Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114
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ABSTRACT |
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During physiological stress, the lung increases production of the amino acid glutamine (Gln) using the enzyme Gln synthetase (GS) to maintain Gln homeostasis. Glucocorticoid hormones are considered the principal mediators of GS expression during stress. However, whereas animal studies have shown that glucocorticoids increase lung GS mRNA levels 500-700%, GS activity levels rise only 20-45%. This discrepancy suggests that a posttranscriptional control mechanism(s) ultimately determines GS expression. We hypothesized that the level of GS protein in the lung is governed by the intracellular Gln concentration through a mechanism of protein destabilization, a feedback regulatory mechanism that has been observed in vitro. To test this hypothesis, Sprague-Dawley rats were treated with a Gln-free diet and the GS inhibitor methionine sulfoximine (MSO) to deplete tissue Gln levels and prevent this feedback regulation. Exposure to Gln-free chow and MSO (100 mg/kg body wt) for 6 days decreased plasma Gln levels 50% (P < 0.01) and decreased lung tissue Gln levels by 70% (P < 0.01). Although lung GS mRNA levels were not influenced by Gln depletion, there was a sevenfold (P < 0.01) increase in GS protein. A parenteral Gln infusion (200 mM, 1.5 ml/h) for the last 2 days of MSO treatment replenished lung Gln levels to 65% of control level and blunted the increase in GS protein levels by 33% (P < 0.05) compared with rats receiving an isomolar glycine solution. The acute effects of glucocorticoid and MSO administration on lung GS expression were also measured. Whereas dexamethasone (0.5 mg/kg) and MSO injections individually augmented lung GS protein levels twofold and fourfold (P < 0.05), respectively, the combination of dexamethasone and MSO produced a synergistic, 12-fold induction (P < 0.01) in lung GS protein over 8 h. The data suggest that, whereas glucocorticoids are potent mediators of GS transcriptional activity, protein stability greatly influences the ultimate expression of GS in the lung.
methionine sulfoximine; glutamate ammonia ligase; parenteral infusion
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INTRODUCTION |
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WHOLE BODY GLUTAMINE (Gln) utilization is thought to increase after surgery, trauma, or infection. This can lead to depletion of plasma Gln levels by as much as 50% in critically ill patients (8, 14). Regional flux studies in patients and animals have demonstrated that the lung contributes significantly to Gln homeostasis (25, 30, 32) and releases increased amounts of the amino acid in response to physiological stresses such as endotoxemia (9), sepsis (5), surgery (26), and trauma (7). Unlike skeletal muscle, which contains substantial quantities of free Gln, the lung primarily contributes Gln synthesized de novo from glutamate (Glu) and ammonia, a reaction catalyzed by the enzyme Gln synthetase (GS; EC 6.3.1.2; Refs. 2, 3, 5). Indeed, the expression of GS in lung tissue is upregulated in response to infection or trauma (5, 9).
Glucocorticoid hormones are considered the primary mediators of GS expression during stress and act on the lung (and skeletal muscle) in a rapid, tissue-specific manner (1). For example, animal studies have shown that lung GS mRNA levels will increase 500-700% hours after exposure to dexamethasone (Dex) (1, 29). In vitro studies (2, 3) with pulmonary-derived cell lines have also demonstrated analogous inductions in GS expression through a direct glucocorticoid receptor-mediated process. Similarly, stress states such as sepsis that produce increased circulating glucocorticoid levels have also been associated with greatly increased (700%) lung GS mRNA levels (21). However, whereas GS mRNA levels are increased more than fivefold in response to endotoxin or direct glucocorticoid administration, lung GS protein and/or activity levels increase less than twofold under the same conditions. In fact, the change in lung GS activity has been measured in animal studies in response to acidosis (7), Dex injection (31), sepsis (cecal ligation and puncture and endotoxin injection; Refs. 5, 9), and laparotomy (7), with comparable inductions in activity ranging from 20 to 45%. The disparity between the increase in lung GS mRNA and that in protein and/or activity suggests that a posttranscriptional control mechanism(s) regulates GS expression.
