Glutamine synthetase expression in muscle is regulated by transcriptional and posttranscriptional mechanisms

Brian I. Labow, Wiley W. Souba, and Steve F. Abcouwer

Surgical Oncology Research Laboratories, Massachusetts General Hospital, and Department of Surgery, Harvard Medical School, Boston, Massachusetts 02114


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Skeletal muscle exports glutamine (Gln) and increases the expression of the enzyme glutamine synthetase (GS) in response to physiological stress. Acute stress or direct glucocorticoid administration raises muscle GS mRNA levels dramatically without a parallel increase in GS protein levels. In the lung, this discrepancy is caused by feedback destabilization of the GS protein by its product Gln. It was hypothesized that muscle GS protein levels increase during stress only when the intracellular Gln pool has been depleted. Adult male rats were injected with the glucocorticoid hormone dexamethasone (DEX) to mimic the acute stress response and with the GS inhibitor methionine sulfoximine (MSO) to deplete muscle Gln stores. DEX increased GS mRNA levels by 2.8-fold but increased GS protein levels by an average of only 40%. MSO diminished muscle GLN levels by 68% and caused GS protein levels to rise in accordance with GS mRNA. Chronic stress was mimicked using 6 days of MSO treatment, which produced anorexia, 23% loss of body weight, and 64% decrease in muscle Gln levels, as well as pronounced increases in both muscle GS mRNA (26-fold) and protein levels (35-fold) without elevation of plasma glucocorticoid levels. Calorie-restricted pair-fed animals exhibited lesser increases in muscle GS mRNA (8-fold) and protein levels (5-fold) without a decline in muscle Gln content. Thus regulation of GS expression in both acute and chronic stress involved both transcriptional and posttranscriptional mechanisms, perhaps affected by muscle Gln content.

glutamate-ammonia ligase; dexamethasone; methionine sulfoximine; starvation; gene expression regulation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DURING STRESS, GLUTAMINE (Gln) export by skeletal muscle increases in association with increased Gln consumption by other tissues such as the gut, liver, and immune system (7, 12). Although Gln comprises >50% of the total amino acid pool in skeletal muscle, the quantities of "free" Gln within muscle are insufficient to account for the amount of Gln released (5). To maintain homeostasis, Gln is synthesized de novo from glutamate (Glu) and ammonia in a process catalyzed by the enzyme glutamine synthetase (GS; EC 6.3.1.2) (11, 24). Once Gln stores are depleted, the conversion of muscle protein and amino acids into Gln provides an important source of Gln during stress, when there is often an increased demand or limited supply of the amino acid.

Glucocorticoid hormones are released rapidly as part of the acute stress response and have been shown to upregulate muscle GS gene expression in vitro and in vivo (22, 23). In addition, muscle GS mRNA levels have been shown to rise in response to endotoxin challenge in a manner that is predominantly, but not completely, adrenal gland dependent (21). This observation has raised the possibility that other mediators besides glucocorticoids may also regulate GS gene expression, but no such mediators have been identified to date. Although muscle GS activity also increases in response to glucocorticoids or acute stress, this increase does not parallel the increase in GS mRNA, suggesting that a posttranscriptional control mechanism(s) contributes to the regulation of GS expression (1, 21). One putative mechanism involves feedback regulation of GS activity by its product Gln. In fact, it has been shown in a skeletal muscle cell line that GS activity increases via a posttranscriptional mechanism in response to decreasing ambient Gln (9). Furthermore, results in our laboratory have demonstrated that GS expression in lung tissue is regulated by protein stability (19). These observations have suggested a model in which GS expression in muscle may be regulated via two general pathways: the first involving direct, glucocorticoid-mediated transcriptional upregulation, and the second involving Gln-mediated destabilization of the GS protein. Therefore, although increased GS transcription during acute stress raises GS mRNA levels, presumably increasing the rate of GS protein synthesis, GS protein levels may not rise proportionately because of the negative feedback of Gln on GS protein stability.

The regulation of GS expression during chronic stress is less well defined. Recent animal studies have demonstrated that both GS mRNA and protein expression can increase markedly in atrophic muscle after denervation (8) or chronic exposure to glucocorticoids (15). In addition to producing significant muscle atrophy, both of these stimuli can induce Gln efflux and deplete muscle Gln stores (4, 17). Furthermore, when rats received the same stimuli but were also infused with a parenteral Gln solution, the induction in both GS mRNA and protein expression was significantly attenuated (15). Collectively, these data suggest that Gln may regulate GS expression during chronic stress at both the transcriptional and posttranscriptional levels.

