Islet Amyloid Polypeptide Toxicity Research Articles

Dysregulation of cholesterol homeostasis is an early signal of β-cell proteotoxicity characteristic of type 2 diabetes.

Type 2 diabetes (T2D) is a common metabolic disease due to insufficient insulin secretion by pancreatic β-cells in the context of insulin resistance. Islet molecular pathology reveals a role for protein misfolding in β-cell dysfunction and loss with islet amyloid derived from islet amyloid polypeptide (IAPP), a protein coexpressed and cosecreted with insulin. The most toxic form of misfolded IAPP is intracellular membrane disruptive toxic oligomers present in β-cells in T2D and in β-cells of mice transgenic for human IAPP (hIAPP). Prior work revealed a high degree of overlap of transcriptional changes in islets from T2D and prediabetic 9- to 10-wk-old mice transgenic for hIAPP with most changes being pro-survival adaptations and therefore of limited therapeutic guidance. Here, we investigated islets from hIAPP transgenic mice at an earlier age (6 wk) to screen for potential mediators of hIAPP toxicity that precede predominance of pro-survival signaling. We identified early suppression of cholesterol synthesis and trafficking along with aberrant intra-β-cell cholesterol and lipid deposits and impaired cholesterol trafficking to cell membranes. These findings align with comparable lipid deposits present in β-cells in T2D and increased vulnerability to develop T2D in individuals taking medications that suppress cholesterol synthesis.NEW & NOTEWORTHY β-Cell failure in type 2 diabetes (T2D) is characterized by β-cell misfolded protein stress due to the formation of toxic oligomers of islet amyloid polypeptide (IAPP). Most transcriptional changes in islets in T2D are pro-survival adaptations consistent with the slow progression of β-cell loss. In the present study, investigation of the islet transcriptional signatures in a mouse model of T2D expressing human IAPP revealed decreased cholesterol synthesis and trafficking as a plausible early mediator of IAPP toxicity.

Islet amyloid polypeptide tagged with green fluorescent protein localises to mitochondria and forms filamentous aggregates in Caenorhabditis elegans

Type 2 diabetes (T2D) is the most common form of diabetes and represents a growing health concern. A characteristic feature of T2D is the aggregation of islet amyloid polypeptide (IAPP), which is thought to be associated with the death of pancreatic β-cells. Inhibiting IAPP aggregation is a promising therapeutic avenue to treat T2D, but the mechanisms of aggregation and toxicity are not yet fully understood. Caenorhabditis elegans is a well-characterised multicellular model organism that has been extensively used to study protein aggregation diseases. In this study, we aimed to develop a simple in vivo model to investigate IAPP aggregation and toxicity based on expression in the C. elegans body wall muscle cells. We show that IAPP tagged with green fluorescent protein (GFP) localises to mitochondria not only in muscle cells but also when expressed in the intestine, in line with previous observations in mouse and human pancreatic β-cells. The IAPP-GFP fusion protein forms solid aggregates, which have a filamentous appearance as seen by electron microscopy. However, the animals expressing IAPP-GFP in the body wall muscle cells do not display a strong motility phenotype, suggesting that the IAPP-GFP aggregates are not considerably toxic. Nevertheless, the mitochondrial localisation and aggregate formation may be useful read-outs to screen for IAPP-solubilizing compounds as a therapeutic strategy for T2D.

Plasma IAPP-Autoantibody Levels in Alzheimer's Disease Patients Are Affected by APOE4 Status.

Pancreas-derived islet amyloid polypeptide (IAPP) crosses the blood-brain barrier and co-deposits with amyloid beta (Aβ) in brains of type 2 diabetes (T2D) and Alzheimer's disease (AD) patients. Depositions might be related to the circulating IAPP levels, but it warrants further investigation. Autoantibodies recognizing toxic IAPP oligomers (IAPPO) but not monomers (IAPPM) or fibrils have been found in T2D, but studies on AD are lacking. In this study, we have analyzed plasma from two cohorts and found that levels of neither immunoglobulin (Ig) M, nor IgG or IgA against IAPPM or IAPPO were altered in AD patients compared with controls. However, our results show significantly lower IAPPO-IgA levels in apolipoprotein E (APOE) 4 carriers compared with non-carriers in an allele dose-dependent manner, and the decrease is linked to the AD pathology. Furthermore, plasma IAPP-Ig levels, especially IAPP-IgA, correlated with cognitive decline, C-reactive protein, cerebrospinal fluid Aβ and tau, neurofibrillary tangles, and brain IAPP exclusively in APOE4 non-carriers. We speculate that the reduction in IAPPO-IgA levels may be caused by increased plasma IAPPO levels or masked epitopes in APOE4 carriers and propose that IgA and APOE4 status play a specific role in clearance of circulatory IAPPO, which may influence the amount of IAPP deposition in the AD brain.

