Evaluating the Effect of Adenine Nucleotide Translocase-1 Acetylation on Mitochondrial Bioenergetics and Metabolic Inflexibility

Abstract

ANT1 in human mitochondria was previously identified as an acetylated protein and lysines 10, 23, and 92 of human ANT1 were noted to be acetylated in most of preparations of mitochondria isolated from human skeletal muscle biopsies. Lysine 23 contributes to a net overall positive charge in the nucleotide binding pocket of ANT1, molecular dynamics modeling showed that acetylation of lysine 23 reduced the positive charge and lowered the affinity of hANT1 from about 3 to 80 M [ADP]. Further computational modeling studies showed that if about 25% or higher of total ANT1 were acetylated, the apparent Km ADP for oxidative phosphorylation would be increased sufficiently to affect ANT1 function, with consequences for glycolysis and fuel selection.Based on previous findings, we decided to evaluate the involvement of altered acetylation of ANT 1 at lysine 23 in regulation of fuel selection and mitochondrial bioenergetics. This thesis consists of three chapters that describe the involvement of altered acetylation of ANT 1 at lysine 23 in regulation of fuel selection. The first chapter describes a luciferase-based assay to evaluate the ADP kinetic parameters of mitochondrial ATP production and the extent of control that ANT has in this pathway. The high sensitivity, reproducibility of this assay enabled to assess ADP kinetic parameters and ANT flux control in isolated mitochondria from skeletal muscle. It is confirmed that under the conditions used to study isolated mitochondria, the majority of flux control over oxidative phosphorylation resides at ANT. The second chapter deals with the question of relationship between acetylation of ANT1 and ANT1 content with metabolic flexibility during insulin stimulation or mild exercise. The findings from human skeletal muscle do not provide evidence that the extent of lysine 23 acetylation of ANT1 is a predictor of fuel selection at low [ADP]. Acetylation of lysine 23 of ANT1 is abundant and variable but perhaps too low to discernibly affect the sensitivity of mitochondria to the respiratory signal provided by [ADP] during conditions of insulin stimulation or mild muscle contraction. However, greater ANT1 content, either as a marker of greater mitochondrial abundance or higher specific abundance of ANT1 per mitochondrial mass, could result in changes in fuel selection by lowering the apparent K0.5ADP, driving skeletal muscle toward higher lipid oxidation during mild exercise. The participants in this study had normal or moderately abnormal glucose metabolism. It is conceivable that patients with more poorly controlled type 2 diabetes and more severe impairments in muscle fuel selection could have higher ANT acetylation. Consequently, it would be helpful to evaluate the extent and effect of ANT1 acetylation in this group. Finally, the last chapter provides actual experimental evidence whether loss of the positive charge at lysine 23 has any effect on the affinity of ANT for ADP. The cellular modeling experiments indicate that removing the positive charge of ANT1 at lysine 23 decreases the ability of ANT1 to bind ADP and result in a higher apparent KmADP for ATP production without changing the Vmax. Although it seems clear from modeling and simulation experiments that acetylation of lysine 23 of human ANT1 affects function, direct biochemical evidence is lacking regarding the extent of this effect. Experiments using ANT incorporated into artificial liposomes may help to answer this question.