Islet Amyloid Formation Research Articles

Efficient Vascular and Neural Engraftment of Stem Cell-Derived Islets.

Pluripotent stem cell-derived islets (SC-islets) now emerge as a new source for beta-cell replacement therapy. While the function of human islet transplants is hampered by excessive cell death post-transplantation, contributing factors include inflammatory reactions, insufficient revascularization and islet amyloid formation, there is a gap in knowledge on the engraftment process of the SC-islets. In this experimental study, we investigated the engraftment capability of SC-islets at three months post-transplantation, and observed that the cell apoptosis rates were lower, but the vascular density was similar in SC-islets to that of human islets. While the human islet transplant vascular structures were a mixture of remnant donor endothelium and ingrowing blood vessels, the SC-islets contained ingrowing blood vessels only. The oxygenation of the SC-islet grafts was twice as high as in the corresponding grafts of human islets, suggesting better vascular functionality. Similar to the blood vessel ingrowth, also the reinnervation of the SC-islets was four- to five-fold higher than the human islets. Both SC-islets and the human islets contained amyloid at one and three months post-transplantation. We conclude that the vascular and neural engraftment of SC-islets is superior to human islets, but that grafts of both origins develop amyloid with potential long-term consequences.

Human islet amyloid polypeptide-induced β-cell cytotoxicity is linked to formation of α-sheet structure.

Type 2 diabetes (T2D) results from insulin secretory dysfunction arising in part from the loss of pancreatic islet β-cells. Several factors contribute to β-cell loss, including islet amyloid formation, which is observed in over 90% of individuals with T2D. The amyloid is comprised of human islet amyloid polypeptide (hIAPP). Here we provide evidence that early in aggregation, hIAPP forms toxic oligomers prior to formation of amyloid fibrils. The toxic oligomers contain α-sheet secondary structure, a nonstandard secondary structure associated with toxic oligomers in other amyloid diseases. De novo, synthetic α-sheet compounds designed to be nontoxic and complementary to the α-sheet structure in the toxic oligomers inhibit hIAPP aggregation and neutralize oligomer-mediated cytotoxicity in cell-based assays. In vivo administration of an α-sheet design to mice for 4 weeks revealed no evidence of toxicity nor did it elicit an immune response. Furthermore, the α-sheet designs reduced endogenous islet amyloid formation and mitigation of amyloid-associated β-cell loss in cultured islets isolated from an hIAPP transgenic mouse model of islet amyloidosis. Characterization of the involvement of α-sheet in early aggregation of hIAPP and oligomer toxicity contributes to elucidation of the molecular mechanisms underlying amyloid-associated β-cell loss.

RIPK3 promotes islet amyloid-induced β-cell loss and glucose intolerance in a humanized mouse model of type 2 diabetes

ObjectiveAggregation of human islet amyloid polypeptide (hIAPP), a β-cell secretory product, leads to islet amyloid deposition, islet inflammation and β-cell loss in type 2 diabetes (T2D), but the mechanisms that underlie this process are incompletely understood. Receptor interacting protein kinase 3 (RIPK3) is a pro-death signaling molecule that has recently been implicated in amyloid-associated brain pathology and β-cell cytotoxicity. Here, we evaluated the role of RIPK3 in amyloid-induced β-cell loss using a humanized mouse model of T2D that expresses hIAPP and is prone to islet amyloid formation. MethodsWe quantified amyloid deposition, cell death and caspase 3/7 activity in islets isolated from WT, Ripk3−/−, hIAPP and hIAPP; Ripk3−/− mice in real time, and evaluated hIAPP-stimulated inflammation in WT and Ripk3−/− bone marrow derived macrophages (BMDMs) in vitro. We also characterized the role of RIPK3 in glucose stimulated insulin secretion (GSIS) in vitro and in vivo. Finally, we examined the role of RIPK3 in high fat diet (HFD)-induced islet amyloid deposition, β-cell loss and glucose homeostasis in vivo. ResultsWe found that amyloid-prone hIAPP mouse islets exhibited increased cell death and caspase 3/7 activity compared to amyloid-free WT islets in vitro, and this was associated with increased RIPK3 expression. hIAPP; Ripk3−/− islets were protected from amyloid-induced cell death compared to hIAPP islets in vitro, although amyloid deposition and caspase 3/7 activity were not different between genotypes. We observed that macrophages are a source of Ripk3 expression in isolated islets, and that Ripk3−/− BMDMs were protected from hIAPP-stimulated inflammatory gene expression (Tnf, Il1b, Nos2). Following 52 weeks of HFD feeding, islet amyloid-prone hIAPP mice exhibited impaired glucose tolerance and decreased β-cell area compared to WT mice in vivo, whereas hIAPP; Ripk3−/− mice were protected from these impairments. ConclusionsIn conclusion, loss of RIPK3 protects from amyloid-induced inflammation and islet cell death in vitro and amyloid-induced β-cell loss and glucose intolerance in vivo. We propose that therapies targeting RIPK3 may reduce islet inflammation and β-cell loss and improve glucose homeostasis in the pathogenesis of T2D.