Previously, our laboratory has determined that GS expression in rat lung tissue during acute and chronic stress is ultimately determined by GS protein stability. The purpose of the present study was to determine whether GS expression in rat skeletal muscle is governed by a similar mechanism. The compound methionine sulfoximine (MSO), an analog of Gln, binds to the GS protein, inhibits GS activity in vitro (10, 20), and has previously been used to deplete plasma and tissue Gln rapidly in vivo (14). An acute stress state was reproduced using injection of the glucocorticoid dexamethasone (DEX), and the effect of DEX alone and in conjunction with MSO on tissue Gln, GS mRNA, and GS protein expression was measured. Chronic stress was produced by repeated treatment with MSO, which produces profound anorexia and weight loss, in addition to tissue Gln depletion (19). The effects of daily MSO injections on muscle GS mRNA and protein expression were compared with pair-fed control animals that were not exposed to MSO.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. All chemicals were purchased from Sigma Chemical (St. Louis, MO) and Fisher Scientific (Pittsburgh, PA) companies. [32P]dCTP was purchased from Amersham (Arlington Heights, IL). MSO (Sigma) was prepared as a 25 g/l stock solution and filter-sterilized immediately before each experiment.

Animal studies. Male Sprague-Dawley rats weighing between 150 and 200 g were obtained from Charles River Labs (Wilmington, MA). All rats were allowed to acclimate to 12:12-h light-dark cycles and standard rat chow and water ad libitum for 6 days before experimentation.

In all experiments, blood and tissue samples were harvested after rats were anesthetized with intraperitoneal ketamine (100 mg/kg body wt) and acepromazine (1.25 mg/kg body wt). In all animals, blood (~500 liters) was obtained via tail venipuncture at the outset of each experiment and at the time of death via direct cardiac puncture. Whole blood samples were centrifuged in heparinized tubes at 500 g for 10 min at 4°C to obtain plasma. Plasma samples were stored at -80°C for no longer than 48 h before Gln and Glu content was measured. All plasma corticosterone levels were measured by Ani Lytics (Gaithersburg, MD).

Skeletal muscle tissue was obtained through a flank incision, and the entire quadriceps muscle mass was excised sharply, immediately frozen in liquid nitrogen, and stored at -80°C until processing. All procedures involving the use of experimental animals were performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals and were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care.

Acute effects of Gln depletion and glucocorticoids on muscle GS expression. Acclimated rats were randomly assigned to one of four groups (n = 6 animals/group). At time zero, animals were injected intraperitoneally with a combination of MSO (100 mg/kg body wt) or an equal volume of PBS vehicle, and either DEX (0.5 mg/kg body wt) or an equal volume of absolute ethanol (EtOH) vehicle. All animals were euthanized 8 h after intraperitoneal injections, and quadriceps muscle tissue and blood were harvested for analysis.

Diets. During chronic Gln depletion, rats received one of two well-defined isocaloric nutritionally complete diets, which were purchased from Teklad Premier (Madison, WI). One diet contained no detectable Gln or Glu (Gln free); the other contained 4% Glu. Complete compositions of these diets have been published previously (16).

Effects of chronic Gln depletion on muscle GS expression. Acclimated rats were assigned randomly to one of four different treatment regimens (n = 5 or 6 animals/group) and phlebotomized. Then one of the following treatment regimens was initiated: 1) 4% Glu diet alone; 2) Gln-free diet alone; 3) Gln-free diet and MSO; and 4) pair feeding. The 4% Glu group received the 4% Glu diet ad libidum for 6 days and received daily intraperitoneal injections with saline (PBS) vehicle. The Gln-free group was treated identically but received the special Gln-free diet ad libidum. The Gln-free and MSO group received the Gln-free diet ad libidum for 6 days and received daily MSO (100 mg MSO ip/kg body wt). The pair-fed group received daily quantities of the Glu-free chow equal to those consumed by the Gln-free and MSO group over the previous 24 h in addition to daily intraperitoneal PBS. Daily weights and chow consumption were measured for each animal. After 6 days of treatment and 8 h after the last intraperitoneal injection, animals were euthanized, and muscle and blood were harvested. This was performed at ~4 PM, which should precede the peak of the corticosterone diurnal cycle by ~3 h.

Northern blotting. Northern blotting was performed as previously described (1). For RNA isolation, 0.1 g of frozen tissue was rapidly thawed and homogenized in 1 ml of Trisolve Reagent (Biotex, Woodlands, TX) using an Omnimixer homogenizer (Omni International, Warrenton, VA). Total RNA was isolated by the one-step acid-phenol guanidinium procedure (6), with Trisolve utilized according to the manufacturer's protocol, followed by an additional acid-phenol, phenol-chloroform-isoamyl alcohol, chloroform extraction and ethanol precipitation in the presence of sodium acetate. Total RNA (~10 g each) was fractionated by electrophoresis through denaturing agarose gels containing 0.2 M formaldehyde, stained with 10 µg/ml ethidium bromide in 50 mM NaOH and 10 mM NaCl, destained in 200 mM sodium acetate pH 5.2, ultraviolet (UV)-transilluminated, and photographed. The RNA was transferred to a nylon membrane (Micron Separations, Westboro, MA) by capillary action and cross-linked to the membrane with a Fisher Biotech UV Crosslinker. An 800-bp segment of rat GS cDNA containing primarily coding sequence was used as a template to generate a [32P]dCTP-labeled probe, using a random-prime labeling kit (Megaprime, Amersham) according to the manufacturer's protocol. Hybridization with radiolabeled probe was performed overnight at 65°C, as described previously (25). After the nylon membrane was washed with a high-stringency buffer [15 mM NaCl, 1 mM NaH2PO4, 0.1 mM EDTA, and 1% sodium dodecyl sulfate (SDS, vol/vol) in distilled water treated with diethyl pyrocarbonate] at 65°C, autoradiographic detection of the hybridized probe was performed by exposing Fuji XAR film for 12-24 h at -80°C. Quantitation of autoradiograph bands was accomplished with a laser densitometer (Molecular Dynamics, Sunnyvale, CA). Membranes were stripped of GS cDNA probe by boiling in 0.1% SDS and were rehybridized with a random-primed probe for beta -actin mRNA. The resulting autoradiographs were then quantified.

