Metabolic stress, IAPP and islet amyloid

review article

review article

Diabetes, Obesity and Metabolism 14 (Suppl. 3): 68–77, 2012. © 2012 Blackwell Publishing Ltd

Metabolic stress, IAPP and islet amyloid J. Montane1 , A. Klimek-Abercrombie1 , K. J. Potter1 , C. Westwell-Roper1 & C. Bruce Verchere1,2 1 Department of Pathology & Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada 2 Department of Surgery, Child & Family Research Institute, University of British Columbia, Vancouver, BC, Canada

Amyloid forms within pancreatic islets in type 2 diabetes from aggregates of the β-cell peptide islet amyloid polypeptide (IAPP). These aggregates are toxic to β-cells, inducing β-cell death and dysfunction, as well as inciting islet inflammation. The β-cell is subject to a number of other stressors, including insulin resistance and hyperglycaemia, that may contribute to amyloid formation by increasing IAPP production by the β-cell. β-Cell dysfunction, evident as impaired glucose-stimulated insulin secretion and defective prohormone processing and exacerbated by metabolic stress, is also a likely prerequisite for islet amyloid formation to occur in type 2 diabetes. Islet transplants in patients with type 1 diabetes face similar stressors, and are subject to rapid amyloid formation and impaired proinsulin processing associated with progressive loss of β-cell function and mass. Declining β-cell mass is predicted to increase metabolic demand on remaining β-cells, promoting a feed-forward cycle of β-cell decline. Keywords: β-cell, amylin, diabetes, insulin Date submitted 2 April 2012; date of final acceptance 1 June 2012

Introduction Declining β-cell mass and function in type 2 diabetes can be attributed to a number of stressors experienced by the islet during the development and progression of disease, including glucolipotoxicity [1,2], islet cholesterol accumulation [3–5], islet inflammation [6–8] and islet amyloid [9–11]. Over 100 years since the first description by Opie of a hyaline lesion in the diabetic islet later identified as amyloid [12], and some 25 years since the identification of its major component as the peptide islet amyloid polypeptide (IAPP) [13,14] or amylin [15], islet amyloid remains somewhat of an enigma in the type 2 diabetes field [16]. Although considerable progress has been made, the mechanisms that lead IAPP to form amyloid deposits in type 2 diabetes as well as the pathways by which aggregation of IAPP causes β-cell death remain largely undefined. Indeed, it even remains a topic of debate as to whether IAPP plays a causative role in islet decline, or whether it is simply an inert marker of other pathophysiological processes that lead to β-cell death and dysfunction [16,17]. Considerable correlative evidence now exists for there being a role for IAPP in β-cell death, including strong association of the degree of amyloid deposition in type 2 diabetic subjects with the presence of β-cell apoptosis and diminished β-cell mass [18]. Aggregates of synthetic human IAPP induce β-cell apoptosis [19], and inhibition of either IAPP aggregation [20] or synthesis [21] can ameliorate β-cell death in a cultured human islet model of rapid in vitro amyloid formation, showing the toxic nature of IAPP aggregates. The possibility that aggregates of IAPP Correspondence to: Bruce Verchere, PhD, Child & Family Research Institute, 950 West 28th Avenue, Vancouver, BC, Canada V5Z 4H4. E-mail: [email protected]

indirectly impact β-cells by inciting islet inflammation [22,23] has recently added a new dimension to our understanding of the mechanism of amyloid-induced β-cell loss. Complicating the field further is recent evidence that the primary toxic moiety in the IAPP aggregation pathway is probably not fullblown amyloid plaques, but rather a prefibrillar, oligomeric aggregate of IAPP molecules [24,25]. Definitive evidence for a causative role for IAPP in β-cell loss in type 2 diabetes awaits the development of inhibitors of islet amyloid formation in vivo and the demonstration that such inhibitors attenuate hyperglycaemia and β-cell loss in animal models of type 2 diabetes and, ultimately, in humans.

