The islet tissue plasminogen activator/plasmin system is upregulated with human islet amyloid polypeptide aggregation and protects beta cells from aggregation-induced toxicity.

AIMS/HYPOTHESIS: Apart from its fibrinolytic activity, the tissue plasminogen activator (tPA)/plasmin system has been reported to cleave the peptide amyloid beta, attenuating brain amyloid deposition in Alzheimer's disease. As aggregation of human islet amyloid polypeptide (hIAPP) is toxic to beta cells, we sought to determine whether activation of the fibrinolytic system can also reduce islet amyloid deposition and its cytotoxic effects, which are both observed in type 2 diabetes. METHODS: The expression of Plat (encoding tPA) and plasmin activity were measured in isolated islets from amyloid-prone hIAPP transgenic mice or non-transgenic control islets expressing non-amyloidogenic mouse islet amyloid polypeptide cultured in the absence or presence of the amyloid inhibitor Congo Red. Plat expression was also determined in hIAPP-treated primary islet endothelial cells, bone marrow-derived macrophages (BMDM) and INS-1 cells, in order to determine the islet cell type(s) producing tPA in response to hIAPP aggregation. Cell-free thioflavin-T assays and MS were used to respectively monitor hIAPP aggregation kinetics and investigate plasmin cleavage of hIAPP. Cell viability was assessed in INS-1 beta cells treated with hIAPP with or without plasmin. Finally, to confirm the findings in human samples, PLAT expression was measured in freshly isolated islets from donors with and without type 2 diabetes. RESULTS: In isolated islets from transgenic mice, islet Plat expression and plasmin activity increased significantly with the process of amyloid deposition (p≤0.01, n=5); these effects were not observed in islets from non-transgenic mice and were blocked by Congo Red (p≤0.01, n=4). In response to hIAPP exposure, Plat expression increased in BMDM and INS-1 cells vs vehicle-treated cells (p≤0.05, n=4), but not in islet endothelial cells. Plasmin reduced hIAPP fibril formation in a dose-dependent manner in a cell-free system, and restored hIAPP-induced loss of cell viability in INS-1 beta cells (p≤0.01, n=5). Plasmin cleaved monomeric hIAPP, inducing a rapid decrease in the abundance of full-length hIAPP and the appearance of hIAPP 1-11 and 12-37 fragments. hIAPP 12-37, which contains the critical amyloidogenic region, was not toxic to INS-1 cells. Finally, PLAT expression was significantly increased by 2.4-fold in islets from donors with type 2 diabetes (n=4) vs islets from donors without type 2 diabetes (n=7) (p≤0.05). CONCLUSIONS/INTERPRETATION: The fibrinolytic system is upregulated in islets with hIAPP aggregation. Plasmin rapidly degrades hIAPP, limiting its aggregation into amyloid and thus protecting beta cells from hIAPP-induced toxicity. Thus, increasing islet plasmin activity might be a strategy to limit beta cell loss in type 2 diabetes.

UTRs and Ago-2/miR-335 Complex Restricts Amylin Translation in Insulinoma and Human Pancreatic ß-Cells.

Amylin promoter and transcriptional factors are well-established, inducible factors in the production of the main amyloidogenic pancreatic hormone, human islet amyloid peptide (hIAPP) or amylin. However, posttranscriptional mechanisms driving hIAPP expression in pancreas remain enigmatic, and hence were explored here. The translational assay revealed that both 5' and 3' untranslated regions (UTRs) of hIAPP restricted expression of the luciferase constructs only in constructs driven by the hIAPP promoter. Bioinformatics analysis revealed several putative seed sequences for a dozen micro RNAs (miRNAs) in hIAPP's 3' UTR. miR-182, miR-335, and miR-495 were the most downregulated miRNAs in stressed human islets exposed to endoplasmic reticulum (ER) or metabolic stressors, thapsigargin (TG) or high glucose (HG). Correspondingly, miR-335 mimics alone or in combination with miR-495 and miR-182 mimics significantly and potently (>3-fold) reduced hIAPP protein expression in HG-treated cultured human islets. siRNA-mediated silencing of Ago2 but not Ago1 significantly stimulated hIAPP expression and secretion from transfected, HG-treated human islets. Conversely, ectopic expression of Ago2 in hIAPP-expressing RIN-m5F cell line driven by CMV promoter reduced hIAPP intracellular protein levels. Collectively, the results point to a novel and synergistic role for hIAPP promoter, 5/3' UTRs and Ago-2/miR-335 complex in post-transcriptional regulation of hIAPP gene expression in normal and metabolically active ß-cells.

