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The Anaplerotic Substrate Alanine Stimulates Acetate Incorporation into Glutamate and Glutamine in Rabbit Kidney Tubules

2002; Elsevier BV; Volume: 277; Issue: 33 Linguagem: Inglês

10.1074/jbc.m111335200

1083-351X

Agnès Conjard, Sylvie Dugelay, Marie-France Chauvin, Daniel Durozard, Gabriel Baverel, Guy Martin,

Muscle metabolism and nutrition

Although acetate, the main circulating volatile fatty acid in humans and animals, is metabolized at high rates by the renal tissue, little is known about the precise fate of its carbons and about the regulation of its renal metabolism. Therefore, we studied the metabolism of variously labeled [13C]acetate and [14C]acetate molecules and its regulation by alanine, which is also readily metabolized by the kidney, in isolated rabbit renal proximal tubules. With acetate as the sole substrate, 72% of the C-1 and 49% of the C-2 of acetate were released as CO2; with acetate plus alanine, the corresponding values were decreased to 49 and 25%. The only other important products formed from the acetate carbons were glutamine, and to a smaller extent, glutamate. By combining 13C NMR and radioactive and enzymatic measurements with a novel model of acetate metabolism, fluxes through the enzymes involved were calculated. Thanks to its anaplerotic effect, alanine caused a stimulation of acetate removal and a large increase in fluxes through pyruvate carboxylase, citrate synthase, and the enzymes involved in glutamate and glutamine synthesis but not in flux through α-ketoglutarate dehydrogenase. We conclude that the anaplerotic substrate alanine not only accelerates the disposal of acetate but also prevents the wasting of the latter compound as CO2. Although acetate, the main circulating volatile fatty acid in humans and animals, is metabolized at high rates by the renal tissue, little is known about the precise fate of its carbons and about the regulation of its renal metabolism. Therefore, we studied the metabolism of variously labeled [13C]acetate and [14C]acetate molecules and its regulation by alanine, which is also readily metabolized by the kidney, in isolated rabbit renal proximal tubules. With acetate as the sole substrate, 72% of the C-1 and 49% of the C-2 of acetate were released as CO2; with acetate plus alanine, the corresponding values were decreased to 49 and 25%. The only other important products formed from the acetate carbons were glutamine, and to a smaller extent, glutamate. By combining 13C NMR and radioactive and enzymatic measurements with a novel model of acetate metabolism, fluxes through the enzymes involved were calculated. Thanks to its anaplerotic effect, alanine caused a stimulation of acetate removal and a large increase in fluxes through pyruvate carboxylase, citrate synthase, and the enzymes involved in glutamate and glutamine synthesis but not in flux through α-ketoglutarate dehydrogenase. We conclude that the anaplerotic substrate alanine not only accelerates the disposal of acetate but also prevents the wasting of the latter compound as CO2. pyruvate carboxylase phosphoenolpyruvate carboxykinase oxalacetate metabolite pyruvate kinase α-ketoglutarate α-ketoglutarate dehydrogenase Acetate is the main circulating volatile fatty acid in humans and other mammalian species. Its blood concentration is low (less than 0.2 mm) in fed and starved humans and starved herbivores but may reach the millimolar range in humans after alcohol consumption and in fed herbivorous species (1Lundquist F. Tygstrup N. Winkler K. Mellemgaard K. Munck- Petersen S. J. Clin. Invest. 