Islet amyloid and type 2 diabetes: from molecular misfolding to islet pathophysiology.

Islet amyloid polypeptide (IAPP, amylin) is secreted from pancreatic islet beta-cells and converted to amyloid deposits in type 2 diabetes. Conversion from soluble monomer, IAPP 1-37, to beta-sheet fibrils involves changes in the molecular conformation, cellular biochemistry and diabetes-related factors. In addition to the recognised amyloidogenic region, human IAPP (hIAPP) 20-29, the peptides human or rat IAPP 30-37 and 8-20, assume beta-conformation and form fibrils. These three amyloidogenic regions of hIAPP can be modelled as a folding intermediate with an intramolecular beta-sheet. A hypothesis is proposed for co-secretion of proIAPP with proinsulin in diabetes and formation of a 'nidus' adjacent to islet capillaries for subsequent accumulation of secreted IAPP to form the deposit. Although intracellular fibrils have been identified in experimental systems, extracellular deposition predominates in animal models and man. Extensive fibril accumulations replace islet cells. The molecular species of IAPP that is cytotoxic remains controversial. However, since fibrils form invaginations in cell membranes, small non-toxic IAPP fibrillar or amorphous accumulations could affect beta-cell stimulus-secretion coupling. The level of production of hIAPP is important but not a primary factor in islet amyloidosis; there is little evidence for inappropriate IAPP hypersecretion in type 2 diabetes and amyloid formation is generated in transgenic mice overexpressing the gene for human IAPP only against a background of obesity. Animal models of islet amyloidosis suggest that diabetes is induced by the deposits whereas in man, fibril formation appears to result from diabetes-associated islet dysfunction. Islet secretory failure results from progressive amyloidosis which provides a target for new therapeutic interventions.     

Amyloidogenicity of naturally occurring full‐length animal IAPP variants

The aggregation of the 37‐amino acid polypeptide human islet amyloid polypeptide (hIAPP), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of human pancreatic β‐islet cells in type 2 diabetes. hIAPP is considered to be one of the most amyloidogenic proteins known. The quick aggregation of hIAPP leads to the formation of toxic species, such as oligomers and fibers, that damage mammalian cells (both human and rat pancreatic cells). Whether this toxicity is necessary for the progression of type 2 diabetes or merely a side effect of the disease remains unclear. If hIAPP aggregation into toxic amyloid is on‐path for developing type 2 diabetes in humans, islet amyloid polypeptide (IAPP) aggregation would likely need to play a similar role within other organisms known to develop the disease. In this work, we compared the aggregation potential and cellular toxicity of full‐length IAPP from several diabetic and nondiabetic organisms whose aggregation propensities had not yet been determined for full‐length IAPP.     

The FGFR D3 domain determines receptor selectivity for fibroblast growth factor 21

Islet amyloid polypeptide (IAPP; also known as amylin) is responsible for islet amyloid formation in type 2 diabetes, and IAPP-induced toxicity is believed to contribute to the loss of β-cell mass associated with the late stages of type 2 diabetes. Islet amyloid formation may also play a role in graft failure after transplantation. IAPP is produced as a prohormone, pro-islet amyloid polypeptide (proIAPP), and processed in the secretory granules of the pancreatic β-cells. Partially processed forms of proIAPP are found in amyloid deposits; most notable is a 48-residue intermediate, proIAPP_1-48, which includes the N-terminal pro-extension, but which has been properly processed at the C-terminus. Incomplete processing may play a role in islet amyloid formation by promoting interactions with sulfated proteoglycans of the extracellular matrix, which, in turn, promote amyloid formation. We show that acid fuchsin (3-(1-(4-amino-3-methyl-5-sulphonatophenyl)-1-(4-amino-3-sulphonatophenyl)methylene)cyclohexa-1,4-dienesulphonic acid), a simple sulfonated triphenyl methyl derivative, is a potent inhibitor of amyloid formation by the proIAPP_1-48 intermediate. The more complicated triphenyl methane derivative fast green FCF {ethyl-[4-[[4-[ethyl-[(3-sulfophenyl)methyl]amino]phenyl]-(4-hydroxy-2-sulfophenyl)methylidene]-1-cyclohexa-2,5-dienylidene]-[(3-sulfophenyl)methyl]azanium} also inhibits amyloid formation by IAPP and the proIAPP processing intermediate. Both compounds inhibit amyloid formation by mixtures of the proIAPP intermediate and the model glycosaminoglycan heparan sulfate. Acid fuchsin also inhibits glycosaminoglycan-mediated amyloid formation by mature IAPP. The ability to inhibit amyloid formation is not simply due to the compounds being sulfonated, since the sulfonated inhibitor of amyloid-β, tramiprosate, is not an inhibitor of amyloid formation by proIAPP_1-48.     

