CALCIUM-INDEPENDENT PLA2BETA IN BETA-CELL APOPTOSIS

糖尿病是最常见的代谢性疾病，细胞凋亡是细胞内减少的原因之一。 糖尿病发展过程中的质量和功能。然而，对细胞有贡献的机制(S) 对细胞凋亡的认识还不是很清楚。我们在第一个授权期的假设是，集团通过钙离子- 独立磷脂酶A2(IPLA2)参与内质网应激诱导的细胞凋亡。我们发现(A)ER 应激诱导iPLA2激活，通过中性鞘磷脂酶(NSMase)产生神经酰胺，以及-细胞 凋亡，(B)这些结果被iPLA2或NSmase抑制，(C)iPLA2缺失的胰岛较少 IPLA2转基因(TG)胰岛对内质网应激诱导的细胞凋亡更敏感；内质网应激(D)增加 内质网和线粒体中的iPLA2蛋白/活性，以及(E)通过 IPLA2-神经酰胺轴；和(F)内质网应激倾向的秋田细胞和糖尿病前期NOD小鼠的胰岛表达 IPLA2水平高于WT细胞和胰岛，(G)STZ诱导的高血糖在iPLA2-TG中加速 小鼠，以及(H)细胞因子诱导胰岛细胞神经酰胺的产生、丢失和凋亡 被iPLA2失活抑制。在第二批资助期内，我们建议研究 IPLA2和iPLA2衍生的脂类介体在以下目的下参与细胞凋亡：目的 1将检测iPLA2的激活和细胞凋亡过程中的脂质变化。高血糖与细胞因子 -部分通过诱导内质网应激促进细胞凋亡，这一过程对iPLA2的依赖将是 评估过了。目的2研究iPLA2和神经酰胺生成途径的诱导机制。角色 脂质介质SREBPs、ROS和GSH对iPLA2和NSMase表达的影响及突变 将检查iPLA2中关于神经酰胺生成的脂肪酶序列。目标3将研究iPLA2在 呃-线粒体串扰。基因调控对iPLA2表达、ROS和GSH水平的影响 NSMase表达、iPLA2突变和细胞器特异性iPLA2表达对细胞活性的影响 线粒体的凋亡途径将被检测。Aim 4将检查iPLA2表达的体内调节 改变胰岛细胞对内质网应激的敏感性。IPLA2缺失小鼠将与秋田小鼠和iPLA2-TG小鼠杂交 用CHOP基因缺失的小鼠和内质网应激的发展监测。AIM 5将确定iPLA2是否有助于 -自身免疫性糖尿病演变过程中的细胞凋亡。细胞因子诱导细胞凋亡的依赖性 IPLA2和一氧化氮通路及链脲佐菌素诱导的细胞凋亡和高血糖 将确定iPLA2的表达。细胞凋亡，流式细胞仪，免疫印迹，实时聚合酶链式反应，酶促 活性、共聚焦显微镜、分子生物学和质谱学方案将在这些方面得到应用 学习。

Diabetes mellitus is the most prevalent metabolic disease, and -cell apoptosis contributes to decreases in -cell mass and function during the evolution of diabetes. However, the mechanism(s) that contribute to -cell apoptosis 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) ER stress induces iPLA2¿ activation, ceramide generation via neutral sphingomyelinase (NSMase), and -cell apoptosis, (b) these outcomes are suppressed by inhibition of iPLA2 or NSmase, (c) iPLA2 -null islets are less and iPLA2 -tansgenic (Tg) islets more sensitive to ER stress-induced apoptosis; that ER stress (d) increases iPLA2 protein/activity in the ER and mitochondria, and (e) activates the mitochondrial apoptotic pathway via the iPLA2 -ceramide axis; and (f) ER stress-prone Akita -cells and islets from pre-diabetic NOD mice express higher levels of iPLA2 than WT cells and islets, (g) STZ-induced hyperglycemia is accelerated in iPLA2 -Tg mice, and (h) cytokines induce ceramide generation, loss in  , and apoptosis in islet-cells that are all suppressed by iPLA2 inactivation. During the 2nd grant period, we propose to examine the mechanism of involvement of iPLA2 and iPLA2 -derived lipid mediators in -cell apoptosis under the following Aims: Aim 1 will examine iPLA2 activation and lipid changes in -cells undergoing apoptosis. Hyperglycemia and cytokines promote -cell apoptosis, in part, by inducing ER stress and the dependence of this process on iPLA2 will be assessed. Aim 2 will examine the mechanism of iPLA2 and ceramide-generating pathway induction. The roles of lipid mediators, SREBPs, ROS, and GSH on iPLA2 and NSMase expression and the affects of mutating the lipase sequence in iPLA2 on ceramide generation will be examined. Aim 3 will examine the role of iPLA2 in ER-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 the mitochondrial apoptotic pathway will be examined. Aim 4 will examine if in vivo modulation of iPLA2 expression alters islet -cell sensitivity to ER stress. iPLA2 -null mice will be crossed with Akita mice and iPLA2 -Tg mice with 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 apoptosis on iPLA2 and nitric oxide pathway and of STZ-induced -cell apoptosis and hyperglycemia on iPLA2 expression will be determined. Apoptosis, flow cytometry, immunoblotting, real time-PCR, enzymatic activity, confocal microscopy, molecular biology, and mass spectrometry protocols will be utilized in these studies.