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

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 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.