On the importance of being amidated: Analysis of the role of the conserved C-terminal amide of amylin in amyloid formation and cytotoxicity

The polypeptide hormone Amylin (also known as islet amyloid polypeptide) plays a role in regulation of glucose metabolism, but forms pancreatic islet amyloid deposits in type 2 diabetes. The process of islet amyloid formation contributes to β-cell dysfunction and the development of the disease. Amylin is produced as a pro-from and undergoes processing prior to secretion. The mature hormone contains an amidated C-terminus. Analysis of an alignment of vertebrate amylin sequences reveals that the processing signal for amidation is strictly conserved. Furthermore, the enzyme responsible for C-terminal amidation is found in all of these organisms. Comparison of the physiologically relevant amidated form to a variant with a free C-terminus (Amylin-COO−) shows that replacement of the C-terminal amide with a carboxylate slows, but does not prevent amyloid formation. Pre-fibrillar species produced by both variants are toxic to cultured β-cells, although hAmylin-COO− is moderately less so. Amyloid fibrils produced by either peptide are not toxic. Prior work (ACS Pharmacol. Translational. Sci. 1, 132–49 (2018)) shows that Amylin- COO− exhibits a 58-fold reduction in activation of the Amylin1 receptor and 20-fold reduction in activation of the Amylin3 receptor. Thus, hAmylin-COO− exhibits significant toxicity, but significantly reduced activity and offers a reagent for studies which aim to decouple hAmylin's toxic effects from its activity. The different behaviours of free and C-terminal amidated Amylin should be considered when designing systems to produce the polypeptide recombinantly.

Regional differences in islet amyloid deposition in the residual pancreas with new-onset diabetes secondary to pancreatic ductal adenocarcinoma.

Islet amyloid deposition and reduced β-cell mass are pathological hallmarks in type 2 diabetes mellitus subjects. To date, the pathological features of the islets in diabetes secondary to pancreatic ductal adenocarcinoma (PDAC) have not been specifically addressed. To provide further insight into the relationship between islet amyloid deposition of the residual pancreas in PDAC patients and to explore whether regional differences (proximal vs distal residual pancreas) are associated with islet amyloid deposition. We retrospectively collected clinical information and pancreatic tissue removed from tumors of 45 PDAC patients, including 14 patients with normal glucose tolerance (NGT), 16 patients with prediabetes and 15 new-onset diabetes (NOD) patients diagnosed before surgery by an oral glucose tolerance test at West China Hospital from July 2017 to June 2020. Pancreatic volume was calculated by multiplying the estimated area of pancreatic tissue on each image slice by the interval between slices based on abdominal computer tomography scans. Several sections of paraffin-embedded pancreas specimens from both the proximal and/or distal regions remote from the tumor were stained as follows: (1) Hematoxylin and eosin for general histological appearance; (2) hematoxylin and insulin for the determination of fractional β-cell area (immunohistochemistry); and (3) quadruple insulin, glucagon, thioflavin T and DAPI staining for the determination of β-cell area, α-cell area and amyloid deposits. Screening for pancreatic histologic features revealed that duct obstruction with islet amyloid deposition, fibrosis and marked acinar atrophy were robust in the distal pancreatic regions but much less robust in the proximal regions, especially in the prediabetes and NOD groups. Consistent with this finding, the remnant pancreatic volume was markedly decreased in the NOD group by nearly one-half compared with that in the NGT group (37.35 ± 12.16 cm3 vs 69.79 ± 18.17 cm3, P < 0.001). As expected, islets that stained positive for amyloid (islet amyloid density) were found in the majority of PDAC cases. The proportion of amyloid/islet area (severity of amyloid deposition) was significantly higher in both prediabetes and NOD patients than in NGT patients (P = 0.002; P < 0.0001, respectively). We further examined the regional differences in islet amyloid deposits. Islet amyloid deposit density was robustly increased by approximately 8-fold in the distal regions compared with that in the proximal regions in the prediabetes and NOD groups (3.98% ± 3.39% vs 0.50% ± 0.72%, P = 0.01; 12.03% vs 1.51%, P = 0.001, respectively). In conclusion, these findings suggest that robust alterations of the distal pancreas due to tumors can disturb islet function and structure with islet amyloid formation, which may be associated with the pathogenesis of NOD secondary to PDAC.

