropathy. However, Boucek et al. (181,182) did not find any significant improvement in intraepidermal nerve fiber density after pancreas transplantation. In contrast, Mehra et al. used corneal confocal microscopy, a noninvasive and well validated imaging technique (183,184), and were able to find significant small nerve fiber repair within 6 months after pancreas transplantation. These latter findings have been recently confirmed (26). Lately, it has been observed that successful pancreas transplantation improved cardiovascular autonomic neuropathy (185). However, the impact of pancreas transplantation on late, serious autonomic neurological complications (gastroparesis, bladder dysfunction) is still unsettled.

Cardiovascular Disease

Patients with diabetes present an increased risk for cardiovascular morbidity and mortality, mainly due to diffuse coronary atherosclerosis and diabetic cardiomyopathy (132). After SPK, cardiovascular events remain a primary cause of morbidity and mortality (186), both in the immediate postoperative period (187) and in the long term (188). Preoperative cardiovascular assessment is mandatory to select patients who may maximally benefit from transplantation (189,190), which could also include myocardial perfusion scintigraphy (191).

In SPK recipients, improvement in macrovascular disease (including cerebral vasculopathy and morphology) and cardiac function has been generally observed. A retrospective study of cardiovascular outcomes after SPK and cadaveric kidney-alone transplantation (192) showed cardiovascular death rate (acute myocardial infarction, acute heart failure, lethal arrhythmias, acute pulmonary edema) of 7.6% in SPK, 20.0% in kidney alone and 16.1% in dialyzed patients. In the same study, SPK was associated with improved left ventricular ejection fraction, left ventricular diastolic function, blood pressure, peak filling rate to peak ejection rate ratio and endothelial dependent dilation of the brachial artery (193,194). A study by Biesenbach et al compared SPK and KTA: after 10 years from the procedure, in the SPK group the authors showed a significant lower frequency of vascular complications which included myocardial infarction (16% vs. 50%), stroke (16% vs. 40%) and amputations (16% vs. 30%). In addition, when the cardiovascular outcomes after SPK or living donor kidney-alone transplantation were compared, it was found that SPK was associated with reduced long-term cardiovascular mortality especially in a long term follow up (195). Less information is available regarding the effects of PTA on the cardiovascular system. In a single center experience with 71 consecutive PTA followed for 5 years, clinical cardiac evaluation and doppler echocardiographic examinations were performed. The authors observed that left ventricular ejection fraction increased significantly, and several parameters of diastolic function improved (13). Most of these findings were confirmed after 8 years from transplant (11). As for the effects of PTx on the peripheral arteries, the available information suggests that this type of transplantation neither aggravates nor improves peripheral vascular disease events or progression (196). However, some authors have reported that SPK is protective against atherosclerotic risk factor and progression, prothrombotic state, endothelial function and carotid intima media thickness independent of significant changes in other risk factor (197).

FIRST WORLD CONSENSUS CONFERENCE ON PANCREAS TRANSPLANTATION

The first WCCPTx was held in Pisa (Italy) October 18-19, 2019. Based on the analysis and discussion of 597 studies, an independent jury provided 49 jury deliberations concerning the impact of pancreas transplantation on the treatment of patients with diabetes, using the Zurich-Danish model, while a group of 51 experts, from 17 countries and 5 continents, provided 110 recommendations for the practice of PTx. Consensus was reached after two online Delphi rounds with a final voting at the consensus conference on Pisa. Each recommendation received a GRADE rating (Grading of Recommendations, Assessment, Development and Evaluations) and was validated using the AGREE II instrument (Appraisal of Guidelines for Research and Evaluation II). Quality of evidence was assessed using the SIGN methodology (Scottish Intercollegiate Guidelines Network).

The WCCPTx conveys several important messages. First, both SPK and PTA can improve long-term patient survival. Second, PAK increases the risk of mortality only in the early period after transplantation, but is associated with improved life expectancy thereafter. Third, all types of PTx dramatically improve of quality of life of recipients. Fourth, depending on severity at baseline, PTX has the potential to improve the course of chronic complications of diabetes. Fifth, SPK transplantation should be performed before initiation of dialysis or shortly thereafter, as time on dialysis has negative prognostic implications for patients with diabetes. As a consequence, kidney grafts should be preferentially allocated to patients listed for an SPK transplant (102-103).

CONCLUSIONS

As shown by the WCCPTx, PTx has a high therapeutic index, when correctly indicated and performed at proficient centers. Therefore, all possible efforts should be made to make this important treatment option available in a timely manner to all suitable recipients.

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Diffuse Hormonal Systems

ABSTRACT

 

Neuroendocrine (NE) cells are rare epithelial cells that, in addition to having an endocrine function, express markers and peptides otherwise associated with neurons and the central nervous system. NE cells can be found as either single cells or small clusters of cells dispersed throughout the parenchymal surface epithelium of different tissues, including the lung, the intestine, and the pancreas. The observation that NE cells, which are dispersed throughout the body in different tissue sites, are often innervated and secrete bioactive compounds that can act both locally and systemically, led to the idea of the diffuse neuroendocrine system, a diffuse hormonal system composed of NE cells. NE cells perform important endocrine functions. Furthermore, NE cells are implicated in several human diseases. In particular, a group of rare tumors that presumably arise from NE cells, neuroendocrine neoplasms (NENs), have sparked a great deal of interest in NE cell biology. NENs can arise in almost all tissues but they show the highest incidence in the lung and the gastroenteropancreatic (GEP) system. In this chapter, we will outline what is currently known about NE cell differentiation and function, focusing specifically on NE cells of the lung, pulmonary neuroendocrine cells (PNECs), and the most prominent NE cells of the GEP system: enteroendocrine cells (EECs) of the small intestine and stomach and pancreatic endocrine cells. We will also discuss the potential role of these specific NE cells in the context of tissue injury. Finally, we will provide a brief overview of NEN biology with regards to NENs arising in the lung and GEP system.  

 

INTRODUCTION

 

Neuroendocrine (NE) cells are epithelial cells that, in addition to having an endocrine function, express markers and peptides otherwise associated with neurons and the central nervous system (1,2). NE cells can be defined by the presence of dense secretory granules and the expression of general NE markers including chromogranin A and synaptophysin. The first identified NE cells were the enterochromaffin (EC) cells of the small intestine, whose distinctive shape and histological properties piqued the interest of scientists during the late nineteenth and early twentieth centuries. In particular, dense secretory granules within NE cells hint to their endocrine function. These secretory granules also make NE cells reactive to chromium and silver, which makes them easy to identify using histological staining methods. Throughout their history, NE cells in the intestine have been referred to in the literature with a variety of names recalling their distinct reactions to histological stains: clear cells (did not pick up conventional stains), chromaffin cells (reacted to chromium salts), argentaffin cells (affinity for silver stains), and Kulchitsky cells (in honor of one of the scientists who studied them) (3,4). 

 

Following the description of NE cells in the intestine, histological studies revealed the presence of NE cells not only throughout the intestinal mucosa, but also in other epithelial tissues (1–8). NE cells can be found as either single cells or small, often innervated clusters of cells dispersed throughout the parenchymal surface epithelium of different tissues, including the small intestine, the lung, and the urogenital tract. As is the case for the cells of the pancreatic islets of Langerhans, the C cells of the thyroid, and adrenal medullary cells, NE cells can also form distinct clusters of cells within endocrine glands.

 

In 1938, drawing on his histological studies of NE cells in the pancreas and intestine, Friederich Feyrter proposed that NE cells comprise a diffuse neuroendocrine system. Nearly 30 years later, Anthony Pearse refined this idea of the diffuse neuroendocrine system by showing that NE cells, much like neurons, are able to metabolize amines and produce polypeptide hormones. Thus, the concept of a diffuse neuroendocrine system that functions as a diffuse hormonal system and is composed of cells dispersed throughout the body that, through the secretion of bioactive compounds, communicate in a coordinated fashion with their surroundings and with the nervous system solidified (8,9). Famously, Pearse also suggested that NE cells are all derived from the neural crest. This hypothesis, however, was later disproved by several elegant lineage tracing experiments. With the exception of the cells of the adrenal medulla, the extra-adrenal paraganglia, and C cells of thyroid, which are indeed derived from the neural crest, different types of NE cells are derived from the epithelial progenitors of their respective tissue sites (3).

 

While the specific function of pancreatic islet cells, the cells of the adrenal medulla, and C cells of the thyroid, for example, have been well-established both in terms of their contribution to specific organ function and the maintenance of homeostasis; the specific functions of other NE cells are less well defined. Furthermore, the list of polypeptide hormones and neuropeptides secreted by NE cells is continuously being updated and further refined. Studies that elucidate the developmental differentiation trajectories of NE cells from different tissues have expanded the list of common NE marker genes so that it no longer includes only hormones and neuropeptides but also lineage specific transcription factors. A summary of common NE markers is provided in Table 1.

 

Table 1. Common NE Markers

NE Marker

Function

Associated NE cell types

ASCL1

Transcription factor

PNECs, some gastric EECs

NEUROD1

Transcription factor

GEP EECs

INSM1

Transcription factor

All NE cells

Chromogranin A (CHGA)*

Secretory protein

All NE cells

Synaptophysin (SYP)*

Synaptic vesicle glycoprotein

All NE cells

NCAM1 (a.k.a. CD56)*

Cell adhesion molecule

All NE cells

UCHL1 (a.k.a. PGP9.5)*

Deubiquitinating enzyme

All NE cells

Neuron Specific Enolase (NSE)*

Metabolic enzyme

All NE cells

* Indicates markers used in clinical diagnosis

 

Much of the interest in NE cell biology has been initiated by observations that have been made regarding their behavior in disease. In particular, a group of rare tumors that presumably arise from NE cells, neuroendocrine neoplasms (NENs), have sparked a great deal of interest in NE cell biology inasmuch as this might relate to the genesis and peculiar clinical behavior of some of these tumors. NENs have been observed in almost all tissues, and consist of well-differentiated neuroendocrine tumors (NETs), tumors that proliferate and progress slowly, and neuroendocrine carcinomas (NECs), poorly differentiated tumors that have a poor prognosis (10). While NENs were initially classified according to the embryological origin of their tissue site of incidence (i.e., foregut, midgut, or hindgut), they are now referred to according to their specific tissue site of origin and, in the case of tumors that elicit hormonal syndromes, according to the primary hormone they secrete.

 

NENs show the highest incidence in the lung and the gastroenteropancreatic (GEP) system (Figure 1). For this reason, as outlined above, while there are many different kinds of NE cells arising in many different tissue sites, for the purposes of this chapter, we will focus on the NE cells of the lung, pulmonary neuroendocrine cells (PNECs), and the most prominent NE cells of the GEP system: enteroendocrine cells (EECs) of the small intestine and stomach, and pancreatic islet cells or pancreatic endocrine cells (pECs). These cells are key components of the body’s diffuse hormonal system (Table 2).

 

Figure 1. Occurrence of the most common types of neuroendocrine neoplasms. The occurrence of the main types of neuroendocrine tumors presented as the percentage of all NENs (315,441). GI-NENs represent the largest subgroup of NENs, followed by lung and pancreatic NENs. Subtypes not listed in this figure include NENs from the thyroid, kidney, adrenal gland, breast, prostate and skin.

 

 

Table 2. Types of NE cells and Their Tissue Site

NE cell type

Tissue site

Predominant hormone

PNEC

lung

numerous (see text)

Alpha cells

Pancreas

Glucagon

Beta cells

Pancreas

Insulin

Gamma/ PP cells

Pancreas

PPY

Delta cells

Pancreas

Somatostatin

Epsilon cells

Pancreas (during development)

Ghrelin

G cells

Stomach, duodenum, pancreas

Gastrin

D cells

Stomach, small intestine

Somatostatin

Enterochromaffin cells (EC) cells

Stomach, small intestine, colon

Serotonin (5-HT)

EC-like (ECL) cells

Stomach

Histamine

X and X/A cells

Stomach (mainly), small intestine

Ghrelin

L-I-N lineage cells

Small intestine (distal), Colon (L cells)

GLP-1, GLP-2, PYY, serotonin (L-cells), CCK, serotonin (I cells), NTS (N cells)

K cells

Small intestine (proximal)

GIP, serotonin

 

PULMONARY NEUROENDOCRINE CELLS (PNECs)

 

Pulmonary neuroendocrine cells (PNECs), the neuroendocrine cells of the lung, are prominent constituents of the diffuse neuroendocrine system. Although PNECs account for only 0.5% of the lung epithelium, their distinct morphological and histological staining properties, which are shared with the majority of NE cells, led to their prominence in early histological studies of the lung (11). First described in 1949 as “helle zellen” (‘bright cells’ in German), it was later appreciated that PNECs contain secretory granules and that they produce and secrete bioactive compounds, including serotonin (12–14). PNECs were thus added to the growing list of NE cells that make up the diffuse neuroendocrine system.

 

PNECs are found both as single cells within the lung parenchyma and as small clusters, called neuroendocrine bodies (NEBs) (Figure 2A) (15). In mammalian lung tissue, NEBs are distinctly located next to airway bifurcation points of the branching airways. Solitary PNECs, on the other hand, show a more divergent pattern of localization between species. Whereas in mice, solitary PNECs are mostly found in the trachea, in human lung tissue, they can be found throughout the airways. A distinct feature of PNECs is their direct innervation (16).

 

Figure 2. Pulmonary Neuroendocrine Cells (PNECs) in the upper airways. (A) Schematic depicting epithelial cell types found in the upper airways. Solitary PNECs and innervated Neuroendocrine Bodies (NEBs) are shown. (B) Diagram of signaling and transcription factor interactions that regulate PNEC differentiation.

Although the precise function of PNECs is not well-defined, their preservation across evolution -- similar cell types are found in fish gills and in all air-breathing vertebrates -- suggest an important physiological function (17–19). Furthermore, the position at airway bifurcation points of PNECs, their contact with the airway lumen, and innervation suggest a role in airway sensing. The bioactive compounds, hormones, and neuropeptides that PNECs secrete are known to affect oxygen sensing, pulmonary blood flow and bronchial tonus, and lung immune responses. The bioactive compounds secreted by PNECs include serotonin, calcitonin, calcitonin gene-related peptide (CGRP), gastrin-releasing peptide (GRP), chromogranin A, gamma aminobutyric acid (GABA), and synaptophysin, clearly suggesting an endocrine function for these cells (20–23). A summary of the hormones and neuropeptides expressed by PNECs and other NE cells discussed in this text is included in Table 3.

