?-cell apoptosis contributes to loss of ?-cells and decreases in ?-cell function in both types 1 and 2 diabetes mellitus. It is therefore important to understand the mechanisms underlying ?-cell apoptosis if this process is to be prevented or delayed. Our hypothesis in the 1st project period (10/04-7/09) was that the group VIA Ca2+-independent phospholipase A2 (iPLA2?) participates in ER stress-induced ?-cell apoptosis. We observed that (a) ER stress promotes accumulation and activity of iPLA2? in the ER and mitochondria, (b) iPLA2? activation during ER stress increases ceramide generation via neutral sphingomyelinase (NSMase)-catalyzed hydrolysis of sphingomyelins triggering the mitochondrial apoptotic pathway and ?-cell apoptosis, (c) these outcomes are suppressed by inhibition of iPLA2? or NSMase, (d) iPLA2?-null islets are less and iPLA2?-transgenic (Tg) islets more sensitive to ER stress, (e) NSMase expression is unaffected in iPLA2?-null islets and amplified in iPLA2?-Tg islets, and (f) ?-cell iPLA2? and NSMase messages are higher in the Akita mouse model of spontaneous ?-cell ER stress that leads to diabetes. We also find that iPLA2? participates in ?-cell apoptosis induced by hyperglycemia and cytokines and that islet iPLA2? and NSMase messages are elevated in pre-diabetic NOD mice. We hypothesize that iPLA2?-mediated ceramide generation and triggering of mitochondrial abnormalities are critical contributory events to ?-cell apoptosis. The studies proposed herein are designed to elucidate the precise role of iPLA2? in this process and whether this pathway, activated by ER stress, is also elicited by hyperglycemia and cytokines.
Our Aims will address the following:
Aim 1, role of iPLA2? activation and iPLA2?-derived lipid mediators. Hyperglycemia and cytokines promote ?-cell apoptosis, in part, by inducing ER stress and the role of iPLA2? and generation of iPLA2?-derived lipid mediators in this process will be assessed;
Aim 2, induction of iPLA2? and ceramide-generating pathway. SREBP-1 and CaMKII? are known to affect iPLA2? and are activated during ER stress. Their roles in iPLA2? induction and the requirement of iPLA2? activity for ceramide generation will be examined;
Aim 3, requirement of iPLA2? mobilization and activation to trigger mitochondrial abnormalities. The affects of genetic modulation, mobilization, and organelle-specific expression of iPLA2? on the mitochondrial apoptotic pathway will be examined;
Aim 4, sensitivity to ER stress following in vivo modulation of iPLA2?. A role of iPLA2? in modulating ER stress in ?-cells will be tested by crossing iPLA2?-null with ER stress-sensitive Akita mice and iPLA2?-Tg with ER stress-resistant CHOP-null mice;
and Aim 5, contribution of iPLA2? to ?-cell apoptosis during the evolution of autoimmune DM. The dependence of cytokine-induced ?-cell apoptosis on an iPLA2?/nitric oxide (NO)-dependent pathway and the contribution of iPLA2? to ?-cell apoptosis in NOD mice will be assessed.
These Aims will be addressed using apoptosis, flow cytometry, immunoblotting, qRT-PCR, enzymatic activity assay, confocal microscopy, molecular biology, and mass spectrometry protocols. Findings from our studies will lead to increased understanding of iPLA2? biology in the ?-cell. The long-range goal of our laboratory is to assess the role of iPLA2? in ?-cell apoptosis during the development and progression of diabetes mellitus.
?-cell apoptosis contributes to decreases in ?-cell mass and ?-cell dysfunction 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. Observations in the PI's laboratory indicate involvement of iPLA2? in the ?-cell apoptotic pathway and we propose to examine in greater detail the role of iPLA2?, as it relates to its expression, activation, and localization, and of iPLA2?-derived lipid mediators in ?-cell apoptosis due to stimuli that contribute to ?-cell death during the evolution of diabetes mellitus. Findings from these studies will extend our knowledge of factors that adversely affect ?-cell health and identify targets for future therapeutic interventions to prevent ?-cell death.
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