oligomycins and alpha-cyano-4-hydroxycinnamate

The different roles of glycolytic and mitochondrial pathways in glucose-induced metabolic activation and insulin secretion were studied in islets of Langerhans. Single islets were perifused with 3 mM glucose together with agents affecting the production or consumption of ATP. Subsequently, glucose was raised to 11 mM and the effects of the agents on metabolic and secretory responses were evaluated. Metabolism was monitored continuously with an oxygen-sensitive microelectrode inserted into the islet. Insulin secretion was determined by assaying insulin in perifusate with ELISA. Inhibitors of mitochondrial ATP production reduced the metabolic and secretory response to glucose. When glycolytic ATP production was reduced, initial but not sustained glucose-stimulated insulin release was observed. Inhibition of mitochondrial pyruvate transport reduced the glucose-induced decline in pO(2). Although mitochondrial metabolism was eventually similar to normal, insulin release was only 20% of normal. Increased energy expenditure also changed the kinetics of the glucose-induced decline in pO(2) and decreased the insulin release by 50%. In conclusion, glucose-induced enhancement of insulin release was only seen when the rise of the sugar concentration triggered a rapid and sustained increase of mitochondrial metabolism. This activation of mitochondrial metabolism required a good metabolic state prior to the glucose challenge.

Topics: Animals; Arsenates; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Respiration; Cells, Cultured; Chelating Agents; Coumaric Acids; Egtazic Acid; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Glucose; Glycolysis; Gramicidin; Insulin; Insulin Secretion; Islets of Langerhans; Male; Mice; Microelectrodes; Mitochondria; Oligomycins; Ouabain; Oxidative Phosphorylation; Oxygen; Uncoupling Agents

A saturable and accumulative transport system for pyruvate has been detected in Phycomyces blakesleeanus NRRL 1555(-) mycelium. It was strongly inhibited by alpha-cyano-4-hydroxycinnamate. l-Lactate and acetate were competitive inhibitors of pyruvate transport. The initial pyruvate uptake velocity and accumulation ratio was dependent on the external pH. The Vmax of transport greatly decreased with increasing pH, whereas the affinity of the carrier for pyruvate was not affected. The pyruvate transport system mediated its homologous exchange, which was essentially pH independent, and efflux, which increased with increasing external pH. The uptake of pyruvate was energy dependent and was strongly inhibited by inhibitors of oxidative phosphorylation and of the formation of proton gradients. Glucose counteracted the inhibitory effect of the pyruvate transport produced by inhibitors of mitochondrial ATP synthesis. Our results are consistent with a pyruvate/proton cotransport in P. blakesleeanus probably driven by an electrochemical gradient of H+ generated by a plasma membrane H+-ATPase.

Topics: Acetates; Anti-Bacterial Agents; Antifungal Agents; Antimycin A; Biological Transport; Coumaric Acids; Glucose; Hydrogen-Ion Concentration; Ionophores; Kinetics; Lactates; Oligomycins; Phycomyces; Pyruvates; Time Factors