Western blotting. For protein isolation, 0.1 g of frozen tissue was homogenized in 400 µl of tissue-homogenizing buffer [50 mM Tris · HCl, pH 7, 330 mM sucrose, and 5 mM MgCl2] using an Omnimixer homogenizer (Omni International). Homogenates were then centrifuged at 12,000 g for 30 min at 4°C. Cytoplasmic fractions (supernatants) were removed and placed on ice, and total protein concentrations were measured using the bicinchoninic acid (BCA) assay (Pierce Chemical, Rockford, IL), according to the manufacturer's protocol. An aliquot of each cytoplasmic fraction containing 20 µg of protein was removed and heat denatured in the presence of beta -mercaptoethanol and SDS and then separated electrophoretically on a 4-20% SDS Tris-glycine Owl Pre-Cast Gel (Owl Scientific, Woburn, MA) under denaturing conditions. Prestained molecular weight markers (Rainbow Markers, Amersham) were included for comparison. The protein was then transferred electrophoretically onto a 0.45-µm polyvinylidine difluoride membrane (Micron Separations) in transfer buffer composed of 10% methanol, 25 mM Tris, and 192 mM glycine. GS protein bands were detected with a murine anti-sheep brain Gln synthetase antibody that cross-reacts with rat GS protein (Transduction Laboratories, Lexington, KY) at a dilution of 1:400 in 2% bovine serum albumin in Tris-buffered saline (TBS). Hybridization was performed overnight at 4°C after the membrane had been blocked for 45 min with 2% bovine serum albumin in TBS. After extensive washing with TBS, the membranes were incubated at room temperature with an alkaline phosphatase-conjugated goat anti-mouse IgG secondary antibody (Promega, Madison, WI) diluted 1:4,000 in 4% nonfat dry milk in TBS for 4 h. After repeated washings in TBS, the GS protein bands were visualized with nitroblue tetrazolium and bromo-chloro-indolyl-phosphate (Proto-blot system, Promega). Band intensity was quantified by laser densitometry as described in Northern blotting.

Glutamine and glutamate analysis. Plasma samples were thawed and diluted 1:2 and 1:10 (vol/vol) into 100 mM NaCl. Ninety-six-well assay plates (Falcon, Oxnard, CA) were loaded with 10 µl of each diluted sample and freshly made Gln and Glu standards prepared in dialyzed fetal calf serum and diluted in saline. A colorimetric L-glutamic acid assay utilizing the nicotinamide-adenine dinucleotide reaction (Boehringer-Mannheim, Indianapolis, IN) was used according to the manufacturer's protocol to quantify Glu. After spectrophotometric analysis with an Anthos HT-2 microplate reader (Anthos, Frederick, MD), Glu concentrations were obtained by comparison with a Glu standard curve. All samples were then treated with 1.5 units of Escherichia coli L-asparaginase (5 mg/ml, Boehringer-Mannheim) and incubated at 37°C in darkness for 45 min to catalyze the hydrolysis of Gln to Glu and ammonia. Gln content was determined by repeating the spectrophotometric analysis and subtracting the first Glu reading from the second.

Tissue Gln and Glu content was obtained by extracting ~0.1 g of frozen tissue in 800 µl of a methanol-acetic acid solution (9:1, vol/vol). Samples were homogenized in the solvent mixture, allowed to stand at room temperature for 30 min, and then centrifuged at 16,000 g. The free amino acid-containing supernatant was removed, lyophilized, and resuspended in 1 ml of PBS and analyzed for Glu and Gln content as we have shown. The resulting tissue pellet was resuspended in 1 ml of a solution containing 0.5 N NaOH and 0.5% SDS. The solubilized protein samples were diluted 1:20 in 100 mM NaCl, and protein content was determined using the BCA assay (Pierce Chemical) according to the manufacturer's protocol. All tissue Glu or Gln concentrations were normalized to total protein content per sample and are expressed as nanomoles per milligram protein.

