A key role for β-cell cytosolic phospholipase A2 in the maintenance of insulin stores but not in the initiation of insulin secretion

Persaud, S. J. and Roderigo-Milne, H. M. and Squires, Paul E. and Sugden, D. and Wheeler-Jones, C. P. D. and Marsh, P. J. and Belin, V. D. and Luther, M. J. and Jones, P. M. (2002) A key role for β-cell cytosolic phospholipase A2 in the maintenance of insulin stores but not in the initiation of insulin secretion. Diabetes, 51 (1). pp. 98-104. ISSN 0012-1797

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Abstract

Cytosolic phospholipase A2 (cPLA2) is a Ca2+-sensitive enzyme that has been implicated in insulin secretion in response to agents that elevate β-cell intracellular Ca2+ (Ca2+i). We generated clones of the MIN6 β-cell line that stably underexpress cPLA2 by transfection with a vector in which cPLA2 cDNA had been inserted in the antisense orientation. Reduced expression of cPLA2 was confirmed by Western blotting. The insulin content of cPLA2-deficient MIN6 cells was reduced by ∼90%, but they showed no decrease in preproinsulin mRNA expression. Measurements of stimulus-dependent changes in Ca2+i indicated that reduced expression of cPLA2 did not affect the capacity of MIN6 cells to show elevations in Ca2+ in response to depolarizing stimuli. Perifusion experiments indicated that cPLA2 under-expressing MIN6 pseudoislets responded to glucose, tolbutamide, and KCl with insulin secretory profiles similar to those of cPLA2 expressing pseudoislets, but that secretion was not maintained with continued stimulus. Analysis of the ultrastructure of cPLA2-deficient MIN6 cells by electron microscopy revealed that they contained very few mature insulin secretory granules, but there was an abundance of non-electron-dense vesicles. These data are consistent with a role for cPLA2 in the maintenance of insulin stores, but they suggest that it is not required for the initiation of insulin secretion from β-cells.

Keywords:calcium ion, complementary DNA, glucose, insulin, phospholipase A2, potassium chloride, tolbutamide, article, calcium cell level, cell line, cell ultrastructure, controlled study, depolarization, electron microscopy, enzyme activity, gene insertion, genetic transfection, insulin release, pancreas islet beta cell, priority journal, protein expression, secretory granule, secretory vesicle, stimulus response, structure analysis, Western blotting, Animals, Clone Cells, Forskolin, Gene Expression Regulation, Enzymologic, Islets of Langerhans, Kinetics, Phospholipases A, Proinsulin, Protein Precursors, Recombinant Proteins, Tetradecanoylphorbol Acetate, Thermodynamics, Tolbutamide, Transfection
Subjects:C Biological Sciences > C990 Biological Sciences not elsewhere classified
Divisions:College of Science > School of Life Sciences
ID Code:14328
Deposited On:13 Jun 2014 11:09

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