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CELLULAR METABOLISM
1Department of Biomedical Sciences (Committee on Molecular Metabolism and Nutrition) and 2Department of Medicine, Martin Boyer Laboratories, Inflammatory Bowel Disease Research Center, The University of Chicago, Chicago, Illinois; and 3Gastrointestinal Diseases Research Unit, Department of Medicine and Anatomy/Cell Biology, Queen’s University, Kingston, Ontario, Canada
Submitted 9 May 2005 ; accepted in final form 13 March 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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IEC-18; NIH/3T3; glutaminase; 6-diazo-5-oxo-L-norleucine; glutathione
Glutamate is a major metabolite of glutamine through the action of a number of glutaminases, and glutamate acts as an intermediate precursor for the tricarboxylic acid cycle via its conversion to 
-ketoglutarate. Glutamate may also be acted upon by glutamate dehydrogenase, forming an important link between cellular nitrogen and carbon cycles. Glutamate is also an intermediate in the cellular production of the antioxidant GSH as well as an intermediate in the production of ornithine, a precursor of some polyamines. However, glutamate is likely not to reproduce all of glutamine’s effects during physiological stress in the whole animal. Glutamate appears to be less efficiently transported across the plasma membrane than glutamine since glutamine is taken up by a larger number of carrier-mediated systems with high capacity (3). Second, glutamine is not exclusively metabolized via glutamate and thus may have effects mediated by other metabolites (28).
One of the first studies on glutamine and its relation to the heat shock/stress response reported that glutamine deprivation correlated with a reduction in the half-life of cytoprotective heat shock proteins in murine L929 cells (18). Many studies in a variety of tissue and cell systems have confirmed the importance of glutamine in the induction of members of the inducible heat shock protein (Hsp) 70 family (4, 6, 12, 22, 24, 27, 31, 32); however, less attention has been given to other heat shock proteins. Some evidence suggests that mechanisms of induction of Hsp25 and Hsp72 may share some similarities. For example, intravenous administration of glutamine caused a rapid and significant increase in both Hsp72 and Hsp25 expression in multiple organs of unstressed rat, including the heart, the lungs, and gastrointestinal tract (41). Furthermore, the presence of glutamine potentiated lipopolysaccharide-mediated heat shock responses in a model of severe physiological stresses (42). Hsp25 is a member of the small heat shock protein family that is expressed nearly in all organs under physiological conditions and that is also rapidly inducible upon stress activation (38). Hsp25 has important chaperone properties similar to the GroE/Hsp60 and the Hsp70 family. Overexpression of Hsp25 has been shown to increase survival in response to a wide variety of potentially lethal agents (15).
Few studies have performed rigorous comparisons of the modulatory effects of various amino acids on the regulation of the stress/heat shock response, particularly with reference to how glutamine is handled intracellularly and which intermediate metabolites convey the potentiating effect on heat shock protein expression. Glutamate, the product of glutaminase action on glutamine, has received limited attention (1, 32, 33). Some of these studies have been performed in neurons where glutamate is cytotoxic and can induce heat shock proteins as well as cell death. None of these studies, those in neurons or Drosophila Kc cells, provides evidence as to why glutamate, even though metabolically related, fails to confer the same regulatory effect afforded by glutamine.
In this study, we demonstrate that glutamine is an essential amino acid in the culture medium that is required for heat induction of Hsp25 in both epithelial and mesenchymal cells. This effect is unique to glutamine and not reproduced by equimolar concentrations of other amino acids tested. Neither ATP nor GSH production from glutamine (or glutamate) appeared to play a role, and the precise mechanism(s) remain unknown. A role for the requisite conversion of glutamine to glutamate was obtained in studies using the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON), which blocked glutamine’s induction of Hsp25.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The normal, diploid, rat small intestinal epithelial cell line IEC-18 (CRL-1589; American Type Culture Collection, Manassas, VA) was used between passages 19 and 30 and grown in DMEM (4.5 g/l high glucose, 1.5 g/l sodium bicarbonate) containing 2 mM L-glutamine, 5% (vol/vol) FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.1 U/ml insulin (media and all supplements were supplied from Invitrogen, Grand Island, NY). For most experiments, IEC-18 cells were plated on 100-mm petri dishes (Corning, Oneonta, NY) in complete growth medium and allowed to attach and grow at 37°C under 5% CO2 environment for 48 h when cells were generally >90% confluent. Cells were then incubated for 24 h in glutamine-free, reduced-serum [1% (vol/vol)] DMEM with other supplements as above, or the medium was replaced with serum-free DMEM containing either 0 or 4 mM glutamine with all other supplements. Cells were then incubated for 2 h at 37°C before being subjected to a heat shock at 42°C in a water bath for 30 min. For immunoblot analysis, cells were harvested after returning to 37°C for 2 h to allow Hsp25 protein synthesis, whereas for Northern blot analysis, cells were scraped promptly after heat shock.