1724-P: Caspase 3/7 Activation and RIPK3 Are Dispensable for Synthetic hIAPP-Induced ß-Cell Death

Islet amyloid is a hallmark of type 2 diabetes that is associated with β-cell loss. Aggregation of the β-cell secretory product human islet amyloid polypeptide (hIAPP) to form islet amyloid promotes β-cell death 1) via direct effects on the β cell and 2) by stimulating islet immune cell cytokine production. We recently showed that β cells are susceptible to TNFα-induced necroptosis, a form of caspase-independent, receptor interacting protein kinase 3 (RIPK3)-mediated cell death. We hypothesized that RIPK3 also promotes β-cell death in response to islet amyloid formation. To test this hypothesis, we evaluated INS-1, NIT-1 and isolated mouse islet cells treated with synthetic hIAPP (20 μM) +/− the pan-caspase inhibitor zVAD (40 μM). We found synthetic hIAPP increased cell death and caspase activity in INS-1, NIT-1 and mouse islet cells over 48 h. Remarkably, inhibition of caspases with zVAD (as measured by caspase 3/7 activity) failed to reduce synthetic hIAPP-mediated cell death in INS-1 (n=3, p=0.99), NIT-1 (n=3, p=0.99) or mouse islet cells (n=4, p=0.98). To evaluate RIPK3 in hIAPP-induced β-cell death, we examined a small molecule RIPK3 inhibitor (GSK’872, 5 μM), gene-edited NIT-1 Ripk3Δ cells (gRNA targeting exon 6), and intact isolated Ripk3−/− mouse islets. We found GSK’872 failed to protect INS-1 (n=3, p=0.32) or NIT-1 (n=3, p=0.30) cells from hIAPP-induced cell death over 48 h. Similarly, neither NIT-1 Ripk3Δ cells (n=2, hIAPP: p=0.82, hIAPP+zVAD: p=0.97) nor Ripk3−/− islets (n=4, hIAPP: p=0.63, hIAPP+zVAD: p=0.86) were protected from synthetic hIAPP-induced cell death either when caspases were active or inhibited. Our data indicate that caspase 3/7 activity and RIPK3 are dispensable for synthetic hIAPP-induced β-cell death in vitro. However, we found that RIPK3 is upregulated in response to endogenous islet amyloid formation in vitro. Future studies will determine whether RIPK3 promotes β-cell loss in part via effects on endogenous amyloid-induced cytokine signaling. Disclosure N.Mukherjee: None. C.J.Contreras: None. L.Lin: None. E.P.Cai: None. S.E.Kahn: Advisory Panel; Anji Pharmaceuticals, Bayer Inc., Boehringer Ingelheim Inc., Eli Lilly and Company, Merck &amp; Co., Inc., Other Relationship; Novo Nordisk. A.T.Templin: None. Funding U.S. Department of Veterans Affairs (IK2BX004659 to A.T.T.); National Institutes of Health (T32DK064466 to C.J.C.); Indiana University School of Medicine (to N.M.)

On the plasticity of amyloid formation: The impact of destabilizing small to large substitutions on islet amyloid polypeptide amyloid formation.

