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Acidosis and astrocyte amino acid metabolism
Neurochemistry International 36 (2000) 329±339
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Acidosis and astrocyte amino acid metabolism Marc Yudko*, Yevgeny Daikhin, Ilana Nissim, Itzhak Nissim Children's Hospital of Philadelphia, Division of Child Development and Rehabilitation, Department of Pediatrics, University of Pennsylvania School of Medicine, 34th St. and Civic Center Blvd., Abramson Building, Philadelphia, PA 19104 4318, USA Received 1 April 1999; received in revised form 23 June 1999; accepted 25 June 1999
Abstract The relationship between acidosis and the metabolism of glutamine and glutamate was studied in cultured astrocytes. Acidi®cation of the incubation medium was associated with an increased formation of aspartate from glutamate and glutamine. The rise of the intracellular content of aspartate was accompanied by a signi®cant decline in the extracellular concentration of both lactate and citrate. Studies with either [2-15N]glutamine or [15N]glutamate indicated that there occurred in acidosis an increased transamination of glutamate to aspartate. Studies with L-[2,3,3,4,4-2H5]glutamine indicated that in acidosis glutamate carbon was more rapidly converted to aspartate via the tricarboxylic acid cycle. Acidosis appears to result in increased availability of oxaloacetate to the aspartate aminotransferase reaction and, consequently, increased transamination of glutamate. The expansion of the available pool of oxaloacetate probably re¯ects a combination of: (a) Restricted ¯ux through glycolysis and less production from pyruvate of acetyl-CoA, which condenses with oxaloacetate in the citrate synthetase reaction; and (b) Increased oxidation of glutamate and glutamine through a portion of the tricarboxylic acid cycle and enhanced production of oxaloacetate from glutamate and glutamine carbon. The data point to the interplay of the metabolism of glucose and that of glutamate in these cells. # 2000 Elsevier Science Ltd. All rights reserved.
1. Introduction It is well accepted that astrocytes are central to brain glutamate handling. These cells, which have abundant high-anity glutamate transporters, probably are the major locus of brain glutamate uptake from the extracellular ¯uid (Hertz et al., 1993; Schousboe et al., 1977). They also are the primary site in brain of the glutamine synthetase reaction, which abets formation of glutamine that neurons subsequently utilize, via the phosphate-dependent glutaminase reaction, to synthesize glutamate (Norenberg and Martinez-Hernandez, 1979; Hertz et al., 1993). Indeed, the production of glutamine in glia and the subsequent reconversion to glutamate in neurons constitutes a metabolic arrangement that often is called the `glutamateglutamine cycle' (Yudko et al., 1992). * Corresponding author. Tel.: +1-215-590-7474; fax: +1-215-5906804 E-mail address: yudko@email.chop.edu (M. Yudko).
The metabolism of glutamate in astrocytes is more varied than the relatively straightforward model that is implied by the notion of a glutamate-glutamine cycle. It now is clear that astrocytes, in addition to forming glutamine from glutamate, also are capable of the robust metabolism of the latter amino acid. Indeed, glial glutamate oxidation is so active that glutamate almost could substitute for glucose as a metabolic fuel in these cells (Yu et al., 1982). The precise metabolic routes by which glutamate carbon enters the tricarboxylic acid cycle has been the subject of some controversy. Three pathways have been implicated: the glutamate dehydrogenase reaction, the malic enzyme pathway, and the aspartate aminotransferase reaction. The relative contributions of an individual pathway probably depends, in large measure, upon the metabolic `set' of the cell as well as the ambient glutamate concentration (McKenna et al., 1994). In our prior studies of brain transamination of glutamate to aspartate in both synaptosomes and astrocytes, we found that ¯ux through this equilibrium pathway probably is determined by the availability of
0197-0186/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 7 - 0 1 8 6 ( 9 9 ) 0 0 1 4 1 - 2
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oxaloacetate, a substrate of the reaction: Glutamate+Oxaloacetate 4 Aspartate+2-oxo-glutarate. The rate of transamination declines if oxaloacetate is utilized for another pathway and is thereby diverted away from the aminotransferase. This occurs, for example, when brain respires on ketone bodies, which are readily metabolized in brain to acetyl-CoA. As the latter ester combines with oxaloacetate to yield citrate in the citrate synthetase pathway, less glutamate can enter the aspartate aminotransferase reaction because of the relative unavailability of oxaloacetate (Yudko et al., 1997). In this study we have explored the relationship between increased H+ concentration and astrocyte metabolism of glutamate. In the kidney acidosis is associated with a rapid and profound increase of ¯ux through glutaminase as well as increased glutamate consumption via both the various glutamate transaminases and the glutamate dehydrogenase pathway (Nissim et al., 1985; Nissim and States, 1989, 1995). The result is increased production of NH3, an important component in the maintenance of body acid-base balance (Nissim et al., 1985). Acidosis also markedly inhibits ¯ux through glycolysis in the brain (Wu and Davis, 1981; Folbergrova et al., 1972a,b; Erecinska et al., 1995; Swanson and Benington, 1996). As a result, there is reduced formation of acetyl-CoA from pyruvate and less oxaloacetate is consumed in the citrate synthetase reaction. More oxaloacetate then is accessible to aspartate aminotransferase. As a consequence, the production of aspartate from glutamate is augmented. Our data are consistent with the formulation of augmented glutamate transamination to aspartate in acidosis. 2. Experimental procedures 2.1. Astrocyte cultures Astrocytes were cultured from young (<24 h) rats by placing the whole brain in a warm buered (pH 7.4) solution containing NaCl (135 mM), KCl (4.0 mM) and Hepes (20 mM). After removing meninges, a wedge of parietal cortex was removed from either side and incubated at 378C for 30 min in the presence of 0.15% trypsin. The cells were then washed three times in 2 ml of the buered saline solution to remove the trypsin. Then was added 2 ml of primary culture medium (80% DMEM, 10% fetal bovine serum, 10% Ham's F-12 nutrient solution). The cell suspension was triturated 15 times with a sterile glass pipette in order to deaggregate the cells. After allowing the tissue debris to settle, the cell suspension was transferred to another 15 ml tube. The cells then were plated at a
density of 2.5 105 cells/ml (3 104 cells/cm2) in 75 cm2 plastic vials. Cells were maintained in an incubator at 378C and with 5% CO2 for 3±4 weeks, when they attained con¯uency. Tests for glial purity routinely showed yields of >95% GFAP-positive cells. 2.2. Experimental conditions After removing the steady-state medium, the cells were washed (4) with a buered (pH 7.4) solution containing 135 mM NaCl, 5 mM NaHCO3, 5 mM KH2PO4, 1 mM MgSO4, 1 mM CaCl2, 10 mM D-glucose and 20 mM HEPES. The steady-state medium then was replaced with 2 ml of warmed (378C) incubation medium of similar ionic composition as the wash medium but at pH 7.4, 7.2, 7.0, 6.4 or 6.0, as indicated below (Results). After 1 h the incubation medium was removed and the cell monolayer was washed quickly (3) with cold buered saline solution. The incubation medium was replaced with 1 ml of cold HCl (5 mM) and the cell monolayer was scraped vigorously. After freezing and thawing to assure liberation of cell contents, total amino acid content of the intracellular compartment was determined. The concentration of citrate and lactate was measured in the medium. In a separate series of studies the metabolism of the -NH2 group of either glutamate or glutamine was studied by washing the cells, as indicated above, and replacing the steady-state medium with an incubation medium (above) that contained either [15N]glutamate, [1-13C]glutamate or [2-15N]glutamine (1 mM each). At the indicated times (Results), the medium was removed, the cell monolayer was washed and the incubation medium was replaced with 2 ml of 5 mM HCl. The internal concentrations of amino acids as well as the 15N label (atom % excess, see below) were determined. In order to study the conversion of L[2,3,3,4,4-2H5]glutamine to [2H1]aspartate, a similar experimental paradigm was employed with the [2H5]glutamine species as precursor. The oxidation of glutamate was studied by adding [1-13C]glutamate (1 mM) to the astrocyte incubation medium. At the indicated times (Results) the medium was removed and treated as indicated (below) to liberate 13CO2. 2.3. Isotopic abundance in amino acids and CO2 To measure isotopic abundance in glutamate and aspartate, 100 microliters of cell extract were taken to dryness and the t-butyldimethylsilyl derivative was determined as described by Frederick et al (1984). Label in [15N]glutamate and [15N]aspartate was determined from the m/z 433/432 and m/z 419/418 ion
