Measuring Mitochondrial Pyruvate Oxidation

Abstract

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.

Two human Mitochondrial Pyruvate Carrier mutations reveal distinct mechanisms of molecular pathogenesis

“…Thus, it is not surprising that deranged pyruvate metabolism contributes to a range of diseases (20). Since the molecular identification of the genes encoding the MPC (30,38), an increasing number of investigations have demonstrated the prominent role of mitochondrial pyruvate uptake in regulating cellular an organismal biology (49,50,58,81,102,103). helices, with both the N-and C-terminus projecting into the mitochondrial matrix (51,64,104).…”

“…While pyruvate-alanine cycles have been demonstrated a bypass mechanism during MPC disruption in in the liver (49,50), mouse embryonic fibroblast (MEF) cells isolated from MPC1 knockout mice did not show alanine or malate interconversion with pyruvate derived from [U-13 C] glucose (106). Furthermore, excess cytosolic pyruvate was secreted from the cells or converted to serine, which can be generated from accumulated glycolytic intermediates (58,120).…”

“…Interestingly, glutamine oxidation and anaplerosis are not always elevated upon MPC disruption (58). Rather, glutamine acts as an alternative substrate for the TCA cycle when mitochondrial pyruvate becomes limited and thus unavailable for MPC bypasses like pyruvatealanine cycling (49,101). Following MPC disruption, metabolic flux downstream of PDH including citrate synthase activity and oxidative IDH activity decreases.…”

“…While MPC function is important in development, the loss of MPC function does not affect cell viability in some differentiated tissues (49,50,102). It may have adverse effects or provide health benefits depending on the approach and tissues studied.…”

“…Mice treated with UK-5099 and mice with whole-body homozygous expression of a hypomorphic MPC2 protein both have impaired glucose tolerance in vivo (87,124). By contrast, disruption of the MPC in liver or POMC neurons improved insulin sensitivity and glycemia during diet-induced diabetes and -obesity but had minimal effect on glucose homeostasis with normal chow treatment (49,50,102). Changes in nutrient requirements during a life span and the pathophysiology of diseases are likely responsible for the various biological outcomes of decreased MPC function.…”