Sensitivity of amyloid formation by human islet amyloid polypeptide to mutations at residue 20

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 Ser20-to-Gly missense 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. Ser20 adopts a normal backbone geometry, and the side chain makes no steric clashes in models of IAPP amyloid fibers, suggesting that the increased 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 (transformed rat insulinoma-1) β-cells. The sensitivity of amyloid formation to the identity of residue 20 was exploited to design a variant that is much slower to aggregate and that inhibits amyloid formation by wild-type IAPP. An S20K mutant forms amyloid with an 18-fold longer lag phase in homogeneous solution. Thioflavin T binding assays, together with experiments using a p-cyanophenylalanine (p-cyanoPhe) variant of human IAPP, show that the designed S20K mutant inhibits amyloid formation by human IAPP. The experiments illustrate how p-cyanoPhe can be exploited to monitor amyloid formation even in the presence of other amyloidogenic proteins.     

Islet amyloid polypeptide (IAPP):cDNA cloning and identification of an amyloidogenic region associated with the species-specific occurrence of age-related diabetes mellitus

We have cloned and sequenced a human islet amyloid polypeptide (IAPP) cDNA. A secretory 89 amino acid IAPP protein precursor is predicted from which the 37 amino acid IAPP molecule is formed by amino- and carboxyterminal proteolytic processing. The IAPP peptide is 43-46% identical in amino acid sequence to the two members of the calcitonin gene-related peptide (CGRP) family. Evolutionary conserved proteolytic processing sites indicate that similar proteases are involved in the maturation of IAPP and CGRP and that the IAPP amyloid polypeptide is identical to the normal proteolytic product of the IAPP precursor. A synthetic peptide corresponding to a carboxyteminal fragment of human IAPP is shown to spontaneously form amyloid-like fibrils in vitro. Antibodies against this peptide cross-react with IAPP from species that develop amyloid in pancreatic islets in conjunction with age-related diabetes mellitus (human, cat, racoon), but do not cross-react with IAPP from other tested species (mouse, rat, guinea pig, dog). Thus, a species-specific structural motif in the putative amyloidogenic region of IAPP is associated with both amyloid formation and the development of age-related diabetes mellitus. This provides a new molecular clue to the pathogenesis of this disease.     

Morin hydrate inhibits amyloid formation by islet amyloid polypeptide and disaggregates amyloid fibers

The polypeptide hormone Islet Amyloid Polypeptide (IAPP, amylin) is responsible for islet amyloid formation in type-2 diabetes and in islet cell transplants, where it may contribute to graft failure. Human IAPP is extremely amyloidogenic and fewer inhibitors of IAPP amyloid formation have been reported than for the Alzheimer's Aβ peptide or for α-synuclein. The ability of a set of hydroxyflavones to inhibit IAPP amyloid formation was tested. Fluorescence detected thioflavin-T-binding assays are the most popular methods for measuring the kinetics of amyloid formation and for screening potential inhibitors; however, we show that they can lead to false positives with hydroxyflavones. Several of the compounds inhibit thioflavin-T fluorescence, but not amyloid formation; a result which highlights the hazards of relying solely on thioflavin-T assays to screen potential inhibitors. Transmission electron microscopy (TEM) and right-angle light scattering show that Morin hydrate (2',3,4',5,7-Pentahydroxyflavone) inhibits amyloid formation by human IAPP and disaggregates preformed IAPP amyloid fibers. In contrast, Myricetin, Kaempferol, and Quercetin, which differ only in hydroxyl groups on the B-ring, are not effective inhibitors. Morin hydrate represents a new type of IAPP amyloid inhibitor and the results with the other compounds highlight the importance of the substitution pattern on the B-ring.     