The murine fibroblast cell line NIH/3T3 (CRL-1658; ATCC) was used in some experiments. Cells were grown in high-glucose (4.5 g/l) DMEM with 4 mM L-glutamine, 10% (vol/vol) FBS, and penicillin and streptomycin under 5% CO2 humidified incubation at 37°C, as recommended by ATCC. Cells were passed when the density was >80% confluent at the ratio of 1:8. The NIH/3T3 was treated for each experiment by reducing serum and glutamine in a similar fashion as described for the IEC-18 cells. Heat shock was performed in the same manner for IEC-18 cells.
Western Blot Analysis
Heat shock protein expression was assessed by Western blot analysis of whole cell lysates. Protein homogenates were prepared by disrupting cells in lysis buffer [containing: 10 mM Tris·HCl, pH 7.4, 5 mM MgSO4, 2.5 U/ml RNase A, 100 U/ml RNase T1 (Ambion, Austin, TX), 50 U/ml DNase I (Amersham Biosciences, Piscataway, NJ), and complete protease inhibitor cocktail (Roche Molecular Biosciences, Indianapolis, IN)]. An aliquot was removed for protein determination by the bicinchoninic acid assay and Laemmli sample buffer [250 mM Tris, pH 7.4, 2% (wt/vol) SDS, 25% (vol/vol) glycerol, 10% (vol/vol) 2-mercaptoethanol, and 0.01% (wt/vol) bromphenol blue] was added to the remainder. Samples were heated at 70°C for 10 min and stored at –80°C. Total protein (15 µg) was separated on a 12.5% SDS-PAGE gel and immediately transferred to a polyvinylidene difluoride membrane (Polyscreen; Perkin-Elmer, Boston, MA) using 1x Towbin buffer [25 mM Tris, pH 8.8, 192 mM glycine, 15% (vol/vol) methanol]. Membranes were subsequently incubated in 5% (wt/vol) nonfat dry milk in Tris-buffered saline [140 mM NaCl, 5 mM KCl, 10 mM Tris, pH 7.4 (TBST)] containing 0.1% (vol/vol) Tween 20 on a constant shaker at room temperature for 1 h. All antibodies were obtained from Stressgen Biotechnologies (Victoria, BC, Canada). Immunoblotting of the membrane was performed using rabbit anti-Hsp25 polyclonal antibody (1:5,000 dilution), mouse anti-Hsp72 C92 monoclonal antibody (1:5,000 dilution), or rat anti-Hsc73 monoclonal antibody (1:10,000 dilution) at 4°C with gentle shaking overnight. Membranes were washed five times in TBST and subsequently incubated with species-appropriate peroxidase-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA) at the dilution of 1:5,000 for Hsp25 and 1:10,000 for Hsp72 and Hsc73 for 1 h at room temperature with gentle agitation. Five additional washes were applied before membranes were developed in SuperSignal PicoWest enhanced chemiluminescence reagents (Pierce Chemical, Rockford, IL) and exposed to Kodak X-Omat Blue XB-1 film (Eastman Kodak, Rochester, NY). Autoradiograph signals of Hsp25 were quantified densitometrically using ImageJ (W. Rasband, Research Services Branch, National Institute of Mental Health, Bethesda, MD) and normalized to the signal intensity of Hsc73 for equal protein loading control in each experiment.
