Activation of Extrinsic Apoptotic Pathway Has an Important Role in Islet Amyloid-Induced Beta-Cell Death in Human Islets

Abstract

Recent studies have shown that islet amyloid - previously known to be a characteristic of type 2 diabetes (T2D) - also forms in human islets during culture and following transplantation into animal models of T1D. Islet amyloid is formed by aggregation of a small beta-cell peptide named islet amyloid polypeptide (IAPP; amylin). Formation of IAPP (proto)fibrils likely contributes to beta-cell death in all three conditions. The molecular mechanism(s) by which IAPP mediates beta-cell death in primary islets has yet to be identified. The extrinsic (cell death receptor) pathway is one of the two major pathways that are primarily responsible for mediating apoptosis in most cellular systems. In the present study, we investigated the role of caspase-8 and −3, two key upstream and effector enzymes in the extrinsic pathway, in amyloid-induced beta-cell death. Freshly isolated human islets obtained from cadaveric donors were dispersed into single cells, tested for their viability (by Alamar blue), and cultured overnight in CMRL in poly-L-lysine coated plates to allow recovery. Islet cells were then treated with fibrillogenic human IAPP (hIAPP; 10 μM), non-fibrillogenic rat IAPP (rIAPP), or vehicle for up to 24 hrs. Activation of caspase-3 , -8, and apoptosis were assessed by immunocytochemistry. The number of double TUNEL/insulinpositive islet cells was significantly higher in hIAPP-treated cells compared to control cells (hIAPP: 15±3%, C: 3±2%, P<0.05, 24 h). This increase in TUNEL-positivity (apoptosis) closely correlated with the elevated number of double insulin/active caspase-8 positive cells (hIAPP: 10±3%, C: 2±1%, P<0.05, 8 h) and double insulin/active caspase-3 positive cells (hIAPP: 16±4%, C: 3±2%, P<0.05, 16 h). Treatment with non-fibrillogenic rat IAPP did not have any detectable effect on beta-cell apoptosis or activation of caspase-8 and −3. A specific peptide inhibitor of caspase-8 markedly reduced the number of TUNEL-positive beta-cells in hIAPP-treated islet cells. Interestingly, very low number of glucagon-positive (alpha cells) were TUNEL-positive in hIAPP-treated iset cells, suggesting that hIAPP-induced cell death is beta-cell specific. We conclude that the cytotoxic effects of hIAPP are mediated, at least partially, through activation of caspase-8 and its downstream enzyme caspase-3 in human islet beta-cells. Specific caspase inhibitors may protect human islets from the cytotoxic effects of amyloid formation during pretransplant culture period and following transplantation.