EFFECTS OF LIVER SPECIFIC KNOCKOUT OF PEPCK ON GLUCOSE METABOLISM

肝脏特异性敲除 PEPCK 对葡萄糖代谢的影响

• 批准号: 7956963
• 负责人: SHAWN M BURGESS
• 金额: $ 1.78万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956963
• 关键词: Animals; Biochemical Pathway; Citric Acid Cycle; Computer Retrieval of Information on Scientific Projects Database; Data; Development; Diabetes Mellitus; Employee Strikes; Energy Metabolism; Enzymes; Fatty Liver; Funding; Gluconeogenesis; Grant; Hepatic; High Pressure Liquid Chromatography; Image; Institution; Investigation; Kidney; Knock-out; Lead; Light; Liver; Measures; Metabolic Pathway; Metabolism; Mitochondria; Modeling; Mouse Strains; Mus; Organ; Oxidation-Reduction; Pathway interactions; Perfusion; Peripheral; Phase; Phosphoenolpyruvate Carboxylase; Play; Positioning Attribute; Production; Research; Research Personnel; Resources; Role; Solutions; Source; Testing; Tissue Extracts; Tissues; Transgenic Mice; United States National Institutes of Health; enzyme activity; fatty acid oxidation; glucose metabolism; hepatic gluconeogenesis; in vivo; instrument; metabolic abnormality assessment; mouse model; oxidation; research study; success

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. Commonly, PEPCK is considered the most important of the control points for gluconeogenesis. However, this fundamental dogma is difficult to test because it would require the combination of in vivo control of enzyme expression and the ability to measure flux through the enzymes of intact tissue. Magnuson and co-workers have generated mice with PEPCK expression ranging from 0-100% of normal mice by using an allelogenic Cre/loxP strategy. We are collaborating with Dr. Burgess in the Advanced Imaging Center to measure fluxes in the intact liver of mice generated at Vanderbilt with graded levels of PEPCK expression. This arrangement offers a unique and important opportunity to understand the control of PEPCK in the intact liver (and eventually kidney) on gluconeogenesis and other peripheral pathways such as fatty acid oxidation. The connection between PEPCK and metabolic pathways besides gluconeogenesis is highlighted by the observation that inhibiting PEPCK expression induces hepatic steatosis and causes large increases in certain intermediate pool sizes. The development of hepatic steatosis in the PEPCK KO mouse seems paradoxical in light of the fact that the enzymes of ¿-oxidation are actually up-regulated. Measuring flux through these metabolic pathways of the liver and kidney of these mice will help us better understand the position of control this enzyme occupies in the gluconeogenic pathway. Our recent data along this line of investigation makes two striking points. First, a total knockout of hepatic PEPCK results in severe alterations of hepatic energy fluxes resulting in dramatically impaired TCA cycle turnover. This is reduction in flux is accompanied by a more reduced mitochondrial redox state which implies that the reduced energy requirements associated with absent gluconeogenesis is to blame. Secondly, our studies in a mouse strain that expresses only 10% of the normal PEPCK levels have shown that these mice do not have dramatic alterations of gluconeogenesis or hepatic energy metabolism (Figure 1). This surprising result clearly suggests that the PEPCK does not play an important practical role in regulated hepatic gluconeogenesis. Further experiments will be performed on mice with 5% and 50% PEPCK expression to better define the curve. This project would not be possible without a strong collaborative relationship with Dr. Burgess who has taken the lead in the metabolic studies of these mice. To assure success, we require access to the 14.1T magnet for 13C and 2H analysis of tissue extracts. We also need to use the shared lab space for organ perfusion experiments and analytical instruments such as the UV spectrophotometer, HPLC and solution phase synthesizer. The combination of transgenic mouse models (PEPCK KO and models of diabetes) and the determination of enzyme activities in intact tissues and whole animals by NMR is a profound new step in this field and will allow us to probe the relationships between the biochemical pathways of gluconeogenesis and energy production.

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。 通常，PEPCK被认为是最重要的控制点的胚胎发育。 然而，这一基本法则难以测试，因为它需要结合体内酶表达的控制和测量通过完整组织的酶的通量的能力。Magnuson及其同事通过使用等位基因Cre/loxP策略产生了PEPCK表达范围为正常小鼠的0-100%的小鼠。我们正在与高级成像中心的Burgess博士合作，测量在范德比尔特产生的具有分级PEPCK表达水平的小鼠的完整肝脏中的通量。 这种安排提供了一个独特的和重要的机会，了解PEPCK的控制在完整的肝脏（最终肾脏）的胚胎发生和其他外周途径，如脂肪酸氧化。 PEPCK和代谢途径之间的联系，除了肝细胞生成，通过观察，抑制PEPCK表达诱导肝脂肪变性，并导致在某些中间池的大小大幅增加突出。 PEPCK KO小鼠中肝脂肪变性的发展似乎自相矛盾，因为氧化酶实际上是上调的。 测量这些小鼠肝脏和肾脏代谢途径的通量将有助于我们更好地了解这种酶在致炎途径中的控制位置。 沿着这条研究路线，我们最近的数据沿着有两个显著的特点。 首先，肝PEPCK的完全敲除导致肝能量通量的严重改变，从而导致显著受损的TCA循环周转。 这是通量的减少伴随着更减少的线粒体氧化还原状态，这意味着与不存在线粒体异生相关的能量需求减少是罪魁祸首。 其次，我们在仅表达正常PEPCK水平的10%的小鼠品系中的研究表明，这些小鼠没有显著的肝再生或肝脏能量代谢改变（图1）。 这一令人惊讶的结果清楚地表明，PEPCK在调节肝再生中不起重要的实际作用。将对PEPCK表达为5%和50%的小鼠进行进一步实验，以更好地定义曲线。如果没有与伯吉斯博士的密切合作关系，这个项目是不可能的，伯吉斯博士在这些小鼠的代谢研究中处于领先地位。 为了确保成功，我们需要使用14.1T磁体对组织提取物进行13 C和2 H分析。 我们还需要使用共享的实验室空间进行器官灌注实验和分析仪器，如紫外分光光度计，HPLC和溶液相合成器。 转基因小鼠模型（PEPCK KO和糖尿病模型）的组合以及通过NMR测定完整组织和整个动物中的酶活性是该领域的一个深刻的新步骤，将使我们能够探索胚胎发生和能量产生的生化途径之间的关系。