Many insects display non-homeostatic regulation over blood sugar level. The concentration of trehalose varies dramatically depending on physiological and nutritional state. In the absence of dietary carbohydrate, blood trehalose in larvae of the lepidopteran insect Manduca sexta is maintained by gluconeogenesis and is dependent on dietary protein consumption. In the present study, the effect of dietary fat on the glucogenic response of insects to increased dietary protein was examined by NMR analysis of (2-13C)pyruvate metabolism. Last instar larvae were maintained on a carbohydrate-free chemically defined artificial diet having variable levels of casein with and without corn oil. Gluconeogenic flux, the ratio of the rate of gluconeogenesis to the rate of glycolysis, was estimated from the 13C distribution in trehalose arising by gluconeogenesis and the 13C enrichment of alanine due to pyruvate cycling. Insects grew well on carbohydrate-free diets and growth increased with increasing dietary protein level. At all dietary protein levels, larvae grew better on diets with fat. Without dietary fat, larvae were glucogenic but displayed low blood trehalose concentrations, <30 mM, regardless of protein consumption. When fat was included in the diet, however, gluconeogenic flux and blood trehalose level increased sharply in response to increased dietary protein level, with trehalose concentrations >50 mM at higher levels of protein consumption. When offered a choice of a high carbohydrate and a high protein diet, larvae maintained on diets with fat displayed a food preference related to blood sugar level. Those with low blood sugar fed on carbohydrate, while those with high blood sugar preferred protein. Trehalose synthesized from (2-13C)pyruvate exhibited asymmetry in the 13C distribution in individual glucose molecules, indicating a disequilibrium at the triose phosphate isomerase-catalyzed step of the gluconeogenic pathway. In trehalose from larvae on diets with fat, the asymmetric 13C distribution was higher than in trehalose from insects on diets lacking fat. This may partially result from isotopic disequilibrium when unenriched glycerol is metabolized to dihydroxyacetone phosphate following fat hydrolysis. The asymmetry in 13C distribution, however, also occurred in insects on diets without fat and decreased with increased gluconeogenic flux suggesting that true disequilibrium between the triose phosphates is the principal reason for the asymmetry.