Amyloids are partially ordered, proteinaceous, β-sheet rich deposits that have been implicated in a wide range of diseases. An even larger set of proteins that do not normally form amyloid in vivo can be induced to do so in vitro. A growing number of structures of amyloid fibrils have been reported and a common feature is the presence of a tightly packed core region in which adjacent monomers pack together in extremely tight interfaces, often referred to as steric zippers. A second common feature of many amyloid fibrils is their polymorphous nature. We examine the consequences of disrupting the tight packing in amyloid fibrils on the kinetics of their formation using the 37 residue polypeptide hormone islet amyloid polypeptide (IAPP, amylin) as a model system. IAPP forms islet amyloid in vivo and is aggressively amyloidogenic in vitro. Six Cryo-EM structures of IAPP amyloid fibrils are available and in all Gly24 is in the core of the structured region and makes tight contacts with other residues. Calculations using the ff14SBonlysc forcefield in Amber20 show that substitutions with larger amino acids significantly disrupt close packing and are predicted to destabilize the various fibril structures. However, Gly to 2-amino butyric acid (2-carbon side chain) and Gly to Leu substitutions actually enhance the rate of amyloid formation. A Pro substitution slows, but does not prevent amyloid formation.

Differential Effects of Aromatic Residues on Amyloid Formation and Cytotoxicity of Human IAPP.

Islet amyloid polypeptide (IAPP) is a 37-residue polypeptide hormone secreted by the pancreatic β-cells. IAPP plays a role in glycemic regulation, but in the pre-type-2 diabetic state, it aggregates to form an islet amyloid. The process of islet amyloid formation contributes to β-cell dysfunction and disease progression. The features of the IAPP sequence that modulate amyloid formation are still not understood. Human IAPP contains three aromatic residues, F15, F23, and Y37. F15 and Y37 are highly conserved, while F23 is more commonly a Leu or Ile in other species. The role of the aromatic residues in modulating the time course of amyloid formation and the cytotoxicity was examined using aromatic to Leu mutations. All three single and double mutants and the triple mutant were studied. F23 plays a dominant role in both amyloid formation and toxicity. An F15L mutant accelerated amyloid formation, a Y37L mutant had little effect, while an F23L replacement slowed amyloid formation by a factor of 2.6. Double mutants, which contained an F23L replacement, had a larger effect than those that did not, and there are non-additive effects between pairs of aromatic residues. F23 also plays a key role in toxicity. Single or multiple mutants that contain the F23L replacement were noticeably less toxic than the wild-type or mutants which did not include the F23L substitution. In contrast, the F15L mutant was more toxic than the wild-type one. The implications for IAPP amyloid formation and for the design of non-aggregating analogues of IAPP are discussed.

Rosiglitazone protects INS-1E cells from human islet amyloid polypeptide toxicity

Human islet amyloid polypeptide (hIAPP or amylin) is a hormone co-secreted with insulin by pancreatic β-cells, and is the main component of islet amyloid. Islet amyloid is found in the pancreas of patients with type 2 diabetes and may be involved in β-cell dysfunction and death, observed in this disease. Thus, counteracting islet amyloid toxicity represents a therapeutic approach to preserve β-cell mass and function. In this sense, thiazolidinediones (TZDs), as rosiglitazone, have shown protective effects against other harmful insults to β-cells. For this reason, we investigated whether rosiglitazone could protect β-cells from hIAPP-induced cell death and the underlying mechanisms mediating such effect. Here, we show that rosiglitazone improved the viability of hIAPP-exposed INS-1E cells. This benefit is not dependent on the insulin-degrading enzyme (IDE) since rosiglitazone did not modulate IDE protein content and activity. However, rosiglitazone inhibited hIAPP fibrillation and decreased hIAPP-induced expression of C/EBP homologous protein (CHOP) (CTL 100.0 ± 8.4; hIAPP 182.7 ± 19.1; hIAPP + RGZ 102.8 ± 9.5), activating transcription factor-4 (ATF4) (CTL 100.0 ± 3.1; hIAPP 234.9 ± 19.3; hIAPP + RGZ 129.6 ± 3.0) and phospho-eukaryotic initiation factor 2-alpha (p-eIF2α) (CTL 100.0 ± 31.1; hIAPP 234.1 ± 36.2; hIAPP + RGZ 150.4 ± 18.0). These findings suggest that TZDs treatment may be a promising approach to preserve β-cell mass and function by inhibiting islet amyloid formation and decreasing endoplasmic reticulum stress hIAPP-induced.

