Isotopomer Analysis of Citric Acid Cycle and Gluconeogenesis in Rat Liver

Abstract

We conducted an extensive mass isotopomer analysis of citric acid cycle and gluconeogenic metabolites isolated from livers of overnight fasted rats perfused with 4 mM glucose, 0.2 mM octanoate, 1 mM [U-13C3]lactate, and 0.2 mM [U-13C3]pyruvate, in the anterograde or retrograde mode. In both perfusion modes, two distinct isotopomer patterns were observed: (i) those of phosphoenolpyruvate, glucose, malate, and aspartate and (ii) those of citrate, alpha-ketoglutarate, glutamate, and glutamine. Key citric acid cycle parameters and, hence, rates of gluconeogenesis, calculated (Lee, W.-N.P. (1989) J. Biol. Chem. 264, 13002-13004 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 inconsistent with Lee's citric acid cycle model. Compared to lactate and pyruvate uptake, which sets an upper limit to glucose production, rates of gluconeogenesis calculated (i) with the phosphoenolpyruvate and citrate data were similar, but those calculated (ii) with the glutamate 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 oxaloacetate carbon skeleton from mitochondria to the cytosol. Also, a good fit of our labeling data was obtained with a model of citric acid cycle and gluconeogenesis which we developed to incorporate the above reactions (Fernandez, C.A., and Des Rosiers, C. (1995) J. Biol. Chem. 270, 10037-10042). The following conclusions can be drawn from the calculated reaction rates: (i) about half of the lactate conversion to glucose occurs via the citrate cleavage pathway, (ii) the flux through the reversal of the isocitrate dehydrogenase reaction is almost as fast as that through the citrate synthase reaction, and (iii) the flux through citrate synthase and alpha-ketoglutarate dehydrogenase is 1.6- and 3.2-fold that through pyruvate carboxylase, respectively.

Assessing the Reversibility of the Anaplerotic Reactions of the Propionyl-CoA Pathway in Heart and Liver

“…The use of uniformly labeled substrates and mass isotopomer distribution analysis provides a greater wealth of information than singly labeled substrates, especially when label passes through the CAC (33,34). In livers and hearts perfused with increasing concentrations of [U- 13 C 3 ]propionate, the mass isotopomer distribution of methylmalonyl-CoA was characterized mostly by the M3 isotopomer, the abundance of which was greater than 90% (Fig.…”

Effects of insulin and cytosolic redox state on glucose production pathways in the isolated perfused mouse liver measured by integrated 2H and 13C NMR

“…Relative flux values through various pathways in isolated perfused rat livers have been measured using a combination of stable isotope tracers and NMR or MS [32,39]. Absolute fluxes through the tricarboxylic acid cycle and associated pathways have been measured by an elegant combination of mass-isotopomer-distribution analysis ('MIDA') and substrate balance across the perfused rat liver [40] or by using a rigorous stoichiometric analysis of metabolite uptake and production [41]. More recently we used the isolated perfused mouse liver in conjunction with NMR spectroscopy to study glucose and energy metabolism in the isolated perfused mouse liver from liver specific PEPCK-null mice [24].…”

Agmatine Stimulates Hepatic Fatty Acid Oxidation

“…13 C enrichment in a given mass isotopomer is expressed by molar percent enrichment (MPE), which is the mol fraction (%) of analyte containing 13 C atoms above natural abundance. The MPE was calculated using the peak area from GC-MS ions corrected for natural abundance as described (33,34). The appearance of 13 C-labeled glutamate, aspartate, or NAG isotopomers was calculated by the product of 13 C enrichment (MPE) in a given isotopomer/100 times concentration (nmol/g wet wt) and is expressed as nanomoles of 13 C-labeled metabolite/g wet wt.…”