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Keywords:

  • brain;
  • [1–13C]glucose;
  • glutamate;
  • glutamine;
  • pyruvate carboxylase;
  • 13C-NMR spectroscopy

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

[1–13C]glucose metabolism in the rat brain was investigated after intravenous infusion of the labelled substrate. Incorporation of the label into metabolites was analysed by NMR spectroscopy as a function of the infusion time: 10, 20, 30 or 60 min. Specific enrichments in purified mono- and dicarboxylic amino acids were determined from 1H-observed/13C-edited and 13C-NMR spectroscopy. The relative contribution of pyruvate carboxylase versus pyruvate dehydrogenase (PC/PDH) to amino acid labelling was evaluated from the enrichment difference between either C2 and C3 for Glu and Gln, or C4 and C3 for GABA, respectively. No contribution of pyruvate carboxylase to aspartate, glutamate or GABA labelling was evidenced. The pyruvate carboxylase contribution to glutamine labelling varied with time. PC/PDH decreased from around 80% after 10 min to less than 30% between 20 and 60 min. This was interpreted as reflecting different labelling kinetics of the two glutamine precursor glutamate pools: the astrocytic glutamate and the neuronal glutamate taken up by astrocytes through the glutamate-glutamine cycle. The results are discussed in the light of the possible occurrence of neuronal pyruvate carboxylation. The methods previously used to determine PC/PDH in brain were re-evaluated as regards their capacity to discriminate between astrocytic (via pyruvate carboxylase) and neuronal (via malic enzyme) pyruvate carboxylation.

Abbreviations used:
acetyl-CoA

, acetyl coenzyme A

CPD

composite pulse decoupling

NAA

N-acetyl-l-aspartate

NOE

nuclear Overhauser enhancement

TCA

tricarboxylic acid

ME

malic enzyme (EC 1.1.1.40), PDH, pyruvate dehydrogenase (EC 1.2.4.1)

PC

pyruvate carboxylase (EC 6.4.1.1).

The glutamate-glutamine cycle is acknowledged as a major pathway of metabolite trafficking in the brain. According to the cycle, the neurotransmitter glutamate released in the synaptic cleft is taken up by astrocytes to be recycled to neurons after conversion into glutamine by the astrocytic glutamine synthetase (Norenberg and Martinez-Hernandez 1979). Glutamine is also generated from astrocytic glutamate synthesized through the anaplerotic pathway which involves the astrocyte-specific enzyme pyruvate carboxylase (PC) (Yu et al. 1983). The activity of this enzyme in brain has been investigated by different authors by using various 13C-labelled precursors. Direct determinations of the relative contributions of PC and pyruvate dehydrogenase (PDH) to glutamine, glutamate and GABA synthesis have been obtained from the 13C-enrichment levels in amino acids after metabolism of [1–13C]glucose (Shank et al. 1993; Hassel et al. 1995; Aureli et al. 1997). In these works, evaluation of the relative PC activity was based on the enrichment difference between Gln (or Glu) C2 and C3 (or C4 and C3 for GABA) owing to the specific entry of the label at oxaloacetate C3 after carboxylation of [3–13C]pyruvate and considering the PC pathway as the unique way to generate different labelling at oxaloacetate C3 and C2 (C3 > C2). Direct determinations have also been obtained by amino acid isotopomer analysis after metabolism of multilabelled precursors (Künnecke et al. 1993; Lapidot and Gopher 1994). Considered all together, these studies give a rather large range of values for the relative contributions of pyruvate carboxylase and pyruvate dehydrogenase (PC/PDH, expressed as the percentage of carbon flux through PC versus PDH pathways) to amino acid labelling. For example, concerning glutamine, the value for PC/PDH determined 15, 30 or 45 min after a single dose intraperitoneal injection of [1–13C]glucose in rats was 33, 36 or 9.8%, respectively (Shank et al. 1993). On the other hand, the PC/PDH value determined 5, 15 or 30 min after a single dose intravenous injection of [1–13C]glucose in mice was 58, 41 or not different from 0% (Hassel et al. 1995). The PC/PDH value was 39%, as determined 15 min after one intraperitoneal [1–13C]glucose injection in rats (Aureli et al. 1997) (and less than 20% after 30, 45 or 60 min according to their data) and 34%, as determined 60 min after [U–13C]glucose infusion in rabbits (Lapidot and Gopher 1994). The results were even more variable for glutamate and GABA. Notwithstanding the different animal models and labelled precursor administration procedures used in these studies, time appears to be an important parameter to be considered. Indeed, there are at least two potential glutamate pools as precursor of glutamine: the astrocytic glutamate connected with the astrocyte tricarboxylic acid (TCA) cycle; and the neuronal glutamate taken up by astrocytes and contributing to the glutamate-glutamine cycle. The neuronal and astrocytic glutamate pool sizes are very different: the glial glutamate pool represents around 9% of total cerebral glutamate (Chapa et al. 2000). Moreover, the labelling kinetics of the two glutamate pools are probably different. Indeed, assuming that glial glutamate is in rapid exchange with glial 2-oxoglutarate, its labelling occurs rapidly after 13C entry into the glial TCA cycle. The situation is different for neuronal glutamate especially since the neurotransmitter pool is stocked in vesicles (Ozkan and Ueda 1998). As a consequence, the glutamate involved in the glutamate-glutamine cycle is initially unlabelled. Therefore, glutamine enrichment in the early phase of labelling might directly be related to that of the glutamate pool connected with the glial TCA cycle. As a function of time, the contribution of neuronal glutamate to glutamine labelling increases, thus obscuring that of glial glutamate. Therefore, measuring a relevant value for PC/PDH through glutamine enrichment, if possible, would require working upon the initial phase of labelling. However, this raises the problem of amino acid enrichment determination when this enrichment is very low, i.e. in the range of natural 13C abundance (1.1%). In such a situation, the signal-to-noise ratio on 13C-NMR spectra is not favourable and it is generally difficult to acquire the data under full relaxation and without nuclear Overhauser enhancement (NOE). As a consequence, correction factors have to be considered in the quantitative analysis. Another possible difficulty arises from signal overlapping, particularly in the spectral region including Gln and Glu C2.

