(Received for publication, February 28, 1996, and in revised form, October 24, 1996)
From the The rabbit kidney does not readily metabolize but
synthesizes glutamine at high rates by pathways that remain poorly
defined. Therefore, the metabolism of variously labeled
[13C]- and [14C]glutamates has been studied
in isolated rabbit kidney tubules with and without acetate.
CO2, glutamine, and alanine were the main carbon and
nitrogenous end products of glutamate metabolism but no ammonia
accumulated. Absolute fluxes through enzymes involved in glutamate
metabolism, including enzymes of four different cycles operating
simultaneously, were assessed by combining mainly the 13C
NMR data with a new model of glutamate metabolism. In contrast to a
previous conclusion of Klahr et al. (Klahr, S.,
Schoolwerth, A. C., and Bourgoignie, J. J. (1972) Am. J. Physiol. 222, 813-820), glutamate metabolism was found to be
initiated by glutamate dehydrogenase at high rates. Glutamate
dehydrogenase also operated at high rates in the reverse direction;
this, together with the operation of the glutamine synthetase reaction,
masked the release of ammonia. Addition of acetate stimulated the
operation of the "glutamate 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.
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
Analytical Methods
Glucose, glycogen, lactate, pyruvate,
glutamate, glutamine, alanine, aspartate, citrate, 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: (Lm 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.
Time course of the metabolism of 5 mM
L-glutamate in rabbit kidney tubules
Centre d'Etudes Métaboliques par
Spectroscopie de Résonance Magnétique (INSERM CRI
950201),
-ketoglutarate
glutamate" cycle and the accumulation of glucose but reduced both the
net oxidative deamination of glutamate and glutamine synthesis. Acetate
considerably increased flux through
-ketoglutarate dehydrogenase and
citrate synthase at the expense of flux through phosphoenolpyruvate
carboxykinase; acetate also caused a large decrease in flux through
alanine aminotransferase, pyruvate dehydrogenase, and the "substrate
cycle" involving oxaloacetate, phosphoenolpyruvate, and pyruvate.
-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.
Fig. 1.
Pathways of glutamate metabolism in rabbit
kidney tubules. Numbers refer to enzymes involved in the different
reactions: 1, aspartate aminotransferase; 2,
phosphoserine aminotransferase; 3, alanine aminotransferase;
4, glutamate dehydrogenase; 5, -ketoglutarate dehydrogenase; 6, citrate synthase; 7, isocitrate
dehydrogenase; 8, phosphoenolpyruvate carboxykinase;
9, phosphoglyceromutase; 10,
glucose-6-phosphatase; 11, 3-phosphoglycerate dehydrogenase; 12, pyruvate kinase; 13, pyruvate carboxylase;
14, pyruvate dehydrogenase; 15, lactate
dehydrogenase; 16, glutamine synthetase; 17,
glutaminase; 18, acetyl-CoA synthetase.
[View Larger Version of this Image (27K GIF file)]
-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).
lm)/(Es
es), where Lm is the amount of
13C measured in the corresponding NMR resonance,
lm is the natural 13C abundance (1.1%)
multiplied by the amount of the metabolite assayed enzymatically,
Es is the 13C enrichment of the C-1 or
C-2, or C-3 or C-5 of glutamate, and es is the
natural 13C abundance.
) 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.
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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.
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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).
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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.
|
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
{Cit
KG
Glu} was converted into glutamate in the
absence and the presence of acetate, respectively. Note also that a
high proportion {Cit
KG
OAA) of the citrate-derived
-ketoglutarate was converted into oxaloacetate. It can be seen
in Table VII that a significant proportion (OAAi) 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).
|
Removal and Metabolic Fate of Glutamate in Rabbit Kidney Tubules
Our study clearly demonstrates that rabbit kidney tubules readily utilize glutamate as substrate; at equimolar concentration, the rate of glutamate removal observed in the present study is about twice that found for glucose in a recent study (6). This demonstration that glutamate is a substrate of rabbit kidney tubules is in agreement with the findings of Krebs (1) that glutamate is a precursor of glutamine in rabbit kidney slices and of Klahr (9) and Klahr et al. (4) who observed also in rabbit kidney slices that glutamate increases the production of glucose. Our results not only confirm that both glutamine and glucose are products of glutamate metabolism but also establish the relative importance of theses two end products. In addition, they establish the relative importance of alanine and CO2 as carbon products of glutamate metabolism. From the results obtained, it is clear that glutamate is a potential energy provider to rabbit renal tubular cells.