Northern Blot Analysis
Cells were rinsed two times in chilled PBS, pelleted, and resuspended in TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). RNA was purified with acid phenol and phenol/chloroform/isoamyl alcohol extractions (Ambion) and precipitated with 3 M sodium acetate, pH 5.5, and ethanol. RNA was rinsed with 75% ethanol, air-dried, and dissolved in nuclease-free water. Total RNA (10 µg) was sized-separated on a formaldehyde-denaturing 1% agarose gel using a MOPS buffer system [30 mM MOPS, pH 7.0, 5 mM sodium acetate, 1 mM EDTA, with 25% (vol/vol) deionized formaldehyde] and subsequently transferred to a nylon membrane Hybond N (Amersham Pharmacia Biotech, Buckinghamshire, UK). RNA blots were prehybridized for 6 h in XOTCH reagent [1% (wt/vol) BSA, 7% (wt/vol) SDS, 10 mM EDTA, 200 mM sodium phosphate, pH 8.0, and 15% (vol/vol) deionized formamide] at 42°C. Hybridization was performed in XOTCH reagent at 42°C overnight using [-32P]dCTP-labeled cDNA probes for Hsp25 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at the concentration of 2 x 106 and 1 x 106 counts·min–1·ml–1, respectively. RNA blots were washed up to a stringency of 0.1x standard saline citrate (15 mM NaCl, 1.5 mM sodium citrate)-0.1% (wt/vol) SDS at 65°C to decrease both nonspecific signal and background. RNA blots were exposed overnight at –80°C to Kodak X-Omat Blue XB-1 film enhanced with Kodak BioMax MS intensifying Screens (Eastman Kodak). Hybridization signals were quantified using ImageJ and normalized to the signal intensity of GAPDH.
Amino Acid Transport Studies
Amino acid transport was performed in six-well plates using duplicate wells for each time point. Each experiment was performed when the density of the IEC-18 was 
2 x 106 cells/well. IEC-18 cells were incubated with a serum- and glutamine-free complete growth medium supplemented with 4 mM L-glutamine, L-glutamate, L-proline, L-arginine, L-histidine, L-alanine, glycine, L-asparagine, L-cysteine, L-leucine, L-methionine, L-phenylalanine, or L-tyrosine and 1 µCi/ml L-[3,4-3H(N)]glutamine, L-[3,4-3H]glutamate, L-[2,3-3H]proline, L-[2,3,4-3H]arginine, L-[2,5-3H]histidine, L-[3-3H]alanine, [2-3H]glycine, L-[3H]asparagine, L-[35S]cysteine, L-[4,5-3H(N)]leucine, L-[methyl-3H]methionine, L-[4-3H]phenylalanine, or L-[ring-3,5-3H]tyrosine, respectively [Perkin-Elmer Life Sciences, Moravek Biochemicals (Brea, CA), and Amersham Biosciences] for 0, 15, 30, 45, 60, 90, 120, 180, and 240 min. At each time point, cells were rinsed two times with 2 ml of iced-cold PBS and air-dried for 1 min before adding 1 ml of 0.1% HNO3 for extraction for 6 h. Cell extracts were diluted in 3 ml scintillation counting fluid (Ultima Gold; Packard, Downers Grove, IL) for measuring 3H or 35S by liquid scintillation spectroscopy.
Intracellular amino acid analysis. Cells were treated for varying conditions as described above. Cells were washed and scraped into ice-cold saline and then washed two times by pelleting (14,000 g for 20 s at 4°C). Cell pellets were extracted with 10% (wt/vol) TCA, vortexed, and allowed to sit on ice for 1 h. Precipitated material was pelleted (14,000 g for 20 s at 4°C), the precipitate was dissolved in 1% (wt/vol) SDS, and protein was measured using the bicinchoninic acid procedure. The supernatant was neutralized using NaOH and was then used for a spectrophotometric determination of glutamine and glutamate. One-half of each sample was reacted with glutaminase to yield glutamate; this reaction, as well as one-half of the original sample, was reacted with NAD+ using glutamate dehydrogenase; and the change of NAD+ to NADH was monitored at 340 nm. The amount of endogenous glutamate in the cellular extracts was subtracted from the value analyzing glutamine plus glutamate to yield cellular glutamine. Values are expressed as nanomoles glutamine or glutamate per milligram cell protein.
Glutaminase Activity
Activity of phosphate-dependent glutaminase was measured in whole cell homogenates using an NADH-linked assay. Briefly, cells were treated with 1 mM DON for either 3 or 5 h. Cells were scraped into ice-cold saline, pelleted, and resuspended in 0.3 M sucrose, 2 mM dithiothreitol, 1 mM EDTA, and 5 mM HEPES, pH 7.4. An aliquot was used for protein determination by the bicinchoninic acid procedure. One hundred micrograms of protein (7–12 µl) were added to 100 µl of incubation mixture of 0.15 M potassium phosphate, 20 mM glutamine, 50 mM Tris, pH 8.6, and 0.2 mM EDTA, incubated at 37°C for 30 min, and stopped by the addition of 10 µl of 3 N HCl. To this reaction, 1 ml of a fresh solution containing 2 mM NAD, 0.25 mM ADP, 0.03% H2O2, 100 µg glutamic dehydrogenase, and 80 mM Tris, pH 9.4, was added, and absorbance at 340 nm was measured after 30 min. A standard curve of glutamate was used in the second set of reactions to calculate the glutaminase activity of the initial reactions, and activity was expressed as micromole glutamate generated per milligram protein per hour.
