Uncoupling of nutrient metabolism from insulin secretion by overexpression of cytosolic phospholipase A(2).
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.
Milne
Helen M
HM
Division of Reproductive Health, Endocrinology and Development, King's College London, London SE1 1UL, UK.
Burns
Chris J
CJ
Squires
Paul E
PE
Evans
Nicholas D
ND
Pickup
John
J
Jones
Peter M
PM
Persaud
Shanta J
SJ
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007473:Ion Channels; D007515:Islets of Langerhans; D026901:Membrane Transport Proteins / Q000235:genetics; D024101:Mitochondrial Proteins / Q000235:genetics; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014044:Tolbutamide / Q000494:pharmacology; D014162:Transfection; D000071246:Uncoupling Protein 2
D007328:Insulin; D007473:Ion Channels; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000071246:Uncoupling Protein 2; D009243:NAD; D009249:NADP; D011189:Potassium Chloride; D014044:Tolbutamide; D010741:Phospholipases A; D005947:Glucose; D002118:Calcium