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

Milne, Helen M. and Burns, Chris J. and Squires, Paul E. and Evans, Nicholas D. and Pickup, John and 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
ID Code:14257
Deposited On:06 Jun 2014 08:18

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