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Objective: Loss of functional β-cell mass is a major cause of diabetes. Thus, identifying regulators of β-cell health is crucial for treating this disease. The Leucine-rich repeat-containing G-protein-coupled receptor (GPCR) 4 (LGR4) is expressed in β-cells and is the fourth most abundant GPCR in human islets. Although LGR4 has regenerative, anti-inflammatory, and anti-apoptotic effects in other tissues, its functional significance in β-cells remains unknown. We have previously identified Receptor Activator of Nuclear Factor Kappa B (NFκB) (RANK) as a negative regulator of β-cell health. In this study, we assessed the regulation of Lgr4 in islets, and the role of LGR4 and LGR4/RANK stoichiometry in β-cell health under basal and stress-induced conditions, in vitro and in vivo. Methods: We evaluated Lgr4 expression in mouse and human islets in response to acute (proinflammatory cytokines), or chronic (high fat fed mice, db/db mice, and aging) stress. To determine the role of LGR4 we employed in vitro Lgr4 loss and gain of function in primary rodent and human β-cells and examined its mechanism of action in the rodent INS1 cell line. Using Lgr4fl/fl and Lgr4fl/fl/Rankfl/fl × Ins1-Cre mice we generated β-cell-specific conditional knockout (cko) mice to test the role of LGR4 and its interaction with RANK in vivo under basal and stress-induced conditions. Results: Lgr4 expression in rodent and human islets was reduced by multiple stressors. In vitro, Lgr4 knockdown decreased proliferation and survival in rodent β-cells, while overexpression protected against cytokine-induced cell death in rodent and human β-cells. Mechanistically, LGR4 protects β-cells by suppressing RANK- Tumor necrosis factor receptor associated factor 6 (TRAF6) interaction and subsequent activation of NFκB. Lgr4cko mice exhibit normal glucose homeostasis but increased β-cell death in both sexes and decreased β-cell proliferation and maturation only in females. Male Lgr4cko mice under stress displayed reduced β-cell proliferation and a further increase in β-cell death. The impaired β-cell phenotype in Lgr4cko mice was rescued in Lgr4/Rank double ko (dko) mice. Upon aging, both male and female Lgr4cko mice displayed impaired β-cell homeostasis, however, only female mice became glucose intolerant with decreased plasma insulin. Conclusions: These data demonstrate a novel role for LGR4 as a positive regulator of β-cell health under basal and stress-induced conditions, through suppressing the negative effects of RANK.

Adipose tissue-derived non-esterified saturated long-chain fatty acid palmitate (PA) decisively contributes to β-cell demise in type 2 diabetes mellitus in part through the excessive generation of hydrogen peroxide (H2O2). The endoplasmic reticulum (ER) as the primary site of oxidative protein folding could represent a significant source of H2O2. Both ER-oxidoreductin-1 (ERO-1) isoenzymes, ERO-1α and ERO-1β, catalyse oxidative protein folding within the ER, generating equimolar amounts of H2O2 for every disulphide bond formed. However, whether ERO-1-derived H2O2 constitutes a potential source of cytotoxic luminal H2O2 under lipotoxic conditions is still unknown. Here, we demonstrate that both ERO-1 isoforms are expressed in pancreatic β-cells, but interestingly, PA only significantly induces ERO-1α. Its specific deletion significantly attenuates PA-mediated oxidative ER stress and subsequent β-cell death by decreasing PA-mediated ER-luminal and mitochondrial H2O2 accumulation, by counteracting the dysregulation of ER Ca2+ homeostasis, and by mitigating the reduction of mitochondrial membrane potential and lowered ATP content. Moreover, ablation of ERO-1α alleviated PA-induced hyperoxidation of the ER redox milieu. Importantly, ablation of ERO-1α did not affect the insulin secretory capacity, the unfolded protein response, or ER redox homeostasis under steady-state conditions. The involvement of ERO-1α-derived H2O2 in PA-mediated β-cell lipotoxicity was corroborated by the overexpression of a redox-active ERO-1α underscoring the proapoptotic activity of ERO-1α in pancreatic β-cells. Overall, our findings highlight the critical role of ERO-1α-derived H2O2 in lipotoxic ER stress and β-cell failure.

IntroductionRecently, the involvement of ferroptotic cell death in the reduction of β-cell mass in diabetes has been demonstrated. To elucidate the mechanisms of β-cell ferroptosis and potential antidiabetic effects of the ferroptosis inhibitor ferrostatin-1 (Fer-1) in vivo, a mouse model of type 1 diabetes (T1D) was used.MethodsAnimals were divided into three groups: control (vehicle-treated), diabetic (streptozotocin-treated, 40 mg/kg, from days 1-5), and diabetic treated with Fer-1 (1 mg/kg, from days 1-21). On day 22, glycemia and insulinemia were measured and pancreases were isolated for microscopic analyses.ResultsDiabetes disturbed general parameters of β-cell mass (islet size, β-cell abundance and distribution) and health (insulin and PDX-1 expression), increased lipid peroxidation in islet cells, and phagocytic removal of iron-containing material. It also downregulated the main players of the antiferroptotic pathway - Nrf2, GPX4, and xCT. In contrast, Fer-1 ameliorated the signs of deterioration of β-cell/islets, decreased lipid peroxidation, and reduced phagocytic activity, while upregulated expression of Nrf2 (and its nuclear translocation), GPX4, and xCT in β-cell/islets.DiscussionOverall, our study confirms ferroptosis as an important mode of β-cell death in T1D and suggests antiferroptotic agents as a promising strategy for the prevention and treatment of diabetes

