ISOTOPOMER ANALYSIS OF CITRIC-ACID CYCLE AND GLUCONEOGENESIS IN RAT-LIVER - REVERSIBILITY OF ISOCITRATE DEHYDROGENASE AND INVOLVEMENT OF ATP-CITRATE LYASE IN GLUCONEOGENESIS

We conducted an extensive mass isotopomer analysis of citric acid cycl e and gluconeogenic metabolites isolated from livers of overnight fast ed rats perfused with 4 mM glucose, 0.2 mM octanoate, 1 mM [U-C-13(3)] lactate, and 0.2 mM [U-C-13(3)]pyruvate, in the anterograde or retrogr ade mode, In both perfusion modes, two distinct isotopomer patterns we re observed: (i) those of phosphoenolpyruvate, glucose, malate, and as partate and (ii) those of citrate, alpha-ketoglutarate, glutamate, and glutamine. Key citric acid cycle parameters and, hence, rates of gluc oneogenesis, calculated (Lee. W.-N. P. (1989) J. Biol. Chem. 264, 1300 2-13004 and Lee, W.-N. P. (1993) J. Biol. Chem. 268, 25522-25526) from our mass isotopomer data did not only vary, but lead to conclusions i nconsistent with Lee's citric acid cycle model, Compared to lactate an d pyruvate uptake, which sets an upper limit to glucose production, ra tes of gluconeogenesis calculated (i) with the phosphoenolpyruvate and citrate data were similar, but those calculated (ii) with the glutama te data amounted to only 60%, which is unlikely, All these conclusions are independent of the perfusion modes, We provide evidence that the following processes contribute to the observed labeling discrepancy: ( i) the reversibility of the isocitrate dehydrogenase reaction and (ii) an active citrate cleavage pathway for the transfer of the oxaloaceta te carbon skeleton from mitochondria to the cytosol. Also, a good fit of our labeling data was obtained with a model of citric acid cycle an d gluconeogenesis which we developed to incorporate the above reaction s (Fernandez, C. A., and Des Rosiers, C. (1995) J. Biol. Chem. 270, 10 037-10042), The following conclusions can be drawn from the calculated reaction rates: (i) about half of the lactate conversion to glucose o ccurs via the citrate cleavage pathway, (ii) the flux through the reve rsal of the isocitrate dehydrogenase reaction is almost as fast as tha t through the citrate synthase reaction, and (iii) the Bur through cit rate synthase and alpha-ketoglutarate dehydrogenase is 1.6 and 3.2-fol d that through pyruvate carboxylase, respectively.