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

J Biol Chem. 2001 Apr 20;276(16):13198-208. doi: 10.1074/jbc.M010423200. Epub 2001 Jan 22.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 1-Methyl-3-isobutylxanthine / pharmacology
  • Adenylyl Cyclases / metabolism
  • Animals
  • Arachidonic Acid / metabolism*
  • Colforsin / pharmacology
  • Enzyme Inhibitors / pharmacology
  • Glucose / pharmacology
  • Group VI Phospholipases A2
  • Humans
  • Insulin / metabolism*
  • Insulin Secretion
  • Insulinoma
  • Kinetics
  • Mice
  • Naphthalenes / pharmacology
  • Pancreatic Neoplasms
  • Phosphatidylcholines / chemistry
  • Phosphatidylcholines / metabolism
  • Phospholipases A / genetics
  • Phospholipases A / metabolism*
  • Phospholipases A2
  • Phospholipids / biosynthesis*
  • Pyrones / pharmacology
  • Rats
  • Recombinant Proteins / metabolism
  • Signal Transduction / physiology*
  • Spectrometry, Mass, Electrospray Ionization
  • Substrate Specificity
  • Transfection
  • Tumor Cells, Cultured

Substances

  • Enzyme Inhibitors
  • Insulin
  • Naphthalenes
  • Phosphatidylcholines
  • Phospholipids
  • Pyrones
  • Recombinant Proteins
  • Colforsin
  • Arachidonic Acid
  • 6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one
  • Phospholipases A
  • Group VI Phospholipases A2
  • PLA2G6 protein, human
  • Phospholipases A2
  • Pla2g6 protein, mouse
  • Adenylyl Cyclases
  • Glucose
  • 1-Methyl-3-isobutylxanthine