Measuring Mitochondrial Pyruvate Oxidation

Pyruvate is a central metabolic intermediate and plays a prominent role in nervous system function. Neurons are highly reliant on pyruvate oxidation for maintenance of cellular energetics. Disorders in pyruvate metabolism may result in severe neurological defects. Pyruvate is imported into the mitochondrial matrix by the mitochondrial pyruvate carrier (MPC) for oxidation by the TCA cycle to support oxidative phosphorylation. Understanding the function of the MPC requires specialized methods for investigating mitochondrial pyruvate metabolism. Multiple instruments are available for measuring respiration of animal tissues, intact cells, permeabilized cells and tissue, and isolated mitochondria. Compared to other platforms, the comparative advantage of the Seahorse extracellular flux analyzer is the ability to sequentially administer treatment compounds and observe the effects on cellular respiration in a multiplexed 96-well format. Here we describe the methods and procedures for: (1) assessing mitochondrial pyruvate oxidation by intact cultured neuronal cells; (2) by mitochondria isolated from HEK293T cells, and (3) by mitochondria isolated from mouse liver, which are useful as a general model of mitochondrial function.