This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Commonly, PEPCK is considered the most important of the control points for gluconeogenesis. However, this fundamental dogma is difficult to test because it would require the combination of in vivo control of enzyme expression and the ability to measure flux through the enzymes of intact tissue. Magnuson and co-workers have generated mice with PEPCK expression ranging from 0-100% of normal mice by using an allelogenic Cre/loxP strategy. We are collaborating with Dr. Burgess in the Advanced Imaging Center to measure fluxes in the intact liver of mice generated at Vanderbilt with graded levels of PEPCK expression. This arrangement offers a unique and important opportunity to understand the control of PEPCK in the intact liver (and eventually kidney) on gluconeogenesis and other peripheral pathways such as fatty acid oxidation. The connection between PEPCK and metabolic pathways besides gluconeogenesis is highlighted by the observation that inhibiting PEPCK expression induces hepatic steatosis and causes large increases in certain intermediate pool sizes. The development of hepatic steatosis in the PEPCK KO mouse seems paradoxical in light of the fact that the enzymes of ?-oxidation are actually up-regulated. Measuring flux through these metabolic pathways of the liver and kidney of these mice will help us better understand the position of control this enzyme occupies in the gluconeogenic pathway. Our recent data along this line of investigation makes two striking points. First, a total knockout of hepatic PEPCK results in severe alterations of hepatic energy fluxes resulting in dramatically impaired TCA cycle turnover. This is reduction in flux is accompanied by a more reduced mitochondrial redox state which implies that the reduced energy requirements associated with absent gluconeogenesis is to blame. Secondly, our studies in a mouse strain that expresses only 10% of the normal PEPCK levels have shown that these mice do not have dramatic alterations of gluconeogenesis or hepatic energy metabolism (Figure 1). This surprising result clearly suggests that the PEPCK does not play an important practical role in regulated hepatic gluconeogenesis. Further experiments will be performed on mice with 5% and 50% PEPCK expression to better define the curve. This project would not be possible without a strong collaborative relationship with Dr. Burgess who has taken the lead in the metabolic studies of these mice. To assure success, we require access to the 14.1T magnet for 13C and 2H analysis of tissue extracts. We also need to use the shared lab space for organ perfusion experiments and analytical instruments such as the UV spectrophotometer, HPLC and solution phase synthesizer. The combination of transgenic mouse models (PEPCK KO and models of diabetes) and the determination of enzyme activities in intact tissues and whole animals by NMR is a profound new step in this field and will allow us to probe the relationships between the biochemical pathways of gluconeogenesis and energy production.
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