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ratios, respectively. Label in [2,3,3,4,4-2H5]glutamate and [2H4]glutamate was determined from the m/z 437/ 432 and m/z 436/432 ratios, respectively. Analysis of isotopic enrichment was performed with selected ion monitoring on a Hewlett-Packard 5970 mass-selective detector on-line with a Hewlett-Packard 5890 gas chromatograph. The electron multiplier was set to 02000 V, the ®lament potential to 70 eV, source temperature and transfer line both were at 2808C and the injector at 2508C. The temperature program was 1208C for 3 min and then 88C/min until 2808C. The column was a 12 M 0.32 mm Ultra-2 capillary from Hewlett-Packard. Calibration curves were run on a weekly basis to assure linearity of response for the (M+1)/(M) ratio. Glial oxidation of [1-13C]glutamate was determined by adding 10 mmol of NaHCO3 as a carrier to 1 ml of the medium removed at the indicated times (Results) during incubation with [1-13C]glutamate (98 atom % excess). This was placed in a glass tube with a rubber stopper. After evacuation of the atmosphere, 0.5 ml of 40% phosphoric acid was injected through the stopper in order to liberate the 13CO2. This atmosphere was removed with a gas-tight syringe and injected into a Sira-12 isotope ratio-mass spectrometer. Selected ion monitoring of the m/z 45/44 ratio was performed in order to monitor enrichment in 13CO2. 2.4. Determination of concentration of lactate and citrate The concentration of lactate and citrate was determined by isotope dilution. To 1.5 ml of medium was added 15 nmol of [2H]citrate and 75 nmol of [1-13C]lactate in aqueous solution (each isotope >98 atom % excess). After acidifying to 2N HCl and saturating with NaCl, the acid fraction was extracted successively with 3 ml each of ethyl acetate, ethyl ether and tetrahydrofuran. The solvents were combined together and back extracted once with 1 ml of 2N HCl. The organic phase then was taken to dryness and the t-butyldimethylsilyl derivative was prepared (Frederick et al., 1984) using acetonitrile as counter solvent. The concentration of citrate and lactate was determined by isotope dilution analysis of the m/z 592/ 591 and m/z 262/261 ratios, respectively. 2.5. Other analyses The concentrations of amino acids are determined by HPLC of their o-phthaldialdehyde derivatives (Jones and Gilligan, 1983). The internal standard was E-aminocaproic acid. Protein was measured with the Bio-Rad protein assay kit (Bio-Rad; Richmond, CA, USA).
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2.6. Calculation of isotopic abundance and data analysis Isotopic abundance (atom % excess) was calculated from the height of each peak according to Rosenblatt et al. (1992). The absolute concentration of 15N in a given metabolite refers to the product/100 of isotopic abundance (atom % excess) and metabolite concentration (nmol/mg protein). The rate of formation of a particular 15N product was determined from the initial linear segment of the rate of synthesis. Values for ¯ux were normalized to the internal labeling (atom % excess) in the precursor species. Statistical signi®cance was determined with the t-test for unpaired data. 3. Results 3.1. Eects of pH on metabolite levels Intracellular amino acid levels after a 1 h incubation in the presence of buer of varying pH are shown in Fig. 1. Internal [aspartate] tended to increase as a function of medium [H+]. At pH 7.4 the aspartate level was 10.1 2 2.9 nmol/mg protein. This value was increased to 216% and 261% of control (pH 7.4) at pH 6.4 and 6.0, respectively. The internal [aspartate] was not altered signi®cantly at pH 7.2 or 7.0. No signi®cant change at any pH was observed with respect to internal [glutamate] (Fig. 1). Intracellular [glutamine] varied much less than did internal [aspartate]: it was not signi®cantly dierent from control except for pH 6.4 and 6.0 (Fig. 1). The foregoing study was repeated in precisely the same manner but with L-glutamine (0.5 mM) as a substrate in the medium. The results are shown in Fig. 2. As the concentration of external H+ was increased, the internal [glutamate] was not signi®cantly altered, but the internal [aspartate] rose sharply, the concentration at pH 6.0 being more than 300% greater than that noted at pH 7.4 (Fig. 2). A signi®cant increase was seen also at pH 6.4. Even at a more `moderate' medium pH of 7.0, the intracellular level of aspartate was nearly twice that measured at physiologic pH. Internal [glutamine] was much less responsive to medium [H+] than was internal [aspartate], although a signi®cant increase was observed at pH 6.4 (Fig. 2). In Table 1 are shown the concentrations (mM) of selected metabolites in medium following a 1 h incubation in the presence of L-glutamine (1 mM). The medium [NH3] was signi®cantly higher at pH 6.4 vs 7.4, presumably re¯ecting greater utilization of glutamine via phosphate-dependent glutaminase (Yudko et al., 1989). The fact that medium [glutamine] is sig-
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Fig. 1. Intra-astrocytic amino acid levels at varying pH. After removing the steady-state medium and washing the astrocytes, the cells were incubated for an additional hour at 378C at pH 7.4, 7.2, 7.0, 6.4 or 6.0. At the end of that period the medium was removed, the cells were washed (3) and the incubation medium was replaced with 2 ml of 5 mM HCl. The cell monolayer was scraped, frozen and thawed to assure lysis of internal contents. Amino acids were measured as described in Methods. Results are expressed as a fraction (%) of the control (pH 7.4) value. Each bar is the mean (2SD) of at least four experiments. P < 0.05 vs pH 7.4.
ni®cantly lower in the presence of higher [H+] (Table 1) is consistent with this formulation. In contrast, medium lactate and citrate are signi®cantly reduced in the acidotic medium. 3.2. Acidosis and glutamate transamination: studies with 15 N and 2H precursors The striking increase of internal [aspartate] in the presence of a high medium [H+] suggested that acidosis might be associated with augmented transamination of glutamate to aspartate. In order to test this possibility, astrocytes were incubated for 1 h in the presence of [2-15N]glutamine (1 mM) and internal labeling of aspartate and glutamate was measured. Labeling (atom % excess; Fig. 3, top panel) in internal [15N]aspartate was increased by 075% with a medium pH of 6.4 vs 7.4 (P < 0.05). Labelling of [15N]gluta-
mate was unaected by the change of medium [H+] (Fig. 3, top panel). Labelling of intra-astrocytic glutamine (not shown in Fig. 3) was similar in pH 6.4 vs pH 7.4: 81.0 vs 84.5 atom % excess, respectively. The absolute concentration of internal 15N-amino acids (Methods) is shown in the bottom panel of Table 1 Concentration (mM) of metabolites in medium after 1 h incubation in presence of [2-15N]glutamine (1.0 mM)a pH
7.4
6.4
NH3 Lactic Citric Gln
110.9211.0 525.0234.1 47.4210.1 758.3224.6
143.629.2 256.2243.5 22.121.0 707.5217.9
a
Results are mean2SD of eight experiments. P < 0.05.
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Fig. 2. Intra-astrocytic amino levels at varying pH in presence of L-glutamine. Incubation procedure is precisely as described in legend to Fig. 1 except that the incubation medium contained L-glutamine (0.5 mM). Amino acid concentrations are expressed as percentage of the control (pH 7.4) value. Each bar is the mean (2SD) of at least four experiments. P < 0.05 vs pH 7.4.
