The hormone islet amyloid polypeptide (IAPP, or amylin) plays a role in glucose homeostasis but aggregates to form islet amyloid in type-2 diabetes. Islet amyloid formation contributes to β-cell dysfunction and death in the disease and to the failure of islet transplants. Recent work suggests a role for IAPP aggregation in cardiovascular complications of type-2 diabetes and hints at a possible role in type-1 diabetes. The mechanisms of IAPP amyloid formation in vivo or in vitro are not understood and the mechanisms of IAPP induced β-cell death are not fully defined. Activation of the inflammasome, defects in autophagy, ER stress, generation of reactive oxygen species, membrane disruption, and receptor mediated mechanisms have all been proposed to play a role. Open questions in the field include the relative importance of the various mechanisms of β-cell death, the relevance of reductionist biophysical studies to the situation in vivo, the molecular mechanism of amyloid formation in vitro and in vivo, the factors which trigger amyloid formation in type-2 diabetes, the potential role of IAPP in type-1 diabetes, the development of clinically relevant inhibitors of islet amyloidosis toxicity, and the design of soluble, bioactive variants of IAPP for use as adjuncts to insulin therapy.

Islet amyloid polypeptide (IAPP, Amylin) is responsible for amyloid formation in type 2 diabetes and in islet cell transplants. The only known natural mutation found in mature human IAPP is a Ser-20 to Gly mis-sense mutation, found with small frequency in Chinese and Japanese populations. The mutation appears to be associated with increased risk of early onset type 2 diabetes. Early measurements in the presence of organic co-solvents showed that S20G-IAPP formed amyloid more quickly than the wild type. We confirm that the mutant accelerates amyloid formation under a range of conditions including in the absence of co-solvents. Ser-20 adopts a normal backbone geometry and the side chain makes no steric clashes in models of IAPP amyloid fibers, suggesting that the increase rate of amyloid formation by the mutant does not result from the relief of steric incompatibility in the fiber state. Transmission electronic microscopy, circular dichroism and seeding studies were used to probe the structure of the resulting fibers. The S20G-IAPP peptide is toxic to cultured rat INS-1 β-cells. The sensitivity of amyloid formation to the identity of residue-20 was exploited to design a variant which is much slower to aggregate and which inhibits amyloid formation by wild type IAPP. A S20K mutant forms amyloid with an 18-fold longer lag phase. Thioflavin-T binding assays, together with experiments using a p-cyanophenylalanine variant of human IAPP, show that the designed S20K mutant inhibits amyloid formation by human IAPP. The experiments illustrate how p-cyanophenylalanine can be exploited to monitor amyloid formation even in the presence of other amyloidogenic proteins.

Ion mobility coupled to mass spectrometry (IM-MS) is widely used to study protein dynamics and structure in the gas phase. Increasing the energy with which the protein ions are introduced to the IM cell can induce them to unfold, providing information on the comparative energetics of unfolding between different proteoforms. Recently, a high-resolution cyclic IM-mass spectrometer (cIM-MS) was introduced, allowing multiple, consecutive tandem IM experiments (IM n ) to be carried out. We describe a tandem IM technique for defining detailed protein unfolding pathways and the dynamics of disordered proteins. The method involves multiple rounds of IM separation and collision activation (CA): IM-CA-IM and CA-IM-CA-IM. Here, we explore its application to studies of a model protein, cytochrome C, and dimeric human islet amyloid polypeptide (hIAPP), a cytotoxic and amyloidogenic peptide involved in type II diabetes. In agreement with prior work using single stage IM-MS, several unfolding events are observed for cytochrome C. IM n -MS experiments also show evidence of interconversion between compact and extended structures. IM n -MS data for hIAPP shows interconversion prior to dissociation, suggesting that the certain conformations have low energy barriers between them and transition between compact and extended forms.

