Studies of insulin secretory responses and of arachidonic acid incorporation into phospholipids of stably transfected insulinoma cells that overexpress group VIA phospholipase A2 (iPLA2β) indicate a signaling rather than a housekeeping role for iPLA2β

A cytosolic 84-kDa group VLA phospholipase A(2) (iPLA(2)beta) that does not require Ca2+ for catalysis has been cloned from several sources, including rat and human pancreatic islet beta -cells and murine P388D1 cells. Many potential iPLA(2)beta functions have been proposed, including a signaling role in beta -cell insulin secretion and a role in generating lysophosphatidylcholine accepters for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA(2)beta function rest in part on effects of inhibiting iPLA(2)beta activity with a bromoenol lactone (BEL) suicide substrate, but EEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A(2). Manipulation of iPLA(2)beta expression by molecular biologic means is an alternative approach to study iPLA(2)beta functions, and we have used a retroviral construct containing iPLA(2)beta cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA(2)beta. Compared with parental INS-I cells or cells transfected with empty vector, both iPLA(2)beta -overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and EEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of iPLA(2)beta affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA(2)beta indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA(2)beta associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA(2)beta in insulinsecreting 13-cells.