Fig. 3. When medium pH was 6.4, the level of intracellular [15N]aspartate was increased more than 4-fold, from 2.7 2 0.5 nmol/mg protein (pH 7.4) to 10.8 2 0.9 nmol/mg protein (pH 6.4) (P < 0.05). The internal [15N]glutamate concentration was not signi®cantly altered by incubation at high medium [H+]. The above data describe the transfer of amino groups from glutamate to aspartate. Glutamate carbon also can be converted to aspartate following oxidation of 2-oxo-glutarate to succinate and then to oxaloacetate in the tricarboxylic acid cycle. In order to gauge the eect of acidosis on this pathway, L[2,3,3,4,4-2H5]glutamine was utilized as a metabolic tracer (Methods). The data are shown in Fig. 4. As indicated in the top panel, labelling of [2H1]aspartate was increased from 7.9 2 1.4 to 11.4 2 4.1 atom % excess (P < 0.05) at pH 6.4 vs 7.4. Labeling in
[2,3,3,4,4-2H5]glutamate was slightly but signi®cantly decreased by incubation at acid pH (Fig. 4, top panel). Labeling of [2H4]glutamate was unchanged (Fig. 4, top). Enrichment in [2,3,3,4,4-2H5]glutamine was unaffected (data not shown in Fig. 4). The absolute concentration of 2H-amino acids is shown in the bottom panel of Fig. 4. The concentration of [2H]aspartate was sharply increased at acid pH: 0.7 2 0.1 (control) vs 1.2 2 0.5 nmol/mg protein (acidosis). The increased rate of aspartate formation was accompanied by a diminished internal concentration of 2H-glutamate. Thus, the concentration of the M+5 species ([2,3,3,4,4-2H5]glutamate), corresponding to glutamate formed directly via glutaminase from labelled glutamine, was 2.7 2 0.3 (control) vs 1.9 20.4 (acidosis) nmol/mg protein (P < 0.05). Similarly, the concentration of the M+4 species, corresponding
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to glutamate formed from the M+5 species following transamination to 2-oxo-glutarate and re-amination of the latter back to glutamate, was 3.520.4 (control) vs 2.1 2 0.4 (acidosis) nmol/mg protein (P < 0.05). The fraction (%) of aspartate compared with glutamate (both M+4 and M+5) was greatly increased at
Fig. 3. Transfer of 15N from [2-15N]glutamine to [15N]glutamate and [15N]aspartate. The astrocytes were incubated essentially as described in the legend to Fig. 1, except that the incubation medium contained 15 L-[2- N]glutamine (1.0 mM). After 1 h the steady-state medium was removed, the cells were washed and the internal labeling (atom % excess) of [2-15N]glutamine was determined as well as the concentration of glutamate and aspartate. Top panel: Label (atom % excess) in intracellular [15N]aspartate and [15N]glutamate after 1 h. Bottom panel: The absolute concentration (nmol 15N-amino acid/mg protein) of [15N]aspartate and [15N]glutamate, corresponding to the product of atom % excess/100 and total amino acid concentration (nmol/mg protein). Each bar is the mean (2SD) of at least four experiments. Open bar: control incubations (pH 7.4). Stippled bar: acidosis (pH 6.4). P < 0.05 vs pH 7.4.
Fig. 4. Metabolism of L-[2,3,3,4,4-2H5]glutamine in astrocytes. Incubations were done precisely as described in the legend to Fig. 3, except that the incubation medium contained L-[2,3,3,4,4-2H5]glutamine (1.0 mM). Each bar is the mean (2SD) of at least 4 experiments. Open bar: control incubations (pH 7.4). Stippled bar: acidosis (pH 6.4). Error bar less than scale for control values of absolute concentration of the M+5 glutamate species. P < 0.05 vs pH 7.4.
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increased medium [H+]: 11.2 2 1.9 (control) vs 29.4 2 10.2 (acidosis) (P < 0.05). The foregoing data all were generated with labeled species of glutamine. In order to determine whether acidosis would similarly aect the metabolism of glutamate, the astrocytes were incubated with [15N]glutamate and the formation of [15N]aspartate and [2-15N]glutamine was determined. Results for intracellular labeling patterns are shown in Fig. 5. Increased medium [H+] appeared to be associated with reduced glial accumulation of [15N]glutamate. At 60 min the internal concentration (nmol/mg protein) was 757.8 2 114.7 (control) vs 514.4 2 70.6 (acidosis) (P < 0.05). Unlike incubations performed with [2-15N]glutamine (see Fig. 3) the intra-astrocytic level of [15N]aspartate was signi®cantly reduced at 60 min by acid pH: 112.3 2 12.7 (control) vs 64.4 2 18.5 (acidosis) (P < 0.05). However, the concentration (mM) of [15N]aspartate in the incubation medium at 60 min was increased at acid pH: 23.0 2 2.8 (control) vs 34.1 2 3.2 (acidosis) (P < 0.01). With an internal aspartate concentration of 0 10 nmol/mg protein, a protein content of 00.2 mg/plate and an incubation medium volume of 2 ml, it follows that >95% of total aspartate (intra-astrocytic+extracellular) is resident in the medium. The overall eect of acidosis, therefore, was to increase total aspartate production by 035%.
Fig. 5. Glial metabolism of [15N]glutamate. Incubations were done as described in the legend to Fig. 3, except that the incubation medium contained [15N]glutamate (1.0 mM) instead of a labeled glutamine species. At the times indicated in the ®gure the incubation medium was removed and the cells were washed and harvested as described in the legend to Fig. 1. Left top panel: absolute concentration of [15N]glutamate (nmol/mg protein). Right top panel: absolute concentration of [15N]aspartate. This parameter was determined as described in the legend to Fig. 3. Bottom panel: concentration of aspartate (mM) in the incubation medium at end of incubation. Each point is the mean (2SD) of at least four experiments. Control and acidosis as indicated by symbols in the ®gure. P < 0.05 compared with pH 7.4.
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One reason that the aspartate accumulated in the medium rather than the intracellular compartment may be that intra-astrocytic aspartate exchanged with external glutamate, the level of which (mM) was greater at increased medium [H+]: 676.0239.9 (control) vs 955.8 270.5 (acidosis) (P < 0.05) (Fig. 5). 3.3. Acidosis and oxidation of glutamate The 15N data are consistent with the formulation that acidosis is associated with enhanced transamination of glutamate N to yield aspartate, a phenomenon similar to that noted in kidney cells (Nissim et al., 1995). The studies with [2,3,3,4,4-2H5]glutamine (Fig. 5) suggests that the 2-oxo-glutarate formed from glutamate is oxidized via the tricarboxylic acid cycle to [2H]oxaloacetate, which, in turn, is transaminated to [2H]aspartate. In order to directly monitor the eects of acidosis on glial glutamate oxidation, a series of studies was done with [1-13C]glutamate (1 mM) as precursor. The results are shown in Fig. 6. Appreciable enrichment was detected in 13CO2 with the intensity of labeling being greater in acidosis vs control. Thus, at 5, 10, 20 and 30 min enrichment in 13CO2 at pH 6.0 was 27.0, 37.5, 31.7 and 29.4% greater in the acidotic vs control samples (Fig. 6, left panel; P < 0.05 at each time point). Furthermore, the increased label in 13CO2 was not attributable to increased intracellular labeling of [1-13C]glutamate. When labeling of 13CO2 was normalized to internal enrichment (atom % excess) with [1-13C]glutamate, a statistically signi®cant dierence was still observed (Fig. 6, right panel).
Fig. 6. Oxidation of L-[1-13C]glutamate in astrocytes. The cells were incubated as described in the legend to Fig. 3, except that the medium contained L-[1-13C]glutamate (1 mM). At the times indicated in the Fig., the medium was removed and the 13CO2 content was analyzed with isotope ratio-mass spectrometry (see Methods for details). Left Panel: Atom % Excess in 13CO2. Values are those obtained following the addition of unlabelled carrier CO2. Right Panel: Ratio (100) of label in 13CO2 in that [1-13C]glutamate. Circles: control (pH 7.4). Squares: acidosis (pH 6.4). Each point is the mean (2SD) of at least four determinations. P < 0.05 compared with pH 7.4.