An INSULIN and IAPP dual reporter enables tracking of functional maturation of stem cell-derived insulin producing cells.

OBJECTIVE: Human embryonic stem cell (hESC; SC)-derived pancreatic ß cells can be used to study diabetes pathologies and develop cell replacement therapies. Although current differentiation protocols yield SCß cells with varying degrees of maturation, these cells still differ from deceased donor human ß cells in several respects. We sought to develop a reporter cell line that could be used to dynamically track SCß cell functional maturation. METHODS: To monitor SCß cell maturation in vitro, we created an IAPP-2A-mScar and INSULIN-2A-EGFP dual fluorescent reporter (INS2A-EGFP/+;IAPP2A-mScarlet/+) hESC line using CRISPR/Cas9. Pluripotent SC were then differentiated using a 7-stage protocol to islet-like cells. Immunohistochemistry, flow cytometry, qPCR, GSIS and electrophysiology were used to characterise resulting cell populations. RESULTS: We observed robust expression of EGFP and mScarlet fluorescent proteins in insulin- and IAPP-expressing cells without any compromise to their differentiation. We show that the proportion of insulin-producing cells expressing IAPP increases over a 4-week maturation period, and that a subset of insulin-expressing cells remain IAPP-free. Compared to this IAPP-free population, we show these insulin- and IAPP-expressing cells are less polyhormonal, more glucose-sensitive, and exhibit decreased action potential firing in low (2.8 mM) glucose. CONCLUSIONS: The INS2A-EGFP/+;IAPP2A-mScarlet/+ hESC line provides a useful tool for tracking populations of maturing hESC-derived ß cells in vitro. This tool has already been shared with 3 groups and is freely available to all.

Exploring the central region of amylin and its analogs aggregation: the influence of metal ions and residue substitutions.

Human amylin (hIAPP) is found in the form of amyloid deposits within the pancreatic cells of nearly all patients diagnosed with type 2 diabetes mellitus (T2DM). However, rat amylin (rIAPP) and pramlintide - hIAPP analogs - are both non-toxic and non-amyloidogenic. Their primary sequences exhibit only slight variations in a few amino acid residues, primarily concentrated in the central region, spanning residues 20 to 29. This inspired us to study this fragment and investigate the impact on the aggregation properties of substituting residues within the central region of amylin and its analogs. Six fragments derived from amylin have undergone comprehensive testing against various metal ions by implementing a range of analytical techniques, including Nuclear Magnetic Resonance (NMR) spectroscopy, Thioflavin T (ThT) assays, Atomic Force Microscopy (AFM), and cytotoxicity assays. These methodologies serve to provide a thorough understanding of how the substitutions and interactions with metal ions impact the aggregation behavior of amylin and its analogs.

Inhibition of YIPF2 Improves the Vulnerability of Oligodendrocytes to Human Islet Amyloid Polypeptide.

