An 84-kDa
group VI phospholipase A2 (iPLA2) that does not require Ca2+ for catalysis has been cloned from Chinese hamster ovary cells, murine P388D1 cells, and pancreatic islet beta-cells. A housekeeping role for iPLA2 in generating
lysophosphatidylcholine (LPC) acceptors for
arachidonic acid incorporation into
phosphatidylcholine (PC) has been proposed because iPLA2 inhibition reduces LPC levels and suppresses arachidonate incorporation and
phospholipid remodeling in P388D1 cells. Because islet beta-cell
phospholipids are enriched in arachidonate, we have examined the role of iPLA2 in arachidonate incorporation into islets and INS-1
insulinoma cells. Inhibition of iPLA2 with a
bromoenol lactone (BEL) suicide substrate did not suppress and generally enhanced [3H]arachidonate incorporation into these cells in the presence or absence of extracellular
calcium at varied time points and BEL concentrations. Arachidonate incorporation into islet
phospholipids involved deacylation-reacylation and not de novo synthesis, as indicated by experiments with varied extracellular
glucose concentrations and by examining [14C]
glucose incorporation into
phospholipids. BEL also inhibited islet cytosolic
phosphatidate phosphohydrolase (PAPH), but the PAPH inhibitor
propranolol did not affect arachidonate incorporation into islet or INS-1 cell
phospholipids. Inhibition of islet iPLA2 did not alter the
phospholipid head-group classes into which [3H]arachidonate was initially incorporated or its subsequent transfer from PC to other
lipids. Electrospray ionization mass spectrometric measurements indicated that inhibition of INS-1 cell iPLA2 accelerated arachidonate incorporation into PC and that inhibition of islet iPLA2 reduced LPC levels by 25%, suggesting that LPC mass does not limit arachidonate incorporation into islet PC. Gas chromatography/mass spectrometry measurements indicated that BEL but not
propranolol suppressed
insulin secretagogue-induced hydrolysis of arachidonate from islet
phospholipids. In islets and INS-1 cells, iPLA2 is thus not required for arachidonate incorporation or
phospholipid remodeling and may play other roles in these cells.