Factors Controlling Metabolic Flux in the Liver by NMR Isotopomer Analysis

肝脏碳水化合物和脂肪代谢异常导致各种代谢疾病中葡萄糖和脂质稳态差。由于这个原因，调节肝脏中这些代谢途径的因素已经被深入研究，但仍然不完全 明白通常，促凋亡酶，特别是磷酸烯醇式丙酮酸羧激酶（PEPCK）的表达被认为控制促凋亡的速率;然而，通过这些途径的通量如何响应于酶表达（即控制强度）而变化仍然知之甚少。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胚胎发生。这些因素之一是通过脂肪氧化产生肝脏能量的速率。例如，肝脏暴露于高水平的脂肪酸导致脂肪生成增加，最近，已经鉴定出平行协调脂肪生成和脂肪氧化的酶的分子因子。 我们已经发现，肝脏TCA循环流量的速率，一个与肝脏能量产生密切相关的途径，与PEPCK酶表达本身相比，与PEPCK流量的相关性更强。为了继续我们对这些途径的研究，我们将测量肝脏中响应于产酶丙酮酸羧化酶（PC）和PEPCK的表达改变的代谢通量，以确定它们影响产酶速率的能力。最后，由于已知向肝脏输送的脂肪增加会增加脂肪生成，并且可能通过PC和PEPCK的流量，因此我们还将测量响应于脂肪可用性改变的肝脏流量。这些研究将采用多学科方法进行，包括基因改变模型、离体器官制备和啮齿动物显微手术。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来证实。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。这项资助的工作将大大提高我们的理解 肝脏中的代谢途径，特别是脂肪生成和脂肪氧化，如何对酶或底物浓度的变化做出反应。最终，这些知识对于通过设计或偶然性来调节这些通路的分子或药理干预的开发和解释至关重要。

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.