Uncoupling of nutrient metabolism from insulin secretion by overexpression of cytosolic phospholipase A(2)

Milne, Helen M., Burns, Chris J., Squires, Paul E., Evans, Nicholas D., Pickup, John, Jones, Peter M. and Persaud, Shanta J. (2005) Uncoupling of nutrient metabolism from insulin secretion by overexpression of cytosolic phospholipase A(2). Diabetes, 54 (1), 116-124. (doi:10.1080/08870440802299543). (PMID:15616018)

Abstract

We have generated MIN6 beta-cells that stably overexpress cytosolic phospholipase A(2) (cPLA(2)) and show a ninefold increase in cPLA(2) activity. Overexpression of cPLA(2) did not affect the capacity of MIN6 cells to show elevations in intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to tolbutamide and KCl, and these depolarizing stimuli produced insulin secretion profiles in cPLA(2)-overexpressing cells similar to those they produced in passage-matched nontransfected MIN6 cells. However, cPLA(2)-overexpressing MIN6 cells did not respond to elevations in extracellular glucose with increases in ATP, [Ca(2+)](i), or insulin secretion. Nontransfected MIN6 cells showed a rapid and sustained increase in NAD(P)H autofluorescence in response to 25 mmol/l glucose, and this was reduced by approximately 95% in MIN6 cells overexpressing cPLA(2). This effect was mimicked in nontransfected MIN6 cells by p-(trifluoromethoxy) phenylylhydrazone, a mitochondrial uncoupler. Quantitative RT-PCR indicated that mRNA for uncoupling protein-2 (UCP-2) was increased in the cPLA(2)-overexpressing MIN6 cells, and this could be prevented by exposure to 100 mumol/l methyl arachidonyl fluorophosphate, a cPLA(2) inhibitor. Glucose caused a decrease in rhodamine 123 fluorescence in control cells, but not in those overexpressing cPLA(2), consistent with the transfected cells being unable to maintain mitochondrial proton gradients as a consequence of UCP-2 upregulation. Our data indicate that overexpression of cPLA(2) results in severe impairment of the calcium and secretory responses of beta-cells to glucose through upregulation of UCP-2 and uncoupling of mitochondrial metabolism from ATP generation.

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Contributors

Author: Nicholas D. Evans

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