1962; 41: 955-996Crossref PubMed Scopus (204) Google Scholar, 2Ballard F.J. Am. J. Clin. Nutr. 1972; 25: 773-779Crossref PubMed Scopus (113) Google Scholar, 3Knowles S.E. Jarrett I.G. Filsell O.H. Ballard F.J. Biochem. J. 1974; 142: 401-411Crossref PubMed Scopus (263) Google Scholar, 4Skutches C.L. Holroyde C.P. Myers R.N. Paul P. Reichard G.A. J. Clin. Invest. 1979; 64: 708-713Crossref PubMed Scopus (135) Google Scholar, 5Pomare E.W. Branch W.J. Cummings J.H. J. Clin. Invest. 1985; 75: 1448-1454Crossref PubMed Scopus (227) Google Scholar, 6Bergman E.N. Physiol. Rev. 1990; 70: 567-590Crossref PubMed Scopus (1576) Google Scholar, 7Pouteau E. Piloquet H. Maugeais P. Champ M. Dumon H. Nguyen P. Krempf M. Am. J. Physiol. 1996; 271: E58-E64PubMed Google Scholar). The sources of blood acetate are on the one hand absorption of the acetate formed as a result of gastrointestinal bacterial fermentation, and on the other hand, the acetate formed and released by various tissues containing acetyl-CoA hydrolase activity (2Ballard F.J. Am. J. Clin. Nutr. 1972; 25: 773-779Crossref PubMed Scopus (113) Google Scholar, 3Knowles S.E. Jarrett I.G. Filsell O.H. Ballard F.J. Biochem. J. 1974; 142: 401-411Crossref PubMed Scopus (263) Google Scholar, 6Bergman E.N. Physiol. Rev. 1990; 70: 567-590Crossref PubMed Scopus (1576) Google Scholar, 8Zhang Y. Agarwal K.C. Beylot M. Soloviev M.V. David F. Reider M.W. Anderson V.E. Tserng K-Y. Brunengraber H. J. Biol. Chem. 1994; 269: 11025-11029Abstract Full Text PDF PubMed Google Scholar). On the basis of experiments performed in vivo with labeled acetate, it has been shown that the turnover of circulating acetate is rapid and that, depending on the species and nutritional state, the oxidation of this compound provides from 6 to 70% of the whole body energy expenditure (3Knowles S.E. Jarrett I.G. Filsell O.H. Ballard F.J. Biochem. J. 1974; 142: 401-411Crossref PubMed Scopus (263) Google Scholar, 4Skutches C.L. Holroyde C.P. Myers R.N. Paul P. Reichard G.A. J. Clin. Invest. 1979; 64: 708-713Crossref PubMed Scopus (135) Google Scholar, 7Pouteau E. Piloquet H. Maugeais P. Champ M. Dumon H. Nguyen P. Krempf M. Am. J. Physiol. 1996; 271: E58-E64PubMed Google Scholar, 9David F. Beylot M. Reider M.W. Aderson V.E. Brunengraber H. Anal. Biochem. 1994; 218: 143-148Crossref PubMed Scopus (13) Google Scholar). This means that acetate is removed and metabolized by peripheral tissues. Indeed, acetyl-CoA synthetase, the enzyme that initiates acetate degradation, has been demonstrated to be active in many tissues including the liver, kidney, heart, brain, adipose tissue, and skeletal muscle (3Knowles S.E. Jarrett I.G. Filsell O.H. Ballard F.J. Biochem. J. 1974; 142: 401-411Crossref PubMed Scopus (263) Google Scholar). It has been found that, besides the heart, the kidney contains a high activity of this enzyme (3Knowles S.E. Jarrett I.G. Filsell O.H. Ballard F.J. Biochem. J. 1974; 142: 401-411Crossref PubMed Scopus (263) Google Scholar). In agreement with this observation, we have shown in a recent study that acetate is readily metabolized by suspensions of rabbit renal proximal tubules (10Dugelay S. Chauvin M.F. Mégnin-Chanet F. Martin G. Laréal M.C. Lhoste J.M. Baverel G. Biochem. J. 1999; 342: 555-566Crossref PubMed Google Scholar). In the same study (10Dugelay S. Chauvin M.F. Mégnin-Chanet F. Martin G. Laréal M.C. Lhoste J.M. Baverel G. Biochem. J. 1999; 342: 555-566Crossref PubMed Google Scholar), we have demonstrated that acetate significantly altered the metabolism of alanine, a major precursor of glutamine in these tubules. For this, we used13C-labeled alanine and unlabeled acetate in combination with enzymatic and 13C NMR spectroscopy measurements to calculate metabolic fluxes related to alanine metabolism. In an attempt to identify precisely the metabolic fate of acetate carbons and gain insight