The Function of Phospholipase A2 in Islet Beta-cells

描述(申请人提供)：胰岛素分泌缺陷或异常的胰岛反应葡萄糖导致糖尿病(DM)。葡萄糖刺激的膜磷脂中花生四烯酸(AA)的水解被认为在葡萄糖刺激的β细胞胰岛素分泌中起作用。某些受体介导的胰岛素促分泌剂，如葡萄糖依赖的促胰岛素多肽(GIP)和CCK-8(CCK-8)也通过激活胰岛磷脂酶A2(PLA2)来刺激AA的释放。我们的长期目标是了解PLA2信号通路在胰岛素分泌中的机制，并表征PLA2的调节及其与β细胞中参与胰岛素分泌机制的其他成分的相互作用。从大鼠和人胰岛中克隆了一种钙非依赖性磷脂酶A2(IPLA2)，iPLA2主要在胰岛β细胞中表达，其催化活性受葡萄糖刺激的胰岛素分泌的已知信号三磷酸腺苷(ATP)的刺激。用溴烯醇内酯(BEL)特异性抑制iPLA2可抑制葡萄糖刺激的胰岛素分泌和AA释放。IPLA2在胰岛素瘤INS-1细胞中的表达显著增加了INS-1细胞对葡萄糖的敏感性，cAMP水平的升高进一步增强了这一作用。我们的假设是，iPLA2是β细胞燃料感应器的一个组成部分，它构成了糖酵解、受体信号和膜磷脂分解途径之间的潜在联系，参与营养和非营养胰岛素促分泌刺激的胰岛素分泌。目的1通过在新分离的胰岛中高表达和低表达iPLA2，研究iPLA2在葡萄糖刺激的胰岛胰岛素分泌中的作用。目的2通过iPLA2三磷酸腺苷结合域的突变和GFP融合蛋白的分析，研究ATP调控的iPLA2的激活和从胞浆到细胞膜的转位。目的3通过药理抑制和磷酸化分析研究iPLA2与cAMP/PKA的相互作用。目的4确定iPLA2基因敲除小鼠体内和从iPLA2-/-小鼠分离的胰岛中iPLA2在体内葡萄糖感应中的作用。这些研究将有助于我们理解iPLA2在胰岛β细胞中的生物学重要性，并将有助于阐明胰岛素分泌促进剂促进胰岛β细胞分泌胰岛素的机制，从而预防2型糖尿病，并为1型糖尿病的细胞治疗提供高质量的胰岛素分泌的β细胞。

DESCRIPTION (provided by applicant): Defective or abnormal insulin secretion by islets in response to glucose results in diabetes mellitus (DM). Glucose-stimulated hydrolysis of arachidonic acid (AA) from membrane phospholipids has been suggested to play a role in glucose-stimulated insulin secretion from beta-cells. Certain receptor-mediated insulin-secretagogues, such as glucose-dependent insulinotropic polypeptide (GIP) and cholecystokinin-8 (CCK-8) also stimulate release of AA by activating islet phospholipase A2 (PLA2). Our long-term objectives are to understand the mechanism of PLA2 signaling pathway in insulin secretion and to characterize the regulation of PLA2 and its interaction with other components involved in the insulin secretion machinery in beta-cells. A Ca2+ -independent PLA2 (iPLA2) has been cloned from rat and human islets, iPLA2 is dominantly expressed in islet beta-cells and its catalytic activity is stimulated by ATP, a well-known signal in glucose-stimulated insulin secretion. Specific inhibition of iPLA2 with bromoenol lactone (BEL) leads to the suppression of both glucose-stimulated insulin secretion and AA release. Expression of iPLA2 in insulinoma INS-1 cells significantly increases glucose sensitivity of INS-1 cells, which is further enhanced by increasing cAMP levels. Our hypothesis is that iPLA2 is one component of the beta-cell fuel-sensing apparatus that constitutes an underlying link among glycolytic, receptor signaling, and membrane phospholipolytic pathways to participate in both nutrient and non-nutrient insulin-secretagogue stimulated insulin secretion. Aim 1 is to examine the function of iPLA2 in glucose-stimulated insulin secretion in islets by overexpressing and underexpressing iPLA2 in freshly isolated islets. Aim 2 is to characterize the ATP-regulated iPLA2 activation and translocation from the cytosol to cellular membranes of beta-cells by mutagenesis of ATP binding domain of iPLA2 and GFP-fusion protein analyses. Aim 3 is to characterize the interaction of iPLA2 with cAMP/PKA by pharmacological inhibition and phosphorylation analyses. Aim 4 is to determine the role of iPLA2 in glucose sensing in vivo in iPLA2-knockout mice and in the islets isolated from iPLA2-/- mice. The proposed studies should contribute significantly to our understanding of the biological importance of iPLA2 in islet beta-cells and will lead to the elucidation of mechanisms by which to increase insulin secretion by beta-cells in response to insulin secretagogues for prevention of type 2 DM and for developing high-quality insulin secreting beta-cells for cell therapy of type 1 DM.