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4. Discussion Altered brain pH, a common metabolic derangement, accompanies various pathologic states, including hypoxia, ischemia, various toxic states and some inborn errors of metabolism. Indeed, acute and severe acidosis may contribute to the brain damage that occurs consequent to tissue ischemia (Siesjo et al., 1996). Relatively mild acidosis may attenuate glutamate neurotoxicity and other forms of ischemic injury (Tombaugh and Sapolsky, 1993; Giard et al., 1990; Sapolsky et al., 1996; Vornov et al., 1996). Various mechanisms have been suggested as causes of acidosismediated brain injury, including increased free radical production (Waterfall et al., 1996; Siesjo et al., 1985), an accentuation of ischemic damage (Hurn et al., 1991; Katsura et al., 1994), injury to brain endothelium (Paljarvi et al., 1983), a release of Ca2+ from internal stores (Abercrombie and Hart, 1986; Baker and Umbach, 1987), and an activation of neuronal apoptosis (Barry and Eastman, 1993). We have shown here that acidosis is associated with increased transamination of glutamate to aspartate, implying increased availability of oxaloacetate to the aspartate aminotransferase pathway. A plausible explanation for an augmented oxaloacetate pool is that less acetyl-CoA is derived from glucose carbon because acidosis inhibits glycolysis. Less oxaloacetate, therefore, is consumed in the citrate synthetase pathway. This is re¯ected in diminished production of both citrate and lactate during acidosis (Table 1). The inhibitory eect of acidosis on glycolysis is rapid and pronounced (Folbergrova et al., 1972a,b, 1975; Miller and Veech, 1975; Wu and Davis, 1981; Erecinska et al., 1995; Swanson and Benington, 1996). It is likely that such inhibition occurs at the level of phosphofructokinase, the rate-limiting step of glycolysis (Erecinska et al., 1995). This enzyme is strongly inhibited by H+ (Trivedi and Danforth, 1966; Dobson et al., 1986) in a manner that is reversible by high concentrations of AMP and Pi, a positive eector of phosphofructokinase (Goldhammer and Paradies, 1979). Our experimental model involved the incubation of astrocytes with a medium of varying pH. Intra-astrocytic pH was not measured, but changes of external [H+] probably are rapidly re¯ected in the internal [H+] (Mellergard et al., 1992; Ouyang et al., 1993). This condition was associated with a marked increase of [aspartate] (Table 1) and a signi®cant increase in the rate of conversion of [15N]glutamate to yield [15N]aspartate (Fig. 3), denoting increased ¯ux through the aspartate aminotransferase reaction. The latter is an equilibrium reaction for which the directionality is sensitive to the relative concentrations of reactants. The increased [aspartate] and augmented production of [15N]aspartate from [15N]glutamate are consistent
with the notion that, during acute acidosis, there was more mitochondrial oxaloacetate available to the aspartate aminotransferase reaction as less oxaloacetate was incorporated into citrate. Flux through citrate synthetase was diminished because of reduced production of acetyl-CoA via the pyruvate dehydrogenase reaction. The latter pathway was relatively quiescent because of an acidosis-induced inhibition of glycolysis (Erecinska et al., 1995). In addition to the acidosis-induced restriction on oxaloacetate utilization for the purpose of citrate synthesis, it is likely that acidosis is associated with increased production of oxaloacetate from glutamine and glutamate, a motif that has been previously observed in renal tissue during acidosis (Nissim et al., 1985, 1989, 1995). We previously demonstrated in synaptosomes that glutamine and glutamate carbon can be an ecient source of brain oxaloacetate (Yudko et al., 1994). This requires that glutamate carbon enter the tricarboxylic acid cycle via transamination to yield 2-oxo-glutarate. These putative metabolic relationships are summarized in Fig. 7, which indicates that the carbon skeletons of oxaloacetate and other intermediates of the tricarboxylic acid cycle can be derived from glutamine and glutamate as well as glucose (Fig. 7). This carbon backbone ordinarily would be furnished by glucose,
Fig. 7. Schema to illustrate inter-relationship of glucose and amino acid metabolism in astrocytes. The rate of transamination of glutamate to aspartate depends upon the accessibility of oxaloacetate (OAA) to the aspartate aminotransferase pathway. When ¯ux through glycolysis is relatively slow, less pyruvate (Pyr) is produced and correspondingly less acetyl-CoA is formed via the pyruvate dehydrogenase pathway. The reduction of internal acetyl-CoA obliges less consumption of oxaloacetate for the synthesis of citrate (Cit) via citrate synthetase. A truncated form of the tricarboxylic acid cycle is thereby created, `beginning' with glutamate (Glu) and extending through 2-oxo-glutarate (AKG), succinate (Succ), succinyl-CoA (Succ-CoA), fumarate (Fum) and oxaloacetate (OAA).
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which enters the tricarboxylic acid cycle via the oxidation of pyruvate in the pyruvate dehydrogenase pathway. During acidosis, however, this carbon ¯ow is limited because of the inhibition of ¯ux through glycolysis. As a consequence, the tricarboxylic acid cycle functions as the `truncated' cycle shown in Fig. 7. Glutamine, rather than glucose, becomes a temporary metabolic `substrate' for the brain (Yu et al., 1982; Erecinska et al., 1988, Yudko et al., 1989, 1994). It must be emphasized that glutamine is not a substrate in the same sense that glucose subserves this role. There is little if any importation of glutamine (or glutamate) from blood to brain (Grill et al., 1992). Thus, essentially all brain glutamine carbon must be derived from glucose. There is no net consumption of peripheral glutamine as a metabolic substrate. However, the data are consistent with the notion that glutamine can temporarily `nourish' neurons and glia when glucose is absent (Erecinska et al., 1988) or, as in the current study, when ¯ux through glycolysis is compromised. The fact that acidosis does not substantially alter astrocyte ATP levels (Swanson and Benington, 1996), although it does inhibit glycolysis, also suggests that these cells can utilize an alternate substrate. The major route of glutamine/glutamate carbon entry into the tricarboxylic acid cycle is transamination to aspartate (Fig. 7). Although this reaction generally is conceived as functioning at equilibrium, in neurons it probably favors the synthesis of aspartate (Erecinska et al., 1990). This apparent vectorality may result in part from the fact that aspartate so produced leaves the mitochondrion via a mitochondrial glutamateaspartate antiporter, which is an essential component of the malate-aspartate shuttle (Cheeseman and Clark, 1988; Fitzpatrick et al., 1983). The fact that considerable aspartate accumulates extracellularly (Results), particularly when glutamate is present in the incubation medium, indicates that at least a portion of the aspartate which exits the mitochondrion leaves the astrocyte, presumably in exchange for external glutamate. In our studies of synaptsomal glutamate transamination we also noted that the major site of aspartate accumulation was in the extracellular space (Erecinska et al., 1988). The acidosis-induced changes of astrocyte metabolism noted here resemble changes previously noted in rat cerebral cortex during hypercapnia (Folbergrova et al., 1972a,b, 1975; Miller and Veech, 1975), when there occurs a sharp drop of pyruvate and lactate concentrations and an apparently increased transamination of glutamate to aspartate. It should be emphasized that the use of glutamine/glutamate as an alternate fuel is only a temporary solution to the problem of inhibited ¯ux through glycolysis. Ultimately, the levels of these amino acids also decline (Miller and Veech, 1975), per-
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haps re¯ecting their continued utilization as a source of energy. The acidosis-induced changes of glial glutamate metabolism are similar in many respects to those noted in the kidney. Acidosis is associated with a marked increase of ¯ux through 2-oxo-glutarate dehydrogenase (Nissim et al., 1985, 1989, 1990). As the intra-renal level of the ketoacid declines, there is a marked intensi®cation of ¯ux through the glutamate dehydrogenase pathway (Nissim et al., 1989, 1990) and a concomitant release of ammonia, which abets acidi®cation of the urine (Nissim et al., 1985). The glutamate dehydrogenase pathway probably is not prominent in the brain (Yudko et al., 1991), particularly in comparison with the aspartate aminotransferase pathway, but the augmented rate of [1-13C]glutamate oxidation (Fig. 6) clearly suggests enhanced ¯ux through 2-oxo-glutarate dehydrogenase. The current data aord some insight into the response of the system to the momentary stress of acidosis. If the latter stress is severe and enduring, it appears that it will contribute to frank tissue necrosis, especially if there is a concomitant failure of energy metabolism (Siesjo et al., 1996). However, the current data also point to the plasticity of glia in terms of recovering from acidosis by rapidly switching to an alternate fuel. The fact that astrocytes also are the main site of synthesis of glutamine (Norenberg and Martinez-Hernandez, 1979), which they subsequently export to neurons as part of the glutamate-glutamine cycle (Hertz et al., 1993), may facilitate this metabolic transition.
Acknowledgements This work was supported by NIH grants HD34900, NS37915, DK53761, HD26979.
