Energy Sensors and the Regulation of the TCA Cycle in the Liver

 描述(由申请人提供)：肝脏在维持血糖水平方面起着至关重要的作用，部分是通过糖异生过程。糖异生是一种能量昂贵的过程，通过肝脏线粒体中的脂肪氧化来支持。TCA循环是一个枢纽，脂肪酸和碳水化合物分解产生的乙酰辅酶A在这里相遇，氧化成二氧化碳，产生3NADH和1FADH2。TCA循环不仅通过形成为电子传递链提供燃料的还原等价物来支持糖异生，而且还通过启动过程提供糖异生底物。底物浓度和氧化还原状态的变化调制了TCA循环通量和退变/退变过程。然而，调节这种代谢反应的机制仍有待阐明。在肥胖和胰岛素抵抗等病理条件下，TCA循环通量和糖异生增加，导致胰岛素抵抗患者经常观察到不适当的内源性葡萄糖产生。尽管如此，在这种疾病状态下，TCA循环是如何调节的还不清楚。许多代谢过程对能量状态很敏感，能量传感器，如AMPK和Sirtuin 3(SIRT3)可能负责协调这些代谢过程对能量电荷变化的反应。AMPK是一种能量传感器，在高AMP：ATP比率时被激活。作为回应，AMPK刺激分解代谢过程，如脂肪酸氧化以补充ATP。特殊目的1将使用体外和体内实验，在有和没有AMPK的调节能力的情况下，确定AMPK是负责TCA循环通量的变化和对底物浓度变化的反应(体外)，还是在肥胖、胰岛素抵抗状态下(在体内)。假设AMPK是TCA循环通量随底物浓度增加而增加所必需的，AMPK-KO小鼠对16周HFD的反应将增加TCA循环通量和消退/退变过程，但固有活性的AMPK将恢复这些过程的通量。SIRT3是一种线粒体脱乙酰酶，通过氧化还原状态(高NAD+/NADH)的改变而激活，并通过脱乙酰化参与代谢过程的线粒体蛋白来做出反应。具体目的2将研究SIRT3是否调节TCA循环通量和促衰老/促衰老过程对氧化还原状态(体外)和肥胖胰岛素抵抗状态(体内)变化的反应。我假设SIRT3将是正常的TCA循环通量和逆转录/引燃过程所必需的。这项研究的发现将扩大我们对能量传感器的作用，以及健康和疾病状态下TCA循环通量和回潮/回潮过程的功能的了解。这些结果可能为制药公司治疗或预防NAFLD和/或胰岛素抵抗提供未来的目标。

 DESCRIPTION (provided by applicant): The liver plays a vital role in maintaining blood glucose levels, in part, through gluconeogenic processes. Gluconeogenesis is an energy costly process that is supported through fat oxidation in hepatic mitochondria. The TCA cycle serves as a hub where acetyl-CoA from the break down of fatty acids and carbohydrates meet for oxidation to CO2 and the production of 3 NADH and 1 FADH2. Not only does the TCA cycle support gluconeogenesis through the formation of reducing equivalents that fuel the electron transport chain, but it also provides gluconeogenic substrates through cataplerotic processes. Changes in substrate concentration and redox state modulate TCA cycle flux and anaplerotic/cataplerotic processes. However, the mechanisms regulating this metabolic response remain to be elucidated. In pathological conditions such as obesity and insulin resistance, TCA cycle flux and gluconeogenesis are elevated, contributing to the inappropriately high endogenous glucose production often observed in insulin resistant individuals. Still, it is no known how the TCA cycle is regulated in this diseased state. Many metabolic processes are sensitive to the energy state, and energy sensors, such as AMPK and Sirtuin 3 (SIRT3) may be responsible for coordinating the response of these metabolic processes to changes in energy charge. AMPK is an energy sensor that is activated in response to high AMP:ATP ratios. In response, AMPK stimulates catabolic processes, such as fatty acid oxidation to replenish ATP. Specific Aim 1 will use ex vivo and in vivo experiments, with and without the regulatory capacity of AMPK to determine whether AMPK is responsible for changes in TCA cycle flux and cataplerotic/anaplerotic processes in response to changes in substrate concentration (ex vivo), or in an obese, insulin resistant state (in vivo). It is hypothesized that AMPK will be necessary for increases in TCA cycle flux in response to increases in substrate concentrations and that TCA cycle flux and anaperotic/cataplerotic processes will be elevated in AMPK-KO mice in response to 16 week HFD, but that constitutively active AMPK will restore the flux of these processes. SIRT3 is a mitochondrial deactylase that is activated by changes in the redox state (high NAD+/NADH) and responds by deacetylating mitochondrial proteins involved in metabolic processes. Specific Aim 2 will examine whether SIRT3 regulates the response of TCA cycle flux and cataplerotic/anaplerotic processes to changes in redox state (ex vivo) and in the obese insulin resistant state (in vivo). I hypothesize that SIRT3 will be required for normal TCA cycle flux and anaplerotic/cataplerotic processes. The findings from this research will expand our knowledge of the role of energy sensors, and the function of TCA cycle flux and anaplerotic/cataplerotic processes in both healthy and diseased states. These results may provide future targets for pharmaceutical companies to treat or prevent NAFLD and/or insulin resistance.