 

Table 3. NE Cell Expressed Hormones

Hormone

Associated

NE cell types

Reported function in

described NE cell types

Calcitonin

PNEC

pulmonary blood flow and bronchial tonus

Calcitonin gene-related peptide (CGRP)

PNEC

vasoregulation, bronchoprotection, immune cell recruitment

Gastrin releasing peptide (GRP)

PNEC

Regulates mucus and cytokine production upon inflammation or inflammation-associated processes

Gamma aminobutyric acid (GABA)

PNEC and pECs

Regulates mucus and cytokine production upon inflammation or inflammation-associated processes

Somatostatin (SST)

PNECs and GEP NE cells

 (D cells)

Inhibits secretion of insulin, glucagon, PYY, serotonin, and gastrin

Vasoactive intestinal polypeptide (VIP)

PNECs and GEP EECs

Acts as a neurotransmitter, an immune regulator, a vasodilator, and a secretagogue

Histamine

PNECs and Gastric EECs

(ECL cells)

Stimulates gastric acid secretion

Ghrelin (GHRL)

PNECs, Gastric EECs (X/A cell), and pECs (Epsilon cells)

Stimulates appetite, promotes gluconeogenesis and increased gastric acid secretion

Cholecystokinin (CCK)

PNECs and GI EECs

 (I cells)

Stimulates gallbladder contraction, pancreatic enzyme secretion, gut motility, satiety, and inhibits acid secretion

Serotonin (5-HT)

PNECs and GI EECs

(EC cells)

Regulates vasodilation and smooth muscle contraction

Secretin (SCT)

GI EECs

Stimulates the release of bicarbonate and water to neutralize gastric acid

Gastric inhibitory peptide (GIP)*

GI EECs (K cells)

Inhibits insulin secretion and to a lesser extent gastric acid secretion

Substance P (neuropeptide)

GI EECs (EC cells)

Regulates intestinal motility and mucosal permeability

Glucagon-like peptide 1 (GLP-1)

Intestinal EECs (L cells)

Stimulates insulin secretion and inhibits glucagon secretion

Neurotensin (NTS)

Intestinal EECs (N cells)

Inhibits gastric acid secretion

Glucagon (GCG)

pECs (alpha cells)

Increases blood glucose levels by stimulating glucose production and inhibiting glycogen storage by the liver

Insulin (INS)

pECs (beta cells)

Stimulates uptake of blood glucose by other tissues

Gastrin

pECs (G cells)

Stimulates gastric acid secretion

Pancreatic polypeptide (PPY)

pECs

(PP/gamma cells)

Inhibits glucagon and somatostatin

* Gastric inhibitory peptide (GIP) is also known as glucose-dependent insulinotropic polypeptide

 

PNECs in Development: Specification, Differentiation, and NEB Formation

 

PNECs are the first differentiated cell type to appear in the developing lung (15). The timing of PNEC differentiation in human lungs has not been comprehensively delineated but several studies have reported the appearance of PNECs in human fetal airways at 8 - 9 weeks of gestation (24). In mice, the fetal epithelial progenitor cells that give rise to mature PNECs appear for the first time at around embryonic day (E) 12.5 (25). These cells are defined by expression of the basic helix loop helix (bHLH) lineage transcription factor, ASCL1, which is required for their specification. Mice that carry null alleles of Ascl1 do not have PNECs (26,27).

 

PNEC lineage specification by expression of Ascl1 is followed by two key events that appear to happen in parallel: the formation of NEBs and the maturation of early Ascl1 expressing cells to fully differentiated PNECs. Using a lineage trace of Ascl1-positive cells in mouse embryonic lung, Kuo and Krasnow observed the first signs of PNEC clustering at around E13.5 to E14, followed by the appearance of bonafide NEBs, which contained mature PNECs at around E15.5 to E16 (25). In human fetal lungs PNEC clustering was first observed at about 9 to 10 weeks of gestation (28).

 

Although one might imagine that the formation of NEBs is likely achieved through proliferation of nascent PNECs, a different mechanism has been shown to be at play. Sparse lineage tracing of early fetal Ascl1-positive PNECs in mice using a multi-color lineage reporter showed that NEBs contained either different colored cells or a single labeled cell and multiple unlabeled cells, arguing that the PNECs in NEBs are not clonal (25). Live cell imaging of fetal mouse lung tissue showed that NEBs are formed through the migration and subsequent aggregation of PNECs at airway branchpoints (25,29). This process appears to be regulated by Slit-Roundabout (ROBO) signaling, a pathway more classically associated with axonal guidance (30). PNECs express the ROBO receptor and the lungs of mice where the Slit-ROBO pathway has been disrupted by mutation of either the Slit ligands or the ROBO receptor itself, have fewer NEBs and more solitary PNECs than the lungs of wildtype mice (21).

 

Concomitant with NEB formation, the transcriptional events initiated by expression of Ascl1 culminate in the emergence of fully differentiated, functional PNECs. In particular, expression of Ascl1 in lung progenitors induces expression of the zinc finger transcription factor, INSM1. PNECs in mice carrying mutant alleles of Insm1 fail to express the mature PNEC markers, CGRP and UCHL1 (ubiquitin C-terminal hydrolase L1, a.k.a. PGP9.5). While the INSM1 targets that mediate the PNEC maturation process have not been delineated, INSM1 has been shown to directly repress the bHLH transcription factor and Notch target gene, Hes1 (Figure 2B) (31).

 

HES1 and other Notch signaling components play crucial roles in repressing the differentiation and specification of PNECs and, thereby, in mediating the NE versus non-NE cell fate choice. The lungs of mice in which Hes1 has been conditionally deleted in early lung progenitors have more ASCL1-positive cells and, in particular, fewer solitary PNECs, showing instead more and larger NEBs compared to the lungs of wildtype mice (29). The increased size of NEBs in Hes1-deficient lungs suggests a mechanism of cell fate specification through Notch-mediated lateral inhibition whereby Notch is activated in PNEC neighboring cells through binding of Notch receptors to the Notch ligands expressed on the surface of PNECs themselves. The activated Notch signaling in these PNEC neighboring cells induces expression of Hes1, which in turn represses the PNEC fate.

 

PNECs express the Notch ligands Dll1, Dll4, Jag1, and Jag2 shortly after their specification during lung development and the cells surrounding PNECs in NEBs express Notch receptors (32). Genetic loss of either all three Notch receptors or Dll1 and Dll4 Notch ligands in the developing mouse lung leads to a dramatic increase in the number and size of NEBs, phenocopying conditional deletion of Hes1 (32,33). In cultures of human airway cells derived from induced pluripotent stem cells, inhibition of Notch signaling leads to increased numbers of PNECs (34,35).

 

As we will discuss in other parts of this text, two other bHLH transcription factors, NEUROG3 and NEUROD1 have been shown to play central roles in the differentiation of EECs and pancreatic islet cells. In contrast, as of yet, there is little evidence that these transcription factors are decisive for PNEC specification or differentiation. To date, Neurog3 expression has not been described in PNECs. While Neurod1 expression has been observed in some PNECs in both fetal and adult mouse lung, a limited number of studies have investigated its specific role in the PNEC lineage (27,36). Neurod1-null mouse lungs from mice less than 2 weeks old showed decreased numbers of solitary PNECs and more NEBs compared to lungs from age-matched wild type mice. However, this difference normalized once mutant mice were 6 weeks old (36). As will be discussed later on in this text, NEUROD1 is a marker of a subtype of the high-grade lung NEN, small cell lung cancer (SCLC), suggesting it might also play a role in normal PNEC biology.

 

Reactive PNEC Proliferations: PNECs in the Response to Lung Injury

 

Multiple studies have shown that PNEC numbers and NEB size are altered in several human disease conditions. Increased numbers of PNECs have been observed in the lungs of patients with COPD, asthma, cystic fibrosis, and some forms of pneumonia. Other pathological conditions associated with increased PNECs include sudden infant death syndrome (SIDS), bronchopulmonary dysplasia (BPD), and congenital diaphragmatic hernias (CDH) (15). Studies in animal models and PNEC culture systems provide experimental evidence that PNECs respond to environmental stimuli by both proliferation and/or secretion of bioactive compounds.

 

The early observation that murine PNECs proliferate in response to a common form of experimental lung injury, naphthalene administration, and that this proliferation precedes epithelial lung regeneration, led to the hypothesis that PNECs are multipotent stem cells that aid in lung regeneration (37). Indeed, lineage tracing studies of PNECs following naphthalene induced lung injury showed that a rare subpopulation of PNECs, termed NEstem, can function as stem cells in this context (38). In response to naphthalene, NEstem cells proliferate and sometimes migrate to the site of injury where they dedifferentiate (lose NE identity) and take on other lung cell fates. The process of dedifferentiation and reprogramming was shown to be mediated by Notch signaling, recalling the role of this pathway in PNEC fate specification, and by EZH2 (38,39). Nonetheless, NEstem cells are not solely responsible for regenerating the lung after injury, as they were shown to contribute only to a small portion of the regenerated surface epithelium (38). Furthermore, genetic ablation of PNECs does not abrogate lung regeneration following naphthalene injury (23,39). 

 

In thinking about PNECs as components of a diffuse hormonal system, two questions arise from the studies of PNECs in the context of naphthalene lung injury. The first is, how do PNECs detect injury? Club cells, which express cytochrome P450 2F2 (Cyp2f2), metabolize naphthalene to a toxic metabolite and the accumulation of this toxic metabolite leads to cell death specifically in these cells (40). Given that PNECs proliferate at time points shortly after peak Club cell injury, it is likely that they are responding to the Club cell injury and not to the naphthalene itself. Consistent with this hypothesis, selective ablation of Club cells using genetic ablation techniques also resulted in PNEC proliferation (41). Nonetheless, injury associated signals that are specific to PNECs and their responses have not been identified.

 

The second question that arises is, besides functioning as stem cells, do PNECs have an endocrine or paracrine/autocrine function in the context of lung injury? Although this question has not been explored in the naphthalene injury model, evidence from other model systems and from human diseases suggest that PNECs respond to some forms of lung injury or disease through the secretion of bioactive compounds. Cigarette smoke, a common culprit of lung injury, provides a good example. Bronchoalveolar lavage (BAL) fluid from smokers has increased levels of peptides secreted by PNECs, implicating these cells in the cellular response to cigarette smoke in humans (42). The PNEC-secreted bioactive compounds associated with this response include GABA and GRP, and both of these molecules have been implicated in inflammation and inflammation-associated processes (42,43). It is likely that PNEC proliferation is also involved in the response to cigarette smoke and its primary component, nicotine. Increased PNECs have been observed in rats exposed to cigarette smoke pre- and postnatally and in rhesus monkeys exposed to nicotine prenatally (44,45).

 

Pointing to a critical role for PNECs in oxygen sensing in the lung, hypoxia and hypoxia-mimicking genetic modifications have been shown to result in higher numbers of PNECs in mice, rats, rabbits, and guinea pigs (22,46–49). Shortly after showing that the distinct dense cored vesicles of PNECs carried serotonin, Lauweryns and Cokelaere went on to show that this serotonin was secreted by PNECs upon exposure to hypoxia (14,49). This finding was further refined and shown to be dependent on changes in intracellular Ca2+ concentrations using cultured rabbit and hamster lung slices (50,51). Serotonin release by PNECs is likely a physiologically relevant functional response to hypoxia as serotonin has been shown to induce vasoconstriction of pulmonary arteries (52).

 

Increased expression in PNECs of the neuropeptide, CGRP, has also been linked to hypoxia (46,53). CGRP has been implicated in promoting alveolar regeneration and in mediating immune cell responses in the lung (21,54). Importantly, results from a study by Shivaraju et al. linked the expression of CGRP by PNECs to the hypoxia-induced regenerative response of epithelial cells in the trachea. When the authors ablated PNECs and exposed mice to hypoxia, they observed a defective regenerative response that could be rescued by intranasal administration of CGRP (46).

 

PNECs appear to also respond to hyperoxia-induced lung injury. Patients with BPD, a chronic lung disease associated with oxygen supplementation of premature infants, have increased numbers of GRP-expressing PNECs (55). In a baboon model of BPD, some of the lung defects associated with the disease could be prevented by treatment of the animals with a GRP blocking antibody, demonstrating that GRP is directly linked to the disease phenotype (56). GRP also plays a role in the lung’s response to viral pneumonia and in the fibrotic response to radiation therapy (57,58).

 

There is clear evidence for a close interplay between PNECs and immune cells. In particular, the effector molecules secreted by PNECs can recruit and activate different populations of immune cells. In one of the first studies to show this, researchers developed a mouse model of CDH, a birth defect that results in pulmonary hypoplasia and pulmonary hypertension (21). CDH is associated with both a heightened immune response and increased numbers of PNECs (59). To study CDH, since point mutations in SLIT and ROBO genes are associated with the disease, Branchfield et al. generated mice with lung-specific deletions of the roundabout receptors, Robo1 and Robo2. When Robo1 and Robo2 were deleted in the entire lung epithelium the authors noted elevated immune cell infiltration in the lung, thus mimicking one of the features of CDH. When Robo1 and Robo2 were deleted only in PNECs, the mice displayed the same phenotype, directly linking the defect to PNECs. Interestingly, ROBO1- and ROBO2-deficient PNECs have increased levels of CGRP and knockout of the gene encoding CGRP partly reversed the immune and lung phenotypes of mice deficient for ROBO1 and ROBO2 in the lung epithelium.  

 

A potential immune regulatory role for PNECs is also suggested by the observation that PNEC numbers are elevated in patients with asthma and that more chromogranin A-positive PNECs are seen in guinea pigs after allergen sensitization and challenge (60). Mice deficient of PNECs due to deletion of Ascl1 in the lung epithelium, show a dampened response to allergen challenge -- reduced goblet cell hyperplasia and reduced immune cell infiltration -- and this was tied directly to reduced levels of PNEC-derived GABA and CGRP, respectively (61).

 

Diseases of Primary PNEC Hyperplasia: NEHI and DIPNECH

 

Up to now we have highlighted instances of increased PNEC number or NEB size that appear to be consequent to or at least associated with some forms of acute or underlying lung injury. These examples are instructive in that they point to a role for PNECs and the molecules they secrete in mediating the response to external stimuli and injury in the lung. Save for lung NENs, which will be discussed in further detail later in this text, there are two notable clinical instances of primary -- as opposed to reactive -- PNEC proliferation that have no known etiology and are not associated with common pathogenic triggers: neuroendocrine cell hyperplasia of infancy (NEHI) and diffuse idiopathic neuroendocrine hyperplasia (DIPNECH).

 

NEHI is a rare pediatric lung disease consisting histologically of hyperplastic GRP-positive and serotonin-positive PNECs in the distal lung epithelium of otherwise normal lung tissue. Symptoms are usually first noted between 6 to 8 months of life and include tachypnea, retractions, crackles and hypoxemia (62). In some cases, patients with NEHI show an inconspicuous, patchy pattern of inflammation or fibrosis, generally assumed to be a consequence of the increased PNEC numbers rather than its cause (63). Interestingly, despite increased PNEC numbers in the lungs of patients with NEHI, from a study on a small patient cohort (5 patients), it appeared that PNECs were not actively proliferating in the lungs of these patients as no Ki67 and GRP double positivity was observed (63). Unfortunately, treatment for patients with NEHI are currently limited to supportive oxygen supplementation and, in some cases, additional nutritional support. The majority of NEHI patients show gradual improvement of symptoms and the disease is not associated with mortality. Nonetheless, recent reports show that some patients experience abnormal lung function persisting into adulthood (62,64,65). While the etiology of this disease remains unknown, there are indications of a genetic basis for the disease. One study identified four families with multiple members diagnosed with NEHI and showing an autosomal dominant pattern of inheritance (66). Another study identified a heterozygous mutation in the NKX2.1 gene in members of a family with a history of childhood lung disease consistent with NEHI (67).

