Studies of insulin secretory responses and of arachidonic acid incorporation into phospholipids of stably transfected insulinoma cells that overexpress group VIA phospholipase A2 (iPLA2beta ) indicate a signaling rather than a housekeeping role for iPLA2beta.
A cytosolic 84-kDa group VIA phospholipase A(2) (iPLA(2)beta) that does not require Ca(2+) for catalysis has been cloned from several sources, including rat and human pancreatic islet beta-cells and murine P388D1 cells. Many potential iPLA(2)beta functions have been proposed, including a signaling role in beta-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA(2)beta function rest in part on effects of inhibiting iPLA(2)beta activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A(2). Manipulation of iPLA(2)beta expression by molecular biologic means is an alternative approach to study iPLA(2)beta functions, and we have used a retroviral construct containing iPLA(2)beta cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA(2)beta. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA(2)beta-overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and BEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of iPLA(2)beta affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA(2)beta indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA(2)beta associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA(2)beta in insulin-secreting beta-cells.
Mass Spectrometry Resource, Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
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Ramanadham
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Wohltmann
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Bohrer
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Hsu
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Turk
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D054522:Group VI Phospholipases A2; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma; D007700:Kinetics; D051379:Mice; D009281:Naphthalenes / Q000494:pharmacology; D010190:Pancreatic Neoplasms; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000096:biosynthesis*; D011753:Pyrones / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D007328:Insulin; D009281:Naphthalenes; D010713:Phosphatidylcholines; D010743:Phospholipids; D011753:Pyrones; D011994:Recombinant Proteins; D005576:Colforsin; D016718:Arachidonic Acid; C068314:6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one; D010741:Phospholipases A; D054522:Group VI Phospholipases A2; C516475:PLA2G6 protein, human; D054467:Phospholipases A2; C516476:Pla2g6 protein, mouse; D000262:Adenylyl Cyclases; D005947:Glucose; D015056:1-Methyl-3-isobutylxanthine