Abstract
Endoplasmic reticulum (ER) stress induces INS-1 cell apoptosis by a pathway involving Ca(2+)-independent phospholipase A(2) (iPLA(2)beta)-mediated ceramide generation, but the mechanism by which iPLA(2)beta 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)beta overexpressing INS-1 cells (OE), relative to empty vector-transfected cells, and is suppressed by iPLA(2)beta 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)beta participates in ER stress-induced apoptosis by activating the intrinsic apoptotic pathway. Consistent with this possibility, we find that ER stress promotes iPLA(2)beta accumulation in the mitochondria, opening of mitochondrial permeability transition pore, and loss in mitochondrial membrane potential (Delta Psi) 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 Delta Psi 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)beta or NSMase, suggesting that iPLA(2)beta-mediated generation of ceramides via sphingomyelin hydrolysis during ER stress affect the mitochondria. In support, inhibition of iPLA(2)beta or NSMase prevents cytochrome c release. Collectively, our findings indicate that the iPLA(2)beta-ceramide axis plays a critical role in activating the mitochondrial apoptotic pathway in insulin-secreting cells during ER stress.
Highlights
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]
Representative quantitation of the bands is presented on the right (D panels). These findings raise the findings indicate that iPLA2 participates in endoplasmic reticulum (ER) stress-induced apoptosis but the precise mechanism by which iPLA2 and ceramides contribute to apoptosis of insulin-secreting cells possibility that a role for iPLA2 in the ER stress-mediated apoptotic process may involve activation of the mitochondria
ER Stress-induced Loss in ⌬⌿ and Cytochrome c Release Are Mediated by Ceramides—In the absence of differences in caspase-12 activation between Vector and overexpressing INS-1 cells (OE) INS-1 cells but amplified cytochrome c release in the OE cells following induction of ER stress, the possibility that iPLA2 activation leads to mitochondrial decompensation through ceramide generation was examined
Summary
Materials—The sources for the material used were as follows: (16:0/[14C]-18:2)-GPC (PLPC, 55 mCi/mmol), rainbow molecular mass standards, and enhanced chemiluminescence (ECL) reagent, Amersham Biosciences; ceramide and other lipid standards, Avanti Polar Lipids, Alabaster, AL; Coomassie reagent, SDS-PAGE supplies and Triton X-100, Bio-Rad; mitochondrial membrane potential detection kit, Cell Technology Inc., Mountain View, CA; paraformaldehyde, Electron Microscopy Sciences, Ft. After washing away excess stain and quenching reagent, the cell pellets were resuspended in 400 l of Hanks’ balanced salt solution containing Ca2ϩ and analyzed for calcein-AM fluorescence by flow cytometry using a BD. Cell Fractionation—Cells were harvested and washed twice (750 ϫ g, 5 min, 4 °C) with 10 volumes of ice-cold PBS. Mitochondrial Permeability Transition Pore (PTP)—The An aliquot (30 g) of cytosolic or mitochondrial protein was PTP opening was measured in intact INS-1 cells by monitoring analyzed by SDS-PAGE (8 or 15%), transferred onto Immobicalcein-AM fluorescence in the absence and presence of CoCl2, lin-P polyvinylidene difluoride membranes, and processed for which quenches cytosolic fluorescence, as described [47]. INS-1 cells were washed twice with PBS and resus- tors and the primary antibody concentrations were as follows: pended (1 ϫ 106 cells/ml) in pre-warmed Hanks’ balanced salt PERK (1:1000), pPERK (1:1000), eIF2a (1:500), peIF2a (1:1000), iPLA2 and Ceramides Link ER and Mitochondria during ER Stress
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
