Fuel-induced amplification of insulin secretion in mouse pancreatic islets exposed to a high sulfonylurea concentration: Role of the NADPH/NADP+ ratio

Abstract

Aims/hypothesis: The aim of this study was to examine whether the cytosolic NADPH/NADP+ ratio of beta cells serves as an amplifying signal in fuel-induced insulin secretion and whether such a function is mediated by cytosolic α-ketoglutarate. Methods: Pancreatic islets and islet cells were isolated from albino mice by collagenase digestion. Insulin secretion of incubated or perifused islets was measured by ELISA. The NADPH and NADP + content of incubated islets was determined by enzymatic cycling. The cytosolic Ca2+ concentration ([Ca2+]c) in islets was measured by microfluorimetry and the activity of ATP-sensitive K + channels in islet cells by patch-clamping. Results: Both 30 mmol/l glucose and 10 mmol/l α-ketoisocaproate stimulated insulin secretion and elevated the NADPH/NADP+ ratio of islets preincubated in the absence of fuel. The increase in the NADPH/NADP+ ratio was abolished in the presence of 2.7 μmol/l glipizide (closing all ATP-sensitive K+ channels). However, α-ketoisocaproate, but not glucose, still stimulated insulin secretion. That glipizide did not inhibit α-ketoisocaproate- induced insulin secretion was not the result of elevated [Ca2+] c, as glucose caused a more marked [Ca2+]c increase. Insulin release triggered by glipizide alone was moderately amplified by dimethyl α-ketoglutarate (which is cleaved to produce cytosolic α-ketoglutarate), but there was no indication of a signal function of cytosolic α-ketoglutarate. Conclusions/interpretation: The results strongly suggest that the NADPH/NADP+ ratio in the beta cell cytosol does not serve as an amplifying signal in fuel-induced insulin release. The study supports the view that amplification results from the intramitochondrial production of citrate by citrate synthase and from the associated export of citrate into the cytosol. © 2007 Springer-Verlag.