Excessive secretion of human islet amyloid polypeptide (hIAPP) is an important pathological basis of diabetic encephalopathy (DE). In this study, we aimed to investigate the potential implications of hIAPP in DE pathogenesis. Brain magnetic resonance imaging and cognitive scales were applied to evaluate white matter damage and cognitive function. We found that the concentration of serum hIAPP was positively correlated with white matter damage but negatively correlated with cognitive scores in patients with type 2 diabetes mellitus. In vitro assays revealed that oligodendrocytes, compared with neurons, were more prone to acidosis under exogenous hIAPP stimulation. Moreover, western blotting and co-immunoprecipitation indicated that hIAPP interfered with the binding process of monocarboxylate transporter (MCT)1 to its accessory protein CD147 but had no effect on the binding of MCT2 to its accessory protein gp70. Proteomic differential analysis of proteins co-immunoprecipitated with CD147 in oligodendrocytes revealed Yeast Rab GTPase-Interacting protein 2 (YIPF2, which modulates the transfer of CD147 to the cell membrane) as a significant target. Furthermore, YIPF2 inhibition significantly improved hIAPP-induced acidosis in oligodendrocytes and alleviated cognitive dysfunction in DE model mice. These findings suggest that increased CD147 translocation by inhibition of YIPF2 optimizes MCT1 and CD147 binding, potentially ameliorating hIAPP-induced acidosis and the consequent DE-related demyelination.

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

Type 2 diabetes (T2D) is a common metabolic disease due to insufficient insulin secretion by pancreatic ß-cells in the context of insulin resistance. Islet molecular pathology reveals a role for protein misfolding in ß-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 ß-cells in T2D and in ß-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-ß-cell cholesterol and lipid deposits and impaired cholesterol trafficking to cell membranes. These findings align with comparable lipid deposits present in ß-cells in T2D and increased vulnerability to develop T2D in individuals taking medications that suppress cholesterol synthesis.NEW & NOTEWORTHY ß-Cell failure in type 2 diabetes (T2D) is characterized by ß-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 ß-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.

XBP1 splicing contributes to endoplasmic reticulum stress-induced human islet amyloid polypeptide up-regulation.

As a pathological hallmark of type 2 diabetes mellitus (T2DM), islet amyloid is formed by the aggregation of islet amyloid polypeptide (IAPP). Endoplasmic reticulum (ER) stress interacts with IAPP aggregates and has been implicated in the pathogenesis of T2DM. To examine the role of ER stress in T2DM, we cloned the hIAPP promoter and analyzed its promoter activity in human ß-cells. We found that ER stress significantly enhanced hIAPP promoter activity and expression in human ß-cells via triggering X-box binding protein 1 (XBP1) splicing. We identified a binding site of XBP1 in the hIAPP promoter. Disruption of this binding site by substitution or deletion mutagenesis significantly diminished the effects of ER stress on hIAPP promoter activity. Blockade of XBP splicing by MKC3946 treatment inhibited ER stress-induced hIAPP up-regulation and improved human ß-cell survival and function. Our study uncovers a link between ER stress and IAPP at the transcriptional level and may provide novel insights into the role of ER stress in IAPP cytotoxicity and the pathogenesis of T2DM.

Pathological evaluation of the pathogenesis of diabetes mellitus and diabetic peripheral neuropathy.

Currently, there are more than 10 million patients with diabetes mellitus in Japan. Therefore, the need to explore the pathogenesis of diabetes and the complications leading to its cure is becoming increasingly urgent. Pathological examination of pancreatic tissues from patients with type 2 diabetes reveals a decrease in the volume of beta cells because of a combination of various stresses. In human type 2 diabetes, islet amyloid deposition is a unique pathological change characterized by proinflammatory macrophage (M1) infiltration into the islets. The pathological changes in the pancreas with islet amyloid were different according to clinical factors, which suggests that type 2 diabetes can be further subclassified based on islet pathology. On the other hand, diabetic peripheral neuropathy is the most frequent diabetic complication. In early diabetic peripheral neuropathy, M1 infiltration in the sciatic nerve evokes oxidative stress or attenuates retrograde axonal transport, as clearly demonstrated by in vitro live imaging. Furthermore, islet parasympathetic nerve density and beta cell volume were inversely correlated in type 2 diabetic Goto-Kakizaki rats, suggesting that diabetic peripheral neuropathy itself may contribute to the decrease in beta cell volume. These findings suggest that the pathogenesis of diabetes mellitus and diabetic peripheral neuropathy may be interrelated.

