Neural control of glutamine in rat skeletal muscles
synthetase
activity
BO FENG, MASAAKI KONAGAYA, YOKO KONAGAYA, JOHN W. THOMAS, CARL BANNER, JOHN MILL, AND STEPHEN R. MAX Department of Neurology and Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201; Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore 21228; and Laboratory of Molecular Biology, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892
FENG, Bo, MASAAKI KONAGAYA, YOKO KONAGAYA, JOHN W. THOMAS, CARL BANNER, JOHN MILL, AND STEPHEN R. MAX. Neural control of glutamine synthetase activity in rat skeletal muscles. Am. J. Physiol. 258 (Endocrinol. Metab. 21): E757-E761, 1990.-The mechanism of glutamine synthetase induction in rat skeletal muscle after denervation or limb immobilization was investigated. Adult male rats were subjected to midthigh section of the sciatic nerve. At 1, 2, and 5 h and 1, 2, and 7 days after denervation, rats were killed and denervated, and contralateral control soleus and plantaris muscles were excised, weighed, homogenized, and assayed for glutamine synthetase. Glutamine synthetase activity increased -twofold 1 h after denervation in both muscles. By 7 days postdenervation enzyme activity had increased to three times the control level in plantaris muscle and to four times the control level in soleus muscle. Increased enzyme activity after nerve section was associated with increased maximum velocity with no change in apparent Michaelis constant. Immunotitration with an antiglutamine synthetase antibody suggested that denervation caused an increase in the number of glutamine synthetase molecules in muscle. However, Northern-blot analysis revealed no increase in the steady-state level of glutamine synthetase mRNA after denervation. A mixing experiment failed to yield evidence for the presence of a soluble factor involved in regulating the activity of glutamine synthetase in denervated muscle. A combination of denervation and dexamethasone injections resulted in additive increases in glutamine synthetase. Thus the mechanism underlying increased glutamine synthetase after denervation appears to be posttranscriptional and is distinct from that of the glucocorticoidmediated glutamine synthetase induction previously described by us.
muscle; denervation
SKELETAL MUSCLE PRODUCES and releases glutamine, which serves as a substrate for the energy metabolism of a number of cells and tissues, including intestine, lymphocytes, kidney, and, possibly, brain (4, 10, 37). The release of glutamine by muscle is increased under conditions of stress, such as fasting and trauma (10). However, the quantity of glutamine contained in muscle proteins is insufficient to account for all the glutamine released by muscle (5, 13). Therefore, glutamine is synthesized de novo in muscle (9, 26). Although the exact
pathway is undefined, glutamine synthetase appears to catalyze the final step (32). Glutamine synthetase expression in skeletal muscle is subject to a number of effecters, notably glucocorticoids (20, 21, 35). In this regard, we recently showed that dexamethasone-mediated induction of glutamine synthetase activity is associated with an increase in the level of glutamine synthetase mRNA in intact skeletal muscles (20) or in L6 skeletal muscle cells in culture (21). Because glucocorticoids cause pronounced muscle atrophy, we postulated that induction of glutamine synthetase may play a role in the progression of muscular atrophy from other causes. Indeed, Rennie et al. (31) recently hypothesized that glutamine may control the rate of muscle protein turnover. Because denervation also causes muscle atrophy, we assessed the effects of nerve section on glutamine synthetase activity. Indeed, denervation causes a significant increase in glutamine synthetase activity in rat skeletal muscles (15, 16). We now report the results of investigations into the mechanism of this increase. MATERIALS
AND METHODS
Adult male rats of the crl:CD(SD)BR strain (Charles River, Wilmington, MA) weighing 236 t 40 (SD) g were used. They were fed Purina Laboratory Chow (no. 5001) and water ad libitum and maintained on a schedule of 12-h light:12-h darkness. Denervation was carried out under ether anesthesia by midthigh section of the sciatic nerve. At 1, 2, and 5 h and 1,2, and 7 days after denervation rats were anesthetized with ether and then decap‘itated. Experimental (i.e., denervated) and contralateral control soleus and plantaris muscles were removed, dissected free of fat and connective tissue, and weighed. They were minced finely with scissors in ice-cold homogenization buffer (0.25 M sucrose, 0.2 mM EDTA, pH 7.4, and 2 mM 2-mercaptoethanol). The minced suspensions were homogenized with a polytron homogenizer (Brinkman Instruments). Homogenates were prepared and centrifuged, and the cytosol (30,000 g supernate) was assayed for glutamine synthetase activity as described by Smith et al. (35), except that the glutamate concentration in our assays was 5 mM (higher concentrations of glutamate resulted E757
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.157) on January 17, 2019.