Rifampicin does not prevent amyloid fibril formation by human islet amyloid polypeptide but does inhibit fibril thioflavin-T interactions: implications for mechanistic studies of beta-cell death

Amyloid formation has been implicated in more than 20 different human diseases, including Alzheimer's disease, Parkinson's disease, and type 2 diabetes. The development of inhibitors of amyloid is a topic of considerable interest, both because of their potential therapeutic applications and because they are useful mechanistic probes. Recent studies have highlighted the potential use of rifampicin as an inhibitor of amyloid formation by a variety of polypeptides; however, there are conflicting reports on its ability to inhibit amyloid formation by islet amyloid polypeptide (IAPP). IAPP is the cause of islet amyloid in type 2 diabetes. We show that rifampicin does not prevent amyloid formation by IAPP and does not disaggregate preformed IAPP amyloid fibrils;, instead, it interferes with standard fluorescence-based assays of amyloid formation. Rifampicin is unstable in aqueous solution and is readily oxidized. However, the effects of oxidized and reduced rifampicin are similar, in that neither prevents amyloid formation by IAPP. Furthermore, use of a novel p-cyanoPhe analogue of IAPP shows that rifampicin does not significantly affect the kinetics of IAPP amyloid formation. The implications for the development of amyloid inhibitors are discussed as are the implications for studies of the toxicity of islet amyloid. The work also demonstrates the utility of p-cyanoPhe IAPP for the screening of inhibitors. The data indicate that rifampicin cannot be used to test the relative toxicity of IAPP fibrils and prefibril aggregates of IAPP.     

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.     

Membrane Curvature-sensing and Curvature-inducing Activity of Islet Amyloid Polypeptide and Its Implications for Membrane Disruption

Islet amyloid polypeptide (IAPP) is a 37-amino acid amyloid protein intimately associated with pancreatic islet β-cell dysfunction and death in type II diabetes. In this study, we combine spectroscopic methods and microscopy to investigate α-helical IAPP-membrane interactions. Using light scattering and fluorescence microscopy, we observe that larger vesicles become smaller upon treatment with human or rat IAPP. Electron microscopy shows the formation of various highly curved structures such as tubules or smaller vesicles in a membrane-remodeling process, and spectrofluorometric detection of vesicle leakage shows disruption of membrane integrity. This effect is stronger for human IAPP than for the less toxic rat IAPP. From CD spectra in the presence of different-sized vesicles, we also uncover the membrane curvature-sensing ability of IAPP and find that it transitions from inducing to sensing membrane curvature when lipid negative charge is decreased. Our in vivo EM images of immunogold-labeled rat IAPP and human IAPP show both forms to localize to mitochondrial cristae, which contain not only locally curved membranes but also phosphatidylethanolamine and cardiolipin, lipids with high spontaneous negative curvature. Disruption of membrane integrity by induction of membrane curvature could apply more broadly to other amyloid proteins and be responsible for membrane damage observed in other amyloid diseases as well.     

The pathogenesis of maturity-onset diabetes mellitus: Is there a link to islet amyloid polypeptide?

The discovery of a novel polypeptide (Islet Amyloid Polypeptide: IAPP) isolated from human and cat islet amyloid and from amyloid of a human insulinoma is reviewed. Structurally, IAPP from the human and cat resembles calcitonin gene-related peptide (CGRP). The structural similarities between the neuropeptide CGRP and IAPP support the premise that IAPP is hormonal in nature. Our immunohistochemical studies also indicate that normal islet B-cells of several mammalian species (including man and cat) give strong immunoreactivity with antiserum directed to a synthetic peptide segment of IAPP. The fact that IAPP is deposited as amyloid in the pancreatic islets of type 2 (noninsulin-dependent) diabetics strongly supports an important but yet unknown link between IAPP and the development of this disease.     

