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• W2004149087 abstract "13C and 1H NMR spectroscopy was used to investigate the metabolism ofl-lactate and d-glucose in C6 glioma cells. The changing of lactate and glucose concentration in the extracellular medium of C6 glioma cells incubated with 5.5 mm glucose and 11 mm lactate indicated a net production of lactate as the consequence of an active aerobic glycolysis. The 13C enrichments of various metabolites were determined after 4-h cell incubation in media containing both substrates, each of them being alternatively labeled in the form of either [3-13C]l-lactate or [1-13C]d-glucose. Using 11 mm[3-13C]l-lactate, the enrichment of glutamate C4, 69%, was found higher than that of alanine C3, 32%, when that of acetyl-CoA C2 was 78%. These results indicated that exogenous lactate was the major substrate for the oxidative metabolism of the cells. Nevertheless, an active glycolysis occurred, leading to a net lactate production. This lactate was, however, metabolically different from the exogenous lactate as both lactate species did not mix into a unique compartment. The results were actually consistent with the concept of the existence of two pools of both lactate and pyruvate, wherein one pool was closely connected with exogenous lactate and was the main fuel for the oxidative metabolism, and the other pool was closely related to aerobic glycolysis. 13C and 1H NMR spectroscopy was used to investigate the metabolism ofl-lactate and d-glucose in C6 glioma cells. The changing of lactate and glucose concentration in the extracellular medium of C6 glioma cells incubated with 5.5 mm glucose and 11 mm lactate indicated a net production of lactate as the consequence of an active aerobic glycolysis. The 13C enrichments of various metabolites were determined after 4-h cell incubation in media containing both substrates, each of them being alternatively labeled in the form of either [3-13C]l-lactate or [1-13C]d-glucose. Using 11 mm[3-13C]l-lactate, the enrichment of glutamate C4, 69%, was found higher than that of alanine C3, 32%, when that of acetyl-CoA C2 was 78%. These results indicated that exogenous lactate was the major substrate for the oxidative metabolism of the cells. Nevertheless, an active glycolysis occurred, leading to a net lactate production. This lactate was, however, metabolically different from the exogenous lactate as both lactate species did not mix into a unique compartment. The results were actually consistent with the concept of the existence of two pools of both lactate and pyruvate, wherein one pool was closely connected with exogenous lactate and was the main fuel for the oxidative metabolism, and the other pool was closely related to aerobic glycolysis. phosphate-buffered saline (0.15 m NaCl, 5.5 mmPi, pH 7.4) 2′,7′-bis(2-carboxyethyl)-5(6)carboxyfluorescein acetomethyl ester 3,3′-dihexyloxacarbocyanine iodine Dulbecco's modified Eagle's medium high performance liquid chromatography specific 13C enrichment acetyl-CoA glutamate C4-specific 13C enrichment/alanine C3-specific 13C enrichment ratio glutamate C4 peak area alanine C3 peak area lactate pyruvate. Neoplastic cells preferentially utilize aerobic glycolysis for their energy needs rather than oxidative phosphorylations, that leads to an important lactate production (1Warburg O. Science. 1956; 123: 309-314Crossref PubMed Scopus (9331) Google Scholar). The reason for this behavior is not yet clearly understood. It has been proposed recently that it would be a means to minimize oxidative stress, in particular during the cell phases linked to biosynthesis and cell division (2Brand K. J. Bioenerg. Biomembr. 