The activity of the GS enzyme is diminished by increasing concentrations of its product Gln (4, 12, 20, 23, 24). In vitro studies (4, 12) that used a variety of cell lines have demonstrated that increasing ambient Gln accelerates GS protein decay. Whereas the precise mechanism by which Gln acts on GS is not known, a variety of molecules have been identified that can block this effect. One such molecule is the Gln analog methionine sulfoximine (MSO) (13, 19, 27). MSO is capable of binding to GS and inhibiting its activity in vitro (13, 19), and in vivo use of MSO produced a 50% decrease in arterial Gln concentrations in rat plasma (15). Furthermore, in vitro data show that MSO is capable of preventing Gln-mediated destabilization of the GS protein (13). Although it is unclear whether MSO stabilizes GS through direct binding or whether its inhibition of Gln synthesis also contributes to this effect, this inhibitor can provide useful insight into the role that protein stability plays in determining GS protein levels in the lung.
If protein stability is a major determinant of GS protein levels in vivo, then the disparity between GS mRNA and GS protein induction in response to an acute stress may be accounted for by the feedback regulatory effect of Gln on GS protein stability. We hypothesized that the increase in GS expression through glucocorticoid-mediated upregulation of transcription does lead to increased translation of GS but that this induction is countered by a Gln-mediated increase in GS protein degradation. If lung GS expression is limited by this feedback mechanism, then interrupting this mechanism by depleting ambient Gln and/or blocking the effect of Gln on GS stability should increase GS protein levels in the lung. Furthermore, induction of GS transcription by glucocorticoids should lead to an even greater induction in GS protein expression if this inhibitory mechanism is blocked. To test this hypothesis, a combination of Gln-free chow and MSO was used to deplete rats of Gln. The effects of Gln depletion alone and in combination with the glucocorticoid hormone Dex on lung GS expression were measured. In addition, indwelling catheters were used to supply parenteral Gln to MSO-treated rats to determine whether exogenous Gln could restore this regulatory pathway in the presence of this drug.
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MATERIALS AND METHODS |
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Reagents. All chemicals were purchased
from Sigma (St. Louis, MO) and Fisher Scientific (Pittsburgh, PA).
[-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.
Diets. During all experiments, rats received one of two well-defined, isocaloric, nutritionally complete diets that were purchased from Teklad Premier (Madison, WI). One diet contained no detectable Gln or Glu (Gln free), whereas the other contained 4% Glu [the approximate content (%weight) of Glu contained in most commercially available rodent diets]. Glu constituted 0 and 22.7% of the total amino acid content in each diet, respectively. Complete compositions of these diets have been previously published (17).
Animal studies. Male Sprague-Dawley rats weighing between 150 and 200 g were obtained from Charles River Laboratories (Wilmington, MA). All rats were allowed to acclimate for 6 days before experimentation with a 12:12-h dark-light cycle and standard rat chow and water ad libitum.
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 was obtained (~500 µl)
via tail venipuncture at the outset of each experiment and at the time
of death via direct cardiac puncture. In some experiments, blood was
also drawn from the tail vein after the third day of treatment. To
obtain plasma, whole blood samples were centrifuged in heparinized
tubes at 500 g for 10 min at 4°C. Plasma samples were stored at 80°C for no longer than 48 h
before Gln and Glu concentrations were measured.
Lung tissue was obtained through a midline sternotomy, and both lungs
were immediately frozen in liquid nitrogen on removal and stored at
80°C until processing. All experimental procedures involving
the use of experimental animals were performed in accordance with the
Guiding Principles in the Care and Use of Laboratory Animals, the
National Research Council Guide, and the Massachusetts General Hospital
Subcommittee on Research Animal Care.
Effects of Gln-free diet and MSO on plasma and tissue Gln and GS expression. Acclimatized rats were randomly assigned to one of four different treatment regimens (n = 5 or 6 animals/group) and phlebotomized, and one of the following treatments was initiated: 1) 4% Glu diet alone, 2) Gln-free diet alone, 3) Gln-free diet + MSO, and 4) pair feeding. The 4% Glu group received the 4% Glu diet ad libitum 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 libitum. The Gln-free + MSO group received the Gln-free diet ad libitum for 6 days and received MSO (100 mg/kg body wt ip) daily. The pair-fed group received daily quantities of the Glu-free diet equal to that consumed by the Gln-free + MSO group over the previous 24 h in addition to daily intraperitoneal PBS.
