Current isotopic approaches underestimate gluconeogenesis in vivo because of Krebs cycle carbon exchange and the inability to measure intramitochondrial precursor specific activity. We therefore applied a new isotopic approach that theoretically overcomes these limitations and permits quantification of Krebs cycle carbon exchange and the individual contributions of gluconeogenesis and glycogenolysis to overall glucose output. [6-3H]Glucose was infused to measure overall glucose output; [2-14C]aceate was infused to trace phosphoenolpyruvate gluconeogenesis and to calculate Krebs cycle carbon exchange as proposed by Katz. Plasma [14C]3-OH-butyrate specific activity was used to estimate intramitochondrial acetyl coenzyme A (CoA) specific activity, and finally the ratio between plasma glucose 14C-specific activity and the calculated intracellular phosphoenolpyruvate 14C-specific activity was used to determine the relative contributions of gluconeogenesis and glycogenolysis to overall glucose output. Using this approach, acetyl CoA was found to enter the Krebs cycle at twice (postabsorptive subjects) and three times (2 1/2 -d fasted subjects) the rate of pyruvate, respectively. Gluconeogenesis in postabsorptive subjects (3.36 ± 0.20 μmol/kg per min) accounted for 28 ± 2% of overall glucose output and increased twofold in subjects fasted for 2 1/2 -d (P < 0.01), accounting for > 97% of overall glucose output. Glycogenolysis in postabsorptive subjects averaged 8.96 ± 0.40 μmol/kg per min and decreased to 0.34 ± 0.08 μmol/kg per min (P < 0.01) after a 2 1/2 -d fast. Since these results agree well with previously reported values for gluconeogenesis and glycogenolysis based on determinations of splanchnic substrate balance and glycogen content of serial liver biopsies, we conclude that the isotopic approach applied herein provides an accurate, noninvasive measurement of gluconeogenesis and glycogenolysis in vivo.
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