 

DIPNECH is a rare syndrome with adult onset consisting histologically of increased PNECs in the small bronchi and bronchioles and confined to the basement membrane, appearing as scattered PNECs, small nodules, or a linear proliferation of PNECs (68). These features are often seen in concomitance with what are referred to as tumorlets, PNEC proliferations that extend beyond the basement membrane but are less than 5 mm in diameter (69). Other histological features include fibrosis, chronic inflammatory cell infiltrate, and constrictive obliterative bronchiolitis. The majority of patients with DIPNECH are women and the disease is not associated with smoking or other lung diseases. Patients diagnosed with DIPNECH often present with symptoms including cough, exertional dyspnea and an obstructive or mixed obstructive/restrictive defect on pulmonary function test. A small number of patients with DIPNECH are diagnosed due to incidental findings (69).

 

DIPNECH was first recognized and formally defined in 1992 by Aguayo et al., who described the symptoms and histological features of 6 DIPNECH patients (42). While DIPNECH is considered a disease of primary rather than reactive PNEC proliferation, cases associated with parathyroid gland hyperplasia, acromegaly and pituitary adenoma, multiple endocrine neoplasia type I syndrome, and pulmonary adenocarcinoma have been reported (70–72). The World Health Organization (WHO) classifies DIPNECH as a preinvasive, possibly preneoplastic condition (73). Most patients with DIPNECH have multiple PNEC nodules, sometimes including both tumorlets and frank low grade lung NET (carcinoid) tumors (70). In contrast to NEHI where Ki67-positive PNECs were not observed, the PNEC proliferations in DIPNECH patients contain some Ki67-positive cells (63,74). While patients with DIPNECH most often follow a clinical course showing stability or slowly progressing functional decline, a small subset of patients have rapidly progressive disease including progression to respiratory failure or metastatic carcinoid tumors (70,75). To date, there is no standard of care for DIPNECH and the most effective treatment strategy for patients with DIPNECH are somatostatin analogues (SSAs), which have shown effectiveness in improving symptoms of cough and dyspnea in some patients (76–78). Considering that it has been well-established that SSAs inhibit the secretion of bioactive compounds from gastrointestinal NETs, the effectiveness of SSAs in treating cough and dyspnea in patients with DIPNECH suggests these symptoms are caused by the secretion of bioactive compounds by DIPNECH PNECs (79).

 

Lung NENs

 

Lung NENs account for 20-25% of all lung cancers and for 25-30% of NENs from all tissue sites (80,81). As is the case for NENs in general, lung NENs comprise both low grade, well-differentiated NETs and high grade, poorly differentiated NECs. Lung NENs can thus be subdivided into the high-grade carcinomas, small cell lung cancer (SCLC) and large cell neuroendocrine carcinomas (LCNEC), and the low-grade tumors, atypical carcinoids (AC), classified as intermediate grade, and typical carcinoids (TC), classified as low grade.   

 

SMALL CELL LUNG CANCER (SCLC)

 

The most common lung NEN, SCLC, accounts for 79% of all lung NENs and 30% of all lung cancers and is also the best studied among lung NENs (82). Consistent with its classification as NEC, SCLC is a highly aggressive tumor with a high rate of metastasis and a 10-year survival rate of only 1-2% (83). Among patients with SCLC, 97% have a history of smoking (18). While rare, patients with SCLC sometimes experience paraneoplastic endocrine syndromes, most commonly a syndrome of inappropriate antidiuretic hormone (SIADH) and ectopic Cushing’s syndrome (84). Studies of SCLC biology have been aided by a collection of tumor-derived cell lines, several patient-derived xenograft (PDX) models, and genetically engineered mouse models (GEMMs) of the disease (85–87). These preclinical model systems have allowed scientists to address questions in two key areas: the cell of origin of SCLC and molecular signatures predictive of therapeutic vulnerabilities.

 

Genetically, SCLC is a relatively homogeneous disease -- RB1 and TP53 are both almost universally lost in patient tumors (88). Conditional simultaneous genetic deletion of Rb1 and p53 in the mouse epithelium results in tumors that recapitulate many of the key features of the human disease at both the histological and molecular levels (87,89–91). Targeting the deletion of Rb1 and p53 to specific epithelial cell types in the lung provided definitive evidence that Cgrp-expressing PNECs are a cell of origin for tumors in this model (23,92). However, PNECs are not the only epithelial lung cell type that can be a cell of origin for mouse SCLC. A separate study showed that an, as of yet, unidentified CGRP-negative cell that is also negative for the canonical markers of two other common lung epithelial cell types gives rise to mouse SCLC lesions that are molecularly distinct from those initiated in CGRP-positive PNECs (93).

 

Genomic analysis of human and mouse SCLC primary tumors and cell lines has revealed commonly mutated genes and pathways, most notably loss of PTEN, NOTCH, and histone modification genes, and amplification of MYC family oncogenes (88,90,91). Several studies focused on metastasis in mouse SCLC have highlighted the role of NFIB in driving progression of some of these tumors, and data from human patients with SCLC support the clinical relevance of these findings (94–96). These studies, in combination with preclinical testing in mouse models and cell lines have suggested some degree of patient stratification. In particular, high expression of MYC is associated with tumor sensitivity to Aurora Kinase inhibitors (97).

 

The standard treatment regimen for patients with SCLC is a combination therapy of a platinum agent combined with etoposide (82). Despite a clinical response to these therapies in the majority of patients, almost all patients will then experience tumor recurrence (83). The analysis of the transcriptomes of both mouse and human SCLC tumors and cell lines has identified 4 molecular subtypes of SCLC, defined by their expression (or lack of expression) of 3 lineage-specific transcription factors: ASCL1 high, NEUROD1 high, POU2F3 high, and a fourth subtype that has low expression of NE transcription factors and has been proposed to be defined by expression of YAP1. A more recent study suggests a classification in which this fourth subtype is defined by expression of immune checkpoint genes and human leukocyte antigens (98,99). It has been hypothesized that the cell of origin for the POU2F3 high subtype of SCLC might be the pulmonary tuft cell, another chemosensory cell type in the lung distinct from PNECs (100,101).

 

Importantly, several studies using preclinical models of SCLC suggest that these molecular classifications can be used to stratify patients according to potential therapeutic vulnerabilities (102). Adding complexity to this schema, single cell RNAseq studies of mouse SCLC and of xenografts derived from circulating tumor cells from SCLC patients suggest that different molecular subtypes might represent different stages of progression where tumors begin in an ASCL1-high state and progress towards a non-NE state and that individual tumors might comprise cells belonging to different subtypes (101,103). Several studies have also shown other forms of intratumor heterogeneity in SCLC that have implications for patient therapy (104–106). Other new therapies suggested for SCLC include tricyclic antidepressants, therapies that target specific metabolic vulnerabilities, and therapies targeting the GNAS/ PKA/PP2A signaling axis (107–109).

 

LARGE CELL NEUROENDOCRINE CARCINOMA (LCNEC)

 

Pulmonary LCNEC is less common than SCLC, accounting for 16% of all lung NENs (82). Like SCLC, pulmonary LCNECs are highly metastatic and are associated with smoking history and with an overall 5-year survival rate ranging from 15% to 25% (110). In contrast to SCLC, however, there are relatively few preclinical models for LCNEC and we know much less about the basic biology of this disease. This might partly explain why guidelines for treating patients with LCNEC are rather rudimentary (82,111). 

 

Pathohistological analysis of tumors from GEMMs of SCLC found that a portion of the mouse tumors in these models had a histological pattern consistent with LCNEC. While these LCNEC tumors only accounted for 10% of the tumors from the GEMM in which only Rb1 and p53 were conditionally deleted in the lung epithelium, they were much more prominent in the GEMM in which Rb1, p53, and Pten were conditionally deleted specifically in CGRP-expressing PNECs (87). A different GEMM, based on loss of Rb1 and expression of mutant p53 alleles, also develops both SCLC and LCNEC mouse tumors (112). These GEMMs have, thus far, not been used to explore the biology specifically of LCNEC and doing so might present some technical challenges. Recently, the first GEMM specifically for LCNEC was reported. In this model, Rb1, p53, Pten, and Rbl1 were simultaneously deleted in the mouse lung epithelium, resulting in a tumor spectrum consisting primarily of LCNEC and low-grade NETs (113).   

 

The majority of insights into LCNEC have been provided by molecular analysis of primary patient tumor samples. The most comprehensive analysis, consisting of whole exome sequencing of 60 LCNEC tumors and RNA-sequencing expression analysis of 69 LCNEC tumors, highlighted the existence of two major molecular subtypes of LCNEC (114). Type I LCNECs had a higher rate of alterations in TP53 and STK11/KEAP1 and an NE expression profile defined by high expression of ASCL1 and DLL3 and low expression of NOTCH. Type II LCNECs had frequent mutations in RB1and TP53, therefore resembling SCLC at the genomic level. The expression profile of type II LCNECs, however, was distinct from SCLC and instead was defined as NE low, with low expression of ASCL1 and DLL3 but high expression of NOTCH.

 

The description of these two molecular subtypes for LCNEC highlights a clinical conundrum relating to the treatment of patients with LCNEC: should they be treated with SCLC chemotherapy regimens or with chemotherapy regimens for non-NE non-small cell lung cancer (NSCLC) (111)? The report of an SCLC-like subtype of LCNEC (type I) and a NSCLC-like subtype of LCNEC (type II), might suggest a way to stratify patients for different chemotherapy regimens. In line with this idea, a retrospective analysis of LCNEC cases found that patients whose tumors either had wildtype RB1 or showed expression of RB1 protein had a better outcome when treated with a NSCLC chemotherapy regimen as opposed to a SCLC chemotherapy regimen (115).   

 

Other therapies beyond traditional chemotherapy regimens are also being explored for patients with LCNEC. One example that relates to patient stratification according to LCNEC subtype, which was defined in part by differential patterns of DLL3 expression, involves therapeutic strategies that use DLL3 to target tumor cells. Given that DLL3 is also expressed by some SCLC tumors, this also represents a potential therapeutic opportunity in SCLC. Although a DLL3-antibody conjugated to the DNA-damaging pyrrolobenzodiazepine dimer toxin did not provide a survival benefit in 2 phase 3 clinical trials, other DLL3 targeting approaches are being developed (116). In addition, several studies have uncovered potentially targetable molecular alterations in some LCNEC tumors, including activating EGFR mutations, FGFR1 amplifications, activating BRAF mutations, ALK rearrangements, and mutations affecting BDNF/TrkB signaling (114,117–119). Given that the majority of these targetable mutations have been identified in LCNEC tumors with wildtype RB1, the question remains as to how best to treat patients with RB1 mutant LCNEC (120).

 

LUNG NETs: TYPICAL AND ATYPICAL CARCINOIDS

 

Lung NETs comprise low grade typical carcinoids (TC) and intermediate grade atypical carcinoids (AC), accounting for 5% and 0.5% of all lung NENs, respectively (82). TC and AC tend to present in younger patients than LCNEC and SCLC, and the majority of patients are women and non-smokers (121). Although the majority of lung NETs are sporadic and non-functional, a small percentage of patients with Lung NETs, 5% of TC and < 2% of AC, present with paraneoplastic syndromes including those associated with adrenocorticotropic hormone (ACTH), growth hormone releasing hormone (GHRH), histamine, and serotonin (122,123). Some of these are more commonly associated with metastatic lesions of TC or AC (123). Approximately 5% of lung NETs are associated with the familial cancer syndrome caused by germline mutations in multiple endocrine neoplasia gene type I (MEN1). Interestingly, 5% to 10% of lung NETs are also associated with tumor multiplicity, a feature which might suggest a connection with either an unappreciated familial predisposition syndrome or with premalignant conditions such as DIPNECH (80,121).

 

The overall 10-year survival rates for stage I TC and AC are comparable ranging from 98% to 91%, respectively. In the case of stage IV tumors, 10-year survival for TC patients is 49% but is only 18% for patients with AC (124). A distinguishing feature of TC and AC is their relatively slow growth. Indeed, the pathological criteria for diagnosing carcinoids are the number of mitoses per mm2 and the presence of absence of necrosis: < 2 mitoses per mm2 and no necrosis for a diagnosis of TC, 2 to 10 mitoses per mm2 and demonstration of necrosis for a diagnosis of AC (82). Morphologically, carcinoids typically contain small cells that show nested, rosette, and trabecular growth patterns with peripheral palisading (125).

 

Complete surgical resection is the most common treatment for patients with TC and AC, and for the majority of these patients’ surgery is associated with a favorable survival prognosis. Unfortunately, however, a fraction of carcinoid tumors metastasizes, and tumor recurrence (even after apparent curative resection) has been reported in 1 to 6% and 14 to 29% of patients with TC and AC, respectively. Due to a highly variable time to relapse for patients with recurrence (0.2 to 12 years), the recommended follow-up period is 15 years (121,126–129). The reported incidence for lymph node metastasis for TC and AC is variable with rates ranging from 12% to 17% for patients with TC and from 35% to 64% for patients with atypical carcinoid (80,130). The incidence of distant metastases for both TC and AC is 3% and 21%, respectively (129,131).

 

The incidence of tumor recurrence and metastasis calls attention to a clinical need for systemic treatment options for patients with TC and AC tumors that are unresectable, as well as for the need for effective adjuvant therapy options that can be offered to patients after surgery. Unfortunately, standard chemotherapy and radiotherapy regimens have proven to be mostly ineffective in this patient population (80). The only treatment option shown to improve progression-free survival in patients with advanced and progressive TC and AC is the mTOR inhibitor everolimus (132).

 

Other therapeutic options for patients with TC and AC that are not currently considered standard of care due to limited clinical trials, include somatostatin (SST) analogues and peptide receptor radionuclide therapy (PRRT), and temozolomide with or without capecitabine. Given that pulmonary carcinoids can express SST receptors, patients with these tumors can be potentially considered for palliative treatment with unlabeled or radiolabeled SST analogues.

 

The lack of clarity regarding standard of care for patients with unresectable, metastatic, or recurrent TC and AC, points to an unmet need for not only new and effective systemic treatment strategies for these patients, but also for clear patient stratification criteria for predicting the probability of a response of a given patient tumor to specific therapeutic options. Furthermore, given the broad range of tumor malignancy for TC and AC, biomarkers that can predict the potential for tumor progression and metastasis or recurrence are also needed. Efforts to address these clinical needs have been hindered by both difficulties in performing molecular characterization of these tumors and by a dearth of preclinical models representative of the disease. Only a handful of cell lines exist for TC and AC and only one GEMM for TC and AC has been reported (133,134).

 

In contrast to SCLC and LCNEC, pulmonary carcinoids have a low tumor mutational burden, have very few recurrent or characteristic mutations, and rarely contain “driver” mutations in known oncogenes. The most commonly mutated gene in pulmonary carcinoids is MEN1, and up to 5 to 13% of patients with germline mutations of this gene are diagnosed with pulmonary carcinoids (135–137). The most commonly mutated class of genes in pulmonary carcinoids are chromatin remodeling genes, a category that includes MEN1, PSIP1, and ARID1A. Though prevalent in SCLC and LCNEC, mutations in RB1 and TP53 are rare in pulmonary carcinoids (135,136). Recurrent copy number alterations have also been identified in pulmonary carcinoids, including in genes that would imply targetable therapeutic vulnerabilities such as, EGFR, MET, PDGFRB, AKT1/PKB, PIK3CA, FRAP1, RICTOR, KRAS, and SRC (136,138).