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www.elsevier.com/locate/neuint
Acidosis and astrocyte amino acid metabolism Marc Yudko*, Yevgeny Daikhin, Ilana Nissim, Itzhak Nissim Children's Hospital of Philadelphia, Division of Child Development and Rehabilitation, Department of Pediatrics, University of Pennsylvania School of Medicine, 34th St. and Civic Center Blvd., Abramson Building, Philadelphia, PA 19104 4318, USA Received 1 April 1999; received in revised form 23 June 1999; accepted 25 June 1999
Abstract The relationship between acidosis and the metabolism of glutamine and glutamate was studied in cultured astrocytes. Acidi®cation of the incubation medium was associated with an increased formation of aspartate from glutamate and glutamine. The rise of the intracellular content of aspartate was accompanied by a signi®cant decline in the extracellular concentration of both lactate and citrate. Studies with either [2-15N]glutamine or [15N]glutamate indicated that there occurred in acidosis an increased transamination of glutamate to aspartate. Studies with L-[2,3,3,4,4-2H5]glutamine indicated that in acidosis glutamate carbon was more rapidly converted to aspartate via the tricarboxylic acid cycle. Acidosis appears to result in increased availability of oxaloacetate to the aspartate aminotransferase reaction and, consequently, increased transamination of glutamate. The expansion of the available pool of oxaloacetate probably re¯ects a combination of: (a) Restricted ¯ux through glycolysis and less production from pyruvate of acetyl-CoA, which condenses with oxaloacetate in the citrate synthetase reaction; and (b) Increased oxidation of glutamate and glutamine through a portion of the tricarboxylic acid cycle and enhanced production of oxaloacetate from glutamate and glutamine carbon. The data point to the interplay of the metabolism of glucose and that of glutamate in these cells. # 2000 Elsevier Science Ltd. All rights reserved.
1. Introduction It is well accepted that astrocytes are central to brain glutamate handling. These cells, which have abundant high-anity glutamate transporters, probably are the major locus of brain glutamate uptake from the extracellular ¯uid (Hertz et al., 1993; Schousboe et al., 1977). They also are the primary site in brain of the glutamine synthetase reaction, which abets formation of glutamine that neurons subsequently utilize, via the phosphate-dependent glutaminase reaction, to synthesize glutamate (Norenberg and Martinez-Hernandez, 1979; Hertz et al., 1993). Indeed, the production of glutamine in glia and the subsequent reconversion to glutamate in neurons constitutes a metabolic arrangement that often is called the `glutamateglutamine cycle' (Yudko et al., 1992). * Corresponding author. Tel.: +1-215-590-7474; fax: +1-215-5906804 E-mail address: yudko@email.chop.edu (M. Yudko).
The metabolism of glutamate in astrocytes is more varied than the relatively straightforward model that is implied by the notion of a glutamate-glutamine cycle. It now is clear that astrocytes, in addition to forming glutamine from glutamate, also are capable of the robust metabolism of the latter amino acid. Indeed, glial glutamate oxidation is so active that glutamate almost could substitute for glucose as a metabolic fuel in these cells (Yu et al., 1982). The precise metabolic routes by which glutamate carbon enters the tricarboxylic acid cycle has been the subject of some controversy. Three pathways have been implicated: the glutamate dehydrogenase reaction, the malic enzyme pathway, and the aspartate aminotransferase reaction. The relative contributions of an individual pathway probably depends, in large measure, upon the metabolic `set' of the cell as well as the ambient glutamate concentration (McKenna et al., 1994). In our prior studies of brain transamination of glutamate to aspartate in both synaptosomes and astrocytes, we found that ¯ux through this equilibrium pathway probably is determined by the availability of
0197-0186/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 7 - 0 1 8 6 ( 9 9 ) 0 0 1 4 1 - 2
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oxaloacetate, a substrate of the reaction: Glutamate+Oxaloacetate 4 Aspartate+2-oxo-glutarate. The rate of transamination declines if oxaloacetate is utilized for another pathway and is thereby diverted away from the aminotransferase. This occurs, for example, when brain respires on ketone bodies, which are readily metabolized in brain to acetyl-CoA. As the latter ester combines with oxaloacetate to yield citrate in the citrate synthetase pathway, less glutamate can enter the aspartate aminotransferase reaction because of the relative unavailability of oxaloacetate (Yudko et al., 1997). In this study we have explored the relationship between increased H+ concentration and astrocyte metabolism of glutamate. In the kidney acidosis is associated with a rapid and profound increase of ¯ux through glutaminase as well as increased glutamate consumption via both the various glutamate transaminases and the glutamate dehydrogenase pathway (Nissim et al., 1985; Nissim and States, 1989, 1995). The result is increased production of NH3, an important component in the maintenance of body acid-base balance (Nissim et al., 1985). Acidosis also markedly inhibits ¯ux through glycolysis in the brain (Wu and Davis, 1981; Folbergrova et al., 1972a,b; Erecinska et al., 1995; Swanson and Benington, 1996). As a result, there is reduced formation of acetyl-CoA from pyruvate and less oxaloacetate is consumed in the citrate synthetase reaction. More oxaloacetate then is accessible to aspartate aminotransferase. As a consequence, the production of aspartate from glutamate is augmented. Our data are consistent with the formulation of augmented glutamate transamination to aspartate in acidosis. 2. Experimental procedures 2.1. Astrocyte cultures Astrocytes were cultured from young (<24 h) rats by placing the whole brain in a warm buered (pH 7.4) solution containing NaCl (135 mM), KCl (4.0 mM) and Hepes (20 mM). After removing meninges, a wedge of parietal cortex was removed from either side and incubated at 378C for 30 min in the presence of 0.15% trypsin. The cells were then washed three times in 2 ml of the buered saline solution to remove the trypsin. Then was added 2 ml of primary culture medium (80% DMEM, 10% fetal bovine serum, 10% Ham's F-12 nutrient solution). The cell suspension was triturated 15 times with a sterile glass pipette in order to deaggregate the cells. After allowing the tissue debris to settle, the cell suspension was transferred to another 15 ml tube. The cells then were plated at a
density of 2.5 105 cells/ml (3 104 cells/cm2) in 75 cm2 plastic vials. Cells were maintained in an incubator at 378C and with 5% CO2 for 3±4 weeks, when they attained con¯uency. Tests for glial purity routinely showed yields of >95% GFAP-positive cells. 2.2. Experimental conditions After removing the steady-state medium, the cells were washed (4) with a buered (pH 7.4) solution containing 135 mM NaCl, 5 mM NaHCO3, 5 mM KH2PO4, 1 mM MgSO4, 1 mM CaCl2, 10 mM D-glucose and 20 mM HEPES. The steady-state medium then was replaced with 2 ml of warmed (378C) incubation medium of similar ionic composition as the wash medium but at pH 7.4, 7.2, 7.0, 6.4 or 6.0, as indicated below (Results). After 1 h the incubation medium was removed and the cell monolayer was washed quickly (3) with cold buered saline solution. The incubation medium was replaced with 1 ml of cold HCl (5 mM) and the cell monolayer was scraped vigorously. After freezing and thawing to assure liberation of cell contents, total amino acid content of the intracellular compartment was determined. The concentration of citrate and lactate was measured in the medium. In a separate series of studies the metabolism of the -NH2 group of either glutamate or glutamine was studied by washing the cells, as indicated above, and replacing the steady-state medium with an incubation medium (above) that contained either [15N]glutamate, [1-13C]glutamate or [2-15N]glutamine (1 mM each). At the indicated times (Results), the medium was removed, the cell monolayer was washed and the incubation medium was replaced with 2 ml of 5 mM HCl. The internal concentrations of amino acids as well as the 15N label (atom % excess, see below) were determined. In order to study the conversion of L[2,3,3,4,4-2H5]glutamine to [2H1]aspartate, a similar experimental paradigm was employed with the [2H5]glutamine species as precursor. The oxidation of glutamate was studied by adding [1-13C]glutamate (1 mM) to the astrocyte incubation medium. At the indicated times (Results) the medium was removed and treated as indicated (below) to liberate 13CO2. 2.3. Isotopic abundance in amino acids and CO2 To measure isotopic abundance in glutamate and aspartate, 100 microliters of cell extract were taken to dryness and the t-butyldimethylsilyl derivative was determined as described by Frederick et al (1984). Label in [15N]glutamate and [15N]aspartate was determined from the m/z 433/432 and m/z 419/418 ion