Statistical analysis. Given the relatively small sample populations within each segment of this study (n = 5 or 6), a normal distribution could not be assumed. Therefore, the two-tailed Mann-Whitney U-test, a nonparametric method of statistical analysis, was utilized to estimate the significance of differences between groups. Statistical significance is defined as a P value of <= 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To test the hypothesis that Gln depletion induces GS expression during acute stress via increased protein stability, rats were injected with a combination of DEX or EtOH vehicle and either MSO or PBS vehicle. After 8 h, plasma Gln levels in rats treated with MSO/EtOH and MSO/DEX fell by similar amounts, 58% (P < 0.01) and 50% (P < 0.01), respectively, compared with PBS/EtOH controls (Fig. 1). Plasma Gln levels in rats treated with PBS/DEX were raised 45% (P < 0.01) compared with PBS/EtOH controls (Fig. 1). Similarly, the mean tissue Gln levels in quadriceps muscle from the MSO/EtOH and MSO/DEX groups were 68% (P < 0.01) and 50% (P < 0.01) below PBS/EtOH controls. Whereas acute DEX treatment raised muscle Gln levels by 20-25% in the PBS/DEX and MSO/DEX groups compared with their respective PBS/EtOH and MSO/EtOH controls, this increase was not statistically significant (Fig. 2). Therefore, in either DEX-stimulated or unstimulated rats, muscle Gln production was effectively inhibited by MSO.


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Fig. 1.   Acute effect of dexamethasone (DEX) and/or methionine sulfoximine (MSO) on plasma glutamine (Gln) concentration. Adult male Sprague-Dawley rats were injected ip with either DEX (0.5 mg/kg body wt) or an equivalent volume of absolute ethanol vehicle (EtOH), and either MSO (100 mg/kg body wt) or an equivalent volume of saline vehicle (PBS). Plasma samples were obtained 8 h later, and the Gln concentration was determined as outlined in MATERIALS AND METHODS. Error bars represent SDs from mean values (n = 6).



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Fig. 2.   Acute effect of DEX and/or MSO on quadriceps muscle Gln content. Adult male Sprague-Dawley rats were injected ip with either DEX (0.5 mg/kg body wt) or an equivalent volume of EtOH and either MSO (100 mg/kg body wt) or an equivalent volume of PBS. The quadriceps muscle mass was harvested 8 h later, and Gln content was measured and normalized to total protein content per sample, as outlined in MATERIALS AND METHODS. Error bars represent SDs from mean values (n = 6).

Total RNA and protein were isolated from quadriceps muscle from each of the four groups of rats 8 h after injections, as described in MATERIALS AND METHODS. Hybridization of all Northern blots with a radiolabeled rat GS cDNA probe revealed a 2.8- and a 1.4-kb GS transcript in all samples (GS mRNA levels refer to the sum of both of these transcripts) (Fig. 3A). Laser densitometry of these autoradiographs and normalization of GS mRNA signals to beta -actin revealed that DEX augmented GS mRNA levels 2.8-fold (P < 0.01) and 3.3-fold (P < 0.01) in the PBS/DEX and MSO/DEX groups, respectively, compared with PBS/EtOH controls (Fig. 4). MSO alone did not significantly increase GS mRNA levels (Fig. 4). In corresponding Western blots, an anti-GS monoclonal antibody detected a single major band with a molecular mass of 45 kDa in all samples (Fig. 3B). Unlike GS mRNA, muscle GS protein was not significantly increased in response to acute DEX treatment alone. Although densitometry and normalization revealed 40 and 10% increases in muscle GS protein in PBS/DEX and MSO/EtOH groups (compared with PBS/EtOH controls), respectively (Fig. 4), neither of these changes was statistically significant. However, the combined effects of MSO and DEX produced a threefold induction (P < 0.01) in GS protein compared with the PBS/EtOH control group (Fig. 4). Thus, whereas GS mRNA levels responded acutely to DEX, GS protein levels were significantly increased at this time point only in response to glucocorticoid stimulation when Gln production was blocked and/or the GS protein was stabilized by MSO.



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Fig. 3.   Quadriceps muscle glutamine synthetase (GS) mRNA and GS protein levels after DEX and/or MSO administration. Adult male Sprague-Dawley rats were injected ip with either DEX (0.5 mg/kg body wt) or an equivalent volume of EtOH and either MSO (100 mg/kg body wt) or an equivalent volume of PBS. Muscle tissue was harvested 8 h later, and total RNA and protein were obtained as outlined in MATERIALS AND METHODS. A: photographs show ethidium bromide-stained gels (top) and autoradiographs of corresponding Northern blots (from a series of rats from each treatment group) hybridized with probes radiolabeled with GS (middle) and beta -actin (bottom). As an internal control, sample run in left lane of each series was an identical pooled sample of equal volumes of total RNA from all members of PBS/EtOH group. B: photographs show Western blots of protein extracts from muscle tissue from a series of rats in each treatment group. All blots were hybridized with a murine anti-sheep brain GS primary antibody and a goat anti-mouse IgG secondary antibody conjugated to alkaline phosphatase, as described in MATERIALS AND METHODS. As an internal control, the sample run in the left lane of each series was an identical pooled sample of equal volumes of total protein from all members of PBS/EtOH group.



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Fig. 4.   Acute induction of muscle GS mRNA or protein after DEX and/or MSO administration. Relative GS mRNA induction (hatched bars) was obtained by densitometric analysis of GS and beta -actin bands from Northern blots (Fig. 3A). Each GS band density was normalized to its corresponding beta -actin band density. Relative induction was obtained by dividing each normalized GS value by the normalized GS value from the pooled PBS/EtOH group sample in left lane (Fig. 3) of each series. Similarly, GS protein induction (solid bars) was obtained by densitometric analysis of GS bands from Western blots (Fig. 3B). Relative induction was obtained by dividing each GS band density by the GS band density from the pooled PBS/EtOH group sample in left lane (Fig. 3) of each series. Error bars represent SDs from mean values (n = 6).

