The mechanisms by which hormones such as glucagon regulate hepatic gluconeogenesis are fairly well established. In addition to evaluating cellular cAMP levels, glucagon has recently been demonstrated to increase cytosolic free-calcium concentrations. In the perfused liver, our recent studies have demonstrated that glucagon's ability to alter a variety of metabolic processes is sensitive to the cellular oxidation-reduction state. Similarly, preliminary data presented in this application demonstrates that in perfused rat livers and perfused rat hepatocytes, the ability of atriopeptins to elevate cGMP levels and stimulate gluconeogenesis is also regulated by the cellular oxidation- reduction state. These findings and the reported presence of atriopeptin receptors in the liver coupled with the demonstration that circulating atriopeptin concentrations are elevated in diabetes and hyperthyroidism (conditions associated with elevated gluconeogenic rats), and decreased under conditions when gluconeogenic rates are low (e.g. hypothyroidism) suggest that atriopeptin may participate in the regulation of hepatic gluconeogenesis. Additionally, the finding that atriopeptins stimulate gluconeogenesis in liver, is the first biological process which can attach significance to the presence of atriopeptin in liver, is the first biological process which can attach significance to the presence of atriopeptin receptors and particulate guanylate cyclase in this tissue. Therefore, it is the overall objective of this proposal to characterize and elucidate the mechanism(s) involved in regulation of energy metabolism by atriopeptins in perfused rat livers and perfused hepatocytes. Among a variety of reasons, studies in the perfused hepatocytes. Among a variety of reasons, studies in the perfused hepatocyte system are proposed to rule out the possibility that atriopeptin- mediated effects in the perfused rat liver are due to alterations in vascular contractile tone and/or result from effects on cell type(s) other than parenchymal cells. Initially, studies will be performed to determine the effect of gluconeogenesis. These and other studies with various pharmacological agents which modulate cellular cGMP content along with measurements of pyruvate kinase activity and the cellular content of fructose 1,6-and 2,6- bisphosphate will indicate the role of cGMP in atriopeptin-elicited effects, and identify at the enzyme level, the mechanism(s) responsible for atriopeptin-mediated stimulation of hepatic gluconeogenesis. Since preliminary data also demonstrates that atriopeptin alters hepatic calcium homeostasis, studies are proposed to (a) investigate the effect of atriopeptins on cytosolic free-calcium concentrations in hepatocytes, (b) elucidate the mechanism(s) by which cellular calcium homeostasis is altered, and (c) determine the role that calcium may play in atriopeptin- stimulated gluconeogenesis. Furthermore, because agonists which alter calcium homeostasis and increase cytosolic free-calcium, also stimulate the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction, studies are proposed to investigate the effect of atriopeptins on this process in perfused livers and perfused hepatocytes. Finally, since several hormones alter hepatic calcium metabolism, via generation of inositol 1,4,5- triphosphate as a second messenger, studies are proposed to investigate the effect of atriopeptins on phosphatidy-linositol metabolism in hepatocyte and also to elucidate the role of protein kinase C activation in atriopeptin-mediated alterations of hepatic metabolism.
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