Data Analysis
Signal intensity of autoradiograms was quantified using ImageJ 1.32 (W. Rasband, Research Services Branch, National Institute of Mental Health). The relative amount of Hsp25 presented in Figs. 1
–9 was a production of signal intensity normalization of Hsp25 to Hsc73 for equal protein loading of the same immunoblot. Results were averaged and plotted with Sigma Plot 7.0 (SPSS, Chicago, IL) with error bars representing means ± SE. Statistical analysis (ANOVA) was performed using SPSS 11.0 (SPSS) for differences among group means. Tukey’s, Scheffé’s, and Bonferroni’s post hoc tests were used to compare differences between group means. Mean differences were shown either at 95% confidence limit or at 99% confidence limit.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Hsp25 increases in heat-shocked IEC-18 cells upon return to 37°C incubation. After heat shock (2 h), Hsp25 was highest in cells preincubated in regular culture medium containing 2 mM glutamine and 5% (vol/vol) FBS (Fig. 1A). Omitting glutamine, but not FBS, significantly reduced heat-induced Hsp25. No statistical difference in the level of heat-induced Hsp25 between DMEM containing both glutamine and FBS and DMEM containing glutamine alone was observed (Fig. 1A). Abundance of Hsc73, a non-heat-inducible, constitutive member of the heat shock protein family, was not affected by heat shock, glutamine, or FBS (Fig. 1A) and was therefore used as a loading control and for normalizing Hsp25 expression. The ratio of Hsp25 to Hsc73 densitometric value was set at one for the condition of 0 mM glutamine/0% FBS. All other ratios of densitometric values were compared with this within each experiment.
To determine whether the effect of glutamine is specific to IEC-18, similar studies were performed in a murine cell line of mesenchymal origin, NIH/3T3. It should be noted that the NIH/3T3 cells were grown in a medium containing 4 mM glutamine and 10% (vol/vol) FBS. The degrees of induction were calculated as for the IEC-18 cells by the ratio of Hsp25 to Hsc73 densitometric values, setting the ratio at 0 mM glutamine/0% FBS to one within each experiment. Heat shock induced a 2.5-fold increase of the ratio of Hsp25 to Hsc73, an effect that also required glutamine but not FBS (Fig. 1B). These results suggest that glutamine serves as an essential component in the culture medium to support the production of Hsp25 in response to heat shock in both epithelial (IEC-18) and mesenchymal (NIH/3T3) cells. Similar results were also obtained using a colonic epithelial cell line derived from the SV40 large-T antigen conditionally immortalized Immortimouse (data not shown), and all subsequent experiments were performed only in the IEC-18 epithelial cells because of the conserved nature of the response.
The concentration dependence of glutamine to support heat-induced Hsp25 was determined over a range of physiological and pharmacological concentrations. Normal serum glutamine is 0.5–0.7 mM, but the uptake of glutamine (primarily by the intestine, immune system, and liver) and the delivery of glutamine to the plasma (predominantly from muscle) are increased during stress (17, 39). Accordingly, serum-free media with glutamine concentrations of 0.1–10 mM were used for the incubation time before heat shock. In addition, to determine whether Hsp25 mRNA expression followed a similar pattern, RNA samples were harvested immediately after heat shock and used for Northern blot analysis. For both Hsp25 protein (Fig. 2A) and Hsp25 mRNA (Fig. 2B), a concentration-dependent increase by glutamine was observed. The optimal glutamine concentration that promotes maximum heat induction of Hsp25 protein in IEC-18 cells coincided with concentrations of glutamine measured under normal physiological concentrations in the range of 0.5–0.7 mM (2, 9, 39).
Although normal IEC-18 culture medium contains 2 mM glutamine, 4 mM glutamine supplementation was used for the rest of the studies. This was justified for the following two main reasons 1) to avoid glutamine insufficiency in some studies when incubation took place >2 h post heat shock and 2) to clearly differentiate the nonglutamine from the glutamine-treated conditions.
