Soluble Islet Amyloid Polypeptide Research Articles

Vaccination Against Amyloidogenic Aggregates in Pancreatic Islets Prevents Development of Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is a chronic progressive disease characterized by insulin resistance and insufficient insulin secretion to maintain normoglycemia. The majority of T2DM patients bear amyloid deposits mainly composed of islet amyloid polypeptide (IAPP) in their pancreatic islets. These—originally β-cell secretory products—extracellular aggregates are cytotoxic for insulin-producing β-cells and are associated with β-cell loss and inflammation in T2DM advanced stages. Due to the absence of T2DM preventive medicaments and the presence of only symptomatic drugs acting towards increasing hormone secretion and action, we aimed at establishing a novel disease-modifying therapy targeting the cytotoxic IAPP deposits in order to prevent the development of T2DM. We generated a vaccine based on virus-like particles (VLPs), devoid of genomic material, coupled to IAPP peptides inducing specific antibodies against aggregated, but not monomeric IAPP. Using a mouse model of islet amyloidosis, we demonstrate in vivo that our vaccine induced a potent antibody response against aggregated, but not soluble IAPP, strikingly preventing IAPP depositions, delaying onset of hyperglycemia and the induction of the associated pro-inflammatory cytokine Interleukin 1β (IL-1β). We offer the first cost-effective and safe disease-modifying approach targeting islet dysfunction in T2DM, preventing pathogenic aggregates without disturbing physiological IAPP function.

Islet Amyloid Polypeptide Promotes Amyloid-Beta Aggregation by Binding-Induced Helix-Unfolding of the Amyloidogenic Core.

Amyloid aggregation of amyloid-beta (Aβ) and islet amyloid polypeptide (IAPP) is associated with Alzheimer's disease (AD) and type-2 diabetes (T2D), respectively. With T2D being the risk factor for AD and the ability of IAPP to cross the blood-brain barrier, the coaggregation of Aβ and IAPP has been explored to understand the cross-talk between the two diseases. Recent studies demonstrated that soluble IAPP could significantly accelerate the aggregation of Aβ while preformed amyloids of IAPP were poor "seeds" for Aβ aggregation. Here, we apply all-atom discrete molecular dynamics simulations to investigate possible molecular mechanisms for the accelerated coaggregation of IAPP and Aβ42 comparing to Aβ42 aggregation alone, which was confirmed by the complementary thioflavin-T fluorescence assay. Our simulation results suggest that peptides in the mixture tend to form heterodimers as the first step toward their coaggregation. Strong interpeptide interactions with IAPP in the heterodimer shift the helical conformation of Aβ42 in its amyloidogenic central hydrophobic core, residues 16-22 (Aβ16-22), to the extended conformation ready to form β-sheets. Our study suggests that the unfolding of Aβ16-22 helix contributes to the aggregation free-energy barrier and corresponds to the rate-limiting conformational change for Aβ42 aggregation. Therefore, we propose that soluble IAPP promotes the aggregation of Aβ42 by binding-induced conformational change of Aβ42 in its amyloidogenic core and thus reduced aggregation free-energy barrier.

Differential Activation of Innate Immune Pathways by Distinct Islet Amyloid Polypeptide (IAPP) Aggregates

Aggregation of islet amyloid polypeptide (IAPP) contributes to beta cell dysfunction in type 2 diabetes and islet transplantation. Like other amyloidogenic peptides, human IAPP induces macrophage IL-1β secretion by stimulating both the synthesis and processing of proIL-1β, a pro-inflammatory cytokine that (when chronically elevated) impairs beta cell insulin secretion. We sought to determine the specific mechanism of IAPP-induced proIL-1β synthesis. Soluble IAPP species produced early during IAPP aggregation provided a Toll-like-receptor-2- (TLR2-) dependent stimulus for NF-κB activation in HEK 293 cells and bone marrow-derived macrophages (BMDMs). Non-amyloidogenic rodent IAPP and thioflavin-T-positive fibrillar amyloid produced by human IAPP aggregation failed to activate TLR2. Blockade of TLR6 but not TLR1 prevented hIAPP-induced TLR2 activation, consistent with stimulation of a TLR2/6 heterodimer. TLR2 and its downstream adaptor protein MyD88 were required for IAPP-induced cytokine production by BMDMs, a process that is partially dependent on autoinduction by IL-1. BMDMs treated with soluble but not fibrillar IAPP provided a TLR2-dependent priming stimulus for ATP-induced IL-1β secretion, whereas late IAPP aggregates induced NLRP3-dependent IL-1β secretion by LPS-primed macrophages. Moreover, inhibition of TLR2 and depletion of islet macrophages prevented up-regulation of Il1b and Tnf expression in human IAPP-expressing transgenic mouse islets. These data suggest participation by both soluble and fibrillar aggregates in IAPP-induced islet inflammation. IAPP-induced activation of TLR2 and secretion of IL-1 may be important therapeutic targets to prevent amyloid-associated beta cell dysfunction.

