Diabetes mellitus is the most prevalent metabolic disease, and -cell apoptosis contributes to decreases in -cellmass and function during the evolution of diabetes. However, the mechanism(s) that contribute to -cellapoptosis are not well-understood. Our hypothesis in the 1st grant period was that the Group VIA Ca2+-independent phospholipase A2 (iPLA2 ) participates in ER stress-induced -cell apoptosis. We find that (a) ERstress induces iPLA2 activation, ceramide generation via neutral sphingomyelinase (NSMase), and -cellapoptosis, (b) these outcomes are suppressed by inhibition of iPLA2 or NSmase, (c) iPLA2 -null islets are lessand iPLA2 -tansgenic (Tg) islets more sensitive to ER stress-induced apoptosis; that ER stress (d) increasesiPLA2 protein/activity in the ER and mitochondria, and (e) activates the mitochondrial apoptotic pathway via theiPLA2 -ceramide axis; and (f) ER stress-prone Akita -cells and islets from pre-diabetic NOD mice expresshigher levels of iPLA2 than WT cells and islets, (g) STZ-induced hyperglycemia is accelerated in iPLA2 -Tgmice, and (h) cytokines induce ceramide generation, loss in , and apoptosis in islet-cells that are allsuppressed by iPLA2 inactivation. During the 2nd grant period, we propose to examine the mechanism ofinvolvement of iPLA2 and iPLA2 -derived lipid mediators in -cell apoptosis under the following Aims:
Aim1 will examine iPLA2 activation and lipid changes in -cells undergoing apoptosis. Hyperglycemia and cytokinespromote -cell apoptosis, in part, by inducing ER stress and the dependence of this process on iPLA2 will beassessed.
Aim 2 will examine the mechanism of iPLA2 and ceramide-generating pathway induction. The rolesof lipid mediators, SREBPs, ROS, and GSH on iPLA2 and NSMase expression and the affects of mutating thelipase sequence in iPLA2 on ceramide generation will be examined.
Aim 3 will examine the role of iPLA2 inER-mitochondria crosstalk. The affects of genetic modulation of iPLA2 expression, ROS and GSH levels,NSMase expression, mutations in iPLA2 , and organelle-specific iPLA2 expression on activation of themitochondrial apoptotic pathway will be examined.
Aim 4 will examine if in vivo modulation of iPLA2 expressionalters islet -cell sensitivity to ER stress. iPLA2 -null mice will be crossed with Akita mice and iPLA2 -Tg micewith CHOP-null mice and the development of ER stress monitored.
Aim 5 will determine if iPLA2 contributes to -cell apoptosis during the evolution of autoimmune DM. The dependence of cytokine-induced -cell apoptosison iPLA2 and nitric oxide pathway and of STZ-induced -cell apoptosis and hyperglycemia oniPLA2 expression will be determined. Apoptosis, flow cytometry, immunoblotting, real time-PCR, enzymaticactivity, confocal microscopy, molecular biology, and mass spectrometry protocols will be utilized in thesestudies.
-cell apoptosis contributes to loss in -cell mass and function during the evolution of diabetes mellitus and is therefore important to understand the mechanisms underlying -cell apoptosis if this process is to be prevented or delayed. Our findings of iPLA2 participation in this process and of stimuli, that contribute to the development of diabetes, activating iPLA2 raises the possibility that iPLA2 contributes to -cell apoptosis during the evolution of diabetes mellitus. Our findings will reveal factors that adversely affect -cell health and identify targets for future therapeutic interventions to prevent -cell death.
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