Uncoupling of Nutrient Metabolism From Insulin Secretion by Overexpression of Cytosolic Phospholipase A₂

Abstract

We have generated MIN6 β-cells that stably overexpress cytosolic phospholipase A₂ (cPLA₂) and show a ninefold increase in cPLA₂ activity. Overexpression of cPLA₂ did not affect the capacity of MIN6 cells to show elevations in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in response to tolbutamide and KCl, and these depolarizing stimuli produced insulin secretion profiles in cPLA₂-overexpressing cells similar to those they produced in passage-matched nontransfected MIN6 cells. However, cPLA₂-overexpressing MIN6 cells did not respond to elevations in extracellular glucose with increases in ATP, [Ca²⁺]_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 ∼95% in MIN6 cells overexpressing cPLA₂. 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 cPLA₂-overexpressing MIN6 cells, and this could be prevented by exposure to 100 μmol/l methyl arachidonyl fluorophosphate, a cPLA₂ inhibitor. Glucose caused a decrease in rhodamine 123 fluorescence in control cells, but not in those overexpressing cPLA₂, 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 cPLA₂ results in severe impairment of the calcium and secretory responses of β-cells to glucose through upregulation of UCP-2 and uncoupling of mitochondrial metabolism from ATP generation.