Factors controlling metabolic flux in the liver by NMR isotopomer analysis

描述(申请人提供)：本项目研究肝脏三羧酸(TCA循环)代谢流量的调节及其在肝脏胰岛素抵抗中的作用。该提案是对上一个供资周期中的关键发现的后续行动，这些发现表明来自三氯乙酸循环的回旋/回旋生物合成通量和三氯乙酸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煽动者的作用。因此，我们提出了一个新的假设，即肝脏TCA循环通量的增加是肝脏胰岛素抵抗病理过程中的一个主要代谢介质，它通过增强生物合成通量(如糖异生)和氧化应激来实现。我们使用条件性KO小鼠、最先进的基于13C和2H示踪剂的核磁共振和MS/MS同位素聚合体方法来验证这一假设，以评估肝脏葡萄糖、脂肪和TCA循环代谢的代谢通量，以及信号、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DYS/调节。我们确定了胰岛素信号的急性基因切除是否重现或加剧了HFD小鼠升高的TCA循环通量，以及急性缺失Foxo1是否改善了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期间也受到底物递送的间接上调，第二个目标是确定促发生物合成和氧化TCA循环通量之间的共同调节机制。虽然ATP、NADH和变构调节剂对这些途径的急性调节是已知的，但我们将测试AMPK和SIRT3是否通过TCA循环途径作为通量的急性分子调节器。最后，如果升高的TCA循环流量加强了肝脏胰岛素抵抗的代谢病理，那么抑制TCA循环途径应该可以防止肝脏并发症的发生。因此，第三个目标是确定基因抑制促癌剂或TCA循环的氧化跨度是否会改善胰岛素抵抗的代谢、氧化应激和炎症反应。

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.