Factors Controlling Metabolic Flux in the Liver by NMR Isotopomer Analysis

描述（申请人提供）：在多种代谢疾病中，肝脏碳水化合物和脂肪代谢异常导致糖和脂质稳态不佳。由于这个原因，调节肝脏中这些代谢途径的因素已被深入研究，但仍未完全了解。通常，糖异生酶的表达，特别是磷酸烯醇丙酮酸羧激酶（PEPCK），被认为控制糖异生的速率；然而，通过这些途径的通量如何随着酶的表达而改变（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控制强度较弱，这意味着其他因素协调控制糖异生。其中一个因素是肝脏通过脂肪氧化产生能量的速率。例如，肝脏暴露于高水平的脂肪酸会导致糖异生增加，最近，分子因素已经被确定为协调糖异生和脂肪氧化的酶。我们发现肝脏TCA循环通量（与肝脏能量产生密切相关的途径）的速率与通过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，我们将测量糖异生酶丙酮酸羧化酶（PC）和PEPCK表达改变时肝脏的代谢通量，以确定它们影响糖异生速率的能力。最后，由于已知肝脏脂肪输送增加会增加糖异生，并可能增加通过PC和PEPCK的通量，我们还将测量肝脏通量，以响应脂肪可用性的改变。这些研究将采用多学科方法进行，包括基因改变模型，分离器官制备和啮齿动物显微外科手术。核磁共振（NMR）同位素分析将用于测量代谢通量，这些技术将通过同时测定肝脏质量平衡得到证实。通过肝脏代谢途径的异常通量参与许多代谢性疾病的发病。该基金资助的工作将大大增强我们对肝脏代谢途径，特别是糖异生和脂肪氧化，如何响应酶或底物浓度变化的理解。最终，这些知识对于通过设计或偶然调节这些途径的分子或药物干预的发展和解释至关重要。

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.