The main fate of glutamate nitrogen in rabbit kidney tubules is glutamine. Both in the presence of glutamate as sole substrate and in the presence of glutamate + acetate, the significant amount of glutamine formed, which cannot be entirely explained by endogenous formation (see Tables I and II), indicates that added glutamate must have contributed not only to the carbon skeleton but also to its amide nitrogen. This clearly demonstrates that some of the glutamate removed must have been deaminated via glutamate dehydrogenase to provide the ammonia needed for the synthesis of the glutamine amide group. This study also establishes that alanine and, to a much smaller extent, aspartate, and serine are nitrogenous products of glutamate metabolism in rabbit kidney tubules. In agreement with the findings of other authors (1, 4, 9), no ammonia accumulated, an observation explainable by the high rate of glutamine synthesis that masked the release of ammonia due to glutamate deamination.
Routes of Glutamate Metabolism in Rabbit Kidney Tubules and Importance of the Information Obtained from the Combination of the 13C NMR Data and the Model
The advantages of using 13C NMR spectroscopy, also
used by other authors for the study of intermediary metabolism of
various organs in vitro and in vivo (8, 20-36),
together with a mathematical model of substrate metabolism (37), have
been underlined in a previous study (6). As will be seen below and in
the accompanying paper (44), further developments of our modeling
approach allowed us to measure not only fluxes in three different
cycles as in our previous study (6) but also unidirectional fluxes at
the level of glutamate dehydrogenase allowing us therefore to measure enzymatic fluxes in a fourth cycle, i.e. the "Glu
KG
Glu" cycle. Note that Chance and co-workers and Shulman and
co-workers (23, 31, 33) were also able to assess the rate of the
exchange between
-ketoglutarate and glutamate in the rat and human
brain in vivo. Unfortunately, flux through glutamine
synthetase was lower than the total glutamine found at the end of the
incubation period. Therefore, we obtained no experimental evidence in
the present study that the glutamine synthesized from glutamate was metabolized by glutaminase which is also active in rabbit kidney cortex
(1-3); otherwise, we would have been able to measure simultaneously enzymatic fluxes through five different cycles operating during glutamate metabolism in rabbit kidney tubules as considered in our
mathematical model of glutamate metabolism in the accompanying paper
(44).
Note that the consistency of our analysis and measurements was satisfactory because there were no contradictions either in the qualitative or in the quantitative distributions of the different labeled glutamate carbons in the different products. For example the symmetrical behavior beyond the succinyl-coenzyme A step on the one hand of C-2 and C-5 and on the other hand of the C-3 and C-4 of glutamate was observed from differently 13C- and 14C-labeled glutamates. In addition the number of experimental results was greater than the number of parameters in the model; therefore, several ways of calculations giving virtually identical results were possible. Furthermore, the consistency of our model is also indicated by the fact that calculated and measured amounts of a few labeled products were also almost identical. Finally, virtually identical fluxes were obtained when they were calculated from the data obtained from differently labeled glutamates.
Role of Glutamate DehydrogenaseIn these experiments, the release of 14CO2 from [1-14C]glutamate, which cannot be explained by the rates of alanine, aspartate, and serine synthesis (see Tables II and III), provides conclusive evidence that glutamate dehydrogenase plays an important role in initiating glutamate metabolism in rabbit kidney tubules. Our data therefore are in agreement with the view of Krebs (1) about the role of this enzyme, but they disagree with the conclusion of Klahr (9) and Klahr et al. (4) who did not notice that the release of ammonia by oxidative deamination of glutamate was masked by the high capacity of rabbit kidney-cortex slices for glutamine synthesis. Additional evidence for a high flux through glutamate dehydrogenase in the direction of oxidative deamination of glutamate is provided by the results drawn from the combination of the 13C NMR data and the model of glutamate metabolism developed in this study (Table VIII).