into the reciprocal effect of alanine on acetate metabolism, we have conducted concomitantly a study in which we incubated rabbit renal proximal tubules with 13C-labeled acetates in the absence and the presence of unlabeled alanine. Thanks to the development of a novel model of acetate metabolism that is of general use (see “”) and to the combination of enzymatic, radioactive, and 13C NMR measurements, we were able to estimate fluxes through enzymes involved in acetate metabolism in rabbit renal proximal tubules. We showed that the addition of alanine, which increased the removal of acetate, also stimulated its metabolism through citrate synthase but not through α-ketoglutarate dehydrogenase. We also demonstrated that acetate carbons were converted to different extents not only into CO2 but also into glutamate and glutamine, especially in the presence of the anaplerotic substrate alanine. Sodium acetate, l-alanine, and glutaminase (grade V) were from Sigma. Other enzymes and coenzymes were purchased from Roche Molecular Biochemicals. [1-14C]acetate (2.05 GBq/mmol) and [2-14C]acetate (1.85 GBq/mmol) were obtained from the Commissariat à l'Energie Atomique (Saclay, France). [1-13C]acetate and [2-13C]acetate were obtained from the Commissariatà l'Energie Atomique and had a 90 and 99% isotopic abundance, respectively. Female rabbits (1.8–2 kg; New Zealand albino strain) were obtained from the Elevage des Dombes (Châtillon-sur-Chalaronne, France) and were fed a standard diet (Usine d'Alimentation Rationnelle, Villemoisson-sur-Orge, France). Kidney cortex tubules were prepared by the treatment of renal cortex slices with collagenase as described by Baverel et al. (11Baverel G. Bonnard M. d'Armagnac de Castanet E. Pellet M. Kidney Int. 1978; 14: 567-575Abstract Full Text PDF PubMed Scopus (38) Google Scholar). Incubations other than those involving radioactive substrates were performed for 60 min at 37 °C in a shaking water bath in 25 ml stoppered Erlenmeyer flasks in an O2/CO2 (19/1) atmosphere. The flasks contained 1 ml of the suspension plus 3 ml of Krebs-Henseleit medium (12Krebs H.A. Henseleit K. Hoppe-Seyler's Z. Physiol. Chem. 1932; 210: 33-66Crossref Scopus (1970) Google Scholar), either supplemented or not with substrates, i.e. 5 mm(final concentration) [1-14C]acetate or [2-14C]acetate (103 Bq/flask), [1-13C]acetate or [2-13C]acetate in the absence and the presence of 5 mm l-alanine. These differently labeled acetates were used in an attempt to completely define the fate of the two acetate carbons. In all experiments, each experimental condition was performed in quadruplicate. Incubation was stopped by the addition of HClO4 (final concentration 2% (v/v)) to each flask. In all experiments, zero-time flasks, with and without substrates, were prepared by the addition of HClO4 before the tubules. When radioactive acetate was present in the medium, incubation, deproteinization, collection, and measurement of the14CO2 formed were performed as described by Baverel and Lund (13Baverel G. Lund P. Biochem. J. 1979; 184: 599-606Crossref PubMed Scopus (71) Google Scholar). After removal of the denaturated protein by centrifugation, the supernatant was neutralized with a mixture of 20% (w/v) KOH and 1% (v/v) H3PO4 (8 m) for metabolite determination and NMR spectroscopy. Lactate, pyruvate, glucose, glutamate, glutamine, ammonia, alanine, citrate, α-ketoglutarate, fumarate, malate, acetate, acetoacetate, and 3-hydroxybutyrate as well as the dry weight of tubules added to the flasks were determined as described previously (11Baverel G. Bonnard M. d'Armagnac de Castanet E. Pellet M. Kidney Int. 1978; 14: 567-575Abstract Full Text PDF PubMed Scopus (38) Google Scholar, 13Baverel G. Lund P. Biochem. J. 