The Function of Phospholipase A2 in Islet Beta-cells

描述（由申请方提供）：胰岛对葡萄糖的反应性胰岛素分泌缺陷或异常导致糖尿病（DM）。已经表明，葡萄糖刺激的花生四烯酸（AA）从膜磷脂的水解在葡萄糖刺激的β细胞的胰岛素分泌中起作用。某些受体介导的胰岛素促分泌素，如葡萄糖依赖性促胰岛素多肽（GIP）和胆囊收缩素-8（CCK-8）也通过激活胰岛磷脂酶A2（PLA 2）刺激AA的释放。我们的长期目标是了解PLA 2信号通路在胰岛素分泌中的机制，并表征PLA 2的调节及其与参与β细胞胰岛素分泌机制的其他组分的相互作用。已经从大鼠和人胰岛中克隆了Ca 2+非依赖性PLA 2（iPLA 2），iPLA 2主要在胰岛β细胞中表达，并且其催化活性由ATP刺激，ATP是葡萄糖刺激的胰岛素分泌中的众所周知的信号。用溴烯醇内酯（BEL）特异性抑制iPLA 2导致葡萄糖刺激的胰岛素分泌和AA释放的抑制。胰岛素瘤INS-1细胞中iPLA 2的表达显著增加INS-1细胞的葡萄糖敏感性，这通过增加cAMP水平进一步增强。我们的假设是，iPLA 2是β细胞燃料传感装置的一个组成部分，其构成糖酵解、受体信号传导和膜磷脂分解途径之间的潜在联系，以参与营养和非营养胰岛素促分泌素刺激的胰岛素分泌。目的1是通过在新鲜分离的胰岛中过表达和低表达iPLA 2来检查iPLA 2在胰岛中葡萄糖刺激的胰岛素分泌中的功能。目的2是通过突变iPLA 2的ATP结合结构域和GFP融合蛋白分析来表征ATP调节的iPLA 2活化和从β细胞的胞质溶胶到细胞膜的易位。目的3通过药理学抑制和磷酸化分析来表征iPLA 2与cAMP/PKA的相互作用。目的4是确定iPLA 2在iPLA 2敲除小鼠和从iPLA 2-/-小鼠分离的胰岛中体内葡萄糖感知中的作用。所提出的研究将显著有助于我们理解胰岛β细胞中iPLA 2的生物学重要性，并将导致阐明响应于胰岛素促分泌素而增加β细胞的胰岛素分泌以预防2型DM和开发用于1型DM的细胞治疗的高质量胰岛素分泌β细胞的机制。

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.