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Novel pathways in fatty-acid induced apoptosis in the pancreatic beta-cell

University of British Columbia

Pancreatic β-cell death is a critical event in the pathogenesis of all forms of diabetes. Type 2 diabetes is caused by the combination of acquired factors such as elevated circulating fatty acids as well as genetic factors. In this study, we show that the free fatty acid palmitate increases markers of endoplasmic stress and apoptosis in pancreatic β-cells. Carboxypeptidase E (CPE), an enzyme involved in the processing of insulin, was identified as the major down-regulated protein spot during palmitate-induced apoptosis using Cy-dye 2D gel proteomics in both the MIN6 β-cell line and human islets. Using MIN6 cells treated with 1.5 mM palmitate complexed to BSA (6:1), a significant decrease in total carboxypeptidase E protein was confirmed through Western blots. The decrease in CPE was seen in the presence of palmitate at both low and high glucose and was not affected in high glucose alone or with the ER-stress inducer, thapsigargin. Palmitate-induced changes in carboxypeptidase E were present after 2 hours, while CHOP, a marker of ER-stress, was not expressed until after 6 hours of incubation, suggesting that the decrease in CPE occurs before ER-stress. This finding, together with experiments using protein synthesis inhibitors and RT-PCR suggested that CPE was likely regulated at the post-translational level. Treatment with the non-metabolizable palmitate, 2-bromopalmitate, did not decrease CPE expression and delayed β-cell death. Addition of the L-type Ca2 + channel blocker nifedipine to palmitate-treated MIN6 cells restored CPE protein levels, reduced ER-stress, and rescued β-cells from cell death. The calpain inhibitor E64D also reversed the palmitate-induced decrease in CPE, further implicating Ca2+-dependent proteolysis pathways. This inhibitor however increased ERstress on its own and did not prevent ER-stress or β-cell death induced by palmitate. Interestingly, islets from CPE mutant mice exhibited increased TUNEL labeling, suggesting elevated apoptosis in vivo. Isolated CPE-deficient islets demonstrated increased CHOP and cleaved caspase-3 levels compared to control mice. The effects of palmitate on ER-stress and apoptosis were not additive to those of CPE deficiency. On the other hand, β-cells over-expressing CPE were resistant to palmitate-induced ERstress and apoptosis. Together, these results show that palmitate markedly lowers CPE protein in the cell in a Ca2+-dependent manner, potentially through Ca2+-dependent proteases, such as the calpains. In addition, a lack of CPE appears to increase the susceptibility of the β-cell to CHOP induction and apoptosis. Conversely, overexpression of CPE protects β-cells from ER-stress and apoptosis induced by palmitate. These findings suggest that CPE represents a novel link between hyperlipidemia and β-cell death in diabetes.

Text

2011-02-16

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http://hdl.handle.net/2429/31378

Physiology

University of British Columbia

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