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 VIA phospholipase A₂ (iPLA ₂β) that does not require Ca²⁺ for catalysis has been cloned from several sources, including rat and human pancreatic islet β-cells and murine P388D1 cells. Many potential iPLA₂β functions have been proposed, including a signaling role in β-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA₂β function rest in part on effects of inhibiting iPLA₂β activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A₂. Manipulation of iPLA₂β expression by molecular biologic means is an alternative approach to study iPLA₂β functions, and we have used a retroviral construct containing iPLA₂β cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA₂β. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA ₂β-overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and BEL 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 iPLA₂β affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA₂β indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA₂β associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA ₂β in insulin-secreting β-cells.