MOLECULAR CONTROL OF GLUCOSE METABOLISM

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. When carbohydrate intake exceeds short-term requirements for energy, it is stored as fat. The liver is the principle organ responsible for the conversion of excess carbohydrate to fat. Ingesting a high carbohydrate diet induces gene transcription of over a dozen enzymes in liver that are involved in glycolysis and fat synthesis. Insulin, secreted by the pancreas in response to carbohydrate promotes lipogenesis by activating lipogenic enzyme expression. However, carbohydrates also stimulate transcription of the same genes independent of insulin. The mechanism by which metabolizable carbohydrate generates a signal to induce the transcription of lipogenic enzyme genes became clearer with discovery of the transcription factor termed carbohydrate response element binding protein (ChREBP). During hypoglycemia, such as might be found during starvation, ChREBP is phosphorylated and remains in the cytosol. However, when blood glucose rises, glycolysis and pentose shunt intermediates including xylulose5-P also rise. Elevated Xylulose5-P levels activate a specific protein phosphatase causing dephosphorylation of ChREBP to its active form which translocates to nucleus. Once in the nucleus ChREBP binds to carbohydrate response elements localized in the promoters of lipogenic and glycolytic enzymes as well as pyruvate kinase (LPK) to activate their transcription. Mice with targeted disruptions of the ChREBP gene (ChREBP-/-) demonstrate significantly reduced fatty acid synthesis and decreased adiposity compared to wild type mice. For instance, liver triglyceride level is reduced by 50% in ChREBP-/- mice fed a high carbohydrate diet compared to wild type mice. Previous metabolite and enzyme activity analysis revealed that the pyruvate level in ChREBP-/- mice is also significantly lower on standard lab chow compared to the WT mice because of the decreased hepatic glycolysis in response to an 80% reduction in LPK activity. Interestingly, on a high carbohydrate diet pyruvate concentration in the mutant mice is the same as that in WT animals, but the source of the pyruvate is not known. In addition, determination of other glycolytic intermediates indicates that lactate (Lac) levels in ChREBP-/- mice are significantly higher than pyruvate on both diets compared to WT mice resulting in decreased NAD/NADH ratios and a more reduced and anaerobic liver. The purpose of this collaboration is (a) to determine the biochemical mechanism for the reduced REDOX state of liver of ChREBP-/-, and (b) to determine the source of pyruvate in the liver of ChREBP-/- mice fed high starch diet. We determined enzyme mRNA levels and enzyme activities of the Krebs cycle, electron shuttle enzymes and metabolite concentrations in the freeze-clamped liver of ChREBP-/- mice, isolated mitochondria and hepatocytes. A limitation of this approach is that changes in enzyme expression often do not match changes in flux. Because NMR isotopomer analysis is an excellent method for measuring fluxes in intact tissue, we took this approach to investigate the metabolism of hepatic pyruvate and lactate in ChREBP-/- mice. Substrate preference experiments were performed by perfusing isolated perfused livers with a mixture 13C labeled FFA, glucose, lactate and pyruvate. Preliminary results from this experiment indicate that FFA oxidation in the TCA cycle is reduced by half, from 80% in WT to 40% in the ChREBP -/- (Figure 1). Concordantly, pyruvate and lactate oxidation were higher in the ChREBP -/- mice compared to controls, in agreement with a 74% reduction in liver PDK3 enzyme activity.