An in vivo13C NMR study on the effect of dietary sugar on pyruvate cycling during glucogenesis from (2- 13C)pyruvate in Manduca sexta L.

Abstract

Pyruvate cycling from (2- 13C)pyruvate was detected in vivo in intact 5th instar Manduca sexta larvae by application of NMR spectroscopy. Cycling was evident from the enrichment of C3 in alanine following transamination of recycled pyruvate in larvae maintained on casein-based diets with or without sucrose. This metabolism is assumed to principally occur in the fat body. Analysis of 13C enriched metabolites released into the hemolymph indicated that isotopic dilution of recycled pyruvate was sufficiently great that further metabolism of the recycled metabolite did not occur to any significant extent under these dietary conditions. The C3/C2 13C-enrichment ratio of alanine, therefore, accurately reflected the relative degree of pyruvate cycling and indicated that the rate of cycling was approximately three-fold lower in larvae maintained on diets lacking sucrose. Moreover, based on the distribution of 13C in trehalose, these larvae displayed significantly greater rates of gluconeogenesis. Enrichment of C1, C2, C5 and C6 were principally due to carboxylation of the isotopically substituted substrate catalyzed by pyruvate carboxylase and, therefore, reflected net carbohydrate synthesis. Trehalose C3 and C4 enrichments were principally due to pyruvate dehydrogenase-catalyzed decarboxylation and reflected incorporation of label following metabolism through the TCA cycle. Pentose cycling following glucogenesis significantly affected the 13C distribution in trehalose in insects on both diets, and the relative intensity of trehalose C6 was, therefore, used for comparing the rates of gluconeogenesis and pyruvate cycling. Based on the 13C enrichment of trehalose C6 relative to C3 of alanine the mean rate of pyruvate cycling relative to the rate of gluconeogenesis was approximately 60% in larvae on the diet lacking sucrose, while the rate of pyruvate cycling in larvae maintained on the diet supplemented with sucrose was greater than the gluconeogenic flux. The results were consistent with the conclusion that pyruvate kinase likely plays an important role in regulating gluconeogenesis in M. sexta larvae.