Islet amyloid formation contributes to β-cell death and dysfunction in type-2 diabetes and to the failure of islet transplants. Amylin (islet amyloid polypeptide, IAPP), a normally soluble 37 residue polypeptide hormone produced in the pancreatic β-cells, is responsible for amyloid formation in type-2 diabetes and is deficient in type-1 diabetes. Amylin normally plays an adaptive role in metabolism, and the development of nontoxic, non-amyloidogenic, bioactive variants of human amylin are of interest for use as adjuncts to insulin therapy. Naturally occurring non-amyloidogenic variants are of interest for xenobiotic transplantation and because they can provide clues toward understanding the amyloidogenicity of human amylin. The sequence of amylin is well-conserved among species, but sequence differences strongly correlate with in vitro amyloidogenicity and with islet amyloid formation in vivo. Bovine amylin differs from the human peptide at 10 positions and is one of the most divergent among known amylin sequences. We show that bovine amylin oligomerizes but is not toxic to cultured β-cells and that it is considerably less amyloidogenic than the human polypeptide and is only a low-potency agonist at human amylin-responsive receptors. The bovine sequence contains several nonconservative substitutions relative to human amylin, including His to Pro, Ser to Pro, and Asn to Lys replacements. The effect of these substitutions is analyzed in the context of wild-type human amylin; the results provide insight into their role in receptor activation, the mode of assembly of human amylin, and the design of soluble amylin analogues.

Antibodies with conformational specificity are important for detecting and interfering with polypeptide aggregation linked to several human disorders. We are developing a motif-grafting approach for designing lead antibody candidates specific for amyloid-forming polypeptides such as the Alzheimer peptide (A␤). This approach involves grafting amyloidogenic peptide segments into the complementarity-determining regions (CDRs) of single-domain (V H ) antibodies. Here we have investigated the impact of polar mutations inserted at the edges of a large hydrophobic A␤42 peptide segment (A␤ residues 17-42) in CDR3 on the solubility and conformational specificity of the corresponding V H domains. We find that V H expression and solubility are strongly enhanced by introducing multiple negatively charged or asparagine residues at the edges of CDR3, whereas other polar mutations are less effective (glutamine and serine) or ineffective (threonine, lysine, and arginine). Moreover, A␤ V H domains with negatively charged CDR3 mutations show significant preference for recognizing A␤ fibrils relative to A␤ monomers, whereas the same V H domains with other polar CDR3 mutations recognize both A␤ conformers. We observe similar behavior for a V H domain grafted with a large hydrophobic peptide from islet amyloid polypeptide (residues 8 -37) that contains negatively charged mutations at the edges of CDR3. These findings highlight the sensitivity of antibody binding and solubility to residues at the edges of CDRs, and provide guidelines for designing other grafted antibody fragments with hydrophobic binding loops.The misfolding and assembly of peptides and proteins into prefibrillar oligomers and amyloid fibrils is linked to several neurodegenerative diseases (1-4). To understand the contributions of polypeptide aggregation to such disorders, it is important to characterize the biochemical properties of aggregates. However, pre-amyloid and amyloid aggregates are difficult to characterize using many traditional biochemical methods that are routinely used for soluble proteins. High-resolution structural analysis of such aggregates is particularly challenging and must be performed using specialized methods such as solid state NMR (5-13) or x-ray crystallography of small peptide fragments (14 -16). These powerful structural methods generally lack the time resolution to probe pre-amyloid intermediates and oligomers unless they can be kinetically trapped.Given these challenges, antibodies with conformational specificity for prefibrillar oligomers and amyloid fibrils have proven valuable for biochemical characterization (17-28). An obvious strength of such antibodies is their ability to detect specific types of aggregates formed both in vivo and in vitro. The ability of conformational antibodies to bridge in vivo and in vitro studies is important for understanding the biochemical mechanisms that contribute to protein misfolding disorders.Several approaches have been used to generate conformational antibodies. The most widely used one is immuni...