Efficacy of IAPP suppression in mouse and human islets by GLP-1 analogue conjugated antisense oligonucleotide.

Insulin resistance is the major risk factor for Type 2 diabetes (T2D). In vulnerable individuals, insulin resistance induces a progressive loss of insulin secretion with islet pathology revealing a partial deficit of beta cells and islet amyloid derived from islet amyloid polypeptide (IAPP). IAPP is co-expressed and secreted with insulin by beta cells, expression of both proteins being upregulated in response to insulin resistance. If IAPP expression exceeds the threshold for clearance of misfolded proteins, beta cell failure occurs exacerbated by the action of IAPP toxicity to compromise the autophagy lysosomal pathway. We postulated that suppression of IAPP expression by an IAPP antisense oligonucleotide delivered to beta cells by the GLP-1 agonist exenatide (eGLP1-IAPP-ASO) is a potential disease modifying therapy for T2D. While eGLP1-IAPP-ASO suppressed mouse IAPP and transgenic human IAPP expression in mouse islets, it had no discernable effects on IAPP expression in human islets under the conditions studied. Suppression of transgenic human IAPP expression in mouse islets attenuated disruption of the autophagy lysosomal pathway in beta cells, supporting the potential of this strategy.

Perturbing the conformation of diabetes-associated oligomers using molecular foldamers.

The islet amyloid polypeptide (IAPP) is a 37 aa peptide hormone secreted from pancreatic β-cells. In patients with Type II Diabetes, IAPP undergoes a toxic gain-of-function that results in the formation of amyloid fibrils. While these fibrils are a hallmark of the disease, soluble oligomeric precursors are most potently linked with IAPP pathogenicity. IAPP oligomers (IAos) disrupt homeostasis through a variety of mechanisms, including mitochondrial stress, aberrant Ca2+ signaling, and the formation of membrane pores. However, the biophysics of IAo pathogenicity is poorly characterized due to their instability in solution. We have shown that sodium dodecyl sulfate (SDS) micelles (which mimic anionic membranes) can stabilize IAos of different sizes in a physiological buffer. However, the morphology and conformational diversity of IAPP at the lipid interface is still unclear. We hypothesize that IAo first assembles on membrane surfaces and forms oligomers, which enables misfolding and their toxic gain-of-function. To gain molecular insight into the formation of IAo at this interface, we employ replica-exchange molecular dynamics (MD) and classical MD simulations. We monitor the conformation of IAo in the presence of various protein/micelle ratios as well as in free detergent. Finally, we introduce a molecular foldamer—shown to rescue IAPP-induced toxicity in cell models—to understand its effect on its conformational landscape. By comparing accessible conformations with and without the foldamer, we hope to identify conformations stabilized by the binding interaction. Molecular foldamers demonstrate the therapeutic potency of conformational modulators towards treating IAPP toxicity in T2D. Furthermore, this general strategy can be applied to other disordered peptides (e.g. amyloid-β) that undergo pathological misfolding.

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.

Membrane-Catalyzed Aggregation of Islet Amyloid Polypeptide Is Dominated by Secondary Nucleation.

