Metabolic fate of alanine in an insect Manduca sexta: effects of starvation and parasitism

Abstract

The fate of [3- 13C]alanine administered to last instar larvae of an insect Manduca sexta was investigated in vivo by 13C-NMR spectroscopy. Following injection of the isotopically substituted substrate and conversion to [3- 13C]pyruvate 13C was principally incorporated into C 2, C 3 and C 4 of glutamate and glutamate in unparasitized ad libitum-fed larvae, insects starved 48 hr prior to injection and larvae parasitized by the insect parasite Cotesia congregata. Selective labeling at C 2 and C 3 of glutamate/glutamine resulted from carboxylation of [3- 13C]pyruvate to [2,3- 13C]oxaloacetate catalyzed by pyruvate carboxylase, randomization of the label in fumarate, and synthesis of glutamate and glutamine after condensation with acetyl CoA to [2 pro R,3- 13C]citrate. In contrast, enrichment at C 4 of glutamate and glutamine resulted from oxidation [3- 13C]pyruvate to[2- 13C]acetyl CoA catalyzed by pyruvate dehyrdogene followed by condensation with oxaloacetate. The ratio of enrichment (C 2 + C 3): C4 provided a measure of the relative contributions of the pyruvate dehydrogenase and pyruvate carboxylase catalyzed pathways of substrate utilization by the tricarboxylic acid cycle. The mean ratio was 0.6 and 0.7 in control and parasitized larvae, respectively, and 2.4 in starved insects. The latter result demonstrated that substrate utilization by the TCA cycle was markedly altered by starvation. In addition, the rate of labeled alanine metabolism was significantly reduced by starvation. The concentrations of glutamate and glutamine in the blood (hemolymph) were similar in all three groups of insects. No evidence for gluconeogenesis was observed in any group. Starved larvae incorporated label into C 6 of glucose and trehalose but no complementary enrichment at C 1 was observed. This result was consistent with the activity of the non-oxidative phase of the phosphate pathway during which labeled glyceraldehyde-3-phosphate arising from [3- 13C]alanine reacts with sedoheptulose-7-phosphate yielding erythrose-4-phosphate and [6- 13C]fructose-6-phosphate catalyzed by transaldolase. Specifically labeled fructose-6-phosphate then gives rise to glucose and trehalose labeled at C 6. Preliminary analysis of the hemolymph of starved insects indicated the presence of several hexose phosphates labeled at C 6. The hemolymph level of trehalose was significantly reduced in both starved and parasitized insects. Lipogenesis from [3- 13C]alanine was evident in unparasitized control larvae but was absent in parasitized and starved insects. The pattern of labeling in fatty acid was consistent with de novo pathway utilizing [2- 13C]acetyl CoA derived by oxidation of [3- 13C]alanine.