Islet Amyloid Polypeptide IAPP or amylin is a 37-amino acid peptide made by islet β-cells. It was first isolated from extracts of amyloid obtained from type 2 diabetic pancreas or insulinoma [13,15]. It has been co-localized by immunogold electron microscopy (EM) to β-cell secretory granules [26] and indeed is secreted along with insulin in response to β-cell secretagogues, including glucose [27]. It appears to reside in the soluble halo portion of the β-cell granule rather than the insulin crystal [28]. IAPP levels in the blood increase after a meal along with insulin [29]. The molar ratio of IAPP : insulin within the β-cell is low, estimated from 0.5 to 5% [27], and is slightly higher in the circulation because IAPP is cleared more slowly than insulin [29]. Regulation of the biosynthesis of IAPP is also co-ordinate with that of insulin. Gene expression of insulin and IAPP in response to glucose parallel each other [30], and as with proinsulin there is strong translational control of proIAPP biosynthesis by glucose [31]. The biological action of IAPP remains not fully understood. Although a number of actions

DIABETES, OBESITY AND METABOLISM

have been shown at supra-physiological concentrations of the hormone [16], the most plausible physiological roles appear to be in delaying gastric emptying [32] and increasing satiety [33]. IAPP activity appears to be mediated via its interaction with receptor complexes comprising the calcitonin receptor and receptor activity-modifying proteins [34]. Hydrophobic amino acids in the mid-region of IAPP are thought to largely confer its tendency to aggregate into β-pleated sheets. Proline substitutions are present in this region of the rodent form of the peptide that maintain it in soluble rather than aggregated form. Other parts of the rodent molecule, particularly in the N-and C-terminal regions, also have fibril forming capacity and may contribute to amyloid formation [35]. IAPP is subject to several post-translational modifications, including disulfide bond formation, amidation at its C-terminus [36], and O-glycosylation at threonines at positions 6 and 9 [37]. Almost all circulating IAPP is likely to be amidated. Enzyme-linked immunosorbent assays (ELISAs) that discriminate between the glycosylated and nonglycosylated forms are available and have allowed estimation of the proportion of glycosylated forms of IAPP in the circulation at about 50% of total IAPP immunoreactivity [37]. Little is known about the biological importance, if any, of these modifications. It is easy to imagine that the addition of large sugar residues to the peptide could have a profound impact on its folding, trafficking or biological activity. One could also speculate that such a modification could sterically inhibit IAPP aggregation within the cell. We recently found pig IAPP to have a unique sequence with a number of non-conservative substitutions in its central amyloidogenic region [38], including an arginine and proline residue at positions 20 and 29, respectively. When we synthesized the porcine peptide we found it had little fibrilforming capacity even at high concentrations, and was not toxic to β-cells. It is interesting to note that neonatal pig islets function well following transplantation [39,40], and pigs seem protected from type 2 diabetes [41]. Could this be in part because, like rodents, they do not make an amyloidogenic form of IAPP? IAPP is first synthesized as a larger precursor form, proIAPP, from which is it processed. As with proinsulin, production of IAPP from proIAPP occurs via sequential cleavage by the prohormone convertases PC1/3 and PC2. Within the β-cell secretory pathway, proIAPP is first cleaved on the C-terminal side of a pair of basic amino acid residues (Lys–Arg) by PC1/3, creating an intermediate form (the N-terminally extended proIAPP intermediate) that is subsequently cleaved by PC2 at another pair of basic residues nearer the N-terminus. The basic residues at the C-terminus of IAPP are removed by carboxypeptidase E, and the peptide is then amidated at the Cterminus by peptidyl-glycine alpha-amidating monooxygenase (PAM). Although the N-terminally extended intermediate form has been detected by western blot in islet extracts, it is not known whether it circulates in humans, as does the proinsulin processing intermediate des-31,32-proinsulin. Since proinsulin processing is impaired in type 2 diabetes [42], and since proIAPP is processed to mature IAPP in parallel with insulin [43], it follows that proIAPP processing is also likely impaired

Volume 14 No. (Suppl. 3) October 2012

review article in type 2 diabetes [44], although this has not been definitively assessed.