1979; 184: 599-606Crossref PubMed Scopus (71) Google Scholar). Serine was measured by high pressure liquid chromatography with the use of the Pico-Tag method (14Cohen S.A. Meys M. Tarvin T.L. A Manual of Advanced Techniques for Amino Acid Analysis. Millipore Corporation, Bedford, MA1989: 1-123Google Scholar). Perchloric acid extracts were neutralized (11Baverel G. Bonnard M. d'Armagnac de Castanet E. Pellet M. Kidney Int. 1978; 14: 567-575Abstract Full Text PDF PubMed Scopus (38) Google Scholar), freeze-dried, and reconstituted in D2O in the presence of [213-C]glycine as internal standard. Data were recorded as described previously (15Chauvin M.F. Mégnin-Chanet F. Martin G. Lhoste J.M. Baverel G. J. Biol. Chem. 1994; 269: 26025-26033Abstract Full Text PDF PubMed Google Scholar, 16Chauvin M.F. Mégnin-Chanet F. Martin G. Mispelter J. Baverel G. J. Biol. Chem. 1997; 272: 4705-4716Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) at 100.6 MHz on a Bruker AM-400 WB spectrometer with a 10-mm broadband probe thermostatically controlled at 8 ± 0.5 °C. Acquisition parameters were as follows: spectral width, 25,000 Hz; tilt angle, 90°; data size, 32,000; repetition time, 30 s; number of scans, 2700; Proton decoupling was carried out during the data acquisition (0.65 s) using a standard WALTZ 16 pulse sequence for inverse-gated proton decoupling (17Shaka A.J. Keeler J. Frenkiel T. Freeman R. J. Magn. Reson. 1983; 52: 335-338Google Scholar). Assignments were made by comparing the chemical shifts obtained with published values (18Canioni P. Alger J.R. Shulman R.G. Biochemistry. 1983; 22: 4974-4980Crossref PubMed Scopus (137) Google Scholar,19Howarth O.W. Lilley D.J. Emsley J.W. Feeney J. Sutcliffe L.H. Progress in Nuclear Magnetic Resonance Spectroscopy. 12. Pergamon Press, Oxford1978: 1-40Google Scholar). Net substrate utilization and product formation were calculated as the difference between the total flask contents (tissue plus medium) at the start (zero-time flasks) and after the period of incubation. The metabolic rates, reported as means ± S.E., are expressed in μmol of substances removed or produced per flask per unit time (60 min). The rates of release of 14CO2 from the14C-labeled acetate species used were calculated by dividing the radioactivity in 14CO2 by the specific radioactivity of the labeled carbon of the acetate species of interest measured in each medium. When [13C]acetate species were the substrate, the transfer of the C-1 or C-2 of acetate to a given position in a given metabolite was calculated from (Lm − lm)/(As− as), in which Lm is the amount of 13C measured in the corresponding NMR resonance, lm is the natural abundance (1.1%) multiplied by the amount of metabolite assayed enzymatically, Asis the total 13C abundance of the C-1 or C-2 of acetate, and as is the natural 13C abundance. To determine the fate of acetate carbons and the metabolic pathways involved, experiments were performed in which rabbit renal proximal tubules were incubated with differently 14C- and13C-labeled acetates with and without alanine. Substrate utilization and product formation were measured by combining enzymatic, radioactive, and 13C NMR spectroscopy measurements. Substrate removal and glutamate plus glutamine accumulation and labeling were approximately linear with time over a 60-min incubation period (n = 2 in duplicate; results not shown). Table I shows that, when acetate was added as the sole exogenous substrate into the incubation medium, it was readily removed by rabbit renal proximal tubules. Although under this condition, no exogenous nitrogenous substrate was provided to the renal cells, they accumulated substantial amounts of glutamine, and to a lesser extent, of glutamate and