 

Transcriptional and methylation analysis of primary pulmonary carcinoids has also revealed distinct subclasses of these tumors. Using multi-omics factor analysis (MOFA), Alcala et al. identified 3 molecular clusters, termed A1, A2, and B (139). While most of the tumors in clusters A1 and A2 were TC, tumors in cluster B were primarily classified as ACs. Tumors in cluster B had high expression of ANGPTL3 and ERBB4, were enriched for mutations in MEN1, and were associated with a worse overall survival. Consistent with a worse prognosis for patients with tumors in cluster B, tumors in this cluster also showed universal downregulation of the orthopedia homeobox protein gene, OTP, whose expression has previously been associated with an improved prognosis in patients with pulmonary carcinoids (126). A separate study performed a similar multi-omic analysis of an independent set of pulmonary carcinoids and also identified 3 molecular subtypes that they termed LC1, LC2, and LC3 (140). The concordance between the molecular subtypes identified in these two studies was shown through integration of the two datasets, further validating the use of these molecular classifications for pulmonary carcinoids (141). 

 

The molecular analysis of pulmonary carcinoids has provided evidence that supports the idea that a fraction of lung NENs may actually fall into a category that lies between G2 ACs and G3 NECs in terms of malignancy. While such a category is recognized in GEP-NENs and is termed well-differentiated G3 NET, its existence has only recently been suggested for lung NENs (142). The study by Alcala et al. identified a subgroup of ACs, termed “supra-carcinoids,” that showed the morphologic characteristics of pulmonary carcinoids, but whose transcriptional profile was closer to that of LCNECs (139). In their analysis of the transcriptional profiles of a series of LCNECs and ACs, Simbolo et al. identified 3 molecular clusters, C1, enriched for LCNECs, C3 enriched for ACs, and C2, which was mixed in terms of number of ACs and LCNECs and which showed intermediate molecular features (143). Finally, an earlier study by Rektman et al. had identified 2 examples of what they referred to as “carcinoid-like” LCNEC tumors -- tumors that showed a clear carcinoid-like morphology and a molecular profile consistent with ACs (low tumor mutational burden and mutation in MEN1) but that had been classified as LCNEC due to a high proliferation rate (118). 

 

The supra-carcinoids in the Alcala et al. study showed a higher expression of MKI67 than other carcinoids in the series, supporting an idea that has been purported in the literature concerning a potential role for percent Ki67 positivity in identifying pulmonary carcinoids more likely to be associated with a poor prognosis (144–146). Typically, ACs show a Ki67 positivity rate of less than 20%. However, some tumors diagnosed as AC show rates between 20 and 50% (147,148). Likewise, as indicated by the “carcinoid-like” LCNEC tumors in the Rekhtman et al. study, some tumors that would otherwise be considered ACs, are diagnosed instead as LCNEC due to having a high proliferation rate (118,149). Furthermore, the comparison of proliferation rates between matched primary stage IV pulmonary carcinoids and metastases indicated an increased proliferation rate in 35% of the metastases, suggesting increased proliferation as a feature of progression (148). This idea is further supported by the observation that Ki67 positivity was heterogeneous in the analyzed tumors with some regions of the tumors showing hot-spots of increased proliferation compared to the rest of the tumor. Beyond Ki67 positivity, a list of defining features of supra-carcinoids or borderline pulmonary carcinoids/neuroendocrine carcinomas has yet to be established.

 

ENDOCRINE CELLS IN THE GASTROENTEROPANCREATIC TRACT

 

Together, the organs connected throughout the mouth to the anus are known as the gastrointestinal (GI) tract, and when the pancreas is included, these organs are collectively referred to as the gastroenteropancreatic (GEP) tract. Throughout the GEP tract endocrine cells can be found as either solitary cells, as is the case in the GI tract, or as innervated clusters, as is the case in the pancreas.

 

Throughout the gastrointestinal (GI) tract the solitary endocrine cells, which have a slender, elongated shape, are referred to as enteroendocrine cells (EECs). This classification helps to distinguish them from endocrine cells of other organs e.g., lung and pancreas. Despite representing only 1% of the gut epithelial cells, the large size of the intestinal epithelium makes it the body’s largest endocrine organ (150,151).

 

Compared to the slender EECs of the GI tract, the pancreatic endocrine cells, dispersed as clusters (known as islets of Langerhans) throughout the organ, have a more pyramidal or round-oval shaped appearance. An adult human has millions of islets, which collectively correspond to roughly 2% of the pancreatic epithelium (152,153). These islets are highly vascularized, a feature that enables pancreatic hormones to travel via the bloodstream to reach their target organs. In fact, the pancreatic hormones act both locally and systemically, eliciting responses throughout the body, consequently affecting the overall metabolic state of the organism. The sections below will provide an overview of the endocrine cells of the GEP tract as components of both the diffuse neuroendocrine system and the body’s diffuse hormonal systems. 

 

GASTRIC ENDOCRINE CELLS

 

The first major organ of the GI tract is the stomach, which, in humans, can be divided into 4 functionally distinct compartments. From proximal to distal; the cardia is the connective region between the esophagus and the stomach, the fundus stores undigested food and gases, the corpus is the largest compartment and performs the digestive action of the stomach, and, finally, the pylorus regulates gastric emptying (Figure 3A) (154,155).

Figure 3. Gastric enteroendocrine cells. (A) Schematic showing the anatomical differences between the murine and human stomach (B) Schematic depicting epithelial cell types found in the gastric pylorus and corpus glands. Solitary gastric enteroendocrine cells are shown in orange. (C) Diagram of signaling and transcription factor interactions that regulate gastric enteroendocrine cell differentiation.

Histologically, the stomach comprises tubular-shaped mucosal invaginations containing a pit region of primarily surface mucous cells, and a gland region. The latter is further subdivided into the isthmus, neck, and base (Figure 3B). The primary differentiated cell types of the stomach are: mucus-producing pit cells, chief cells, which secrete digestive enzymes, acid-secreting parietal cells, gastric tuft cells, whose function is ill defined, and gastric EECs. These cells are continuously formed throughout life, albeit at different rates, by progenitor cells located in the isthmus of the gland region (156,157). With the exception of the cardia, which primarily contains pit cells and scattered parietal cells, gastric EECs can be found in all of the compartments of the stomach. In the corpus and fundus EECs are located in the lower third of the glands. EECs in the pylorus are located in the neck region (158).                                                  

 

Gastric EECs are divided into the following 5 main subtypes defined by their predominantly expressed hormone: G cells (gastrin), D cells (somatostatin), enterochromaffin (EC) (serotonin), EC-like (ECL) cells (histamine), and X/A cells (ghrelin) (Figure 3C) (159). While some gastric EEC subtypes overlap with those found in the intestine, comparison of duodenal EECs and gastric EECs by single cell RNA sequencing (scRNA-seq) suggested a distinct gastric EEC expression profile. These differences are most likely reflective of the tissue specific microenvironment of these EECs, the stimuli they are exposed to, and their different functions (160).     

 

As will be outlined in subsequent parts of this text, differentiation of intestinal EECs requires expression of the master bHLH transcription factor, NEUROG3. While NEUROG3 is important for gastric EECs, its role appears to be EEC subtype specific. Most gastric EECs are also dependent on ASCL1, the same transcription factor that initiates PNEC specification. Studies from two different groups, which independently generated Neurog3 null mice showed that while these mice lacked D-cells and G-cells and had decreased numbers of EC cells, ECL and X/A cells were unaffected (161,162). Ascl1 null mice displayed a similar but not identical phenotype, showing lack of D, G and EC cells and severely decreased numbers of X/A cells (163). ECL cells were not examined in Ascl1 null mice. Thus, some gastric EECs, such as D- and G- cells are dependent on both NEUROG3 and ASCL1, while others, such as X/A cells, are dependent on ASCL1 but not NEUROG3. EC cells appear to be entirely dependent on ASCL1, and only partially dependent on NEUROG3 (163). Ascl1 is not expressed in intestinal EECs from the mouse. Nonetheless, scRNA-seq of human intestinal EECs identified expression of ASCL1, suggesting a role for this transcription factor not only in gastric EECs but also in human intestinal EECs (164).

 

The EEC hormone most clearly associated with gastric function is gastrin, secreted by G-cells located in the pyloric compartment of the stomach. These cells are also found in the duodenum, but their function has been best studied in the stomach (160,164). Gastrin secreted into the bloodstream by G-cells binds to its receptor expressed by ECL cells in the corpus thereby stimulating them to secrete histamine, which, in turn, stimulates neighboring parietal cells to secrete gastric acid. Given that parietal cells themselves also express the gastrin receptor, gastrin release by G-cells can also directly stimulate parietal cells to secrete gastric acid (165–167). The other hormones produced by the gastric EECs are also secreted by other gastroenteropancreatic endocrine cells (GEP-ECs) and will be discussed in later sections.

 

INTESTINAL AND COLONIC ENDOCRINE CELLS

 

Architecture and Cell Types of the Intestine

 

The gut can be divided into the small and large intestine (also known as the colon). The two primary functions performed by the small intestinal epithelium are 1) to form a barrier against the continuous chemical and mechanical insults induced by the undigested food, microorganisms, and toxins present in the intestinal lumen, and 2) to absorb nutrients from ingested food (150). This latter process occurs with an exceptionally high efficiency made possible by the large surface area generated by the intestinal epithelium’s folded structure. Protrusions known as villi contain differentiated non-mitotic cells, while invaginations known as crypts contain proliferative, self-renewing stem cells and their epithelial niche cells, Paneth cells. The colon lacks villi and its primary function is water absorption and movement of the stool. The continuous damage experienced by the intestinal epithelium necessitates a high cellular turnover to maintain organ function. The intestinal stem cells, marked by the expression of leucine-rich G-protein-coupled receptor 5 (LGR5), continuously replace lost cells via rapid division which regenerates the epithelium within 4-5 days (150).

 

The differentiated cells that originate from the LGR5+ stem cells can be divided into two main functional categories, absorptive (enterocytes and microfold cells) and secretory (EECs, goblet, Paneth, and tuft cells) (Figure 4A). Intestinal EECs secrete hormones in response to stimuli such as nutrients from digested food and metabolites produced by the gut microbiota (168,169). The stimuli that EECs respond to can be both mechanical and chemical, and the hormones they produce are secreted both locally and into the bloodstream, thereby allowing them to act not just locally but also systemically. Gut hormones regulate important functions such as digestion, nutrient absorption, appetite, and gastric as well as gut motility (170).

 

Figure 4. Intestinal enteroendocrine lineage specification. (A) Schematic of the intestinal epithelium. Solitary enteroendocrine cells (EECs) are depicted in purple. (B) Diagram of signaling and transcription factor interactions that regulate intestinal enteroendocrine cell differentiation. Differentiated EEC subtypes are highlighted with yellow circles.

Factors Regulating Commitment to the Secretory Lineage  

 

The rapid division of LGR5-positive stem cells gives rise to progenitor cells, the majority of which differentiate as they migrate upwards along the crypt-villus axis. The following section describes the differentiation of intestinal EECs and highlights some of the essential regulatory factors and signaling pathways that direct this process.

 

NOTCH SIGNALING  

 

The first step in becoming an EEC is commitment to the secretory lineage, a process that is initiated by the transcription factor, ATOH1 (Protein atonal homolog 1). At the crypt bottom, active Notch signaling prevents the differentiation of stem and progenitor cells. Cell-cell contact between Notch ligand (Dll1 and Dll4) expressing Paneth cells and stem/progenitor cells results in the expression of the Notch target gene, HES1, which in turn represses the secretory cell fate by repressing ATOH1 (171,172). Hence, commitment to the secretory lineage requires inactivation of Notch signaling.

 

Inactivation of Notch signaling is concomitant with loss of contact with Paneth cells. Due to the high ratio of progenitor to Paneth cells in the crypt, not all progenitors can simultaneously be in touch with a Paneth cell and this results in stochastic loss of Paneth cell-stem cell contact. Additionally, as new progenitors are continuously generated by the stem cells in the crypt, older progenitors are pushed upward along the crypt-villus axis, causing them to lose contact with the Notch ligand-presenting Paneth cells. The resultant loss of active Notch in these cells enables expression of ATOH1 and commitment to the secretory lineage (173,174). Mice lacking Atoh1 do not have any secretory cells (173). In contrast, mice with null alleles of Hes1 have excessive numbers of secretory cells.

 

Factors Regulating Commitment to the EEC Lineage         

 

Transient expression of NEUROG3 commits Atoh1-expressing secretory progenitors to the EEC fate. Whereas Neurog3 knockout mice completely lack EECs, overexpression of Neurog3 leads to increased numbers of EECs and decreased numbers of goblet cells (162,175,176). Homozygous NEUROG3 mutations have been identified in children with generalized malabsorption and reduced numbers of intestinal EECs (177). Downstream targets of NEUROG3 include other transcription factors important for EEC differentiation such as NEUROD1, PAX4/6, NKX2.2, INSM1, and PDX1 (162,178–181). NEUROG3 is also implicated in cell cycle control: Neurog3 expression in mouse pancreatic endocrine progenitors leads to upregulation of the cell cycle inhibitor, Cdkn1a, and consequent cell cycle exit (182). Consistent with the idea that cell cycle exit biases secretory progenitors to the EEC lineage, inhibition of either epidermal growth factor receptor (EGFR) or mitogen activated protein kinase (MAPK) signaling induced quiescence of intestinal stem cells in organoid culture. When this quiescence was reversed by reactivation of these pathways, the resulting organoids had an increased proportion of EECs (183).

 

Specification of the Different EEC Lineages  

 

While most progenitors generated in the crypt immediately begin migrating upwards, those primed to become EECs remain in the crypt for anywhere between 48h and 60h (184). During this time, these cells become committed to one of several divergent differentiation trajectories, each of which results in a different EEC subtype. Prior to leaving the crypt, EEC-committed progenitors have already started to express and secrete their lineage-defining hormones. The time required to produce a specific hormone varies between the different EEC lineages and this may explain why some EEC-committed progenitors remain in the crypt longer than others (184). 

 

Altogether, intestinal EECs produce more than 20 different hormones. The earliest classification of EECs was based on immunostainings and consisted of the following 8 EEC lineages as defined by the main hormone they were found to express: enterochromaffin (EC) cells that secrete serotonin (5-hydroxytryptamine, 5-HT), I cells that secrete cholecystokinin (CCK), K cells that secrete gastric inhibitory peptide (GIP), L cells that secrete glucagon-like peptide 1 (GLP-1), X cells that secrete ghrelin (GHRL), S cells that secrete secretin (SCT), D cells that secrete somatostatin (SST), and N cells that secrete neurotensin (NTS) (185).

