Uncoupling of nutrient metabolism from insulin secretion by overexpression of cytosolic phospholipase A2

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 A2. Diabetes, 54 (1). pp. 116-124. ISSN 0012-1797

Full content URL: http://dx.doi.org/10.2337/diabetes.54.1.116

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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.

Keywords:Insulin, Phospholipase A2, Pancreatic beta cells, Glucose, messenger RNA, COX cyclo-oxygenase, cPLA2 cytosolic phospholipase A2, [Ca2+] intracellular Ca2+ concentration, FCCP p-(trifluoromethoxy) phenylylhydrazon, KATP channel, ATP-sensitive K+ channel, KIC, MAFP methyl arachidonyl fluorophosphate, [3H]PC, UCP-2 uncoupling protein-2
Subjects:B Subjects allied to Medicine > B990 Subjects Allied to Medicine not elsewhere classified
C Biological Sciences > C910 Applied Biological Sciences
Divisions:College of Science > School of Life Sciences
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ID Code:14257
Deposited On:06 Jun 2014 08:18

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