Glucogenesis in an insect, Manduca sexta L., estimated from the 13C isotopomer distribution in trehalose synthesized from [1,3- 13C 2]glycerol

Abstract

Glucogenesis from [3- 13C]alanine and [1,3- 13C 2]glycerol was demonstrated in the insect Manduca sexta by examining the 13C enrichment of trehalose, a non-reducing disaccharide of glucose synthesized in the insect fat body and released into the blood or hemolymph. In insects maintained on a low carbohydrate diet, trehalose synthesized from [3- 13C]alanine was selectively enriched at C1 and C6, and C2 and C5. The 13C-labelling pattern indicated the carboxylation of [3- 13C]pyruvate, formed by transamination of the [3- 13C]alanine followed by randomization of the label at the fumarate step of the tricarboxylic acid cycle and glucose synthesis via the gluconeogenic pathway. 13C enrichment of trehalose was absent in similarly maintained insect larvae administered 3-mercaptopicolinic acid, an inhibitor of hepatic phosphoenolpyruvate carboxykinase. Insects on the low carbohydrate diet also synthesized trehalose from [1,3- 13C 2]glycerol. 13C multiplets were observed in trehalose C3 and C4 demonstrating the synthesis of three 13C enriched glucose isotopomers from the 13C-labelled glycerol. The relative contributions of 13C-labelled glycerol and unlabelled 3 carbon substrates to the synthesis of the 13C enriched trehalose isotopomers were determined from the multiplet structure at C3, and calculation of minimal rates of glucogenesis were based on the 13C enrichment of C4. The C4/C3 13C enrichment ratio in trehalose synthesized from [1,3- 13C 2]glycerol was close to unity, and total glucogenesis was calculated after estimation of the expected contribution of unlabelled trehalose synthesis from 3 carbon substrates by comparison of the ratio of unlabelled and labelled contributions to the 13C enriched trehalose isotopomers with the 13C enrichment of [1,3- 13C 2]glycerol-3-phosphate. The estimated total rates of glucogenesis varied from 0.33 to 2.80 μmol glucose/g fresh weight/h. The blood sugar level of M. sexta was also highly variable. Although the potential importance of glucogenesis from 3 carbon substrates to the maintenance of blood sugar was not established by the present investigation, insects maintained on the low carbohydrate diet had similar blood trehalose levels to those previously reported by others for insects maintained on a natural food.