β-Cell Death in Diabetes: Past Discoveries, Present Understanding, and Potential Future Advances.

β-cell death is regarded as a major event driving loss of insulin secretion and hyperglycemia in both type 1 and type 2 diabetes mellitus. In this review, we explore past, present, and potential future advances in our understanding of the mechanisms that promote β-cell death in diabetes, with a focus on the primary literature. We first review discoveries of insulin insufficiency, β-cell loss, and β-cell death in human diabetes. We discuss findings in humans and mouse models of diabetes related to autoimmune-associated β-cell loss and the roles of autoreactive T cells, B cells, and the β cell itself in this process. We review discoveries of the molecular mechanisms that underlie β-cell death-inducing stimuli, including proinflammatory cytokines, islet amyloid formation, ER stress, oxidative stress, glucotoxicity, and lipotoxicity. Finally, we explore recent perspectives on β-cell death in diabetes, including: (1) the role of the β cell in its own demise, (2) methods and terminology for identifying diverse mechanisms of β-cell death, and (3) whether non-canonical forms of β-cell death, such as regulated necrosis, contribute to islet inflammation and β-cell loss in diabetes. We believe new perspectives on the mechanisms of β-cell death in diabetes will provide a better understanding of this pathological process and may lead to new therapeutic strategies to protect β cells in the setting of diabetes.

Islet amyloid toxicity: From genesis to counteracting mechanisms.

Islet amyloid polypeptide (IAPP or amylin) is a hormone co-secreted with insulin by pancreatic β-cells and is the major component of islet amyloid. Islet amyloid is found in the pancreas of patients with type 2 diabetes (T2D) and may be involved in β-cell dysfunction and death, observed in this disease. Thus, investigating the aspects related to amyloid formation is relevant to the development of strategies towards β-cell protection. In this sense, IAPP misprocessing, IAPP overproduction, and disturbances in intra- and extracellular environments seem to be decisive for IAPP to form islet amyloid. Islet amyloid toxicity in β-cells may be triggered in intra- and/or extracellular sites by membrane damage, endoplasmic reticulum stress, autophagy disruption, mitochondrial dysfunction, inflammation, and apoptosis. Importantly, different approaches have been suggested to prevent islet amyloid cytotoxicity, from inhibition of IAPP aggregation to attenuation of cell death mechanisms. Such approaches have improved β-cell function and prevented the development of hyperglycemia in animals. Therefore, counteracting islet amyloid may be a promising therapy for T2D treatment.

The role of mitochondrial apoptotic pathway in islet amyloid-induced β-cell death

Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to β-cell death in type 2 diabetes. We previously showed that extracellular hIAPP aggregates promote Fas-mediated β-cell apoptosis. Here, we tested if hIAPP aggregates can trigger the mitochondrial apoptotic pathway (MAP). hIAPP aggregation in Ad-hIAPP transduced INS-1 and human islet β-cells promoted cytochrome c release, caspase-9 activation and apoptosis, which were reduced by Bax inhibitor. Amyloid formation in hIAPP-expressing mouse islets during culture increased caspase-9 activation in β-cells. Ad-hIAPP transduced islets from CytcKA/KA and BaxBak βDKO mice (models of blocked MAP), had lower caspase-9-positive and apoptotic β-cells than transduced wild-type islets, despite comparable amyloid formation. Blocking Fas (markedly) and Bax or caspase-9 (modestly) reduced β-cell death induced by extracellular hIAPP aggregates. These findings suggest a role for MAP in amyloid-induced β-cell death and a potential strategy to reduce intracellular amyloid β-cell toxicity by blocking cytochrome c apoptotic function.

Quantitative, High-Throughput Assays for Proteolytic Degradation of Amylin.