Type II diabetes is characterized by the loss of pancreatic β-cells. This loss is thought to be a consequence of membrane disruption, caused by the aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils. However, the molecular mechanisms of IAPP aggregation in the presence of membranes have remained unclear. Here, we use kinetic analysis to elucidate the aggregation mechanism of IAPP in the presence of mixed zwitterionic and anionic lipid membranes. The results converge to a model in which aggregation on the membrane is strongly dominated by secondary nucleation, that is, the formation of new nuclei on the surface of existing fibrils. The critical nucleus consists of a single IAPP molecule, and anionic lipids catalyze both primary and secondary nucleation, but not elongation. The fact that anionic lipids promote secondary nucleation implies that these events take place at the interface between the membrane and existing fibrils, demonstrating that fibril growth occurs at least to some extent on the membrane surface. These new insights into the mechanism of IAPP aggregation on membranes may help to understand IAPP toxicity and will be important for the development of therapeutics to prevent β-cell death in type II diabetes.

Silybins inhibit human IAPP amyloid growth and toxicity through stereospecific interactions

Type 2 Diabetes is a major public health threat, and its prevalence is increasing worldwide. The abnormal accumulation of islet amyloid polypeptide (IAPP) in pancreatic β-cells is associated with the onset of the disease. Therefore, the design of small molecules able to inhibit IAPP aggregation represents a promising strategy in the development of new therapies. Here we employ in vitro, biophysical, and computational methods to inspect the ability of Silybin A and Silybin B, two natural diastereoisomers extracted from milk thistle, to interfere with the toxic self-assembly of human IAPP (hIAPP). We show that Silybin B inhibits amyloid aggregation and protects INS-1 cells from hIAPP toxicity more than Silybin A. Molecular dynamics simulations revealed that the higher efficiency of Silybin B is ascribable to its interactions with precise hIAPP regions that are notoriously involved in hIAPP self-assembly i.e., the S20-S29 amyloidogenic core, H18, the N-terminal domain, and N35. These results highlight the importance of stereospecific ligand-peptide interactions in regulating amyloid aggregation and provide a blueprint for future studies aimed at designing Silybin derivatives with enhanced drug-like properties.

Structural Dissection of the First Events Following Membrane Binding of the Islet Amyloid Polypeptide

The islet amyloid polypeptide (IAPP) is the main constituent of the amyloid fibrils found in the pancreas of type 2 diabetes patients. The aggregation of IAPP is known to cause cell death, where the cell membrane plays a dual role: being a catalyst of IAPP aggregation and being the target of IAPP toxicity. Using ATR-FTIR spectroscopy, transmission electron microscopy, and molecular dynamics simulations we investigate the very first molecular steps following IAPP binding to a lipid membrane. In particular, we assess the combined effects of the charge state of amino-acid residue 18 and the IAPP-membrane interactions on the structures of monomeric and aggregated IAPP. Distinct IAPP-membrane interaction modes for the various IAPP variants are revealed. Membrane binding causes IAPP to fold into an amphipathic α-helix, which in the case of H18K-, and H18R-IAPP readily moves beyond the headgroup region. For all IAPP variants but H18E-IAPP, the membrane-bound helix is an intermediate on the way to amyloid aggregation, while H18E-IAPP remains in a stable helical conformation. The fibrillar aggregates of wild-type IAPP and H18K-IAPP are dominated by an antiparallel β-sheet conformation, while H18R- and H18A-IAPP exhibit both antiparallel and parallel β-sheets as well as amorphous aggregates. Our results emphasize the decisive role of residue 18 for the structure and membrane interaction of IAPP. This residue is thus a good therapeutic target for destabilizing membrane-bound IAPP fibrils to inhibit their toxic actions.

Surface-Catalyzed Secondary Nucleation Dominates the Generation of Toxic IAPP Aggregates.

The aggregation of the human islet amyloid polypeptide (IAPP) is associated with diabetes type II. A quantitative understanding of this connection at the molecular level requires that the aggregation mechanism of IAPP is resolved in terms of the underlying microscopic steps. Here we have systematically studied recombinant IAPP, with amidated C-terminus in oxidised form with a disulphide bond between residues 3 and 7, using thioflavin T fluorescence to monitor the formation of amyloid fibrils as a function of time and IAPP concentration. We used global kinetic analyses to connect the macroscopic measurements of aggregation to the microscopic mechanisms, and show that the generation of new aggregates is dominated by the secondary nucleation of monomers on the fibril surface. We then exposed insulinoma cells to aliquots extracted from different time points of the aggregation process, finding the highest toxicity at the midpoint of the reaction, when the secondary nucleation rate reaches its maximum. These results identify IAPP oligomers as the most cytotoxic species generated during IAPP aggregation, and suggest that compounds that target secondary nucleation of IAPP could be most effective as therapeutic candidates for diabetes type II.