In the present study, we investigated the metabolism of [1–13C]glucose in rat brain after different times of intravenous infusion. The mono- and dicarboxylic amino acids in brain extracts were separated and their enrichment determined by NMR spectroscopy. Relative carbon enrichments in amino acids were determined from a series of 13C-NMR spectra acquired either under fast pulsing conditions with NOE or full relaxation without NOE. Specific enrichments were determined from 1H-observed/13C-edited spectra of the purified amino acids. From the NMR data, the relative contributions of PC and PDH to glutamine labelling were determined on the basis of the difference between C2 and C3 enrichments. PC/PDH was found to be higher in the initial than in the late phase of [1–13C]glucose infusion. The results are discussed in the light of how PC activity and pyruvate carboxylation in the brain may be discriminated.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animal preparation

The experimental protocols used in this study were approved by appropriate institutional review committees and met the guidelines of the appropriate governmental agency (authorization n°7368).

Female Wistar rats (210–230 g) were used for the experiments. Animals were fed ad libitum. They were anesthetized with an intraperitoneal injection of chloralhydrate (8%, 0.5 mL/100 g). A solution of [1–13C]glucose (750 ml, 99% enrichment) was then infused in a tail vein. The infusion flow was monitored to follow a time-decreasing exponential from 15 to 1.23 mL/h (at time 60 min) in order to maintain a 9–10 ml hyperglycaemia (Bouzier 2000). After 10, 20, 30 or 60 min infusion, the rats were killed by cervical dislocation. Their head was then immediately split in two by giving a hammer stroke onto a sharp knife maintained on the cranium according to the longitudinal axis. The two cerebral hemispheres were then rapidly removed and dipped in liquid nitrogen. Three animals were used for each infusion time.

Metabolite extraction

The frozen cerebral tissue was pulverized under liquid nitrogen with a mortar and pestle. A volume of 5 mL of 0.9 l perchloric acid was then added drop by drop at the nitrogen surface and the frozen droplets were immediately pulverized. The mixture was transferred to a Dounce's homogenizer and homogenized at 4°C after thawing. After centrifugation (10 000 g, 20 min), the supernatant was neutralized with KOH, centrifuged to eliminate perchlorate salts and freeze-dried. For NMR spectroscopy, each sample was dissolved in 500 µL D2O.

Amino acid purification

After a first series of NMR analyses, the crude extracts corresponding to the same infusion time (n = 3) were pooled and diluted with H2O to 25 mL. Traces of radiolabelled amino acids (3H-labelled Ala and 14C-labelled Asp and Glu) were added as markers to check the purification steps. The solution was acidified to pH 3 by adding concentrated HCl and was filtered through a column (1 cm diameter, 2.5 cm height) of AG 50WX8 (Bio-Rad Laboratories, Hercules, CA, USA) previously equilibrated with 25 ml formic acid. After rinsing the column with 15 mL of 25 ml formic acid and 15 mL of water to eliminate the acidic and neutral metabolites, the fixed cationic compounds were eluted with 50 ml NH4OH. The fractions containing the amino acids were pooled, neutralized with formic acid and lyophilized. The resulting powder was dissolved in a small volume of a 0.25 l formic acid solution adjusted to pH 5 with NH4OH and filtered through a Dowex 1 × 8 column (1 cm diameter, 37 cm height) previously equilibrated with the same solution also used as mobile phase. The mono- and dicarboxylic amino acids were eluted in different fractions. The fractions containing each type of amino acids were pooled and lyophilized. The amino acids were finally desalted by a new run through the AG 50WX8 column and conditioned for NMR analyses.

