Dan Luciani

Glucose-stimulated insulin secretion from pancreatic β-cells depends on mitochondrial oxidative metabolism. Mitochondrial dysfunction is believed to be a significant factor in the development of type 2 diabetes (T2D). Mitochondria exist as dynamic networks and the control of mitochondrial biomass and fusion/fission dynamics is essential for cellular health and function. The anti-apoptotic protein Bcl-xL has recently been demonstrated to dampen β-cell mitochondrial metabolism and studies in other cell types suggest Bcl-xL regulates mitochondrial biomass and dynamics. We hypothesize that Bcl-xL is important for β-cell adaptation to metabolic stress by regulating mitochondrial dynamics and mass. To quantitatively study mitochondrial structural changes, we developed an image analysis pipeline for 2D/3D confocal imaging of mitochondria in FIJI. We applied the pipeline to primary islet cells and found that glucose stimulation is correlated with a more fragmented mitochondrial morphology. In vitro Bcl-xL overexpression causes β-cell mitochondria to lose their tubular network structure and aggregate. These changes to network morphology and kinetics are associated with decreased total mitochondrial volume and a marked impairment of β-cell O2 consumption. β-cell specific Bcl-xL knockout islet cells demonstrated increased basal activity and decreased average mitochondrion size, suggesting that they behave more similarly to β-cells undergoing glucose stimulation. Challenging β-cells with prolonged high glucose culture increased the size and overall connectivity of their mitochondrial network. In Bcl-xL knockout β-cells this increase in total mitochondrial mass and networking was significantly amplified, but was associated with reduced morphological and functional glucose-responsiveness of the individual mitochondrion. In conclusion, our in vitro data demonstrate that Bcl-xL affects mitochondrial networking, function, and adaptation to stress in pancreatic β-cells.

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Chronic nutrient oversupply, such as seen in obesity, increases metabolic load and oxidative stress in the insulin-secreting β-cells. This progressively impairs β-cell function and survival, contributing to the development of type 2 diabetes. Bcl-xL is an antiapoptotic protein of the Bcl-2 family. Recent studies have shown additional non-apoptotic functions of Bcl-xL in suppressing glucose signaling of non-diabetic β-cells. Conceivably, this metabolic dampening may be beneficial to counter β-cell dysfunction during nutrient excess of type 2 diabetes. To test the hypothesis that Bcl-xL protects β-cell function during metabolic stress via regulation of mitochondrial physiology, we examined the effects of gene deletion and overexpression of Bcl-xL in β-cells. In normal conditions, islets of β-cell-specific Bcl-x knockout (BclxβKO) mice tend to be metabolically more active compared to BclxβWT islets. This metabolic effect of Bcl-xL is further enhanced after prolonged high glucose culture, where BclxβKO islets display a pre-toxic state of metabolic amplification with dysregulated intracellular Ca²+ and insulin secretion. Islets overexpressing Bcl-xL display suppressed intracellular Ca²+ responses, in agreement with our knockout studies. Interestingly, cells expressing Bcl-xL at high levels have increased mitochondrial aggregates. We also demonstrated that Bcl-xL suppresses superoxide levels and cell death induced by ribose, but not islet-cell death under glucolipotoxic conditions. In conclusion, we propose that endogenous Bcl-xL protects β-cells from high glucose-induced failure by dampening mitochondrial activity, as well as suppressing oxidative stress-induced cell death.

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Stress-induced failure and death of pancreatic β-cells are integral steps in the pathogenesis of type 1 and type 2 diabetes. Better understanding of the molecular interactions that influence β-cell function and stress signaling may therefore identify therapeutic targets to protect endogenous β-cells or transplanted islet grafts. B7-H4 is a negative co-stimulatory molecule that is expressed on the cell membranes of antigen presenting cells and down-regulates the immune response. Interestingly, pancreatic β-cells also express high levels of B7-H4 mRNA and moderate levels of B7-H4 protein. Of note, various tumor cells have up-regulated levels of B7-H4, which has been linked to metabolic and anti-apoptotic effects. This raises the intriguing possibility that B7-H4 may also regulate β-cell function, stress signaling, and survival independent of immune-regulation. In this study, we used mice with β-cell-specific overexpression of B7-H4, as well as B7-H4 knockout mice to examine the possible roles of B7-H4 in β-cell physiology and responses to endoplasmic reticulum (ER) stress. Cytosolic Ca²+ imaging showed that B7-H4 transgenic islets had increased sensitivity to sub-maximal glucose stimulation. Additional experiments indicated no differences in ER Ca²+ uptake/release or glucose metabolism, but revealed that B7-H4 transgenic islets are sensitized to tolbutamide and are resistant to diazoxide, suggesting changes at the ATP-sensitive potassium channels. The B7-H4-induced amplification of glucose-stimulated Ca²+ did not translate into detectable differences in in vitro insulin secretion or in vivo glucose tolerance, suggesting secondary control between rise in intracellular calcium and exocytosis of insulin granules. ER stress was induced in vitro using thapsigargin, and gene expressions were compared by real time quantitative PCR. Moderate ER stress induced the expression of key unfolded protein response genes, BiP, CHOP, and XBP1s to significantly higher levels in B7-H4 transgenic islets compared with wild type. However, the death of dispersed B7-H4 and wild type islet-cells did not differ following more severe and prolonged ER stress. Together, our findings demonstrate that over-expression of B7-H4 amplifies β-cell glucose-stimulated Ca²+ responses and the unfolded protein response during ER stress, revealing novel roles for B7-H4 in the pancreatic β-cell.

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Functional failure and loss of pancreatic β-cells are critical events in the pathogenesis of diabetes and there is mounting evidence that suggests chronic endoplasmic reticulum (ER) stress contributes significantly to β-cell dysfunction and apoptotic death. Two core pro-apoptosis proteins, Bax and Bak, mediate the execution of mitochondrial apoptosis and have also been suggested to regulate aspects of ER physiology and stress signalling. In this study we set out to determine the relative contributions of Bax and Bak in the execution of β-cell death and examine their putative roles in β-cell ER-stress signalling under diabetogenic conditions. We generated mice in which the single or combined knockout of Bax and Bak could be induced in the pancreatic β-cell. Physiological islet function assessed both in vivo and in vitro was not affected by the knockout of Bax and/or Bak. However, Bax and Bak knockout improved β-cell survival under stress conditions. Single knockout, double knockout, and wild-type cells were assayed for ER-stress and cell death following treatment with staurosporine, thapsigargin, and culture under glucolipotoxic conditions. Time-course kinetic cell death analysis demonstrated that single and double knockout cells were protected from staurosporine, and further revealed that Bax-Bak double knockout was required for significant protection from death under glucolipotoxic conditions. ER-stress signalling was evaluated by quantitative PCR for XBP1s and CHOP. Interestingly, spliced XBP1 expression was augmented in Bax-Bak double knockout islets in the early phase of ER-stress signalling compared to wild-type controls. Stress-induced CHOP expression increased in a time-dependent manner but was not significantly different between Bax-Bak double knockout and wild-type islets. These results suggest that Bax and Bak regulate the IRE1α arm of the ER-stress response upstream of apoptosis by suppressing maximal XBP1 splicing. Under glucolipotoxic conditions, pancreatic insulin content, insulin secretion, and insulin transcription were unaffected by Bax and Bak knockout, indicating Bax and Bak do not mediate their protective effects towards β-cell death by retaining islet function. Together these data demonstrate that Bax and Bak have both individual and combined contributions to β-cell death under various stress conditions and suggest novel non-apoptotic roles regulating early ER-stress signalling in the β-cell.

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