INTRODUCTION

Phosphate-activated glutaminase (hereafter referred to as glutaminase), the enzyme which initiates glutamine degradation and releases both glutamate and ammonia, is present in the rabbit kidney (1-3). However, its activity is low when compared with that in the kidney of other species (2, 4). This is not surprising because the rabbit, like other herbivorous species, excretes an alkaline urine (2) that does not need to contain large amounts of ammonium ions. This also explains why rabbit kidney tubules do not readily use glutamine as substrate (5, 6). By contrast, the rabbit kidney has a high capacity to trap ammonia and synthesize glutamine (1, 2, 4-8) by pathways that are far from being fully defined. In his initial work on glutamine synthesis in mammalian tissues (1), Krebs demonstrated that addition of glutamate to the incubation medium significantly increased glutamine synthesis by rabbit kidney cortex slices; since no ammonia accumulated under this condition, he concluded that the ammonia released by the glutamate dehydrogenase reaction was immediately utilized for the synthesis of glutamine (1). Later, Klahr (9) and Klahr et al. (4) have also shown that glucose synthesis by rabbit kidney cortex slices is increased in the presence of glutamate. Since the latter authors observed glucose synthesis without concomitant accumulation of ammonium ions, they concluded, unlike Krebs (1), that glutamate metabolism in rabbit kidney slices is initiated by transamination rather than by oxidative deamination by glutamate dehydrogenase (4). More recently, Watford et al. (10) stated that rabbit kidney tubules do not readily use glutamate or glutamine as gluconeogenic substrate, possibly because of inhibition of glutamate dehydrogenase by ammonia (4). However, none of the above authors measured glutamate uptake in their study. Recent studies in our laboratory (5, 6) have also shown that endogenous glutamate is converted at significant rates into glutamine in isolated rabbit kidney-cortex tubules which contain a high activity of glutamine synthetase (1, 7-9), the enzyme responsible for glutamine synthesis.

Thus, all the above observations suggest that both rabbit renal cortical slices and tubules have the capacity to remove glutamate, but the extent to which the rabbit renal cortex can utilize glutamate as substrate and the pathways involved in glutamate metabolism by this tissue remain uncertain. 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).

Our results clearly establish that rabbit kidney tubules readily utilize glutamate as substrate and convert it mainly into glutamine, alanine, and CO₂ and to a lesser extent into glucose and serine. Modeling of the data obtained by ¹³C NMR spectroscopy indicates that, during glutamate utilization via mainly glutamate dehydrogenase, glutamine synthetase, and alanine aminotransferase, glutamate synthesis also occurs at high rates. In addition, our study demonstrates that addition of acetate reduces net removal of glutamate by inhibiting not only net flux through glutamate dehydrogenase in the oxidative deamination direction but also by inhibiting glutamine, alanine, and serine synthesis. Finally, the rate of the cycle involving pyruvate, oxaloacetate, and phosphoenolpyruvate that was high with glutamate as sole substrate is drastically reduced in the presence of acetate, whereas glucose synthesis is stimulated.

EXPERIMENTAL PROCEDURES

Reagents

Glutaminase (grade V) was from Sigma. Other enzymes and coenzymes were purchased from Boehringer Mannheim (Meylan, France). L-[1-¹⁴C]Glutamate (2.04 GBq/mmol) and L-[1,5-¹⁴C]glutamate (2.15 GBq/mmol) were obtained from Amersham Corp. (Les Ulis, France). L-[U-¹⁴C]Glutamate (9.25 GBq/mmol) and NaH¹⁴CO₃ (2.07 GBq/mmol) were supplied by Dositek (Orsay, France).

L-[1,2-¹³C]Glutamate, L-[3-¹³C]glutamate, and L-[5-¹³C]glutamate (isotopic enrichment of 99% for the three labeled compounds) were obtained from Merck, Sharp and Dohme (Montreal, Canada).

Rabbits

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 (U.A.R., Villemoisson-sur-Orge, France). All experiments were performed with kidney tubules from fed rabbits. Access to water was not limited.

Preparations of Kidney Tubules and Incubations

Kidney cortex tubules were prepared by collagenase treatment of renal cortex slices as described by Baverel et al. (11). Incubations other than those involving radioactive substrates were performed for 30 or 60 min at 37 °C in a shaking water bath, in 25-ml stoppered Erlenmeyer flasks in an atmosphere of O₂/CO₂ (19/1). The flasks contained 1 ml of the tubule suspension plus 3 ml of Krebs-Henseleit medium (12) either unsupplemented or supplemented with substrates, i.e. 5 mM (final concentration) L-[1-¹⁴C]glutamate (10³ Bq/flask), L-[1,5-¹⁴C]glutamate (2. 10³ Bq/flask), L-[U-¹⁴C]glutamate (5. 10³ Bq/flask), L-[1,2-¹³C]glutamate, L-[3-¹³C]glutamate, L-[5-¹³C]glutamate, in the absence and the presence of 10 mM (final concentration) acetate or 25 mM NaH¹⁴CO₃ (10⁶ Bq/flask). These differently labeled glutamate were used in an attempt to define the fate of all the glutamate carbons which depends on the metabolic pathways involved (see Fig. 1). It should be mentioned that the C-1 of glutamate could be released as ¹⁴CO₂ by the -ketoglutarate dehydrogenase reaction. The C-2 and C-5 on one hand, and the C-3 and C-4 on the other hand, were expected to behave symmetrically beyond the stage of succinyl-CoA. Therefore, the [¹⁴C]glutamates used allowed us to measure and/or calculate easily the contribution of each glutamate carbon to the release of CO₂. Finally, ¹⁴C-labeled bicarbonate was used to measure the pyruvate carboxylase-mediated incorporation of the bicarbonate carbon into the C-1 of alanine and of glutamate + glutamine. In all experiments, each experimental condition was performed in quadruplicate. Incubation was stopped by adding perchloric acid (final concentration 2%, v/v) to each flask. Metabolite assays were conducted on the neutralized supernatant. In all experiments, zero time flasks, with and without substrates, were prepared by adding perchloric acid before the tubules. When radioactive glutamate or bicarbonate was present in the medium, incubation, deproteinization, collection, and measurement of the ¹⁴CO₂ formed were performed as described by Baverel and Lund (13). 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, measurement of bicarbonate fixation, and NMR spectroscopy measurements.