IAPP Marks Mono-hormonal Stem-cell Derived ß Cells that Maintain Stable Insulin Production in vitro and in vivo.

Islet transplantation for treatment of diabetes is limited by availability of donor islets and requirements for immunosuppression. Stem cell-derived islets might circumvent these issues. SC-islets effectively control glucose metabolism post transplantation, but do not yet achieve full function in vitro with current published differentiation protocols. We aimed to identify markers of mature subpopulations of SC-ß cells by studying transcriptional changes associated with in vivo maturation of SC-ß cells using RNA-seq and co-expression network analysis. The ß cell-specific hormone islet amyloid polypeptide (IAPP) emerged as the top candidate to be such a marker. IAPP+ cells had more mature ß cell gene expression and higher cellular insulin content than IAPP- cells in vitro. IAPP+ INS+ cells were more stable in long-term culture than IAPP- INS+ cells and retained insulin expression after transplantation into mice. Finally, we conducted a small molecule screen to identify compounds that enhance IAPP expression. Aconitine up-regulated IAPP and could help to optimize differentiation protocols.

Neuroinflammation induced by amyloid-forming pancreatic amylin: Rationale for a mechanistic hypothesis.

Amylin is a systemic neuroendocrine hormone co-expressed and co-secreted with insulin by pancreatic ß-cells. In persons with thype-2 diabetes, amylin forms pancreatic amyloid triggering inflammasome and interleukin-1ß signaling and inducing ß-cell apoptosis. Here, we summarize recent progress in understanding the potential link between amyloid-forming pancreatic amylin and Alzheimer's disease (AD). Clinical data describing amylin pathology in AD alongside mechanistic studies in animals are reviewed. Data from multiple research teams indicate higher amylin concentrations are associated with increased frequency of cognitive impairment and amylin co-aggregates with ß-amyloid in AD-type dementia. Evidence from rodent models further suggests cerebrovascular amylin accumulation as a causative factor underlying neurological deficits. Analysis of relevant literature suggests that modulating the amylin-interleukin-1ß pathway may provide an approach for counteracting neuroinflammation in AD.

The processing intermediate of human amylin, pro-amylin(1-48), has in vivo and in vitro bioactivity.

Amylin is released by pancreatic beta-cells in response to a meal and its major soluble mature form (37 amino acid-peptide) produces its biological effects by activating amylin receptors. Amylin is derived from larger propeptides that are processed within the synthesizing beta-cell. There are suggestions that a partially processed form, pro-amylin(1-48) is also secreted. We tested the hypothesis that pro-amylin(1-48) has biological activity and that human pro-amylin(1-48) may also form toxic pre-amyloid species. Amyloid formation, the ability to cross-seed and in vitro toxicity were similar between human pro-amylin(1-48) and amylin. Human pro-amylin(1-48) was active at amylin-responsive receptors, though its potency was reduced at rat, but not human amylin receptors. Pro-amylin(1-48) was able to promote anorexia by activating neurons of the area postrema, amylin's primary site of action, indicating that amylin can tolerate significant additions at the N-terminus without losing bioactivity. Our studies help to shed light on the possible roles of pro-amylin(1-48) which may be relevant for the development of future amylin-based drugs.

The Inhibition Effect of Epigallocatechin-3-Gallate on the Co-Aggregation of Amyloid-ß and Human Islet Amyloid Polypeptide Revealed by Replica Exchange Molecular Dynamics Simulations.