Combined effects of Dex and MSO on GS expression. Acclimatized rats were randomly assigned to one of four groups (n = 6 animals/group). At time 0, animals were injected intraperitoneally with a combination of either MSO (100 mg/kg body wt) or an equal volume of PBS vehicle and either Dex (0.5 mg/kg body wt; MSO-Dex or PBS-Dex) or an equal volume of absolute ethanol (EtOH) vehicle (MSO-EtOH or PBS-EtOH). All animals were killed 8 h after intraperitoneal injections.
Effect of parenteral Gln on GS expression after MSO treatment. Animals underwent placement of indwelling central internal jugular venous catheters (Silastic tubing, 0.51-mm ID) under anesthesia 48 h before completing their period of acclimation, and an infusion of sterile 0.9% NaCl was initiated. Animals were connected to infusion lines via a swing-swivel mechanism and remained freely moving at all times. At time 0, one-half of the animals in the experiment (n = 10) began to receive daily intraperitoneal injections with MSO (100 mg MSO/kg body wt) and Gln-free chow ad libitum. The other half (n = 10) were pair fed with the same diet and injected intraperitoneally daily with proportionate volumes of PBS. After 4 days of treatment, one-half of the animals within each group (n = 5) were switched from central venous infusions of normal saline to either a solution of 200 mM Gln in 0.9% NaCl or an isomolar glycine (Gly) solution at a rate of 1.5 ml/h. After 2 more days of treatment and infusions, all rats were killed, and blood and lung tissue were harvested for analysis.
Northern blotting. Northern blotting
was performed as previously described in detail (1). For RNA isolation,
0.1 g of frozen tissue was rapidly thawed and homogenized in 1 ml of
Trisolve reagent (Biotex, Woodlands, TX) with an
Omnimixer homogenizer (Omni International, Warrenton, VA). Total RNA
was isolated by the one-step acid guanidinium-phenol procedure (11)
utilizing Trisolve according to the manufacturer's
protocol, followed by an additional acid-phenol,
phenol-chloroform-isoamyl alcohol, chloroform extraction and EtOH
precipitation in the presence of sodium acetate. Total RNA (~10 µg
each) was fractionated by electrophoresis through denaturing agarose
gels containing 0.2 M formaldehyde, stained with 10 µg/ml of ethidium
bromide in 50 mM NaOH and 10 mM NaCl, destained in 200 mM sodium
acetate, pH 5.2, ultraviolet 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 ultraviolet cross-linker. An 800-bp segment of rat GS cDNA
containing primarily coding sequence (a gift from Dr. John F. Mill,
Laboratory of Molecular Biology, National Institute of Neurological
Disorders and Stroke, Bethesda, MD) was used as a template
to generate an [-32P]dCTP-labeled
probe with a random-prime labeling kit (Megaprime, Amersham, Arlington
Heights, IL) according to the manufacturer's protocol. Hybridization
with radiolabeled probe was performed overnight at 65°C as
previously described (28). After the nylon membrane was washed with a
high-stringency buffer [15 mM NaCl, 1 mM
NaH2PO4,
0.1 mM EDTA, and 1% (vol/vol) SDS] 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 the constitutively expressed
-actin
mRNA. The resulting autoradiographs were then quantified as described
above.
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)
with an Omnimixer homogenizer. 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 with the bicinchoninic
acid assay (Pierce Chemical, Rockford, IL) according to the
manufacturer's protocol. An aliquot of each cytoplasmic fraction
containing 20 µg of protein was removed, heat denatured in the
presence of -mercaptoethanol and SDS, and separated
electrophoretically on a 4-20% acrylamide gradient SDS-Tris-Gly gel (Owl Scientific, Woburn, MA) under denaturing conditions. Prestained molecular-mass 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 Gly. GS protein bands were
detected with a murine anti-sheep brain GS 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 being washed extensively with TBS, the membranes were
incubated with a goat anti-mouse IgG secondary antibody conjugated to
alkaline phosphatase (Promega, Madison, WI) diluted 1:4,000 in 4%
nonfat dry milk in TBS for 4 h at room temperature. After repeated
washings in TBS, the GS protein bands were visualized with nitro blue
tetrazolium and 5-bromo-1-chloro-3-indolyl phosphate (Promega Protoblot
System). Band intensity was quantified by laser densitometry.
