Calcium-independent Phospholipase A2 (iPLA2β)-mediated Ceramide Generation Plays a Key Role in the Cross-talk between the Endoplasmic Reticulum (ER) and Mitochondria during ER Stress-induced Insulin-secreting Cell Apoptosis

Abstract

Endoplasmic reticulum (ER) stress induces INS-1 cell apoptosis by a pathway involving Ca2؉ -independent phospholipase A 2 (iPLA 2 ␤)-mediated ceramide generation, but the mechanism by which iPLA 2 ␤ and ceramides contribute to apoptosis is not well understood. We report here that both caspase-12 and caspase-3 are activated in INS-1 cells following induction of ER stress with thapsigargin, but only caspase-3 cleavage is amplified in iPLA 2 ␤ overexpressing INS-1 cells (OE), relative to empty vector-transfected cells, and is suppressed by iPLA 2 ␤ inhibition. ER stress also led to the release of cytochrome c and Smac and, unexpectedly, their accumulation in the cytosol is amplified in OE cells. These findings raise the likelihood that iPLA 2 ␤ participates in ER stress-induced apoptosis by activating the intrinsic apoptotic pathway. Consistent with this possibility, we find that ER stress promotes iPLA 2 ␤ accumulation in the mitochondria, opening of mitochondrial permeability transition pore, and loss in mitochondrial membrane potential (⌬⌿) in INS-1 cells and that these changes are amplified in OE cells. ER stress also led to greater ceramide generation in ER and mitochondria fractions of OE cells. Exposure to ceramide alone induces loss in ⌬⌿ and apoptosis and these are suppressed by forskolin. ER stress-induced mitochondrial dysfunction and apoptosis are also inhibited by forskolin, as well as by inactivation of iPLA 2 ␤ or NSMase, suggesting that iPLA 2 ␤-mediated generation of ceramides via sphingomyelin hydrolysis during ER stress affect the mitochondria. In support, inhibition of iPLA 2 ␤ or NSMase prevents cytochrome c release. Collectively, our findings indicate that the iPLA 2 ␤-ceramide axis plays a critical role in activating the mitochondrial apoptotic pathway in insulin-secreting cells during ER stress.Diabetes mellitus is the most prevalent human metabolic disease resulting from the loss and/or dysfunction of ␤-cells in pancreatic islets. Type 1 diabetes mellitus (T1DM) 2 is caused by autoimmune ␤-cell destruction (1) and apoptosis plays a prominent role in the loss of ␤-cells during development of T1DM (1, 2). Type 2 diabetes mellitus (T2DM) results from a progressive decline in ␤-cell function and chronic insulin resistance (3, 4) that is also associated with decreases in ␤-cell mass due to increased ␤-cell apoptosis (5, 6). Autopsy studies indicate that the ␤-cell mass in obese T2DM subjects is smaller than that in obese non-diabetic subjects (7,8) and that the loss in ␤-cell function in non-obese T2DM is associated with decreases in ␤-cell mass (5, 6). ␤-Cell mass is regulated by a balance between ␤-cell replication/neogenesis and ␤-cell death resulting from apoptosis (9, 10). Findings in rodent models of T2DM (10, 11) and in human T2DM (5, 6) indicate that the decrease in ␤-cell mass in T2DM is not attributable to reduced ␤-cell proliferation or neogenesis but to increased ␤-cell apoptosis. Emerging evidence also suggests that cytokine-mediated ␤-cell apoptosis is a contributor to ␤-cell death...

Targeting cyclophilin D and the mitochondrial permeability transition enhances β-cell survival and prevents diabetes in Pdx1 deficiency

“…Recent findings have established that opening of the MPTP is a component of the ER stress response, including in β-cells (32,33). Sachdeva et al recently reported that Pdx1 deficiency enhances β-cell susceptibility to ER stress-associated apoptosis (34).…”

The BCL-2 Protein BAK Is Required for Long-chain Ceramide Generation during Apoptosis

“…Ceramide also regulates ER calcium levels and activates ER stress surveillance machinery (37)(38)(39)(40). Moreover, ceramide generation occurs following the induction of ER stress with thapsigargin (38).…”

“…Ceramide also regulates ER calcium levels and activates ER stress surveillance machinery (37)(38)(39)(40). Moreover, ceramide generation occurs following the induction of ER stress with thapsigargin (38). The connection between BAK and ceramide generation established here raises the possibility that BAK may regulate ER calcium and ER stress responses by elevating cellular ceramide.…”

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