Alzheimer's disease and Type 2 diabetes are two epidemiologically linked diseases which are closely associated with the misfolding and aggregation of amyloid proteins amyloid-ß (Aß) and human islet amyloid polypeptide (hIAPP), respectively. The co-aggregation of the two amyloid proteins is regarded as the fundamental molecular mechanism underlying their pathological association. The green tea extract epigallocatechin-3-gallate (EGCG) has been extensively demonstrated to inhibit the amyloid aggregation of Aß and hIAPP proteins. However, its potential role in amyloid co-aggregation has not been thoroughly investigated. In this study, we employed the enhanced-sampling replica exchange molecular dynamics simulation (REMD) method to investigate the effect of EGCG on the co-aggregation of Aß and hIAPP. We found that EGCG molecules substantially diminish the ß-sheet structures within the amyloid core regions of Aß and hIAPP in their co-aggregates. Through hydrogen-bond, π-π and cation-π interactions targeting polar and aromatic residues of Aß and hIAPP, EGCG effectively attenuates both inter-chain and intra-chain interactions within the co-aggregates. All these findings indicated that EGCG can effectively inhibit the co-aggregation of Aß and hIAPP. Our study expands the potential applications of EGCG as an anti-amyloidosis agent and provides therapeutic options for the pathological association of amyloid misfolding disorders.

On the importance of being amidated: Analysis of the role of the conserved C-terminal amide of amylin in amyloid formation and cytotoxicity.

The polypeptide hormone Amylin (also known as islet amyloid polypeptide) plays a role in regulation of glucose metabolism, but forms pancreatic islet amyloid deposits in type 2 diabetes. The process of islet amyloid formation contributes to ß-cell dysfunction and the development of the disease. Amylin is produced as a pro-from and undergoes processing prior to secretion. The mature hormone contains an amidated C-terminus. Analysis of an alignment of vertebrate amylin sequences reveals that the processing signal for amidation is strictly conserved. Furthermore, the enzyme responsible for C-terminal amidation is found in all of these organisms. Comparison of the physiologically relevant amidated form to a variant with a free C-terminus (Amylin-COO-) shows that replacement of the C-terminal amide with a carboxylate slows, but does not prevent amyloid formation. Pre-fibrillar species produced by both variants are toxic to cultured ß-cells, although hAmylin-COO- is moderately less so. Amyloid fibrils produced by either peptide are not toxic. Prior work (ACS Pharmacol. Translational. Sci. 1, 132-49 (2018)) shows that Amylin- COO- exhibits a 58-fold reduction in activation of the Amylin1 receptor and 20-fold reduction in activation of the Amylin3 receptor. Thus, hAmylin-COO- exhibits significant toxicity, but significantly reduced activity and offers a reagent for studies which aim to decouple hAmylin's toxic effects from its activity. The different behaviours of free and C-terminal amidated Amylin should be considered when designing systems to produce the polypeptide recombinantly.

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 ß-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 ß-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.

Human islet amyloid polypeptide-induced ß-cell cytotoxicity is linked to formation of α-sheet structure.

Type 2 diabetes (T2D) results from insulin secretory dysfunction arising in part from the loss of pancreatic islet ß-cells. Several factors contribute to ß-cell loss, including islet amyloid formation, which is observed in over 90% of individuals with T2D. The amyloid is comprised of human islet amyloid polypeptide (hIAPP). Here we provide evidence that early in aggregation, hIAPP forms toxic oligomers prior to formation of amyloid fibrils. The toxic oligomers contain α-sheet secondary structure, a nonstandard secondary structure associated with toxic oligomers in other amyloid diseases. De novo, synthetic α-sheet compounds designed to be nontoxic and complementary to the α-sheet structure in the toxic oligomers inhibit hIAPP aggregation and neutralize oligomer-mediated cytotoxicity in cell-based assays. In vivo administration of an α-sheet design to mice for 4 weeks revealed no evidence of toxicity nor did it elicit an immune response. Furthermore, the α-sheet designs reduced endogenous islet amyloid formation and mitigation of amyloid-associated ß-cell loss in cultured islets isolated from an hIAPP transgenic mouse model of islet amyloidosis. Characterization of the involvement of α-sheet in early aggregation of hIAPP and oligomer toxicity contributes to elucidation of the molecular mechanisms underlying amyloid-associated ß-cell loss.

TPE conjugated islet amyloid polypeptide probe for detection of peptide oligomers.