Gln and Glu analysis. Plasma samples were thawed and diluted 1:2 and 1:10 (vol/vol) in 100 mM NaCl. Ninety-six-well assay plates were loaded with 10 µl of each diluted sample and freshly made Gln and Glu standards prepared in dialyzed fetal calf serum. A colorometric L-glutamic acid assay utilizing the NAD reaction (Boehringer Mannheim, Mannheim, Germany) was used according to the manufacturer's protocol to quantify Glu. After the spectrophotometric analysis was completed with an 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 U of Escherichia coli L-asparaginase (5 mg/ml; Boehringer Mannheim) and incubated at 37°C in darkness for 45 min. L-Asparaginase has multiple activities, including the ability to catalyze the hydrolysis of Gln to Glu and ammonia. Gln content was determined by repeating the spectrophotometric analysis after treatment with L-asparaginase and subtracting the first Glu reading from the second.
Tissue Gln and Glu contents were obtained by extracting ~0.1 g of frozen tissue with 800 µl of a methanol-acetic acid solution (9:1 vol/vol). Samples were homogenized in the solvent mixture and 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, resuspended in 1 ml of PBS, and analyzed for Glu and Gln contents as above. The 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 with the bicinchoninic acid assay according to the manufacturer's protocol. All tissue Glu and Gln concentrations were normalized to total protein content per sample and are expressed as nanomoles per milligram of protein.
Statistical analysis. Given the
relatively small sample populations within each segment of this study
(n = 5 or 6 rats), 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. Significance was defined as
a P value 0.05.
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RESULTS |
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To determine whether Gln deprivation could alter lung GS expression, changes in plasma and lung Gln content as well as in lung GS mRNA and protein levels were measured in rats treated with a 6-day course of a specific amino acid diet containing either 0 or 4% Glu and daily injections of MSO or PBS vehicle. Animals injected with MSO exhibited anorexia (75% decrease in chow consumption) and some 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 an average of 20% of body weight, which was not significantly different from the pair-fed control group, which lost 18% of body weight on average (data not shown). The other two groups receiving either the 4% Glu or the Gln-free chow and vehicle intraperitoneally consumed similar quantities of their respective chow and gained 10 and 5% body weight, respectively (data not shown). The difference in weight gain was not significant.
The mean initial plasma Gln concentration was 0.59 ± 0.08 mM (Fig. 1). Whereas all groups had a decrease in plasma Gln by day 3, this decrease was only significant for the Gln-free + MSO group in which the mean plasma Gln concentration fell to 0.34 ± 0.07 mM (P < 0.01). After 6 days of treatment, plasma Gln levels in rats from the three groups not receiving MSO had returned to initial levels. Rats in the group receiving MSO had a mean final plasma Gln concentration of 0.30 ± 0.05 mM, which was significantly lower than all other groups on day 6 (P < 0.01). Gln and Glu contents in lung tissue were measured after 6 days of treatment in these four groups of rats (Fig. 2). The group receiving the Gln-free diet + MSO was the only group to show a significantly lower level of Gln: 2.0 nmol/mg protein compared with 7.0-8.0 nmol/mg protein in the other three groups (P < 0.01). There was no significant difference in lung Glu levels (25-32 nmol/mg protein) among the four groups. Thus a combination of a Gln-deficient diet and MSO was effective in reducing Gln content in both plasma and lung.
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To determine whether Gln depletion affected lung GS expression, RNA and
protein were isolated from whole lung tissue from each of the four
groups of rats and analyzed by Northern and Western blotting
techniques, respectively (Fig. 3).
Hybridization of all Northern blots with a radiolabeled rat GS cDNA
probe revealed a 2.8- and 1.4-kb GS transcript in all samples (GS mRNA
levels refer to the sum of both of these transcripts). Laser
densitometry of these autoradiographs and normalization of GS mRNA
signals to -actin revealed that there was no significant change in
lung GS mRNA levels with Gln deprivation alone or in combination with MSO treatment (Fig. 4). In corresponding
Western blots, an anti-GS monoclonal antibody detected a major band
with a molecular mass of 45 kDa in all samples. In contrast to GS mRNA,
GS protein levels were markedly increased by Gln deprivation and MSO
treatment. Densitometric analysis revealed that lung tissue from rats
treated with a Gln-free diet + MSO had a sevenfold higher level of GS protein compared with the other three treatment groups
(P < 0.01) in which GS protein
levels did not differ significantly from one another (Fig.
4).