serine. The fact that substantial amounts of glutamine were also synthesized in the absence of added substrate (Table I) means that, in the presence of acetate, these amino acids were formed, at least in part, from endogenous sources. Some acetoacetate was also formed in the presence of acetate as the sole exogenous substrate (Table I).Table IEffect of 5 mM L-alanine on the metabolism of 5 mM acetate in rabbit kidney tubulesExperimental conditionMetabolite removal (−) or production (μmol/hr)AcetateAlanineGlutamineGlutamateLactatePyruvateSerineAcetoacetate5 mm Acetate−8.26 ± 0.300.00 ± 0.011.57 ± 0.050.34 ± 0.03−0.10 ± 0.050.03 ± 0.010.00 ± 0.010.45 ± 0.025 mm Acetate + 5 mm Alanine−10.91 ± 0.44 ***−12.28 ± 0.72 ***4.64 ± 0.31 ***2.38 ± 0.08 ***0.46 ± 0.04 ***0.17 ± 0.01 **0.35 ± 0.140.13 ± 0.01 ***No added substrate0.15 ± 0.23−0.02 ± 0.012.32 ± 0.01−0.07 ± 0.01−0.15 ± 0.020.20 ± 0.23−0.02 ± 0.010.03 ± 0.01 Open table in a new tab As shown in Table I, and in agreement with our recent results (10Dugelay S. Chauvin M.F. Mégnin-Chanet F. Martin G. Laréal M.C. Lhoste J.M. Baverel G. Biochem. J. 1999; 342: 555-566Crossref PubMed Google Scholar), alanine was also metabolized at high rates by the renal tubules when this amino acid was added as substrate together with acetate. Alanine addition caused a 32% stimulation of acetate removal and a great increase in glutamate and glutamine accumulation. In the presence of alanine, acetoacetate accumulation was also significantly reduced, and small amounts of pyruvate and lactate accumulated. Nitrogen balance calculations indicate that, in the presence of alanine, no significant room was left for glutamine and glutamate synthesis from endogenous substrates. Under none of the experimental conditions studied did we observe any substantial accumulation of glucose, ammonia, β-hydroxybutyrate, or intermediates of the tricarboxylic acid cycle. Table II shows that, with acetate as the sole substrate, 72% of the C-1 and 49% of the C-2 of the acetate removed were released as CO2. This clearly indicates that, under this condition, the remainder of the C-1 and C-2 of the acetate removed was incorporated into the non-volatile carbon products found to accumulate, namely acetoacetate, glutamine, and glutamate.Table IIEffect of 5 mM alanine on the release of 14CO2 from 5 mM [1-14C]acetate and [2-14C]acetate in rabbit kidney tubulesExperimental condition14CO2 from [1-14C]acetate14CO2 from [2-14C]acetate5 mm [14C] acetate5.96 ± 0.124.05 ± 0.055 mm [14C] acetate + 5mmalanine5.31 ± 0.112.70 ± 0.05* Open table in a new tab The release of 14CO2 from [1-14C]acetate did not change upon the addition of alanine; under this condition, it represented only 49% of the C-1 of the acetate removed. By contrast, the presence of alanine significantly diminished the production of 14CO2 from [2-14C]acetate; under the latter condition, the production of CO2 accounted for only 25% of the C-2 of the acetate removed. Fig.1, A and B, shows the 13C NMR spectra of perchloric acid extracts obtained after 60 min of incubation of renal tubules with [2-13C]acetate in the absence and the presence, respectively, of alanine. As all the C-1 and C-2 of the14C-labeled acetates removed could not be accounted for by the production of 14CO2 (Table II), it is not surprising that a significant amount of the C-2 of [2-13C]acetate removed was recovered in glutamine and glutamate, especially in the presence of alanine. No substantial amount of lactate, acetoacetate, or serine was found to be labeled. Using these spectra and those obtained with [1-13C]acetate as substrate without and with alanine (results not shown), we calculated the amount of labeled products after correction for the 13C natural abundance (Tables