Analytical Methods

Glucose, glycogen, lactate, pyruvate, glutamate, glutamine, alanine, aspartate, citrate, -ketoglutarate, fumarate, malate, glycerol, and glycerol 3-phosphate were determined by usual enzymatic methods, and the dry weight of tubules added to the flasks was determined as described previously (11, 13).

These were recorded at 100.6 MHz on a Bruker AM-400 WB spectrometer using a 10-mm broadband probe thermostated at 8 ± 0.5 °C. Magnet homogeneity was adjusted using the deuterium lock signal. Supernatants, obtained from four flasks for each experimental condition with either L-[1,2-¹³C]glutamate, or L[3-¹³C]glutamate, or L-[5-¹³C]glutamate, with or without acetate as substrates, were pooled and lyophilized. Then, the freeze-dried material was redissolved in 3 ml of ²H₂O and centrifuged (5000 × g, 4 °C, 15 min). In order to obtain absolute quantitative results, special care was taken for data acquisition. Relaxation times, saturation, and nuclear Overhauser effects were minimized, and resolution was optimized. To reduce long relaxation times, 20 µl of a ²H₂O solution containing 13 mM sodium EDTA and 11 mM gadolinium nitrate and [2-¹³C]glycine as internal standard were added to each milliliter of sample. Under this condition, all the T1 relaxation times determined for the carbons of interest in our experiments and measured using the inversion-recovery method were less than 10 s. Acquisition parameters were as follows: spectral width, 25000 Hz; tilt angle, 90°; data size, 32K; 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 (14). We did not use the nuclear Overhauser enhancement during proton decoupling to avoid the use of corresponding correction factors. A 2-Hz line broadening was applied prior to Fourier transform. Peak areas were determined by measuring the integral heights. Chemical shifts were expressed as parts/million relative to tetramethylsilane. Assignments were made by comparing the chemical shifts obtained with those given in the literature (15, 16).

Calculations

Net substrate utilization and product formation were calculated as the difference between the total flasks 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 micromoles of substances removed or produced/g dry wt/unit of time (30 or 60 min).

The rates of release of ¹⁴CO₂ from the ¹⁴C-labeled glutamate species used were calculated by dividing the radioactivity in ¹⁴CO₂ by the specific radioactivity of each labeled carbon of the glutamate species of interest measured in each medium.

Fixation of ¹⁴CO₂ from [¹⁴C]bicarbonate was calculated by dividing the acid-stable radioactivity present in the incubation medium after incubation by the specific radioactivity of the total "bicarbonate + CO₂" pool measured in the zero time flasks. The formation of [1-¹⁴C]glutamine + [1-¹⁴C]glutamate was measured by the method of Squires and Brosnan (17).

When a [¹³C]glutamate species was the substrate, the transfer of the C-1 or C-2 or C-3 or C-5 of glutamate to a given position in a given metabolite was calculated by the following formula: (L_m  l_m)/(E_s  e_s), where L_m is the amount of ¹³C measured in the corresponding NMR resonance, l_m is the natural ¹³C abundance (1.1%) multiplied by the amount of the metabolite assayed enzymatically, E_s is the ¹³C enrichment of the C-1 or C-2, or C-3 or C-5 of glutamate, and e_s is the natural ¹³C abundance.

RESULTS

As shown in Table I, rabbit kidney tubules metabolized glutamate at high rates; glutamate utilization was linear with time over 60 min. Glutamine, which was the main nitrogenous product found, accumulated linearly with time; since glutamine synthesis is an ATP-consuming reaction, this means that our tubules were metabolically viable (their ATP content was 6.5 ± 0.4, 7.4 ± 0.6, and 7.5 ± 0.5 µmol/g dry wt after 0, 30, and 60 min of incubation, respectively; n = 4; these mean values take into account the values found both in the absence and the presence of acetate because they were not significantly different). Significant amounts of alanine also accumulated at both incubation times, but alanine accumulation was not linear with time; nitrogen balance calculations reveal that alanine nitrogen accounted for 31.5 and 15.3% of the nitrogen removed as glutamate at 30 and 60 min, respectively. Small amounts of aspartate, glucose, and lactate were also found to accumulate but no significant accumulation of pyruvate, glycogen, glycerol 3-phosphate, glycerol, acetate, ketone bodies, or tricarboxylic acid cycle intermediates occurred. As found by Krebs (1) and Klahr (9) and Klahr et al. (4) in rabbit kidney slices, no ammonia accumulated; on the contrary, a small amount of the ammonia brought by the tubules at zero time was utilized as substrate during the incubation.