Mechanism of Islet Amyloid Formation A number of hypotheses have been put forward to explain why islet amyloid forms in persons with type 2 diabetes. The simplest explanation is perhaps that increased production of IAPP, associated with increased secretory demand on the β-cell due to insulin resistance and worsening hyperglycaemia, exceeds some threshold IAPP level beyond which IAPP aggregation occurs. The role of IAPP over-production in IAPP amyloid formation is discussed in more detail in a later section. As detailed later, we believe it is more likely that, in addition to increased IAPP production, defects in β-cell handling of IAPP or its precursors associated with the impaired β-cell function of type 2 diabetes must also be present. An additional question of some debate and addressed below is whether IAPP aggregation initiates intracellularly within the granule or extracellularly in the interstitial space next to β-cells. There is little doubt that massive overproduction of IAPP can lead to IAPP aggregation. Aggregation of synthetic human IAPP in vitro, as with any amyloidogenic peptide, is concentration dependent [45], indicating that the concentration of IAPP present in vivo, whether in the granule or outside the cell, is also likely of importance. Over-expression of human IAPP in cells can lead to IAPP aggregation and amyloid formation within the cell. Thus, when COS cells were transfected to express human proIAPP, IAPP-immunoreactive amyloid fibrils were observed within the cell by EM. Complicating this finding, however, COS cells would not be expected to have all of the cellular machinery needed to process and traffic (pro)IAPP; indeed they do not have secretory granules or prohormone convertases. When we expressed human proIAPP by adenovirus at high levels in INS-1 cells, a β-cell line equipped for processing and regulated secretion of IAPP, cell death was not increased; however, at higher multiplicities of infection when human proIAPP expression was markedly increased and proIAPP processing was noticeably impaired, β-cell death was observed [46]. One can envision a number of ways in which IAPP aggregation might normally be prevented in the cell and how the capacity of the β-cell to maintain IAPP in soluble monomeric form might be exceeded in conditions of increased IAPP production. The concentration of IAPP in the halo of the secretory granule may be in the low millimolar range [47], well in excess of the micromolar concentrations required for IAPP aggregation in vitro. Alterations in the granule milieu probably change in type 2 diabetes; for example, under conditions of increased secretory stress, granules take on a more immature phenotype, possibly because there is less time for insulin aggregation into the crystal. Granule components including insulin and zinc have been explored as potential inhibitors of IAPP aggregation. Insulin, in particular, appears to be a potent inhibitor of IAPP aggregation [48], and it is possible that an increase in the amount of IAPP relative to insulin enables IAPP aggregation. The pH of β-cell granules under secretory stress is also likely to be elevated, again because of less time for granule maturation or possibly because the deficient energy

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review article state of the diabetic β-cell may preclude proper acidification of the granule, as this requires the activity of an ATP-dependent proton pump on the granule membrane [49]. Clues to the mechanism of islet amyloid formation have also come from findings in other amyloid diseases. Notably, in all amyloidoses including Alzheimer’s disease, other molecular components are present in the amyloid plaque in addition to the major protein component. Thus, in the case of Alzheimer’s disease, amyloid deposits have been shown to contain heparan sulphate proteoglycans (HSPGs), apolipoprotein E (apoE), amyloid P component, and perhaps other molecules as well. Similarly, amyloid in the pancreatic islet is comprised not only of its major protein component IAPP, but also has been shown histologically to be associated with HSPGs [50,51], apoE [52,53] and other molecules [54]. HSPGs in particular have been an invariant finding in all amyloids, and accumulating evidence strongly points to a role for HSPGs in amyloid formation. Heparin analogues have been developed as inhibitors of amyloid formation [55]. Heparin-like molecules strongly stimulate IAPP aggregation, as with other amyloidogenic peptides [56]. An inhibitor of glycosaminoglycan synthesis was also found to decrease amyloid formation [57], although it was associated with cell toxicity. We proposed one possible mechanism by which heparan sulphate could initiate islet amyloid formation, through binding of unprocessed proIAPP or an incompletely processed proIAPP intermediate via a series of positively charged amino acid residues in its N-terminal cleavage site to negatively charged sulphate moieties on heparan sulphate [58]. Although apoE is present in islet amyloid [59], transgenic mice expressing human IAPP but lacking apoE are not protected from islet amyloid formation [53], suggesting that apoE does not play a critical role. Defective processing of the precursors of amyloidogenic peptides has been strongly implicated in a number of amyloids, including the amyloid-β (Aβ) protein of Alzheimer’s disease and islet amyloid in type 2 diabetes. In Alzheimer’s disease, increased cleavage of the amyloid precursor protein (APP) by γ secretase promotes accumulation of the amyloidogenic Aβ 1–42 . Indirect and direct evidence for a role for defective proIAPP processing in islet amyloid formation has come from several findings. First, immunoreactivity for the N-terminal flanking region of proIAPP has been reported to be present in islet amyloid by immunostaining [60]. Second, proIAPP processing has been found to be impaired in vitro under conditions in which amyloid occurs, including high glucose culture [21,61]. Third, insulinomas, which are known to have impaired proinsulin processing, also have IAPP-derived amyloid. Evidence that insulinomas secrete incompletely processed proIAPP has come from one clinical study where a larger circulating form of IAPP immunoreactivity was observed in the blood of a patient with an endocrine tumour of the pancreas [62]. Defective proinsulin and proIAPP processing in insulinomas could arise by their secretion via the constitutive secretory pathway, because the de-differentiated state of tumour cells leads to impaired sorting to the regulated pathway. As PC1/3 and PC2 are normally sorted to granules and not found in constitutive vesicles, processing in the constitutive pathway is via furin, which does not cleave proIAPP

