CALCIUM-INDEPENDENT PLA2BETA IN BETA-CELL APOPTOSIS

糖尿病是最常见的代谢性疾病，细胞凋亡导致细胞凋亡减少， 在糖尿病的发展过程中的质量和功能。然而，有助于细胞增殖的机制 细胞凋亡还没有被很好地理解。我们在第一个授权期的假设是，通过Ca 2 +- 非依赖性磷脂酶A2（iPLA 2 <$）参与内质网应激诱导的细胞凋亡。我们发现（a）ER 应激诱导iPLA 2激活，通过中性鞘磷脂酶（NSMase）产生神经酰胺， 细胞凋亡，（B）这些结果被iPLA 2或NSmase的抑制所抑制，（c）iPLA 2无效的胰岛较少 和iPLA 2-转基因（Tg）胰岛对ER应激诱导的细胞凋亡更敏感; ER应激（d）增加 在ER和线粒体中的iPLA 2蛋白/活性，和（e）通过细胞凋亡激活线粒体凋亡途径。 iPLA 2-神经酰胺轴;和（f）来自糖尿病前期NOD小鼠的ER应激倾向性秋田细胞和胰岛表达 iPLA 2的水平高于WT细胞和胰岛，（g）STZ诱导的高血糖症在iPLA 2-Tg中加速 小鼠，和（h）细胞因子诱导神经酰胺产生，  和胰岛细胞凋亡， 通过iPLA 2失活抑制。在第二个资助期内，我们建议研究 iPLA 2和iPLA 2衍生的脂质介质参与细胞凋亡，目的如下： 1将检测经历凋亡的细胞中iPLA 2的活化和脂质变化。高血压和细胞因子 促进细胞凋亡，部分是通过诱导ER应激，这一过程对iPLA 2的依赖性将是重要的。 评估。目的2将研究iPLA 2和神经酰胺生成途径诱导的机制。的角色 脂质介质、SREBPs、ROS和GSH对iPLA 2和NSMase表达的影响，以及 将检查iPLA 2中的脂肪酶序列对神经酰胺生成的影响。目标3将研究iPLA 2在以下方面的作用： ER-线粒体串扰。iPLA 2表达、ROS和GSH水平的遗传调节的影响， NSMase表达、iPLA 2突变和细胞器特异性iPLA 2表达对细胞凋亡的影响。 将检查线粒体凋亡途径。目的4将检查是否在体内调节iPLA 2表达 改变胰岛细胞对内质网应激的敏感性。将iPLA 2缺失小鼠与秋田小鼠和iPLA 2-Tg小鼠杂交 用CHOP-缺失小鼠进行，并监测ER应激的发展。目标5将确定iPLA 2是否有助于 - 自身免疫性DM演变过程中的细胞凋亡。苦参碱诱导细胞凋亡的依赖性 对iPLA 2和一氧化氮通路的影响以及STZ诱导的细胞凋亡和高血糖对 将测定iPLA 2表达。细胞凋亡，流式细胞术，免疫印迹，真实的时间PCR，酶 活性，共聚焦显微镜，分子生物学和质谱协议将用于这些 问题研究

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.