Islet amyloid polypeptide (IAPP), also known as amylin, is a polypeptide hormone co-secreted with insulin by pancreatic ß-cells. In general, IAPP is soluble and lacks a defined structure. However, under certain conditions, these peptides tend to aggregate into soluble oligomers, eventually forming insoluble amyloid fibrils with typical cross-ß-sheet structures. Amylin aggregates, therefore, have been regarded as one of the hallmarks of type II diabetes (T2D). Among these aggregated species, oligomers were shown to exhibit significant cytotoxicity, leading to impaired ß-cell function and reduced ß-cell mass. Monitoring of oligomer appearance during IAPP fibrillation is of particular interest. In this study, we successfully grafted an aggregation-induced emission molecule, tetraphenylethylene (TPE), at the N-terminus of IAPP. By mixing a small amount of TPE-labeled IAPP with unlabeled IAPP, we were able to detect an increase in TPE fluorescence during the nucleation phase of IAPP aggregation in vitro. It may enable real-time monitoring of IAPP oligomer formation and is further applied in the diagnosis of T2D.

Regional differences in islet amyloid deposition in the residual pancreas with new-onset diabetes secondary to pancreatic ductal adenocarcinoma.

BACKGROUND: Islet amyloid deposition and reduced ß-cell mass are pathological hallmarks in type 2 diabetes mellitus subjects. To date, the pathological features of the islets in diabetes secondary to pancreatic ductal adenocarcinoma (PDAC) have not been specifically addressed. AIM: To provide further insight into the relationship between islet amyloid deposition of the residual pancreas in PDAC patients and to explore whether regional differences (proximal vs distal residual pancreas) are associated with islet amyloid deposition. METHODS: We retrospectively collected clinical information and pancreatic tissue removed from tumors of 45 PDAC patients, including 14 patients with normal glucose tolerance (NGT), 16 patients with prediabetes and 15 new-onset diabetes (NOD) patients diagnosed before surgery by an oral glucose tolerance test at West China Hospital from July 2017 to June 2020. Pancreatic volume was calculated by multiplying the estimated area of pancreatic tissue on each image slice by the interval between slices based on abdominal computer tomography scans. Several sections of paraffin-embedded pancreas specimens from both the proximal and/or distal regions remote from the tumor were stained as follows: (1) Hematoxylin and eosin for general histological appearance; (2) hematoxylin and insulin for the determination of fractional ß-cell area (immunohistochemistry); and (3) quadruple insulin, glucagon, thioflavin T and DAPI staining for the determination of ß-cell area, α-cell area and amyloid deposits. RESULTS: Screening for pancreatic histologic features revealed that duct obstruction with islet amyloid deposition, fibrosis and marked acinar atrophy were robust in the distal pancreatic regions but much less robust in the proximal regions, especially in the prediabetes and NOD groups. Consistent with this finding, the remnant pancreatic volume was markedly decreased in the NOD group by nearly one-half compared with that in the NGT group (37.35 ± 12.16 cm3 vs 69.79 ± 18.17 cm3, P < 0.001). As expected, islets that stained positive for amyloid (islet amyloid density) were found in the majority of PDAC cases. The proportion of amyloid/islet area (severity of amyloid deposition) was significantly higher in both prediabetes and NOD patients than in NGT patients (P = 0.002; P < 0.0001, respectively). We further examined the regional differences in islet amyloid deposits. Islet amyloid deposit density was robustly increased by approximately 8-fold in the distal regions compared with that in the proximal regions in the prediabetes and NOD groups (3.98% ± 3.39% vs 0.50% ± 0.72%, P = 0.01; 12.03% vs 1.51%, P = 0.001, respectively). CONCLUSION: In conclusion, these findings suggest that robust alterations of the distal pancreas due to tumors can disturb islet function and structure with islet amyloid formation, which may be associated with the pathogenesis of NOD secondary to PDAC.

Diabetes mellitus drug discovery: insights into targeting feline and human amylin with small molecules.

