Isotopolog perturbation techniques for metabolic networks:: Metabolic recycling of nutritional glucose in Drosophila melanogaster

Drosophila melanogaster strain Oregon-R* was grown on standard medium supplemented with [U-C-13(6)]glucose. One to two days after hatching, flies were extracted with water. Glucose was isolated chromatographically from the extract and was analyzed by C-13 NMR spectroscopy. All C-13 signals of the isolated glucose were multiplets arising by (CC)-C-13-C-13 coupling. Based on a comprehensive analysis of the coupling constants and heavy isotope shifts in glucose, the integrals of individual C-13 signal patterns afforded the concentrations of certain groups of C-13 isotopologs. These data were deconvoluted by a genetic algorithm affording the abundances of all single-labeled and of 15 multiply labeled isotopologs. Among the latter group, seven isotopologs were found at concentrations >0.1 mol % with [1,2-C-13(2)]glucose as the most prominent species. The Multiply C-13-labeled glucose isotopologs are caused by metabolic remodeling of the proffered glucose via a complex network of catabolic and anabolic processes involving glycolysis and/or passage through the pentose phosphate, the Cori cycle and/or the citrate cycle. The perturbation method described can be adapted to a wide variety of experimental systems and isotope-labeled precursors.