 

At the time that this classification was first proposed, it was believed that EECs belonging to a given subtype predominantly expressed the hormone that defined that EEC subtype. Thus, for example, it was believed that EC cells only predominantly expressed serotonin, and L cells only predominantly expressed GLP-1. However, new techniques for performing hormone co-stainings using multiple antibodies, fluorescent hormone reporter mice, and transcriptome-based sequencing of EECs have all led to the observation that some EECs express multiple subtype-defining hormones (186–190). The question thus arose, did these multihormonal EECs represent previously unidentified and distinct EEC subtypes? Or were they simply cells caught in a transition state along the EEC subtype differentiation trajectory? The latter would suggest that EECs are capable of hormonal plasticity and are therefore capable of transitioning from expression of one lineage defining hormone to another.

 

RESOLVING LINEAGE IDENTITY AND HORMONE SWITCHING  

 

One of the first lines of evidence that EECs might undergo hormone switching was the observation that, while serotonin-producing EC cells were rapidly labeled after a single pulse of radioactive thymidine, secretin-producing S cells were labeled much later and only after multiple injections of the isotope (191,192). Thus, it was concluded that serotonin-expressing cells but not secretin-expressing cells had the ability to self-renew and that secretin cells did not differentiate before reaching the villus. Based on this data, one could postulate that serotonin-expressing cells might become secretin-expressing cells once they reach the villus.

 

A lineage relationship between EECs localized in the crypt and EECs localized in the villus was first suggested in 1990 by Roth and Gordon based on an immunohistochemical study in which it was observed that cells expressing substance P (encoded by the Tac1 gene) but not secretin were found in the crypt, cells expressing both substance P and secretin were found in the bottom of the villus, and cells expressing secretin but not substance P were found exclusively at the top of the villus. The majority of substance P-expressing and secretin-expressing cells co-expressed serotonin. The authors thus concluded that these hormones were sequentially expressed along the crypt villus axis (186). The fact that substance P-expressing cells were labeled by the thymidine analogue, BrdU, faster than secretin-producing cells further supported this idea of sequential expression of substance P and secretin by the same EEC (193). Functional evidence for dynamic hormone-switching in EECs was provided first by cell ablation studies showing that ablation of one EEC subtype led to decreased numbers of other EEC subtypes (194). Subsequently the generation of a novel mouse Neurog3 reporter allele, Neurog3Chrono, enabled more definitive delineation of dynamic hormone-expression patterns of single EECs (184).

 

In the Neurog3Chrono mouse, two fluorescent reporter proteins, an unstable mNeonGreen and highly-stable tdTomato, are expressed concurrently with endogenous Neurog3. As a result, the ratio of red to green fluorescence of a given EEC provides real-time information about the age of that cell relative to when it expressed Neurog3 (184). ScRNAseq of EECs from Neurog3Chrono mice provided definitive evidence that many EECs switch the hormone they produce throughout the course of their lives and furthermore suggested a more simplified EEC subtype classification consisting of five mature EEC lineages. One of these five lineages was the one proposed by Roth and Gordon of substance P expressing cells that give rise to serotonin-expressing cells and then to secretin-expressing cells. This lineage was also confirmed independently by lineage tracing of Tac1-expressing cells in the mouse intestinal epithelium (195). 

 

Two key observations from the Neurog3Chrono study substantiated a simplified EEC lineage classification. First, all EEC lineages except for SST-expressing D-cells began to express secretin upon entering the villus, thus rendering the S-cell lineage obsolete. Second, L-, I- and N- cells were shown to belong to a single lineage. Prior to this, observations from a mouse model of L-cell ablation had led the Schwartz lab to propose that L- and N- cells were part of the same lineage (194). The Neurog3Chrono study showed that, when located at the bottom of the crypt, L-cells secrete GLP-1 but, upon reaching the upper regions of the crypt, begin to express the I-cell defining hormone, CCK, and, finally, upon reaching the villus region, they begin to express the N-cell defining hormone, NTS. Thus, the EEC lineages or subtypes could be reduced to the following 5 main lineages: enterochromaffin (EC) cells that secrete Serotonin, K cells that secrete GIP, X cells that secrete GHRL, D cells that secrete SST, and LIN cells that secrete GLP-1, CCK and NTS (Figure 4B) (184).

 

ENTEROCHROMAFFIN (EC) CELLS

 

EC cells are the most prevalent EEC subtype and they can be found in all regions of the intestine (196,197). They are slender, triangularly-shaped cells that can have protrusions extending towards the luminal surface of the intestine (198,199). EC cells are defined by their expression of both serotonin and tryptophan hydroxylase 1 (TPH1), an enzyme that catalyzes the rate limiting step in the biosynthesis of serotonin (189). Serotonin in EC cells is stored in pleomorphic granules and is released in response to chemical and mechanical stimuli. Although commonly associated with brain development and regulation of mood and stress, 95% of the body’s serotonin is produced by the intestine where it regulates functions such as motility, fluid secretion, and vasodilation (200–202). The response of EC cells to different types of stimuli is mediated, in part, through their expression of various receptors, including the olfactory receptor, OLFR558, which acts as a sensor for microbial metabolites, and the transient receptor potential A1 (TRPA1), a receptor-operated ion channel that detects dietary irritants (203). A subset of EC cells expresses the mechanosensitive channel Piezo2, which converts mechanical forces to secretion of fluids and serotonin (204).

 

Given the important functions exerted by serotonin, it is not surprising that EC cells are implicated in several GI pathologies (202). Consistent with the observation that most SI-NETs express serotonin, intestinal EC cells are thought to be the cell of origin for these tumors. An early hint that SI-NETs might indeed arise from EC cells was provided by a Immunohistochemical study in which serial sections of an entire ileal SI-NET tumor were stained for several EEC markers, including serotonin. The authors observed aggregates of proliferating EC cells within crypts in close proximity to the tumor, which the authors speculated were indicative of where the tumor had originated (205). A later study made the same observation in a larger cohort of SI-NET samples from eight different patients, supporting the idea that aberrant proliferation of EC cells within the crypt is associated with SI-NET formation (206).

More recently, it has been suggested that the cell of origin of SI-NETs is not necessarily a fully differentiated EC cell, but rather an EC cell that expresses not only the EC cell markers, TPH1 and CHGA, but also markers of reserve stem cells (207). As we will discuss later in this text (see, EECs can act as reserve niche and stem cells), lineage tracing of both progenitor cells and of cells expressing mature EC cell markers in the mouse intestinal epithelium has shown that EC cells can adopt a stem cell fate upon injury (208). This observation suggests a plausible scenario whereby, if an EC cell were to acquire a genomic (or other) aberration capable of driving NET genesis, it could be long lived enough to indeed give rise to a tumor. Consistent with this hypothesis, Sei et al. identified human EC cells co-expressing the EC cell marker TPH1, and markers associated with both canonical and reserve stem cells, within crypt EC cell microtumors in tissue sections from patients with familial SI-NETs (209,210).

 

WHAT CAUSES HORMONE SWITCHING?  

 

Growth factor gradients change from high WNT, high EGF, high NOTCH, and low BMP, at the crypt bottom, to increasingly high BMP, low WNT, low EGF, and low NOTCH along the villus. Thus, upon leaving the crypt, EEC progenitors are exposed to increasingly different signaling environments. It was therefore attractive to speculate that these signaling gradients could induce hormone switching in EECs.

 

Adult stem cell (ASC) derived mouse and human intestinal organoids lack mesenchymal cells and are therefore not exposed to growth factor gradients but instead experience a constant environment determined by the media composition (211). Consequently, the ASC-derived intestinal organoid system provides researchers with a controlled, in vitro setting in which the signaling environment that intestinal cells experience can be modulated. Under expansion conditions that mimic the crypt environment and are optimized to promote stem cell maintenance, the EECs in mouse small intestinal organoids display a crypt hormone profile. However, when BMP4 was added to the culture media to mimic the villus environment, the EECs in the organoids expressed Secretin suggesting they had taken on a villus-like profile (195). The molecular mechanisms governing the hormone switching from GLP-1 to CCK to NTS observed in the L-I-N lineage remain to be unraveled but are likely to similarly involve cellular signals that differ along the crypt-villus axis.

 

Non-Neoplastic EEC Hyperplasia  

 

The previous sections described the formation of intestinal EECs. These cells and the hormones they secrete play a central role in regulating processes that are important for maintaining organismal function and energy homeostasis. It is therefore not surprising that EECs are implicated in a number of human disease conditions. Increased plasma level of EEC hormones and, in some cases, direct evidence of increased numbers of specific intestinal EEC subtypes have been reported in inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), lymphocytic colitis, Celiac disease, H. pylori infection, and Giardia infection (212–215). A genome-wide association study (GWAS) identified a strong association between a single nucleotide polymorphism (SNP) in the promoter of the EEC transcription factor, PHOX2B, and Crohn’s Disease (CD), a form of IBD (216). The mechanism leading to intestinal EEC hyperplasia in these conditions is not clear, though there is some evidence that inflammatory cytokines are involved (213). Consistent with this idea, treatment of mice with IFN and TNF led to increased numbers of Chromogranin A-positive colonic EECs (217). Likewise, IL-13 has been linked to the response of EC cells to enteric parasite infection (218).

 

It is plausible that the observed changes in EEC numbers in these conditions are not necessarily or not only a result of the disease pathology, but are also mediators of the pathology itself. There is clear evidence for interplay between EECs, the immune system, sensory neurons, and commensal bacteria (168,219). Together with goblet cells, EECs have been shown to secrete the cytokine IL-17C in patients with IBD (220). EEC hormones have been shown to have immunomodulatory functions (219). Some EECs are directly innervated and one study showed that serotonin-expressing intestinal ECs form synapses with nerve fibers through which they can modulate nerve fiber activity (203). EECs express functional toll-like receptors (TLR) and can thereby interact with commensal bacteria by responding to the metabolites they produce (221). Furthermore, as EEC hormones are known to also act systemically, the consequences of disease associated alterations in their abundance or function are not limited to the GI tract. Most notably, GLP-1 and GIP, which amplify glucose stimulated insulin secretion, are less effective in patients with type 2 diabetes (T2D) and GLP-1 receptor agonists are currently used to treat T2D and obesity (185).

 

The most well-documented examples of non-neoplastic EEC hyperplasia are hyperplasias of gastric and duodenal EECs. These include: ECL cell hyperplasia and G-cell hyperplasia. ECL-cell hyperplasia is associated with chronic excessive gastrin production, hypergastrinemia, resulting most commonly from achlorhydria due to chronic atrophic gastritis (CAG), gastrin-producing tumors in Zollinger-Ellison syndrome (ZES), and long-term proton-pump inhibitor (PPI) treatment (222). Lineage tracing of gastrin/cholecystokinin-2 receptor (CCK2R)-expressing cells in mice showed that CCK2R-expressing ECL cells in the isthmus but not the base of the stomach proliferated in response to PPI-induced hypergastrinemia (223). ECL cell hyperplasia only rarely progresses to neoplastic gastric ECL NETs. G-cell hyperplasia is most commonly observed as a secondary change associated with CAG in patients with pernicious anemia. In rare cases, it has also been observed in patients with peptic ulcer disease in conjunction with decreased numbers of a different gastric EEC, the SST producing D-cell. G-cell hyperplastic lesions do sometimes progress to G-cell NETs, gastrinomas (222).

 

EECs CAN ACT AS A RESERVE NICHE AND STEM CELLS

 

The microenvironment in which LGR5-positive intestinal stem cells reside is known as the stem cell niche and includes Paneth cells. The stem cell niche presents the stem cells with cellular signals that prevent them from differentiating and help preserve their self-renewal capacity. Loss of Paneth cells or severe injury such as irradiation, chemotherapy treatment, or surgery can cause loss of intestinal stem cells. In these situations, several different epithelial cell populations have been shown to act as reserve stem cells (224–228). The first studies indicating that EECs could act as reserve stem cells followed the fate of secretory progenitors (224–228). As this progenitor population also contained progenitor cells fated to become Paneth or goblet cells, the contribution specifically of EECs could not be explicitly determined. A later study that used lineage tracing of a population of EEC committed secretory progenitors expressing markers of differentiated EECs showed that these cells can act as reserve stem cells, capable of both contributing to intestinal regeneration after irradiation and generating organoids in vitro (228).

 

Lineage tracing of mouse intestinal cells expressing either NEUROD1 or Tryptophan hydroxylase (TPH1; the rate limiting enzyme in Serotonin biosynthesis and considered an EC cell marker) showed that these cells, similar to the EEC committed secretory progenitors mentioned above, had the ability to contribute to homeostasis and to regeneration following irradiation induced injury (208). Given that the EEC-committed progenitors in the earlier study expressed Neurod1, and that a subset of these cells also expressed Tph1, it is unclear whether the Tph1 lineage-traced cells with regenerative capacity in this study were indeed mature differentiated EECs or the same multi-potent EEC committed secretory progenitors described before. More recently, it was shown that, upon genetic Paneth cell ablation, mature EECs replaced the ablated Paneth cells, serving as Notch-presenting niche cells for intestinal stem cells (229).

 

PANCREATIC ENDOCRINE CELLS

 

Located in the upper left abdomen behind the stomach, the adult pancreas, although being one anatomical entity, originates from two individual buds that arise from either side of the distal foregut endoderm during early embryonic development. As development progresses, these buds fuse to form the final glandular composite organ consisting of two compartments, one exocrine and one endocrine, which contain functionally and morphologically distinct cell types. Acinar cells secrete digestive enzymes which are transported to the duodenum by mucin-secreting ductal cells, and together these two cell types comprise the exocrine compartment. The endocrine compartment comprises 5 different pancreatic endocrine cell (pEC) types, each of which secretes a specific hormone: alpha (glucagon), beta (insulin), gamma/ PP (pancreatic polypeptide), delta (SST) and epsilon cells (ghrelin). As is the case for EEC-derived hormones, pancreatic hormones act both locally and systemically. Indeed, some pancreatic hormones are transported throughout the body via the bloodstream and instruct other organs to, among other things, release or store glucose from the blood. Hence, the pECs play a key role in nutrient metabolism, digestion, and glucose homeostasis (230).

 

Development and Differentiation of Pancreatic Endocrine Cells

 

In contrast to the GI tract where EECs are generated throughout the life of the organism, the pECs are almost exclusively generated during the fetal development of the organ (231). This process is orchestrated by an interplay of regulatory transcription factors. Whereas some of these transcription factors are expressed constitutively, others play a transient yet essential role in mediating pEC lineage commitment and differentiation (see table 4). Murine models have made invaluable contributions to our understanding of pEC generation. In rodents, pancreatic development is divided into 3 transition phases, each outlined below (Figure 5).

