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

Jeffrey I. Messer, Matthew R. Jackman, Wayne T. Willis

Research output: Contribution to journalArticle

47 Scopus citations

Abstract

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 (Jo) 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 (ΔGP), established with a creatine kinase energy clamp, and also at saturating [ADP]. The apparent Km,malate for mitochondrial Jo 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 ΔGP declined. V max for both substrates also increased as ΔGP fell, reflecting thermodynamic control of Jo. Reported in vivo skeletal muscle [malate] are >10-fold greater than the Km,malate determined in this study. In marked contrast, the Km,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 (ΔGP-Jo) relationship, the ΔGP-Jo 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.

Original languageEnglish (US)
Pages (from-to)C565-C572
JournalAmerican Journal of Physiology - Cell Physiology
Volume286
Issue number3 55-3
DOIs
StatePublished - Mar 2004

Keywords

  • Anaplerosis
  • Bioenergetics
  • Fatigue
  • Fuel limitation
  • Krebs cycle
  • Metabolic control analysis

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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