Simultaneous Degradation and Synthesis of GlutamateThe fact
that, in the presence of glutamate + acetate, the release of
14CO2 from [1-14C]glutamate, or
half the release of 14CO2 from
[1,5-14C]glutamate, or one-fifth of the release of
14CO2 from [U-14C]glutamate was
higher than the removal of glutamate measured enzymatically (Tables II
and III) strongly suggests that glutamate removal was accompanied by
glutamate synthesis. That this was indeed the case not only in the
presence of glutamate + acetate but also in the presence of glutamate
as sole substrate was unequivocally demonstrated by the conversion of
the C-3 of added [3-13C]glutamate to the C-1, C-2, C-4,
and C-5 of glutamate (Table IV). As shown in Table VIII, flux through
glutamate dehydrogenase in the direction of the reductive amination of
-ketoglutarate was high both in the absence and the presence of
acetate; however, as expected since glutamate was removed in net
amounts under both experimental conditions, flux in the oxidative
deamination direction exceeded that in the reductive amination
direction. The net flux through glutamate dehydrogenase, which was
therefore in favor of glutamate deamination (Table VIII), gives the
availability of ammonia for glutamine synthesis. It can be seen that,
both in the absence and the presence of acetate, the ammonia derived
from added glutamate and available for glutamine synthesis was not sufficient to explain all the glutamine found to accumulate (Table II);
in agreement with the nitrogen balance calculations mentioned above,
this means that the glutamine synthesis from endogenous substrates
observed in the absence of exogenous substrates still occurred (at
least partially) in the presence of glutamate alone and in the presence
of glutamate + acetate (Table II).
The fact that glutamate carbons other than those labeled in the added
glutamates became labeled during incubation (Tables IV, V, VI) implies
that the -ketoglutarate derived from added glutamate as a result of
glutamate deamination or transamination was further metabolized in the
tricarboxylic acid cycle to yield newly synthesized glutamate. Thus, at
any incubation time point, the glutamate added at the start of
incubation was mixed with small amounts of variously labeled glutamates
synthesized by rabbit kidney tubules through various pathways. Thanks
to the NMR data we were able to identify these pathways and to quantify
them by combining the NMR data and the model developed. An interesting observation made from the data of Table IV is that part of the [3-13C]glutamate found at the end of incubation, which is
equal to the [2-13C]glutamate found, was synthesized by
the tubules; the difference between the [3-13C]glutamate
found and the [2-13C]glutamate found gives the amount of
added [3-13C]glutamate that was not removed by the
tubules or that was removed to give
[3-13C]
-ketoglutarate subsequently reconverted into
[3-13C]glutamate by the glutamate dehydrogenase reaction.
That our way of differentiating the [3-13C]glutamate not
removed in net amounts from the [3-13C]glutamate
synthesized and accumulated (Table IV) is correct is suggested by the
relatively good agreement between the [3-13C]glutamate
minus the [2-13C]glutamate found (Table IV) on the one
hand and the [5-13C]glutamate (Table V) or
[1,2-13C]glutamate (Table VI) found on the other hand,
both with glutamate alone or with glutamate + acetate.
Note that the two reactions of the glutamate
-ketoglutarate
interconversion could occur within one or different populations of
mitochondria or within different cells. In this respect, it should also
be emphasized here that no evidence was found for different pools of
glutamate as will be discussed in the next section.
Although we recently presented some evidence that, in the presence of glucose plus NH4Cl, glutamine accumulation resulting from the action of glutamine synthetase was accompanied by glutamine degradation by glutaminase (6), we obtained in the present study no evidence that glutaminase operated; therefore, we were unable to quantify flux through the latter enzyme which is present along the entire length of the rabbit proximal tubule (3).
In this study, we were able to distinguish between the direct (from added glutamate) and indirect (from synthesized glutamate) synthesis of glutamine (see Tables IV, V, VI and "Results" ). This raises the question of whether or not glutamine was synthesized from a single or from two different glutamate pools. In this respect, the observations made from Tables IV, V, VI that (i) acetate increased the accumulation of [2-13C]- and [3-13C]glutamate from [3-13C]glutamate but not the indirect accumulation of [2-13C]- and [3-13C]glutamine from the latter glutamates, and (ii) acetate caused a decrease in the direct accumulation of glutamine (Tables IV, V, VI) but not in the indirect accumulation of this amino acid should be examined with great caution. At first sight, the latter observations might be interpreted as indicative of the existence of two different glutamate pools from which two different glutamine pools would be synthesized. In fact, the absence of a (2-fold) decrease in the indirect accumulation of [2-13C]- and [3-13C]glutamine in the presence of acetate can simply be explained by a large (2-fold) increase in the net synthesis of glutamate as reflected by the accumulation of [2-13C]glutamate + [2-13C]glutamine (see Table IV). Thus, there is no evidence in the present study for the existence of two different glutamate or glutamine pools in rabbit kidney tubules.