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[43]. ProIAPP sorting to granules is normally quite efficient [43], but appears to be impaired in immature β-cells and β-cell lines [63,64], raising the possibility that de-differentiated β-cells do not sort proIAPP well. In addition, missorting of proIAPP to the constitutive pathway has been reported in human islets exposed to high glucose culture [63,65]. It could be speculated that missorting of proIAPP might lead to its release at a membrane site that would not facilitate its entry into the vasculature, and have more time to accumulate in islet interstitial space. Finally, and most importantly, two groups have provided direct evidence that impaired N-terminal processing of proIAPP can lead to islet amyloid formation and cell death, at least in culture models. Paulsson et al. transfected various secretory cell lines to express human proIAPP and observed fibril formation in those cells lacking PC2, the enzyme critical for the final N-terminal cleavage step in proIAPP processing [66]. We crossed human IAPP transgenic mice with mice lacking PC2 and found that islets from these mice rapidly formed amyloid associated with β-cell death in culture, and amyloid and β-cell death could both be prevented by adenoviral expression of PC2 to rescue N-terminal proIAPP processing [46]. Using GH3 cells that lack both PC1/3 and PC2 and expressing human proIAPP by recombinant adenovirus, we also showed that initiating the first C-terminal cleavage step by adenoviral expression of PC1/3 actually increased cell death, whereas expressing both PC1/3 and PC2 to complete processing resulted in decreased death [46], supporting the idea that the N-terminally extended proIAPP intermediate form (rather than the full length proIAPP peptide) is an important molecule in islet amyloid formation. Whether IAPP aggregation initiates intracellularly or extracellularly is still a matter of debate. Fibril-like structures have been described by EM in granules in studies from human autopsy, transplanted human islets, and a human IAPP expressing mouse model [16,67]. Unequivocal identification of intracellular aggregates by EM in human tissue is limited by speed of organ procurement and quality of fixation, and the need to rule out that any aggregation occurred postmortem during these procedures. Oligomeric aggregates have also been localized to intracellular sites within the β-cell using the anti-oligomer antibody, A11 [68]. The limitations of these studies and the antibody used have been debated elsewhere [25,69]. Indirect support also came from a study by Butler and colleagues who immunized transgenic mice with Aβ-derived oligomers to induce anti-oligomer antibodies; when this approach had no effect on β-cell death they concluded that aggregation was probably occurring within the cell, inaccessible to the antibody [68]. It is also possible that early oligomeric aggregates are present in granules but cannot be detected by EM in vivo with current approaches, given the difficulty in studying these aggregates in vitro. Although full-blown islet amyloid plaques reside extracellularly, the argument has been made that fibrils may still form intracellularly and then be secreted or exteriorized after cell death. In favour of an extracellular site of initiation, EM analysis of human IAPP transgenic mouse islets from mice that had hyperglycaemia associated with thioflavin S-positive amyloid deposits showed IAPP-immunoreactive fibrillar aggregations



between the β-cell membrane and islet capillary [70], supporting an extracellular site of aggregation. The apparent accumulation adjacent to capillaries is notable, as islet amyloid in humans, non-human primates and transgenic mice is frequently observed as ring-like structures around islet capillaries, similar to congophilic cerebral angiopathy in the brain. Endothelial cells are a rich source of HSPGs, and one hypothesis compatible with a number of the findings described here is that, following its secretion, the N-terminal proIAPP intermediate binds to HSPGs on the endothelial cell or β-cell membrane. That extracellular aggregation of IAPP plays an important role is further supported by our finding that peptide inhibitors of IAPP aggregation, which would not be expected to cross the β-cell plasma membrane, are potent inhibitors of amyloid formation and toxicity in cultured human islets [20]. Finally, Kahn and colleagues, as detailed later, showed that the inhibitors of β-cell secretion diazoxide and somatostatin markedly suppress amyloid formation in a cultured islet model [71], suggesting that secretion is critical for amyloid formation to occur. Identifying the site of amyloid initiation is of importance for the development of therapies, because drugs that target an intracellular site of aggregation initiation must cross the β-cell membrane. From the available data it seems that even if the early IAPP aggregation events occur within the β-cell, extracellular aggregation and toxicity occurs and is a valid therapeutic target.

