Pyruvate and citric acid cycle carbon requirements in isolated skeletal muscle mitochondria

Exercise and Sport Research Institute, Arizona State University, Tempe, Arizona 85287-0404 Submitted 14 April 2003 ; accepted in final form 14 September 2003 Carbohydrate depletion precipitates fatigue in skeletal muscle, but, because pyruvate provides both acetyl-CoA for mainline oxidation and anaplerotic carbon to the citric acid cycle (CAC), the mechanism remains obscure. Thus pyruvate and CAC kinetic parameters were independently quantified in mitochondria isolated from rat mixed skeletal muscle. Mitochondrial oxygen consumption rate ( J o ) was measured polarographically while either pyruvate or malate was added stepwise in the presence of a saturating concentration of the other substrate. These substrate titrations were carried out across a physiological range of fixed extramitochondrial ATP free energy states ( G P ), established with a creatine kinase energy clamp, and also at saturating [ADP]. The apparent K m,malate for mitochondrial J o ranged from 21 to 32 µM, and the apparent K m,pyruvate ranged from 12 to 26 µM, with both substrate K m values increasing as G P declined. V max for both substrates also increased as G P fell, reflecting thermodynamic control of J o . Reported in vivo skeletal muscle [malate] are >10-fold greater than the K m,malate determined in this study. In marked contrast, the K m,pyruvate determined is near the [pyruvate] reported in muscle approaching exhaustion associated with glycogen depletion. When data were evaluated in the context of a linear thermodynamic force-flow ( G P - J o ) relationship, the G P - J o slope was essentially insensitive to changes in [malate] in the range observed in vivo but decreased markedly with declining [pyruvate] across the physiological range. Mitochondrial respiration is particularly sensitive to variations in [pyruvate] in the physiological range. In contrast, physiological [malate] exerts very little, if any, influence on mitochondrial pyruvate oxidation measured in vitro. bioenergetics; fatigue; anaplerosis; Krebs cycle; fuel limitation; metabolic control analysis Address for reprint requests and other correspondence: W. T. Willis, Dept. of Kinesiology, Arizona State Univ., Tempe, AZ 85287-0404 (E-mail: waynewillis{at}asu.edu ).