1997; 29: 355-364Crossref PubMed Scopus (126) Google Scholar). With regard to a tumor, aerobic glycolysis may increase lactate concentration inside the tumor itself and in its close vicinity (3Argiles J.M. Azcon-Bieto J. Mol. Cell. Biochem. 1988; 81: 3-17Crossref PubMed Scopus (97) Google Scholar, 4Vaupel P. NMR Biomed. 1992; 5: 220-225Crossref PubMed Scopus (55) Google Scholar). Consequently, local changes in metabolic activities could be generated as an increased local lactate concentration provides a possible source for cell energy requirements in competition with glucose or other substrates. There is no data concerning the competition between lactate and glucose as fuel for the oxidative metabolism in glioma cells. It has been only reported that, in vitro, glioma cells utilize glucose, but that once it is depleted, they will take up pyruvate and lactate generated by aerobic glycolysis for further metabolism (5Schwartz J.P. Lust W.D. Lauderdale V.R. Passonneau J.V. Mol. Cell. Biochem. 1975; 9: 67-72Crossref PubMed Scopus (17) Google Scholar).To study the possible competition between glucose and lactate metabolism in glioma cells, we investigated the fate of each of these substrates when both were present in cell medium by using either [3-13C]l-lactate or [1-13C]d-glucose. We used the C6 glioma cell clone for which the metabolism of [1-13C]glucose has been investigated already (6Portais J.C. Schuster R. Merle M. Canioni P. Eur. J. Biochem. 1993; 217: 457-468Crossref PubMed Scopus (97) Google Scholar, 7Portais J.C. Voisin P. Merle M. Canioni P. Biochimie (Paris). 1996; 78: 155-164Crossref PubMed Scopus (62) Google Scholar). The initial glucose concentration (5.5 mm) was chosen higher than the apparentK m for transport (1.7 mm) (8Lust W.D. Schwartz J.P. Passonneau J.V. Mol. Cell. Biochem. 1975; 8: 169-176Crossref PubMed Scopus (28) Google Scholar), whereas the different lactate concentrations used (11, 5.5, and 1.1 mm) were higher or in the same range than the apparentK m for transport (1 mm) (9Dringen R. Peters H. Wiesinger H. Hamprecht B. Dev. Neurosci. 1995; 17: 63-69Crossref PubMed Scopus (47) Google Scholar). The13C enrichment of extra and intracellular lactate and that of various intracellular metabolites was analyzed after a 4 h cell incubation. The results evidence that when lactate concentration was higher than K m, cells preferentially utilize lactate as carbon source for oxidative phosphorylations notwithstanding a high rate of glycolysis. Metabolite enrichments forced the consideration of different intracellular pools for both lactate and pyruvate.EXPERIMENTAL PROCEDURESl-Lactate (sodium salt) was from Sigma. [3-13C]l-Lactate (sodium salt) (99% enrichment) was from Eurisotop. [1-13C]d-Glucose (99% enrichment) was from the Commissariat à l'Energie Atomique (Saclay, France).Cell Incubation with Glucose and LactateC6 cells were cultured in 10-cm diameter dishes (6Portais J.C. Schuster R. Merle M. Canioni P. Eur. J. Biochem. 1993; 217: 457-468Crossref PubMed Scopus (97) Google Scholar). At the end of the growth period, confluent cells taken at least 24 h after the last medium change were washed twice with phosphate-buffered saline (PBS)1 and then incubated in 10 ml of Dulbecco's modified Eagle's medium (DMEM) free of glutamine and pyruvate, but containing 5.5 mmd-glucose and/or 11 mml-lactate. To determine glucose consumption and lactate production (or consumption), 200 μl of medium were sequentially removed after different incubation times. At the end of the incubation (24 h), cells were rinsed twice with PBS. After addition of 5 ml/dish of 0.9 m perchloric acid, the insoluble part of the cellular layer was removed and pelleted by centrifugation. Similar experiments were performed with C6 cells incubated in the presence of 5.5 mm[1-13C]glucose