The Function of Phospholipase A2 in Islet Beta-cells

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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.