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.