and 11 mml-lactate. At the indicated time, 200 μl of medium were removed for glucose and lactate determinations. The remaining medium in each dish was conditioned for 1H NMR analysis (see below).For cell incubation with [1-13C]d-glucose or [3-13C]l-lactate, approximately 3 × 108 cells (10–15 culture dishes) were used for each experiment. After 4-h incubation, 4-ml aliquots of the medium were removed and subsequently freeze-dried. Cells were then rinsed twice with PBS and their metabolites extracted with 0.9 mperchloric acid (10Pianet I. Merle M. Labouesse J. Canioni P. Eur. J. Biochem. 1991; 195: 87-95Crossref PubMed Scopus (26) Google Scholar).Sample Preparation for NMR AnalysesTo facilitate the analysis of the lactate methyl signal in the 1H NMR spectra of incubation media and cell extracts, the amino acids contained in the samples were eliminated by fixation on a Dowex 50W-X8 column, and glutamate in cell extracts was further purified (11Merle M. Martin M. Villégier A. Canioni P. Eur. J. Biochem. 1996; 239: 742-751Crossref PubMed Scopus (29) Google Scholar).A solution of Ala and Glu in D2O (around 100 mmfor each amino acid) was prepared and used as standard solution for both the quantitative analysis of 13C NMR spectra and the high performance liquid chromatography (HPLC) determination of amino acids in cell extracts.NMR SpectroscopySpectra were obtained with a Bruker DPX400 wide-bore spectrometer equipped with a 5-mm broad-band probe.1H NMR spectra were acquired with 3-μs pulses (41° flip angle), 1-s acquisition time, 6-s relaxation delay, 2.4-kHz sweep width, and 32K memory size. Residual water signal was suppressed by homonuclear presaturation. 13C NMR spectra were acquired at 100.6 MHz. Measurements were conducted at 25 °C under bilevel broad-band gated proton decoupling and D2O lock, using 11.5-μs pulses (59° flip angle), 21.7-kHz sweep width, and 64K memory size. Two relaxation delays, 0.1 and 10 s, were used to check the effect of different acquisition conditions on the quantitative analysis of carbon enrichments.Determination of Metabolite 13C EnrichmentsThe specific 13C enrichments (SE) of Glu C2 and lactate C3 were determined from satellite peak areas resulting from1H-13C spin-coupling on 1H NMR spectra (12Martin M. Portais J.C. Labouesse J. Canioni P. Merle M. Eur. J. Biochem. 1993; 217: 617-625Crossref PubMed Scopus (38) Google Scholar).The ratio (E4/A3) between SE Glu C4 and SE Ala C3 was determined from both peak areas on the 13C NMR spectra and the HPLC determinations of the amino acid contents in cell perchloric acid extracts and in the amino acid standard solution according to Equation 1. This procedure wherein both samples were analyzed by both methods was chosen to enhance the guarantee of the determination by eliminating the risk of bias linked to the use of a standard solution containing not rigorously adjusted Ala and Glu concentrations.E4/A3=SE Glu C4SE Ala C3=(A Glu C4)e(A Ala C3)e×(A Ala C3)s(A Glu C4)s×[Ala]e[Glu]e×[Glu]s[Ala]s(Eq. 1) In this equation, (A Glu C4)e and (A Ala C3)e correspond to peak areas on the 13C NMR spectrum of cell extract, (A Glu C4)s and (A Ala C3)s correspond to peak areas on the spectrum of the amino acid standard solution, [Ala]e and [Glu]ecorrespond to Ala and Glu contents in cell extract, and [Glu]s and [Ala]s correspond to the amino acid contents in the standard solution.The criterion for the relative contributions of lactate and glucose to Glu and Ala syntheses (IL/G) was defined as follows, IL/G=(SE Glu C4)L/(SE Ala C3)L/(SE Glu C4)G/(SE Ala C3)G(Eq. 2) where L or G indicates the incubation with either [3-13C]lactate or [1-13C]glucose as labeled substrate, respectively.The specific enrichment of acetyl-CoA (Ace-CoA) C2 was determined according to the method based on the analysis of the homonuclear spin-coupling pattern of glutamate carbons in the 13C NMR spectrum (13Malloy C.R. Thompson J. Jeffrey F. Sherry A. Biochemistry. 