 

Table 4. NE Cell Lineage Transcription Factors

Transcription Factor

Associated NE cell types

Role (specifically in NE cells)

ASCL1

PNECs and gastric EECs

Lineage specification (26,27,163)

NEUROG3

GEP NE cells

Lineage specification (transiently expressed) (161,162,175,176,232,233)

NEUROD1

PNECs*, Intestinal EECs (L-I-N lineage), and pECs (beta cells)

Specification of some NE cell subtypes (not defined in lung, secretin-expressing cells and I-cells in the intestine, beta cells in the pancreas) (27,36,255,407,408)

ATOH1

(a.k.a. MATH1)

Intestinal EEC precursors

Specification of the intestinal secretory cell fate (precedes EEC lineage commitment) (173)

INSM1

PNECs and GEP EECs**

NE cell maturation (31,160,180)

GFI1

PNECs and Intestinal EECs

Maturation of CGRP+ PNECs (409); lineage specification (via inhibition of EEC fate in intestinal secretory progenitors) (410,411)

PDX1

GEP EECs (G cells, some intestinal EECs, and pECs

Patterning and specification of gastric and duodenal EECs (G cells, SST- and GIP-expressing duodenal EECs) (412,413); Maintenance of beta cell maturity (414,415)

ISL1

GEP EECs (some gastric EECs**, non EC intestinal EECs, and pECs)

Specification of non-EC intestinal EECs (416); maturation and survival of pECs in development (417,418); maintenance of adult beta cell function (419)

RFX3

pECs

pEC differentiation during development (420); differentiation and maintenance of mature beta cells in the adult (421)

RFX6

GEP EECs (gastric EECs**, some intestinal EECs, and pECs)

Differentiation of intestinal EECs (K, L, X, I, and EC cells) (184,422); pEC specification and differentiation in development (423,424); Regulation of insulin expression in beta-cells (425)

PAX4

GEP EECs (some gastric EECs, duodenal EECs, some pECs)

Differentiation of EECs in the duodenum, of EC cells and D cells in the stomach (178), and of beta and delta cells in the pancreas (426,427)

PAX6

GEP EECs (some gastric EECs, some duodenal EECs, some pECs)

Differentiation of D and G cells in the stomach, of K cells in the duodenum (178), and of alpha and epsilon cells in the pancreas (428,429)

NKX2.2

Some intestinal EECs and some pECs

Regulation of cell fate within the intestinal EEC population (promotes EC, L-I-N, and K lineages) (179) and within the pEC population (promotes beta cell, alpha cell, and PP cell lineages) (252)

NKX6.1

Some gastric EECs and some pECs

Differentiation of EC cells in the stomach**(430) and of beta cells in the pancreas (253)

NKX6.2

Some pECs

Differentiation of alpha, beta, and PP cells in the pancreas (431,432)

NKX6.3

Some gastric EECs

Differentiation of G cells in the stomach (433)

MAFA

Some pECs

Maintenance of beta cell identity in the adult pancreas (257,434)

MAFB

Some pECs

Differentiation of alpha and beta cells in the developing pancreas (246,247); Maturation of alpha and beta cells in the adult pancreas (247,256)

ARX

GEP NE cells (some gastric EECs, some intestinal EECs, and some pECs)

Differentiation of G cells in the stomach (435), of L-, I-, and K cells in the intestine (435), and of alpha cells in the developing pancreas (244,245); Maintenance of alpha cell identity in the adult pancreas (436)

FOXA2

Some intestinal EECs and some pECs

Differentiation of L cells and D cells in the intestine (437); Maintenance of adult beta cell maturation in the pancreas (438,439); Maturation of alpha cells in the developing pancreas (440)

HHEX

Some intestinal EECs and some pECs

Differentiation of and maintenance of delta cell identity in the pancreas (262)(implicated in delta cells of the intestine (184,195))

* Role in PNECs no well defined

** Role in gastric EECs implicated by expression pattern

 

Figure 5. Rodent pancreas development. Schematic depicting the development of the murine pancreas. Islets of Langerhans containing the hormone-secreting endocrine cells are shown in blue and green.

PRIMARY TRANSITION

 

The first transition phase starts at around embryonic day (E) 9.0-9.5 with the formation of the pancreatic buds and ends at around E12.5 with the start of branching morphogenesis. During this period, the dorsal bud gives rise to the first pancreatic endocrine progenitor cells. As is the case for intestinal EECs, the formation of pECs depends on the expression of the bHLH transcription factor NEUROG3, which is transiently expressed at a relatively low level starting at ~E9.0-E11.0. Mice with targeted disruption of Neurog3 lack pECs and die postnatally of diabetes (232). Consistent with this data in mice, human induced pluripotent stem cells (iPSCs) with null alleles of NEUROG3 fail to differentiate into pECs (233). Following this first pulse of Neurog3 expression, a limited number of pancreatic progenitors becomes committed to the pEC lineage, and some of these cells begin to express glucagon (234).

 

SECONDARY TRANSITION

 

Branching morphogenesis continues throughout the first half of the second transition, (~E12.5-E15.5), during which the plexus expands and segregates into tip and trunk domains. From E16.5-E18.5 the epithelial plexus is remodeled into a ductal network with a tree-like structure. Inter- and intralobular ducts branch off from the main pancreatic duct, which connects the pancreas to the common bile duct. During this period, multipotent pancreatic progenitor cells, which make up the majority of cells at the beginning of this transition phase, become either unipotent, fated to become acinar cells at the tip, or bipotent, with the potential to form endocrine and ductal cells. These lineage commitments coincide with a second, more pronounced wave of Neurog3 expression that peaks at around E14.5-E15.5, and then rapidly decreases at around E17.5(235). This second wave of Neurog3 expression commits progenitors to the pEC fate and primes them towards one of the five pEC types.

 

TERTIARY TRANSITION

 

During the tertiary transition, individual progenitor cells that have now become fated to the pEC fate by the second, higher pulse of Neurog3 expression delaminate from the ductal network through what is thought to be asymmetric cell division and/or epithelial to mesenchymal transition (EMT). These pECs then migrate throughout the mesenchyme until they encounter other pEC-fated cells, aggregate with the help of Neural cell adhesion molecule (NCAM), and form oval shaped proto-islets (236,237). This process is reminiscent of the way, during lung development, PNECs form NEBs. The formation of fully mature islets requires their vascularization by endothelial cells and their innervation by neurons, both processes that take place from E16.5 onwards, and during the first weeks postnatally.

 

POSTNATAL

 

At birth the majority (more than 80%) of the pECs originates from endocrine progenitors and the remainder results from proliferation of preexisting pECs within the islets.

 

DIFFERENCES IN PANCREATIC ENDOCRINE CELL EVELOPMENT BETWEEN RODENTS AND HUMANS

 

Although studies of human pEC development are limited, they have uncovered a few main differences compared to rodents. First, there seems to be only one pulse of NEUROG3 during the formation of human pECs (compared to two in the mouse), which occurs at around 8 weeks of gestation. Second, the premature proto-islet structures described above are formed earlier in human development (at 12 weeks post conception) (238,239). Finally, the mature islet architecture differs between rodents and humans (Figure 6). Rodent islets have a central core of beta cells, which are also the most common of all the islet cells (60–80%). The remainder of the endocrine cells, alpha cells (15–20% of the cells of the islet), delta cells (<10% of islet cells), and gamma/ PP cells (<1% of cells) surround the beta cells in a circular structure known as the mantle (240). Human islets do not display the same organized structure as rodent islets. Instead, there is a salt and pepper pattern where the different endocrine cells are randomly scattered within the islets. The proportion of the different pECs also differs as human islets have proportionally less beta but more alpha cells compared to rodent islets, consisting of circa 30% alpha cells, 60% beta cells, about 10% delta cells, and rare <1% gamma/PP cells and epsilon cells (241,242).

 

Figure 6. Differences between the murine and human pancreas. Schematic showing the anatomical differences between the murine and human pancreas in terms of both organ and islet architecture.

 

Pancreatic Islet Cells and Hormones – All with Their Own Function

 

This section describes the molecular and cellular characteristics of the different subtypes of pECs and their specific hormones and function during adult homeostasis (Figure 7).

Figure 7. Pancreatic endocrine cells. Diagram of signaling and transcription factor interactions that regulate pancreatic endocrine cell differentiation.

ALPHA CELLS, GLUCAGON

 

Alpha cells secrete glucagon and are the second most common pEC subtype. Glucagon contributes to maintaining homeostatic blood glucose levels by stimulating glucose production and inhibiting glycogen storage by the liver. The glucagon receptor, GCGR, is widely expressed by multiple organs besides the liver, and glucagon mediates multiple other physiological processes including amino acid metabolism, glomerular filtration by the kidney, lipolysis, and gastric motility (243).

 

The key factor promoting commitment of pEC progenitors to alpha cells is the transcription factor ARX. Deletion of Arxin Pdx1-expressing progenitors in the mouse leads to a complete loss of alpha cells, accompanied by a compensatory increase in the number of beta and delta cells, which results in the same total numbers of pECs in mutant and wildtype mice (244). Similarly, overexpression of Arx in the embryonic mouse pancreas or in developing islet cells results in an increase in the number of alpha and PP cells while diminishing the number of beta and delta cells (245). While ARX is required for alpha cell specification, the transcription factor, MAFB, is required for their final maturation and hormone expression. Mice with null alleles of MafB show a 50% reduction in insulin and glucagon positive cells (246). Moreover, adult mice in which MafB has been conditionally deleted in either Neurog3-expressing pEC progenitors or in mature pECs had reduced numbers of glucagon-positive cells (247).

 

BETA CELLS, INSULIN

 

The most well-known and abundant cell type of the Islets of Langerhans is the beta cell. Beta cells produce insulin which lowers the blood glucose levels via direct and indirect effects on target tissues. Binding of insulin to its receptor facilitates glycolysis in liver hepatocytes and skeletal muscle cells and promotes lipogenesis in the liver and white adipose tissue. Additionally, insulin inhibits hepatic gluconeogenesis and glucagon production by alpha cells (248–251).

 

Some of the most important transcription factors involved in the differentiation of beta cells are NKX2.2, NKX6.1, NEUROD1, MAFA, and MAFB. In mice lacking the homeodomain transcription factor NKX2.2, beta cell precursors fail to fully mature or produce insulin and, as a consequence, the mutant mice develop hyperglycemia and die shortly after birth (252). NKX2.6 lies downstream of NKX2.2, and in mice lacking Nkx6.1 beta cell neogenesis during the second transition is blocked, and the development of fully differentiated beta cells is prevented (253). NEUROD1 is also required for beta cell maturation (254). Neurod1 null mice have reduced beta cell numbers and fail to develop mature islets (255). Differentiating beta cells express both MafA and MafB, but fully mature beta cells in the mouse only express MafA. Conditional deletion of MafB in the mouse pancreas delayed beta cell maturation (247). Notably, MAFB is expressed in adult human beta cells and while human pluripotent stem cells with MAFB knockout are capable of pEC differentiation, they favor delta cell and PP cell specification at the expense of beta cells (256). MAFA is not essential for beta cell development but is required for insulin secretion and for maintenance of the beta cell identity (257).

 

DELTA CELLS, SOMATOSTATIN

 

Pancreatic delta cells, which make up ~5-6% of all islet cells, are responsible for the production of SST. Like insulin and glucagon, SST is a peptide hormone and cleavage of the precursor pro-hormone gives rise to two active isoforms, a long form that acts primarily in the central nervous system and a short form that acts on the organs of the GEP system. Delta cells predominantly secrete the short isoform of SST in response to a variety of stimuli including acetylcholine, glutamate, GLP-1, and urocortin3 produced by beta cells, ghrelin produced by epsilon cells, and high blood glucose levels (258). Within the islets, SST binds to one of its five different receptors on the surface of beta cells and alpha cells, thereby inhibiting the secretion of insulin and glucagon (259,260). SST also acts on cholangiocytes in the liver, inhibiting their secretion of fluids and thereby mediating bile flow (261).

 

The delta cell-specific transcription factor, haematopoietically expressed homeobox (HHEX), is required for both differentiation of delta cells during embryogenesis and maintenance of their identity in the adult. Deletion of Hhex in mouse pEC progenitors during development led to loss of delta cells by E16.5. Likewise, deletion of Hhex in adult mouse pancreatic delta cells led to a reduced number of delta cells and reduced secretion of SST (262). The phenotype of mice lacking HHEX in the pancreas speaks to the hormonal interplay between pECs: reduced SST secretion in mutant mice led to increased hormone secretion from alpha and beta cells in response to different stimuli. Indeed, despite not being expressed in beta cells, HHEX has been repeatedly identified through GWAS studies as a locus conferring susceptibility to T2D (263).

 

EPSILON CELLS, GHRELIN  

 

Epsilon cells, described for the first time in 2002, were the last islet cell type to be discovered. The number of pancreatic epsilon cells is highest during embryogenesis, comprising up to 10% of islet cells at mid-gestation, but their numbers then decrease such that they make up only a bit more than 1% of islet cells in the adult pancreas (264). Scattered throughout the islets, epsilon cells produce the growth hormone secretagogue ghrelin. The name of this hormone comes from the Proto-Indo-European root “ghre-”, meaning "to grow," which seems appropriate for a hormone that was discovered in efforts to identify the ligand for the growth hormone secretagogue receptor (GHS-R) (265). Also known as the hunger hormone, ghrelin is a 28 amino acid peptide that needs to be post-translationally acetylated before it can bind to its receptor, GHS-R, and exert its function on its target cells in an expanding list of target organs (266).

 

Consistent with its stimulatory effect on food intake, ghrelin concentrations are highest during fasting. This function is primarily mediated by ghrelin secreted by gastric X cells. The other hallmark functions of ghrelin are stimulating fat deposition and stimulating growth hormone release from the pituitary. The list of functions attributed to ghrelin is still growing and includes regulating glucose and energy homeostasis, cardioprotection, muscle atrophy, and bone metabolism (267). Secretion of ghrelin is stimulated by glucagon and is inhibited by glucose, insulin, leptin, and GLP-2(268–270). In the pancreas, ghrelin secreted by epsilon cells binds to the GHS-R on delta cells, thereby inducing them to secrete SST, which in turn inhibits the secretion of insulin by beta cells (271,272). There is also some evidence that ghrelin promotes the survival and proliferation of beta cells (264).

 

A complex interaction between NKX2.2 and NEUROD1 appears to be involved in epsilon cell specification during development (264). However, the transcription factor most closely linked specifically to epsilon cells is PAX6, which appears to inhibit epsilon cell formation. Ghrelin-expressing cells are increased in the developing pancreas of Pax6 knock-out mice at the cost of alpha cells (273). Likewise, when Pax6 was deleted in the adult pancreas of mice, the same increase in epsilon cell numbers was observed, concomitant with loss of beta cells, alpha cells, and delta cells (274). A later study demonstrated that, upon loss of Pax6 expression, beta and alpha cells began to express ghrelin (275).

 

PP (GAMMA) CELLS, PANCREATIC POLYPEPTIDE  

 

PP cells comprise less than 1-2% of the islets and the majority are located in the head of the pancreas. Perhaps due to their scarcity in the islets, not much is known about the genetic determinants of PP cell specification and maturation. Signals from the vagus nerve, enteric neurons, and arginine following meal intake stimulate PP cells to release pancreatic polypeptide (PP) (276). This hormone has the opposite effect of ghrelin and is referred to as the satiety hormone (277). Transgenic mice that overexpress PP show reduced weight gain and decreased fat mass, and long-acting PP analogues are being explored for the treatment of human obesity (278,279). Within the pancreatic islet, PP indirectly affects insulin secretion by inhibiting glucagon and SST secretion via a receptor expressed on alpha and delta cells, respectively (280,281).

 

Other members belonging to the same peptide hormone family as PP are neuropeptide Y (NPY) and peptide YY (PYY) which are produced in the GI tract. PYY is produced by L cells in the intestine and colon and its function mimics that of PP in the pancreas (282).