Glutamine and Glutamate Facilitate Heat Induction of Hsp25
To determine the specificity of glutamine’s effects on heat-induced Hsp25, effects of other metabolically related amino acids were investigated. Cells were pretreated with arginine, glutamate, histidine, or proline, which all may enter the tricarboxylic acid cycle. Studies were performed exactly as for glutamine. In addition, alanine and glycine were used in the same experiment, since both amino acids are highly abundant in the plasma (2, 20, 35) and have been shown to enhance both mRNA and protein abundance of Hsp70 after heat stress (27). The same concentration of these amino acids was used (4 mM, which had been used for glutamine and shown to be effective to support the heat induction of Hsp25). Neither arginine, glutamate, histidine, proline, alanine, nor glycine supported heat induction of Hsp25 (Fig. 3A). Glutamine was included as a control and supported the heat induction of Hsp25. No changes were observed in Hsc73; therefore, the use of the Hsp25-to-Hsc73 ratio (set to 1 for no added amino acid in Fig. 3A) remained valid.
Because it could be argued that the apparent lack of effect of other amino acids on Hsp25 induction could be the result of differences in intracellular accumulation, we therefore measured amino acid uptake for each amino acid. Cells were incubated with a 4 mM concentration of each amino acid nonradioactively labeled individually, and the same amino acid (3H labeled) was added at 1 µCi/ml. Samples were taken up to 4 h. Proline uptake was the greatest among all the amino acids studied, even exceeding uptake of glutamine (Fig. 3B). In contrast, arginine and glutamate were poorly taken up by the cells compared with glutamine at any time point using the same concentration (4 mM) of each amino acid. Alanine, glycine, and histidine uptakes were similar to that obtained with glutamine. Thus differences in Hsp25 responses to supplementation with different amino acids (see Fig. 3A) could not be explained by lack of uptake for most amino acids, although for glutamate and arginine this argument is valid.
The ability of other amino acids, including sulfur-containing (cysteine and methionine), branch-chained (leucine), aromatic group-containing (phenylalanine and tyrosine), and amide group-containing amino acids (asparagine) to support heat induction of Hsp25 was determined next. IEC-18 cells were preincubated with 4 mM cysteine-, asparagine-, leucine-, methionine-, phenylalanine-, tyrosine-, or glutamine-supplemented medium for 2 h and heat shocked 30 min at 42°C, and Hsp25 protein abundance was measured after 2 h. Neither cysteine, asparagine, leucine, methionine, phenylalanine, nor tyrosine supported heat induction of Hsp25. Glutamine was included as a positive control (Fig. 4A). As above, uptake of these amino acids was measured to determine whether lack of uptake might explain the lack of effect. For this set of experiments, [35S]cysteine was used instead of [3H]cysteine. Asparagine uptake was the greatest in this group, taken up more rapidly than glutamine. All other amino acids were taken up less well (50–70% of glutamine at 2 h in Fig. 4B), indicating that differences in uptake were likely not the basis for the lack of effect on promotion of heat induction of Hsp25.
Glutamine Conversion to Glutamate is Required to Maximally Support Heat Induction of Hsp25
The role of glutamate was examined further, since glutamate is the immediate downstream metabolite of glutamine, a reaction that requires the enzyme glutaminase. The failure of glutamate to facilitate the Hsp25 response (see Fig. 3A) could be because of the limited uptake capacity for glutamate by IEC-18 cells (Fig. 3B). Therefore, attempts were made to increase cellular uptake of glutamate by increasing the concentration of glutamate in the preincubation medium as well as duration of uptake. Glutamate uptake studies were performed as above, however, with an increase in the nonradioactive glutamate to 20 and 40 mM. An increase in glutamate to 40 mM increased uptake but was still less than the transport at each time point of 4 mM glutamine, a concentration one-tenth that of glutamate (Fig. 5).
To determine whether increased glutamate uptake could promote heat induction of Hsp25, cells were preincubated with 0–30 mM of glutamate-supplemented DMEM for 2 h, followed by 30 min heat shock and recovery for 2 h. Glutamine (4 µM) was included as a positive control. Increasing the glutamate media concentration supported heat induction of Hsp25 but to a level less than that of glutamine (Fig. 6A). The most effective concentration, 10 mM glutamate, was chosen, and the incubation time increased for up to 6 h. Increasing the incubation time with glutamate before heat shock and recovery further promoted heat induction of Hsp25 (Fig. 6B).