Table I. Time course of the metabolism of 5 mM L-glutamate in rabbit kidney tubules Kidney tubules (55.5 ± 2.7 mg dry wt per flask) were incubated as described under "Experimental Procedures." Results (µmol/g dry wt/h) for metabolite removal () or production are reported as means ± S.E. for four experiments performed in duplicate. Added substrate Incubation Metabolite removal () or production Glutamate NH4+ Glutamine Alanine Aspartate Glucose Lactate min 5 mM glutamate 30  139.8  ± 9.9  3.9  ± 0.6 64.4  ± 3.8 44.2  ± 4.3 11.9  ± 1.6 3.8  ± 0.6 3.6  ± 0.4 None 30  3.2  ± 0.8  0.6  ± 0.6 18.5  ± 1.7  1.0  ± 0.4 0.1  ± 0.4  0.0  ± 0.1  0.7  ± 0.3 5 mM glutamate 60  256.3  ± 22.1  0.8  ± 1.2 126.5  ± 8.8 40.6  ± 12.7 4.2  ± 0.7 8.4  ± 1.8 3.6  ± 1.1 None 60 0.4  ± 0.4 7.4  ± 1.8 30.5  ± 2.3  1.2  ± 0.7 1.2  ± 0.3  0.1  ± 0.1  1.0  ± 0.1

Nitrogen balance calculations also indicate that the nitrogen found as glutamine, alanine, and aspartate exceeded the nitrogen removed as glutamate and ammonia; this means that the glutamine synthesis observed from endogenous sources in the absence of exogenous substrate (see Table I) still occurred, at least partially, in the presence of glutamate.

To test whether acetate, a substrate that circulates at high levels in the rabbit blood (18) and is readily metabolized by rabbit kidney tissue (19), can alter the synthesis of glutamine in rabbit kidney tubules by providing additional carbons, increasing concentrations of acetate (0.5-10 mM) were added to the incubation medium containing 5 mM glutamate. Fig. 2 shows that acetate addition induced a dose-dependent inhibition of both glutamate removal and glutamine accumulation; alanine accumulation was reduced only at the highest concentration of acetate used, whereas an increase in glucose accumulation was observed at all acetate concentrations employed.

In order to determine the precise pathways of glutamate metabolism (see Fig. 1) and the effect of 10 mM acetate (that had the greatest effect of the concentrations used) on these pathways, we performed a series of experiments in which we combined enzymatic, radioactive, and ¹³C NMR spectroscopy measurements using specifically or uniformly ¹⁴C- and/or ¹³C-labeled glutamate in the absence and the presence of unlabeled acetate. Table II shows that 10 mM acetate, which was avidly removed by rabbit kidney tubules, caused effects that have already been partially commented above (see Fig. 2). This table provides the values (obtained by enzymatic methods) necessary to the ¹³C (see the "Calculation" section) and flux calculations in which the glutamate removal is a key parameter. In addition, this table allows us to compare the formation of end products with calculated fluxes through the corresponding enzymes. Note also that the presence of glutamate stimulated the utilization of acetate.

Table II. Effect of 10 mM acetate on the metabolism of 5 mM L-glutamate in rabbit kidney tubules Kidney tubules (57.9 ± 2.5 mg dry wt per flask) were incubated for 60 min as described under "Experimental Procedures." Results (µmol/g dry wt/h) for metabolite removal () or production are reported as means ± S.E. for four experiments performed in quadruplicate. The paired Student's t test was used to measure the statistical difference against the control without acetate: *, p < 0.05; **, p < 0.01; ***, p < 0.001. The radioactivity and 13C NMR data corresponding to these experiments are reported in Tables III, IV, V, VI. Experimental condition Metabolite removal () or production Glutamate Acetate NH4+ Glutamine Alanine Aspartate Lactate Glucose 5 mM glutamate  218.4  ± 5.1 1.9  ± 0.5  1.8  ± 0.7 107.1  ± 1.4 57.4  ± 10.1 7.2  ± 0.8 2.5  ± 1.1 5.5  ± 1.9 5 mM glutamate + 10 mM acetate  90.2  ± 5.5***  406.1  ± 10.6***  2.8  ± 1.4 78.5  ± 7.2* 11.2  ± 0.9* 8.2  ± 0.7 2.1  ± 0.2 14.5  ± 1.5** No added substrate 3.1  ± 4.0  0.3  ± 0.1  0.5  ± 0.9 41.1  ± 1.4  1.1  ± 0.2 1.4  ± 0.2  2.5  ± 0.4 0.0  ± 0.1 10 mM acetate 19.0  ± 2.2*  302.5  ± 1.5***  1.9  ± 3.1 35.2  ± 1.0  2.6  ± 0.7  0.2  ± 0.4*  2.7  ± 0.4* 2.0  ± 1.0