 

Pancreatic Endocrine Cells in Injury Repair

 

Injury to the pancreatic epithelium in the form of surgery, inflammation or metabolic trauma disrupts homeostasis by causing extensive cell loss. Failure in injury repair can result in organ dysfunction and cause clinical symptoms.

 

The youthful pancreas has a high potential to regenerate damaged tissue following injury (283,284). In contrast to the GI tract, however, where this capacity to regenerate is maintained throughout life, the regenerative capacity of pECs is limited in adult tissue (285,286). Through observations made in rodent injury models, mostly looking at beta cells, it has become clear that the endocrine pancreas employs multiple strategies to regain homeostasis following injury.

 

REPLICATION OF PRE-EXISTING BETA CELLS  

 

The ability of pre-existing beta cells to replicate in response to injury was first shown by the Melton lab. Using a beta-cell-specific lineage trace they found that the newly generated beta cells that arose following partial pancreatectomy originated from pre-existing ones (287). Using the same injury model, but an unbiased thymidine analogue based labelling technique, a different group came to the same conclusion (288).

 

NEOGENESIS OF BETA CELLS FROM A STEM/PROGENITOR CELL    

 

An alternative hypothesis is that beta cell regeneration in the adult pancreas involves a process called neogenesis, which involves the formation of de novo beta cells derived from a stem or progenitor cell expressing Neurog3. Initial studies of neogenesis were based on in vivo pulse labelling with the thymidine analogue, BrdU. In one study, IFN-gamma induced beta cell depletion resulted in the appearance of budding duct/islet-like structures containing proliferative ductal cells as well as newly formed acinar and endocrine cells (289). Similar observations were made in two different models of pancreatic injury in rats, where regeneration involved marked proliferation of ductal cells, some of which expressed ductal and endocrine markers, followed by the formation of new pECs and islet structures (290,291). Researchers from the Heimberg lab found that, subsequent to pancreatitis induced by pancreatic duct ligation in mice, the region of the pancreas undergoing regeneration contained NEUROG3-positive cells, a portion of which also expressed ductal markers (292). Finally, a lineage trace of ductal cells in mice showed that, following injury, ductal, acinar, and endocrine cells all contained the lineage trace (293). Altogether, these studies suggest that, following injury, new pECs can be derived from a progenitor cell in the pancreas ductal epithelium, whose differentiation to the pEC fate is dependent on transient expression of Neurog3.

 

TRANS-DIFFERENTIATION OF NON-BETA CELLS INTO BETA CELLS        

 

Finally, a landmark study from the lab of Pedro L. Herrera, provided evidence for yet another strategy employed by the pancreatic endocrine compartment to regenerate.  Prior to inducing genetic ablation of adult beta cells, the authors induced a lineage trace of glucagon-producing alpha cells. In the initial months following beta cell ablation, mice required supplementation with insulin. However, at 6 months post-ablation, mice no longer required supplemental insulin and their pancreata showed increased beta cell mass and beta cells that expressed both insulin and glucagon. At early time points following ablation, the majority of the regenerated beta cells in these mice contained the alpha cell lineage trace, arguing that the beta cells had resulted from trans-differentiation of the alpha cells (294). This provided new evidence of endocrine cell plasticity.

 

Pancreatic Endocrine Cell Hyperplasia

 

In the previous section we discussed observations relating to pEC regeneration in the context of conditions that lead to direct loss of pECs. Given defining pEC characteristics such as direct innervation and the role of pECs in mediating a number of physiological processes, it is likely that pECs also respond to stimuli produced in the context of other pathological conditions. As we have discussed in previous sections of this text, EEC and PNEC hyperplasia have each been observed in the context of inflammation or injury of their respective tissue sites. Likewise, an increase in the number pECs has been observed as a response to some pathological conditions relating to the pancreas (295,296). Moreover, focal endocrine hyperplasias have been observed incidentally in the pancreas of up to 10% of screened adults at autopsy (297).

 

The pEC mass is normally 1% and 3% of the total pancreatic mass in adults and infants, respectively. If this increases to more than 2% or 10% of the total pancreatic mass in adults or infants, respectively, it is defined as pEC hyperplasia (222,297,298). Some instances of pEC hyperplasia involve a general increase in the size of pancreatic islets that results in an increase in overall islet cell numbers but not in a change in the relative frequencies of one pEC subtype versus another. However, the majority of pEC hyperplasia are associated with an increase in the number of a specific subtype of pEC, most commonly alpha and beta cells. Hyperplasia of other pEC subtypes including the rare gamma/PP cells have also been reported. Morphologically, pEC hyperplasia either appears as large islets (larger than 250 mm in diameter) or as budding structures protruding from the ductal epithelium (297). The latter budding structures are reminiscent of the structures described above in mouse models of pEC injury and are suggestive of pEC neogenesis.

 

BETA CELL HYPERPLASIA

 

Beta cell hyperplasia is commonly observed in patients with insulin resistance and early T2D and is likely a physiological response to these conditions. Other clinical conditions in which beta cell hyperplasia is implicated include persistent hyperinsulinemic hypoglycemia of infancy (PHHI) and non-insulinoma pancreatogenous hypoglycemia syndrome (NIPHS) (299–302). These conditions are associated with dysregulated insulin secretion and hypoglycemia in infants and neonates or in adults, respectively.

 

The pancreas of patients with PHHI is characterized by the presence of either focal beta cell hyperplasia, resulting in a focal increase in islet size or, more commonly, of diffuse beta cell hyperplasia in which enlarged islets and small, irregularly shaped endocrine cell clusters are found throughout the pancreas (297). The percentage of beta cells present within the islets of patients with PHHI is increased such that they account for 70-90% of the islet (222). Increased proliferation of not just beta cells but also of ductal and centroacinar cells has been reported in the pancreas of patients with PHHI (303). PHHI is caused by mutations in ABCC8 and KCNJ11, which encode for subunits of the ATP-sensitive potassium channel involved in insulin secretion, as well as by mutations in genes affecting beta cell metabolism such as glucokinase (GCK), glutamate dehydrogenase (GLUD1), and short chain fatty acid hyroxyacyl dehydrogenase (SCHAD) (304).

 

NIPH is defined by postprandial hypoglycemia and, unlike PHHI, the genetic cause has not been clearly identified. The pancreas of patients with NIPH exhibits an increase in both number and size of the islets, and contains endocrine cells budding from the ductal epithelium (305,306). Symptoms of both PHHI and NIPH can be resolved through either partial or near total pancreatectomy.

 

ALPHA CELL HYPERPLASIA

 

Alpha cell hyperplasia (ACH) is a rare condition most commonly caused by mutations in the gene that encodes for the glucagon receptor, GCGR (307,308). The number of islets in patients with ACH is increased and the islets vary in size, are often larger, and contain a high proportion of alpha cells (309). Patients with ACH often, though not always, also present with hyperglucagonemia, and multiple pancreatic NETs. The multifocality of ACH and pancreatic NET lesions in these patients, and the observed presence of large islets showing signs of morphological transition from ACH to glucagonomas suggests that ACH lesions can progress to frank glucagonomas (308,309). Interestingly, these patients do not display features of glucagonoma syndrome due to their GCGR mutations. Mice with germline null mutations in Gcgr, or with liver-specific deletion of Gcgr also develop ACH that can progress to glucagonomas. Pharmacologic interruption of glucagon signaling in mice also leads to ACH. In these models, ACH appears to be primarily driven by alpha cell proliferation, though it is possible that transdifferentiation of other pECs or of ductal cells is also involved. It is interesting to note that the clear link between GCGR function and ACH implies a signaling feedback loop that regulates both the number of glucagon-producing alpha cells and their secretion of glucagon. One of the signals that is likely to contribute to ACH is amino acids. Serum amino acid levels are increased in patients with ACH and transcriptomic analysis of mouse models of ACH have shown altered expression patterns for genes involved in amino acid catabolism and transport (310).

 

GAMMA/PPCELL HYPERPLASIA

 

Compared to alpha and beta cell hyperplasia even less is known about PP cell hyperplasia, which occurs very rarely. As with alpha and beta cell hyperplasia the number and size of the pancreatic islets in patients with PP cell hyperplasia is increased and contain a high proportion of PP cells (311–313). In 50% of the reported PP hyperplasia cases, the patients had suffered from gastrinoma or ZES. In addition, there is no correlation between PP cell number and PP serum levels. It is possible that PP hyperplasia arises as an effect of gastrinomas (297). There are also no genetic changes directly related to the onset of this condition.

 

GEP NEUROENDOCRINE NEOPLASMS (GEP-NENs)

 

GEP-NENs encompass all NENs that arise along the GEP tract and account for 55 to 70% of NENs from all tissue sites (82,314,315). GEP-NENs comprise both well-differentiated NETs and poorly differentiated NECs. In addition, some GEP-NENs occur as GEP-mixed neuroendocrine/non-neuroendocrine neoplasms (GEP-MiNEN) that can be either well- or poorly- differentiated and are characterized by their mixed morphology showing endocrine and non-endocrine features. Based on proliferation (measured by mitotic count and Ki67 index) GEP-NETs are further subdivided into low (G1), intermediate (G2), and high (G3) grade NETs (82). G3 well-differentiated NETs are associated with a poor prognosis and show a decidedly more aggressive clinical behavior than G1 and G2 GEP-NETs. G3 well-differentiated NETs are more commonly observed in the pancreas than in other GEP tissue sites. GEP-NECs can be subdivided into small cell NEC and large cell NECs.

 

GEP-NECs

 

GEP-NECs comprise 10-20% of all NENs, with roughly 38% arising in the GI tract (colon, anus, rectum) and 23% in the pancreas (316). GEP-NETs and GEP-NECs display different mutational profiles and are therefore considered separate disease entities. Indeed, the most common site for NECs is the large bowel, whereas the most common site for NETs is the ileum (316). In addition, the observations that some GI-NECs show features resembling adenocarcinomas or squamous cell carcinomas and that up to 40% of NECs show non-endocrine features, have led some researchers to hypothesize that GEP-NECs are more closely related to non-endocrine tumor types than to high grade (G3) NETs (82,317,318).

 

The importance of the two genes RB1 and TP53 in the genesis of NECs is highlighted by the fact that these genes are commonly mutated in both lung-NECs and GEP-NECs. TP53 mutations have been identified in 20-73% of all GEP-NECs (319–323) while RB1 mutations have been identified in 44-86% of all GEP-NECs (322,324–326). The limited number of studies that have performed genomic analyses of GEP-NECs have also identified other genes that are commonly mutated in GEP-NECs, including KRAS, SMAD4 and APC (318,327,328). In addition, GEP-NECs are characterized by frequent and severe chromosomal abnormalities (79).

As discussed previously, generating a GEMM of SCLC was achieved through conditional tissue-specific deletion of Rb1 and p53 in the mouse lung epithelium (89). In contrast, attempts to generate GEMMs of RB1; TP53 mutant GEP-NECs by deletion of Rb1 and p53 in targeted mouse GEP tissues have proven to be less straightforward and have given mixed results (329). Conditional deletion of Rb1 and p53 in renin-expressing mouse pECs led to the generation of highly aggressive, metastatic, glucagon-producing tumors (330). Given the expression of glucagon in combination with the aggressive course of the tumors developed by this GEMM, however, it is unclear whether they are a more suitable model for sporadic glucagonomas or for pancreatic NECs. In human patients, pancreatic NECs are almost exclusively non-functional, i.e., they do not secrete symptom-inducing hormones (331). In the RIP-Tag2 GEMM, instead, SV40 T-antigen is expressed in beta cells, thereby effectively abrogating the Rb1 and p53 pathways in these pECs. RIP-Tag2 mice primarily develop aggressive insulinomas and, to a lesser extent, poorly differentiated pancreatic NECs (332). The RIP-Tag2 model has been instrumental in delineating stepwise aspects of pancreatic NEN tumorigenesis and in identifying therapeutic strategies for these tumors in patients (329). Nonetheless, whether this GEMM can be used as a reliable model for pancreatic NECs is unclear. 

 

In addition to GEMMs, cell lines and, more recently, GEP-NEC patient-derived tumor organoid lines have been generated (333,334). Patient-derived tumor organoids, 3D long-term cultures of tumor cells, can be expanded long-term, can be cryopreserved, and have been shown to be representative of the patient tumor tissue from which they were derived at both the genetic and phenotypic levels (335). Recently, a genetically engineered organoid model of GEP-NECs was generated by CRISPR/Cas9 mediated compound knock-out of RB1 and TP53 combined with overexpression of 6 transcription factors in otherwise normal colon organoids (333). Interestingly, in the absence of transcription factor overexpression, compound knock-out of RB1 and TP53 was not sufficient to generate GEP-NECs from these cells.   

 

Together with genomic analyses of GEP-NECs, studies using preclinical models of GEP-NECs have been informative. Nonetheless, to date, strategies for stratifying patients in terms of the molecular characteristics of their tumors and the likely response of their tumors to specific therapies are lacking. Currently, the primary treatment strategies for GEP-NECs use platinum agents combined with etoposide, based on the relative effectiveness of this approach in treating SCLC, which is a more common and better studied NEC subtype (336–338). Overall, the response rate for GEP-NECs to first line therapy is 40-60%. Upon relapse or the tumors becoming refractory, there are no well-established second-line therapies. This is reflected in a median survival of 38 months in patients with localized disease and only 5 months in patients with metastatic disease (339). Other potential treatment strategies for patients with GEP-NECs that are currently undergoing clinical trials are the mTOR inhibitor everolimus and some forms of immunotherapy (336–338).                                               

 

GEP-NETs

 

GEP-NETs represent 80-90% of all GEP-NENs and comprise many different tumor types. GEP-NETs are classified as functional or nonfunctional depending on whether they secrete symptom-causing hormone peptides. Functional GEP-NETs, which can arise throughout the GEP-tract, include gastrinomas and insulinomas (10). GEP-NETs are highly heterogeneous with regards to their biological behavior and clinical presentation, course, and prognosis. The most common tissue sites of primary GEP-NETs are the small intestine, the rectum, the colon, the pancreas (12.1%), and the appendix (315). In general, G1 and G2 GEP-NETs are associated with high 5-year survival rates ranging from 75 to 79% and from 62 to 74%, respectively. The 5-year survival rate for patients with G3 well-differentiation NETs, on the other hand, shows more variability between NETs of the intestine (40%) and NETs of the pancreas (7%) (340). Notably, the recognition of the category of G3 well-differentiated pancreatic NET by the WHO in 2017 has had important implications for patients and clinicians, as it highlighted the fact that some pancreatic NETs, despite showing morphological features and differentiation more commonly associated with low-grade NETs, show aggressive behavior more similar to that of NECs (82,331).

 

The most prevalent sites of origin for GEP-NETs show regional differences that are likely reflective of differences in both environmental and genetic factors. GI-NETs arising from the small intestine or colon are most common in the USA, small intestinal or pancreatic NETs are more common in Europe, while in Asia gastric and rectal NETs are most prevalent (341). The most well studied GEP-NETs are pancreatic NETs and SI-NETs.

