METABOLIC-FATE OF ALANINE IN AN INSECT MANDUCA-SEXTA - EFFECTS OF STARVATION AND PARASITISM

The fate of [3-C-13]alanine administered to last instar larvae of an i nsect Manduca sexta was investigated in vivo by C-13-NMR spectroscopy. Following injection of the isotopically substituted substrate and con version to [3-C-Cl]pyruvate C-13 was principally incorporated into C2, C3 and C4 of glutamate and glutamine in unparasitized ad libitum-fed larvae, insects starved 48 hr prior to injection and larvae parasitize d by the insect parasite Cotesia congregata. Selective labeling at C2 and C3 of glutamate/glutamine resulted from carboxylation of [3-C-13]p yruvate to [2,3-C-13]oxaloacetate catalyzed by pyruvate carboxylase, r andomization of the label in fumarate, and synthesis of glutamate and glutamine after condensation with acetyl CoA to [2 pro R,3-C-13]citrat e. In contrast, enrichment at C4 of glutamate and glutamine resulted f rom oxidation [3-C-13]pyruvate to [2-C-13]acetyl CoA catalyzed by pyru vate dehyrdogenase followed by condensation with oxaloacetate. The rat io of enrichment (C2 + C3): C4 provided a measure of the relative cont ributions of the pyruvate dehydrogenase and pyruvate carboxylase catal yzed pathways of substrate utilization by the tricarboxylic acid cycle . The mean ratio was 0.6 and 0.7 in control and parasitized larvae, re spectively, and 2.4 in starved insects. The latter result demonstrated that substrate utilization by the TA cycle was markedly altered by st arvation. In addition, the rate of labeled alanine metabolism was sign ificantly reduced by starvation. The concentrations of glutamate and g lutamine in the blood (hemolymph) were similar in all three groups of insects. No evidence for gluconeogenesis was observed in any group. St arved larvae incorporated label into C6 of glucose and trehalose but n o complementary enrichment at C1 was observed. This result was consist ent with the activity of the non-oxidative phase of the pentose phosph ate pathway during which labeled glyceraldehyde-3-phosphate arising fr om [3-C-13]alanine reacts with sedoheptulose-7-phosphate yielding eryt hrose-4-phosphate and [6-C-13]fructose-6-phosphate catalyzed by transa ldolase. Specifically labeled fructose-6-phosphate then gives rise to glucose and trehalose labeled at C6. Preliminary analysis of the hemol ymph of starved insects indicated the presence of several hexose phosp hates labeled at C6. The hemolymph level of trehalose was significantl y reduced in both starved and parasitized insects. Lipogenesis from [3 -C-13]alanine was evident in unparasitized control larvae but was abse nt in parasitized and starved insects. The pattern of labeling in fatt y acid was consistent with de novo pathway utilizing [2-C-13]acetyl Co A derived by oxidation of[3-C-13]alanine.