The rabbit kidney does not readily metabolize but synthesizes glutamine at high rates by pathways that remain poorly defined. Therefore, the metabolism of variously labeled [13 C]-and [ 14 C]glutamates has been studied in isolated rabbit kidney tubules with and without acetate. CO 2 , glutamine, and alanine were the main carbon and nitrogenous end products of glutamate metabolism but no ammonia accumulated. Absolute fluxes through enzymes involved in glutamate metabolism, including enzymes of four different cycles operating simultaneously, were assessed by combining mainly the 13 C NMR data with a new model of glutamate metabolism. In contrast to a previous conclusion of Klahr et al. (Klahr, S., Schoolwerth, A. C., and Bourgoignie, J. J. (1972) Am. J. Physiol. 222, 813-820), glutamate metabolism was found to be initiated by glutamate dehydrogenase at high rates. Glutamate dehydrogenase also operated at high rates in the reverse direction; this, together with the operation of the glutamine synthetase reaction, masked the release of ammonia. Addition of acetate stimulated the operation of the "glutamate 3 ␣-ketoglutarate 3 glutamate" cycle and the accumulation of glucose but reduced both the net oxidative deamination of glutamate and glutamine synthesis. Acetate considerably increased flux through ␣-ketoglutarate dehydrogenase and citrate synthase at the expense of flux through phosphoenolpyruvate carboxykinase; acetate also caused a large decrease in flux through alanine aminotransferase, pyruvate dehydrogenase, and the "substrate cycle" involving oxaloacetate, phosphoenolpyruvate, and pyruvate.

Based on the same principles as those utilized in a recent study for modeling glucose metabolism (Martin, G., Chauvin, M. F., Dugelay, S., and Baverel, G. (1994) J. Biol. Chem. 269, 26034 -26039), a method is presented for determining metabolic fluxes involved in glutamate metabolism in mammalian cells. This model consists of five different cycles that operate simultaneously. It includes not only the tricarboxylic acid cycle, the "oxaloacetate 3 phosphoenolpyruvate 3 pyruvate 3 oxaloacetate" cycle and the "oxaloacetate 3 phosphoenolpyruvate 3 pyruvate 3 acetyl-CoA 3 citrate 3 oxaloacetate" cycle but also the "glutamate 3 ␣-ketoglutarate 3 glutamate" and the "glutamate 3 glutamine 3 glutamate" cycles. The fates of each carbon of glutamate, expressed as ratios of integrated transfer of this carbon to corresponding carbons in subsequent metabolites, are described by a set of equations. Since the data introduced in the model are micrograms of atom of traced carbon incorporated into each carbon of end products, the calculation strategy was determined on the basis of the most reliable parameters determined experimentally. This model, whose calculation routes offer a large degree of flexibility, is applicable to data obtained by 13 C NMR spectroscopy, gas chromatography ؊ mass spectrometry, or 14 C counting in a great variety of mammalian cells.In the accompanying paper (16), we have conducted a study on glutamate metabolism in isolated rabbit kidney tubules. For the interpretation of the data obtained, we have constructed a mathematical model that is based on the incorporation of 13 C and 14 C into various metabolites and allows the calculation of reaction rates of gluconeogenesis, tricarboxylic acid cycle, and the pathways of glutamate and glutamine synthesis and degradation occurring simultaneously in mammalian cells. This model, which is applicable to data obtained by 13 C NMR, gas chromatography-mass spectrometry, and 14 C counting, is described in the present paper.

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