Much of our knowledge of diabetes is derived from studies of rodent models. An alternative approach explores evolutionary solutions to physiological stress by studying organisms that face challenging metabolic environments. Polar bears eat an enormously lipid-rich diet without deleterious metabolic consequences. In contrast, transgenic rodents expressing the human neuropancreatic polypeptide hormone amylin develop hyperglycemia and extensive pancreatic islet amyloid when fed a high fat diet. The process of islet amyloid formation by human amylin contributes to β-cell dysfunction and loss of β-cell mass in type-2 diabetes. We show that ursine amylin is considerably less amyloidogenic and less toxic to β-cells than human amylin, consistent with the hypothesis that part of the adaptation of bears to metabolic challenges might include protection from islet amyloidosis-induced β-cell toxicity. Ursine and human amylin differ at four locations: H18R, S20G, F23L, and S29P. These are interesting from a biophysical perspective since the S20G mutation accelerates amyloid formation but the H18R slows it. An H18RS20G double mutant of human amylin behaves similarly to the H18R mutant, indicating that the substitution at position 18 dominates the S20G replacement. These data suggest one possible mechanism underpinning the protection of bears against metabolic challenges and provide insight into the design of soluble analogs of human amylin.

Type 2 diabetes is associated with upregulation of neprilysin, a peptidase capable of cleaving glucoregulatory peptides such as glucagon-like peptide-1 (GLP-1). In humans, use of the neprilysin inhibitor sacubitril in combination with an angiotensin II receptor blocker was associated with increased plasma GLP-1 levels and improved glycemic control. Whether neprilysin inhibition per se is mediating these effects remains unknown. We sought to determine whether pharmacological neprilysin inhibition on its own confers beneficial effects on glycemic status and beta-cell function in a mouse model of reduced insulin secretion, and whether any such effects are dependent on GLP-1 receptor (GLP-1R) signaling. High-fat-fed male wild-type (Glp1r+/+) and GLP-1R knockout (Glp1r−/−) mice were treated with low-dose streptozotocin (STZ) to recapitulate type 2 diabetes-associated beta-cell dysfunction, or vehicle as control. Mice were continued on high fat diet alone or supplemented with the neprilysin inhibitor sacubitril for 8 weeks. At the end of the study period, beta-cell function was assessed by oral or intravenous glucose tolerance test. Fasting and fed glucose were significantly lower in wild-type mice treated with sacubitril, although active GLP-1 levels and insulin secretion during oral glucose challenge were unchanged. In contrast, insulin secretion in response to intravenous glucose was significantly enhanced in sacubitril-treated wild-type mice, and this effect was blunted in Glp1r−/− mice. Similarly, sacubitril enhanced insulin secretion in vitro in islets from STZ-treated Glp1r+/+ but not Glp1r−/− mice. Together, our data suggest the insulinotropic effects of pharmacological neprilysin inhibition in a mouse model of beta-cell dysfunction are mediated via intra-islet GLP-1R signaling.

Aims/hypothesis 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. Methods 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 48 h 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. Results Treatment with a low concentration of IL-1β (4 pg/ml) for 48 h 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 hIAPPexpressing 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 48 h in IL-1β-treated vs control islets (IAPP 0.409 ± 0.082 vs 0.165 ± 0.051 pmol/5 islets; insulin 87.5 ± 8.81 vs 48.3 ± 17.3 pmol/5 islets), and this effect was blocked by co-treatment with IL-1β neutralising antibody. Conclusions/interpretation 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. Keywords hIAPP. IL-1β. Islet amyloid. Type 2 diabetes Abbreviations Fmoc 9-Fluorenylmethyloxycarbonyl hIAPP Human islet amyloid polypeptide IAPP Islet amyloid polypeptide mIAPP Mouse islet amyloid polypeptide NLRP3 NACHT, LRR and PYD domains-containing protein 3 RIP Rat insulin promoter TFA Trifluoroacetic acid UPR Unfolded protein response Electronic supplementary material The online version of this article (