Studies of phospholipid metabolism, proliferation, and secretion of stably transfected insulinoma cells that overexpress group VIA phospholipase A2.
A cytosolic 84 kDa Group VIA phospholipase A2 (iPLA2beta) that does not require Ca2+ for catalysis was cloned from Chinese hamster ovary (CHO) cells, murine P388D1 cells, pancreatic islet beta-cells, and other sources. Proposed iPLA2beta functions include participation in phosphatidylcholine (PC) homeostasis by degrading excess PC generated in CHO cells that overexpress CTP:phosphocholine cytidylyltransferase (CT), which catalyzes the rate-limiting step in PC biosynthesis; participation in biosynthesis of arachidonate-containing PC species in P388D1 cells by generating lysophosphatidylcholine (LPC) acceptors for arachidonate incorporation; and participation in signaling events in insulin secretion from islet beta-cells. To further examine iPLA2beta functions in beta-cells, we prepared stably transfected INS-1 insulinoma cell lines that overexpress iPLA2beta activity eightfold compared to parental INS-1 cells or to INS-1 cells transfected with an empty retroviral vector that did not contain iPLA2beta cDNA. The iPLA2beta-overexpressing cells exhibit a twofold increase in CT activity compared to parental cells but little change in rates of [3H]choline incorporation into or disappearance from PC. Electrospray ionization (ESI) tandem mass spectrometric measurements indicate that iPLA2beta-overexpressing cells have 1.5-fold higher LPC levels than parental INS-1 cells but do not exhibit increased rates of [3H]arachidonate incorporation into phospholipids, and incorporation is unaffected by a bromoenol lactone (BEL) suicide substrate inhibitor of iPLA2beta. The rate of appearance of arachidonate-containing phosphatidylethanolamine species visualized by ESI mass spectrometry is also similar in iPLA2beta-overexpressing and parental INS-1 cells incubated with supplemental arachidonic acid, and this process is unaffected by BEL. Compared to parental INS-1 cells, iPLA2beta-overexpressing cells proliferate more rapidly and exhibit amplified insulin secretory responses to a protein kinase C-activating phorbol ester, glucose, and a cAMP analog. These findings suggest that iPLA2beta plays a signaling role in beta-cells that differs from housekeeping functions in PC biosynthesis and degradation in P388D1 and CHO cells.
Ma
Z
Z
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Bohrer
A
A
Wohltmann
M
M
Ramanadham
S
S
Hsu
F F
FF
Turk
J
J
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D016466:CHO Cells; D002455:Cell Division; D002794:Choline / Q000378:metabolism; D019916:Choline-Phosphate Cytidylyltransferase / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D015870:Gene Expression; D005822:Genetic Vectors; D007340:Insulinoma / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D010190:Pancreatic Neoplasms / Q000201:enzymology* / Q000473:pathology; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010741:Phospholipases A / Q000235:genetics* / Q000378:metabolism; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D014162:Transfection; D014316:Tritium; D014407:Tumor Cells, Cultured
D008244:Lysophosphatidylcholines; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D014316:Tritium; D016718:Arachidonic Acid; D019916:Choline-Phosphate Cytidylyltransferase; D010741:Phospholipases A; D054467:Phospholipases A2; D002794:Choline