Amyloidogenicity and cytotoxicity of recombinant mature human islet amyloid polypeptide (rhIAPP)

Pancreatic amyloid plaques formed by the pancreatic islet amyloid polypeptide (IAPP) are present in more than 95% of type II diabetes mellitus patients, and their abundance correlates with the severity of the disease. IAPP is currently considered the most amyloidogenic peptide known, but the molecular bases of its aggregation are still incompletely understood. Detailed characterization of the mechanisms of amyloid formation requires large quantities of pure material. Thus, availability of recombinant IAPP in sufficient amounts for such studies constitutes an important step toward elucidation of the mechanisms of amyloidogenicity. Here, we report, for the first time, the successful expression, purification and characterization of the amyloidogenicity and cytotoxicity of recombinant human mature IAPP. This approach is likely to be useful for the production of other amyloidogenic peptides or proteins that are difficult to obtain by chemical synthesis.     

Amyloid formation by pro-islet amyloid polypeptide processing intermediates: examination of the role of protein heparan sulfate interactions and implications for islet amyloid formation in type 2 diabetes

Amyloid formation has been implicated in a wide range of human diseases including Alzheimer's disease, Parkinson's disease, and type 2 diabetes. In type 2 diabetes, islet amyloid polypeptide (IAPP, also known as amylin) forms cytotoxic amyloid deposits in the pancreas, and these are believed to contribute to the pathology of the disease. The mechanism of islet amyloid formation is not understood; however, recent proposals have invoked a role for incompletely processed proIAPP. In this model, incompletely processed proIAPP containing the N-terminal pro region is excreted and binds to heparan sulfate proteoglycans (HSPGs) of the basement membrane thereby establishing a high local concentration which can act as a seed for amyloid formation. Here we report biophysical proof-of-principle experiments designed to test the viability of this model. The model predicts that interactions with HSPGs should accelerate amyloid formation by the proIAPP processing intermediate, and this is indeed what is observed. Interaction with heparan sulfate leads to the rapid formation of an intermediate state with partial helical content which then converts, on a slower time scale, to amyloid fibrils. TEM shows that fibrils formed by the proIAPP processing intermediate in the presence and in the absence of heparan sulfate have the classic features of amyloid. Fibrils formed by the proIAPP processing intermediate are competent to seed amyloid formation by mature IAPP. The seeding experiments support a second major premise of the model, namely, that fibrils formed by the processing intermediate are capable of seeding amyloid formation by the mature peptide.     

Mutational Analysis of Preamyloid Intermediates: The Role of His-Tyr Interactions in Islet Amyloid Formation

Islet amyloid polypeptide (IAPP or Amylin) is a 37-residue, C-terminally amidated pancreatic hormone, cosecreted with insulin that forms islet amyloid in type 2 diabetes. Islet amyloid formation is complex and characterizing preamyloid oligomers is an important topic because oligomeric intermediates are postulated to be the most toxic species produced during fibril formation. A range of competing models for early oligomers have been proposed. The role of the amidated C-terminus in amyloid formation by IAPP and in stabilizing oligomers is not known. Studies with unamidated IAPP have provided evidence for formation of an antiparallel dimer at pH 5.5, stabilized by stacking of His-18 and Tyr-37, but it is not known if this interaction is formed in the physiological form of the peptide. Analysis of a set of variants with a free and with an amidated C-terminus shows that disrupting the putative His-Tyr interaction accelerates amyloid formation, indicating that it is not essential. Amidation to generate the physiologically relevant form of IAPP accelerates amyloid formation, demonstrating that the advantages conferred by C-terminal amidation outweigh increased amyloidogenicity. The analysis of this variant argues that IAPP is not under strong evolutionary pressure to reduce amyloidogenicity. Analysis of an H18Q mutant of IAPP shows that the charge state of the N-terminus is an important factor controlling the rate of amyloid formation, even though the N-terminal region of IAPP is believed to be flexible in the amyloid fibers.     