Table III presents the release of ¹⁴CO₂ from various ¹⁴C-labeled glutamates (5 mM) as well as the formation of 1-[¹⁴C]glutamate + 1-[¹⁴C]glutamine (referred to as [1-¹⁴C]Glx) and of [1-¹⁴C]alanine in the presence of glutamate (5 mM) + NaH¹⁴CO₃ (25 mM). With glutamate as sole substrate, it can be seen that a large proportion of the C-1 of this amino acid was released as CO₂ by the -ketoglutarate dehydrogenase reaction; since the alanine and aspartate formed by the alanine and aspartate aminotransferase reactions can account for only 34% (64.6: 190.6) of the -ketoglutarate that has been decarboxylated, our data clearly indicate that most (66%) of the -ketoglutarate synthesis from glutamate occurred thanks to the glutamate dehydrogenase reaction. The difference between the releases of ¹⁴CO₂ from [1,5-¹⁴C]glutamate and [1-¹⁴C]glutamate gives the release of ¹⁴CO₂ from [5-¹⁴C]glutamate which is equal to a mean value of 153 µmol/g dry wt. Since the releases as CO₂ of the C-2 and of the C-5 of glutamate, which occur beyond the stage of succinate and fumarate (two symmetrical molecules), are assumed to be equal, one can deduce that the release of ¹⁴CO₂ from [2-¹⁴C]glutamate is also equal to 153 µmol/g dry wt. Thus, the C-1, C-2, and C-5 of glutamate accounted for about 80% (497.2: 635.2) of the total release of CO₂ from glutamate measured as the ¹⁴CO₂ release from [U-¹⁴C]glutamate (see Table III). Therefore, it appears that only a small proportion (about 10%) of the C-3 and of the C-4 of glutamate, which are assumed to behave symmetrically beyond the stage of succinyl-CoA, was released as CO₂ in rabbit kidney tubules. Another important observation made when glutamate was the only exogenous substrate is that a significant amount of ¹⁴CO₂ was fixed from [¹⁴C]bicarbonate and recovered in the form of the C-1 of glutamate + glutamine and of the C-1 of alanine (Table III). This means that the pyruvate carboxylase reaction operates during glutamate metabolism and leads to the formation of [4-¹⁴C]oxaloacetate which gives [1-¹⁴C]citrate and then [1-¹⁴C]-ketoglutarate (via the tricarboxylic acid cycle) which yields [1-¹⁴C]glutamate and [1-¹⁴C]glutamine by the successive operation of glutamate dehydrogenase (operating in the reductive amination direction) and glutamine synthetase. This demonstrates that glutamate synthesis occurred even in the presence of a high concentration of glutamate which was removed in net amounts. Thus, our enzymatic measurements reflect only the net removal of glutamate, which is simultaneously removed and synthesized, and therefore underestimate the unidirectional removal of this substrate.

Table III. Effect of 10 mM acetate on the release of 14CO2 from [1-14C]-, [1,5-14C]-, and [U-14C]glutamate and on the accumulation of [1-14C]glutamate plus glutamine and [1-14C]alanine during the incorporation of 14CO2 during 5 mM glutamate metabolism in rabbit kidney tubules Kidney tubules (57.9 ± 2.5 mg dry wt per flask) were incubated for 60 min as described under "Experimental Procedures." Results (µmol/g dry wt/h) are reported as means ± S.E. for four experiments performed in quadruplicate. Substrate utilization and product formation, measured enzymatically, are reported in Table II. Statistical difference was measured by the paired Student's t test against the control without acetate: *, p < 0.05; **, p < 0.01. Glu = glutamate; Glx = glutamate plus glutamine; Ala = alanine. Experimental condition 14CO2 from [1-14C]Glu 14CO2 from [1,5-14C]Glu 14CO2 from [U-14C]Glu [1-14C]Glx from Glu + H14CO3 [1-14C]Ala from Glu + H14CO3 5 mM [14C]glutamate 190.6  ± 4.5 343.9  ± 13.3 635.2  ± 43.0 53.4  ± 1.5 25.0  ± 5.4 5 mM [14C]glutamate + 10 mM acetate 191.2  ± 12.2 316.3  ± 12.3** 579.4  ± 21.6 86.7  ± 4.2* 5.2  ± 0.2*

Since the C-1 of alanine was labeled in the presence of glutamate plus ¹⁴C-bicarbonate, this reveals that part of the oxaloacetate synthesized by the pyruvate carboxylase reaction underwent equilibration with fumarate to give [1-¹⁴C]malate and then [1-¹⁴C]oxaloacetate. This [1-¹⁴C]oxaloacetate was converted into [1-¹⁴C]alanine by the combined action of phosphoenolpyruvate carboxykinase, pyruvate kinase, and alanine aminotransferase (see Fig. 1). Thus, the above observations demonstrate that the "substrate cycle" involving pyruvate, oxaloacetate, and phosphoenolpyruvate was operating during glutamate metabolism in rabbit kidney tubules.

Table III also reveals that, as far as the release of CO₂ from glutamate is concerned, only the release of ¹⁴CO₂ from [1,5-¹⁴C]glutamate was significantly inhibited by the addition of acetate. Since the release of ¹⁴CO₂ from [1-¹⁴C]glutamate did not change under this condition, this indicates that the oxidation of the C-5, and therefore of the C-2 of glutamate, was inhibited by acetate. Simple calculations reveal that acetate did not alter the oxidation of the C-3 and C-4 of glutamate. Surprisingly (at least at first sight), in the presence of glutamate plus acetate, the release of ¹⁴CO₂ from [1-¹⁴C]glutamate (Table III), which gives an estimate of the minimal removal of glutamate, was considerably higher than the net removal of glutamate measured enzymatically (see Table II). This reflects the fact that acetate considerably stimulated the synthesis of glutamate; further evidence for this is provided by the observation that acetate increased the accumulation of [1-¹⁴C]glutamate + [1-¹⁴C]glutamine from glutamate plus [¹⁴C]bicarbonate (Table III). Therefore, under this experimental condition (glutamate + acetate), the unidirectional removal of glutamate was masked to a large extent by the simultaneous synthesis of glutamate, a fact which could not be demonstrated solely by enzymatic measurement of glutamate removal.

Finally, Table III shows that, in the absence and the presence of acetate, 44 and 46%, respectively, of the alanine that accumulated was labeled on its C-1 in the presence of [¹⁴C]bicarbonate. This suggests that a large fraction of the oxaloacetate giving rise to alanine equilibrated with fumarate.

