A testable """"""""metabolic hypothesis"""""""" for the postprandial induction of insulin secretion is that B cell metabolism of nutrient fuels, especially glucose, -(1)-> alteration of intracellular intermediates -(2)-> closure of metabolically regulated K+ channels _(3)-> cell depolarization -(4)-> action potential initiation and the opening of voltage dependent Ca2+ channels - (5)-> Ca2+ entry -(6)-> insulin granule exocytosis. Over the past five years important advances have been made in identifying """"""""molecular players"""""""" in this hypothesis including an ATP1 sensitive K+ channel, whose closure depolarizes the cell; voltage dependent Ca2+, and K+ channels, which probably contribute to the action potential; and increases in cytosolic Ca2+ under conditions where B cells depolarize. We propose to further verify this hypothesis by investigating several dynamic links (4- 6) which connect cell depolarization to granule exocytosis by simultaneously applying several new """"""""integrative"""""""" techniques to single normal adult human islet B cells harvested from cadaver donors. These techniques are """"""""perforated patch"""""""" current or voltage clamp recording; microspectrofluometry, using Ca2+- sensitive dyes; and membrane capacitance measurements by """"""""phase detection"""""""" techniques. First we shall examine the ionic channel currents underlying patterns of metabolite induced electrical activity, especially Ca2+ channels. Second, we shall correlate changes in cytosolic Ca2+ with patterns of electrical activity induced by nutrient fuel secretogogues. Third, we shall attempt to correlate patterns of electrical activity and Ca2+ entry with patterns of insulin granule exocytosis as reflected by changes in membrane capacitance. The last approach will require us to establish optimal conditions for insulin secretion using the """"""""reverse hemolytic plaque assay"""""""" and to verify that capacitance changes seen reflect real-time incorporation of insulin granule membrane into the cell surface. The experiments should further our understanding of stimulus-secretion coupling in the normal human B cell and serve as a basis for studies on the pathophysiology of diminished insulin secretion in non-insulin dependent diabetes mellitus.
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