BACKGROUND: Type 2 diabetes (T2D) is a health concern for both humans and cats, with cases rising over the past decade. Around 70% of patients from either species exhibit pancreatic aggregates of islet amyloid polypeptide (IAPP), a protein that proves toxic upon misfolding. These misfolded protein aggregates congregate in the islets of Langerhans of the pancreas, diminishing the capability of ß-cells to produce insulin and further perpetuating disease. OBJECTIVE: Our team's drug discovery program is investigating newly synthesized compounds that could diminish aggregates of both human and feline IAPP, potentially disrupting the progression of T2D. MATERIAL AND METHODS: We prepared 24 compounds derived from diaryl urea, as ureas have previously demonstrated great potential at reducing accumulations of misfolded proteins. Biophysical methods were employed to analyze the anti-aggregation activity of these compounds at inhibiting and/or disrupting IAPP fibril formation in vitro. RESULTS: The results demonstrate that compounds 12 and 24 were most effective at reducing the fibrillization and aggregation of both human and feline IAPP. When compared with the control for each experiment, samples treated with either compound 12 or 24 exhibited fewer accumulations of amyloid-like fibrils. CONCLUSION: Urea-based compounds, such as compounds 12 and 24, may prove crucial in future pre-clinical studies in the search for therapeutics for T2D.

Hybrid insulin peptide isomers spontaneously form in pancreatic beta-cells from an aspartic anhydride intermediate.

Hybrid insulin peptides (HIPs) form in beta-cells when insulin fragments link to other peptides through a peptide bond. HIPs contain nongenomic amino acid sequences and have been identified as targets for autoreactive T cells in type 1 diabetes. A subgroup of HIPs, in which N-terminal amine groups of various peptides are linked to aspartic acid residues of insulin C-peptide, was detected through mass spectrometry in pancreatic islets. Here, we investigate a novel mechanism that leads to the formation of these HIPs in human and murine islets. Our research herein shows that these HIPs form spontaneously in beta-cells through a mechanism involving an aspartic anhydride intermediate. This mechanism leads to the formation of a regular HIP containing a standard peptide bond as well as a HIP-isomer containing an isopeptide bond by linkage to the carboxylic acid side chain of the aspartic acid residue. We used mass spectrometric analyses to confirm the presence of both HIP isomers in islets, thereby validating the occurrence of this novel reaction mechanism in beta-cells. The spontaneous formation of new peptide bonds within cells may lead to the development of neoepitopes that contribute to the pathogenesis of type 1 diabetes as well as other autoimmune diseases.

Is islet amyloid polypeptide indeed expressed in the human brain?

AIMS: This study aims to study the association between pancreatic islet amyloid polypeptide (IAPP) and Alzheimer's disease neuropathological change (ADNC) in brain biopsies obtained from subjects with idiopathic normal pressure hydrocephalus (iNPH) and in post-mortem (PM) brain samples obtained from aged individuals. METHODS: For the immunohistochemical (IHC) analyses, two IAPP antibodies (Abs), monoclonal and polyclonal, and Abs directed towards ADNC were applied. RESULTS: The iNPH cohort included 113 subjects. Amyloid-ß (Aß) was detected in 50% and hyperphosphorylated τ (HPτ) in 47% of the cases. Concomitant pathology was seen in 32%. The PM cohort included 77 subjects. Aß was detected in 69% and HPτ in 91% of the cases. Combined Aß/HPτ pathology was seen in 62%. Reactivity for the monoclonal IAPP was not detected in the brain tissue in either of the cohorts. Reactivity for the polyclonal IAPP was observed in all 77 PM brain samples. CONCLUSIONS: There was no specific expression of IAPP in human brain tissue; hence, an association between IAPP and ADNC is not assessable. Of note, the observed reactivity of the polyclonal IAPP Ab was not reproduced with a specific monoclonal Ab; thus, we considered the observed staining with the polyclonal Ab to be unreliable. When using IHC, several pitfalls, especially the choice of an Ab, always need to be considered. Polyclonal Abs cross-react with other epitopes and proteins, thus leading to false-positive results. This seems to be the case for the polyclonal IAPP Abs in the human brain.