Rodent Models of Islet Amyloid Formation Because the sequence of mature IAPP in rodents is not amyloidogenic, studying IAPP aggregation and islet amyloid formation in vivo has relied upon the development of transgenic mice with β-cell expression of the human form of the peptide [72]. In most cases, these models have used fragments of the rat insulin promoter to drive expression of human proIAPP at high levels within the β-cell. These models have provided some insight into the mechanism of islet amyloid formation in vivo, but because of marked differences in phenotype among the various transgenic lines generated by different groups, no clear picture has emerged. Thus, in some models, β-cell human proIAPP expression leads to hyperglycaemia associated with islet amyloid similar to what is seen in type 2 diabetes, although some inciting environmental or additional genetic condition seems invariably to be needed, such as administration of a high fat diet [70], crossing to the ob/ob [73] or obese Agouti viable yellow [74] background, or administration of steroids plus growth hormone [75]. In these models, the pathophysiology seems to resemble most closely that of human type 2 diabetes. Transgenic β-cell expression of human proIAPP in rats (RIPHAT rat) results in extensive islet amyloid in hemizygous rats that precedes hyperglycaemia, re-capitulating what may occur in humans. In other models, human IAPP expression is associated with early hyperglycaemia and rapid β-cell death but no visible amyloid. In these models, the phenotype has been interpreted as being caused by intracellular oligomer formation and rapid β-cell death. The phenotype observed in these different transgenic lines may be strongly influenced by background strain [76].


review article Mechanism of IAPP Toxicity The precise mechanism by which IAPP aggregates induce β-cell death is unknown. Synthetic human IAPP at concentrations above the threshold for IAPP aggregation (low micromolar) is toxic to β-cells, inducing β-cell apoptosis. A form of the peptide that is capable of aggregating is critical for toxicity, as rodent and porcine IAPP (which are soluble) are not toxic. Toxicity of IAPP aggregates to β-cells requires interaction with the cell membrane and may involve creation of pores in βcell membranes, allowing entry of calcium and induction of apoptotic pathways. Candidate mediators of IAPP-induced apoptosis include caspases, the JNK pathway [77], oxidative [78] and ER stress [24], and induction of Fas [79]. Induction of β-cell death by IAPP aggregates probably involves similar mechanisms to that of other amyloidogenic peptides, for example, the toxic mechanism of aggregates of Aβ on neurons in Alzheimer’s disease. Indeed, IAPP aggregates are toxic to not just β-cells but others cells as well, including neurons [19]. Nonetheless, amyloid-induced islet cell loss in type 2 diabetes appears to be β-cell specific [18]. One explanation for this may be that β-cells are more sensitive to IAPP-induced death than αcells [80]. The β-cell specificity of IAPP-induced islet cell death probably also arises from the close proximity of extracellular aggregates to the β-cell, or alternatively if aggregation initiates intracellularly, because IAPP is expressed specifically in β-cells. Mounting evidence indicates that smaller ‘oligomeric’ aggregates of IAPP are the most toxic aggregated form. Amyloid fibrils generated from synthetic human IAPP in vitro lose their toxicity to β-cells over time, as fibrils elongate. This possibility has led one to suggest that full-blown amyloid plaques are inert [24]. While fibrillar aggregates may be less able to directly cause membrane disruption and β-cell death, it seems probable that amyloid within the islet, even if not overtly toxic to β-cells, is disruptive to islet function and delivery of substrate to the β-cell from the islet vasculature. Amyloid has been shown to correlate with β-cell apoptosis and reduced β-cell mass in humans [18]. We have used thioflavin S positive amyloid as an end-point measure of IAPP aggregation in a cultured human islet model and found that inhibition of amyloid formation correlates well with that of β-cell death [20,21]. If the pathway from IAPP monomer through toxic prefibrillar aggregate to full-blown amyloid is a linear one, as we believe [45,81], then measuring amyloid by light microscopy (with thioflavin S or congo red) should provide a reasonable surrogate end-point measure of any toxic species. Some evidence has been provided to suggest that oligomer formation is an ‘off-pathway’ phenomenon [82], but the difficulties in demonstrating this experimentally have been shown by others [81] and debated at length elsewhere [25,69]. Complicating our understanding of the pathway of IAPP aggregation is evidence that the earliest aggregates may not be β-pleated sheet-derived oligomers but α-helical structures [83]. Delineation of the IAPP aggregation pathway and characterization of the toxic species is of considerable importance to the field and the development of therapeutics, because if an inhibitor of amyloid formation acted downstream of the formation of toxic aggregates, it could prevent full-blown amyloid from forming yet cause a ‘back-up’ of the pathway and an increase in the proportion of toxic species.

