This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of this project is to elucidate the in vivo function of mitochondrial transport proteins involved in metabolic redox-shuttles in two mammalian cell types, hepatocytes and adipocytes;the three specific membrane proteins being examined are dicarboxylate carrier (malate transport), citrate/isocitrate carrier (citrate and isotictrate transport), and 2-oxoglutarate carrier (a-ketoglutarate transport). These proteins are key components of shuttles that transport electrons across the mitochondrial membrane. Recent studies have demonstrated the importance of these redox-shuttles in hepatic metabolism for de novo glucose production (gluconeogenesis), and fat metabolism for de novo lipid biosynthesis (lipogenesis). Improving our understanding of in vivo regulation of gluconeogenesis and lipogenesis is central to finding a cure for diabetes. Because the mitochondrial membrane is essentially impermeable to pyridine nucleotide coenzymes NADH and NADPH (which serve as electron carriers in the cell), once reduced these coenzymes must be reoxidized in the compartment where they were generated. Electrons from NAD(P)H can also be transported across the mitochondrial membrane via specific redox-shuttles, e.g. the pyruvate/malate, pyruvate/citrate, and pyruvate/isocitrate shuttles. While key enzymes and membrane carriers for these shuttles have been identified in the human genome, it is still difficult to measure the activity and determine the function of these shuttles in vivo. We have pioneered the development of a large number of metabolic flux analysis techniques for quantitative dissection of cellular metabolic networks using stable isotopes (e.g. 13C and 2H) and mass spectrometry. The research approach here is to apply these state-of-the-art techniques to determine responses of hepatocytes and adipocytes to knockdown (by RNAi) of specific membrane carriers that alter redox metabolism. Metabolite and flux responses, including comprehensive intracellular flux maps and specific fluxes of gluconeogenesis and lipogenesis, are used as a guide to develop quantitative models that predict the relative importance of specific mitochondrial transport proteins in the regulation of hepatocyte and adipocyte cell function.
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