Factors Controlling Metabolic Flux in the Liver by NMR Isotopomer Analysis

描述(申请人提供)：在多种代谢性疾病中，肝脏碳水化合物和脂肪代谢异常会导致糖和脂平衡不良。因此，调节肝脏中这些代谢途径的因素已经得到了深入的研究，但仍不完全清楚。通常情况下，糖异生酶的表达，特别是磷酸烯醇式丙酮酸羧激酶(PEPCK)，被认为控制糖异生的速度；然而，通过这些途径的通量如何对酶表达的响应(即控制强度)仍然知之甚少。我们的工作表明，在PEPCK表达水平分级的小鼠中，PEPCK的控制力很弱，这意味着其他因素协调了糖异生的控制。其中一个因素是肝脏通过脂肪氧化产生能量的速度。例如，肝脏暴露于高水平的脂肪酸会导致糖异生增加，最近，人们发现了协调糖异生和脂肪氧化的酶的分子因素。我们发现，肝脏TCA循环通量的速率与肝脏能量产生密切相关，与通过PEPCK的通量的相关性比PEPCK酶表达本身更强。为了继续我们对这些途径的研究，我们将测量肝脏中的代谢通量，以响应糖异生酶丙酮酸羧化酶(PC)和PEPCK的表达变化，以确定它们影响糖异生速率的能力。最后，由于已知向肝脏输送的脂肪增加会增加糖异生，推测通过PC和PEPCK的流量也会增加，我们也将测量肝脏流量对脂肪供应改变的反应。这些研究将使用多学科方法进行，包括基因改变模型、分离器官准备和啮齿动物显微手术。核磁共振(核磁共振)同位素分析将被用来测量代谢通量，这些技术将被同时的肝脏质量平衡测定所证实。肝脏代谢途径的异常通量参与了多种代谢性疾病的发病。由这笔赠款资助的工作将极大地增强我们对肝脏代谢途径，特别是糖异生和脂肪氧化，如何应对酶或底物浓度变化的理解。归根结底，这些知识对于分子或药物干预的发展和解释至关重要，这些干预通过设计或偶然的方式调节这些途径。

DESCRIPTION (provided by applicant): Abnormal liver carbohydrate and fat metabolism contribute to poor glucose and lipid homeostasis in a variety of metabolic diseases. For this reason, factors that regulate these metabolic pathways in the liver have been intensely studied, yet remain incompletely understood. Commonly, the expression of gluconeogenic enzymes, in particular phosphoenolpyruvate carboxykinase (PEPCK), are thought to control the rate of gluconeogenesis; however, how flux through these pathways change in response to enzyme expression (i.e. control strength) remains poorly understood. Our work demonstrates that in mice with graded levels of PEPCK expression, PEPCK control strength is weak, implying that other factors coordinate control of gluconeogenesis. One of these factors is the rate of hepatic energy production via fat oxidation. For instance, exposure of liver to high levels of fatty acids results in increased gluconeogenesis, and more recently, molecular factors have been identified that coordinate the enzymes of gluconeogenesis and fat oxidation in parallel. We've found that the rate of hepatic TCA cycle flux, a pathway intimately linked to hepatic energy production, correlates more strongly with flux through PEPCK than PEPCK enzyme expression itself. To continue our studies of these pathways we will measure metabolic fluxes in liver in response to altered expression of the gluconeogenic enzymes pyruvate carboxylase (PC) and PEPCK to determine their capacity to influence the rate of gluconeogenesis. Finally, since elevated fat delivery to liver is known to increase gluconeogenesis, and presumably flux through PC and PEPCK, we will also measure hepatic fluxes in response to altered fat availability. These studies will be performed using a multidisciplinary approach comprised of gene altered models, isolated organ preparations and rodent micro-surgery. Nuclear magnetic resonance (NMR) isotopomer analysis will be used to measure metabolic fluxes and these techniques will be corroborated by simultaneous hepatic mass balance determinations. Aberrant fluxes through metabolic pathways of the liver participate in the morbidity of numerous metabolic diseases. Work funded by this grant will substantially enhance our understanding of how metabolic pathways in the liver, specifically gluconeogenesis and fat oxidation, respond to changes in enzyme or substrate concentration. Ultimately, this knowledge is critical for development and interpretation of molecular or pharmacological interventions that modulate these pathways, either by design or happenstance.