1990; 29: 6756-6761Crossref PubMed Scopus (115) Google Scholar) as follows. (SE Ace­CoA C2)=(A Glu doublet C43)e(A Glu C3)e×(A Glu C3)s(A Glu C4)s(Eq. 3) In this equation, (A Glu doublet C43)e and (A Glu C3)e correspond to the area of the Glu C4 resonance doublet and that of the Glu C3 resonance in the spectrum, respectively.Determination of Metabolite Contents in SamplesGlucose and lactate were determined by enzymatic assays (Sigma kits). Proteins dissolved in a mixture of 0.5 m Tris base, 2% SDS were determined using bovine serum albumin as standard (14Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). The amounts of amino acids (particularly Glu and Ala) in cell perchloric acid extracts and in the standard solution used for the quantitative analysis of NMR spectra were determined by HPLC (15Henrikson R.L. Meredith S.L. Dev. Neurosci. 1984; 1: 226-237Google Scholar). In each case, an internal standard of norleucine (10 nmol) was added to the sample to overcome any bias in the determination.Flow CytometryThe homogeneity of the cell culture was investigated by flow cytometry using a Coulter Elite flow cytometer equipped with a 2025 Spectra Physics argon laser. Viable cells were determined using propidium iodide (16St. John P.A. Kell W.M. Mazzetta J.S. Lange G.D. Barker J.L. J. Neurosci. 1986; 6: 1492-1512Crossref PubMed Google Scholar). The pH of cell cytoplasm was determined using 2′,7′-bis(2-carboxyethyl)-5(6)carboxyfluorescein acetomethyl ester (BCECF-AM) (17Musgrove E. Rugg C. Hedley D. Cytometry. 1986; 7: 347-355Crossref PubMed Scopus (105) Google Scholar). Cells were suspended in DMEM (107 cells/ml) and incubated 30 min at 37 °C with 10 μm BCECF-AM. A 100-μl volume of the cell suspension was then mixed with 900 μl of DMEM containing 5.5 mm glucose and 11 mm lactate before flow cytometry measurement. The standard curve for pH determination was drawn from measurements performed after dilution of 100 μl of the cell suspension in 900 μl of phosphate-buffered saline at different pH values (obtained by mixing in various proportions 135 mmKH2PO4 + 20 mm NaCl with 110 mm K2HPO4 + 20 mm NaCl) and 10-min incubation with 6 μm nigericin. Mitochondrial membrane potential was investigated with 3,3′-dihexyloxacarbocyanine iodine (DiOC6(3)) (18Liu Z. Bushnell W.R. Brambl R. Plant Physiol. 1987; 84: 1385-1390Crossref PubMed Google Scholar). The suspension of cells in DMEM containing 5.5 mmglucose and 11 mm lactate (106 cells/ml) was incubated 10 min at 37 °C with 0.1 μmDiOC6(3) before measurement.Checking of the Relevance of the Proposed Model of Glucose and Lactate Metabolism CompartmentationA model of lactate and glucose metabolic compartmentation was proposed (Fig. 5). It involved the fractional amounts of Glu or Ala synthesized from lactate (Lac), glucose, and other carbon sources: Glu1, Glu2, and Glu3 or Ala1, Ala2, and Ala3, respectively. According to the model, Glu C4 and Ala C3 enrichments would be as follows, (SE Glu C4)L=(SE Lac C3)×Glu1+1.1×(Glu2+Glu3)(Eq. 4) (SE Ala C3)L=(SE Lac C3)×Ala1+1.1×(Ala2+Ala3)(Eq. 5) with [3-13C]lactate as labeled substrate. In these expressions, SE Lac C3 was the mean medium lactate enrichment between 0- and 4-h incubation, starting from either 11 or 5.5 mm[3-13C]lactate, respectively, and 1.1 corresponded to the percent 13C natural abundance,(SE Glu C4)G=(SE Lac C3)×Glu1+(SE Pyr C3)×Glu2+1.1×Glu3(Eq. 6) (SE Ala C3)G=(SE Lac C3)×Ala1+(SE Pyr C3)×Ala2+1.1×Ala3(Eq. 7) with [1-13C]glucose as initial labeled substrate. In these expressions, SE Lac C3 was the mean medium lactate enrichment between 0- and 4-h incubation, starting from either 11 or 5.5 mm lactate and 5.5 mm[1-13C]glucose, and the specific enrichment of glycolytic pyruvate (Pyr) was evaluated from the yield of glucose C1 decarboxylation.These expressions were used to check if there was a set of Gluiand Alai (i = 1–3) values able to agree with the experimental data. The data used in the iterative procedure (11Merle M. Martin M. Villégier A. Canioni P. Eur. J. Biochem. 