This proposal addresses the hypothesis that oscillatory behavior of glycolysis may underlie glucose-stimulated insulin secretion in pancreatic islets. According to the proposed model, the increased glycolytic flux in the islets due to increased glucose concentration initiates oscillations in glycolysis and the ATP/ADP ratio. The oscillations in the ATP/ADP ratio in turn cause oscillations in the activity of ATP-sensitive K-channels, the membrane potential, and intracellular free Ca2+ that lead to insulin release. The model could account for the observed pulsatile release of insulin in vivo and in isolated islets, and the loss of this pulsatility in Type II diabetes. It could also explain why glucose stimulation of insulin secretion requires metabolism of the glucose, yet no specific-metabolite has been pinpointed as the effector--because the oscillatory process itself provides the coupling. The model is supported by recent observations of (a) oscillations in intracellular free Ca2+ in single rat islets, (b) NADH oscillations (indicative of glycolytic oscillations) in islet extracts, and (c) induction by an oscillating ATP/ADP ratio of oscillations in free Ca2+ of a suspension of permeabilized clonal pancreatic B-cells and in the activity of ATP-sensitive K-channels. The proposed studies will test and further refine this model. Cultured rat islets will be examined using fluorescence microscopy. Intracellular free Ca2+ and pH will be monitored with fluorescent probes, and NADH by endogenous fluorescence. At different phases of the Ca2+ oscillation cycle, islets will be assayed for glycolytic intermediates and adenine nucleotides. The dependence of the amplitudes and/or frequencies of these oscillating parameters on the glucose concentration will be examined. The mechanism of the regulation of the metabolic oscillations will be determined from detailed analysis of changes in metabolite profiles in islet extracts, together with studies of the kinetic properties of islet phosphofructokinase and pyruvate kinase. The effects of factors shown to modulate glycolytic oscillations in muscle extracts (e.g., fructose-2,6-P2, glucose-1,6-P2, citrate) will be examined. Finally, studies. will be extended to the effects of non-glucose secretagogues, such an glyceraldehyde, carbamylcholine and sulfonylurea, on glycolytic oscillations in islets.
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