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Normal rats were injected intraperitoneally with a combination of either MSO or PBS vehicle and either Dex or EtOH vehicle to determine whether glucocorticoid and MSO treatment could act synergistically to increase lung GS expression. Lung tissue was harvested 8 h after the injections and analyzed for Gln content, GS mRNA, and GS protein. Rats treated with MSO and EtOH had the lowest lung Gln levels (9.7 nmol/mg protein; P < 0.05), which was 43% less than the PBS-EtOH control group (16.9 nmol/mg protein; Fig. 5). Dex increased lung Gln levels in both PBS-Dex (19.6 nmol/mg protein) and MSO-Dex (14.7 nmol/mg protein) groups by 16 and 51% over their PBS-EtOH and MSO-EtOH counterparts, respectively. However, this difference only reached significance in MSO-treated animals. Indeed, when rats were injected with both MSO and Dex, lung Gln levels were only 15% less than in PBS-EtOH control animals.
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Northern blot analysis confirmed that Dex increased GS mRNA levels in rat lung (Fig. 6A). The PBS-Dex and MSO-Dex groups had lung GS mRNA levels that were increased 4.5- and 4-fold over the PBS-EtOH and MSO-EtOH groups, respectively (P < 0.01; Fig. 7). MSO alone was not associated with any significant change in lung GS mRNA expression in either Dex- or EtOH-treated animals. However, lung GS protein was increased in response to either Dex or MSO (Fig. 6B). The PBS-Dex and MSO-EtOH groups had GS protein levels twofold and fourfold greater, respectively, than PBS-EtOH controls (P < 0.05; Fig. 7). The combined effects of MSO and Dex led to a 12-fold increase in GS protein in the MSO-Dex group (P < 0.01), which was significantly greater than the sum of their separate effects (P < 0.01).
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To determine whether the effects of MSO on lung GS protein were due to its effect on Gln levels, rats were infused with either a Gln solution or an isomolar Gly control solution for the last 48 h of a 6-day course of Gln depletion. Rats treated with MSO and parenteral Gly had a mean plasma Gln concentration of 0.35 ± 0.06 mM on day 6. This plasma concentration was 44% less than the mean PBS-Gly control group level of 0.63 ± 0.10 mM (P < 0.01). However, Gln infusions into MSO-treated rats (MSO-Gln) restored plasma Gln levels to 0.67 ± 0.09 mM. When PBS-treated rats received parenteral Gln (PBS-Gln), the mean plasma Gln concentration rose to 1.09 ± 0.14 mM, or 73% higher than PBS-Gly controls (P < 0.01). Plasma Glu levels ranged from 0.10 to 0.22 mM, without any significant difference among treatment groups.
A comparison of lung Gln content between the MSO-Gly (9.3 nmol/mg protein) and the PBS-Gly (29.8 nmol/mg protein) groups revealed a 69% lower Gln concentration in the MSO-treated group (P < 0.01; Fig. 8). When MSO-treated animals received parenteral Gln for 48 h (MSO-Gln), lung Gln levels rose to 22.2 nmol/mg of protein or 239% over MSO-Gly animals (P < 0.01) and were only 35% less than PBS-Gly control levels. A comparison of the PBS-Gln and PBS-Gly groups shows that PBS-injected animals infused with Gln had a 31% higher mean lung Gln level (39 nmol/mg of protein), but this difference did not achieve significance. Thus, whereas Gln infusions into PBS-treated control animals did not significantly increase lung Gln content, infusions into MSO-treated rats restored Gln levels to 75% of control levels (PBS-Gly).
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As in previous experiments, MSO therapy did not significantly affect lung GS mRNA levels in animals receiving parenteral Gly or Gln (Figs. 9A and 10). Similarly, neither parenteral Gln nor Gly infusions changed GS mRNA levels in lung tissue from rats injected with either PBS or MSO. When GS protein levels were compared in MSO-Gly and PBS-Gly groups, there was an eightfold increase in GS protein with MSO treatment (P < 0.01; Figs. 9B and 10). However, in those animals that received parenteral Gln in addition to MSO treatment (MSO-Gln), GS protein levels increased only fivefold or 33% less than in the MSO-Gly group (P < 0.05). Thus partial restoration of lung Gln content led to partial inhibition of the effect of MSO on GS protein levels. There was no significant difference in lung GS protein levels between PBS-treated animals receiving parenteral Gln or Gly.