Distinct actions of oligomers and fibrils of islet amyloid polypeptide on Toll-like receptor 2 and NLRP3 inflammasome activation (INM7P.423)

Abstract Aggregation of islet amyloid polypeptide (IAPP) to form amyloid contributes to beta cell dysfunction in type 2 diabetes. Human but not non-amyloidogenic rodent IAPP activates TLR2 and NLRP3 to induce IL-1β secretion by islet macrophages. It is unclear which form of IAPP aggregate induces IL-1β and whether this pathway is relevant to disease pathogenesis. We therefore evaluated the contributions of soluble and fibrillar IAPP to IL-1β secretion by bone marrow-derived macrophages (BMDMs) and assessed the effects of IL-1 receptor antagonist (IL-1Ra) on glucose homeostasis in transgenic mice with beta cell expression of human IAPP. BMDMs treated with soluble but not fibrillar IAPP provided a TLR2-dependent priming stimulus for ATP-induced IL-1β secretion, suggesting that pre-fibrillar species interact with TLR2. Fibrillar but not soluble IAPP aggregates were required for IL-1β secretion in LPS-primed BMDMs and are therefore likely the principal stimulus for NLRP3. Moreover, IL-1Ra (50 mg/kg/d for 8 weeks) improved glucose tolerance in obese human IAPP transgenic mice (AUC=1360±110 vs. 880±70; p<0.05) with no effect in wild-type littermates. These data provide the first in vivo evidence that IAPP-induced islet dysfunction in type 2 diabetes is mediated by IL-1, and suggest that both soluble and fibrillar IAPP aggregates participate in this process. Islet inflammation and amyloid formation are therefore important therapeutic targets to improve beta cell function in type 2 diabetes.

Selectively N‐Methylated Soluble IAPP Mimics as Potent IAPP Receptor Agonists and Nanomolar Inhibitors of Cytotoxic Self‐Assembly of Both IAPP and Aβ40

The selective incorporation of N-methyl groups in the highly amyloidogenic and cytotoxic sequence of the type 2 diabetes islet amyloid polypeptide (IAPP) generates a unique class of soluble and nontoxic IAPP mimics. These polypeptides combine potent IAPP receptor agonism with nanomolar-affinity inhibitory potential on the amyloid formation and cell-damaging effects of both IAPP and the Alzheimer's β-amyloid peptide (Aβ40).

Small Molecule Inhibitors of Islet Amyloid Polypeptide Fibrillogenesis and Cytotoxicity

Protein fiber formation is associated with diseases ranging from Alzheimer's to type II diabetes. The fiber formation is a complex reaction, which includes a number of conformational and oligomeric intermediate states. In recent years, it has become clear that it is these states, and not the end product (i.e. fibers) of amyloid formation that are the toxic agents associated with disease. These insights indicate that small molecule screens must to be directed at assembly mechanism in order to maximize the prospects for success. Islet amyloid polypeptide (IAPP) is a 37 residue peptide hormone co-secreted with insulin by the ß-cells of the pancreas. In patients with type II diabetes, this protein aggregates as amyloid in a process that is correlated with ß-cell dysfunction and the loss of ß-cell mass. In in vitro studies, the addition of soluble IAPP has been shown to be toxic to many ß- and non-ß-cell lines. IAPP fibrillogenesis, as is the case for many other amylodiogenic proteins, can be catalyzed by lipid bilayers. Paradoxically, while amyloid fibers are ß-sheet rich, membrane-stabilized states are α-helical. We have identified a small molecule alpha helix mimetic, IS5, which inhibits bilayer catalysis of fibrillogenesis, and rescues IAPP-induced toxicity in cell culture. Importantly, IAPP:IS5 interactions localize to the putative α-helical region of IAPP, revealing that α-helical states are on pathway to fiber formation. Normally, IAPP is not amyloidogenic as its cosecreted partner, insulin, prevents self-assembly. Here, we show that IS5 inhibition is synergistic with insulin. IS5 therefore represents a new approach to amyloid inhibition as the target is an assembly intermediate that may additionally restore functional IAPP expression.

