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.