III andIV).Table IIIEffect of 5 mM alanine on the metabolism of 5 mM [2-13C]acetate in rabbit kidney tubulesExperimental conditionRate of labeled product accumulationGlutamateGlutamineC2C3C4C2C3C4[2-13C]Acetate0.35 ± 0.030.29 ± 0.020.53 ± 0.050.44 ± 0.020.42 ± 0.010.82 ± 0.02[2-13C]Acetate + Alanine0.47 ± 0.030.41 ± 0.021.81 ± 0.080.66 ± 0.030.74 ± 0.123.06 ± 0.29********** Open table in a new tab Table IVEffect of 5 mm alanine on the metabolism of 5 mm [1-13C]acetate in rabbit kidney tubulesExperimental conditionRate of labeled product accumulationGlutamateGlutamineC1C5C1C5[1-13C]Acetate0.10 ± 0.010.36 ± 0.070.16 ± 0.020.64 ± 0.06[1-13C]Acetate + Alanine0.29 ± 0.011.98 ± 0.110.43 ± 0.012.90 ± 0.28****** Open table in a new tab With [2-13C]acetate as substrate (Table III), the high labeling of the C-4 of glutamate and glutamine indicates that the C-2 of acetate gave the C-2 of acetyl-CoA, and then gave the C-4 of citrate, α-ketoglutarate, glutamate, and glutamine, via the successive operation of acetyl-CoA synthetase, citrate synthase, aconitase, isocitrate dehydrogenase, alanine, or (in the absence of alanine) other amino acid aminotransferases and glutamine synthetase. The fact that virtually equal amounts of C-2 and C-3 of glutamate plus glutamine were labeled is consistent with the previous observations made by other authors with acetate and other substrates in kidney and other tissues (20Yang D. Previs S.F. Fernandez C.A. Dugelay S. Soloviev M.V. Hazey J.W. Argawal K.C. Levine W.C. David F. Rinaldo P. Beylot M. Brunengraber H. Am. J. Physiol. 1996; 270: E882-E889PubMed Google Scholar, 21Carvalho R.A. Babcock E.E. Jeffrey F.M. Sherry A.D. Malloy C.R. Magn. Reson. Med. 1999; 42: 197-200Crossref PubMed Scopus (15) Google Scholar, 22Jucker B.M. Lee J.Y. Shulman R.G J. Biol. Chem. 1998; 273: 12187-12194Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 23Large V. Brunengraber H. Odeon M. Beylot M. Am. J. Physiol. 1997; 272: E51-E58PubMed Google Scholar, 24Jans A.W. Leibfritz D. NMR Biomed. 1989; 1: 171-176Crossref PubMed Scopus (23) Google Scholar). This is also in agreement with the view that the C-2 of acetate passed through succinate and fumarate, two symmetrical molecules, during the first tricarboxylic acid cycle turn, leading to the formation of either [2-13C]oxalacetate or [3-13C]oxalacetate and then to either [3-13C]citrate or [2-13C]citrate and α-ketoglutarate during the second tricarboxylic acid cycle turn, yielding finally glutamate and glutamine labeled on their C-2 and C-3 after the transaminase and glutamine synthetase reactions. Consistent with the increase in the synthesis of glutamine plus glutamate shown in Table I, the addition of alanine caused an increase in the incorporation of the C-2 of acetate into the C-2, C-3, and C-4 of glutamate and glutamine (Table III). It should be noted that a fraction of the glutamate and glutamine molecules formed from [2-13C]acetate were simultaneously labeled on their C-3 and C-4 as revealed by the doublets that indicate 13C-13C couplings between these two glutamine and glutamate carbons (Fig. 1). However, as the spectral resolution was not sufficient to quantify these doublets in a reliable manner, no attempt was made to quantify them to determine the relative proportions of labeled and unlabeled oxalacetate and acetyl-CoA molecules contributing to the synthesis of glutamate and glutamine. Table IV shows as expected that, with [1-13C]acetate as substrate and both in the absence and the presence of alanine, the labeling of the C-5 of glutamate and glutamine was close to the labeling of the C-4 of these two amino acids observed when [2-13C]acetate was the substrate (see Table III for comparison). The labeling of the C-1 of