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review article IAPP and Inflammation Islet inflammation is being increasingly recognized as a major contributor to β-cell decline in type 2 diabetes. An increase in the number of islet macrophages has been shown in the islet in type 2 diabetes [6], and inhibition of IL-1 action by the IL-1 receptor (IL-1R) antagonist anakinra was found to improve glycaemia and β-cell function in patients with type 2 diabetes [8]. Recent evidence from our group [22] and others [23] strongly implicates amyloid-induced inflammation as an indirect pathway by which IAPP aggregation causes β-cell dysfunction and death. Masters et al. [23] showed that human IAPP aggregates activate the NLRP3 inflammasome in bone marrow-derived dendritic cells, leading to processing of proIL1β induced by other stimuli such as high glucose or oxidated low-density lipoprotein (LDL). We found that IAPP aggregates induce the release of chemokines such as MCP-1 from β-cells, and that these chemokines may promote monocyte recruitment to the islet [22]. We also found that bone-marrow derived macrophages were activated by IAPP aggregates to produce pro-inflammatory cytokines, including IL-1β, by a mechanism dependent on the activity of MyD88, an intracellular mediator of IL-1R and toll-like receptor (TLR) function [22]. Thus, we found that IAPP is capable of activating both NF-κB, perhaps via a TLR-mediated pathway as described for other amyloidogenic peptides, to stimulate pro-IL1β synthesis (signal 1), in addition to the NLRP3 inflammasome to stimulate proIL1β processing and secretion (signal 2). Interestingly, in both studies, early prefibrillar aggregates were much more effective at inducing pro-inflammatory cytokine release than were fibrillar aggregates [22,23]. IAPP aggregates therefore probably recruit macrophages to the islet in type 2 diabetes or act on resident islet macrophages to promote a pro-inflammatory milieu. To understand whether IAPP-induced inflammation might be of importance to β-cell dysfunction in vivo, we used transplantation of human IAPP expressing transgenic islets as a model of rapid islet amyloid formation in vivo and observed that these islets developed amyloid associated with macrophage infiltration [22]. Recipients of human IAPPexpressing islet grafts also had impaired glucose tolerance compared to recipients of wild-type islets, and treatment with IL-1 receptor antagonist (IL-1Ra) improved glycaemia in these graft recipients. Histological examination of the grafts revealed decreased inflammation in IL-1Ra-treated recipients and, notably, decreased amyloid formation, suggesting that amyloid-induced inflammation might further stimulate IAPP aggregation in a feed-forward cycle. These recent reports thus link two important pathologies in type 2 diabetes – amyloid and inflammation – and open a new direction in islet amyloid pathophysiology.

The ‘Hyper-stimulated’ β-Cell and Islet Amyloid In prediabetes when impaired fasting glucose and glucose tolerance is present, and in insulin resistance associated with obesity, there is increased secretory demand on the β-cell. Because IAPP and insulin synthesis and secretion

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are largely co-regulated, it follows that concomitant with the hyperinsulinaemia of obesity and type 2 diabetes that precedes β-cell failure, there is increased IAPP production and secretion. Indeed, increased circulating IAPP levels – socalled hyper-amylinaemia – are present in insulin resistant states [84]. Thus, in the context of the hyper-stimulated βcell, one might predict that simply by mass action, increased availability of IAPP to form aggregates could lead to amyloid formation. Indeed, under conditions of increased production of IAPP, amyloid formation has been observed to occur not only in transgenic rodents, cell models and in cultured islets, but in humans as well. For example, in an autopsy study of pancreas from a lipodystrophic patient who exhibited profound insulin resistance associated with ectopic fat deposition and compensatory hyperinsulinaemia, extensive islet amyloid was observed [85]. Increased islet amyloid was found in cats treated with sulphonylureas, suggesting that increased IAPP secretion can enhance amyloid deposition in vivo [86], and suggesting a possible contributing mechanism to sulphonylurea failure in type 2 diabetic patients. If secretory stress and increased production of IAPP indeed contributes to islet amyloid formation, it follows that relieving such stress, for example, by administration of insulin sensitizers to rest β-cells, would be predicted to decrease amyloid load. Indeed, when rosiglitazone or metformin was administered to human IAPP transgenic mice fed a high-fat diet, both agents were found to markedly reduce the prevalence and severity of islet amyloid [87]. Although the simplest explanation for these data is that these compounds relieved secretory stress on the β-cell, resulting in decreased IAPP production and secretion, complicating this interpretation are data showing direct effects of these compounds on β-cells. Thiazolidinediones, in particular, play a role in regulating islet cholesterol via PPARγ upregulation of ABCA1 and β-cell cholesterol levels [3,4]. In transgenic rodent models of human IAPP expression in β-cells, doubling transgene copy number by the generation of homozygous animals has, in most cases, enhanced the phenotype, although not always resulting in the typical pathology observed in type 2 diabetes. As mentioned earlier, on the FVB background, homozygous human IAPP transgenic mice develop rapid β-cell death associated with ER stress and no visible amyloid. Although this has been interpreted as evidence for intracellular aggregation and oligomeric IAPP inducing cell death, it is plausible that in this background strain high levels of human IAPP production result in an unfolded protein response and β-cell death. It seems clear, then, that marked overproduction of human IAPP in transgenic mice can induce a phenotype of β-cell loss, but it is less clear how well this recapitulates what is happening in diabetic humans. Indeed, simple overproduction of IAPP as a path to amyloid formation seems simplistic, and it seems likely that other mechanisms must also be at play. Obese, insulin resistant humans with high circulating levels of IAPP do not necessarily develop amyloid. In transgenic rodent models of β-cell overexpression of human IAPP, there have been a number of reported instances where increasing expression was not sufficient to induce amyloid formation. In our hands, mice homozygous for the human IAPP transgene, with twice the number of copies of the transgene


