RAGE binds preamyloid IAPP intermediates and mediates pancreatic ß cell proteotoxicity.

Islet amyloidosis is characterized by the aberrant accumulation of islet amyloid polypeptide (IAPP) in pancreatic islets, resulting in ß 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) ß 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, ß 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 ß cell area, ß 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 ß cell dysfunction in metabolic disease.

The EndoC-ßH1 cell line is a valid model of human beta cells and applicable for screenings to identify novel drug target candidates.

OBJECTIVE: To characterize the EndoC-ßH1 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-ßH1 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-ßH1 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-ßH1 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-ßH1 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-ßH1 cell line was successfully used as a screening platform for identification of novel anti-diabetic drug candidates.

Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics.

Islet amyloidosis by IAPP contributes to pancreatic ß-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 ß-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive ß-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced ß-cell death.

XIAP inhibition of ß-cell apoptosis reduces the number of islets required to restore euglycemia in a syngeneic islet transplantation model.

Clinical pancreatic islet transplantation has great promise as a treatment for type 1 diabetes but despite recent advances, it is still limited by the need for lifelong immunosuppression, restricted availability of donor islets, and uncertainty regarding long-term graft survival. Using a syngeneic, suboptimal islet transplantation model, we asked whether adenoviral overexpression of an anti-apoptotic protein, the X-linked inhibitor of apoptosis protein (XIAP) would protect transplanted islet cells from death and reduce the number of islets required for successful transplantation. Transplantation of 100 XIAP-expressing islets into the kidney capsule of syngeneic Balb/c mice restored euglycemia in 86% of recipients, where transplantation of 100 islets transduced with a control adenovirus expressing LacZ restored euglycemia in only 27% of recipients. Analysis of islet grafts by insulin/TUNEL double immunostaining revealed fewer apoptotic beta-cells in recipients of XIAP- compared with LacZ-expressing grafts (0.8±0.5 vs. 2.4±0.8 double-positive cells/graft), suggesting that XIAP enhances graft success by inhibiting ß-cell apoptosis in the immediate post-transplant period. In summary, XIAP overexpression inhibits beta cell apoptosis in syngeneic islet transplants, thereby reducing the number of islets and decreasing the number of days required to restore euglycemia. These data raise the possibility that ex vivo XIAP gene transfer in islets prior to transplantation has the potential to increase the number of donor islets available for transplantation and may enhance graft function and long-term transplant success.

The flavanol (-)-epigallocatechin 3-gallate inhibits amyloid formation by islet amyloid polypeptide, disaggregates amyloid fibrils, and protects cultured cells against IAPP-induced toxicity.

Islet amyloid polypeptide (IAPP, amylin) is the major protein component of the islet amyloid deposits associated with type 2 diabetes. The polypeptide lacks a well-defined structure in its monomeric state but readily assembles to form amyloid. Amyloid fibrils formed from IAPP, intermediates generated in the assembly of IAPP amyloid, or both are toxic to ß-cells, suggesting that islet amyloid formation may contribute to the pathology of type 2 diabetes. There are relatively few reported inhibitors of amyloid formation by IAPP. Here we show that the tea-derived flavanol, (-)-epigallocatechin 3-gallate [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate] (EGCG), is an effective inhibitor of in vitro IAPP amyloid formation and disaggregates preformed amyloid fibrils derived from IAPP. The compound is thus one of a very small set of molecules which have been shown to disaggregate IAPP amyloid fibrils. Fluorescence-detected thioflavin-T binding assays and transmission electron microscopy confirm that the compound inhibits unseeded amyloid fibril formation as well as disaggregates IAPP amyloid. Seeding studies show that the complex formed by IAPP and EGCG does not seed amyloid formation by IAPP. In this regard, the behavior of IAPP is similar to the reported interactions of Aß and α-synuclein with EGCG. Alamar blue assays and light microscopy indicate that the compound protects cultured rat INS-1 cells against IAPP-induced toxicity. Thus, EGCG offers an interesting lead structure for further development of inhibitors of IAPP amyloid formation and compounds that disaggregate IAPP amyloid.

The sulfated triphenyl methane derivative acid fuchsin is a potent inhibitor of amyloid formation by human islet amyloid polypeptide and protects against the toxic effects of amyloid formation.

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

The X-linked inhibitor of apoptosis protein enhances survival of murine islet allografts.