Note that it is conceivable that the glutaminase reaction also operated especially in the presence of glutamate as sole substrate when the glutamine concentration reached a level higher (Table II) than that at which the glutaminase reaction has been demonstrated to function in rabbit kidney tubules (5); however, the extent to which this reaction operated, if at all, remains uncertain because of the presence of high concentrations of glutamate, a well established end product inhibitor of renal glutaminase (38). It should also be mentioned here that any operation of the glutaminase reaction in the present study would imply that our value of flux through glutamine synthetase is underestimated.
Other Pathways of Glutamate MetabolismAn interesting observation is that the "substrate cycle" involving the operation of phosphoenolpyruvate carboxykinase, pyruvate kinase, and pyruvate carboxylase, which has recently been shown to operate during glucose metabolism in rabbit kidney tubules (6), was also found in the present study to operate at high rates during glutamate metabolism (Tables VII and VIII). Note that the operation of pyruvate carboxylase, which resulted in the labeling of the C-4 of oxaloacetate and then of the C-1 of glutamate and glutamine in the presence of glutamate + [14C]bicarbonate (Table III), cannot explain the labeling of the C-1 of alanine, glutamate, and glutamine as a result of the refixation of the 13CO2 released from the differently 13C-labeled glutamates used as substrates; as a matter of fact, simple calculations taking into account the total "CO2 + bicarbonate" pool of our flasks reveal that the 13CO2 released was considerably diluted by unlabeled CO2 and could account for only negligible labelings of the C-1 of the three latter amino acids.
Of the pyruvate synthesized by pyruvate kinase, a significant fraction
was metabolized by pyruvate dehydrogenase; this reveals the existence
of an additional cycle involving the successive operation of the latter
enzyme, the tricarboxylic acid cycle from citrate to oxaloacetate,
phosphoenolpyruvate carboxykinase, and pyruvate kinase. Thus, glutamate
metabolism in rabbit kidney tubules involves four different cycles; in
addition to the latter cycle, the "Glu
KG
Glu", the
tricarboxylic acid and the "OAA
PEP
Pyr
OAA" cycles are
all operative at their own rate which was determined by the enzyme
operating at the lowest rate.
The fact that, with glutamate as sole substrate, flux through alanine aminotransferase (Table VIII) was found slightly higher than alanine accumulation (Table II) can be simply explained by the fact that some of the alanine brought by the tubules at the start of incubation was metabolized and replaced by the alanine synthesized from glutamate; this suggests that bidirectional fluxes also occurred at the level of alanine aminotransferase, a fact that is not surprising for a near-equilibrium enzyme.
That some glucose was synthesized from glutamate in our experiments is in agreement with the existence of key gluconeogenic enzymes in the rabbit proximal tubule (39). The small amount of [13C]serine found suggests that, like in the rat kidney (40), 3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase are also functional in rabbit kidney tubules. It should be mentioned here that, given the very small amounts of glucose synthesized from glutamate in this study, no account was taken of the possible utilization of glucose previously demonstrated in rabbit kidney tubules (6, 41).
Effect of Acetate on Glutamate Metabolism
Our data indicate that the rabbit kidney, like the kidney of other
species (42, 43), has a high capacity to utilize acetate as substrate
(Table II). Furthermore, they show that acetate has a number of
important regulatory effects on the metabolism of glutamate in rabbit
kidney tubules (Table VIII). The acetate-induced stimulation of flux
through glutamate dehydrogenase in the oxidative deamination direction
is a priori surprising because one would rather expect an
inhibition of this unidirectional flux as a result of the expected
shift of the mitochondrial redox potential toward a more reduced state
secondary to acetate oxidation; one partial explanation may be an
increased availability of glutamate for its oxidative deamination as a
result of the inhibition of glutamate utilization by the glutamine
synthesis and glutamate transamination pathways. The stimulation by
acetate of the unidirectional flux through glutamate dehydrogenase in
the reductive amination direction may have resulted from (i) an
increased supply of -ketoglutarate secondary to the stimulation
of flux through citrate synthase caused by acetate and (ii) the more
reduced state of the mitochondrial NADH/NAD+ couple
mentioned above.
The inhibition of flux through glutamine synthetase caused by acetate cannot be attributed to a diminution of glutamate availability which on the contrary increased. Similarly, the provision of ATP (which can be calculated from the data of Table VIII), which is needed for the glutamine synthetase reaction (1), was not decreased but rather was more than doubled in the presence of acetate. This inhibition can be explained by a significant reduction of the release of ammonia, a substrate of the glutamine synthetase reaction, by glutamate dehydrogenase (Table VIII).
