Glucose, the most potent insulin secretagogue, stimulates insulin secretion by aerobic glycolysis, but other secretagogues stimulate insulin release exclusively by mitochondrial metabolism. It is well known that in the intact pancreatic β-cell, either kind of secretagogue can induce oscillations in metabolism (e.g., glycolysis, ATP/ADP, NAD(P)/NAD(P)H ratios) that occur with a periodicity similar to oscillations in membrane electrical potential and insulin secretion. In this study, pancreatic islet cytosol or mitochondrial fractions were incubated in the presence of physiological concentrations of substrates. Repeated additions of physiological effectors caused oscillations in the activities of the three enzymes studied. Succinate dehydrogenase activity in islet mitochondrial extracts was made to oscillate by adding oxaloacetate (5 μmol/l) to inhibit the enzyme. The enzyme was reactivated by adding acetyl-CoA (3 μmol/l), which combines with oxaloacetate in the citrate synthase reaction and lowers the concentration of oxaloacetate, thus beginning another oscillation. Pyruvate kinase activity was made to oscillate by adding fructose bisphosphate (10 μmol/l). Fructose bisphosphate was degraded to triose phosphates fairly rapidly, and, as it was degraded, there was a parallel decrease in pyruvate kinase activity. The enzyme was reactivated and made to oscillate with subsequent additions of fructose bisphosphate. The mitochondrial glycerol phosphate dehydrogenase was made to oscillate by adding EGTA to chelate calcium, which activates the enzyme. When the concentration of free calcium was raised to >0.1 μmol/l by adding more calcium, the activity of the enzyme increased. Repeated additions of chelator and calcium caused the enzyme activity to oscillate. The results with these three enzymes and physiological concentrations of naturally occurring effectors raise the possibility that the activities of not only these enzymes but of numerous enzymes oscillate in vivo in response to levels of allosteric effectors and substrates. If this is the case, pacemaker activity may result from complex effects distributed across multiple regulatory sites in both the cytosol and mitochondria, rather than from a single enzyme acting as a primary pacemaker.

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