Factors controlling metabolic flux in the liver by NMR isotopomer analysis

项目描述（申请人提供）：本项目研究肝脏三羧酸（TCA）循环代谢通量的调节及其在肝脏胰岛素抵抗中的作用。该提案是在上一个资助周期的关键发现的基础上提出的，该发现表明，TCA循环的倒转/倒转生物合成通量和TCA循环的氧化通量是相互调节的，并且这些途径在肝脏胰岛素抵抗期间升高。这些发现挑战了氧化代谢受损作为肝脏胰岛素抵抗的诱因的作用。因此，我们提出了一个新的假设，即肝脏TCA循环通量升高是肝脏胰岛素抵抗病理的主要代谢介质，通过增强生物合成通量（如糖异生）和氧化应激。我们使用条件KO小鼠来验证这一假设，采用最先进的基于13C和2H示踪剂的NMR和MS/MS同位素方法来评估葡萄糖，脂质和TCA循环代谢的肝脏代谢通量，以及信号传导，基因表达和胰岛素敏感性的标准评估。第一个目的是测试胰岛素作用是否介导TCA循环通量的调节。我们确定胰岛素信号的急性基因切除是否会重现或加剧HFD小鼠中升高的TCA循环通量，以及FOXo1的急性缺失是否会改善HFD期间的TCA循环通量和氧化应激。由于这些途径在胰岛素抵抗过程中也受到底物递送的间接上调，因此第二个目的是确定突变生物合成和氧化TCA循环通量之间的共同调节机制。虽然已知ATP、NADH和变弹性调节剂对这些途径的急性调节，但我们将测试AMPK和Sirt3是否在TCA循环途径中作为通量的急性分子调节剂。最后，如果TCA循环通量升高会增强肝脏胰岛素抵抗的代谢病理，那么抑制TCA循环途径应该可以预防肝脏并发症的发生。因此，第三个目的是确定基因抑制TCA循环的突变期或氧化期是否会改善代谢、氧化应激和胰岛素抵抗的炎症反应。

DESCRIPTION (provided by applicant): This project investigates the regulation of metabolic flux in the hepatic tricarboxylic acid (TCA cycle) and its role in hepatic insulin resistance. The proposal follows up on key findings in the last funding cycle which indicate that anaplerotic/cataplerotic biosynthetic flux from the TCA cycle and oxidative flux of the TCA cycle are reciprocally regulated and that these pathways are elevated during hepatic insulin resistance. These findings challenge the role of impaired oxidative metabolism as an instigator of hepatic insulin resistance. Thus, we propose a novel hypothesis that elevated hepatic TCA cycle flux is a principal metabolic mediator of pathologies of hepatic insulin resistance by potentiating biosynthetic flux (e.g. gluconeogenesis) and oxidative stress. We test this hypothesis using conditional KO mice, state of the art 13C and 2H tracer based NMR and MS/MS isotopomer methods to evaluate hepatic metabolic fluxes of glucose, lipid and TCA cycle metabolism, and standard evaluation of signaling, gene expression and insulin sensitivity. The first aim is to test whether insulin action mediates dys/regulation of TCA cycle fluxes. We determine if acute genetic excision of insulin signaling recapitulates or exasperates elevated TCA cycle flux in HFD mice and whether acute deletion of FOXo1 ameliorates TCA cycle flux and oxidative stress during a HFD. Since these pathways are also subject indirect upregulation by substrate delivery during insulin resistance, the second aim is to determine the mechanism of co-regulation between cataplerotic biosynthesis and oxidative TCA cycle flux. While the acute regulation of these pathways by ATP, NADH and allosteric regulators are known, we will test whether AMPK and Sirt3 act as acute molecular regulators of flux through TCA cycle pathways. Finally, if elevated TCA cycle flux potentiates metabolic pathologies of hepatic insulin resistance, then suppressing TCA cycle pathways should prevent the onset of hepatic complications. Thus, the third aim is to determine if genetic suppression of the cataplerotic or the oxidative span of the TCA cycle results in improved metabolic, oxidative stress and inflammatory response to insulin resistance.