NMR spectroscopy

Spectra were obtained with a Bruker DPX400 wide-bore spectrometer equipped with a 5-mm broad-band probe. Measurements were performed at 25°C. Proton-decoupled 13C-NMR spectra of crude extracts were acquired under the following conditions: 6 µs pulse (?58° flip angle), 1.48 s acquisition time, 0.1 s relaxation delay, 22 150 Hz sweep width and 64 K memory, CPD (composite pulse decoupling) gated proton decoupling and D2O lock. The number of scans was 20 000–40 000. Proton-decoupled 13C-NMR spectra of purified amino acids were acquired both by using the above fast pulsing conditions involving NOE enhancement and by using the following conditions: 9 µs pulse (?87° flip angle), 0.74 s acquisition time, 10 s relaxation delay, 22 150 Hz sweep width and 32 K memory size. Proton decoupling worked only during acquisition. The number of scans was 20 000–40 000. 1H-NMR spectra were acquired using: 8 µs pulse (?71° flip angle), 4.09 s acquisition time, 10 s relaxation delay, 4000 Hz sweep width and 64 K memory size. Residual water signal was suppressed by homonuclear presaturation. The number of scans was 128 or 256. 1H-observed/13C-edited spectra were obtained as in Bouzier et al. (2000). The acquisition sequences involved a proton spin echo with calibrated 90° and 180° pulses separated by a 3.94-ms delay corresponding to the 1/2 JCH value for the 1H–13C scalar coupling in amino acid -CH2- and -CH3 (JCH = 126–132 Hz) either with or without 13C spin inversion with a 9.25 µs (?90° flip angle) composite pulse. Other parameters were: 3.28 s acquisition time, 15 s relaxation delay, 4990 Hz sweep width and 64 K memory size. Measurements were conducted under carbon decoupling and water proton presaturation. The number of scans was 256, 512 or 1024; the free induction decays corresponding to the two types of data were alternatively acquired by blocks of 32 or 64 scans.

NMR spectrum analyses

13C-NMR spectra of crude extracts were used to investigate the relative enrichments of metabolite carbons as a function of infusion time taking the resonance signal of natural 13C in creatine and inositol (not enriched after a 60-min [1–13C]glucose infusion) as internal reference. The specific enrichment of brain glucose C1 was determined from these spectra by comparing glucose C1α and β peak areas to those of other carbons (C3 to C6), taking into account 13C natural abundance peak areas on a glucose spectrum acquired under the same conditions.

13C-decoupled 1H-NMR spin echo spectra of the crude extracts were used to determine the relative amino acid brain contents according to the number of protons giving rise to resonance peaks. The specific enrichments of metabolite carbons were determined from the ratios between homologous peak areas in the 13C-edited and spin echo spectra, respectively. Each determination was done by superposition of a given resonance peaks in the two spectra using the Dual routine from Bruker, after careful line width, phase and baseline adjustment.

13C-NMR spectra of purified amino acids acquired under fast pulsing conditions were used to determine the relative enrichments of amino acid carbons. Correction factors for saturation and NOE were determined from 13C-NMR spectra of the same samples acquired under full relaxation without NOE. For each spectrum type, peak areas were calculated using the highest peak as reference (peak area = 1 for Gln C4 or Glu C4 peak in mono- or dicarboxylic amino acid spectra, respectively). For a given carbon, the correction factor was then defined as the mean ratio (± SD) between the areas of its peak in each couple of spectra corresponding to the four different infusion times. Using these correction factors, the relative enrichments in compounds were then calculated from spectra recorded under fast pulsing conditions, considering the same relative error for the enrichment and the correction factor. Finally, specific enrichments in compounds were evaluated using the specific enrichment value of the most enriched carbon (Gln and Gln C4, GABA C2) and that of Asp C3 determined from 1H-observed/13C-edited spectra.

The percentage of [3–13C]pyruvate metabolized through PC relative to the flux through PDH was evaluated according to the method previously used by Shank et al. (1993). The method is based on the fact that the enrichment difference between Gln (or Glu) C2 and C3 (or between GABA C4 and C3) reflects [3–13C]pyruvate metabolism via PC, whereas Gln (or Glu) C4 (or GABA C2) enrichment reflects [3–13C]pyruvate metabolism through PDH. For Gln and Glu, the expression used to calculate PC/PDH was: PC/PDH = 100 × (C2 – C3)/(C4–1.1), where Ci represents the specific enrichment of carbon i and 1.1 represents the natural 13C abundance. For GABA, the expression was: PC/PDH = 100 × (C4 – C3)/(C2–1.1).

The enrichment (at the C2 position) of acetyl-CoA entering the TCA cycle from which the amino acid labelling originated was evaluated according to the method described by Malloy et al. (1990). The method is based on the fact that the percentage of Gln (or Glu) isotopomers labelled at C3 and C4 relative to all isotopomers labelled at C3 reflects acetyl-CoA C2 enrichment. The evaluation requires the measurement of the C43 doublet area contributing to the C4 resonance on 13C-NMR spectra. Acetyl-CoA C2 enrichment was then calculated according to the expression: 100 × (C43 doublet area/C4 area) × [C4/(C3 − 1.1)].