Fig. 3, A and B, shows the ¹³C NMR spectra of perchloric extracts obtained after 60 min of incubation of rabbit kidney tubules in the presence of [3-¹³C]glutamate either in the absence or the presence of acetate. As expected, these figures show that the main non-volatile carbon products of glutamate metabolism in the absence of acetate were glutamine and alanine and that acetate inhibited the accumulation of both alanine and glutamine. The fact that carbons of glutamate and glutamine other than that originally labeled in the added glutamate became labeled during incubation confirms that glutamate synthesis occurred during glutamate utilization as concluded above on the basis of enzymatic and radioactive measurements (see Tables II and III). Irrespective of the labeled glutamate species used as substrate (the spectra obtained with [5-¹³C]- and [1,2-¹³C]glutamate as substrate are not shown), acetate stimulated the synthesis and accumulation of labeled glutamate carbons but reduced the accumulation of labeled glutamine. In all spectra, small amounts of labeled serine carbons could also be identified.

From the spectra obtained with [3-¹³C]-, [5-¹³C]-, and [1,2-¹³C]glutamate as substrate (in the absence and in the presence of acetate), in which virtually all the significant resonances could be identified, we calculated the amounts of labeled products after correction for the ¹³C natural abundance as described under "Experimental Procedures." With [3-¹³C]glutamate as substrate in the absence and the presence of acetate (Table IV), the labeled C-2 of alanine was of the same order of magnitude as the labeled C-3 of alanine, and the labeled C-4 of glutamate and glutamine was virtually equal to the labeled C-5 of glutamate and glutamine; this is in agreement with the view that the C-3 and C-4 of the glutamate converted into -ketoglutarate by either glutamate dehydrogenase or by alanine or aspartate or phosphoserine aminotransferase (see Fig. 1), and further metabolized in the tricarboxylic acid cycle, passed through the stage of succinate and fumarate, two symmetrical molecules. The passage through these symmetrical molecules is also consistent with the fate of the C-2 and C-5 of the glutamate converted into the -ketoglutarate that was further metabolized in the tricarboxylic acid cycle because the labelings of the C-1 (singlet) of glutamate, glutamine, and alanine in the presence of [5-¹³C]glutamate as substrate were approximately the same as those obtained when [1,2-¹³C]glutamate was the substrate (see Tables V and VI).

Table IV. Effect of 10 mM acetate on the metabolism of 5 mM [3-13C]glutamate in rabbit kidney tubules Kidney tubules (57.9 ± 2.5 mg dry wt per flask) were incubated for 60 min as described under "Experimental Procedures." Results (µmol/g dry wt/h) for 13C-labeled products accumulated are reported as means ± S.E. for four experiments performed in quadruplicate. Substrate utilization and product formation, measured enzymatically, are reported in Table II. The paired Student's t test was used to measure the statistical difference against the control with glutamate alone: *, p < 0.05; **, p < 0.01; ***, p < 0.001; Glu = glutamate. Experimental condition Amount of labeled products Glutamate Lactate Aspartate C1 C2 C3 C4 C5 C1 C2 C3 C2 C3 [3-13C]Glu 1.2  ± 0.2 16.1  ± 2.0 134.4  ± 15.5 4.1  ± 0.3 3.5  ± 0.8   2.0  ± 0.6 1.8  ± 0.5 3.0  ± 0.6 2.9  ± 0.4 [3-13C]Glu + acetate 7.6  ± 0.6** 49.4  ± 1.4*** 230.2  ± 16.4*** 1.4  ± 0.3* 1.2  ± 0.1   1.0  ± 0.1 1.0  ± 0.1 3.1  ± 0.4 3.1  ± 0.1 Experimental condition Glutamine Alanine Serine C1 C2 C3 C4 C5 C1 C2 C3 C2 C3 [3-13C]Glu 1.0  ± 0.1 10.8  ± 1.0 77.8  ± 5.4 2.8  ± 1.0 2.9  ± 1.1 1.0  ± 0.2 26.0  ± 4.6 23.4  ± 3.9 5.8  ± 0.2 7.2  ± 0.2 [3-13C]Glu + acetate 0.1  ± 0.1*** 11.3  ± 0.4 45.9  ± 0.6*   0.1  ± 0.1 0.3  ± 0.1 2.7  ± 0.2* 2.7  ± 0.3* 1.9  ± 0.1*** 3.0  ± 0.3*

Table V. Effect of 10 mM acetate on the metabolism of 5 mM [5-13C]glutamate in rabbit kidney tubules Kidney tubules (57.9 ± 2.5 mg dry wt per flask) were incubated for 60 min as described under "Experimental Procedures." Results (µmol/g dry wt/h) for 13C-labeled products accumulated are reported as means ± S.E. for four experiments performed in quadruplicate. Substrate utilization and product formation, measured enzymatically, are reported in Table II. The paired Student's t test was used to measure the statistical difference against the control with glutamate alone: *, p < 0.05; **, p < 0.01; ***, p < 0.001; Glu = glutamate. Experimental condition Amount of labeled products Glutamate Glutamine Alanine Aspartate Serine C1 C5 C1 C5 C1 C1 C1 [5-13C]Glu 7.2  ± 1.4 131.7  ± 18.8 5.6  ± 0.1 73.3  ± 4.0 11.2  ± 2.2 1.1  ± 0.1 6.6  ± 0.4 [5-13C]Glu + acetate 22.0  ± 1.8*** 168.5  ± 21.4** 6.3  ± 0.5 43.8  ± 6.0 2.0  ± 0.2* 1.5  ±  0.1* 1.7  ± 0.2*

