Limitations of the mass isotopomer distribution analysis of glucose to study gluconeogenesis - Heterogeneity of glucose labeling in incubated hepatocytes

We previously reported (Previs, S, F,, Fernandez, C, A, Yang, D,, Soloviev, M, V., David, F,, and Brunengraber, H. (1995) J, Biol. Chem. 270, 19806-19815) that glucose made in isolated livers from starved rats perfused with physiological concentrations of lactate, pyruvate, and either [2-C-13]- or [U-C-13(3)]glycerol had a mass isotopomer distribution incompatible with glucose being made from a homogeneously labeled pool of triose phosphates, Similar data were obtained in live rats infused with [U-C-13(3)]glycerol, We ascribed the labeling heterogeneity to major decreases in glycerol concentration and enrichment across the liver, We concluded that [C-13]glycerol is unsuitable for tracing the contribution of gluconeogenesis to total glucose production. We now report isotopic heterogeneity of gluconeogenesis in hepatocytes, even when all cells are in contact with identical concentrations and enrichments of gluconeogenic substrates, Total rat hepatocytes were incubated with concentrations of glycerol, lactate, and pyruvate that were kept constant by substrate infusions. To modulate competition between substrates, the (glycerol)/(lactate + pyruvate) infusion ratio ranged from 0.23 to 3.60, Metabolic and isotopic steady states were achieved in all cases. The apparent contribution of gluconeogenesis to glucose production (f) was calculated from the mass isotopomer distribution of glucose. When all substrates were C-13-labeled, f was 97%, as expected in glycogen-deprived hepatocytes, As the infusion ratio ([C-13]glycerol)/(lactate C pyruvate) increased, f increased from 73% to 94%, In contrast, as the infusion ratio (glycerol)/([C-13]lactate + [C-13]pyruvate) increased, f decreased from 93% to 76%, In all cases, f increased with the rate of supply of the substrate that was labeled. Variations in f show that the C-13 labeling of triose phosphates was not equal in all hepatocytes, even when exposed to the same substrate concentrations and enrichments, We also showed that zonation of glycerol kinase activity is minor in rat liver. We conclude that zonation of other processes than glycerol phosphorylation contributes to the heterogeneity of triose phosphate labeling from glycerol in rat liver.