Model Applicable to NMR Studies for Calculating Flux Rates in Five Cycles Involved in Glutamate Metabolism

Abstract

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.

The Rabbit Kidney Tubule Simultaneously Degrades and Synthesizes Glutamate

“…Unfortunately, flux through glutamine synthetase was lower than the total glutamine found at the end of the incubation period. Therefore, we obtained no experimental evidence in the present study that the glutamine synthesized from glutamate was metabolized by glutaminase which is also active in rabbit kidney cortex (1-3); otherwise, we would have been able to measure simultaneously enzymatic fluxes through five different cycles operating during glutamate metabolism in rabbit kidney tubules as considered in our mathematical model of glutamate metabolism in the accompanying paper (44).…”

“…As will be seen below and in the accompanying paper (44), further developments of our modeling approach allowed us to measure not only fluxes in three different cycles as in our previous study (6) but also unidirectional fluxes at the level of glutamate dehydrogenase allowing us therefore to measure enzymatic fluxes in a fourth cycle, i.e. the "Glu 3 ␣KG 3 Glu" cycle.…”

“…In an attempt to clarify this subject, we decided to study glutamate metabolism in rabbit kidney tubules; for this, we conducted a study involving metabolic balance of substrate, incorporation of label into metabolites of glutamate, and modeling of glutamate metabolism (44).…”

Complexity of glutamine metabolism in kidney tubules from fed and fasted rats

“…For this, we have incubated isolated kidney tubules in the presence of 13 C-labelled and 14 C-labelled glutamine, and have taken advantage of the use of 13 C NMR spectroscopy, which has allowed us to unravel the complexity of the renal metabolism of various substrates in previous studies from this laboratory [34][35][36][37]. With the data obtained, which were combined with a model of glutamate and glutamine metabolism developed previously [38], we were able to estimate the fluxes through enzymes of glutamine metabolism in kidney tubules from fed and fasted rats.…”

Nuclear Magnetic Resonance Spectroscopy: A Non-Invasive Probe of Kidney Metabolism and Function

“…As an example, 31 P NMR studies have compared the effect of organ preservation solutions (Ringers, Eurocollins and University of Wisconsin), and storage conditions, on high-energy cellular phosphates in rat and rabbit models, providing new insights into reperfusion injury and kidney viability in transplantation [1, 20]. In a similar manner, 13 C NMR spectroscopy, using 13 C-labelled substrates (such as glycerol, citrate, alanine), has been used with success to trace substrate utilisation and metabolic fluxes through enzymes involved in glucose metabolism, gluconeogenesis, and other cycles operating simultaneously [18, 21]. By monitoring the relative enrichment of 13 C into substrates synthesised by these cycles, the effect of diabetes, induced by streptozotocin in rats, to increase the rate of gluconeogenesis in this model and that of chronic acidosis has been shown [18].…”