Statistical analysis

Data are represented as the mean ± SD. The data were analysed by lnova and the level of significance was set at p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Brain perchloric acid extracts prepared after 10, 20, 30 and 60 min infusion of [1–13C]glucose were analysed by 13C, 1H and 1H-observed/13C-edited NMR spectroscopy. Typical spectra acquired with the latter method using the 3.91 ms delay corresponding to the 1/2 JCH value for JCH ? 127  Hz are shown in Fig. 1. Spectra acquired using a 3.44-ms delay corresponding to the 1/2 JCH value for JCH?145  Hz were tentatively recorded in order to determine the enrichment of amino acid C2. Unfortunately, owing to overlapping, enrichment determination was not possible from crude extract spectra. Therefore, quantitative analysis of spectra was restricted to carbon-bound protons with a coupling constant close to 127 Hz (methyl, methylene groups). 13C-enrichment values are reported in Table 1. This table shows that creatine C4 (CH3) enrichment corresponded to natural 13C abundance (1.1%), indicating that this carbon was not enriched from [1–13C]glucose under our experimental conditions. N-Acetyl-lnova-aspartate (NAA) C6 (CH3) was also found not to be enriched after 10 and 20 min, whereas a low enrichment was evaluated beyond. Such a low labelling of NAA C6 from [1–13C]glucose has already been observed and was related to the slow NAA turnover (Tyson and Sutherland 1998). Enrichments in other compounds increased with time. Enrichment values were determined with a mean relative standard error of 12.3%. A part of the errors reflected the variability between experiments with different animals (n = 3 for each incubation time); however, the presence of numerous overlapping peak clusters in spectra probably represented a risk of inaccuracy (Fig. 1). Indeed, phase and base line adjustments were of importance for enrichment evaluation and peak overlapping (Glu and Gln C3, Glu C4 and GABA C2…) likely induced systematic errors. For example, lactate C3 enrichment was probably underestimated owing to overlapping with unenriched Thr C4. To overcome these drawbacks, the homologous extracts were pooled and the amino acids were separated from the neutral and acidic compounds by ion exchange chromatography. Thereafter, the fractions containing the mono- or dicarboxylic amino acids were analysed again. Carbon-specific enrichments determined from 1H-observed/13C-edited NMR spectra acquired using the 3.91 ms delay (Fig. 2) are reported in Table 1. As above, it was not possible to determine Gln C2 enrichment from purified monocarboxylic amino acid 1H-observed/13C-edited NMR spectra acquired using the 3.44 ms delay owing to resonance overlapping for the proton bound to Gln C2 and Ala C2. As shown in Table 2, the values were generally in good agreement with the previous determinations, particularly for carbons whose bound proton resonance did not overlap another resonance (e.g. protons bound to Ala C3). For the 60 min infusion time, Glu C3 enrichment was found to be higher than with the crude extract (9.40% as compared with 6.95%), which indicated an underestimation of the value from the crude extract spectra as the Glu C3 proton resonance overlapped those of protons bound to Gln C3 and NAA C6, two carbons less enriched than Glu C3. On the contrary, GABA C2 enrichment was lower than with the crude extract (17.2% as compared with 20.2%), which could be explained by resonance overlapping for the protons bound to GABA C2 and Glu C4, the latter carbon being the most enriched. The evaluations from purified amino acids were thus considered to be the most relevant.

image

Figure 1. Determination of 13C-specific enrichment in brain metabolites. Spectra correspond to a perchloric acid extract of brain metabolites prepared after 60 min [1–13C] glucose infusion. A – Spin-echo 1H-NMR spectrum. B –1H-observed/13C-edited NMR spectrum with an expansion factor of 2. The specific enrichment for a given carbon corresponds to the percentage of its peak area in spectrum B relative to that in spectrum A. This figure illustrates the difficulty of accurate enrichment determination owing to numerous overlapping peaks.

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Table 1.  Specific 13C-enrichments (%) of metabolites in crude perchloric acid brain extracts and in purified amino acids as a function of [1–13C]glucose infusion time
 [1–13C]glucose infusion time (min)
10203060
  1. Specific 13C-enrichments were determined from 1H-observed/13C-edited NMR spectra. For amino acids, the first line gives the mean ±SD value of the determinations from crude extract spectra (n = 3). The second line gives the value determined from the spectrum of the pool of purified amino acids; no SD value referring to the different brains was reported because the extracts were pooled in the purification procedure; however, the error related to the NMR analysis was less than 5%. Values are corrected to the nearest 0.05 (or 0.5 for lactate).

Creatine C4 0.95 ± 0.05 0.95 ± 0.05 1.15 ± 0.20 1.05 ± 0.10
Asp C3 2.30 ± 0.60 5.55 ± 1.05 9.95 ± 0.6015.50 ± 1.00
 1.95 5.80 9.7516.50
Gln C4 2.50 ± 0.60 6.55 ± 0.55 8.10 ± 1.1513.50 ± 1.50
 2.10 6.15 8.4013.50
Glu C4 4.55 ± 0.80 9.15 ± 1.9014.50 ± 1.5020.50 ± 1.50
 3.75 9.2014.5019.00
GABA C2 2.90 ± 0.55 6.45 ± 0.7013.00 ± 2.5020.00 ± 1.00
 2.80 7.8512.5017.00
Gln C3 1.40 ± 0.20 2.55 ± 0.30 4.30 ± 0.60 6.65 ± 0.50
 1.40 2.95 3.60 6.50
Glu C3 1.40 ± 0.15 2.85 ± 0.80 4.45 ± 0.90 6.95 ± 0.90
 1.20 2.65 4.75 9.40
NAA C6 1.05 ± 0.10 1.10 ± 0.10 1.35 ± 0.20 1.65 ± 0.15
GABA C3 1.15 ± 0.15 1.85 ± 0.30 3.35 ± 0.35 6.60 ± 0.35
 1.25 2.10 4.10 7.00
Ala C3 7.00 ± 0.4514.00 ± 1.5018.00 ± 3.5021.50 ± 1.50
 6.8014.0018.5022.50
Lactate C316.0 ± 1.517.5 ± 1.522.0 ± 2.525.5 ± 1.0
image