Amylin is a pancreatic peptide hormone that regulates glucose homeostasis but also aggregates to form islet amyloid in type-2 diabetes. Given its role in both health and disease, there is renewed interest in proteolytic degradation of amylin by insulin-degrading enzyme (IDE) and other proteases. Here, we describe the development and detailed characterization of three novel assays for amylin degradation, two based on a fluoresceinated and biotinylated form of rodent amylin (fluorescein-rodent amylin-biotin, FrAB), which can be used for any amylin protease, and another based on an internally quenched fluorogenic substrate (FRET-based amylin, FRAM), which is more specific for IDE. The FrAB-based substrate can be used in a readily implemented fluorescence-based protocol or in a fluorescence polarization (FP)-based protocol that is more amenable to high-throughput screening (HTS), whereas the FRAM substrate has the advantage of permitting continuous monitoring of proteolytic activity. All three assays yield highly quantitative data and are resistant to DMSO, and the FRAM and FP-based FrAB assay are ideally suited to HTS applications.

Role of lysine residue of islet amyloid polypeptide in fibril formation, membrane binding, and inhibitor binding

The aggregation of islet amyloid polypeptide (IAPP) is implicated in the pathogenesis of type 2 diabetes (T2D). In T2D, this peptide aggregates to form amyloid fibrils; the mechanism responsible for islet amyloid formation is unclear. However, it is known that the aggregation propensity of IAPP is highly related to its primary sequence. Several residues have been suggested to be critical in modulating IAPP amyloid formation, but role of the sole lysine residue at position 1 (Lys-1) in IAPP has not been discussed. In our previous study, we found that glycated IAPP can form amyloid faster than normal IAPP and induce normal IAPP to expedite the aggregation process. To gain more insight into the contribution of Lys-1 in the kinetics of fibril formation, we synthesized another two IAPP variants, K1E-IAPP and K1Nle-IAPP, in which the Lys residue was mutated to glutamate and norleucine, respectively. Interestingly, we observed that the negative or neutral charged side chain at this position was preferred for amyloid formation. The findings suggested this residue may take part in the inter- or intra-molecular interaction during IAPP aggregation, even though it was proposed not to be in part of fibril core structure. Our data also revealed that the inhibitory mechanism of some inhibitors for IAPP aggregation require reaction with Lys-1. Modifications of Lys-1, such as protein glycation, may affect the effectiveness of the inhibitory action of some potential drugs in the treatment of amyloidosis.

Low concentration IL-1β promotes islet amyloid formation by increasing hIAPP release from humanised mouse islets in vitro.

Aggregation of the beta cell secretory product human islet amyloid polypeptide (hIAPP) results in islet amyloid deposition, a pathological feature of type 2 diabetes. Amyloid formation is associated with increased levels of islet IL-1β as well as beta cell dysfunction and death, but the mechanisms that promote amyloid deposition in situ remain unclear. We hypothesised that physiologically relevant concentrations of IL-1β stimulate beta cell islet amyloid polypeptide (IAPP) release and promote amyloid formation. We used a humanised mouse model of endogenous beta cell hIAPP expression to examine whether low (pg/ml) concentrations of IL-1β promote islet amyloid formation in vitro. Amyloid-forming islets were cultured for 48h in the presence or absence of IL-1β with or without an IL-1β neutralising antibody. Islet morphology was assessed by immunohistochemistry and islet mRNA expression, hormone content and release were also quantified. Cell-free thioflavin T assays were used to monitor hIAPP aggregation kinetics in the presence and absence of IL-1β. Treatment with a low concentration of IL-1β (4pg/ml) for 48h increased islet amyloid prevalence (93.52 ± 3.89% vs 43.83 ± 9.67% amyloid-containing islets) and amyloid severity (4.45 ± 0.82% vs 2.16 ± 0.50% amyloid area/islet area) in hIAPP-expressing mouse islets in vitro. This effect of IL-1β was reduced when hIAPP-expressing islets were co-treated with an IL-1β neutralising antibody. Cell-free hIAPP aggregation assays showed no effect of IL-1β on hIAPP aggregation in vitro. Low concentration IL-1β did not increase markers of the unfolded protein response (Atf4, Ddit3) or alter proIAPP processing enzyme gene expression (Pcsk1, Pcsk2, Cpe) in hIAPP-expressing islets. However, release of IAPP and insulin were increased over 48h in IL-1β-treated vs control islets (IAPP 0.409 ± 0.082 vs 0.165 ± 0.051pmol/5 islets; insulin 87.5 ± 8.81 vs 48.3 ± 17.3pmol/5 islets), and this effect was blocked by co-treatment with IL-1β neutralising antibody. Under amyloidogenic conditions, physiologically relevant levels of IL-1β promote islet amyloid formation by increasing beta cell release of IAPP. Neutralisation of this effect of IL-1β may decrease the deleterious effects of islet amyloid formation on beta cell function and survival.