Table VI. Effect of 10 mM acetate on the metabolism of 5 mM [1,2-13C]glutamate in rabbit kidney tubules Kidney tubules (57.9 ± 2.5 mg dry wt per flask) were incubated for 60 min as described under "Experimental Procedures." Results (µmol/g dry wt/h) for 13C-labeled products accumulated are reported as means ± S.E. for four experiments performed in quadruplicate. Substrate utilization and product formation, measured enzymatically, are reported in Table II. The paired Student's t test was used to measure the statistical difference against the control with glutamate alone: *, p < 0.05; **, p < 0.01; Glu = glutamate. Experimental condition Amount of labeled products Glutamate Glutamine Alanine Serine C2-1 C1-2 C1 C2-1 C1-2 C1 C1 C1 [1,2-13C]Glu 131.5  ± 18.0 133.7  ± 19.3 7.7  ± 1.5 72.0  ± 3.1 72.9  ± 3.7 5.1  ± 0.1 12.7  ± 3.4 6.9  ± 0.2 [1,2-13C]Glu + acetate 165.8  ± 21.9* 164.0  ± 20.9** 27.1  ± 2.6** 40.7  ± 4.8 40.6  ± 6.2* 6.5  ± 0.5 2.0  ± 0.1* 1.6  ± 0.1*

Since the C-2 and the C-3 of the oxaloacetate derived from the added [3-¹³C]glutamate were converted at the same rates to the C-3 and C-2 (respectively) of the glutamate synthesized and therefore to the C-3 and C-2 of the glutamine derived from the latter glutamate, one may calculate that with glutamate as sole substrate, a mean value of 67 µmol/g dry wt of glutamine arose directly from the added [3-¹³C]glutamate, whereas 10.8 µmol/g dry wt of [3-¹³C]glutamine derived from synthesized [3-¹³C]glutamate (see Table IV); in the presence of [3-¹³C]glutamate plus acetate, the corresponding values are 34.6 and 11.3 µmol/g dry wt, respectively. The latter values obtained for estimating the direct accumulation of glutamine from added [3-¹³C]glutamate in the absence and the presence of acetate are in relatively good agreement with those obtained by measuring the accumulation of [5-¹³C]glutamine from [5-¹³C]glutamate (Table V) as well as by measuring the multiplets (spin-spin ¹³C coupling of C-1 and C-2) resulting from the synthesis of [1,2-¹³C]glutamine from [1,2-¹³C]glutamate (Table VI).

Table IV also shows that the addition of acetate stimulated the accumulation of the [2-¹³C]glutamate and [3-¹³C]glutamate synthesized from [3-¹³C]glutamate but not that of the [2-¹³C]glutamine and [3-¹³C]glutamine synthesized from the corresponding synthesized glutamates. Another interesting observation drawn from Table IV is that, in the presence of acetate, the indirect accumulation of [2-¹³C]glutamine and [3-¹³C]glutamine (from synthesized [2-¹³C]glutamate and [3-¹³C]glutamate, respectively) did not significantly fall as did the direct accumulation of [3-¹³C]glutamine from added [3-¹³C]glutamate (Table IV). Note that the direct accumulation of [3-¹³C]glutamine from added [3-¹³C]glutamate was virtually identical to the accumulation of [5-¹³C]glutamine from added [5-¹³C]glutamate (Table V) or of [1,2-¹³C]glutamine from added [1,2-¹³C]glutamate (Table VI).

Table IV also shows that, with [3-¹³C]glutamate as sole substrate, the C-2 and the C-3 of aspartate, lactate, alanine, and serine became labeled to the same extent. This reveals that the C-3 of glutamate, after having passed through the stage of succinate and fumarate, two symmetrical molecules, and then through the stage of malate and oxaloacetate, was metabolized by the action of phosphoenolpyruvate carboxykinase, pyruvate kinase, lactate dehydrogenase, aspartate, alanine, and phosphoserine aminotransferases. The metabolism of [3-¹³C]glutamate also resulted in the labeling of the C-4 and C-5 of glutamate and glutamine (Table IV). This indicates that the labeled C-3 of glutamate led to the labeling of the C-2 and C-1 of acetyl-CoA that were converted to the C-4 and C-5 of citrate and then to the C-4 and C-5 of glutamate and glutamine. Thus, the pyruvate derived from glutamate underwent oxidative decarboxylation by the pyruvate dehydrogenase reaction. Addition of acetate did not alter the labeling of the C-2 and C-3 of aspartate but reduced the labeling of the C-2 and C-3 of lactate, alanine, and serine as well as the labeling of the C-4 and C-5 of glutamate and glutamine (Table IV). Since the sum of the labeled C-2 and C-3 of alanine (Table IV) was close to the accumulation of alanine found in Table II, virtually all the carbons of the alanine found were derived from glutamate.

Very little C-1 of glutamate and glutamine became labeled during the metabolism of [3-¹³C]glutamate alone, but significant labeling of the C-1 of glutamate but not of glutamine occurred upon addition of acetate (Table IV). Such a labeling pattern results from prior labeling of the C-4 of oxaloacetate leading via the combined action of the tricarboxylic acid cycle and the glutamate dehydrogenase reaction to the labeling of the C-1 of glutamate.

Tables V and VI show that, as expected, the C-2 and C-5 of glutamate behaved in a symmetrical way when the [1,2-¹³C]- or [5-¹³C]-ketoglutarate synthesized from [1,2-¹³C]glutamate or [5-¹³C]glutamate was further metabolized in the tricarboxylic acid cycle to lead to the accumulation of alanine and to the synthesis of glutamate which accumulated as glutamate and glutamine. Both in the absence and the presence of acetate, the labelings of the C-1 (singlet) of glutamate and glutamine and of alanine were almost identical when [5-¹³C]glutamate or [1,2-¹³C]glutamate was the substrate. Tables V and VI also show that, with [5-¹³C]glutamate and [1,2-¹³C]glutamate as substrate, both in the absence and the presence of acetate, the labeling of the C-1 (singlet) of glutamate, glutamine, and alanine was virtually half that of the C-2 of glutamate, glutamine, and alanine when [3-¹³C]glutamate was the substrate (see Table IV).