Figure 2. Determination of 13C-specific enrichment in brain amino acids. Spectra correspond to purified amino acids from perchloric acid extracts of brain metabolites after 20 min [1–13C]glucose infusion. Coupled spectra A and B on the one hand, and C and D on the other hand, correspond to 1H-observed/13C-edited (plotted with an expansion factor of 4) and spin-echo spectra of the di- and monocarboxylic amino acids, respectively. Peak assignments: protons bound to: 1 Thr C4, 2 Ala C3, 3 GABA C3, 4 Gln C3, 5 GABA C2, 6 Gln C4, 7 creatine C4 (CH3), 8 Gly C2, 9 Gln C2, 10 creatine C2, 11 Thr C3, 12 Glu C3, 13 Glu C4, 14 Asp C3, 15 Glu C2 and 16 Asp C2.

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Table 2.  Specific 13C-enrichments (%) of purified amino acids as a function of [1–13C]glucose infusion time
 [1–13C]glucose infusion time (min)
10203060
  1. Relative 13C-enrichments in amino acids were determined from 13C-NMR spectra of purified amino acid pools acquired either under saturation with NOE or full relaxation without NOE conditions as described in the Materials and methods section. Correction factors for saturation and NOE were determined after peak area integration using as reference the most intense peak on each spectrum (Gln and Glu C4 for mono- and dicarboxylic amino acids spectra, respectively). This explains why no error on Gln and Glu C4 was reported. Specific enrichments were thus calculated taking into consideration Gln and Glu C4, GABA C2 and Asp C3 enrichment values determined by 1H-observed/13C-edited NMR spectroscopy.

Gln C42.086.15 8.4213.50
C31.17 ± 0.142.44 ± 0.30 3.20 ± 0.40 5.78 ± 0.73
C21.99 ± 0.183.75 ± 0.34 5.08 ± 0.46 9.23 ± 0.84
Glu C43.769.2014.4019.10
C31.15 ± 0.082.61 ± 0.18 4.99 ± 0.34 9.00 ± 0.62
C21.16 ± 0.082.87 ± 0.19 5.36 ± 0.36 9.68 ± 0.65
GABA C22.79 ± 0.257.80 ± 0.7212.72 ± 1.1617.2 ± 1.58
C31.21 ± 0.182.50 ± 0.38 4.43 ± 0.67 6.99 ± 1.06
C41.45 ± 0.062.72 ± 0.12 4.48 ± 0.20 8.37 ± 0.38
Asp C31.95 ± 0.075.80 ± 0.22 9.75 ± 0.3816.7 ± 0.65
C21.89 ± 0.156.05 ± 0.48 9.79 ± 0.7816.09 ± 1.28

1H-decoupled 13C-NMR spectra were acquired by using two different conditions: either under full relaxation without NOE or under fast pulsing with NOE conditions. Spectra of the mono- and dicarboxylic amino acids obtained under the latter conditions are shown in Figs 3 and 4, respectively. In Fig. 3, spectra were normalized relative to the intensity of the creatine signal, as this unenriched compound copurified with monocarboxylic amino acids. In Fig. 4, spectra were normalized relative to the ratio between glutamate C4 and inositol carbon (unenriched) peak areas in 13C-NMR spectra of the crude perchloric acid extracts. Spectra in Figs 3 and 4 show the time increase in alanine, GABA, glutamine, aspartate and glutamate 13C-enrichment and the progressive emergence of coupling figures which indicated compound labelling on adjacent carbons.

image

Figure 3. [1–13C]glucose infusion time dependence of brain monocarboxylic amino acid labelling. 13C-NMR spectra A–D correspond to 10, 20, 30 and 60 min [1–13C]glucose infusions, respectively. Spectra are normalized relative to creatine peaks (6 and 8). Peak assignments: 1 Ala C3; 2 GABA C3, 3 Gln C3, 4 Gln C4, 5 GABA C2, 7 GABA C4 and 9 Gln C2.

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image

Figure 4. [1–13C]glucose infusion time dependence of brain dicarboxylic amino acid labelling. 13C-NMR spectra A–D correspond to 10, 20, 30 and 60 min [1–13C]glucose infusions, respectively. Spectra are normalized relative to the ratio between glutamate C4 and inositol carbon (unenriched) peak areas in 13C NMR spectra of the crude perchloric acid extracts. Peak assignments: 1 Glu C3, 2 Glu C4, 3 Asp C3, 4 Asp C2 and 5 Glu C2.

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The specific enrichments in amino acids are reported in Table 2. For each incubation time, Gln C2 was more enriched than Gln C3. Comparatively, the specific enrichments of the homologous carbons in dicarboxylic amino acids (Glu C2 and C3, Asp C3 and C2) were very close to each other whatever the incubation time. Concerning GABA, the results suggested a higher enrichment at C4 than C3 after 10 min infusion, but the difference was debatable considering the error bars. There was no difference between GABA C4 and C3 enrichments after 20, 30 and 60 min infusion.

Glutamine C4 enrichment was lower than glutamate C4 enrichment even after 60 min infusion (Tables 1 and 2) in agreement with previous studies (Lapidot and Gopher 1994; Bouzier et al. 1999). However, at this time, these carbon enrichments are known to be close to steady-state (Lapidot and Gopher 1994; Mason et al. 1992; Shen et al. 1999). Such an isotopic dilution has been explained by a brain influx of unlabelled blood glutamine (Shen et al. 1999).