To determine whether Gln deprivation would increase GS expression in skeletal muscle during chronic stress, rats were treated with a 6-day course of an elemental diet containing either 0 or 4% Glu and daily injections of either MSO or PBS vehicle. Animals injected with MSO exhibited anorexia (75% decrease in daily chow consumption) and loss of grooming but did not show other behavioral or physiological changes, such as lethargy or diarrhea. Over 6 days of treatment, the group receiving MSO lost 23% of their body weight on average and displayed visible muscle wasting (Fig. 5). Rats in the pair-fed group were fed quantities of chow identical to those in the MSO-treated group and also exhibited signs of wasting (17% loss in body weight). Animals receiving the 4% Glu chow and the isocaloric Glu-free chow, along with daily injections of saline vehicle, gained 8 and 3% of their initial body weights, respectively.


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Fig. 5.   Effect of chronic Gln deprivation with a Gln-free diet and MSO on body weight. Rats were randomly assigned to 1 of 4 treatment groups: 4% glutamate (Glu) diet (), Gln-free diet (star ), Gln-free diet + MSO (), and pair fed (triangle ), as outlined in MATERIALS AND METHODS. The pair-fed group received daily quantities of the Glu-free chow equal to that consumed by the Gln-free+MSO group over the previous 24 h. Daily weights and food intake were recorded for each group. Error bars represent SDs from mean values (n = 5 or 6).

The mean initial plasma Gln level (day 0) for all animals was 0.59 ± 0.08 mM. After 6 days of treatment, rats that received MSO had a mean plasma Gln concentration of 0.30 ± 0.09 mM. Animals fed 4% Glu or Gln-free chow and pair-fed animals had mean plasma Gln levels of 0.54 ± 0.07, 0.57 ± 0.09, and 0.60 ± 0.10 mM, respectively, which did not differ significantly from the mean initial plasma concentrations. Similarly, muscle Gln content was lowest in animals that received both Gln-free chow and MSO. Chronic exposure to MSO and Gln-free chow decreased mean muscle Gln content by 64% (P < 0.01), from 15 ± 3 nmol/mg protein in 4% Glu controls to 5.3 ± 2 nmol/mg protein in Gln-free and MSO-treated animals (Fig. 6). Rats in the Gln-free and pair-fed groups had mean muscle Gln concentrations of 12 ± 3 and 14 ± 3 nmol/mg protein, respectively, but neither of these was statistically different from 4% Glu controls. Muscle Glu content was similar in all treatment groups and ranged from 3.7 to 6.2 nmol/mg protein.


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Fig. 6.   Chronic effects of Gln deprivation with a Gln-free diet and MSO on quadriceps muscle Gln and Glu content. After 6 days of treatment, quadriceps muscle was excised from animals in each group, and total Glu (solid bars) and Gln (stippled bars) content was measured and normalized to total protein content per sample, as outlined in MATERIALS AND METHODS. Error bars represent SDs from mean values (n = 5 or 6).

Northern and Western blot analyses were used to quantify the effects of Gln depletion and MSO treatment on GS expression during the chronic stress of starvation (Fig. 7, A and B). In contrast to the acute response to MSO treatment, rats that were injected repeatedly with MSO had a significant increase in muscle GS mRNA expression. Specifically, rats that received Gln-free chow and MSO for 6 days had a 26-fold increase (P < 0.01) in mean muscle GS mRNA, whereas rats that received Gln-free chow alone had a 2-fold increase, which did not reach statistical significance. However, rats in the pair-fed group also had a statistically significant 8-fold increase (P < 0.01) in GS mRNA. Although MSO treatment significantly lowered muscle Gln levels and produced the greatest increase in muscle GS mRNA levels, there was also an induction in GS mRNA levels in the pair-fed group in which no significant change in tissue Gln content was observed. In corresponding Western blots, a similar pattern in GS protein induction was observed (Fig. 7B). Rats receiving Gln-free chow and MSO had a 35-fold increase (P < 0.01) in GS protein compared with the 4% Glu-fed control group, whereas there was no significant increase in muscle GS protein levels in rats that received Gln-free chow alone (Fig. 8). Rats in the pair-fed group had a GS protein level that was 5-fold greater (P < 0.01) than the 4% Glu-fed group. Therefore, as with muscle GS mRNA levels, GS protein levels were dramatically elevated in MSO-treated rats. However, GS protein levels also rose significantly in muscle tissue from calorie-restricted (pair-fed) animals, even though muscle Gln content was not depressed.



