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

Abstract

Drosophila melanogaster strain Oregon-R* was grown on standard medium supplemented with [U-13 C6]glucose. One to two days after hatching, flies were extracted with water. Glucose was isolated chromatographically from the extract and was analyzed by 13 C NMR spectroscopy. All 13 C signals of the isolated glucose were multiplets arising by 13 C 13 C coupling. Based on a comprehensive analysis of the coupling constants and heavy isotope shifts in glucose, the integrals of individual 13 C signal patterns afforded the concentrations of certain groups of 13 C 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 [1,2-13 C2]glucose as the most prominent species. The multiply 13 Clabeled glucose isotopologs are caused by metabolic remodeling of the proffered glucose via a complex network of catabolic and anabolic processes involving glycolysis and͞or passage through the pentose phosphate, the Cori cycle and͞or the citrate cycle. The perturbation method described can be adapted to a wide variety of experimental systems and isotope-labeled precursors.stable isotope ͉ nuclear magnetic resonance ͉ genetic algorithm ͉ insect ͉ X group

Metabolic flux analysis in complex isotopolog space. Recycling of glucose in tobacco plants

“…3; isotopolog sets of this type are subsequently designated X groups. On the basis of known 13 C coupling constants, specific spectral signatures can be assigned to the sum of abundances of the isotopologs which are members of a specific X group (for details, see Eisenreich et al, 2004). The assignment of specific signatures in the spectrum of glucose to the cognate X groups is summarised in Tables 1-3 and Fig.…”

“…For example, the isotopolog species {100000} and {000001} can arise from the citric acid cycle (cf. Eisenreich et al, 2004) or from fully or partially labelled glucoses. The formal analysis of the observed 13 C isotopologs including the data simulation using the rule-based 4F software shows that extensive cycling of labelled glucose molecules takes place in the plant system.…”

“…The abundances of individual isotopologs were then obtained by computational deconvolution of X group abundance using a genetic algorithm (Holland, 1975;Rechenberg, 1973) implemented by the GeneHunter library (WardSystems Inc., Frederick, MD). A detailed description of the methods used is given in Eisenreich et al (2004).…”

“…Errors of the NMR experiment are propagated via the glucose isotopolog abundances to the fraction of the executed rules in two steps: (I) For each X group an estimated experimental error of 10% is propagated via the final X group state matrix (described in Eisenreich et al, 2004) to the single glucose isotopologs. (II) Metabolic rules are related to isotopologs by metabolic transfer rules (Table 4) and the error deviation of each isotopolog can therefore be propagated to the relative fraction of a specific circular pathway in the simulation.…”

13 C isotopologue perturbation studies of Listeria monocytogenes carbon metabolism and its modulation by the virulence regulator PrfA

“…In cells and organisms, such a perturbation will rapidly spread through the entire metabolic network. The quantitative analysis of that relaxation process can afford qualitative as well as quantitative information on metabolic processes in the experimental system under study (1)(2)(3)(4)(5)(6)(7).…”