Fuel-induced amplification of insulin secretion in mouse pancreatic islets exposed to a high sulfonylurea concentration: role of the NADPH/NADP+ ratio.
OBJECTIVE
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 alpha-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 alpha-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 micromol/l glipizide (closing all ATP-sensitive K+ channels). However, alpha-ketoisocaproate, but not glucose, still stimulated insulin secretion. That glipizide did not inhibit alpha-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 alpha-ketoglutarate (which is cleaved to produce cytosolic alpha-ketoglutarate), but there was no indication of a signal function of cytosolic alpha-ketoglutarate.


CONCLUSIONS
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.
Panten
U
U
Institute of Pharmacology and Toxicology, Technical University of Braunschweig, Mendelssohnstrasse 1, 38106, Brunswick, Germany. u.panten@tu-bs.de
Rustenbeck
I
I
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005913:Glipizide / Q000494:pharmacology; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects*; D007656:Ketoglutaric Acids / Q000494:pharmacology; D051379:Mice; D009249:NADP / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D013453:Sulfonylurea Compounds / Q000494:pharmacology*
D007004:Hypoglycemic Agents; D007328:Insulin; D007656:Ketoglutaric Acids; D013453:Sulfonylurea Compounds; D009249:NADP; D000255:Adenosine Triphosphate; D002118:Calcium; D005913:Glipizide