It is noteworthy that, despite the increased provision of oxaloacetate
via the tricarboxylic acid cycle secondary to the acetate-induced stimulation of flux through -ketoglutarate dehydrogenase, there was
no increase in aspartate accumulation and a large reduction of flux
through phosphoenolpyruvate carboxykinase (Table VIII). This can be
explained by the increased availability of acetyl-CoA due to acetate
utilization which directed oxaloacetate to the citrate synthase
reaction at the expense of the aspartate aminotransferase and the
phosphoenolpyruvate carboxykinase reactions. The inhibition of
phosphoenolpyruvate synthesis caused by acetate led to a reduction of
flux through pyruvate kinase and therefore of the synthesis of pyruvate
which explains the observed diminution of fluxes through both pyruvate
carboxylase and pyruvate dehydrogenase as well as the fall of alanine
accumulation. It should be mentioned that, in the presence of acetate,
the small fraction of the phosphoenolpyruvate synthesized that was
further metabolized in the gluconeogenic pathway was directed to the
synthesis of glucose at the expense of the synthesis of
3-phosphohydroxypyruvate (see fluxes through 3-phosphoglycerate
dehydrogenase and glucose-6-phosphatase, Table VIII). This effect may
result from an increased flux through glyceraldehyde-3-phosphate dehydrogenase due to an increased availability of NADH in the cytosol
as a result of acetate oxidation. This is suggested by the unchanged
lactate accumulation despite a reduced availability of pyruvate in the
presence of acetate. Another possible explanation for the increased
accumulation of glucose in the presence of acetate is that the latter
compound inhibited the reutilization of the glucose synthesized since
it is known that glucose is utilized by rabbit kidney tubules (6, 41)
and that acetate inhibits glucose utilization in these
tubules.2 Unfortunately, the results
obtained in the present study do not allow us to test the latter
hypothesis because the glucose synthesis and the 13C
resonances of glucose were too small to draw reliable conclusions on
the possible existence of glucose reutilization.
Physiological Significance
It is clear that in the rabbit kidney, like in the kidney of other herbivorous animals, which excretes an alkaline urine (2) and contains a high glutamine synthetase activity, glutamine synthesis represents an important mechanism whereby the ammonia synthesized by the glutamate dehydrogenase and possibly glutaminase reactions can be trapped. It is therefore of physiological, biochemical, and nutritional interest to characterize not only the metabolism of glutamate, a substrate of glutamine synthetase and a regulator of glutaminase, but also the regulation of glutamate metabolism by important circulating substrates such acetate, which represents most of the volatile fatty acids in the rabbit blood (18).
It should be emphasized here that the glutamate concentration used in the present study is much higher than the blood glutamate concentration found in the rabbit (0.2 mM).2 We used such a high glutamate concentration and a large amount of kidney tubules to increase the rate of glutamate removal and the rate of 13C-labeled product formation so that it was possible to measure substrate removal and product formation in a reliable manner to compensate for the poor sensitivity of 13C NMR spectroscopy. This led us also to use a 10 mM acetate concentration so that this substrate, which is avidly metabolized by rabbit kidney tubules (Table II) and is present at millimolar concentrations in the rabbit blood (18), was available in sufficient amounts until the end of the 60-min incubation period.
It should be underlined that the effects of acetate observed in the
present study are probably of physiological relevance; indeed, the
metabolism of sodium acetate leads to the production of bicarbonate
which should be eliminated in the urine, thus leading to urine
alkalinization and therefore to a decreased need for renal ammonia
production and excretion. In the present study, the decreased flux
through glutamate dehydrogenase (Table VIII), which releases ammonia,
caused by the presence of acetate fits well with a physiological
adaptation to the metabolic alkalosis caused by acetate oxidation. In
this respect, one would also expect an increased flux through glutamine
synthetase and an increased glutamine synthesis which, on the contrary,
were found to decrease in this study (Table II and VIII). The absence
of this expected increase in glutamine synthesis is probably related to
the decreased availability of ammonia mentioned above. Whether acetate,
which leads to an increased flux through the tricarboxylic acid and the
"Glu
KG
Glu" cycles and to a decreased flux through the "OAA
PEP
Pyr
OAA" and the "OAA
PEP
Pyr
AcCoA
citrate
OAA" cycles under in vitro
conditions, exerts similar actions under in vivo conditions
when there is an increased supply of glutamine precursors and acetate
to the rabbit kidney during the post-prandial period deserves further
study.