MOLECULAR CONTROL OF GLUCOSE METABOLISM

葡萄糖代谢的分子控制

• 批准号: 7600836
• 负责人: KOSAKU UYEDA
• 金额: $ 1.51万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7600836
• 关键词: Agreement; Animal Sources; Binding Proteins; Biochemical; Blood Glucose; Carbohydrates; Cell Nucleus; Citric Acid Cycle; Collaborations; Computer Retrieval of Information on Scientific Projects Database; Cytosol; Diet; Disruption; Electrons; Enzyme Gene; Enzymes; Fatty Acids; Fatty acid glycerol esters; Freezing; Funding; Genes; Genetic Transcription; Glucose; Glycolysis; Grant; Hepatic; Hepatocyte; Hypoglycemia; Institution; Insulin; Intake; Label; Liver; Localized; Measures; Messenger RNA; Metabolism; Methods; Mitochondria; Molecular; Mus; Mutant Strains Mice; Obesity; Organ; Oxidation-Reduction; Pancreas; Pentosephosphate Pathway; Protein Binding; Protein Dephosphorylation; Protein phosphatase; Purpose; Pyruvate; Pyruvate Kinase; Pyruvates; Research; Research Personnel; Resources; Response Elements; Signal Transduction; Source; Standards of Weights and Measures; Starch; Starvation; Tissues; Triglycerides; United States National Institutes of Health; Wild Type Mouse; enzyme activity; feeding; glucose metabolism; lipid biosynthesis; oxidation; preference; promoter; research study; response; transcription factor

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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 当碳水化合物的摄入量超过短期能量需求时，它会以脂肪的形式储存起来。 肝脏是负责将多余的碳水化合物转化为脂肪的主要器官。 摄入高碳水化合物的饮食会诱导肝脏中参与糖酵解和脂肪合成的十几种酶的基因转录。 胰腺分泌的胰岛素对碳水化合物有反应，通过激活脂肪生成酶的表达促进脂肪生成。 然而，碳水化合物也刺激不依赖胰岛素的相同基因的转录。 随着被称为碳水化合物反应元件结合蛋白（ChREBP）的转录因子的发现，代谢性碳水化合物产生信号以诱导脂肪生成酶基因转录的机制变得更加清楚。 在低血糖期间，例如可能在饥饿期间发现，ChREBP被磷酸化并保留在胞质溶胶中。 然而，当血糖升高时，糖酵解和戊糖分流中间产物包括木酮糖5-P也升高。 升高的木酮糖5-P水平激活特异性蛋白磷酸酶，导致ChREBP去磷酸化为其活性形式，其易位至细胞核。 一旦进入细胞核，ChREBP就与位于脂肪生成酶和糖酵解酶以及丙酮酸激酶（LPK）的启动子中的碳水化合物反应元件结合以激活它们的转录。 与野生型小鼠相比，具有ChREBP基因的靶向破坏（ChREBP-/-）的小鼠表现出显著减少的脂肪酸合成和减少的肥胖。 例如，与野生型小鼠相比，喂食高碳水化合物饮食的ChREBP-/-小鼠的肝脏甘油三酯水平降低了50%。 先前的代谢物和酶活性分析显示，与WT小鼠相比，ChREBP-/-小鼠中的丙酮酸水平在标准实验室食物中也显著较低，因为响应于LPK活性降低80%的肝糖酵解降低。 有趣的是，在高碳水化合物饮食中，突变小鼠中的丙酮酸浓度与WT动物中的相同，但丙酮酸的来源未知。此外，其他糖酵解中间体的测定表明，与WT小鼠相比，ChREBP-/-小鼠中的乳酸盐（Lac）水平在两种饮食中均显著高于丙酮酸盐，导致NAD/NADH比值降低，肝脏还原和厌氧程度更高。 这项合作的目的是（a）确定ChREBP-/-肝脏REDOX状态降低的生化机制，以及（B）确定喂食高淀粉饮食的ChREBP-/-小鼠肝脏中丙酮酸的来源。我们确定了酶的mRNA水平和酶活性的克雷布斯循环，电子穿梭酶和代谢物浓度的ChREBP-/-小鼠，分离的线粒体和肝细胞的冷冻夹持的肝脏。 这种方法的局限性在于酶表达的变化通常与通量的变化不匹配。 由于NMR同位素分析是测量完整组织中通量的一种很好的方法，因此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13 C标记的FFA、葡萄糖、乳酸盐和丙酮酸盐的混合物灌注分离的灌注肝脏进行底物偏好实验。 该实验的初步结果表明，TCA循环中的FFA氧化减少了一半，从WT中的80%减少到ChREBP -/-中的40%（图1）。 一致地， 与对照组相比，ChREBP -/-小鼠中丙酮酸和乳酸氧化更高，与肝脏PDK 3酶活性降低74%一致。