MOLECULAR CONTROL OF GLUCOSE METABOLISM

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。这项合作的目的是(A)确定ChREBP-/-肝脏氧化还原状态降低的生化机制，以及(B)确定喂食高淀粉饲料的ChREBP-/-小鼠肝脏中丙酮酸的来源。我们测定了ChREBP-/-小鼠冷冻肝脏、分离的线粒体和肝细胞中Krebs循环酶的mRNA水平和酶活性、电子传递酶和代谢物浓度。这种方法的一个局限性是，酶表达的变化通常与通量的变化不匹配。由于核磁共振同位素分析是测量完整组织中通量的一种很好的方法，我们采用这种方法来研究ChREBP-/-小鼠肝脏丙酮酸和乳酸的代谢。通过用13C标记的FFA、葡萄糖、乳酸和丙酮酸的混合物灌流离体肝进行底物选择性实验。本实验的初步结果表明，TCA循环中的FFA氧化减少了一半，从WT的80%减少到ChREBP-/-的40%。一致的是，与对照组相比，ChREBP-/-组小鼠的丙酮酸和乳酸氧化程度更高，与肝脏PDK3酶活性降低74%的情况一致。

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. 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 -/-. 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.