(130b) Elucidation of Perturbed Gluconeogenesis Flux in Fao Rat Hepatoma Cell Line Using Multiple Isotopic Tracers

Type II diabetes is a growing concern in the world. Hyperglycemia caused by hepatic glucose production is one of the symptoms caused by abnormal regulation of liver metabolism. Here, we investigated fluxes of gluconeogenesis (GNG) in a liver cell line using multiple stable isotope tracers and gas chromatography/mass spectrometry and elucidated the effects of representative regulators (cAMP, dexamethasone, insulin) on GNG flux, and the effect of specific inhibitors of dicarboxylate, citrate and 2-oxoglutare carriers on central metabolism. To stimulate glucose production, overnight-starved Fao rat hepatoma cells were cultured in glucose-free medium in the presence gluconeogenic precursors (glycerol, glutamine, lactate and pyruvate) and isotopic tracers ([U-13C] glycerol, [U-13C] glutamine and [U-13C] lactate). We performed metabolic flux analysis with mass isotopomer distributions of intracellular metabolites and Metran software based on the elementary metabolite unit framework. Using labeling of extracellular glucose secreted from cells and [U-13C] glycerol, we showed that GNG flux was enhanced by 8-bromo cAMP, dibutyryladenosine cAMP and dexamethasone, while insulin significantly suppressed hepatic GNG. Interestingly, the two cAMP analogues showed differential stimulation of GNG pathway. Also, we discovered that the hepatoma cells utilize non-oxidative pentose phosphate pathway in GNG. Secondly, we estimated metabolic fluxes of pyruvate and citric acid cycling with MID of intracellular metabolites and [U-13C] glutamine and [U-13C] lactate tracers. We quantified the effects on malic enzyme, phosphoenolpyruvate carboxylase, pyruvate dehydrogenase and isocitrate dehydrogenase by blocking specific mitochondrial metabolite transporters with inhibitors. In conclusion, in this study we investigated perturbations of GNG pathway to understand pathological metabolic fluxes observed in type II diabetes. Our results provide valuable information regarding the metabolic disease and could be used as a basis for drug screening.