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Fig. 7.   Quadriceps muscle GS mRNA and GS protein levels after chronic Gln depletion. After 6 days of treatment (Gln-free diet, 4% Glu diet, Gln-free diet + MSO, or pair feeding), the entire quadriceps muscle was excised, and total RNA and protein were obtained as outlined in MATERIALS AND METHODS. A: photographs show ethidium bromide-stained gels (top) and autoradiographs of corresponding Northern blots (from a series of rats from each treatment group) hybridized with radiolabeled probes of GS (middle) and beta -actin (bottom). As an internal control, sample run in left lane of each series was an identical pooled sample of equal volumes of total RNA from all members of 4% Glu group. B: photographs show Western blots of protein extracts from muscle tissue from a series of rats in each treatment group. All blots were hybridized with a murine anti-sheep brain GS primary antibody and a goat anti-mouse IgG secondary antibody conjugated to alkaline phosphatase, as described in MATERIALS AND METHODS. As an internal control, sample run in left lane of each series was an identical pooled sample of equal volumes of total protein from all members of 4% Glu group.



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Fig. 8.   Induction of muscle GS mRNA or protein after chronic Gln deprivation. Relative GS mRNA induction (hatched bars) was obtained by densitometric analysis of GS and beta -actin bands from Northern blots (Fig. 7A). Each GS band density was normalized to its corresponding beta -actin band density. Relative induction was obtained by dividing each normalized GS value by the normalized GS value from pooled 4% Glu group sample in 1st lane of each series. Similarly, relative GS protein induction (solid bars) was obtained by densitometric analysis of GS bands from Western blots (Fig. 7B). Relative induction was obtained by dividing each GS band density by the GS band density from pooled 4% Glu group sample in 1st lane of each series. Error bars represent SDs from mean values (n = 5 or 6).

To determine whether increased endogenous glucocorticoid release could account for the increase in GS mRNA and protein levels observed in the MSO-treated and pair-fed groups, corticosterone (the predominant glucocorticoid hormone in rats) levels were measured in plasma samples taken from each animal, as described in MATERIALS AND METHODS (Fig. 9). The mean plasma corticosterone concentration in the 4% Glu control group was 580 ± 180 ng/ml. Animals that received Glu-free chow alone or in conjunction with MSO had the lowest corticosterone levels, 360 ± 117 and 370 ± 100 ng/ml, respectively. Rats in the pair-fed group had the highest mean corticosterone level (680 ± 164 ng/ml), but this difference was not statistically significant compared with the 4% Glu control group. Therefore, stress hormone levels did not correlate with the elevations in GS mRNA and protein in the MSO-treated and pair-fed groups.


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Fig. 9.   Effect of Gln deprivation with a Gln-free diet and MSO on plasma stress hormone levels. After 6 days of treatment, plasma samples were obtained from every animal in each group (4% Glu diet, Gln-free diet, Gln-free diet + MSO, and pair fed), as outlined in MATERIALS AND METHODS, and corticosterone levels were assayed. Error bars represent SDs from mean values (n = 5 or 6).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The upregulation of muscle GS expression in response to stress is essential for maintenance of Gln homeostasis in a variety of organ systems (7, 12). However, the molecular mechanisms that regulate GS expression in muscle during stress remain incompletely understood. Previous work in our laboratory has suggested that Gln depletion may regulate GS expression in lung tissue through increased stability of the GS protein (19). The present study demonstrates that, during acute stress, Gln depletion may indeed increase muscle GS protein levels through a similar mechanism. During chronic stress, such as starvation, Gln depletion may potentiate the induction of GS mRNA and protein in muscle; however, increased GS expression can also occur without any sustained alteration in either tissue Gln or circulating glucocorticoids.

Acute exposure to DEX raised plasma Gln levels by 45% in PBS/DEX rats compared with PBS/EtOH controls. This elevation in plasma Gln may be accounted for by an increased release of Gln into the circulation by tissues such as lung (26) and muscle (2), an effect of glucocorticoids that has been previously described. Conversely, a single dose of MSO significantly lowered both plasma and muscle Gln after 8 h in both DEX-stimulated and PBS-treated animals. This dramatic decline in plasma and muscle Gln content not only confirms that MSO was able to inhibit Gln synthesis effectively, but it also demonstrates the extremely high turnover rate of Gln in vivo and the need for continuous synthesis of this amino acid to maintain homeostasis.

Although muscle Gln was profoundly depleted in both MSO/DEX and MSO/EtOH groups, only MSO/DEX rats had a significant (3.3-fold) increase in GS mRNA levels. Thus, although the effect of the glucocorticoid DEX on GS mRNA was evident, acute depletion of the muscle Gln pool by itself did not stimulate GS mRNA accumulation, nor did it significantly increase muscle GS protein levels. This result differs somewhat from lung GS expression in these rats, in which acute depletion of tissue Gln with MSO raised GS protein levels without an increase in GS mRNA (19). The reason for this disparity is unknown. Higher normal levels of Gln within muscle (compared with lung) may necessitate a greater depletion of intracellular Gln before GS protein levels are affected. It is also likely that the accumulation of MSO-stabilized GS protein would be very gradual in the absence of DEX because of a relatively slow rate of protein synthesis from the small-pool GS mRNA template. During this acute stress, steady state may not be obtained. Because GS mRNA levels are so low in unstressed rats, an increase in GS protein level due to MSO alone would be very gradual. Using an in vitro lung cell model, we have found that MSO alone does stimulate a slow accumulation of GS protein without GS mRNA accumulation; when DEX and MSO treatments are combined, the accumulation of GS protein is much faster (Labow and Abcouwer, unpublished observations).