review article


and increased production, did not develop amyloid, nor did animals treated with nicotinic acid to generate insulin resistance [88]. These mice had marked increases in circulating IAPP and insulin and increased islet size, suggesting IAPP overproduction associated with β-cell compensation for the insulin resistance. Yet these mice remained normoglycaemic and had only trace amounts of detectable amyloid. These results suggest that increased IAPP production alone is unlikely the primary cause of islet amyloid formation and that some underlying β-cell dysfunction must also be present.

The Hyper-stimulated β-Cell in Cultured Islets Amyloid forms rapidly in cultured islets, whether from human donors or from transgenic rodents expressing human IAPP. Hull and colleagues showed clearly that amyloid formation in cultured human IAPP-expressing islets was stimulated by higher glucose concentrations [89]. In the HIP-RAT, we similarly see a striking increase in amyloid associated with the glucose concentration in culture (figure 1). In our studies aimed at developing ways to inhibit amyloid formation, we routinely use cultured human and human IAPP-expressing islets and culture these islets in high glucose concentrations. Amyloid appears to form in culture even in ‘normoglycemic’ conditions – 11 mM for mouse islets and 5.5 mM for human – but is clearly increased with increasing glucose. Of course, glucose stimulation of IAPP synthesis and secretion may not be the sole driver of amyloid formation in this model. Isolated and cultured islets are subject to βcell dysfunction, and elevated glucose in culture may in addition induce a number of other changes in the β-cell, including potential defects in prohormone processing and trafficking. A recent report from Kahn and colleagues pointed to an important role for the amount of IAPP being produced and secreted, at least in a transgenic islet culture model. Increasing IAPP secretion in cultured human IAPP transgenic islets by addition of KCl or exendin-4 markedly increased islet amyloid formation, whereas inhibition of IAPP secretion with diazoxide or somatostatin almost completely suppressed amyloid deposition [71]. As KCl-induced depolarization primarily stimulates β-cell secretion and not synthesis, and conversely diazoxide blocks β-cell secretion with little impact on synthesis, these data indirectly suggest that IAPP secretion is needed for amyloid formation to occur, and by extension that aggregation occurs extracellularly. Indeed, the finding that intracellular IAPP content is increased when secretion is blocked with KCl or diazoxide during high glucose culture speaks against intracellular accumulation of IAPP as a cause of amyloid formation. In further support of this possibility, our finding that peptide inhibitors of IAPP aggregation – which probably do not cross the β-cell membrane – not only suppress islet amyloid formation but also amyloid-associated cell death [20], supports the idea that IAPP aggregation and toxicity to β-cells occur extracellularly. These data do not rule out, however, initiation of IAPP aggregation within the


A

B

Figure 1. Effect of glucose on islet amyloid formation in cultured islets from human IAPP expressing transgenic rats. Islets were isolated from rats with β-cell expression of human islet amyloid polypeptide and cultured for 7 days in medium containing either 11 or 22 mM glucose. Sections of paraffin-embedded islets were (A) stained for amyloid (thioflavin S; green), insulin (red), or nuclei (DAPI; blue) and (B) amyloid (thioflavin S-positive) area quantified as a proportion of total islet area. *p < 0.05 by Student’s t-test.

cell, but rather point to extracellular events being important contributors to aggregation and toxicity. Amyloid formation during pretransplant culture of human islets may limit the quality and viability of islet material available for transplant. Low (22 ◦ C) temperature culture of human islets may slow amyloid formation by decreasing β-cell metabolism and IAPP production and secretion. Culture of islets with inhibitors of IAPP aggregation or synthesis might also be of benefit prior to transplantation.