Enrichments of either aspartate C3 and C2 or glutamate C2 and C3 or GABA C3 and C4 were the same, reflecting the neuronal metabolism. However, aspartate C2 and C3 were much more enriched than the homologous carbons in glutamate or GABA, thus emphasizing the complex compartmentation of the neurotransmitter metabolism.

For each infusion time, the percentage of [3–13C]pyruvate metabolized through PC relative to the flux through PDH was evaluated. Moreover, when possible, we calculated the enrichment (at the C2 position) of acetyl-CoA entering the TCA cycle from which amino acid labelling originated. As reported in Table 3, a large contribution of PC to glutamine enrichment was evidenced at the initial phase of labelling: PC/PDH was around 80% after 10 min; beyond this time, the value dropped to less than 30%. In comparison, no significant contribution of PC to glutamate or GABA labelling was demonstrated (though the result for GABA at 10 min might appear ambiguous). Beyond 20 min, the enrichment of acetyl-CoA C2 (determined from both Glu and Gln labelling, Table 3) was close to the theoretical maximum (half that of glucose C1) considering the specific enrichment of glucose C1 which was maintained at a constant level throughout the infusion (Table 3). This indicated that glucose was the main primary substrate of the TCA cycles leading to glutamate and glutamine labelling and confirmed that the lower enrichment of glutamine C4 than that of glutamate C4 was the result of an isotopic dilution occurring downstream the acetyl-CoA node (i.e. due to blood glutamine influx).

Table 3.  Percentage of [3–13C]pyruvate metabolized through pyruvate carboxylase relative to the flux through pyruvate dehydrogenase (PC/PDH), enrichment of acetyl-CoA C2 (%) entering the tricarboxylic acid cycle responsible for amino acid labelling, and enrichment of brain glucose C1 (%)
 [1–13C]glucose infusion time (min)
10203060
  1. PC/PDH was evaluated as described in Materials and methods using the carbon enrichment values reported in Table 2. Acetyl-CoA C2 enrichment was determined after measuring the C43 doublet contributing to the Glu or Gln C4 resonance on purified amino acid 13C-NMR spectra. The relative error on the determination corresponds to the relative error on Glu or Gln C3 enrichment. N.D. not determined.

Glutamine
 PC/PDH84 ± 3326 ± 1326 ± 1328 ± 13
 Acetyl-CoA C2 enrichment (%)N.D.22.2 ± 2.724.3 ± 3.024.0 ± 3.0
Glutamate
 PC/PDH0.4 ± 6.03.2 ± 4.62.8 ± 2.63.8 ± 7.0
 Acetyl-CoA C2 enrichment (%)N.D.19.6 ± 1.423.7 ± 1.624.4 ± 1.7
GABA
 PC/PDH12.7 ± 12.73.3 ± 7.50.4 ± 7.58.6 ± 8.9
 Acetyl-CoA C2 enrichment (%)N.D.N.D.N.D.N.D.
 Glucose C1 enrichment (%)45.4 ± 4.540.0 ± 10.048.5 ± 7.046.0 ± 9.0

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Pyruvate carboxylase activity and pyruvate carboxylation

Pyruvate carboxylase is an astrocyte-specific enzyme (Yu et al. 1983). When primary astrocytes are incubated with [1–13C]glucose, its activity is evidenced by different enrichments at glutamine C2 and C3 (Martin et al. 1993). This asymmetrical labelling demonstrates that the pyruvate carboxylation product ([3–13C]oxaloacetate) does not equilibrate with fumarate after its entry into the TCA cycle. A 39% partial cycling between oxaloacetate and fumarate was deduced from glutamine isotopomer analysis (Merle et al. 1996a,b).

Pyruvate carboxylation has also been found to occur in primary neurons by evaluating carbon enrichments in amino acids or by analyzing glutamate isotopomer after incubating cerebellar granule cells with either [2 or 3–13C]pyruvate (Brand et al. 1992) or [1–13C]glucose (Martin et al. 1993), respectively. Glutamate labelling was characterized by equivalent enrichments at C2 and C3, which thus implied complete equilibration of the pyruvate carboxylation product with fumarate (Martin et al. 1993).

Pyruvate carboxylation was more recently reported to occur in neurons both in vitro and in vivo and was proposed to be mediated by malic enzyme (ME) (Hassel and Brathe 2000a,b). The occurrence of ME in neurons is well documented (Cruz et al. 1998), particularly that of the mitochondrial enzyme (Vogel et al. 1998) which presents a high activity in synaptic terminals (McKenna et al. 2000), and it may have a cataplerotic function. ME could make TCA cycle intermediates available (Bukato et al. 1995) and thereby could be involved in the neuronal pyruvate recycling pathway described by Cerdan et al. (1990) and could contribute to the regeneration of NADPH required for mitochondrial glutathione disulphide reduction (Vogel et al. 1999). However, the pyruvate carboxylation carbon flux in primary neurons was found to correspond to 23–33% of the flux through PDH (Brand et al. 1992; Merle et al. 1996b; Hassel and Brathe 2000a), thus demonstrating the intrinsic anaplerotic capacity of ME in immature neurons. In the adult brain, although the reversibility of the ME reaction makes neuronal pyruvate carboxylation possible according to the metabolic needs (as discussed by Hassel 2001), the anaplerotic role of the enzyme remains controversial.

