Quantitative analysis of intermediary metabolism in hepatocytes incubated in the presence and absence of glucagon with a substrate mixture containing glucose, ribose, fructose, alanine and acetate

Abstract

Hepatocytes were isolated from the livers of fed rats and incubated, in the presence and absence of 100 nM-glucagon, with a substrate mixture containing glucose (10 mM), fructose (4 mM), alanine (3.5 mM), acetate (1.25 mM) and ribose (1 mM). In any given incubation 1 substrate was labeled with 14C. Incorporation of 14C into glucose, glycogen, CO2, lactate, alanine, glutamate, lipid glycerol and fatty acids was measured after 20 and 40 min of incubation under quasi-steady-state conditions. These data and the measured O2 consumption were analyzed with the aid of a structural metabolic model incorporating all reactions of the glycolytic, gluconeogenic and pentose phosphate pathways, and associated mitochondrial and cytosolic reactions. A considerable excess of experimental measurements over independent flux parameters and a number of independent measurements of changes in metabolite concentrations allowed for a stringent test of the model. A satisfactory fit to the data was obtained for each condition. Significant findings included: control cells were glycogenic and glucagon-treated cells glycogenolytic during the 2nd interval; an ordered (last in, first out) model of glycogen degradation was required in order to fit the experimental data; the pentose shunt contributed .apprx. 15% of the carbon for gluconeogenesis in both control and glucagon-treated cells; net flux through the lower Embden-Meyerhof pathway was in the glycolytic direction except during the 20-40 min interval in glucagon-treated cells; the increased gluconeogenesis in response to glucagon was correlated with a drcreased pyruvate kinase flux and lactate output; fluxes through pyruvate kinase, pyruvate carboxylase and phosphoenolpyruvate carboxykinase were not coordinately controlled; Krebs cycle activity did not change with glucagon treatment; flux through the malic enzyme was towards pyruvate formation except for control cells during interval II and futile cycling at each of the 5 substrate cycles examined (including a previously undescribed cycle at acetate/acetyl-CoA) consumed .apprx. 26% of cellular ATP production in control hepatocytes and 21% in glucagon-treated cells.