Role of calcium-independent phospholipase A₂β in human pancreatic islet β-cell apoptosis

Abstract

Death of β-cells due to apoptosis is an important contributor to β-cell dysfunction in both type 1 and type 2 diabetes mellitus. Previously, we described participation of the Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)β) in apoptosis of insulinoma cells due to ER stress. To examine whether islet β-cells are similarly susceptible to ER stress and undergo iPLA(2)β-mediated apoptosis, we assessed the ER stress response in human pancreatic islets. Here, we report that the iPLA(2)β protein is expressed predominantly in the β-cells of human islets and that thapsigargin-induced ER stress promotes β-cell apoptosis, as reflected by increases in activated caspase-3 in the β-cells. Furthermore, we demonstrate that ER stress is associated with increases in islet iPLA(2)β message, protein, and activity, iPLA(2)β-dependent induction of neutral sphingomyelinase and ceramide accumulation, and subsequent loss of mitochondrial membrane potential. We also observe that basal activated caspase-3 increases with age, raising the possibility that β-cells in older human subjects have a greater susceptibility to undergo apoptotic cell death. These findings reveal for the first time expression of iPLA(2)β protein in human islet β-cells and that induction of iPLA(2)β during ER stress contributes to human islet β-cell apoptosis. We hypothesize that modulation of iPLA(2)β activity might reduce β-cell apoptosis and this would be beneficial in delaying or preventing β-cell dysfunction associated with diabetes.

Group VIA Phospholipase A2 (iPLA2β) Modulates Bcl-x 5′-Splice Site Selection and Suppresses Anti-apoptotic Bcl-x(L) in β-Cells

“…Thapsigargininduced ER stress resulted in induction of iPLA 2 ␤ (Fig. 1A) as we have demonstrated previously (17,18). To investigate the impact of ER stress on anti-apoptotic Bcl-x(L), protein and RNA were harvested from cells treated with thapsigargin for 13 h, and Bcl-x protein levels and RNA splicing, respectively, were assessed.…”

“…The iPLA 2 ␤ is a member of the phospholipase A 2 family of enzymes, which hydrolyze the sn-2 fatty acid from membrane phospho-lipids to release a free fatty acid and a lysolipid (16). In pancreatic islets, iPLA 2 ␤ is predominantly localized in ␤-cells (17)(18)(19), and our studies reveal that expression and activity of iPLA 2 ␤ are increased when ␤-cells undergo ER stress-induced apoptosis (12,17,18,20,21). Activation of iPLA 2 ␤ also induces neutral sphingomyelinase 2 (NSMase2), resulting in accumulation of pro-apoptotic ceramides (12,17,18,22).…”

“…In pancreatic islets, iPLA 2 ␤ is predominantly localized in ␤-cells (17)(18)(19), and our studies reveal that expression and activity of iPLA 2 ␤ are increased when ␤-cells undergo ER stress-induced apoptosis (12,17,18,20,21). Activation of iPLA 2 ␤ also induces neutral sphingomyelinase 2 (NSMase2), resulting in accumulation of pro-apoptotic ceramides (12,17,18,22). Various strategies (selective inhibitors, siRNA, and genetic-modulation) indicate a role for iPLA 2 ␤ and subsequent NSMase2-derived ceramides in ER stress-induced ␤-cell apoptosis (12, 17, 18, 20 -23).…”

“…Alternative splicing of the Bcl-x pre-mRNA generates Bcl-x(S), which does not encode an anti-apoptotic protein (28). In view of previous studies linking ceramide to alternative splicing of Bcl-x pre-mRNA and iPLA 2 ␤ to ceramide accumulation and ER stress-induced apoptosis of ␤-cells (12,17,18,22,37,41), we hypothesized that iPLA 2 ␤ might regulate Bcl-x splicing in ␤-cells. To test this, we amplified Bcl-x mRNA splice variants in ␤-cells.…”

“…Our studies indicate that activation of iPLA 2 ␤ in ␤-cells undergoing ER stress promotes loss of mitochondrial membrane potential and resultant apoptosis of the ␤-cells (12,17,18,23). The intrinsic apoptosis pathway is regulated by members of the Bcl-2 family of proteins that can be pro-or anti-apoptotic, depending on the spectrum of Bcl-2 homology (BH) domains that they contain.…”