In the light of the above considerations, the methods used to determine PC/PDH in brain astrocytes need re-evaluation. Since pyruvate carboxylation in primary neurons from a labelled substrate generates glutamate equally labelled at C2 and C3 (Martin et al. 1993; Zwingmann et al. 2000a), a difference in brain glutamine (or glutamate) C2 and C3 (or GABA C4 and C3) enrichments evidences glial PC activity. Therefore, the method for evaluating PC/PDH based on the difference in carbon enrichment seems relevant. However, owing to a possible cycling of oxaloacetate to fumarate, the method may under evaluate the contribution of PC. In comparison, the method used by Lapidot and Gopher (1994) based on amino acid isotopomer analysis after metabolism of [U-13C]glucose appears unable to discriminate between neuronal and glial pyruvate carboxylation. For PC/PDH evaluation, they considered as equivalent the glutamate (or glutamine) isotopomers labelled at C2 and C3 and those labelled at C1, C2 and C3, whereas the occurrence of the latter implied fumarase activity. In their metabolic model, they assumed total cycling of oxaloacetate to fumarate. As a consequence, the contribution of PC deduced from their study might be overestimated. The same remark holds for the work of Künnecke et al. (1993) who used [1,2–13C2]glucose, a precursor which cannot generate per se asymmetrical labelling of oxaloacetate C2 and C3 after pyruvate carboxylation (in fact, the authors assumed randomization of the oxaloacetate label in the malate-fumarate equilibrium).

The experimental discrimination between neuronal (via ME) and astrocytic (via PC) pyruvate carboxylation in brain appears to be an interesting challenge. If oxaloacetate and malate, the two products of pyruvate carboxylation, are really characterized by different yields of equilibration with fumarate, isotopomer analysis after [U–13C]glucose metabolism (as in Lapidot and Gopher 1994) might provide useful information. Indeed, if we assume that oxaloacetate does not equilibrate with fumarate whereas malate does so fully, then glutamate and glutamine isotopomers labelled at C1, C2 and C3 would be specific of ME activity. According to the data of Lapidot and Gopher (1994), it appears that in the rabbit brain and near steady state, 90% of the glutamate and glutamine label which entered through pyruvate carboxylation was randomized, thereby suggesting a neuronal metabolism. The result would probably be different in rats or mice because at steady state, glutamine C2 and C3 labelling from [1–13C]glucose metabolism are far from the same.

The issues of the occurrence of neuronal anaplerosis and the discrimination between neuronal and glial pyruvate carboxylation seem of importance for the general understanding of brain metabolism. For example, in two recent studies that aimed at providing an overview of brain metabolism using mathematical modelling, neuronal pyruvate carboxylation was not considered. Sibson et al. (2001) used [2–13C]glucose as substrate to determine the rate of different brain metabolic pathways. Although they considered the possibility of oxaloacetate C2 and C3 label equilibration in their model, they assumed that glutamate and glutamine C2 and C3 labelling from [2–13C]glucose specifically resulted from glial PC activity, i.e. the contribution of any neuronal pyruvate carboxylation to amino acid labelling was thus ignored. Under this assumption, the contribution of astrocytic anaplerosis to glutamine synthesis might be overestimated. On the contrary, Gruetter et al. (2001) used a model to analyse [1–13C]glucose metabolism in human brain wherein reverse flux from oxaloacetate to fumarate was neglected. Evaluation of the pyruvate carboxylation flux was thus based on the difference in glutamine C2 and C3 enrichments. Therefore, this flux might be underestimated. Notwithstanding the important intrinsic differences in modelling, flux values estimated from these two studies were unexpectedly broadly similar. These two studies were based mainly on NMR data acquired in vivo. Considering the importance of the early labelling phase as shown in the present study, the signal to noise ratio and time resolution in data from in vivo experiments appear to be crucial parameters for obtaining relevant data.

Time dependence of PC/PDH

Under the present experimental conditions, PC/PDH was around 80% after 10 min [1–13C]glucose infusion and around 25–30% during the 20–60-min period, as deduced from glutamine labelling. In contrast, no significant contribution of PC to glutamate and GABA labelling was evidenced. From a general point of view, these results are in agreement with previous findings of a much higher contribution of PC to glutamine metabolism than to that of other amino acids (Shank et al. 1993; Hassel et al. 1995; Aureli et al. 1997). However, a time-dependent decrease of PC contribution to glutamine labelling was already noted by Hassel et al. (1995) and the same phenomenon could be deduced from the data of Aureli et al. (1997). This decrease was interpreted as reflecting the scrambling of the label between the oxaloacetate C2 and C3 positions due to equilibration of oxaloacetate with the symmetrical fumarate (Hassel et al. 1995). More likely, this dependence can be due to the different labelling kinetics of the two glutamine precursor glutamate types, i.e. the glial and the neuronal glutamate. The glial glutamate is in direct connection with the astrocytic TCA cycle and then rapidly incorporates the label entered in that cycle via PC and PDH. The neuronal glutamate corresponds to the transmitter taken up by the astrocytes after its release from the neuronal vesicles, so it is not directly connected to the neuronal TCA cycle. With time, labelling of the small astrocytic glutamate pool is masked by labelling of the large neuronal glutamate pool, and glutamine labelling from its astrocytic precursor is obscured by the occurrence of the glutamate-glutamine cycle. An estimate of the relative flux on the two pathways of glutamine labelling can be obtained from the time evolution of the PC/PDH value. Indeed, assuming that the value at 10 min (84%) results only from the astrocytic TCA cycle activity and that the value at 60 min (28%) results from the steady state where both astrocytic and neuronal glutamate contribute to glutamine labelling, the relative contributions of the astrocytic TCA cycle and the glutamate-glutamine cycle to glutamine labelling might be about 1/3 and 2/3, respectively.