When [3-¹³C]glutamate was the substrate in the absence and the presence of acetate, the labeling (in µmol/g dry wt) of the different carbons of glucose ( +  anomers) was as follows: C-1, 2.2 ± 0.5 and 3.4 ± 0.2; C-6, 1.8 ± 0.5 and 2.6 ± 0.1; C-2, 1.7 ± 0.6 and 2.6 ± 0.4; C-5, 2.5 ± 0.9 and 4.5 ± 0.2, respectively; the C-3 and C-4 were not enriched. When [5-¹³C]- and [1,2-¹³C]glutamate were the substrates, the C-3 and C-4 of glucose were enriched only in the presence of acetate; the values were 2.0 ± 0.4 and 1.1 ± 0.1 for the C-3, respectively; the corresponding values for the C-4 were 2.4 ± 0.5 and 1.7 ± 0.2, respectively. Thus, in agreement with the data of Table II, it appears that acetate tended to stimulate the conversion of glutamate carbons into glucose carbons, although the ¹³C resonances of the glucose carbons were small.

Table VII contains the proportions of each metabolite converted into the next one(s). It should be emphasized here that these proportions which constitute the basis of our model do not give direct access to fluxes. Fluxes also take into account the formation of the substrate of the reaction of interest. Table VII shows that with glutamate as sole substrate, the proportion of oxaloacetate synthesized that was converted into phosphoenolpyruvate was much greater than that converted into citrate, whereas the contrary was true in the presence of acetate. Under the control condition without acetate, almost all the phosphoenolpyruvate synthesized was converted into pyruvate, a very small fraction being converted into 3-phosphoglycerate; addition of acetate increased the proportion of phosphoenolpyruvate converted into 3-phosphoglycerate at the expense of that converted into pyruvate. Most of the pyruvate synthesized was converted into oxaloacetate both in the absence and the presence of acetate; addition of acetate significantly changed the proportions of pyruvate converted into oxaloacetate, alanine, and lactate. Table VII also shows that the proportion of 3-phosphoglycerate synthesized from glutamate as sole substrate that was converted into 3-phosphohydroxypyruvate and then into serine was much greater than that converted into glucose; the reverse was observed upon addition of acetate. The proportion (KG¹  OAA)¹ of -ketoglutarate that was directly converted into oxaloacetate was diminished in the presence of acetate.

Table VII. Various proportions through pathways of glutamate metabolism in the absence or in the presence of 10 mM acetate in rabbit kidney tubules Values, given as means ± S.E. for four experiments, were calculated from those of Tables III, IV, V, VI. The various proportions are shown in Fig. 2 of the accompanying paper. The paired Student's t test was used to measure the statistical difference against the control with glutamate as sole substrate: *, p < 0.05; **, p < 0.01; ***, p < 0.001. TCA, tricarboxylic acid cycle; Lac, lactate. Proportion of Converted to Parameter notation Parameter value Without acetate With acetate Proportions of the direct conversion   OAA Cit (OAA  Cit) 0.22  ± 0.01 0.80  ± 0.01***   OAA PEP (OAA  PEP) 0.78  ± 0.01 0.18  ± 0.01***   OAA Asp (OAA  Asp) 0.01  ± 0.01 0.02  ± 0.01*   PEP Pyr (PEP  Pyr) 0.96  ± 0.01 0.69  ± 0.05*   PEP 3P-glycerate (PEP  3PG) 0.04  ± 0.01 0.31  ± 0.05*   Pyr OAA (Pyr  OAA) 0.81  ± 0.02 0.66  ± 0.06*   Pyr Ala (Pyr  Ala) 0.11  ± 0.03 0.15  ± 0.03*   Pyr AcCoA (Pyr  AcCoA) 0.07  ± 0.01 0.14  ± 0.03   Pyr Lac (Pyr  Lac) 0.01  ± 0.01 0.05  ± 0.01*** 3P-glycerate Glc (3PG  Glc) 0.35  ± 0.08 0.71  ± 0.01* 3P-glycerate Ser (3PG  Ser) 0.65  ± 0.08 0.29  ± 0.01*  KG OAA (KG  OAA) 0.73  ± 0.06 0.52  ± 0.02* Proportions taking into account the recycling through "Glu  KG  Glu" and "Glu  Gln  Glu" cycles   Added Glu  KG {Glu  KG} 0.74  ± 0.09 0.95  ± 0.12*   Added Glu Accumulated Glu {Glu  Glu} 0.27  ± 0.04 0.41  ± 0.04***   Added Glu Accumulated Gln {Glu  Gln} 0.20  ± 0.02 0.10  ± 0.01* Cit-derived KG Glu {CitKG  Glu} 0.33  ± 0.09 0.70  ± 0.06** Cit-derived KG OAA {CitKG  OAA} 0.88  ± 0.02 0.76  ± 0.02 Other parameters Parameter notation Parameter value Without acetate With acetate OAA inversion (OAAi) 0.32  ± 0.04 0.46  ± 0.02 Recycling factor through   citric acid cycle (TCA  ) = (OAA  Cit)·{CitKG  OAA} 0.19  ± 0.01 0.61  ± 0.02**   "OAA  PEP  Pyr  PEP" cycle (Pyr  OAA) = (OAA  PEP)·(PEP  Pyr)·(Pyr  OAA) 0.60  ± 0.03 0.08  ± 0.01**   "OAA  PEP  Pyr  AcCoA  Cit  OAA" cycle (AcCoA  OAA) = (OAA  PEP)·(PEP  Pyr)·(Pyr  AcCoA)·{CitKG  OAA} 0.05  ± 0.01 0.01  ± 0.01 Recycling ratio of KG through   "Glu  KG  Glu" and "Glu  Gln  Glu" cycles {KG  Glu + Gln} = [1-(Glu  Gln)]/[1-(Glu  KG)-(Glu  Gln)] 1.21  ± 0.07 1.46  ± 0.07*** Glu-derived AcCoA condensed with OAA not derived from Glu (AcCoA +   Cit) 0.19  ± 0.01 0.19  ± 0.03 Glu-derived OAA condensed with AcCoA not derived from Glu (OAA +   Cit) 0.78  ± 0.05 0.98  ± 0.01*