DON, a Glutaminase Inhibitor, Decreases Glutamine’s Effect to Promote Hsp25 Expression
As additional evidence that glutamine’s effect may be in part the result of conversion to glutamate, an inhibitor of glutaminase, DON was used. DON inhibits glutaminase through covalent modification, resulting in cellular glutamate depletion (8, 19). IEC-18 cells were pretreated with varying concentrations of DON for 1 h before glutamine was added to the incubation medium to the final concentration of 4 mM for two more hours, followed by 30 min heat shock and 2 h recovery. A concentration-dependent inhibition of glutamine’s promotion of heat-induced Hsp25 was observed at both the protein level (Fig. 7A, samples taken at 2 h after heat shock) and the mRNA level (Fig. 7B, samples taken immediately after heat shock).
No alterations in cellular morphology were observed after DON; however, to confirm that DON did not decrease cellular viability over this time period, trypan blue exclusion was determined. DON did not alter the percentage of trypan blue-positive cells, <3% under all conditions tested, including 72 h-postplating IEC-18 cells maintained in DMEM containing 0 mM glutamine with 1% (vol/vol) FBS or DMEM containing 0 or 4 mM glutamine without FBS in the experiments. To confirm that DON exerted no inhibitory effect on uptake of glutamine, transport studies were performed. Glutamine was used at a concentration of 4 mM for these studies to be consistent with the previous transport studies shown above, and DON was included at 1 mM during the flux period only. Because no effect of DON on glutamine uptake was observed, we conclude that this step was not involved in DON’s ability to block glutamine’s support of heat-induced Hsp25 (Fig. 8, top).
To ensure that DON by itself did not stimulate Hsp25 under non-heat-shocked conditions and to demonstrate that the effect of glutamine also occurred for another inducible heat shock protein, cells were grown and changed to glutamine-free DMEM with 1% (vol/vol) FBS for 24 h before media change to 0 or 4 mM glutamine 2 h before heat shock. For the conditions used for 2 h treatment before heat shock, parallel plates of cells were used where DON (1 mM) was added 3 h before the media change. For cells treated with DON, this compound was included in all subsequent media. DON by itself did not result in increased Hsp25 using this protocol in the absence of glutamine (Fig. 8, bottom, lane 1) nor did DON support heat induction of Hsp25 (Fig. 8, bottom, lane 2). DON inclusion, as previously shown, inhibited the glutamine support of heat induction of Hsp25 and also the induction of Hsp72, which is not expressed by IEC-18 cells under unstimulated conditions (Fig. 8, bottom, lane 4 vs. lane 8).
To further investigate the effect of DON, intracellular glutamine and glutamate were measured after DON treatment. Cells were treated as above for glutamine’s support of heat-induced Hsp25. Cells were taken after the 24 h in 0 mM glutamine-1% (vol/vol) FBS (condition 1, 106.7 ± 6.4 glutamine and 65.7 ± 3.1 nmol/mg protein for glutamate), and one plate of cells was treated with DON for the last 3 h (condition 2, 118.3 ± 6.3 glutamine and 49.1 ± 1.3 nmol/mg protein for glutamate). Media were then changed to 0 or 2 mM glutamine in FBS-free media with or without DON and then taken for intracellular glutamine and glutamate content [condition 3 is 0 mM glutamine without DON (99.4 ± 7.9 glutamine and 66.0 ± 4.0 nmol/mg protein for glutamate); condition 4 is 0 mM glutamine with DON (glutamine 102.4 ± 8.0 and 40.6 ± 1.3 nmol/mg protein for glutamate); condition 5 is 2 mM glutamine without DON (glutamine 151.1 ± 7.2 and 87.4 ± 1.6 nmol/mg protein for glutamate); and condition 6 is 2 mM glutamine with DON (glutamine 160.6 ± 4.7 and 44.6 ± 3.6 nmol/mg protein for glutamate)]. From these results a number of conclusions may be drawn. First, inclusion of glutamine back in the medium increased both intracellular glutamine and glutamate (note glutamine levels from conditions 1–5, P < 0.05 by ANOVA using a Bonferroni correction). Second, DON decreased intracellular glutamate and caused a modest increase in cellular glutamine content (compare groups 1 and 2, 3 and 4, 5 and 6, all comparisons P < 0.05 using ANOVA with a Bonferroni correction). The data do not support a clear correlation between cellular glutamine or glutamate and support of heat induction of Hsp25. Cellular glutamate levels do not dramatically decrease in those conditions where the supplementation is insufficient to support heat induction of Hsp25. It is possible that conversion of glutamine to glutamate, yielding ammonia, may play some role in the response, particularly since the glutaminase inhibitor DON is so effective. Because glutamate does recapitulate part of the effect of glutamine, the conversion by glutaminase cannot be solely responsible.
