Type 1 diabetes is preceded by islet β-cell dysfunction, but the mechanisms leading to β-cell dysfunction have not been rigorously studied. Because immune cell infiltration occurs prior to overt diabetes, we hypothesized that activation of inflammatory cascades and appearance of endoplasmic reticulum (ER) stress in β-cells contributes to insulin secretory defects. Prediabetic nonobese diabetic (NOD) mice and control diabetes-resistant NOD-SCID and CD1 strains were studied for metabolic control and islet function and gene regulation. Prediabetic NOD mice were relatively glucose intolerant and had defective insulin secretion with elevated proinsulin:insulin ratios compared with control strains. Isolated islets from NOD mice displayed age-dependent increases in parameters of ER stress, morphologic alterations in ER structure by electron microscopy, and activation of nuclear factor-κB (NF-κB) target genes. Upon exposure to a mixture of proinflammatory cytokines that mimics the microenvironment of type 1 diabetes, MIN6 β-cells displayed evidence for polyribosomal runoff, a finding consistent with the translational initiation blockade characteristic of ER stress. We conclude that β-cells of prediabetic NOD mice display dysfunction and overt ER stress that may be driven by NF-κB signaling, and strategies that attenuate pathways leading to ER stress may preserve β-cell function in type 1 diabetes.

Significance: Elevated circulating proinsulin and a poor biological response to insulin are observed early in individuals with type 2 diabetes. Genome-wide association studies have recently identified genes associated with proinsulin processing, and clinical observations suggest that elevated proinsulin and its intermediates are markers of dysfunctional insulin-secreting β cells. Here, we propose a previously unidentified mechanism in the regulation of an enzyme that is involved in proinsulin processing called carboxypeptidase E (CPE). Disruption of insulin signaling in β cells reduces expression of a scaffolding protein, eukaryotic translation initiation factor 4 gamma 1, that is required for the initiation of translation and occurs via regulation of two transcription factors, namely, pancreatic and duodenal homeobox 1 and sterol regulatory element-binding protein 1. Together, these effects lead to reduced levels of CPE protein and poor proinsulin processing in β cells.

Saturated free fatty acids (FFAs) have complex effects on the islet β-cell, acutely promoting adaptive hyperplasia but chronically impairing insulin release. The acute effects of FFAs remain incompletely defined. To elucidate these early molecular events, we incubated mouse β-cells and islets with palmitate and then studied mRNA translation by polyribosomal profiling and analyzed signaling pathways by immunoblot analysis. We found that palmitate acutely increases polyribosome occupancy of total RNA, consistent with an increase in mRNA translation. This effect on translation was attributable to activation of mammalian target of rapamycin (mTOR) pathways via L-type Ca2+ channels but was independent of insulin signaling. Longer incubations led to depletion of polyribosome-associated RNA, consistent with activation of the unfolded protein response (UPR). Pharmacologic inhibition of mTOR suppressed both the acute effects of palmitate on mRNA translation and the chronic effects on the UPR. Islets from mice fed a high-fat diet for 7 days showed increases in polyribosome-associated RNA and phosphorylation of S6K, both consistent with activation of mTOR. Our results suggest that palmitate acutely activates mRNA translation and that this increase in protein load contributes to the later UPR.

β-cell death is regarded as a major event driving loss of insulin secretion and hyperglycemia in both type 1 and type 2 diabetes mellitus. In this review, we explore past, present, and potential future advances in our understanding of the mechanisms that promote β-cell death in diabetes, with a focus on the primary literature. We first review discoveries of insulin insufficiency, β-cell loss, and β-cell death in human diabetes. We discuss findings in humans and mouse models of diabetes related to autoimmune-associated β-cell loss and the roles of autoreactive T cells, B cells, and the β cell itself in this process. We review discoveries of the molecular mechanisms that underlie β-cell death-inducing stimuli, including proinflammatory cytokines, islet amyloid formation, ER stress, oxidative stress, glucotoxicity, and lipotoxicity. Finally, we explore recent perspectives on β-cell death in diabetes, including: (1) the role of the β cell in its own demise, (2) methods and terminology for identifying diverse mechanisms of β-cell death, and (3) whether non-canonical forms of β-cell death, such as regulated necrosis, contribute to islet inflammation and β-cell loss in diabetes. We believe new perspectives on the mechanisms of β-cell death in diabetes will provide a better understanding of this pathological process and may lead to new therapeutic strategies to protect β cells in the setting of diabetes.

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.