Aggregation of islet amyloid polypeptide (IAPP) to form amyloid contributes to β-cell dysfunction in type 2 diabetes, yet the identity and temporal persistence of the toxic species is unresolved. Competing models attribute toxicity to mature fibrils, fibril growth, or transient prefibrillar intermediates formed during the lag phase or via secondary nucleation. Here, we directly test these models by combining time-resolved β-cell functional assays with concurrent biophysical measurements of IAPP aggregation across multiple perturbations and sequence variants. Across 22 independent experiments spanning more than a 450-fold range in lag times, we find that maximal toxicity occurs during the lag phase and declines as fibrils accumulate. The duration of β-cell dysfunction scales linearly with lag phase length, establishing aggregation kinetics as a quantitative predictor of the onset, peak, and termination of toxicity. Perturbations that alter aggregation kinetics, including concentration, temperature, and sequence, predictably shift the temporal window of toxicity. The diabetes-associated S20G variant produces higher peak toxicity over a compressed time window, whereas the slower-aggregating variants examined prolong toxicity without increasing its magnitude. These results resolve competing models by demonstrating that transient lag-phase intermediates, rather than growth phase processes or mature fibrils, dominate β-cell dysfunction, and establish aggregation kinetics as a predictor of the timing and duration of cellular exposure to toxic intermediates.

HLA-DQA1*03:90 differs from HLA-DQA1*03:03 in exon 1 at codon -13 resulting in an Alanine to Serine substitution.

Abstract Aggregation of islet amyloid polypeptide (IAPP) contributes to pancreatic β-cell dysfunction in diabetes, yet the identity and temporal persistence of the toxic species remain unresolved. Because amyloid formation proceeds through distinct kinetic phases, relating cytotoxicity to these phases provides a direct strategy to test competing mechanistic models. Here, we combine time-resolved β-cell functional assays with concurrent biophysical measurements to quantitatively link IAPP aggregation kinetics to cellular dysfunction across multiple perturbations and sequence variants. We demonstrate that maximal toxicity occurs during the lag phase and establish aggregation kinetics as a predictive framework for the onset and duration of IAPP proteotoxicity. Across 22 independent experiments spanning more than a 450-fold range in lag times, the duration of β-cell dysfunction scales linearly with lag phase length. Perturbations that alter aggregation kinetics, including changes in concentration, temperature, and sequence, predictably shift both the onset and duration of toxicity. Early lag-phase intermediates of the diabetes-associated S20G-IAPP mutant are more toxic than corresponding species formed by wild-type h-IAPP. Together, these results show that aggregation kinetics quantitatively define the temporal window of IAPP proteotoxicity and support a model in which transient pre-fibrillar intermediates, rather than mature fibrils, dominate β-cell dysfunction.

HLA-DRB1*03:201 differs from HLA-DRB1*03:01 in exon 3 at codon 178 resulting in a proline to serine substitution.

Amylin, a member of the calcitonin family, acts via amylin receptors in the hindbrain and hypothalamus to suppress appetite. Native ligands of these receptors are peptides with short half-lives. Conjugating fatty acids to these peptides can increase their half-lives. The long-acting human amylin analog, NN1213, was generated from structure-activity efforts optimizing solubility, stability, receptor affinity, and selectivity, as well as in vivo potency and clearance. In both rats and dogs, a single dose of NN1213 reduced appetite in a dose-dependent manner and with a long duration of action. Consistent with the effect on appetite, studies in obese rats demonstrated that daily NN1213 dosing resulted in a dose-dependent reduction in body weight over a 21-day period. Magnetic resonance imaging indicated that this was primarily driven by loss of fat mass. Based on these data, NN1213 could be considered an attractive option for weight management in the clinical setting.

HLA-DRB1*03:201 differs from HLA-DRB1*03:01 in exon 3 at codon 178 resulting in a proline to serine substitution.

HLA‐DRB1*08:112 differs from HLA‐DRB1*08:01 in exon 2 at amino acid 62; asparagine to lysine substitution.

HLA-DRB1*08:112 differs from HLA-DRB1*08:01 in exon 2 at amino acid 62; asparagine to lysine substitution.

HLA-DQA1*01:65 differs from HLA-DQA1*01:03 in exon 1 at amino acid -7 a valine to methionine substitution.

HLA‐DRB1*08:112 differs from HLA‐DRB1*08:01 in exon 2 at amino acid 62; asparagine to lysine substitution.

HLA-DRB1*08:112 differs from HLA-DRB1*08:01 in exon 2 at amino acid 62; asparagine to lysine substitution.

HLA-DQA1*01:65 differs from HLA-DQA1*01:03 in exon 1 at amino acid -7 a valine to methionine substitution.

HLA‐DQA1*01:65 differs from HLA‐DQA1*01:03 in exon 1 at amino acid ‐7 a valine to methionine substitution.

HLA-DQA1*01:65 differs from HLA-DQA1*01:03 in exon 1 at amino acid -7 a valine to methionine substitution.

Islet amyloidosis is characterized by the aberrant accumulation of islet amyloid polypeptide (IAPP) in pancreatic islets, resulting in &bgr; cell toxicity, which exacerbates type 2 diabetes and islet transplant failure. It is not fully clear how IAPP induces cellular stress or how IAPP-induced toxicity can be prevented or treated. We recently defined the properties of toxic IAPP species. Here, we have identified a receptor-mediated mechanism of islet amyloidosis–induced proteotoxicity. In human diabetic pancreas and in cellular and mouse models of islet amyloidosis, increased expression of the receptor for advanced glycation endproducts (RAGE) correlated with human IAPP–induced (h-IAPP–induced) &bgr; cell and islet inflammation, toxicity, and apoptosis. RAGE selectively bound toxic intermediates, but not nontoxic forms of h-IAPP, including amyloid fibrils. The isolated extracellular ligand–binding domains of soluble RAGE (sRAGE) blocked both h-IAPP toxicity and amyloid formation. Inhibition of the interaction between h-IAPP and RAGE by sRAGE, RAGE-blocking antibodies, or genetic RAGE deletion protected pancreatic islets, &bgr; cells, and smooth muscle cells from h-IAPP–induced inflammation and metabolic dysfunction. sRAGE-treated h-IAPP Tg mice were protected from amyloid deposition, loss of &bgr; cell area, &bgr; cell inflammation, stress, apoptosis, and glucose intolerance. These findings establish RAGE as a mediator of IAPP-induced toxicity and suggest that targeting the IAPP/RAGE axis is a potential strategy to mitigate this source of &bgr; cell dysfunction in metabolic disease.

Objective: To characterize the EndoC-beta H1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti diabetic drug candidates. Methods: EndoC-beta H1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation. Results: Transplantation of EndoC-beta H1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-beta H1 pseudoislets compared to monolayer cultures for both glucose and incretins. Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate. By screening of various proteins and peptides, we found Bombesin (BB) receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP) to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation. ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST) was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion. Conclusions: Overall, the EndoC-beta H1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active cytokine induced caspase 3/7 apoptotic pathway and is responsive to ER stress initiation factors. The cells' ability to proliferate can be further increased by already known compounds as well as by novel peptides and proteins. Based on its robust performance during the functionality assessment assays, the EndoC-beta H1 cell line was successfully used as a screening platform for identification of novel anti-diabetic drug candidates. (C) 2017 Novo Nordisk A/S. Published by Elsevier GmbH.