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

Zhongmin Ma,Sasanka Ramanadham,Mary Wohltmann,Alan Bohrer,Fong-Fu Hsu,John Turk

doi:10.1074/jbc.m010423200

Abstract

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.

Highlights

A second cytosolic Phospholipases A2 (PLA2) has been cloned (8 –10) that does not require Ca2ϩ for catalysis, and it is classified as a group VIA PLA2 and has been designated iPLA2 [3, 4]
Transfection of INS-1 insulinoma cells with a retroviral construct containing the rat iPLA2␤ cDNA followed by selection of G418-resistant cells resulted in the isolation of two stably transfected clones that expressed severalfold more iPLA2␤ activity than parental INS-1 cells (Fig. 1A)
This is consistent with a report that propranolol amplifies insulin secretion from ␤-cells [78]. These findings indicate that the suppression by bromoenol lactone (BEL) of insulin secretion from iPLA2-X and control INS-1 cells stimulated with glucose and cAMP-elevating agents is not attributable to inhibition of phosphatidate phosphohydrolase-1 (PAPH-1)

Summary

EXPERIMENTAL PROCEDURES

Materials—ECL detection reagents and 1-palmitoyl-2-[14C]linoleoylsn-glycero-3-phosphocholine (55 mCi/mmol) were purchased from Amersham Pharmacia Biotech. Determination of INS-1 Cell cAMP Content—After experimental incubations, medium was removed from each well, and ice-cold ethanol (0.4 ml) containing IBMX (100 ␮M) was added. Incubation of INS-1 Cells with Arachidonic Acid to Induce Phospholipid Remodeling—INS-1 cells were cultured in RPMI medium containing penicillin, streptomycin, fungizone, and gentamicin (0.1% w/v each). Measurement of Nonesterified Arachidonic Acid in INS-1 Cells by Isotope Dilution Gas Chromatography Negative Ion Electron Capture Mass Spectrometry—Parental and iPLA2␤-overexpressing INS-1 cells that had been incubated with supplemental arachidonic acid for 24 h as described above were washed with 0.1% BSA in KRB four times to remove unincorporated arachidonic acid and were preincubated for 30 min in KRB medium containing 10 ␮M BEL or BEL-free vehicle. Statistical Analyses—Student’s t test was used to compare two groups, and multiple groups were compared by one-way analysis of variance with post hoc Newman-Keul’s analyses

RESULTS

BEL reduced the basal content of nonesterified arachidonate to

DISCUSSION