Stimulation of insulin secretion by glucose involves increased sugar metabolism by Beta-cells. However, it is yet to be convincingly demonstrated that enhanced metabolism of glucose occurs during the first phase insulin secretory response due to high glucose or during stimulation of insulin secretion by neurotransmitters. Also, the metabolic processes linking increased sugar metabolism and increased Ca2+ levels initiating insulin secretion are unknown. The goal of this research is to test whether increased metabolism of glucose is an even common to both first and second phases of glucose-stimulated insulin secretion and in insulin secretion stimulated by carbachol and isoproterenol, the action of which depend on the presence of glucose. The biochemical mechanisms by which glycolysis is activated and the relationship between alteration of the energy state of Beta-cells and stimulation of insulin secretion will be determined. Beta-cells will be studied using radiation-induced (RIN) insulinomas transplantable in rats and in clonal RIN m5F and HIT cells as models of Beta-cells of normal islets. Usage and futile cycling of glucose will be determined in perfused RIN insulinomas at times selected to coincide with onset of secretion and during first and second phase insulin secretion. The method introduced by Hawkins will be used. Sites in the glycolytic pathway that may be transiently limiting will be determined by the classical approach of determining levels of glycolytic intermediates as they are influenced by secretagogues. Allosteric regulators of glycolysis (e.g., fructose-2,6-P2 and glucose-1,6-P2) and glycolytic enzymes will be determined in RIN insulinomas. Phosphofructokinase and glucose-1,6-P2 synthase, enzymes that may determine the rate of glycolysis during transition states will be purified from RIN insulinomas and from rat islets to identify isozymic forms and kinetic properties likely to determine enzyme activities in intact Beta-cells. The relationship between the cellular energy state and initiation of insulin secretion by glucose or by glyceraldehyde will be determined by measuring metabolite indicators of the energy state and by measuring parameters regulating the energy state. The effects of decreasing creatine-P + creatine or of low 02 tension on the energy state and insulin secretion will be determined. Thus essential data will be generated for developing a model of glycolysis in Beta-cells and it will be determined whether an alteration in the Beta-cell energy state is an essential event in the metabolic sequence initiated by high glucose and stimulating insulin secretion.
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