Analysis of Amylin Consensus Sequences Suggests That Human Amylin Is Not Optimized to Minimize Amyloid Formation and Provides Clues to Factors That Modulate Amyloidogenicity.

The neuropancreatic polypeptide hormone amylin forms pancreatic islet amyloid in type-2 diabetes. Islet amyloid formation contributes to β-cell death in the disease and to the failure of islet transplants, but the features which influence amylin amyloidogenicity are not understood. We constructed an amino acid sequence alignment of 202 sequences of amylin and used the alignment to design consensus sequences of vertebrate amylins, mammalian amylins, and primate amylins. Amylin is highly conserved, but there are differences between human amylin and each consensus sequence, ranging from one to six substitutions. Biophysical analysis shows that all of the consensus sequences form amyloid but do so more slowly than human amylin in vitro. The rate of amyloid formation by the primate consensus sequence is 3- to 4-fold slower than human amylin; the mammalian consensus sequence is approximately 20- to 25-fold slower, and the vertebrate consensus sequence is approximately 6-fold slower. All of the consensus sequences are moderately less toxic than human amylin toward a cultured β-cell line, with the vertebrate consensus sequence displaying the largest reduction in toxicity of 3- to 4-fold. All of the consensus sequences activate a human amylin receptor and exhibit only modest reductions in activity, ranging from 3- to 4-fold as judged by a cAMP production assay. The analysis argues that there is no strong selective evolutionary pressure to avoid the formation of islet amyloid and provides information relevant to the design of less amyloidogenic amylin variants.

Islet Amyloid in Patients With Diabetes Due to Exocrine Pancreatic Disorders, Type 2 Diabetes, and Nondiabetic Patients.

Amyloid deposits are a typical finding in pancreatic islets from patients with type 2 diabetes. Whether this is linked to the pathogenesis of type 2 diabetes is currently unknown. Therefore, we compared the occurrence of islet amyloid in patients with type 2 diabetes, diabetes secondary to pancreatic disorders, and nondiabetic individuals. Pancreatic tissue from 15 nondiabetic patients, 22 patients with type 2 diabetes, and 11 patients with diabetes due to exocrine pancreatic disorders (chronic pancreatitis, pancreatic carcinoma) were stained for insulin, amyloid, and apoptosis. β-cell area, amyloid deposits, and β-cell apoptosis were quantified by morphometric analysis. The proportion of islets containing amyloid deposits was significantly higher in both type 2 diabetes and diabetes due to exocrine pancreatic disorders than in healthy subjects. Islets with both amyloid and apoptosis were observed more frequently in type 2 diabetes and significantly more so in diabetes due to exocrine pancreatic disorders. In both diabetic groups, apoptotic ß-cells were found significantly more frequently in islets with more prominent amyloid deposits. The occurrence of amyloid deposits in both type 2 diabetes and diabetes secondary to exocrine pancreatic disorders suggests that islet amyloid formation is a common feature of diabetes mellitus of different etiologies and may be associated with a loss of pancreatic ß-cells.

PAM haploinsufficiency does not accelerate the development of diet- and human IAPP-induced diabetes in mice.