Design of a mimic of nonamyloidogenic and bioactive human islet amyloid polypeptide (IAPP) as nanomolar affinity inhibitor of IAPP cytotoxic fibrillogenesis.

Protein aggregation into cytotoxic oligomers and fibrils in vivo is linked to cell degeneration and the pathogenesis of >25 uncurable diseases, whereas the high aggregation propensity and insolubility of several bioactive polypeptides and proteins in vitro prevent their therapeutic use. Aggregation of human islet amyloid polypeptide (IAPP) into pancreatic amyloid is strongly associated with the pathogenesis of type II diabetes. IAPP is a 37-residue polypeptide that acts as a neuroendocrine regulator of glucose homeostasis. However, IAPP misfolds and self-associates into cytotoxic aggregates and fibrils even at nanomolar concentrations. Because IAPP aggregation causes beta-cell death and prohibits therapeutic application of IAPP in diabetes, we pursued a minimalistic chemical design approach to generate a molecular mimic of a nonamyloidogenic and bioactive IAPP conformation that would still be able to associate with IAPP and thus inhibit its fibrillogenesis and cytotoxicity. We show that the double N-methylated full length IAPP analog [(N-Me)G24, (N-Me)I26]-IAPP (IAPP-GI) is a highly soluble, nonamyloidogenic, and noncytotoxic IAPP molecular mimic and an IAPP receptor agonist. Moreover, IAPP-GI binds IAPP with low nanomolar affinity and completely blocks IAPP cytotoxic self-assembly and fibrillogenesis with activity in the low nanomolar concentration range. Importantly, IAPP-GI dissociates cytotoxic IAPP oligomers and fibrils and is able to reverse their cytotoxicity. Bifunctional soluble IAPP mimics that combine bioactivity with the ability to block and reverse IAPP cytotoxic self-assembly are promising candidates for the treatment of diabetes. Moreover, our amyloid disease inhibitor design concept may be applicable to other protein aggregation diseases.

Increased beta-cell apoptosis prevents adaptive increase in beta-cell mass in mouse model of type 2 diabetes: evidence for role of islet amyloid formation rather than direct action of amyloid.

Nondiabetic obese humans adapt to insulin resistance by increasing beta-cell mass. In contrast, obese humans with type 2 diabetes have an approximately 60% deficit in beta-cell mass. Recent studies in rodents reveal that beta-cell mass is regulated, increasing in response to insulin resistance through increased beta-cell supply (islet neogenesis and beta-cell replication) and/or decreased beta-cell loss (beta-cell apoptosis). Prospective studies of islet turnover are not possible in humans. In an attempt to establish the mechanism for the deficit in beta-cell mass in type 2 diabetes, we used an obese versus lean murine transgenic model for human islet amyloid polypeptide (IAPP) that develops islet pathology comparable to that in humans with type 2 diabetes. By 40 weeks of age, obese nontransgenic mice did not develop diabetes and adapted to insulin resistance by a 9-fold increase (P < 0.001) in beta-cell mass accomplished by a 1.7-fold increase in islet neogenesis (P < 0.05) and a 5-fold increase in beta-cell replication per islet (P < 0.001). Obese transgenic mice developed midlife diabetes with islet amyloid and an 80% (P < 0.001) deficit in beta-cell mass that was due to failure to adaptively increase beta-cell mass. The mechanism subserving this failed expansion was a 10-fold increase in beta-cell apoptosis (P < 0.001). There was no relationship between the extent of islet amyloid or the blood glucose concentration and the frequency of beta-cell apoptosis. However, the frequency of beta-cell apoptosis was related to the rate of increase of islet amyloid. These prospective studies suggest that the formation of islet amyloid rather than the islet amyloid per se is related to increased beta-cell apoptosis in this murine model of type 2 diabetes. This finding is consistent with the hypothesis that soluble IAPP oligomers but not islet amyloid are responsible for increased beta-cell apoptosis. The current studies also support the concept that replicating beta-cells are more vulnerable to apoptosis, possibly accounting for the failure of beta-cell mass to expand appropriately in response to obesity in type 2 diabetes.

