The overall objective of this study is to elucidate the mechanism of action of glucagon and adrenergic hormones with respect to their ability to stimulate gluconeo-genesis and ureagenesis in liver. Measurements of glutamate and aspartate concentrations in isolated hepatocytes and perfused livers combined with estimates of carbon flux suggest that the kinetic parameters of the glutamate-aspartate mitochondrial transporter may be altered by glucagon and Alpha-adrenergic agonists. These hormones stimulate: 1 ) gluconeogenesis from lactate, 2) ureagenesis, and 3) reducing equivalent transport from cytosol to mitochondria. All three processes in the liver involve transport of aspartate from the mitochondria into the cytosol. Thus, attention will be focussed on this aspect of mitochondrial metabolism. We will also evaluate the possibility that the Alpha-glycerophosphate shuttle is stimulated by the hormones secondary to an increase in cytosolic free Ca++. Free Ca++ will be monitored in the cell cytosol with """"""""quin 2"""""""", a Ca++ indicator fluorescent dye which can be trapped inside liver cells. In order to determine whether the effects of Ca++ are mediated by Ca++ dependent protein kinases, we will compare the sensitivity of mitochondrial metabolism in intact cells to the monitored levels of free cytosolic Ca++ in the presence and absence of trifluoperazine, a calmodulin antagonist. Cellular metabolites of the malate-aspartate shuttle and the Alpha-glycerophosphate shuttle will be measured in the presence and absence of hormones with varied levels of substrates of gluconeogenesis (i.e. lactate), ureagenesis (i.e. NH3), and reducing equivalent transport (i.e. ethyl alcohol). The dependence of flux through the glutamate-aspartate transporter and through the alpha-glycerophosphate shuttle on metabolites will be evaluated over a wide range of fluxes in order to identify the target of the hormone action. Similar experiments will be carried out in perfused hearts. The components of the malate-aspartate shuttle will be monitored in the presence and absence of norepinephrine while varying perfusate lactate concentration and heart work. Further experiments will be designed to identify more precisely the mechanism by which the hormones stimulate target proteins and may involve isolation of specific enzymes or carrier proteins.
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