Allotransplantation of pancreatic islets represents a promising approach to treat type 1 diabetes. Destruction of beta-cells in islet allografts involves multiple immune mechanisms that lead to activation of caspases and apoptotic cell death. The X-linked inhibitor of apoptosis (XIAP) inhibits apoptosis induced by a variety of triggers, primarily by preventing the activation of caspases. To determine whether XIAP would protect beta-cells from apoptosis, we used a recombinant adenovirus to overexpress XIAP in transformed murine beta-cells and in freshly isolated islets. In vitro cytokine-induced beta-cell death was decreased to baseline levels in XIAP-transduced MIN-6 and NIT-1 cell lines compared with controls. To evaluate the potential of XIAP overexpression to prevent in vivo allogeneic graft rejection, we transduced Balb/c islets ex vivo with XIAP before transplantation into CBA mice with streptozotocin-induced diabetes. We observed that almost all mice receiving allografts of XIAP-expressing islets maintained normoglycemia until the experiment was terminated (45-72 days posttransplant), whereas control mice receiving islets transduced with adenovirus expressing LacZ were hyperglycemic by approximately 17 days posttransplantation due to graft rejection. Immunohistochemistry revealed preservation of beta-cells and clearance of infiltrating immune cells in the XIAP-expressing islet grafts. The in vitro allogeneic response of splenocytes isolated from recipients of XIAP-expressing grafts 8 weeks posttransplant was similar to that seen in nonprimed allogeneic mice, suggesting that XIAP overexpression may lead to the acceptance of islet allografts in diabetic recipients. Long-term protection of islet allografts by XIAP overexpression may enhance the survival of islet transplants in diabetes.

Synergistic inhibition of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human pancreatic beta cells by Bcl-2 and X-linked inhibitor of apoptosis.

To better understand the cytokine death-signal transduction pathways in human beta cells, we investigated the inhibitory effects of Bcl-2 (protooncogene bcl-2) and X-linked inhibitor of apoptosis (XIAP) on TRAIL (TNF-related apoptosis-inducing ligand)-induced human beta-cell destruction. A panel of Bcl-2-overexpressing transfectants of the human beta-cell lines NES2Y and CM was developed by transfection with a pEFpGKpuro vector containing Bcl-2 or an empty vector as a control. TRAIL-induced cytotoxicity and apoptosis of Bcl-2-overexpressing beta cells were clearly decreased, in comparison with wild-type cells and the empty vector transfectants. XIAP-overexpressing CM, NES2Y, and primary islet cells were generated by exposing cells to recombinant adenovirus-expressing XIAP (AdXIAP) or AdLacz as a control. TRAIL-induced cytotoxicity and apoptosis of CM, NES2Y, and primary islet cells infected with AdXIAP were clearly reduced compared with controls. Interestingly, cytotoxicity induced by TRAIL in human beta cells transfected with both Bcl-2 and AdXIAP was much less than that observed in human beta cells transfected with either Bcl-2 or XIAP alone (p < 0.005 in CM and p < 0.03 in NES2Y). Overexpression of both Bcl-2 and XIAP inhibited TRAIL-induced activation of caspases as well as TRAIL-mediated damage of mitochondrial function in cells, suggesting possible regulatory mechanisms. These results indicate that Bcl-2 and XIAP synergistically inhibit TRAIL-mediated death pathways in human beta cells.

Increasing the yield of human mononuclear cells and low serum conditions for in vitro generation of macrophages with M-CSF.

The yield of mononuclear cells extracted from peripheral blood using standard protocols is frequently inadequate when working with material of limited availability. In addition, the regular usage of autologous and fetal calf serum (FCS) to generate human macrophages in vitro may complicate antigen uptake, processing and presentation on HLA molecules. We optimized the yield of mononuclear cells from 34+/-3% to 65+/-5% by collecting both the interface and more than half of the overlayering supernatant, followed by three washes at 4 degrees C. Monocytes from 12 individuals were cultured 1-4 days with 0-100 ng/ml macrophage colony-stimulating factor (M-CSF) at either 1% (low) or 5% (v/v) FCS. Regardless of number of days in culture, maximal (50-100 ng/ml) M-CSF stimulation and low FCS induced 65+/-5% esterase-positive cells in all individuals compared to 52+/-7% without M-CSF (p<0.001). M-CSF increased the mean proportion of esterase-positive cells in both 1% and 5% FCS with a negative interaction found between 5% FCS and M-CSF (p<0.05). All cells were positive for CD14 and HLA class II but cell number did not increase. The generation of human macrophages by M-CSF at low FCS should prove useful in studies where higher FCS concentrations may interfere with the assay.