1996; 239: 742-751Crossref PubMed Scopus (29) Google Scholar) were the ratios E4/A3 = (SE Glu C4)L/(SE Ala C3)L or (SE Glu C4)G/(SE Ala C3)Gdetermined for the experiments carried out at 11 or 5.5 mmlactate using either [3-13C]lactate or [1-13C]glucose as labeled substrate, respectively, (values reported in Table I), the ratios (SE Glu C4)L/(SE Glu C4)G and (SE Ala C3)L/(SE Ala C3)G determined from the 13C NMR spectra (Fig. 2) and the enrichment of Glu C4 obtained after 4 h cell incubation with 5.5 mm glucose and 11 mm[3-13C]lactate. The procedure was not applied to experiments carried out at 1.1 mm lactate because this concentration was not saturating (9Dringen R. Peters H. Wiesinger H. Hamprecht B. Dev. Neurosci. 1995; 17: 63-69Crossref PubMed Scopus (47) Google Scholar).Table I13C enrichments of metabolites after 4 h C6 glioma cell incubation with either [3-13C]lactate or [1-13C]glucoseIncubation conditionLabeled substrateE4/A3IL/GSE Ace-CoA C2SE lactate C3Cell lactateMedium lactate%%5.5 mmglucose[3-13C]Lactate, n = 42.15 ± 0.3577.5 ± 6.0 (p < 0.02)1-bIndicates significantly different values by Student'st test between SE Ace CoA C2 and cellular SE Lac C3.59.0 ± 8.590.0 ± 4.0+(p < 0.015)1-aIndicates significantly different values by Student'st test between E4/A3 values obtained with labeled glucose and lactate.1.75 ± 0.2511 mm lactate[1-13C]Glucose, n = 31.20 ± 0.30ND1-cND, not determined.10.0 ± 2.04.90 ± 1.505.5 mmglucose[3-13C]Lactate, n = 31.60 ± 0.5060.0 ± 5.557.5 ± 6.571.0 ± 4.5+(p < 0.4)1-aIndicates significantly different values by Student'st test between E4/A3 values obtained with labeled glucose and lactate.1.35 ± 0.205.5 mmlactate[1-13C]Glucose, n = 31.20 ± 0.35ND14.5 ± 2.511.5 ± 1.55.5 mmglucose[3-13C]Lactate, n = 20.805 ± 0.140ND19.0 ± 1.027.0 ± 1.5+1.00 ± 0.101.1 mm lactate[1-13C]Glucose,n = 20.805 ± 0.200ND25.5 ± 1.527.0 ± 0.55.5 mmglucose[3-13C]Lactate, n = 31.60 ± 0.4068.5 ± 1.5 (p < 0.3)1-bIndicates significantly different values by Student'st test between SE Ace CoA C2 and cellular SE Lac C3.64.5 ± 5.582.0 ± 4.0+(p < 0.4)1-aIndicates significantly different values by Student'st test between E4/A3 values obtained with labeled glucose and lactate.1.30 ± 0.2011 mmlactate,[1-13C]Glucose, n = 21.20 ± 0.50ND8.95 ± 0.707.90 ± 1.053-h preincubationE4/A3 represents the ratio between the enrichments of Glu C4 and Ala C3, IL/G the ratio between E4/A3 for the incubation with [3-13C]lactate (L), and E4/A3 for the incubation with [1-13C]glucose (G). SE Ace-CoA C2 and SE Lac C3 represent the specific enrichment of acetyl-CoA C2 and lactate C3, respectively.1-a Indicates significantly different values by Student'st test between E4/A3 values obtained with labeled glucose and lactate.1-b Indicates significantly different values by Student'st test between SE Ace CoA C2 and cellular SE Lac C3.1-c ND, not determined. Open table in a new tab Figure 213 C NMR spectra of perchloric acid extracts of cells incubated for 4 h in the presence of glucose and lactate, each of the two substrates being alternately labeled in the form [1-13C]glucose or [3-13C]lactate. Cells were incubated in medium containing 5.5 mm glucose and either 11 mm(spectra A and B), 5.5 mm(spectra C and D), or 1.1 mm(spectra E and F) lactate, respectively. Each couple