A Method for Multiplexed Measurement of Mitochondrial Pyruvate Carrier Activity

• Published in: The Journal of biological chemistry Vol. 291; no. 14; pp. 7409 - 7417
• Main Authors: Gray, Lawrence R., Rauckhorst, Adam J., Taylor, Eric B.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-04-2016; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Anion Transport Proteins - analysis; Anion Transport Proteins - metabolism; kinetics; Metabolism; method; Mice; mitochondria; Mitochondria, Liver - metabolism; Mitochondrial Membrane Transport Proteins - analysis; Mitochondrial Membrane Transport Proteins - metabolism; mitochondrial pyruvate carrier; Monocarboxylic Acid Transporters; pyruvate; pyruvate transport; Pyruvic Acid - chemistry; Pyruvic Acid - metabolism; Rats; Rats, Sprague-Dawley; transport; uptake; method; pyruvate transport; uptake; mitochondria; mitochondrial pyruvate carrier; metabolism; transport; kinetics; pyruvate
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.711663

The discovery that the MPC1 and MPC2 genes 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 [14C]pyruvate uptake that markedly decreases sample requirements and increases throughput relative to previous techniques. This method was applied to determine the mouse liver MPC Km (28.0 ± 3.9 μm) and Vmax (1.08 ± 0.05 nmol/min/mg), which have not previously been reported. Km and Vmax of the rat liver MPC were found to be 71.2 ± 17 μm and 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.