Objective: Type 1 diabetes (T1D) is characterized by autoimmune-associated β-cell loss, insulin insufficiency, and hyperglycemia. Although TNFα signaling is associated with β-cell loss and hyperglycemia in non-obese diabetic mice and human T1D, the molecular mechanisms of β-cell TNF receptor signaling have not been fully characterized. Based on work in other cell types, we hypothesized that receptor interacting protein kinase 1 (RIPK1) and receptor interacting protein kinase 3 (RIPK3) regulate TNFα-induced β-cell death in concert with caspase activity. Methods: We evaluated TNFα-induced cell death, caspase activity, and TNF receptor pathway molecule expression in immortalized NIT-1 and INS-1 β-cell lines and primary mouse islet cells in vitro. Our studies utilized genetic and small molecule approaches to alter RIPK1 and RIPK3 expression and caspase activity to interrogate mechanisms of TNFα-induced β-cell death. We used the β-cell toxin streptozotocin (STZ) to determine the susceptibility of Ripk3+/+ and Ripk3−/− mice to hyperglycemia in vivo. Results: Expression of TNF receptor signaling molecules including RIPK1 and RIPK3 was identified in NIT-1 and INS-1 β cells and isolated mouse islets at the mRNA and protein levels. TNFα treatment increased NIT-1 and INS-1 cell death and caspase activity after 24–48 h, and BV6, a small molecule inhibitor of inhibitor of apoptosis proteins (IAPs) amplified this TNFα-induced cell death. RIPK1 deficient NIT-1 cells were protected from TNFα- and BV6-induced cell death and caspase activation. Interestingly, small molecule inhibition of caspases with zVAD-fmk (zVAD) did not prevent TNFα-induced cell death in either NIT-1 or INS-1 cells. This caspase-independent cell death was increased by BV6 treatment and decreased in RIPK1 deficient NIT-1 cells. RIPK3 deficient NIT-1 cells and RIPK3 kinase inhibitor treated INS-1 cells were protected from TNFα+zVAD-induced cell death, whereas RIPK3 overexpression increased INS-1 cell death and promoted RIPK3 and MLKL interaction under TNFα+zVAD treatment. In mouse islet cells, BV6 or zVAD treatment promoted TNFα-induced cell death, and TNFα+zVAD-induced cell death was blocked by RIPK3 inhibition and in Ripk3−/− islet cells in vitro. Ripk3−/− mice were also protected from STZ-induced hyperglycemia and glucose intolerance in vivo. Conclusions: RIPK1 and RIPK3 regulate TNFα-induced β-cell death in concert with caspase activity in immortalized and primary islet β cells. TNF receptor signaling molecules such as RIPK1 and RIPK3 may represent novel therapeutic targets to promote β-cell survival and glucose homeostasis in T1D.

Background/Aims: VCP-interacting membrane selenoprotein (VIMP), an ER resident selenoprotein, is highly expressed in β-cells, however, the role of VIMP in β-cells has not been characterized. In this study, we studied the relationship between VIMP deficiency and β-cell survival in MIN6 insulinoma cells. Methods: To determine the role of VIMP in β-cells, lentiviral VIMP shRNAs were used to knock down (KD) expression of VIMP in MIN6 cells. Cell death was quantified by propidium iodide (PI) staining followed by flow cytometric analyses using a FACS Caliber and FlowJo software. Cell apoptosis and proliferation were determined by TUNEL assay and Ki67 staining, respectively. Cell cycle was analyzed after PI staining. Results: The results show that 1) VIMP suppression induces β-cell apoptosis, which is associated with a decrease in Bcl-xL, and the β-cell apoptosis induced by VIMP suppression can be inhibited by overexpression of Bcl-xL; 2) VIMP knockdown (KD) decreases cell proliferation and G1 cell cycle arrest by accumulating p27 and decreasing E2F1; 3) VIMP KD suppresses unfolded protein response (UPR) activation by regulating the IRE1α and PERK pathways; 4) VIMP KD increases insulin secretion. Conclusion: These results suggest that VIMP may function as a novel regulator to modulate β-cell survival, proliferation, cell cycle, UPR and insulin secretion in MIN6 cells.

Abstract Disease heterogeneity is as major issue in Type II Diabetes Mellitus (T2DM), and this patient inter-variability might not be sufficiently reflected by measurements of glycated haemoglobin (HbA1c). Β-cell dysfunction and β-cell death are initiating factors in development of T2DM. In fact, β-cells are known vanish prior to the development of T2DM, and autopsy of overt T2DM patients have shown a 60% reduction in β-cell mass. As the decline in β-cell function and mass have been proven to be pathological traits in T2DM, methods for evaluating β-cell loss is becoming of more interest. However, evaluation of β-cell death or loss is currently invasive and unattainable for the vast majority of diabetes patients. Serological markers, reflecting β-cell loss would be advantageous to detect and monitor progression of T2DM. Biomarkers with such capacities could be neo-epitopes of proteins with high β-cell specificity containing post translational modifications. Such tools may segregate T2DM patients into more appropriate treatment groups, based on their β-cell status, which is currently not possible. Presently individuals presenting with adequately elevated levels of both insulin and glucose are classified as T2DM patients, while an important subdivision of those is pending, namely those patients with sufficient β-cell capacity and those without. This may warrant two very different treatment options and patient care paths. Serological biomarkers reflecting β-cell health status may also assist development of new drugs for T2DM and aid physicians in better characterization of individual patients and tailor individual treatments and patient care protocols.