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ratios, respectively. Label in [2,3,3,4,4-2H5]glutamate and [2H4]glutamate was determined from the m/z 437/ 432 and m/z 436/432 ratios, respectively. Analysis of isotopic enrichment was performed with selected ion monitoring on a Hewlett-Packard 5970 mass-selective detector on-line with a Hewlett-Packard 5890 gas chromatograph. The electron multiplier was set to 02000 V, the ®lament potential to 70 eV, source temperature and transfer line both were at 2808C and the injector at 2508C. The temperature program was 1208C for 3 min and then 88C/min until 2808C. The column was a 12 M 0.32 mm Ultra-2 capillary from Hewlett-Packard. Calibration curves were run on a weekly basis to assure linearity of response for the (M+1)/(M) ratio. Glial oxidation of [1-13C]glutamate was determined by adding 10 mmol of NaHCO3 as a carrier to 1 ml of the medium removed at the indicated times (Results) during incubation with [1-13C]glutamate (98 atom % excess). This was placed in a glass tube with a rubber stopper. After evacuation of the atmosphere, 0.5 ml of 40% phosphoric acid was injected through the stopper in order to liberate the 13CO2. This atmosphere was removed with a gas-tight syringe and injected into a Sira-12 isotope ratio-mass spectrometer. Selected ion monitoring of the m/z 45/44 ratio was performed in order to monitor enrichment in 13CO2. 2.4. Determination of concentration of lactate and citrate The concentration of lactate and citrate was determined by isotope dilution. To 1.5 ml of medium was added 15 nmol of [2H]citrate and 75 nmol of [1-13C]lactate in aqueous solution (each isotope >98 atom % excess). After acidifying to 2N HCl and saturating with NaCl, the acid fraction was extracted successively with 3 ml each of ethyl acetate, ethyl ether and tetrahydrofuran. The solvents were combined together and back extracted once with 1 ml of 2N HCl. The organic phase then was taken to dryness and the t-butyldimethylsilyl derivative was prepared (Frederick et al., 1984) using acetonitrile as counter solvent. The concentration of citrate and lactate was determined by isotope dilution analysis of the m/z 592/ 591 and m/z 262/261 ratios, respectively. 2.5. Other analyses The concentrations of amino acids are determined by HPLC of their o-phthaldialdehyde derivatives (Jones and Gilligan, 1983). The internal standard was E-aminocaproic acid. Protein was measured with the Bio-Rad protein assay kit (Bio-Rad; Richmond, CA, USA).
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2.6. Calculation of isotopic abundance and data analysis Isotopic abundance (atom % excess) was calculated from the height of each peak according to Rosenblatt et al. (1992). The absolute concentration of 15N in a given metabolite refers to the product/100 of isotopic abundance (atom % excess) and metabolite concentration (nmol/mg protein). The rate of formation of a particular 15N product was determined from the initial linear segment of the rate of synthesis. Values for ¯ux were normalized to the internal labeling (atom % excess) in the precursor species. Statistical signi®cance was determined with the t-test for unpaired data. 3. Results 3.1. Eects of pH on metabolite levels Intracellular amino acid levels after a 1 h incubation in the presence of buer of varying pH are shown in Fig. 1. Internal [aspartate] tended to increase as a function of medium [H+]. At pH 7.4 the aspartate level was 10.1 2 2.9 nmol/mg protein. This value was increased to 216% and 261% of control (pH 7.4) at pH 6.4 and 6.0, respectively. The internal [aspartate] was not altered signi®cantly at pH 7.2 or 7.0. No signi®cant change at any pH was observed with respect to internal [glutamate] (Fig. 1). Intracellular [glutamine] varied much less than did internal [aspartate]: it was not signi®cantly dierent from control except for pH 6.4 and 6.0 (Fig. 1). The foregoing study was repeated in precisely the same manner but with L-glutamine (0.5 mM) as a substrate in the medium. The results are shown in Fig. 2. As the concentration of external H+ was increased, the internal [glutamate] was not signi®cantly altered, but the internal [aspartate] rose sharply, the concentration at pH 6.0 being more than 300% greater than that noted at pH 7.4 (Fig. 2). A signi®cant increase was seen also at pH 6.4. Even at a more `moderate' medium pH of 7.0, the intracellular level of aspartate was nearly twice that measured at physiologic pH. Internal [glutamine] was much less responsive to medium [H+] than was internal [aspartate], although a signi®cant increase was observed at pH 6.4 (Fig. 2). In Table 1 are shown the concentrations (mM) of selected metabolites in medium following a 1 h incubation in the presence of L-glutamine (1 mM). The medium [NH3] was signi®cantly higher at pH 6.4 vs 7.4, presumably re¯ecting greater utilization of glutamine via phosphate-dependent glutaminase (Yudko et al., 1989). The fact that medium [glutamine] is sig-
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Fig. 1. Intra-astrocytic amino acid levels at varying pH. After removing the steady-state medium and washing the astrocytes, the cells were incubated for an additional hour at 378C at pH 7.4, 7.2, 7.0, 6.4 or 6.0. At the end of that period the medium was removed, the cells were washed (3) and the incubation medium was replaced with 2 ml of 5 mM HCl. The cell monolayer was scraped, frozen and thawed to assure lysis of internal contents. Amino acids were measured as described in Methods. Results are expressed as a fraction (%) of the control (pH 7.4) value. Each bar is the mean (2SD) of at least four experiments. P < 0.05 vs pH 7.4.
ni®cantly lower in the presence of higher [H+] (Table 1) is consistent with this formulation. In contrast, medium lactate and citrate are signi®cantly reduced in the acidotic medium. 3.2. Acidosis and glutamate transamination: studies with 15 N and 2H precursors The striking increase of internal [aspartate] in the presence of a high medium [H+] suggested that acidosis might be associated with augmented transamination of glutamate to aspartate. In order to test this possibility, astrocytes were incubated for 1 h in the presence of [2-15N]glutamine (1 mM) and internal labeling of aspartate and glutamate was measured. Labeling (atom % excess; Fig. 3, top panel) in internal [15N]aspartate was increased by 075% with a medium pH of 6.4 vs 7.4 (P < 0.05). Labelling of [15N]gluta-
mate was unaected by the change of medium [H+] (Fig. 3, top panel). Labelling of intra-astrocytic glutamine (not shown in Fig. 3) was similar in pH 6.4 vs pH 7.4: 81.0 vs 84.5 atom % excess, respectively. The absolute concentration of internal 15N-amino acids (Methods) is shown in the bottom panel of Table 1 Concentration (mM) of metabolites in medium after 1 h incubation in presence of [2-15N]glutamine (1.0 mM)a pH
7.4
6.4
NH3 Lactic Citric Gln
110.9211.0 525.0234.1 47.4210.1 758.3224.6
143.629.2 256.2243.5 22.121.0 707.5217.9
a
Results are mean2SD of eight experiments. P < 0.05.
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Fig. 2. Intra-astrocytic amino levels at varying pH in presence of L-glutamine. Incubation procedure is precisely as described in legend to Fig. 1 except that the incubation medium contained L-glutamine (0.5 mM). Amino acid concentrations are expressed as percentage of the control (pH 7.4) value. Each bar is the mean (2SD) of at least four experiments. P < 0.05 vs pH 7.4.