of spectra (A and B, C and D, and E and F) corresponds to the incubations with [3-13C]lactate (13C-Lac) or [1-13C]glucose (13C-Glc), respectively. Each spectrum was obtained after zero filling to 64K, 1-Hz line-broadening, and Fourier transformation on the free induction decay corresponding to at least an overnight acquisition using 0.1-s relaxation delay. Spectra were plotted such as the signal intensity of 13C natural abundance of inositol carbons used as an internal reference was of the same range, thus making it possible to visualize the relative enrichment of metabolites depending on the labeled substrate. Peak assignments were: 1, alanine C3 (17.6 ppm); 2, lactate C3 (21.7 ppm); 3 γ-Glu, glutathione C3 (27.1 ppm);4, glutamate C3 (27.7 ppm); 5, γ-Glu, glutathione C4 (32.1 ppm); 6, glutamate C4 (34.1 ppm);7, inositol (C4-C6, C2, C1-C3, and C5 at 71.5, 72.7, 73.0, and 74.8 ppm, respectively). For spectra A and B, the ratio between homologous peak areas was 12.3 for Glu C4 and 9.2 for Ala C3, respectively; for spectra C and D, the ratio was 6.8 and 4.5, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)StatisticsThe results are expressed as means ± S.D.; when indicated, the statistical significance of value differences was assessed by Student's t test.DISCUSSIONIn a previous work it has been shown that incubating C6 cells for 4 h with 5.5 mm [1-13C]glucose led to the following specific enrichments: 36.7% for lactate and Ala C3, 27.3% for Ace-CoA C2, and 22.5% for Glu C4, in agreement with a monocompartmental model of cell carbon metabolism (6Portais J.C. Schuster R. Merle M. Canioni P. Eur. J. Biochem. 1993; 217: 457-468Crossref PubMed Scopus (97) Google Scholar). According to its network, the decrease in enrichment from glucose C1 to Glu C4 was the consequence of both the hexose monophosphate shunt activity and several isotopic dilutions occurring at different branch points (pyruvate, Ace-CoA, and 2-oxoglutarate) due to carbon entry from endogenous and exogenous unlabeled sources. In the reported study, E4/A3 was thus less than 1. In contrast, the present study evidenced that when C6 cells were incubated with 5.5 mm glucose and 11 or 5.5 mm [3-13C]lactate, E4/A3 was higher than 1 (Table I). This result was concomitant with a higher enrichment of Ace-CoA C2 than lactate C3 at 11 mm[3-13C]lactate and to similar enrichments at 5.5 mm. These results were unexpected, because [3-13C]lactate was the precursor of [3-13C]pyruvate and thereafter [2-13C]Ace-CoA, keeping in mind the occurrence of isotopic dilutions at the pyruvate and Ace-CoA nodes. Obviously, such an enrichment pattern could not fit with a metabolic network wherein Ace-CoA derived from a unique pool of pyruvate at equilibrium with alanine and lactate through alanine aminotransferase and lactate dehydrogenase activity, respectively. Data analysis needed therefore to reconsider the route of metabolite enrichment upstream from Ace-CoA. Downstream, the isotopic dilution (from Ace-CoA C2 to Glu C4) was very similar under the two incubation conditions (from 77.5 to 69.5% in the present study using 11 mm [3-13C]lactate and from 27.3 to 22.5% in Ref. 6Portais J.C. Schuster R. Merle M. Canioni P. Eur. J. Biochem. 