A realistic model of carbon flow in hepatocytes incubated with a substrate mixture containing glucose, ribose, fructose, alanine, and acetate has been developed and used to obtain a quantitative description of metabolite flows in the presence and absence of glucagon. To increase our understanding of the structural organization of intermediary metabolism in hepatocytes, we will carry out paired experiments (in the presence and absence of vasopressin) in which aspartate and glutamate will be added to the above substrate mixture. These experiments will not only provide further information on the handling of gluconeogenic and ureagenic amino acids, but will allow quantitative comparison of the effects of vasopressin with those of glucagon on the entire pattern of metabolite flow along the major pathways of intermediary metabolism. We also intend to incubate hepatocytes isolated from fasted rats with a substrate mixture containing erucoate, oleate, dodecanoate, hexanoate, propioante, acetate, lactate, and pyruvate, with one of these substrates labeled with 14C in any given flask. Approximately 80 independent measurements of label flow into CO2, glucose, and the 1 and 2, 3, 4 positions of acetoacetate will be made, a number sufficiently in excess of the number of independent flux parameters to be computed to yield a stringent test of a proposed model of fatty acid metabolism and thus a quantitative estimate of Beta-oxidation in the mitochondrial and peroxisomal pathways and of Omega-oxidation for each of the fatty acids in the mixture. These studies will be extended by: 1) using hepatocytes isolated from rats treated with bezafibrate, a hypolipidemic drug known to increase peroxisomal Beta-oxidation enzymes; and 2) using a substrate mixture containing glucose, ribose, fructose, alanine, acetate, glutamate, oleic, and erucic acids to obtain a quantitative description of metabolite flow along the pathways of the combined models developed above.