As discussed above, the 80% contribution of PC to glutamine enrichment in the early labelling phase represents a minimal evaluation if the possibility of partial cycling of oxaloacetate to fumarate is considered. Moreover, the masking effect due to the contribution of neuronal glutamate to glutamine labelling might not be negligible even after 10 min Since the contribution of substrates other than pyruvate to acetyl-CoA synthesis was rather low (from the glutamine enrichment pattern, acetyl-CoA C2 enrichment was in the range 22–24% while glucose C1 enrichment was around 46%), this means that most, if not all, of the glial TCA cycle activity was devoted to anaplerosis.

Most of the net glutamine synthesis is assumed to be related to brain ammonia detoxification (Sibson et al. 2001), which results in a net flux of brain glutamine exportation to blood. However, the release of glutamine is associated with an entry of blood glutamine into the brain, which leads to different labelling of Glu and Gln C4 at steady state. From the data in Table 2, it may be estimated that after 60 min infusion, blood glutamine represents at least 30% of the brain glutamine (a value of 26% was reported in human by Shen et al. 1999). In view of the various glutamine sources from the whole body, labelling of blood glutamine is likely to be slow. For example, blood glutamine C4 enrichment was only 7% after 1 h [1–13C]glucose infusion in glioma-bearing rats (Bouzier et al. 1999). Therefore, in the presteady-state phase of brain glutamine labelling, the consequence of the blood glutamine influx is a simple isotopic dilution not affecting the evaluation of PC/PDH. However at steady state, the blood glutamine enrichment pattern (which may differ from that of brain-synthesized glutamine) may interfere with that of brain glutamine, depending on the origin (kidney, liver…) of the labelled glutamine in the blood.

If the anaplerotic flux through the astrocytic TCA cycle is considered to represent the net glutamine synthesis, then this could lead to an overestimation of the latter, at least under normoammonemic conditions. Indeed, if the enrichment pattern of 2-oxoglutarate is transferred to astrocytic glutamate (by isotopic exchange) and then to glutamine, the net glutamine synthesis may be lower than pyruvate carboxylation, depending on the anaplerotic needs of the cells. For example, in primary astrocytes, anaplerosis was found to be much more due to the synthesis of citrate than to that of glutamine (Martin et al. 1997). Moreover, the anaplerotic flux may be partly required for the replenishment of neurotransmitter amino acid pools if some of them re-enter the TCA cycle (e.g. to provide the molecules for pyruvate recycling).

Glutamine release from brain is particularly necessary under hyperammonemic conditions to avoid the occurrence of a brain oedema generated by an excess of glutamine acting as an osmoregulator (Olafsson et al. 1995; Zwingmann et al. 2000b). Under such conditions, astrocytic anaplerosis could be more directly associated with ammonia detoxification via glutamine synthesis and efflux from the brain. A 1.5-fold increase in PC/PDH activity under hyperammonemic conditions was reported by Gopher and Lapidot (1991). In their experiments, PC/PDH was evaluated from glutamine labelling 40 min after [U–13C]glucose infusion. Considering the already high PC/PDH value determined in the present study under normoammonemic conditions, it may be proposed that the increase in PC/PDH reflects both an intrinsic increase in the ratio (i.e. PC versus PDH activity) and an increase in the astrocytic TCA cycle flux, the consequence of the latter being an increase in the contribution of the astrocytic glutamate to glutamine synthesis, compared with the neuronal glutamate. This assumption is strengthened by the results of Zwingmann et al. (1998) who found both an increase in PC/PDH and an increase in glutamate and glutamine labelling in primary astrocytes incubated with [1–13C]glucose under hyperammonemia.

Although the PC catalyzed reaction is acknowledged as the main anaplerotic pathway in the brain, the occurrence of cytosolic ME in astrocytes (Kurz et al. 1993) raises the question of its contribution to anaplerosis, as discussed by Vogel et al. (1999) and Zwingmann et al. (2000a). However, in the present study, the contribution of this pathway cannot be evaluated if the malate generated by the cytosolic ME equilibrates with fumarate after its entry into mitochondria and TCA cycle.

In conclusion, this study emphasizes the importance of the presteady-state phase of amino acid labelling in the investigation of brain metabolism using labelled substrates. Under our experimental conditions, astrocytic TCA cycle activity and anaplerosis were found to be in the same range, thereby indicating that TCA cycle function is more closely related to anaplerotic than to energy requirements.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by grants from the Association pour la Recherche contre le Cancer, the Ligue Nationale contre le Cancer and the Région Aquitaine.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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