IAPP-induced beta cell stress recapitulates the islet transcriptome in type 2 diabetes

Aims/hypothesisType 2 diabetes is characterised by islet amyloid and toxic oligomers of islet amyloid polypeptide (IAPP). We posed the questions, (1) does IAPP toxicity induce an islet response comparable to that in humans with type 2 diabetes, and if so, (2) what are the key transcriptional drivers of this response?MethodsThe islet transcriptome was evaluated in five groups of mice: beta cell specific transgenic for (1) human IAPP, (2) rodent IAPP, (3) human calpastatin, (4) human calpastatin and human IAPP, and (5) wild-type mice. RNA sequencing data was analysed by differential expression analysis and gene co-expression network analysis to establish the islet response to adaptation to an increased beta cell workload of soluble rodent IAPP, the islet response to increased expression of oligomeric human IAPP, and the extent to which the latter was rescued by suppression of calpain hyperactivation by calpastatin. Rank-rank hypergeometric overlap analysis was used to compare the transcriptome of islets from human or rodent IAPP transgenic mice vs humans with prediabetes or type 2 diabetes.ResultsThe islet transcriptomes in humans with prediabetes and type 2 diabetes are remarkably similar. Beta cell overexpression of soluble rodent or oligomer-prone human IAPP induced changes in islet transcriptome present in prediabetes and type 2 diabetes, including decreased expression of genes that confer beta cell identity. Increased expression of human IAPP, but not rodent IAPP, induced islet inflammation present in prediabetes and type 2 diabetes in humans. Key mediators of the injury responses in islets transgenic for human IAPP or those from individuals with type 2 diabetes include STAT3, NF-κB, ESR1 and CTNNB1 by transcription factor analysis and COL3A1, NID1 and ZNF800 by gene regulatory network analysis.Conclusions/interpretationBeta cell injury mediated by IAPP is a plausible mechanism to contribute to islet inflammation and dedifferentiation in type 2 diabetes. Inhibition of IAPP toxicity is a potential therapeutic target in type 2 diabetes.Graphical abstract

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.

IAPP toxicity activates HIF1\u03b1/PFKFB3 signaling delaying \u03b2-cell loss at the expense of \u03b2-cell function

The islet in type 2 diabetes (T2D) is characterized by amyloid deposits derived from islet amyloid polypeptide (IAPP), a protein co-expressed with insulin by β-cells. In common with amyloidogenic proteins implicated in neurodegeneration, human IAPP (hIAPP) forms membrane permeant toxic oligomers implicated in misfolded protein stress. Here, we establish that hIAPP misfolded protein stress activates HIF1α/PFKFB3 signaling, this increases glycolysis disengaged from oxidative phosphorylation with mitochondrial fragmentation and perinuclear clustering, considered a protective posture against increased cytosolic Ca2+ characteristic of toxic oligomer stress. In contrast to tissues with the capacity to regenerate, β-cells in adult humans are minimally replicative, and therefore fail to execute the second pro-regenerative phase of the HIF1α/PFKFB3 injury pathway. Instead, β-cells in T2D remain trapped in the pro-survival first phase of the HIF1α injury repair response with metabolism and the mitochondrial network adapted to slow the rate of cell attrition at the expense of β-cell function.

All-atom structure ensembles of islet amyloid polypeptides determined by enhanced sampling and experiment data restraints.