Peptide hormones are first synthesised as larger, inactive precursors that are converted to their active forms by endopeptidase cleavage and post-translational modifications, such as amidation. Recent, large-scale genome-wide studies have suggested that two coding variants of the amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), are associated with impaired insulin secretion and increased type 2 diabetes risk. We aimed to elucidate the role of PAM in modulating beta cell peptide amidation, beta cell function and the development of diabetes. PAM transcript and protein levels were analysed in mouse islets following induction of endoplasmic reticulum (ER) or cytokine stress, and PAM expression patterns were examined in human islets. To study whether haploinsufficiency of PAM accelerates the development of diabetes, Pam+/- and Pam+/+ mice were fed a low-fat diet (LFD) or high-fat diet (HFD) and glucose homeostasis was assessed. Since aggregates of the PAM substrate human islet amyloid polypeptide (hIAPP) lead to islet inflammation and beta cell failure, we also investigated whether PAM haploinsufficiency accelerated hIAPP-induced diabetes and islet amyloid formation in Pam+/- and Pam+/+ mice with beta cell expression of hIAPP. Immunostaining revealed high expression of PAM in alpha, beta and delta cells in human pancreatic islets. Pam mRNA and PAM protein expression were reduced in mouse islets following administration of an HFD, and in isolated islets following induction of ER stress with thapsigargin, or cytokine stress with IL-1β, IFN-γ and TFN-α. Despite Pam+/- only having 50% PAM expression and enzyme activity as compared with Pam+/+ mice, glucose tolerance and body mass composition were comparable in the two models. After 24weeks of HFD, both Pam+/- and Pam+/+ mice had insulin resistance and impaired glucose tolerance, but no differences in glucose tolerance, insulin sensitivity or plasma insulin levels were observed in PAM haploinsufficient mice. Islet amyloid formation and beta cell function were also similar in Pam+/- and Pam+/+ mice with beta cell expression of hIAPP. Haploinsufficiency of PAM in mice does not accelerate the development of diet-induced obesity or hIAPP transgene-induced diabetes.

Analysis of Baboon IAPP Provides Insight into Amyloidogenicity and Cytotoxicity of Human IAPP

The polypeptide hormone islet amyloid polypeptide (IAPP) forms islet amyloid in type 2 diabetes, a process which contributes to pancreatic β-cell dysfunction and death. Not all species form islet amyloid, and the ability to do so correlates with the primary sequence. Humans form islet amyloid, but baboon IAPP has not been studied. The baboon peptide differs from human IAPP at three positions containing K1I, H18R, and A25T substitutions. The K1I substitution is a rare example of a replacement in the N-terminal region of amylin. The effect of this mutation on amyloid formation has not been studied, but it reduces the net charge, and amyloid prediction programs suggest that it should increase amyloidogenicity. The A25T replacement involves a nonconservative substitution in a region of IAPP that is believed to be important for aggregation, but the effects of this replacement have not been examined. The H18R point mutant has been previously shown to reduce aggregation in vitro. Baboon amylin forms amyloid on the same timescale as human amylin in vitro and exhibits similar toxicity toward cultured β-cells. The K1I replacement in human amylin slightly reduces toxicity, whereas the A25T substitution accelerates amyloid formation and enhances toxicity. Photochemical cross-linking reveals that the baboon amylin, like human amylin, forms low-order oligomers in the lag phase of amyloid formation. Ion-mobility mass spectrometry reveals broadly similar gas phase collisional cross sections for human and baboon amylin monomers and dimers, with some differences in the arrival time distributions. Preamyloid oligomers formed by baboon amylin, but not baboon amylin fibers, are toxic to cultured β-cells. The toxicity of baboon oligomers and lack of significantly detectable toxicity with exogenously added amyloid fibers is consistent with the hypothesis that preamyloid oligomers are the most toxic species produced during IAPP amyloid formation.

Role of pancreatic amyloidosis in pathogenesis of type 2 diabetes mellitus

The occurrence of type 2 diabetes mellitus (T2DM) in developed countries is currently becoming epidemic primarily due to the changes in quality of life. This disease typically makes progress for a long time, and its clinical pattern is mostly related to various micro- and macrovascular complications. In addition to said complications the pancreas itself often undergoes damage associated with amyloid formation in islets of Langerhans which results in a toxic effect on the hormone-producing islet cells. Ultimately, hormone overproduction in T2DM shifts to hormone deficiency. The crucial causative factor of the development of pancreatic amyloidosis in T2DM is а short peptide hormone, amylin (IAPP), which is cosecreted with insulin and considered to be a kind of insulin antagonist. The present review reports structure, functions and amyloidogenic properties of human IAPP. The basic concepts of molecular and cellular aspects concerning pathogenesis of pancreatic amyloidosis have been provided. Patterns of the development of pancreatic amyloid lesions and possible approaches for early diagnosis and treatment of this significant complication of T2DM have been discussed.