glutamate and glutamine is in agreement with the conversion of the C-1 of acetate into the C-4 of oxalacetate during the first tricarboxylic acid cycle turn and then the formation of the C-1 of citrate and α-ketoglutarate during the second tricarboxylic acid cycle turn before the transamination of α-ketoglutarate into glutamate followed by glutamine synthesis. The fact that the labeling of the C-1 of glutamate and glutamine when [1-13C]acetate was the substrate was smaller than the labeling of either the C-2 or the C-3 of glutamate and glutamine when [2-13C]acetate was the substrate is not surprising because, in the presence of [1-13C]acetate, the label found in the C-1 and the C-4 of oxalacetate after the first tricarboxylic acid cycle turn was lost as CO2 during the second turn via the isocitrate dehydrogenase and the α-ketoglutarate dehydrogenase step, respectively. As already seen with the incorporation of the C-2 of acetate into the C-2, C-3, and C-4 of glutamate and glutamine (Table III), incorporation of the C-1 of acetate into the C-1 and C-5 of glutamate and glutamine was increased in the presence of alanine. Table V shows the calculated proportions of metabolites converted into the next one(s). It should be mentioned here that, with the model used, these proportions allowed us to calculate enzymatic fluxes only when they were combined with the utilization of the substrate(s) of interest. As can be seen in Table V, some proportions could not be calculated when acetate was the sole substrate. This is because, under this condition, the 13C resonances were not great enough to allow us to quantify the corresponding conversions. The fraction of pyruvate converted into oxalacetate, which was close to unity in the absence of alanine, was significantly decreased in the presence of alanine, whereas a significant fraction of pyruvate was converted into acetyl-CoA only in the presence of alanine.Table VVarious proportions through pathways of acetate metabolism in the absence or in the presence of 5 mm alanine in rabbit kidney tubulesProportion ofConverted toParameter notationParameter valueWithout alanineWith alanineOAACita0.94 ± 0.01OAAPEP5-aPEP, P-enolpyruvate.1−a0.06 ± 0.01PEPPyrr1.000.66 ± 0.12PyrOAAc0.99 ± 0.030.60 ± 0.04**PyrAcCoAd0.000.35 ± 0.04**AcCoACit1−u0.94 ± 0.010.99 ± 0.01***AcCoAAcAcu0.06 ± 0.010.01 ± 0.01***αKGOAAs″0.83 ± 0.010.55 ± 0.01***αKGaccumulated Glx1−s″0.17 ± 0.010.45 ± 0.01***Proportion5-bOf recycling at each turn.Parameter notationParameter value with alanineIn TCA5-cTCA, tricarboxylic acid.cycleg =a·s″0.51 ± 0.02in OAA → PEP → Pyr → AcCoA → OAA cyclez = (1 −a)·r·d·(1 − u)·s″0.01 ± 0.01in OAA → PEP → Pyr → OAA cycleh = (1 −a)·r·c0.02 ± 0.01Equivalent recycling factor5-dIntroduced to explain total OAA formation.F0.61 ± 0.025-a PEP, P-enolpyruvate.5-b Of recycling at each turn.5-c TCA, tricarboxylic acid.5-d Introduced to explain total OAA formation. Open table in a new tab The decrease in the fraction of acetyl-CoA converted into acetoacetate caused by the addition of alanine was fully compensated by an increase in the acetyl-CoA converted into citrate. The addition of alanine also significantly increased the proportion of α-ketoglutarate converted into glutamate plus glutamine at the expense of the proportion of α-ketoglutarate converted into oxalacetate (Table V). Table V also shows that the proportions of the oxalacetate recycled at each turn of the three different cycles could be quantified only in the presence of alanine. Table VI shows the absolute values of fluxes through enzymes involved in acetate metabolism both in the absence and the presence of alanine. Fluxes through acetyl-CoA synthetase were identical to the values of acetate removal reported in Table