Mutational Analysis of Preamyloid Intermediates: The Role of His-Tyr Interactions in Islet Amyloid Formation

Islet amyloid polypeptide (IAPP or Amylin) is a 37-residue, C-terminally amidated pancreatic hormone, cosecreted with insulin that forms islet amyloid in type 2 diabetes. Islet amyloid formation is complex and characterizing preamyloid oligomers is an important topic because oligomeric intermediates are postulated to be the most toxic species produced during fibril formation. A range of competing models for early oligomers have been proposed. The role of the amidated C-terminus in amyloid formation by IAPP and in stabilizing oligomers is not known. Studies with unamidated IAPP have provided evidence for formation of an antiparallel dimer at pH 5.5, stabilized by stacking of His-18 and Tyr-37, but it is not known if this interaction is formed in the physiological form of the peptide. Analysis of a set of variants with a free and with an amidated C-terminus shows that disrupting the putative His-Tyr interaction accelerates amyloid formation, indicating that it is not essential. Amidation to generate the physiologically relevant form of IAPP accelerates amyloid formation, demonstrating that the advantages conferred by C-terminal amidation outweigh increased amyloidogenicity. The analysis of this variant argues that IAPP is not under strong evolutionary pressure to reduce amyloidogenicity. Analysis of an H18Q mutant of IAPP shows that the charge state of the N-terminus is an important factor controlling the rate of amyloid formation, even though the N-terminal region of IAPP is believed to be flexible in the amyloid fibers.     

Amyloidogenicity and cytotoxicity of islet amyloid polypeptide

Insoluble amyloid formation by islet amyloid polypeptide (IAPP) in the islets of Langerhans of the pancreas is a major pathophysiological feature of noninsulin dependent diabetes mellitus (NIDDM) or type II diabetes. Because in vivo formed amyloid colocalizes with areas of cell degeneration and IAPP amyloid aggregates are cytotoxic per se, the process of IAPP amyloid formation has been strongly associated with the progressive pancreatic cell degeneration and thus much of the pathology of type II diabetes. IAPP is a pancreatic polypeptide of 37 residues that, in its soluble form, is believed to play a role as a regulator of glucose homeostasis. The molecular cause and mechanism of the conversion of soluble IAPP into insoluble amyloid aggregates in vivo and its role in disease progress still remain to be clarified. Nevertheless, in the past few years significant progress has been made in understanding the amyloidogenesis pathway of IAPP in vitro and gaining insight into the structural and conformational "requirements" of IAPP amyloidogenesis and related cytotoxic effects. Importantly, several of the studies have revealed significant similarities of the above features of IAPP to other amyloidogenic polypeptides such as the beta-amyloid polypeptide Abeta. This suggests that, at the molecular level, amyloidogenesis, and possibly related cell degeneration and disease pathogenesis by completely different polypeptide sequences, may obey to common structural and conformational "rules" and follow similar molecular pathways. This review describes studies on the structural and conformational features of IAPP amyloid formation and cytotoxicity, and the application of the obtained knowledge for the understanding of the molecular mechanism of the IAPP amyloidogenesis pathway and the related cytotoxicity.     

Heparan Sulfate Proteoglycans Are Important for Islet Amyloid Formation and Islet Amyloid Polypeptide-induced Apoptosis

Deposition of β cell toxic islet amyloid is a cardinal finding in type 2 diabetes. In addition to the main amyloid component islet amyloid polypeptide (IAPP), heparan sulfate proteoglycan is constantly present in the amyloid deposit. Heparan sulfate (HS) side chains bind to IAPP, inducing conformational changes of the IAPP structure and an acceleration of fibril formation. We generated a double-transgenic mouse strain (hpa-hIAPP) that overexpresses human heparanase and human IAPP but is deficient of endogenous mouse IAPP. Culture of hpa-hIAPP islets in 20 mm glucose resulted in less amyloid formation compared with the amyloid load developed in cultured islets isolated from littermates expressing human IAPP only. A similar reduction of amyloid was achieved when human islets were cultured in the presence of heparin fragments. Furthermore, we used CHO cells and the mutant CHO pgsD-677 cell line (deficient in HS synthesis) to explore the effect of cellular HS on IAPP-induced cytotoxicity. Seeding of IAPP aggregation on CHO cells resulted in caspase-3 activation and apoptosis that could be prevented by inhibition of caspase-8. No IAPP-induced apoptosis was seen in HS-deficient CHO pgsD-677 cells. These results suggest that β cell death caused by extracellular IAPP requires membrane-bound HS. The interaction between HS and IAPP or the subsequent effects represent a possible therapeutic target whose blockage can lead to a prolonged survival of β cells.     