Fig. 3. When medium pH was 6.4, the level of intracellular [15N]aspartate was increased more than 4-fold, from 2.7 2 0.5 nmol/mg protein (pH 7.4) to 10.8 2 0.9 nmol/mg protein (pH 6.4) (P < 0.05). The internal [15N]glutamate concentration was not signi®cantly altered by incubation at high medium [H+]. The above data describe the transfer of amino groups from glutamate to aspartate. Glutamate carbon also can be converted to aspartate following oxidation of 2-oxo-glutarate to succinate and then to oxaloacetate in the tricarboxylic acid cycle. In order to gauge the eect of acidosis on this pathway, L[2,3,3,4,4-2H5]glutamine was utilized as a metabolic tracer (Methods). The data are shown in Fig. 4. As indicated in the top panel, labelling of [2H1]aspartate was increased from 7.9 2 1.4 to 11.4 2 4.1 atom % excess (P < 0.05) at pH 6.4 vs 7.4. Labeling in
[2,3,3,4,4-2H5]glutamate was slightly but signi®cantly decreased by incubation at acid pH (Fig. 4, top panel). Labeling of [2H4]glutamate was unchanged (Fig. 4, top). Enrichment in [2,3,3,4,4-2H5]glutamine was unaffected (data not shown in Fig. 4). The absolute concentration of 2H-amino acids is shown in the bottom panel of Fig. 4. The concentration of [2H]aspartate was sharply increased at acid pH: 0.7 2 0.1 (control) vs 1.2 2 0.5 nmol/mg protein (acidosis). The increased rate of aspartate formation was accompanied by a diminished internal concentration of 2H-glutamate. Thus, the concentration of the M+5 species ([2,3,3,4,4-2H5]glutamate), corresponding to glutamate formed directly via glutaminase from labelled glutamine, was 2.7 2 0.3 (control) vs 1.9 20.4 (acidosis) nmol/mg protein (P < 0.05). Similarly, the concentration of the M+4 species, corresponding
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to glutamate formed from the M+5 species following transamination to 2-oxo-glutarate and re-amination of the latter back to glutamate, was 3.520.4 (control) vs 2.1 2 0.4 (acidosis) nmol/mg protein (P < 0.05). The fraction (%) of aspartate compared with glutamate (both M+4 and M+5) was greatly increased at
Fig. 3. Transfer of 15N from [2-15N]glutamine to [15N]glutamate and [15N]aspartate. The astrocytes were incubated essentially as described in the legend to Fig. 1, except that the incubation medium contained 15 L-[2- N]glutamine (1.0 mM). After 1 h the steady-state medium was removed, the cells were washed and the internal labeling (atom % excess) of [2-15N]glutamine was determined as well as the concentration of glutamate and aspartate. Top panel: Label (atom % excess) in intracellular [15N]aspartate and [15N]glutamate after 1 h. Bottom panel: The absolute concentration (nmol 15N-amino acid/mg protein) of [15N]aspartate and [15N]glutamate, corresponding to the product of atom % excess/100 and total amino acid concentration (nmol/mg protein). Each bar is the mean (2SD) of at least four experiments. Open bar: control incubations (pH 7.4). Stippled bar: acidosis (pH 6.4). P < 0.05 vs pH 7.4.
Fig. 4. Metabolism of L-[2,3,3,4,4-2H5]glutamine in astrocytes. Incubations were done precisely as described in the legend to Fig. 3, except that the incubation medium contained L-[2,3,3,4,4-2H5]glutamine (1.0 mM). Each bar is the mean (2SD) of at least 4 experiments. Open bar: control incubations (pH 7.4). Stippled bar: acidosis (pH 6.4). Error bar less than scale for control values of absolute concentration of the M+5 glutamate species. P < 0.05 vs pH 7.4.
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increased medium [H+]: 11.2 2 1.9 (control) vs 29.4 2 10.2 (acidosis) (P < 0.05). The foregoing data all were generated with labeled species of glutamine. In order to determine whether acidosis would similarly aect the metabolism of glutamate, the astrocytes were incubated with [15N]glutamate and the formation of [15N]aspartate and [2-15N]glutamine was determined. Results for intracellular labeling patterns are shown in Fig. 5. Increased medium [H+] appeared to be associated with reduced glial accumulation of [15N]glutamate. At 60 min the internal concentration (nmol/mg protein) was 757.8 2 114.7 (control) vs 514.4 2 70.6 (acidosis) (P < 0.05). Unlike incubations performed with [2-15N]glutamine (see Fig. 3) the intra-astrocytic level of [15N]aspartate was signi®cantly reduced at 60 min by acid pH: 112.3 2 12.7 (control) vs 64.4 2 18.5 (acidosis) (P < 0.05). However, the concentration (mM) of [15N]aspartate in the incubation medium at 60 min was increased at acid pH: 23.0 2 2.8 (control) vs 34.1 2 3.2 (acidosis) (P < 0.01). With an internal aspartate concentration of 0 10 nmol/mg protein, a protein content of 00.2 mg/plate and an incubation medium volume of 2 ml, it follows that >95% of total aspartate (intra-astrocytic+extracellular) is resident in the medium. The overall eect of acidosis, therefore, was to increase total aspartate production by 035%.
Fig. 5. Glial metabolism of [15N]glutamate. Incubations were done as described in the legend to Fig. 3, except that the incubation medium contained [15N]glutamate (1.0 mM) instead of a labeled glutamine species. At the times indicated in the ®gure the incubation medium was removed and the cells were washed and harvested as described in the legend to Fig. 1. Left top panel: absolute concentration of [15N]glutamate (nmol/mg protein). Right top panel: absolute concentration of [15N]aspartate. This parameter was determined as described in the legend to Fig. 3. Bottom panel: concentration of aspartate (mM) in the incubation medium at end of incubation. Each point is the mean (2SD) of at least four experiments. Control and acidosis as indicated by symbols in the ®gure. P < 0.05 compared with pH 7.4.
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One reason that the aspartate accumulated in the medium rather than the intracellular compartment may be that intra-astrocytic aspartate exchanged with external glutamate, the level of which (mM) was greater at increased medium [H+]: 676.0239.9 (control) vs 955.8 270.5 (acidosis) (P < 0.05) (Fig. 5). 3.3. Acidosis and oxidation of glutamate The 15N data are consistent with the formulation that acidosis is associated with enhanced transamination of glutamate N to yield aspartate, a phenomenon similar to that noted in kidney cells (Nissim et al., 1995). The studies with [2,3,3,4,4-2H5]glutamine (Fig. 5) suggests that the 2-oxo-glutarate formed from glutamate is oxidized via the tricarboxylic acid cycle to [2H]oxaloacetate, which, in turn, is transaminated to [2H]aspartate. In order to directly monitor the eects of acidosis on glial glutamate oxidation, a series of studies was done with [1-13C]glutamate (1 mM) as precursor. The results are shown in Fig. 6. Appreciable enrichment was detected in 13CO2 with the intensity of labeling being greater in acidosis vs control. Thus, at 5, 10, 20 and 30 min enrichment in 13CO2 at pH 6.0 was 27.0, 37.5, 31.7 and 29.4% greater in the acidotic vs control samples (Fig. 6, left panel; P < 0.05 at each time point). Furthermore, the increased label in 13CO2 was not attributable to increased intracellular labeling of [1-13C]glutamate. When labeling of 13CO2 was normalized to internal enrichment (atom % excess) with [1-13C]glutamate, a statistically signi®cant dierence was still observed (Fig. 6, right panel).
Fig. 6. Oxidation of L-[1-13C]glutamate in astrocytes. The cells were incubated as described in the legend to Fig. 3, except that the medium contained L-[1-13C]glutamate (1 mM). At the times indicated in the Fig., the medium was removed and the 13CO2 content was analyzed with isotope ratio-mass spectrometry (see Methods for details). Left Panel: Atom % Excess in 13CO2. Values are those obtained following the addition of unlabelled carrier CO2. Right Panel: Ratio (100) of label in 13CO2 in that [1-13C]glutamate. Circles: control (pH 7.4). Squares: acidosis (pH 6.4). Each point is the mean (2SD) of at least four determinations. P < 0.05 compared with pH 7.4.