Islet Transplantation and Amyloid Human islet transplants are subject to rapid islet amyloid formation associated with β-cell loss when transplanted either into the kidney capsule [38,90] or the liver [91]. Islets from human IAPP-expressing transgenic mouse donors also develop amyloid, and fare less well than do grafts from non-transgenic donors. Accumulation of amyloid associated with β-cell loss was recently reported in intrahepatic islet grafts in non-human primates [92]. Notably, study of islet graft tissue from the liver of a type 1 diabetic recipient of an islet transplant obtained at autopsy revealed marked amyloid deposition, yet no amyloid was detectable in the islets at time of transplantation [93]. Collectively, these data suggest that islet amyloid may be an unrecognized contributor to islet transplant failure.

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review article Why does amyloid form rapidly in cultured and transplanted islets? In cultured human islets amyloid can be detected by 1 week of culture and following transplantation into immune-deficient diabetic mice marked amyloid deposition is observed at 8 weeks of age. One explanation may be that following isolation and removal from the pancreas, islets are not vascularly perfused and secreted IAPP cannot diffuse rapidly outside of the islet and therefore has more opportunity to aggregate in the extracellular space. β-Cell dysfunction is also likely present in both cultured and transplanted islets, in part due to the stress of the isolation procedure, lack of innervation and vascular delivery of nutrients. Transplanted islets in humans have defects in β-cell function including decreased glucose-stimulated insulin release [94] and impaired proinsulin processing [95], similar to patients with type 2 diabetes. It is therefore possible that the same β-cell defects that underlie amyloid formation in type 2 diabetes also contribute to the lesion in transplanted islets. Because insufficient islets are obtained from a single donor in clinical islet transplants, multiple donors are required for most diabetic recipients to achieve euglycaemia. It follows that an insufficient β-cell mass will be present in most graft recipients, at least until additional transplants are performed. Insufficient transplanted β-cell mass is likely to lead to increased secretory demand and metabolic stress on the transplanted islet, increasing (pro)IAPP production and exacerbating defects in β-cell function that may underlie amyloid deposition.

Summary Islet amyloid formation in type 2 diabetes and in transplanted islets probably occurs as a combination of both defective handling by the β-cell of IAPP and its precursors, as


well as increased production of IAPP driven by β-cell ‘hyper-stimulation’ in the presence of insulin resistance, hyperglycaemia and insufficient β-cell mass. A feed-forward cycle probably results, wherein increasing hyperglycaemia worsens β-cell dysfunction and increases the drive for IAPP production and secretion. Aggregates of IAPP incite islet inflammation, likely by attracting macrophages and inducing production of pro-inflammatory cytokines. These cytokines, in particular IL-1β, contribute to this feed-forward cycle by inducing further β-cell dysfunction and islet amyloid formation. This model (figure 2) suggests several potential points of intervention in diabetes as means of preventing islet amyloid formation and associated β-cell death, including insulin sensitizers, anti-inflammatory drugs and inhibitors of IAPP synthesis and aggregation.

Acknowledgements The authors wish to acknowledge funding support from the Canadian Institutes of Health Research (CIHR; MOP-14862) and the Canadian Diabetes Association (CDA; OG-3-11-3413CV) for the studies described in this manuscript. C. Bruce Verchere is an investigator of the Child & Family Research Institute and the Irving K Barber Chair in Diabetes Research at the University of British Columbia. J. Montane is supported by a post-doctoral fellowship from the Juvenile Diabetes Research Foundation, A. Klimek-Abercrombie by the CIHR Transplant Training Program, C. Westwell-Roper by a Vanier Canada Graduate Scholarship, and K. J. Potter by a Rx&D/CIHR MD PhD Studentship and CDA.

Conflict of Interest The authors do not declare any conflict of interest relevant to this manuscript.

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Figure 2. Model of drivers for islet amyloid formation and β-cell dysfunction and death in type 2 diabetes and islet transplants. The model predicts a feed-forward cycle of metabolic stress caused by insulin resistance and hyperglycaemia exacerbating amyloid formation and islet inflammation, which will lead to β-cell dysfunction and death, and in turn worsening hypergycaemia and metabolic stress on remaining β-cells.

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