Islet amyloid polypeptide: identification and chromosomal localization of the human gene

Islet or insulinoma amyloid polypeptide (IAPP) is a 37 amino acid polypeptide isolated from pancreatic amyloid. Here, we describe the isolation and partial characterization of the human gene encoding IAPP. The DNA sequence predicts that IAPP is excised from a larger precursor protein and that its carboxy-terminus is probably amidated. The predicted normally occurring IAPP is identical to the reported polypeptides isolated from pancreatic amyloid, except for the amidated carboxy-terminus. IAPP specific polyadenylated RNAs of 1.6 kb and 2.1 kb are present in human insulinoma RNA. The human IAPP gene is located on chromosome 12.     

Sphingomyelinase dependent apoptosis following treatment of pancreatic beta-cells with amyloid peptides Aß1-42 or IAPP

Amyloid peptides interfere with survival of pancreatic beta-cells. In some cells apoptosis is paralleled by ceramide-dependent alterations of ion channel activity. The purpose of the present study was to elucidate the dependence of amyloid peptides Aß_1-42 and islet amyloid polypeptide (IAPP)-induced cell death on ceramide formation and ion channel activity in murine pancreatic islet cells. As disclosed by TUNEL (terminal dUTP nick-end labelling) and cleaved caspase 3 staining, apoptotic cell death was induced by Aß_1-42, IAPP and exogenously added C2-ceramide in islet cells from wild type mice. In islet cells from acid sphingomyelinase-deficient mice (ASMKO) Aß_1-42 and IAPP but not exogenously added N-acetyl-d-sphingosine (C2-ceramide, 20 μM) failed to stimulate apoptosis. Immunofluorescent staining revealed a stimulatory effect of Aß_1-42 on ceramide formation. According to patch clamp experiments, administration of Aß_1-42 and IAPP significantly decreased outwardly rectifying whole cell currents in wild type but not in ASMKO islet cells. C2-ceramide but not inactive di-ceramide (20 μM) mimicked the inhibitory effect on Kv channel current. In conclusion, amyloid peptides induce apoptosis of pancreatic islet cells at least in part through activation of acid sphingomyelinase resulting in production of ceramide and subsequent inhibition of ion channel activity.     

The Sulfated Triphenyl Methane Derivative Acid Fuchsin Is a Potent Inhibitor of Amyloid Formation by Human Islet Amyloid Polypeptide and Protects against the Toxic Effects of Amyloid Formation

Islet amyloid polypeptide (IAPP), also known as amylin, is responsible for amyloid formation in type 2 diabetes. The formation of islet amyloid is believed to contribute to the pathology of the disease by killing β-cells, and it may also contribute to islet transplant failure. The design of inhibitors of amyloid formation is an active area of research, but comparatively little attention has been paid to inhibitors of IAPP in contrast to the large body of work on β-amyloid, and most small-molecule inhibitors of IAPP amyloid are generally effective only when used at a significant molar excess. Here we show that the simple sulfonated triphenyl methane derivative acid fuchsin, 3-(1-(4-amino-3-methyl-5-sulfonatophenyl)-1-(4-amino-3-sulfonatophenyl) methylene) cyclohexa-1,4-dienesulfonic acid, is a potent inhibitor of in vitro amyloid formation by IAPP at substoichiometric levels and protects cultured rat INS-1 cells against the toxic effects of human IAPP. Fluorescence-detected thioflavin-T binding assays, light-scattering, circular dichroism, two-dimensional IR, and transmission electron microscopy measurements confirm that the compound prevents amyloid fibril formation. Ionic-strength-dependent studies show that the effects are mediated in part by electrostatic interactions. Experiments in which the compound is added at different time points during the lag phase after amyloid formation has commenced reveal that it arrests amyloid formation by trapping intermediate species. The compound is less effective against the β-amyloid peptide, indicating specificity in its ability to inhibit amyloid formation by IAPP. The work reported here provides a new structural class of IAPP amyloid inhibitors and demonstrates the power of two-dimensional infrared spectroscopy for characterizing amyloid inhibitor interactions.