The glucocorticoid DEX increased GS mRNA levels in quadriceps muscle 2.8- and 3.3-fold over 8 h in both MSO-treated and PBS control groups, demonstrating that MSO did not interfere with this glucocorticoid-mediated upregulation of GS transcription. However, densitometric analysis of corresponding Western blots indicated that GS protein levels were, on average, increased only 40% in response to DEX alone (PBS/DEX). This increase did not meet statistical significance. It is possible that an increased expression of GS protein of the same or similar magnitude as GS mRNA induction may occur at a somewhat later time after DEX injection. However, other studies examining GS expression at various times have also shown that acute exposure to glucocorticoids or acute stress states associated with increased endogenous glucocorticoid release (e.g., sepsis, trauma) produces a marked tissue-specific increase in muscle GS mRNA within hours, without a similar increase in GS protein levels or activity (1, 21, 23). In the case of endotoxemia, an increase in muscle GS protein levels lagged behind the increase in GS mRNA levels by 6-12 h, implying that the upregulation in protein was not simply due to increased translation from the expanded GS mRNA pool (21).

In lung tissue, glucocorticoid stimulation in the setting of Gln depletion by MSO led to a synergistic increase in GS protein levels (19). This suggested that, in lung tissue, protein stability ultimately governs GS expression during acute stress. Similarly, in the present study, when DEX was administered in conjunction with MSO (MSO/DEX), the induction in GS protein paralleled the increase in GS mRNA (~3-fold). Although Gln depletion can stabilize the GS protein (10), MSO may also bind to GS and stabilize the protein directly (10). Therefore, one cannot conclude that the increase in GS protein in the MSO/DEX group is causally related to a fall in muscle Gln levels. Instead, this may reflect an increase in protein stability as a result of either MSO binding or Gln depletion, or both. In either case, it suggests that increased protein stability can have an impact on muscle GS expression during acute stress. However, because GS translation rates were not measured, the possibility that MSO acts by stimulating the synthesis of GS protein, rather than by inhibiting its degradation, cannot be ruled out. In this case, feedback inhibition may occur through an undefined transacting factor that limits the translation of GS mRNA into protein.

In a model of chronic stress, adult male rats were deprived of Gln and repeatedly treated with MSO, which caused severe anorexia and visible muscle wasting (23% decrease in body weight). Control animals received the chronic stress of calorie restriction brought about by pair feeding with anorexic MSO-treated animals. In this chronic stress model, pair-fed control rats experienced a similar decrease in body weight (17%) and had similar changes in body morphology. Thus this model represented chronic stress with and without Gln depletion by MSO. Despite similar losses in body weight, rats treated with MSO had a 64% decrease in muscle Gln, whereas the pair-fed group had no significant change in muscle Gln content compared with control rats fed a 4% Glu diet ad libidum. The same pattern was observed in plasma, in which only the MSO-treated group had a significant decline in Gln concentration (51%) over the course of treatment. Therefore, the stress of severe calorie restriction and loss of muscle mass alone was not responsible for the decline in plasma or muscle Gln content observed in MSO-treated rats. Instead, this decrease probably reflects the importance of endogenous GS activity in maintaining plasma and intracellular Gln levels in muscle.

Although Gln depletion did not have a significant affect on GS expression in the acute stress model, animals that were treated with MSO and Gln-free chow for 6 days had a profound increase in muscle GS expression. Animals receiving MSO and Gln-free chow had a 26-fold and 35-fold induction in GS mRNA and protein, respectively, compared with 4% Glu controls. Although animals receiving isocaloric Gln-free chow ad libitum did not have a significant increase in muscle GS expression, pair-fed rats that were calorie restricted parallel to the MSO-treated group had an eightfold and fivefold induction in GS mRNA and protein, respectively. An earlier study demonstrated that muscle GS activity is increased in fasted rats (3). Studies in our laboratory have found that acute fasting causes depletion of muscle Gln pools and upregulation of GS mRNA levels that are partially attributable to an increase in plasma glucocorticoid levels (K. M. Elgadi, W. W. Souba, and S. F. Abcouwer, unpublished observations). To determine whether elevated endogenous glucocorticoid release might account for the increase in GS expression in the two chronically stressed (Gln-free+MSO and pair-fed) groups, plasma corticosterone (the dominant glucocorticoid hormone in rats) was assayed. Although rats in the pair-fed group did have a slightly higher mean plasma corticosterone level compared with 4% Glu controls, this difference did not reach statistical significance. Furthermore, rats receiving MSO had the greatest induction in GS mRNA and protein expression but the lowest mean corticosterone level, suggesting that this response was not glucocorticoid mediated.

