Studies of phospholipid metabolism, proliferation, and secretion of stablytransfected insulinoma cells that overexpress group VIA phospholipase A(2)

A cytosolic 84 kDa Croup VIA phospholipase A(2) (iPLA(2)beta) that does not require Call for catalysis was cloned from Chinese hamster ovary (CHO) cel ls, murine P388D1 cells, pancreatic islet beta -cells, and other sources, P roposed iPLA(2)beta functions include participation in phosphatidylcholine (PC) homeostasis by degrading excess PC generated in CHO cells that overexp ress CTP:phosphocholine cytidylyltransferase (CT), which catalyzes the rate -limiting step in PC biosynthesis; participation in biosynthesis of arachid onate-containing PC species in P388D1 cells by generating lysophosphatidylc holine (LPC) acceptors for arachidonate incorporation; and participation in signaling events in insulin secretion from islet beta -cells. To further e xamine iPLA(2)beta functions in beta -cells, we prepared stably transfected INS-I insulinoma cell lines that overexpress iPLA(2)beta activity eightfol d compared to parental INS-1 cells or to INS-1 cells transfected with an em pty retroviral vector that did not contain iPLA(2)beta cDNA. The iPLA(2)bet a -overexpressing cells exhibit a twofold increase in CT activity compared to parental cells but little change in rates of [H-3]choline incorporation into or disappearance from PC. Electrospray ionization (ESI) tandem mass sp ectrometric measurements indicate that iPLA(2)beta -overexpressing cells ha ve 1.5-fold higher LPC levels than parental INS-1 cells but do not exhibit increased rates of [H-3]arachidonate incorporation into phospholipids, and incorporation is unaffected by a bromoenol lactone (BEL) suicide substrate inhibitor of iPLA(2)beta. The rate of appearance of arachidonate-containing phosphatidylethanolamine species visualized by ESI mass spectrometry is al so similar in iPLA(2)beta -overexpressing and parental INS-1 cells incubate d with supplemental arachidonic acid, and this process is unaffected by BEL . Compared to parental INS-1 cells, iPLA(2)beta -overexpressing cells proli ferate more rapidly and exhibit amplified insulin secretory responses to a protein kinase C-activating phorbol ester, glucose, and a cAMP analog. Thes e findings suggest that iPLA(2)beta plays a signaling role in beta -cells t hat differs from housekeeping functions in PC biosynthesis and degradation in P388D1 and CHO cells.