The Function of Phospholipase A2 in Islet Beta-cells

磷脂酶 A2 在胰岛 Beta 细胞中的功能

• 批准号: 7071174
• 负责人: ZHONGMIN ALEX MA
• 金额: $ 31.55万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-15 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7071174
• 关键词: adenosine triphosphate; biological signal transduction; blood glucose; cell line; cyclic AMP; enzyme activity; enzyme mechanism; fluorescence microscopy; genetically modified animals; green fluorescent proteins; insulin; intracellular transport; laboratory mouse; laboratory rat; pancreatic islet function; phospholipase A2; phosphorylation; protein kinase A; protein transport; radioimmunoassay; western blottings

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.

描述（由申请人提供）：胰岛对葡萄糖的胰岛素分泌缺陷或异常导致糖尿病（DM）。葡萄糖刺激下膜磷脂中花生四烯酸（AA）的水解被认为在葡萄糖刺激下β细胞分泌胰岛素中起作用。某些受体介导的胰岛素分泌因子，如葡萄糖依赖性胰岛素促胰岛素多肽（GIP）和胆囊收缩素-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细胞胰岛素分泌机制中其他成分的相互作用。从大鼠和人胰岛中克隆了一个Ca2+独立PLA2 (iPLA2)， iPLA2主要在胰岛β细胞中表达，其催化活性受到ATP的刺激，ATP是葡萄糖刺激胰岛素分泌的一个众所周知的信号。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细胞中的表达可显著提高INS-1细胞的葡萄糖敏感性，cAMP水平的升高可进一步增强这种敏感性。我们的假设是iPLA2是β细胞燃料感应装置的一个组成部分，该装置构成糖酵解、受体信号传导和膜磷脂水解途径之间的潜在联系，参与营养性和非营养性胰岛素分泌剂刺激的胰岛素分泌。目的1是通过在新鲜分离的胰岛中过表达和过表达iPLA2来检测iPLA2在葡萄糖刺激胰岛胰岛素分泌中的功能。目的2是通过iPLA2的ATP结合域诱变和gfp融合蛋白分析，表征ATP调节的iPLA2活化和从细胞质到细胞膜的易位。目的3是通过药理抑制和磷酸化分析来表征iPLA2与cAMP/PKA的相互作用。目的4是确定iPLA2在iPLA2敲除小鼠体内和从iPLA2-/-小鼠分离的胰岛中葡萄糖传感中的作用。拟议的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，并将导致阐明β细胞响应胰岛素分泌剂增加胰岛素分泌以预防2型糖尿病的机制，以及开发用于1型糖尿病细胞治疗的高质量胰岛素分泌β细胞。