TY - JOUR
T1 - Studies of Insulin Secretory Responses and of Arachidonic Acid Incorporation into Phospholipids of Stably Transfected Insulinoma Cells That Overexpress Group VIA Phospholipase A2 (iPLA2β) Indicate a Signaling Rather Than a Housekeeping Role for iPLA2β
AU - Ma, Zhongmin
AU - Ramanadham, Sasanka
AU - Wohltmann, Mary
AU - Bohrer, Alan
AU - Hsu, Fong Fu
AU - Turk, John
PY - 2001/4/20
Y1 - 2001/4/20
N2 - A cytosolic 84-kDa group VIA phospholipase A2 (iPLA 2β) that does not require Ca2+ for catalysis has been cloned from several sources, including rat and human pancreatic islet β-cells and murine P388D1 cells. Many potential iPLA2β functions have been proposed, including a signaling role in β-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA2β function rest in part on effects of inhibiting iPLA2β activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A2. Manipulation of iPLA2β expression by molecular biologic means is an alternative approach to study iPLA2β functions, and we have used a retroviral construct containing iPLA2β cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA2β. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA 2β-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 iPLA2β affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA2β indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA2β associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA 2β in insulin-secreting β-cells.
AB - A cytosolic 84-kDa group VIA phospholipase A2 (iPLA 2β) that does not require Ca2+ for catalysis has been cloned from several sources, including rat and human pancreatic islet β-cells and murine P388D1 cells. Many potential iPLA2β functions have been proposed, including a signaling role in β-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA2β function rest in part on effects of inhibiting iPLA2β activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A2. Manipulation of iPLA2β expression by molecular biologic means is an alternative approach to study iPLA2β functions, and we have used a retroviral construct containing iPLA2β cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA2β. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA 2β-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 iPLA2β affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA2β indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA2β associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA 2β in insulin-secreting β-cells.
UR - http://www.scopus.com/inward/record.url?scp=0035918201&partnerID=8YFLogxK
U2 - 10.1074/jbc.M010423200
DO - 10.1074/jbc.M010423200
M3 - Article
C2 - 11278673
AN - SCOPUS:0035918201
SN - 0021-9258
VL - 276
SP - 13198
EP - 13208
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 16
ER -