Of the added glutamate removed and not recycled in the tricarboxylic acid cycle, {Glu¹  KG}, about 74 and 95% was converted into -ketoglutarate in the presence and the absence of acetate, respectively (Table VII); such a proportion, which may appear surprisingly high in view of the significant proportion of glutamate converted into glutamine, takes into account the operation of the "Glu  KG  Glu" and of the "Glu  Gln¹  Glu" cycles but not the operation of the other cycles (see Fig. 1).

The following proportions presented inside specific braces, {}, also take into account the recycling through the "Glu  KG  Glu" and the "Glu  Gln  Glu" cycles but not through the other cycles. The proportion {^vGlu  Glu} of the added glutamate removed that accumulated as glutamate was increased in the presence of acetate. On the contrary, the proportion {Glu  Gln} of added glutamate removed that accumulated as glutamine was significantly reduced by the addition of acetate.

Note that 33 and 70% of the citrate-derived -ketoglutarate {_CitKG  Glu} was converted into glutamate in the absence and the presence of acetate, respectively. Note also that a high proportion {_CitKG  OAA) of the citrate-derived -ketoglutarate was converted into oxaloacetate. It can be seen in Table VII that a significant proportion (OAA_i) of the oxaloacetate synthesized was inverted as a result of the equilibration with fumarate thanks to the reversible part of the tricarboxylic acid cycle catalyzed by malate dehydrogenase and fumarase. This means that, both in the absence and the presence of acetate, most of the oxaloacetate synthesized by the pyruvate carboxylase reaction underwent equilibration with fumarate. It can also be seen in Table VII that the proportion (TCA) of oxaloacetate recycled at each turn of the tricarboxylic acid cycle that was relatively small with glutamate as sole substrate, dramatically increased in the presence of acetate. By contrast, the proportion (Pyr¹  OAA) of the oxaloacetate that was metabolized in the "OAA PEP¹  Pyr  OAA" cycle, which was high in the absence of acetate, was considerably diminished in the presence of acetate. Both in the absence and the presence of acetate, a very small proportion (AcCoA¹  OAA) of the oxaloacetate synthesized was metabolized in the "OAA  PEP  Pyr  AcCoA  Cit  OAA" cycle.

Table VII shows that the recycling ratio {KG  Glu + Gln} of -ketoglutarate through the "Glu  KG  Glu" and "Glu  Gln  Glu" cycles, which corresponds to the proportion of the citrate-derived -ketoglutarate that passed through these 2 cycles, was increased in the presence of acetate. A significant proportion (AcCoA +  Cit) of glutamate-derived acetyl-CoA was condensed with oxaloacetate that did not originate from glutamate both in the absence and the presence of acetate (Table VII). Finally, a very large proportion (OAA +   Cit) of glutamate-derived oxaloacetate was condensed with acetyl-CoA that did not originate from glutamate, especially in the presence of acetate.

Table VIII shows the absolute values of fluxes through enzymes involved in glutamate metabolism in the absence and the presence of acetate. It can be seen that high unidirectional fluxes occurred from glutamate to -ketoglutarate and from -ketoglutarate to glutamate at the level of glutamate dehydrogenase under both experimental conditions. Acetate considerably increased these two unidirectional fluxes, resulting in the diminution of the net difference (which was small) in favor of glutamate utilization by the latter enzyme. This effect caused by acetate was accompanied by an inhibition of flux through glutamine synthetase which is in agreement with the reduction of glutamine accumulation shown in Table II. Note that, both in the absence and the presence of acetate, flux through -ketoglutarate dehydrogenase was severalfold greater than the sum of fluxes through glutamate dehydrogenase (net flux in the direction of oxidative deamination of glutamate), alanine, aspartate, and phosphoserine aminotransferases, which lead to -ketoglutarate synthesis; this clearly indicates that most of the -ketoglutarate oxidatively decarboxylated was synthesized by the tricarboxylic acid cycle. This is in agreement with the high flux found through citrate synthase. It is also clear from the data of Table VIII that, upon addition of acetate, the increased flux through -ketoglutarate dehydrogenase can be entirely accounted for by the increase in flux through citrate synthase which occurred at the expense of the very large flux through phosphoenolpyruvate carboxykinase. Table VIII also shows that the acetate-induced diversion of oxaloacetate from phosphoenolpyruvate formation to citrate synthesis was logically accompanied by a large decrease in flux through pyruvate kinase and therefore in a decreased availability of pyruvate leading to a diminution of fluxes through alanine aminotransferase and pyruvate dehydrogenase. The increased glucose synthesis (see flux through glucose-6-phosphatase) leading to competition between the gluconeogenic pathway and pyruvate synthesis for utilizing the phosphoenolpyruvate available in decreased amounts in the presence of acetate may also explain in part the decreased availability of pyruvate and of 3-phosphohydroxypyruvate (see flux through 3-phosphoglycerate dehydrogenase in Table VIII).

Table VIII