Structure-based design and study of non-amyloidogenic, double N-methylated IAPP amyloid core sequences as inhibitors of IAPP amyloid formation and cytotoxicity

Pancreatic amyloid is formed by the aggregation of the 37-residue islet amyloid polypeptide (IAPP) in type II diabetes patients and is cytotoxic. Pancreatic amyloid deposits are found in more than 95 % of type II diabetes patients and their formation is strongly associated with disease progression. IAPP amyloid forms via a conformational transition of soluble IAPP into aggregated β-sheets. We recently identified IAPP(22-27) (NFGAIL) as a minimum length sequence sufficient to self-associate into β-sheet-containing amyloid fibrils. Here, we have used the NFGAIL model of the IAPP amyloid core as a structural template to design non-amyloidogenic derivatives of amyloidogenic sequences of IAPP that are able to interact with the native sequences and inhibit amyloid formation. The design of the derivatives was based on a simple, structure-based minimalistic and selective N-methylation approach. Accordingly, a minimum number of two amide bonds on the same side of the β-strand of the amyloid core was N-methylated. This was expected to eliminate the two intermolecular backbone NH to CO hydrogen bonds which are critical for the extension of the β-sheet dimers into multimers and amyloid. Other β-strand “contact sides” remained intact allowing for the derivatives to interact with the native sequences. Double N-methylated derivatives of amyloidogenic and cytotoxic partial IAPP sequences generated included F(N-Me)GA(N-Me)IL, NF(N-Me)GA(N-Me)IL, SNNF(N-Me)GA(N-Me)IL, and SNNF(N-Me)GA(N-Me)ILSS and were found to be devoid of β-sheet structure, amyloidogenicity and cytotoxicity according to Fourier transform-infrared spectroscopy (FT-IR), Congo red (CR) staining, electron microscopy (EM), and cell viability tests. The derivatives were able to interact with the native sequences and inhibit amyloid formation as shown by circular dichroism spectroscopy (CD), FT-IR and EM. Moreover, SNNF(N-Me)GA(N-Me)ILSS inhibited cytotoxicity of SNNFGAILSS and is thus the first reported inhibitor of IAPP amyloid formation and cytotoxicity. Our results demonstrate the validity of the design approach for IAPP and suggest that it may find application in understanding the structural features of amyloid formation and in the development of inhibitors of amyloid formation and cytotoxicity of other amyloidogenic polypeptides as well.

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.

Contribution of advanced glycosylation to the amyloidogenicity of islet amyloid polypeptide.

The formation of amyloid within the islets of Langerhans is associated with the development of type II diabetes mellitus and occurs by the aggregation and insolubilization of islet amyloid polypeptide (IAPP). Recent in vitro studies suggest that amyloid formation follows a nucleation-dependent polymerization mechanism, i.e. aggregation is initiated by pre-formed aggregates or nucleation seeds. Modification of the Alzheimer's disease amyloid peptide by advanced glycosylation end products (AGEs), which form spontaneously by the non-enzymatic addition of glucose to protein amino groups, has been shown to enhance peptide aggregation in vitro. To explore the possibility that AGEs contribute to islet amyloid formation, we prepared AGE-modified IAPP (AGE-IAPP) in vitro and studied its properties by biochemical and biophysical techniques. AGE modification induced the formation of high-molecular-mass IAPP aggregates and amyloid formation was demonstrated by Congo red green-gold birefringence and by the presence of a characteristic fibrillar structure by electron microscopy. AGE-IAPP also showed an increase in cytotoxicity toward the astroglioma cell line HTB14. When added to soluble IAPP, AGE-IAPP seeds accelerated IAPP aggregation and abolished the nucleation period required for the polymerization of unseeded IAPP. Circular dichroism spectropolarimetry indicated that AGE-IAPP seeds may act as a template to stabilize the beta-sheet conformation of IAPP, thereby promoting its aggregation. Our studies demonstrate that AGE modification of IAPP results in high-molecular mass, fibrillar amyloid structures that nucleate IAPP amyloid formation and suggest a model for intra-islet amyloid deposition that may occur by the progressive advanced glycosylation of IAPP in vivo.

B Cell Granule Peptides Affect Human Islet Amyloid Polypeptide (IAPP) Fibril Formationin Vitro

Islet amyloid polypeptide (IAPP) forms fibrils spontaneously. We examined whether other B cell granule peptides affect formation of β-pleated sheet fibrilsin vitrofrom human IAPP. Quantitative radioassay (radioactivity of soluble IAPP after adding125I-IAPP) and thioflavine fluorescence spectroscopy showed that insulin, C-peptide, and pancreastatin inhibited fibril formation by 60–100% at ratio 100:1 (peptide:IAPP), whereas at 1:10 or 1:100, i.e., IAPP in excess, a potentiated IAPP fibril formation was induced by the peptides. Semi-quantitative analysis by electron microscopy yielded similar effects. Thus, spontaneous IAPP fibrillisation is influenced by other B cell secretory granule peptides in a molar ratio dependent manner.