I. Fluxes through 3-ketothiolase were also identical to the acetoacetate accumulations presented in Table Ibecause there was no evidence of β-hydroxybutyrate accumulation. On the addition of alanine, flux through acetyl-CoA synthetase was stimulated, whereas that through 3-ketothiolase was inhibited. In the absence of alanine, the minimum flux through pyruvate carboxylase (1.35 μmol/h; in Table VI, see {PC} − {PEPCK})1 logically matches the exit of α-ketoglutarate from the tricarboxylic acid cycle to form glutamate and glutamine (in Table VI, see Glx accumulated). It should be mentioned that the small 13C labeling data obtained did not provide evidence for the existence of the OAA → P-enolpyruvate → Pyr → OAA cycle. Whatever the precise value in the presence of acetate as the sole substrate, the anaplerotic flux, mainly represented by {PC} − {PEPCK}, was stimulated by the addition of alanine.Table VIEffect of 5 mm alanine on fluxes through pathways of acetate metabolism in rabbit kidney tubulesExperimental conditionAcetyl-CoA synthetase3-Keto thiolase{PC}{PEPCK}{PC} − {PEPCK}{PK}{PC} − {PK}5 mmacetate8.26 ± 0.300.45 ± 0.021.35 ± 0.641.35 ± 0.645 mm acetate + 5 mmalanine10.91 ± 0.440.13 ± 0.017.97 ± 0.400.94 ± 0.107.03 ± 0.360.59 ± 0.077.38 ± 0.45************Experimental conditionPyruvate dehydrogenaseCitrate synthaseα-ketoglutarate dehydrogenasenet {αKG → Glu} = Glx accumulatedGlutamine synthetase5 mmacetate0.007.81 ± 0.316.46 ± 0.271.35 ± 0.061.00 ± 0.075 mm acetate + 5 mmalanine4.89 ± 0.9515.67 ± 0.828.64 ± 0.577.03 ± 0.364.64 ± 0.31******** Open table in a new tab Table VI also shows that the differences of fluxes between pyruvate carboxylase and phosphoenolpyruvate carboxykinase on the one hand and between pyruvate carboxylase and pyruvate kinase on the other hand were identical in the presence of acetate as the sole substrate. This means that, under this condition, fluxes through phosphoenolpyruvate carboxykinase and through pyruvate kinase were also identical, indicating that there was no output of phosphoenolpyruvate from the OAA → P-enolpyruvate → Pyr → OAA cycle. A flux through pyruvate dehydrogenase was observed only in the presence of alanine; under this condition, the absence of labeling of the C-5 of glutamate and glutamine from [2-13C]acetate indicates that pyruvate dehydrogenase metabolized to a significant extent only unlabeled pyruvate formed from the added unlabeled alanine. Similarly, flux through lactate dehydrogenase was derived from the accumulation of lactate shown in Table I because no 13C-labeled lactate was found to accumulate. Table VI also shows that the addition of alanine caused a doubling of flux through citrate synthase but no statistically significant increase in flux through α-ketoglutarate dehydrogenase because the increased synthesis of α-ketoglutarate was accompanied by an augmented conversion of α-ketoglutarate into glutamate and glutamine. This is in agreement with the increased labeling of glutamate and glutamine carbons from the 13C-labeled acetates (Fig. 1 and Tables III and IV). The increased accumulation of glutamate plus glutamine and the stimulation of flux through glutamine synthetase are also in agreement with the results presented not only in Tables I, III, and IV but also in Fig. 1. Thanks to enzymatic, radioactive, and 13C NMR measurements in combination with an original model of acetate metabolism that is of general use, this study not only establishes the fate of acetate in rabbit renal proximal tubules in the absence and the presence of alanine but also provides a precise quantification of fluxes through the enzymes related to acetate metabolism. Confirming previous results (10Dugelay S. Chauvin M.F. Mégnin-Chanet F. Martin G. Lare