Studies of phospholipid metabolism, proliferation, and secretion of stably transfected insulinoma cells that overexpress group VIA phospholipase A2

• Published in: Lipids 2001-07, Vol.36 (7), p.689-700
• Main Authors: Ma, Zhongmin, Bohrer, Alan, Wohltmann, Mary, Ramanadham, Sasanka, Hsu, Fong‐Fu, Turk, John
• Format: Article
• Language: English
• Subjects: Animals; Arachidonic Acid - metabolism; Biosynthesis; Catalysis; Cell Division; Cells; CHO Cells; Choline - metabolism; Choline-Phosphate Cytidylyltransferase - genetics; Choline-Phosphate Cytidylyltransferase - metabolism; Cricetinae; Gene Expression; Genetic Vectors; Incorporation; Insulinoma - enzymology; Insulinoma - metabolism; Insulinoma - pathology; Ionization; Kinases; Kinetics; Lysophosphatidylcholines - metabolism; Mass Spectrometry; Mice; Pancreatic Neoplasms - enzymology; Pancreatic Neoplasms - pathology; Phosphatidylcholines - metabolism; Phosphatidylethanolamines - metabolism; Phospholipases A - genetics; Phospholipases A - metabolism; Phospholipases A2; Phospholipids - metabolism; Retroviridae - genetics; Suicide; Transfection; Tritium; Tumor Cells, Cultured
• ISSN: 0024-4201; 1558-9307
• DOI: 10.1007/s11745-001-0774-9

A cytosolic 84 kDa Group VIA phospholipase A2 (iPLA2β) that does not require Ca2+ for catalysis was cloned from Chinese hamster ovary (CHO) cells, murine P388D1 cells, pancreatic islet β‐cells, and other sources. Proposed iPLA2β 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 (IPC) acceptors for arachidonate incorporation; and participation in signaling events in insulin secretion from islet β‐cells. To further examine iPLA2β functions in β‐cells, we prepared stably transfected INS‐1 insulinoma cell lines that overexpress iPLA2β activity eightfold compared to parental INS‐1 cells or to INS‐1 cells transfected with an empty retroviral vector that did not contain iPIA2β cDNA. The iPLA2β‐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 iPLA2β‐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 iPLA2β. The rate of appearance of arachidonate‐containing phosphatidylethanolamine species visualized by ESI mass spectrometry is also similar in iPLA2β‐overexpressing and parental INS‐1 cells incubated with supplemental arachidonic acid, and this process is unaffected by BEL. Compared to parental INS‐1 cells, iPLA2β‐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 iPLA2β plays a signaling role in β‐cells that differs from housekeeping functions in PC biosynthesis and degradation in P388D1 and CHO cells.