Exploring the accurate structure ensembles are critical to understand the functions of intrinsically disordered proteins (IDPs). As a well-known IDP, islet amyloid polypeptide (IAPP) plays important roles in the development of human type II diabetes (T2D). The toxicity of human IAPP (hIAPP) is induced by the amyloidosis of the peptide, however, its aggregation mechanism remains ambiguous. The characterization of structure ensemble of hIAPP, as well as the differences between hIAPP and its non-amyloidogenic homologous such as rat IAPP (rIAPP), would greatly help to illuminate the amyloidosis mechanism of IAPP. In this study, the atomic structure ensembles of hIAPP and rIAPP were characterized by all-atom molecular dynamics (MD) simulations combined with enhanced sampling technology and experiment data restraints. The obtained structure ensembles were firstly compared with those determined by the conventional MD (cMD) and enhanced sampling without experiment data restraints. The results showed that the enhanced sampling and experiment data restraints would improve the simulation accuracy. The transient N-terminal α-helix structures were adopted by the sub-states of both hIAPP and rIAPP, however, the C-terminal helical structures were only present on hIAPP. The hydrophobic residues in the amyloid-core region of hIAPP are exposed to the solvent. The structure ensemble differences between hIAPP and rIAPP revealed in this work provide potential explain to the amyloidogenic mechanism and would be helpful for the design of drugs to combat T2D.

Zinc-Induced Conformational Transitions in Human Islet Amyloid Polypeptide and Their Role in the Inhibition of Amyloidosis.

Type-2 diabetes mellitus (T2DM) is a disease hallmarked by improper homeostasis within the islets of Langerhans of the pancreas. The most critical species affected is insulin, which is produced by the β-cells of the islets, but there are a number of other species copackaged and cosecreted within the insulin granules. This includes zinc, which exists in high (millimolar) concentrations within the β-cells, and islet amyloid polypeptide (IAPP), which is an amyloid peptide thought to induce β-cell apoptosis through self-association into toxic amyloid oligomers. Zinc is essential in the packaging of crystalline insulin within the vesicles but it can also bind and interact with IAPP. This implies a complex relationship between all three species and diabetes, particularly in the structure and function of toxic IAPP aggregates. Atypical (low or high) concentrations of zinc generally appear to correlate with increased hIAPP aggregation, whereas physiological zinc concentrations have an inhibitory effect. To better understand how zinc ions alter the monomer and oligomer structure of hIAPP in vitro, we employ a combination of ion mobility mass spectrometry and atomic force microscopy. We observe an increase in the extended β-hairpin conformation of hIAPP when it is bound to zinc. With sufficiently low concentrations of zinc this could result in an association site for zinc-free hIAPP, promoting amyloid aggregation. At high zinc concentrations, we see the appearance of a secondary zinc association site whose coordination could account for the loss of inhibition at high zinc concentrations. Generally, it appears that zinc preferentially stabilizes the β-hairpin conformation of hIAPP and the population of zinc-bound hIAPP in solution determines what effect this has on amyloid aggregation.

In Vivo Mitigation of Amyloidogenesis through Functional-Pathogenic Double-Protein Coronae.

Amyloid diseases are global epidemics with no cure available. Herein, we report a first demonstration of in vivo mitigation of amyloidogenesis using biomimetic nanotechnology. Specifically, the amyloid fragments (ba) of β-lactoglobulin, a whey protein, were deposited onto the surfaces of carbon nanotubes (baCNT), which subsequently sequestered human islet amyloid polypeptide (IAPP) through functional-pathogenic double-protein coronae. Conformational changes at the ba-IAPP interface were studied by Fourier transform infrared, circular dichroism, and X-ray scattering spectroscopies. baCNT eliminated the toxic IAPP species from zebrafish embryos, as evidenced by the assays of embryonic development, cell morphology, hatching, and survival as well as suppression of oxidative stress. In addition to IAPP, baCNT also displayed high potency against the toxicity of amyloid-β, thereby demonstrating the broad applicability of this biomimetic nanotechnology and the use of an embryonic zebrafish model for the high-throughput screening of a range of amyloidogenesis and their inhibitors in vivo.

Nanoscale inhibition of polymorphic and ambidextrous IAPP amyloid aggregation with small molecules.

Understanding how small molecules interface amyloid fibrils on the nanoscale is of importance for developing therapeutic treatment against amyloid-based diseases. Here we show, for the first time, that human islet amyloid polypeptide (IAPP) in the fibrillar form is polymorphic and ambidextrous possessing multiple periodicities. Upon interfacing with small molecule epigallocatechin gallate (EGCG), IAPP aggregation was rendered off pathway assuming the form of soft and disordered clusters, while mature IAPP fibrils displayed kinks and branching but conserved the twisted fibril morphology. These nanoscale phenomena resulted from competitive interactions between EGCG and the IAPP amyloidogenic region, as well as end capping of the fibrils by the small molecule. This information is crucial to delineating IAPP toxicity implicated in type 2 diabetes and developing new inhibitors against amyloidogenesis.