It has also been suggested that Gln may regulate GS gene expression during chronic stress. Recent studies using chronic stimuli such as denervation (8) or repeated exposure to glucocorticoids (15) have shown a marked induction in both muscle GS mRNA and protein. Both of these stimuli are characterized by increased Gln efflux from muscle tissue, which can decrease tissue Gln levels (4, 17). Furthermore, parenteral infusions of Gln were shown to inhibit the upregulation of GS mRNA and protein in rats chronically stimulated with glucocorticoids, suggesting that Gln itself might regulate GS expression directly (15). However, tissue Gln content was either not measured or was not significantly altered in these studies. Therefore, it has been difficult to ascribe a direct regulatory effect of Gln on GS gene expression during chronic stress.

In the present study, a control group receiving dietary Gln was not included in this chronic stress experiment, because rat dietary Gln intake does not compensate for inhibition of de novo synthesis. In fact, dietary intake of Gln (~1 g/day) is much less than total Gln turnover (probably >2.5 g/day). Although parenteral glutamine intake at a rate greater than 2 g/day should compensate for inhibition of de novo synthesis and raise plasma levels, we have found that this still does not appreciably replete muscle Gln content in MSO-treated rats (data not shown). Thus, although it is desirable to determine whether the repletion of muscle Gln causes reversal of MSO's effect on GS protein levels, we have not been able to accomplish this in vivo.

Muscle Gln levels were profoundly diminished over the 6-day period of caloric deprivation in rats receiving MSO and Gln-free chow. However, whereas pair-fed rats had no significant change in tissue Gln content, muscle GS mRNA and protein levels were still increased significantly, although to a much lesser degree (8-fold and 5-fold, respectively). The reason for this increase in GS protein levels in the absence of muscle Gln depletion may be an increased demand for Gln efflux. It is important to point out that the increase in GS protein during chronic starvation is similar to, but not quite as great as, the increase in GS mRNA. During chronic starvation, there is an increased demand for Gln output from muscle, both to supply Gln that is not being derived from the diet and to supply Gln to be used for increased gluconeogenesis in the liver (13, 18). Therefore, GS activity would likely increase so that de novo synthesis of Gln (from precursors derived from proteolysis) can meet the increased demand. Perhaps, as soon as the de novo synthesis falls behind demand, muscle Gln levels start to fall, causing the specific GS protein degradation rate to decrease until a new steady state is reached, where the rates of GS protein synthesis and degradation are in balance with a higher level of GS protein content. Greater GS activity would in turn allow Gln levels to rise back to normal levels. Once a new steady state is reached, GS protein synthesis is greater because more mRNA template is available for translation; the specific rate of GS protein degradation is normal, but total GS protein degradation is correspondingly greater because there is a larger pool of protein to degrade.

Simple first-order kinetics including a rate of GS protein degradation, which is a linear function of Gln content, predicts that at steady state the ratio of GS protein to GS mRNA is inversely proportional to the intracellular Gln content. In chronic starvation, the inductions of GS protein levels and GS mRNA levels are coordinate, because the Gln content does not change and the ratio of GS protein to GS mRNA is retained. Of course, this predicts that the ratio of GS protein to GS mRNA should more than double in the muscles of animals chronically treated with MSO, because Gln levels are reduced by one-half. Although this is not the case, the GS protein does increase more than the GS mRNA level in these animals. This discrepancy may reflect the fact that the rate of GS protein degradation is not a simple linear function of Gln content.

MSO itself may have contributed to the elevation of muscle GS mRNA in chronically treated rats. However, lung tissue from the same rats showed no increase in GS mRNA level (19). Likewise, MSO treatment of lung cells in culture had no effect on GS mRNA level (unpublished observations). Furthermore, acute exposure to MSO had no effect on muscle GS mRNA levels. Collectively, these data suggest that MSO is unlikely to be directly responsible for the induction in muscle GS mRNA in this chronic stress model. However, a partial or synergistic effect of MSO cannot be excluded. Likewise, although Gln depletion may potentiate an increase in muscle GS expression, lesser increases in muscle GS expression can also occur during a chronic stress, such as starvation, independent of tissue Gln concentration.

In summary, GS expression in skeletal muscle tissue is regulated by both transcriptional and posttranscriptional mechanisms. Gln appears to affect GS expression in muscle differently during acute stress compared with chronic Gln and/or caloric deprivation. During acute stress, glucocorticoids can increase muscle GS mRNA levels rapidly, while GS protein levels are limited, possibly through a feedback mechanism that involves control of protein stability by tissue Gln. During a chronic stress such as starvation, Gln may directly impact GS gene expression or potentiate the effects of another mediator, although significant increases in GS expression can occur without changes in tissue Gln levels or circulating glucocorticoids. The precise mechanism underlying this induction remains unknown.


    ACKNOWLEDGEMENTS

We acknowledge the expert editorial assistance of Julianne Wattles.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grant R01-HL-44986 (W. W. Souba) and the Edward D. Churchill Fellowship, Massachusetts General Hospital Dept. of Surgery (B. I. Labow).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. F. Abcouwer, Surgical Oncology Research Laboratories, Massachusetts General Hospital, Jackson Bldg., Rm. 91855, Fruit St., Boston, MA 02114 (E-mail: abcouwer.steven{at}mgh.harvard.edu).

Received 5 October 1998; accepted in final form 3 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 276(6):E1136-E1145
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