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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. 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 Lgr4 fl/fl and Lgr4 fl/fl/ Rank fl/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. 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. Lgr4 cko mice exhibit normal glucose homeostasis but increased β-cell death in both sexes and decreased β-cell proliferation and maturation only in females. Male Lgr4 cko mice under stress displayed reduced β-cell proliferation and a further increase in β-cell death. The impaired β-cell phenotype in Lgr4 cko mice was rescued in Lgr4 / Rank double ko (dko) mice. Upon aging, both male and female Lgr4 cko mice displayed impaired β-cell homeostasis, however, only female mice became glucose intolerant with decreased plasma insulin. 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. [Display omitted] • Lgr4 mRNA is reduced in islets/β-cells under T1D- and T2D-related stress in mice and humans. • Lgr4 knockdown in vitro impairs β-cell survival and proliferation; LGR4 overexpression protects against cytokines. • LGR4 inhibits RANK/NFκB activation to protect β-cells against death. • Lgr4cko mice display impaired β-cell health; aged female mice are glucose intolerant with reduced plasma insulin. • Rank/Lgr4 double ko rescues β-cell impairments, highlighting RANK/LGR4 stoichiometry in β-cell health. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. • RIPK1 regulates TNFα-induced β-cell death independent of caspase activity. • cIAP2 is upregulated in response to TNFα to counter regulate β-cell death. • RIPK3 mediates TNFα-induced β-cell death when caspases are inhibited. • Loss of RIPK3 protects mice from streptozotocin-induced hyperglycemia. [ABSTRACT FROM AUTHOR]

A hallmark of type 2 diabetes mellitus (T2DM) is the presence of extracellular amyloid deposits in the islets of Langerhans. These deposits are formed by the human islet amyloid polypeptide, hIAPP (or amylin), which is a hormone costored and cosecreted with insulin. Under normal conditions, the hormone remains in solution but, in the pancreas of T2DM individuals, it undergoes misfolding giving rise to oligomers and cross-β amyloid fibrils. Accumulating evidence suggests that the amyloid deposits that accompany type 2 diabetes mellitus are not just a trivial epiphenomenon derived from the disease progression. Rather, hIAPP aggregation induces processes that impair the functionality and viability of β-cells and may lead to apoptosis. The present review article aims to summarize a few aspects of the current knowledge of this amyloidogenic polypeptide. In the first place, the physicochemical properties which condition its propensity to misfold and form aggregates. Secondly, how these properties confer hIAPP the capacity to interfere with some signaling of the pancreatic β-cell, interact with membranes, form channels or affect natural ion channels, including calcium channels. Finally, how misfolded hIAPP cytotoxicity results in apoptosis. A number of pathophysiological changes of the T2DM islet can be related to the amyloidogenic properties of hIAPP. However, in a certain way, the in vivo aggregation of the polypeptide also reflects a failure of chaperones and, in general, of cellular proteostasis, supporting the view that T2DM may also be considered as a conformational disorder. [ABSTRACT FROM AUTHOR]