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: 7724113
• 负责人: MARK A MAGNUSON
• 金额: $ 1.05万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724113
• 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; Mitochondria; Modeling; Mouse Strains; Mus; Organ; Oxidation-Reduction; Pathway interactions; Perfusion; Peripheral; Phase; Phosphoenolpyruvate Carboxylase; Play; Positioning Attribute; Production; Range; Research; Research Personnel; Resources; Role; Solutions; Source; Testing; Tissue Extracts; Tissues; Transgenic Organisms; 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; size; 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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 通常，PEPCK被认为是糖异生最重要的控制点。然而，这一基本教条很难检验，因为它需要体内控制酶表达和通过完整组织的酶测量通量的能力相结合。Magnuson和他的同事已经使用等位基因Cre/loxP策略培育出PEPCK表达从正常小鼠的0-100%不等的小鼠。我们正在与先进成像中心的Burgess博士合作，测量Vanderbilt公司生产的PEPCK表达水平分级的小鼠完整肝脏中的流量。这一安排为了解完整肝脏(最终是肾脏)中PEPCK对糖异生和其他外周途径(如脂肪酸氧化)的控制提供了一个独特而重要的机会。抑制PEPCK的表达会导致肝脏脂肪变性，并导致某些中间池大小的大幅增加，这突显了PEPCK与糖异生以外的代谢途径之间的联系。在PEPCK KO小鼠中，肝脏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。通过测量这些小鼠的肝和肾的代谢途径的通量将有助于我们更好地了解该酶在糖异生途径中所占的控制地位。我们最近沿着这条线调查的数据提出了两个引人注目的观点。首先，完全敲除肝脏PEPCK会导致肝脏能量通量的严重改变，导致TCA循环周转严重受损。这就是说，通量的减少伴随着线粒体氧化还原状态的进一步降低，这意味着与缺糖异生相关的能量需求减少是罪魁祸首。其次，我们对只表达正常PEPCK水平10%的小鼠的研究表明，这些小鼠没有明显的糖异生或肝脏能量代谢变化(图1)。这一令人惊讶的结果清楚地表明，PEPCK在调节肝脏糖异生方面并没有发挥重要的实际作用。进一步的实验将在PEPCK表达为5%和50%的小鼠身上进行，以更好地确定曲线。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，伯吉斯博士领导了这些小鼠的新陈代谢研究。为了确保成功，我们需要使用14.1T磁铁进行组织提取物的13C和2H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、高效液相色谱仪和液相合成仪。转基因小鼠模型(PEPCK、KO和糖尿病模型)与核磁共振检测完整组织和整体动物中的酶活性的结合是该领域的一项深刻的新进展，将使我们能够探索糖异生的生化途径与能量产生之间的关系。