title: A Method for Multiplexed Measurement of Mitochondrial Pyruvate Carrier Activity.
abstract: The discovery that theMPC1andMPC2genes encode the protein components of the mitochondrial pyruvate carrier (MPC) has invigorated studies of mitochondrial pyruvate transport and its regulation in normal and disease states. Indeed, recent reports have demonstrated MPC involvement in the control of cell fate in cancer and gluconeogenesis in models of type 2 diabetes. Biochemical measurements of MPC activity are foundational for understanding the role of pyruvate transport in health and disease. We developed a 96-well scaled method of [(14)C]pyruvate uptake that markedly decreases sample requirements and increases throughput relative to previous techniques. This method was applied to determine the mouse liver MPCKm(28.0 ± 3.9 μm) andVmax(1.08 ± 0.05 nmol/min/mg), which have not previously been reported.KmandVmaxof the rat liver MPC were found to be 71.2 ± 17 μmand 1.42 ± 0.14 nmol/min/mg, respectively. Additionally, we performed parallel pyruvate uptake and oxidation experiments with the same biological samples and show differential results in response to fasting, demonstrating the continued importance of a direct MPC activity assay. We expect this method will be of value for understanding the contribution of the MPC activity to health and disease states where pyruvate metabolism is expected to play a prominent role.
authors.affiliation: From the Department of Biochemistry, the Fraternal Order of the Eagles Diabetes Research Center, the Abboud Cardiovascular Research Center, and the Pappajohn Biomedical Institute, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242.
authors.forename: Lawrence R
authors.initials: LR
authors.lastname: Gray
authors.affiliation: From the Department of Biochemistry, the Fraternal Order of the Eagles Diabetes Research Center, the Abboud Cardiovascular Research Center, and the Pappajohn Biomedical Institute, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Rauckhorst
authors.affiliation: From the Department of Biochemistry, the Fraternal Order of the Eagles Diabetes Research Center, the Abboud Cardiovascular Research Center, and the Pappajohn Biomedical Institute, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242 eric-taylor@uiowa.edu.
authors.forename: Eric B
authors.initials: EB
authors.lastname: Taylor
mesh_terms: D000818:Animals; D027321:Anion Transport Proteins / Q000032:analysis / Q000378:metabolism; D051379:Mice; D008930:Mitochondria, Liver / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000032:analysis / Q000378:metabolism; D027501:Monocarboxylic Acid Transporters; D019289:Pyruvic Acid / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D027321:Anion Transport Proteins; C000595573:MPC1 pyruvate carrier protein, mouse; C000595574:MPC2 pyruvate carrier protein, mouse; D033681:Mitochondrial Membrane Transport Proteins; D027501:Monocarboxylic Acid Transporters; D019289:Pyruvic Acid
keywords: kinetics; metabolism; method; mitochondria; mitochondrial pyruvate carrier; pyruvate; pyruvate transport; transport; uptake
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