1993; 217: 457-468Crossref PubMed Scopus (97) Google Scholar).Before proposing an explanation to the experimental data, it was critical to emphasize their reliability. First, from spectrum B in Fig. 2, it appears that using 5.5 mm[1-13C]glucose and 11 mm lactate, metabolite enrichment was very poor. This suggested that despite the high rate of cell glucose consumption, the contribution of this compound as Ace-CoA precursor was very low. It could be proposed that the label was lost through the decarboxylation step of the hexose monophosphate shunt. This proposal did not, however, agree with the data in Fig. 1, which indicated that almost all of the label was recovered as [3-13C]lactate. Moreover, the decarboxylation yield can be estimated from the enrichments of lactate C3 in cell media. Indeed, considering the enrichments obtained after 4-h incubation with 5.5 mm glucose and 11 mm lactate, 90 and 4.9% using either lactate or glucose as labeled substrate (Table I), if L0 was the fraction of initial lactate remaining after 4 h and Lg the fraction of lactate formed from glycolysis, lactate enrichment (%) was expressed by 90 = 99 × L0 + 1.1 × Lg when starting from [3-13C]lactate (99 and 1.1% being the initial enrichment and the natural 13C abundance, respectively). As L0 + Lg = 1, it came L0 = 0.91. Then it was possible to express lactate enrichment when starting from labeled [1-13C]glucose: 4.9 = 1.1 × L0 + 1.1 × Lg/2 + A × Lg/2, where A represents the enrichment of the carbon corresponding to glucose C1 (it would be equal to 99 without decarboxylation). The value found was A = 83, indicating only 17% of decarboxylation, which could not explain the poor label incorporation into cell metabolites. The same calculation led to 26, 28.5, and 20.5% decarboxylation when using 5.5 and 1.1 mmlactate and for the experiment, including 3-h preincubation, respectively.Second, during 4-h cell incubation with 5.5 mm glucose and 11 mm lactate, medium glucose decrease and lactate increase were near-linear, indicating that substrate consumption was close to steady state. It has been shown previously that 4-h cell incubation in a medium containing [1-13C]glucose (but no initial lactate) was sufficient to ensure isotopic steady state, particularly for Glu and Ala (6Portais J.C. Schuster R. Merle M. Canioni P. Eur. J. Biochem. 1993; 217: 457-468Crossref PubMed Scopus (97) Google Scholar, 7Portais J.C. Voisin P. Merle M. Canioni P. Biochimie (Paris). 1996; 78: 155-164Crossref PubMed Scopus (62) Google Scholar). In the present study, an isotopic steady-state,stricto sensu, could not occur after only 4 h because medium lactate enrichment was time-decreasing (or increasing) due to the release of unlabeled (or labeled) lactate generated from unlabeled (or labeled) glucose. The demonstration of the existence of different pyruvate and lactate pools (as discussed below) was indeed dependent on this condition. From a theoretical point of view, isotopic steady state could only be expected after total glucose consumption, i.e.after 30–40-h incubation. Nevertheless, starting from 11 mm [3-13C]lactate, the decrease in medium lactate enrichment after 4 h was rather moderate (from 99 to 90%), making it possible to consider that the changes in metabolite enrichment were very slow at this time. The value of IL/G, the criterion for the relative contributions of lactate and glucose to Glu and Ala syntheses, appeared dependent on lactate concentration. It was higher than 1 only at 11 and 5.5 mm lactate (Table I). This indicated primarily that the relative contributions of lactate and glucose to cell metabolism could be only investigated if their enrichments remained very different. This was obviously not the case using 1.1 mm lactate as the exogenous lactate was strongly diluted by glycolytic lactate (27% enrichment after 4 h). The isotopic dilution effects were also apparent when comparing the enrichments obtained at 11 and 5.5 mm lactate (Table I). In particular, Ace-CoA C2 and lactate C3 enrichments were closer at 5.5 mm lactate. As a consequence, data interpretation was focused on the results obtained at 11 mm lactate.From the Glu C4 enrichment (69.5%) and the ratio E4/A3 (2.15, Table I), it follows that Ala C3 enrichment was around 32% when using [3-13C]lactate. Therefore, the fact that the enrichment of Ace-CoA C2 was higher than both that of Ala C3 and intracellular lactate C3 dictated to consider at least two pools of pyruvate. Moreover, the pool corresponding to the Ace-CoA precursor had to display an enrichment of at least 77.5% and thus to derive from an intracellular pool of lactate at least at the same enrichment. As intracellular lactate enrichment was measured to be less (59%), this implied also the existence of two intracellular lactate pools. This situation led to consider that the exogenous lactate was the main mitochondrial substrate (via pyruvate). Therefore, the metabolic pathway including the different steps on the conversion of extracellular lactate into mitochondrial Ace-CoA had to be compartmentalized toward glycolysis. This phenomenological compartmentation is depicted in Fig. 5, wherein compartments 1 and 2 correspond to lactate and glucose metabolism, respectively. However, the fact that E4/A3 remained close to 1 (Table I) when using [1-13C]glucose also needed to be explained. Indeed, if lactate and glucose were the sole carbon sources for Glu and Ala syntheses, the expected value for E4/A3 would be less than 1 when using [1-13C]glucose. The means to explain why the value remained close to 1 was to consider the contribution of unenriched endogenous and exogenous carbon sources to amino acid syntheses. This led to include a third compartment in Fig. 5containing Glu and Ala pools not originating from glucose or lactate metabolism. It was thus interesting to test the relevance of the model by estimating Glu1, Glu2, and Glu3or Ala1, Ala2, and Ala3, the fractional amounts of Glu or Ala synthesized from lactate, glucose, and the other sources, respectively. The procedure involved Equations (Eq. 4), (Eq. 5), (Eq. 6)(see “Experimental Procedures”). In Equation 4 (or 5), lactate C3 enrichment was 95% (or 85%), corresponding to the mean medium lactate enrichment between 0 and 4 h, starting from 11 (or 5.5) mm [3-13C]lactate, respectively. In Equation 6 (or 7), glycolytic pyruvate C3 enrichment was 42.5% (or 37.7%), corresponding to 17% (or 26%) glucose C1 decarboxylation (as discussed above), and the mean enrichment of medium lactate C3 was 3% (or 6.3%), starting from 11 (or 5.5) mm lactate, respectively. The best set of parameter values was: Glu1 = 0.72, Glu2 = 0.09, Glu3 = 0.19, Ala1 = 0.42, Ala2 = 0.10, and Ala3= 0.48. These parameters were determined with a mean relative error of 13.5 ± 5.8% for the variables used in the iterative procedure, indicating a rather good fitting.Instead of intracellular compartmentation of lactate and glucose metabolism, it could be proposed that the different metabolite labeling pattern as a function of the labeled substrate was the consequence of the presence of a mixed population of cells, one that was primarily oxidative in nature and principally oxidizing lactate and some glucose, with no net lactate production from glucose, and the other glycolytic and thus producing lactate, with no oxidation of lactate,i.e. intercellular compartmentation. Such markedly different metabolic behavior could not be the consequence of a lack in oxygen supply to the glycolytic cells, because all cells were incubated in the same conditions, but rather by impairment of their mitochondria. Therefore, a difference in membrane potential between functional and impaired mitochondria would be expected. F" @default.
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• W2004149087 date "1998-10-01" @default.
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• W2004149087 title "Compartmentation of Lactate and Glucose Metabolism in C6 Glioma Cells" @default.
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