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DISCUSSION |
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Although glucocorticoid hormones remain the only systemic mediator to regulate GS expression, these hormones often raise GS mRNA levels without a parallel increase in GS protein (6, 21, 29). One explanation for this discrepancy has come from in vitro data showing that GS protein stability is markedly affected by the ambient Gln concentration (4, 12). In the present study, the potential impact of increased protein stability alone and in conjunction with the glucocorticoid Dex on lung GS expression has been demonstrated in vivo.
Plasma and lung Gln levels were measured in rats after 3 and 6 days on a Gln-free diet alone and in conjunction with the GS inhibitor MSO. The mean initial plasma Gln level for all rats was 0.59 mM, which is similar to previously reported values (10, 22). Animals fed the Gln-free or 4% Glu diet and not receiving MSO ate similar amounts of chow and gained 5 and 10% of body weight, respectively, over 6 days. However, the difference in body weight between these two groups did not reach significance, and even in a study (17) with these exact diets for up to 28 days, a difference in mean body weight was not detected. In both diet groups, there was no significant change in plasma or lung Gln levels over the course of the experiment, and this is also consistent with published work (17). The stability of plasma Gln levels in animals ingesting food deficient in this amino acid clearly demonstrates the compensatory ability of tissues such as skeletal muscle to synthesize and/or release Gln into the blood when Gln is not present in the diet.
In contrast, animals injected with MSO (in addition to a Gln-free diet) lost 20% of their body weight and had a 50% decrease in plasma Gln and a 70% decrease in lung Gln levels. Rats that were pair fed with the MSO-treated group lost a similar amount of body weight (18%) but did not show a similar decline in either plasma or lung Gln. Thus anorexia alone is not responsible for the decline in plasma and lung Gln content in MSO-treated rats. The fact that MSO treatment was able to diminish both plasma and lung Gln concentrations demonstrates that this drug was effectively inhibiting Gln synthesis and suggests that GS activity is rate limiting for Gln synthesis. It is known that Gln stores do turn over quite rapidly in vivo and that Gln homeostasis depends to a large extent on de novo synthesis (30). Therefore, one would expect a rapid decline in plasma and tissue Gln levels after MSO treatment, which has been shown by others (15) to effectively inhibit GS activity in vivo.
Quantification of Northern blots did not reveal any significant change in lung GS mRNA levels in animals that were Gln depleted with MSO. In contrast, Western blots revealed a sevenfold increase in GS protein in Gln-depleted rats. These results show that in the lung a large increase in GS protein may be achieved via a completely posttranscriptional mechanism. MSO could act simply through inhibition of Gln formation, increasing lung GS protein levels indirectly by preventing feedback destabilization of the enzyme by its product. However, it has been reported that MSO binding to GS can directly block degradation of the protein (13, 19). Therefore, one cannot conclude that the increase in GS protein after treatment with MSO is causally related to a fall in lung Gln levels, inasmuch as it may also reflect increased protein stability due to a direct effect of MSO on the protein. In either case, the increase in lung GS expression that can be achieved by preventing destabilization of the protein is clear.
When Dex was injected in combination with either PBS or MSO, lung Gln content was increased relative to corresponding EtOH-injected groups. In fact, animals in the MSO-Dex group had lung Gln levels that were only 15% below the PBS-EtOH control group. This response to Dex may reflect higher circulating plasma Gln levels as a result of increased peripheral release from skeletal muscle, independent of de novo synthesis (18). Alternatively, increased tissue Gln levels may reflect incomplete inhibition of increased GS activity after Dex treatment. Lung GS mRNA levels were increased 8 h after Dex administration, with a 4- and 4.5-fold induction in GS mRNA levels in the PBS-Dex and MSO-Dex groups, respectively, and this response to Dex is similar to other reports (1, 6, 7). Although MSO did not affect GS mRNA levels significantly, GS protein levels rose in response to Dex as well as to MSO. The increase in GS protein in response to Dex is also in agreement with another report (29) that demonstrated a maximal effect of glucocorticoids on both protein and mRNA within hours of exposure. However, it is clear that alterations in protein stability can also augment GS protein levels rapidly as well as appreciably. Furthermore, the synergistic 12-fold increase in lung GS protein after exposure to both Dex and MSO suggests that an appreciable increase in lung GS protein can occur when the conditions of physiological stress and Gln depletion occur simultaneously in vivo.