To further investigate the potential role of glutaminase activity, cells were treated with DON, and the activity of phosphate-dependent glutaminase was determined. Cells were split and treated as described for Fig. 9. Some cells were harvested before the 24-h treatment with media with 0 glutamine/0 FBS. Some cells were treated with DON for the last 3 h in this media, and then cells were incubated with media with 0 FBS and either 2 or 0 mM glutamine without or with DON. The phosphate-dependent glutaminase activity in cells in complete media before treatment with DON was 1.24 ± 0.08 µmol·mg protein–1·h–1 (n = 3). After 24 h in 0 glutamine/0 FBS, activity was 1.07 ± 0.08, and treatment for the last 3 h decreased the activity to 0.26 ± 0.03 (P < 0.001 by paired Student’s t-test). After 2 h in 2 mM glutamine/0 FBS media, activity was 1.11 ± 0.05 and with 0 glutamine/0 FBS activity was 1.15 ± 0.04. Inclusion of DON in these 2-h incubations decreased activity; in 2 mM glutamine/0 FBS activity was 0.13 ± 0.01, and in 0 glutamine/0 FBS media the activity was 0.14 ± 0.02. The changes in intracellular glutamate in Fig. 9 after DON are not as significant as those for glutaminase, which may relate to the rate of glutamate metabolism in the cells.
GSH nor ATP Mediates Glutamine’s Effect on Heat-induced Hsp25
Glutamine is a precursor for a number of cellular pathways, including ATP and GSH generation. ATP is required for cellular energy use, whereas GSH may be protective against oxidative stress (11), both potentially important for glutamine’s actions. Thus these two downstream outcomes of glutamine metabolism were investigated further. Cellular GSH and ATP were measured in cells incubated in the following three different conditions over time: 1) DMEM alone, 2) DMEM with 4 mM glutamine, and 3) DMEM with 2 mM glutamine and 5% FBS. ATP was measured by a luminescent protocol employing firefly luciferase (Roche Molecular Biochemicals), and cell GSH was measured in a spectrophotometric assay measuring cellular mercaptans, predominantly GSH (Calbiochem, San Diego, CA). Cell GSH declined over 4 h in DMEM alone (from 41 ± 7 to 24 ± 6 mmol/mg protein, n = 3), increased upon glutamine supplementation (to 124 ± 11 mmol/mg protein), and with glutamine and serum supplementation increased greater (to 182 ± 36 mmol/mg protein). To establish the importance of GSH in heat induction of Hsp25, an inhibitor of gluathione synthetase, buthionine sulfoxamine (BSO) was used. BSO decreased basal and glutamine-supplemented GSH in a concentration-dependent manner, being most effective at 200 µM (data not shown). At this concentration, BSO did not, however, block glutamine’s support of heat induction of Hsp25, suggesting that GSH generation from glutamine does not play a role.
ATP was measured over 4 h in the three media described for GSH determination. In DMEM alone, cell ATP decreased from 11.8 ± 2.4 to 9.2 ± 2.1 µmol/mg protein (n = 3). With glutamine supplementation, cell ATP was maintained near the same level (11.7 ± 2.6 µmol/mg protein), whereas inclusion of FBS with the glutamine caused increased cell ATP (to 13.3 ± 3.5 µmol/mg protein). Thus, unless heat induction of Hsp25 is sensitive to a threshold level of ATP >10 µmol/mg protein, ATP generation stimulated by glutamine also appears not to play a role.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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and oxidant-induced cell death, leading to inflammation and injury (7, 21, 43). The effect of glutamine on the heat shock response might contribute to the protective role of glutamine against inflammation and end-organ complications during physiological stress (17, 37, 39).
Because of the many pathways through which intracellular glutamine may be metabolized, the precise mechanism(s) underlying many of glutamine’s effects, including the support of heat induction of Hsp25, have been difficult to identify. Glutamine is metabolized via a number of pathways, including glutaminase, to yield glutamate, which is further metabolized to -ketoglutarate and used for generation of ATP in the tricarboxylic acid cycle. It can also be metabolized to ornithine, with further metabolism of ornithine to putrescine and polyamines, or ornithine may also be converted to proline or to arginine. Glutamate is also metabolized to 
-aminobutyrate, to folate polyglutamates, or to GSH. Independent of this metabolism to glutamate, glutamine may also be converted to NAD+, histidine and tryptophan, nucleosides and amino sugars and is important in the synthesis of many glycoproteins. The relative roles of each pathway may vary because of expression of these pathways in different cell types. The observation that the glutaminase inhibitor DON blocks much of glutamine’s effects and that glutamate can partially replace glutamine for heat induction of Hsp25 suggests that a significant portion of glutamine’s actions is the result of its metabolism to glutamate. In our series of experiments, we examined the effects of two potential downstream metabolic effectors of glutamine supplementation, ATP and GSH. No apparent role for GSH or ATP generation was determined, so the specific pathway(s) involved remain unresolved.
