Pro-apoptotic bax and bak control beta-cell death and early endoplasmic reticulum stress signalling

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.Medicine, Faculty ofGraduat