The IAPP Toxicity on Rats, Raccoons and Degus in HeLa Cells

Type 2 diabetes is the most common form of diabetes, where the body suffers from insulin resistance. The islet amyloid polypeptide (IAPP) is a secretory product of beta cells in pancreatic islets of Langerhans that also inhibits glucagon and insulin secretion. The aggregation of IAPP is present in pancreatic islet amyloid deposits seen especially in Type 2 diabetes, in humans and other mammalian species. The objective of this study was to determine the effects of the animal toxicity on mammalian cells. In this work, the human IAPP sequence and the effect on mammalian cells was compared to the IAPP from degu, rat, and raccoon. Molecular techniques such as MTT and LDH Cytotoxicity assay were used to test the effect of human and animal IAPP on HeLa cells. The MTT results show a consistent drop in cell viability as the concentration of IAPP from a human, degu, rat and raccoon increases. LDH cytotoxicity shows the highest toxicity in human IAPP compared to the rest of the studied animals. Although when mixing both the human and animal IAPPs, the toxicity of the human IAPP decreased whereas the viability had no significant effect. Further molecular analysis is necessary to determine the correlation between animal IAPP toxicity and the incidence of Type 2 diabetes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of Dog, Cat, Guinea Pig, and Human IAPP toxicity in HeLa cells

Type 2 diabetes is one of the leading causes of death in the U.S. Type 2 Diabetes is the most generic form of diabetes, where your body suffers from insulin resistance. Islet Amyloid Polypeptide (IAPP) is a regulatory peptide which inhibits glucagon and insulin secretion. The aggregation of IAPP is present in pancreatic islet amyloid deposits seen especially in Type 2 diabetes in humans and other mammalian species. Our objective was to see how the changes in IAPP sequence correlates to the toxicity of IAPP on mammalian cells. Here, we focus on dog, cat, and guinea pig IAPP. Molecular techniques such as MTT assay and LDH Cytotoxicity assay were used to test the effect animal IAPP has on HeLa cells. As well as to see the effect mixing human and animal IAPP has on the HeLa cells. MTT assay results showed that the animal IAPP volumes had no significant effect on mammalian cell viability while the human IAPP was consistently toxic to the cells. Cell viability percentages stayed relatively constant despite increasing IAPP volume. In comparison, human IAPP showed a decreasing cell viability trend with increasing IAPP volume. However, when dog IAPP and human IAPP were mixed, cell viability was increased. The same effect occurred with the mixing of cat IAPP and human IAPP. LDH Cytotoxicity assay results showed that percent toxicity increased with increasing IAPP volumes for human, cat and guinea pig. Dog IAPP cell toxicity stayed constant as IAPP volume increased. When dog IAPP and cat IAPP were separately mixed with human IAPP, a decrease in cell toxicity is seen. Mixing guinea pig IAPP with human IAPP had no effect on cell viability or cell toxicity. Further experiments are necessary to determine a correlation between these specific animal IAPPs and cell toxicity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Translocon Declogger Ste24 Protects against IAPP Oligomer-Induced Proteotoxicity

Aggregates of human islet amyloid polypeptide (IAPP) in the pancreas of patients with type 2 diabetes (T2D) are thought to contribute to β cell dysfunction and death. To understand how IAPP harms cells and how this might be overcome, we created a yeast model of IAPP toxicity. Ste24, an evolutionarily conserved protease that was recently reported to degrade peptides stuck within the translocon between the cytoplasm and the endoplasmic reticulum, was the strongest suppressor of IAPP toxicity. By testing variants of the human homolog, ZMPSTE24, with varying activity levels, the rescue of IAPP toxicity proved to be directly proportional to the declogging efficiency. Clinically relevant ZMPSTE24 variants identified in the largest database of exomes sequences derived from T2D patients were characterized using the yeast model, revealing 14 partial loss-of-function variants, which were enriched among diabetes patients over 2-fold. Thus, clogging of the translocon by IAPP oligomers may contribute to β cell failure.