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Studies of phospholipid metabolism, proliferation, and secretion of stably transfected insulinoma cells that overexpress group VIA phospholipase A 2
Author(s) -
Ma Zhongmin,
Bohrer Alan,
Wohltmann Mary,
Ramanadham Sasanka,
Hsu FongFu,
Turk John
Publication year - 2001
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/s11745-001-0774-9
Subject(s) - transfection , chinese hamster ovary cell , phosphatidylethanolamine , insulinoma , biology , phosphatidylcholine , phospholipase , phospholipid , cell culture , microbiology and biotechnology , biochemistry , enzyme , pancreas , genetics , membrane
A cytosolic 84 kDa Group VIA phospholipase A 2 (iPLA 2 β) that does not require Ca 2+ for catalysis was cloned from Chinese hamster ovary (CHO) cells, murine P388D1 cells, pancreatic islet β‐cells, and other sources. Proposed iPLA 2 β 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 iPLA 2 β functions in β‐cells, we prepared stably transfected INS‐1 insulinoma cell lines that overexpress iPLA 2 β activity eightfold compared to parental INS‐1 cells or to INS‐1 cells transfected with an empty retroviral vector that did not contain iPIA 2 β cDNA. The iPLA 2 β‐overexpressing cells exhibit a twofold increase in CT activity compared to parental cells but little change in rates of [ 3 H] choline incorporation into or disappearance from PC. Electrospray ionization (ESI) tandem mass spectrometric measurements indicate that iPLA 2 β‐overexpressing cells have 1.5‐fold higher LPC levels than parental INS‐1 cells but do not exhibit increased rates of [ 3 H]arachidonate incorporation into phospholipids, and incorporation is unaffected by a bromoenol lactone (BEL) suicide substrate inhibitor of iPLA 2 β. The rate of appearance of arachidonate‐containing phosphatidylethanolamine species visualized by ESI mass spectrometry is also similar in iPLA 2 β‐overexpressing and parental INS‐1 cells incubated with supplemental arachidonic acid, and this process is unaffected by BEL. Compared to parental INS‐1 cells, iPLA 2 β‐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 iPLA 2 β plays a signaling role in β‐cells that differs from housekeeping functions in PC biosynthesis and degradation in P388D1 and CHO cells.