The requirement for glutamine for support of heat induction of Hsp25 is not specific for epithelial cells, since a similar requirement is found in mesenchymal cells (NIH/3T3). In both cases, glutamine is needed for the production of Hsp25 protein and Hsp25 mRNA transcript, an effect that is concentration-dependent and occurs within physiologically relevant concentrations (up to 0.6 mM) of plasma glutamine. We have also determined the effect of concentrations above the physiological range that might be considered pharmacological and perhaps achievable using glutamine supplementation during stress. Glutamine supplementation during stress provides therapeutic benefit. In a number of conditions, glutamine supplementation increases macrophage and lymphocyte activity, improves nitrogen balance, maintains intestinal barrier function, prevents decreases in villus height, and prevents mortality (17, 37, 41–43).
The present studies demonstrate that glutamine’s effects on the heat-induced Hsp25 response are unique among amino acids. Only glutamate, a direct metabolite of glutamine, demonstrated the ability to augment heat-induced Hsp25 production. These results should be considered carefully, since extracellular glutamate was only able to support heat induction of Hsp25 when concentrations were raised to drive uptake of glutamate to effective intracellular concentrations. This difference is almost certainly attributed to the known pathways for glutamine and glutamate uptake by intestinal epithelial cells (3). Glutamine can be taken up by several membrane transporters, both specific to glutamine and shared by other amino acids, accounting for its efficient uptake at physiologically relevant extracellular concentrations. In contrast, glutamate uptake is relatively inefficient because it is transported only by one known specific transporter (EAAC1; see Ref. 16). From these data, it appears that effective intracellular glutamate levels are primarily achieved through glutamine uptake and subsequent metabolism by glutaminase. However, it should be emphasized that conversion of glutamine to glutamate may mediate a significant portion of the ability of glutamine to support heat induction of Hsp25, since inhibition by the glutaminase inhibitor DON abrogates much of the glutamine effect.
In examining potential mechanisms that might underlie the glutamine’s effect on heat-induced Hsp25, we examined the possibilities of increased metabolism and generation of GSH; however, in both cases, no evidence was found to support a role. It should be noted that there is abundant glucose in the medium that should support maintenance of cellular ATP. Additionally, other amino acids that are metabolized by the tricarboxylic acid cycle, albeit perhaps less avidly than glutamine, failed to recapitulate the glutamine effect. Although we are unable to show the mechanisms through which glutamate exerts its effects, we speculate that glutamate may affect transcriptional regulation of Hsp25 through either a glutamate response element or induction/activation of a specific, yet to be characterized, transcriptional effector pathway. The capacity to sense amino acids has been shown to alter transcriptional regulation in budding yeast through modulation of transcription factors of the GCN family (25). Another possibility is that an intracellular receptor may exist for glutamine and/or glutamate. For glutamate, the existence of a classical membrane receptor communicating via second messenger pathways has been very well established but has not been found in nonneural cells. Whether such a receptor exists for glutamine is presently unknown. The effect of glutamine is not unique, since previous investigations have determined an essential role for glutamine in the activation of the transcription factor Elk-1, which is pivotal in epithelial cell proliferation (29, 30). Although the mechanism of glutamine action and the specific nature of this amino acid were not determined, these studies demonstrate that glutamine may activate certain mitogen-activated protein kinases. This may in turn increase activity of the Elk-1 transcription factor and possibly regulate c-Jun and AP-1 activity. Because sites for some of these transcription factors are in the mouse and human Hsp25/27 promoter, this could account for glutamine’s effects in heat induction of Hsp25.
In summary, our investigations demonstrate the essential requirement of glutamine for heat-induced Hsp25 response. This response is observed in both epithelial and mesenchymal cell types and requires conversion of glutamine to glutamate through the actions of glutaminase. Effective intracellular glutamate levels are primarily achieved through glutamine metabolism, since specific uptake mechanisms for glutamate are not as well expressed as the number of high-affinity, high-capacity transport systems for glutamine. We believe these findings underscore the importance of glutamine as a nutritionally important amino acid in stress conditions and provide rational for glutamine supplementation during periods of physiological stress when glutamine depletion may occur.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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