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4. Discussion Altered brain pH, a common metabolic derangement, accompanies various pathologic states, including hypoxia, ischemia, various toxic states and some inborn errors of metabolism. Indeed, acute and severe acidosis may contribute to the brain damage that occurs consequent to tissue ischemia (Siesjo et al., 1996). Relatively mild acidosis may attenuate glutamate neurotoxicity and other forms of ischemic injury (Tombaugh and Sapolsky, 1993; Giard et al., 1990; Sapolsky et al., 1996; Vornov et al., 1996). Various mechanisms have been suggested as causes of acidosismediated brain injury, including increased free radical production (Waterfall et al., 1996; Siesjo et al., 1985), an accentuation of ischemic damage (Hurn et al., 1991; Katsura et al., 1994), injury to brain endothelium (Paljarvi et al., 1983), a release of Ca2+ from internal stores (Abercrombie and Hart, 1986; Baker and Umbach, 1987), and an activation of neuronal apoptosis (Barry and Eastman, 1993). We have shown here that acidosis is associated with increased transamination of glutamate to aspartate, implying increased availability of oxaloacetate to the aspartate aminotransferase pathway. A plausible explanation for an augmented oxaloacetate pool is that less acetyl-CoA is derived from glucose carbon because acidosis inhibits glycolysis. Less oxaloacetate, therefore, is consumed in the citrate synthetase pathway. This is re¯ected in diminished production of both citrate and lactate during acidosis (Table 1). The inhibitory eect of acidosis on glycolysis is rapid and pronounced (Folbergrova et al., 1972a,b, 1975; Miller and Veech, 1975; Wu and Davis, 1981; Erecinska et al., 1995; Swanson and Benington, 1996). It is likely that such inhibition occurs at the level of phosphofructokinase, the rate-limiting step of glycolysis (Erecinska et al., 1995). This enzyme is strongly inhibited by H+ (Trivedi and Danforth, 1966; Dobson et al., 1986) in a manner that is reversible by high concentrations of AMP and Pi, a positive eector of phosphofructokinase (Goldhammer and Paradies, 1979). Our experimental model involved the incubation of astrocytes with a medium of varying pH. Intra-astrocytic pH was not measured, but changes of external [H+] probably are rapidly re¯ected in the internal [H+] (Mellergard et al., 1992; Ouyang et al., 1993). This condition was associated with a marked increase of [aspartate] (Table 1) and a signi®cant increase in the rate of conversion of [15N]glutamate to yield [15N]aspartate (Fig. 3), denoting increased ¯ux through the aspartate aminotransferase reaction. The latter is an equilibrium reaction for which the directionality is sensitive to the relative concentrations of reactants. The increased [aspartate] and augmented production of [15N]aspartate from [15N]glutamate are consistent
with the notion that, during acute acidosis, there was more mitochondrial oxaloacetate available to the aspartate aminotransferase reaction as less oxaloacetate was incorporated into citrate. Flux through citrate synthetase was diminished because of reduced production of acetyl-CoA via the pyruvate dehydrogenase reaction. The latter pathway was relatively quiescent because of an acidosis-induced inhibition of glycolysis (Erecinska et al., 1995). In addition to the acidosis-induced restriction on oxaloacetate utilization for the purpose of citrate synthesis, it is likely that acidosis is associated with increased production of oxaloacetate from glutamine and glutamate, a motif that has been previously observed in renal tissue during acidosis (Nissim et al., 1985, 1989, 1995). We previously demonstrated in synaptosomes that glutamine and glutamate carbon can be an ecient source of brain oxaloacetate (Yudko et al., 1994). This requires that glutamate carbon enter the tricarboxylic acid cycle via transamination to yield 2-oxo-glutarate. These putative metabolic relationships are summarized in Fig. 7, which indicates that the carbon skeletons of oxaloacetate and other intermediates of the tricarboxylic acid cycle can be derived from glutamine and glutamate as well as glucose (Fig. 7). This carbon backbone ordinarily would be furnished by glucose,
Fig. 7. Schema to illustrate inter-relationship of glucose and amino acid metabolism in astrocytes. The rate of transamination of glutamate to aspartate depends upon the accessibility of oxaloacetate (OAA) to the aspartate aminotransferase pathway. When ¯ux through glycolysis is relatively slow, less pyruvate (Pyr) is produced and correspondingly less acetyl-CoA is formed via the pyruvate dehydrogenase pathway. The reduction of internal acetyl-CoA obliges less consumption of oxaloacetate for the synthesis of citrate (Cit) via citrate synthetase. A truncated form of the tricarboxylic acid cycle is thereby created, `beginning' with glutamate (Glu) and extending through 2-oxo-glutarate (AKG), succinate (Succ), succinyl-CoA (Succ-CoA), fumarate (Fum) and oxaloacetate (OAA).
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which enters the tricarboxylic acid cycle via the oxidation of pyruvate in the pyruvate dehydrogenase pathway. During acidosis, however, this carbon ¯ow is limited because of the inhibition of ¯ux through glycolysis. As a consequence, the tricarboxylic acid cycle functions as the `truncated' cycle shown in Fig. 7. Glutamine, rather than glucose, becomes a temporary metabolic `substrate' for the brain (Yu et al., 1982; Erecinska et al., 1988, Yudko et al., 1989, 1994). It must be emphasized that glutamine is not a substrate in the same sense that glucose subserves this role. There is little if any importation of glutamine (or glutamate) from blood to brain (Grill et al., 1992). Thus, essentially all brain glutamine carbon must be derived from glucose. There is no net consumption of peripheral glutamine as a metabolic substrate. However, the data are consistent with the notion that glutamine can temporarily `nourish' neurons and glia when glucose is absent (Erecinska et al., 1988) or, as in the current study, when ¯ux through glycolysis is compromised. The fact that acidosis does not substantially alter astrocyte ATP levels (Swanson and Benington, 1996), although it does inhibit glycolysis, also suggests that these cells can utilize an alternate substrate. The major route of glutamine/glutamate carbon entry into the tricarboxylic acid cycle is transamination to aspartate (Fig. 7). Although this reaction generally is conceived as functioning at equilibrium, in neurons it probably favors the synthesis of aspartate (Erecinska et al., 1990). This apparent vectorality may result in part from the fact that aspartate so produced leaves the mitochondrion via a mitochondrial glutamateaspartate antiporter, which is an essential component of the malate-aspartate shuttle (Cheeseman and Clark, 1988; Fitzpatrick et al., 1983). The fact that considerable aspartate accumulates extracellularly (Results), particularly when glutamate is present in the incubation medium, indicates that at least a portion of the aspartate which exits the mitochondrion leaves the astrocyte, presumably in exchange for external glutamate. In our studies of synaptsomal glutamate transamination we also noted that the major site of aspartate accumulation was in the extracellular space (Erecinska et al., 1988). The acidosis-induced changes of astrocyte metabolism noted here resemble changes previously noted in rat cerebral cortex during hypercapnia (Folbergrova et al., 1972a,b, 1975; Miller and Veech, 1975), when there occurs a sharp drop of pyruvate and lactate concentrations and an apparently increased transamination of glutamate to aspartate. It should be emphasized that the use of glutamine/glutamate as an alternate fuel is only a temporary solution to the problem of inhibited ¯ux through glycolysis. Ultimately, the levels of these amino acids also decline (Miller and Veech, 1975), per-
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haps re¯ecting their continued utilization as a source of energy. The acidosis-induced changes of glial glutamate metabolism are similar in many respects to those noted in the kidney. Acidosis is associated with a marked increase of ¯ux through 2-oxo-glutarate dehydrogenase (Nissim et al., 1985, 1989, 1990). As the intra-renal level of the ketoacid declines, there is a marked intensi®cation of ¯ux through the glutamate dehydrogenase pathway (Nissim et al., 1989, 1990) and a concomitant release of ammonia, which abets acidi®cation of the urine (Nissim et al., 1985). The glutamate dehydrogenase pathway probably is not prominent in the brain (Yudko et al., 1991), particularly in comparison with the aspartate aminotransferase pathway, but the augmented rate of [1-13C]glutamate oxidation (Fig. 6) clearly suggests enhanced ¯ux through 2-oxo-glutarate dehydrogenase. The current data aord some insight into the response of the system to the momentary stress of acidosis. If the latter stress is severe and enduring, it appears that it will contribute to frank tissue necrosis, especially if there is a concomitant failure of energy metabolism (Siesjo et al., 1996). However, the current data also point to the plasticity of glia in terms of recovering from acidosis by rapidly switching to an alternate fuel. The fact that astrocytes also are the main site of synthesis of glutamine (Norenberg and Martinez-Hernandez, 1979), which they subsequently export to neurons as part of the glutamate-glutamine cycle (Hertz et al., 1993), may facilitate this metabolic transition.
Acknowledgements This work was supported by NIH grants HD34900, NS37915, DK53761, HD26979.
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