Parenteral infusions of Gln have been used to study the impact of Gln on GS expression in skeletal muscle depleted of Gln through chronic glucocorticoid administration (16). In that study, parenteral Gln increased plasma Gln but did not raise muscle Gln levels. In the present study, both plasma and lung Gln levels were increased after parenteral Gln infusion. Although infusions of Gln effectively restored the plasma Gln concentration to normal in MSO-treated rats, this was not the case for lung Gln levels. Thus, whereas the uptake of extracellular Gln by the lung significantly augmented intracellular Gln levels in the presence of MSO, normal circulating plasma levels of Gln could not entirely replace normal Gln synthesis. This may reflect the inability of the lung to import Gln at a rate comparable to endogenous Gln production or alternatively could reflect competitive inhibition of Gln uptake by MSO.
It should be noted that the "control" value for lung Gln content in PBS-Gly rats was higher than either the PBS-EtOH or 4% Glu control values in the previous two sets of experiments. This difference may reflect some variability in the Gln and Glu assays. All samples from a given experiment were analyzed for Gln and Glu contents at the same time with freshly prepared Gln and Glu standards. No comparisons of lung tissue Gln concentrations were made between experiments or to other studies. However, the range of control lung Gln levels in these experiments may also reflect real differences in lung Gln levels among the three control groups. Differences in diet (chow vs. elemental amino acid diet), stress state (one pair of injections vs. daily injections for 1 wk with or without an indwelling catheter), or the use of a concentrated, parenteral Gly solution could all conceivably impact tissue Gln levels.
Lung GS mRNA levels were not influenced by either MSO or parenteral Gln, whereas lung GS protein levels were increased eightfold in Gln-depleted rats receiving MSO and Gly compared with PBS-Gly control levels. When MSO-treated rats also received parenteral Gln, GS protein levels were only induced fivefold, or 33% less than in the MSO-Gly group. These results show that the effect of MSO on lung GS protein is partially reversible by exogenous Gln and suggest that MSO may exert its effect on GS protein levels (at least partially) through diminished ambient Gln. However, parenteral Gln did not completely restore GS protein levels to those of the PBS-Gly or PBS-Gln group. This may be due to the fact that, whereas lung Gln levels in MSO-treated rats were raised by parenteral Gln, they did not completely reach those in either the PBS-Gly or PBS-Gln group. Similarly, the duration of Gln repletion may have been insufficient to allow GS protein levels to decay to steady-state levels. It is also possible that MSO stabilizes the GS protein even in the setting of relatively high ambient Gln conditions. In this case, GS protein levels would be somewhat elevated even if lung Gln levels were equal to PBS-Gly control values. Regardless, the data demonstrate that lung GS upregulation can alternatively be potentiated and suppressed in response to protein-stabilizing (i.e., MSO or Gln depletion) and -destabilizing influences (i.e., Gln), respectively. It should also be noted that Gln infusions into PBS-injected animals did not lower GS protein levels appreciably. Perhaps it is because lung Gln content was not sufficiently elevated by Gln infusion (as noted, the increase was not significant). If lung Gln levels were truly elevated, then our model would predict that this increase should accelerate GS protein turnover and cause GS protein levels to fall. A greater increase in lung Gln content is clearly needed to test this hypothesis. However, transport limitations may prevent plasma Gln delivery from raising intracellular lung Gln content sufficiently in the absence of MSO.
In summary, whereas glucocorticoid hormones remain the only mediators known to upregulate GS expression in the lung, they often raise GS mRNA levels, with little effect on GS protein levels or activity. Because it is the GS protein that determines the capacity of the lung to synthesize Gln, control mechanisms that regulate protein levels are of primary importance. We have demonstrated that protein stability may provide one such control mechanism in vivo. Because Gln depletion increases GS protein stability and occurs during severe stress or critical illness, we believe this mechanism to be relevant. Furthermore, the combined effects of protein stability and glucocorticoid hormones can act synergistically to augment GS protein expression, allowing the lung to adjust GS activity to meet actual Gln demand.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-44986 (to W. W. Souba) and the Edward D. Churchill Fellowship, Massachusetts General Hospital Department of Surgery (to B. I. Labow).
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests: S. F. Abcouwer, Massachusetts General Hospital, Cox Bldg., Rm. 626, 100 Blossom St., Boston, MA 02114.
Received 6 May 1998; accepted in final form 7 July 1998.
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