 

Gastric NETs (G-NETs) are relatively rare and only account for 4 to 6% of NENs (342,343). These tumors are classified into one of four categories according to their clinical characteristics. Most G-NETs are type I tumors, which are associated with CAG and arise as multiple small nodules. These tumors rarely metastasize. Type II G-NETs are very similar to type I tumors but are commonly associated with MEN1 syndrome (in conjunction with gastrin producing pancreatic NETs) or ZES. Type I and type II G-NETs arise as a consequence of excessive gastrin. Type III G-NETs are sporadic tumors that are not associated with other gastric conditions and present as large, solitary lesions. Finally, type IV tumors, G-NECs, are both the very rare and the most malignant. These tumors arise sporadically and are poorly differentiated. While type I, II, and III G-NETs are ECL cell tumors, type IV G-NECs arise from other endocrine cell types (344).

 

Small intestinal NETs (SI-NETs) are the most common neoplasm arising in the small intestine (345). They include NETs arising in the jejunum and ileum, with the ileum being the major site of incidence (346). A unique feature of ileal SI-NETs is that they are multifocal in 10-20% of cases, with the different tumors arising independently (347). Moreover, ileal SI-NETs have a high rate of metastasis, with >50% of ileal SI-NET patients presenting with metastases at the time of diagnosis (348). Finally, the majority of SI-NETs produce serotonin, causing many of these to induce carcinoid syndrome, the most common functional hormonal syndrome of patients with NETs (349). Clinical symptoms of carcinoid syndrome include watery diarrhea, flushing, hypotension, breathlessness, wheezing, and loss of appetite, all attributable to not just increased serum levels of serotonin but also of prostaglandins, histamine, bradykinin, and tachykinins (9,350). Carcinoid syndrome is most often observed when NETs, having metastasized to the liver, secrete their biologically active compounds directly into the systemic circulation (351). Carcinoid syndrome can cause carcinoid heart disease in which elevated serum serotonin levels cause fibrosis in the heart. Symptoms of carcinoid syndrome attributable to serotonin can be ameliorated through the administration of serotonin-inhibiting therapies (340,352).

 

Of note, duodenal NETs are sometimes considered separately from SI-NETs as they more closely resemble gastric and pancreatic NETs in terms of their mutational profile (353). Whereas the serotonin producing EC cells are thought to be the cell of origin of SI-NETS, duodenal NETs more commonly express gastrin and somatostatin (described in more detail later in this chapter) and are therefore thought to arise from G and D cells (354,355).

 

Pancreatic NETs (PanNETs) are the best studied subtype of GEP-NET and are the only subtype for which the category of high grade G3 well-differentiated NET has been officially recognized (356). While some well-differentiated PanNETs are functional and consist of a single hormone-producing cell type, the majority of PanNETs are non-functional and contain a mixture of cells expressing markers for the different pEC types (357,358). PanNETs are thought to arise from differentiated endocrine cells of the islets of Langerhans. However, a recent study based on next generation DNA methylation analysis suggests that they may also arise from the exocrine pancreas (359).

 

Familial GEP-NETS

 

While the majority of the GEP-NETs arise sporadically, approximately 5% occur as part of a hereditary cancer predisposition syndrome (360). The most common of these syndromes are associated with the development of duodenal and PanNETs and are caused by germline mutation in one of five different genes: MEN1, tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 2 (TSC2), Von Hippel–Lindau (VHL), and neurofibromatosis type 1 (NF1) (361). Of these, MEN1 is the gene most strongly implicated in PanNETs. MEN1 encodes for the protein menin, which acts as a scaffold for both transcription factors and chromatin-modifying enzymes (362–364). Thus, although its exact function is yet to be determined, menin has been suggested to play a role in multiple processes including DNA damage repair, cell cycle regulation, histone methylation, and mTOR pathway activity (365).

 

Familial SI-NETs are far rarer and have been associated with germline mutations in CDKN1B, inositol polyphosphate multikinase (IPMK), and MutY DNA glycosylase gene (MUTYH) (366–369). Notably, germline mutations in CDKN1B have been shown to be causative of the MEN4 familial cancer syndrome in which patients develop parathyroid, pituitary, and, more rarely, SI-NETs (323). Finally, there is one example of a single consanguineous family in which several family members were affected by hypergastrinemia and consequent gastric ECL NETs. These familial G-NETs were shown to be caused by germline inactivating mutations in the gene for a proton pump expressed by parietal cells and involved in gastric secretion, ATP4a (370).

 

Genetics of Sporadic GEP-NETs

 

Although rare, familial cancer syndromes associated with NETs have pointed to genes and pathways that are also important for the genesis of sporadic NETs. Genetic studies have revealed that somatic inactivation by mutation, chromosomal alteration, or epigenetic silencing of genes such as MEN1, TSC2, and VHL, each associated with a familial NET syndrome, are found in approximately 40%, 35%, and 25% of sporadic pancreatic NETs (371,372). Familial SI-NET syndromes are rarer, and of the causative genes for these syndromes, only mutations in CDKN1Bhave also been identified in 9% of sporadic SI-NETs (373). Of note, the largest whole genome analysis of pancreatic NETs to date, uncovered germline mutations in CDKN1B and MUTYH in pancreatic NETs from patients that had no family history of the disease, therefore implicating alterations in these genes also in the genesis of pancreatic NETs and, perhaps, of GEP-NETs in general (371,372). Incidentally, germline variants in pancreatic NET samples in this cohort were also identified in checkpoint kinase 2 (CHEK2), and BRCA2 (371,372).    

 

Molecular analysis of pancreatic NETs also uncovered alterations in genes that are not implicated in familial NET syndromes. In particular, Jiao et al. identified recurrent mutations in PTEN and PIK3CA in pancreatic NETs. This study also identified recurrent mutations in the chromatin remodeling enzymes DAXX (death domain associated protein) and ATRX (α thalassemia/mental retardation syndrome X-linked), in 25% and 18% of pancreatic NETs (374). DAXX and ATRX function together in a complex that deposits histone H3.3 at different sites including telomeres and mutations in ATRX/DAXX were mutually exclusive in pancreatic NETs (372). ATRX/DAXX mutations in pancreatic NETs were correlated with chromosomal instability and the activation of a telomerase independent telomere maintenance mechanism, alternative lengthening of telomeres (ALT) (375,376). Perhaps it is thus not entirely surprising that ATRX/DAXX mutations are more often found in G3 pancreatic NETs than G1/G2 pancreatic NETs and are associated with reduced patient survival (323,375). Other mutations found in G3 pancreatic NETs and associated with shorter survival times are mutations in ARID1A and CDKN2a (377,378).

 

Whereas several driver mutations have been identified in pancreatic NETs, identifying a clear genetic driver of SI-NETs has been more difficult. Instead, the development of sporadic SI-NETs seems to be dependent on chromosomal aberrations. Loss of chromosome (chr) 18 is observed in more than 60% of SI-NETs. While the functional importance of this chromosomal loss in SI-NETs has not been determined, one study identified allelic loss of BCL2, CDH19, DCC, and SMAD4 (all on chr 18) in 44% of SI-NETs (379). Loss of chr 9 and 16 or gain of chr 4, 5, 7, 14 or 20 are also recurrently observed in SI-NETs, albeit at lower frequencies (353,380–383). Targeted mutational and copy number analysis of metastatic SI-NETs has identified recurrent mutations in APC, CDKN2C, BRAF, KRAS, PIK3CA, and TP53, though at relatively low frequencies (ranging from 4 to 10%) (328,379,384).

 

Although genomic studies demonstrate that SI-NETs and pancreatic NETs have distinct genomic profiles, a common pathway alteration stands out -- activation of mTOR/PI3K signaling. In pancreatic NETs the pathway is recurrently activated by frequent loss of negative regulators of the pathway (PTEN, TSC1/2) and by recurrent activating mutations in PIK3CA. Likewise, this pathway is implicated in SI-NETs by the observation of recurrent activating mutations in KRAS, BRAF, and PIK3CA as well as, more commonly, of frequent copy number gains in components of this pathway, including SRC and mTOR itself (323). Frequent alteration of CDKN family genes also appears to be a common feature of both GEP-NET subtypes, suggesting deregulation of the cell cycle is also important for NETs. Finally, though not discussed in this text, aberrant methylation patterns are likely to contribute to GEP-NETs in general and some studies have suggested that methylation patterns can be used to stratify GEP-NETs with implications for patient prognosis (385–387).

 

Treatments for GEP-NETs

 

The main alternatives used to treat GEP-NETs for which surgical resection is not possible are hormone analogs, PRRT, the mTOR inhibitor everolimus, and for pancreatic NETs, sunitinib. Chemotherapy mainly targets proliferative cells and is therefore predominantly used on NECs and G2 pancreatic NETs.

 

Somatostatin analogues (SSAs) such as octreotide and lanreotide bind to the somatostatin receptors (SSTR1-5) and mimic the natural hormone’s ability to exert an inhibitory function on the cell’s hormone secretion. To prolong their effect, these analogues have been synthetically engineered to have an increased half-life compared to the endogenous hormone. Administration of SSAs is common in the treatment of functional GEP-NETs as they prevent the tumors from secreting excessive amounts of hormones and thus alleviate clinical symptoms (388,389). Although GEP-NETs can express all five somatostatin receptors, the majority express SSTR2. Binding of SSAs to SSTR2 has been shown to decrease proliferation and lead to disease stabilization in GEP-NET patients (390–392). In addition, SSAs can also be coupled to radioactive isotopes and used for targeted radiological therapy. The internalization of the radioactive isotope by the cancer cells causes DNA damage and cell death (393,394).

 

As discussed previously, the mTOR-AKT pathway plays an important role in GEP-NETs. Consistent with this, inhibitors of this pathway such as everolimus have been shown to have a positive effect (132,395,396). Cell type-specific drugs have also been used in the treatment of pancreatic NETs and one of the oldest drugs is streptozotocin, a beta cell specific cytotoxic agent that has been used for almost four decades (397). Streptozotocin selectively enters beta cells via the glucose transporter, GLUT2, causing DNA damage resulting in cell death (398). Finally, inhibitors of receptor tyrosine kinases, vascular endothelial growth factor and its receptor have been effective in the treatment of GEP-NETs, most notably, pancreatic NETs as they are often highly vascularized (399,400).

 

CONCLUSIONS

 

When comparing neuroendocrine cells from different tissues, multiple recurrent themes can be identified. For one, their development relies on the expression of bHLH transcription factors ASCL1 and NEUROG3. Animal models have highlighted the differential roles played by these two transcription factors in the formation and differentiation of NE cells of the diffuse NE system. Whereas ASCL1 drives PNEC lineage commitment in the lung, NEUROG3 is essential for the formation of the intestinal EECs and pECs. Both ASCL1 and NEUROG3 appear to be important for the development of gastric EECs. Another interesting difference can be seen in the expression pattern of these transcription factors. Whereas NEUROG3 is transiently expressed during the formation of GEP endocrine cells, ASCL1 is constitutively expressed in lung progenitor and mature PNECs. The mechanistic differences and biological reason behind these differential dependencies on either ASCL1 or NEUROG3 remain to be determined.

 

Second, NE cells of the diffuse NE system appear to be involved in the response to some forms of injury, as indicated by their increased numbers in some disease conditions. Importantly, comparing NE cells during development or homeostasis to NE cells in disease conditions has the potential to uncover aberrations in common pathways and regulatory factors that contribute to or mediate the pathological state. This kind of analysis is likely to provide candidate targets for the development of new (targeted) therapies.

 

Another recurrent theme in the biology of neuroendocrine cells from different tissues, is the crosstalk between NE cells and their environment. This crosstalk can be seen both in the response of NE cells to environmental stimuli and in the direct influence they can exert both locally and systemically through the bioactive compounds they secrete. Finally, the ideas of NE cell plasticity and NE cell heterogeneity are ones that have not been fully explored but might be important to their function. As an example of the former, although EECs are named after their predominantly expressed hormone, they can also be multi-hormonal and even change their hormone expression depending on their location within the tissue. While EEC hormonal switching is just starting to be discovered within the intestinal tract, it is likely that a similar degree of plasticity in hormone expression exists for other NE cell types. Indeed, some hints of plasticity between endocrine cells can also be seen in pancreatic endocrine cells, where some studies imply trans-differentiation of alpha cells to beta cells under certain conditions. Regarding NE cell heterogeneity, it is interesting to note that, whereas the initial studies of NE cells were driven by observations about their shared features, with the advancement of single-cell sequencing technologies, the more recent era of NE cell research has highlighted a previously underappreciated heterogeneity of different tissue-specific NE cell populations (34,35,164,183,401,402). NE cell heterogeneity highlights the plasticity and dynamic nature of these cells as they respond to external stimuli and microenvironmental signals in a context-specific manner.

 

With regards to NENs, next generation sequencing efforts have started to characterize the mutational landscape of NENs. So far, these have been mostly focused on pancreatic and lung NENs. Similar studies of NENs originating from other organs would be of value as they could provide new insights into NEN biology. Importantly, candidates discovered in genome-wide genomic studies need to be validated to determine whether they represent drivers or passengers throughout disease progression. Additionally, their exact mechanistic function is of importance if they are to become targets for drug development. For NENs with a lower mutational burden and few recurrent mutations, epigenome, non-coding or protein level changes may play a more significant role in disease initiation and progression. These possibilities are just starting to be explored.

 

A significant challenge in studying NENs is the development of model systems that can be used to study both basic as well as translational NEN biology. Currently, the most commonly used models include GEMMs, cell lines, and patient derived xenografts (PDX). Although these model systems have provided invaluable knowledge about NEN biology they also have their limitations. GEMMs have been instrumental in the study of SCLC and some very specific subtypes of pancreatic NETs (RIP-Tag2 mice and Men1 mutant mice). Nonetheless, the current GEMMs for different NENs do not cover the entire range of different tumor types encompassed by NENs. Furthermore, species differences require comparisons and/or end stage studies in human derived model systems.

 

Cell lines, which while being robust and relatively cheap to culture, suffer from genetic changes occurring over time and may not recapitulate the full tumor heterogeneity.

 

PDX models circumvent those limitations while providing analysis in an in vivo environment, but have a very low engraftment rate of <10% and tedious logistics (403). Organoids derived from healthy tissue provide researchers with the means to study normal NE cell differentiation via the establishment of differentiation protocols (183,195) and offer the potential to model NEN disease progression by stepwise genome engineering, as has been achieved for other tumor types (333,404–406). Moreover, in vitro drug screens on patient-derived tumor organoids have the potential to aid personalized treatment. However, to date only a handful of NEN organoid lines have been established. Of note, most of those organoid lines represent NECs, rather than NETs (333,334).

 

Although NEN biology is starting to be explored in more detail, much remains to be discovered. A combination of both basic and translational research will be needed to provide the biological insights necessary to significantly be able to improve or establish novel clinical treatment options for NENs.

 

ACKNOWLEDGEMENTS

 

We thank Lisanne den Hartigh for help with making figure 5 and Joep Beumer for helpful discussions. Figures 1 - 4 and 6-7 were created with BioRender.com. This work was supported by an EMBO long-term fellowship (AALTF 332-2018 to A.A.R.) and funding from the Neuroendocrine Tumor Research Foundation under a Petersen Accelerator Award (H.C. and T.D.). 

 

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