Pyruvate supply and respiration in isolated skeletal muscle mitochondria

The primary hypotheses addressing the coincidence of fatigue and carbohydrate depletion in working skeletal muscle consider either pyruvate availability or citrate cycle inter- mediate levels. In this study we evaluated the role of pyruvate concentration on the relationship between ΔG_ATP and VO₂. Mitochondria were isolated from rat hindlimb muscle and incubated in KCl-based media at 37°C. Various energy states were fixed by manipulating added phosphocreatine/creatine ratios in the presence of 25 U creatine kinase. VO₂ was assessed polarographically. Malate (1.0 mM) was provided and pyruvate was stepwise added to yield 10 uM to 100 uM concentrations. Across all [pyruvate]. there was a strong linear relationship between ΔG_ATP and VO₂. However, the slope of the relationship was highly dependent on [pyruvate]. The slope at 100 uM pyruvate was more than 4-fold greater than the slope at 10 uM pyruvate. These results indicate that, for a given VO₂, a decrease in [pyruvate], in the physiological range, is associated with a fall in extramitochondrial energy state (ΔG_ATP).