E758
MUSCLE
GLUTAMINE
in no increase in enzyme activity). Protein was determined by the procedure of Lowry et al. (18) by use of crystalline bovine serum albumin as standard. For mRNA analyses, muscles were frozen and pulverized in liquid nitrogen in a mortar and pestle that had been treated with diethyl pyrocarbonate (19). The powders were stored at -70°C. Total cellular RNA was isolated from the frozen muscle powders by means of the guanidine isothiocyanate procedure of Chirgwin et al. (6). Equal amounts of RNA, as determined both spectrophotometrically and through ethidium bromide staining, were fractionated by electrophoresis through denaturing agarose gels containing formaldehyde (29)) transferred to nitrocellulose filters, and hybridized with a radioactive cDNA probe (20). In some experiments (1 h and 7 days postdenervation) a radioactive RNA probe was used (21). The ,&tubulin probe was as described (20). Northern autoradiograms were quantified using an LKB laser densitometer. Immunotitration of glutamine synthetase from denervated and control soleus muscles was performed by the method of Shimke (34) by use of a rabbit antiglutamine synthetase antiserum prepared with purified rat liver glutamine synthetase as antigen (22). The incubation conditions of Miller and Carrino (22) were used. After immunoprecipitation, glutamine synthetase activity was assayed in the supernate. Where indicated (experiment of Fig. 5), dexamethasone was injected for 5 days (5 rng- kg-‘. day-l SC) into normal rats or into rats with a denervated plantaris muscle. Statistical analysis was carried out with paired t tests @-tailed).
SYNTHETASE
M. plantaris
M. soleus
I
1
I
I
.
1
2
5
24
48
Hours
after
,
168
Denervation
1. Effect of denervation on glutamine synthetase specific activity (expressed as percent control) in rat soleus and plantaris muscles (M). Data are means -I- SE of 5 determinations. Glutamine synthetase activity in denervated muscles was significantly different from that in contralateral control muscles in every case, P < 0.001. Experimental procedures are described in text. FIG.
RESULTS
Denervation of soleus and plantaris muscles caused a significant increase in the activity of glutamine synthetase (Fig. 1). A twofold increase was apparent as early as 1 h after nerve section; by 7 days after denervation, glutamine synthetase activity was increased threefold in plantaris muscle and fourfold in soleus (Fig. 1). Increased glutamine synthetase activity in plantaris muscles was associated with an increase in maximum velocity ( Vmax)(48 h denervated = 21.9 nmol . h-l. mg protein-‘; control = 10.49 nmol .h-l. mg protein-‘), with no change in apparent Michaelis constant (K,; 48 h denervated = 1.48 x low3 M; control = 1.9 x 10m3M) (Fig. 2). The increase in Vmaxsuggested an increase in the amount of enzyme, possibly via enhanced synthesis. Indeed, immunotitration experiments by use of an antiglutamine synthetase antiserum showed the equivalence point for the enzyme to be the same from denervated (24 h) and control muscles (Fig. 3A), and that more antibody is required to precipitate glutamine synthetase in denervated than in control muscles (Fig. 3B). These results suggest a twofold increase in the number of glutamine synthetase molecules in 24-h denervated vs. control muscles, in excellent correspondence to the increase in enzyme activity (Fig. 1). Thus denervated muscles appear to contain an increased number of enzyme molecules
1 /
-2
V
0
2
4 6 8 1 /[glutamate] ( 109 FIG. 2. Effect of glutamate concentration on glutamine synthetase activity of denervated (48 h) and control rat plantaris muscles: Lineweaver-Burk plot. Experimental procedures are described in text. CTL, control; DEN, denervation.
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.157) on January 17, 2019.
MUSCLE
0
ctl
l
den
GLUTAMINE
0 0
E759
SYNTHETASE
synthetase mRNA after denervation. A similar result was obtained with Northern blots prepared 1 h or 7 days after denervation (Fig. 4). Stripping and rehybridization of the 24-h Northern blot with a radioactive probe for ,& tubulin revealed no difference between denervated and control muscles (Fig. 4). Therefore the glutamine synthetase mRNA results are not caused by unequal loading of the gels. The half-life of glutamine synthetase mRNA has been estimated to be 6-8 h (33). Therefore, it is unlikely that our determinations would have missed a transient change in mRNA level. To compare further the mechanism of the denervationmediated increase in glutamine synthetase with that of dexamethasone (20, 21), rats with denervated or unoperated muscles were given dexamethasone (5 mg kg-‘. day-l SC) for 5 days. The dexamethasone-treated-plusdenervated muscles were compared with denervated muscles and with muscles from rats receiving dexamethasone alone. The effects of dexamethasone and denervation were additive, indicating that different mechanisms are involved (Fig. 5). A mixing experiment was performed to investigate the possible presence of soluble factors influencing enzyme activity. Mixing equal portions of supernatant fractions from denervated (24 h) and control soleus muscles resulted in a specific activity equal to the average of the activities when these fractions were assayed separately (control, 13.3 nmol . h-l. mg protein-‘; denervated, 45.0 nmol . h-l . mg protein-l; control + denervated, 26.4 nmol . h-’ . mg protein-‘). Thus denervated muscles do not appear to contain a soluble activator molecule; conversely, control muscles do not appear to contain a soluble inhibitory molecule. l
600
total
activity
(cpm)
.B
v
denervalion
DISCUSSION
mtibody
added
(pi)
3. Immunotitration of glutamine synthetase 24 h postdenervation. A: increasing amounts of cytosol were added to tubes containing fixed amount of antiserum. B: increasing amounts of antiserum were added to tubes containing fixed amount of cytosol. Glutamine synthetase activity was assayed in supernate after immunoprecipitation. Separate groups of muscles were used for these experiments. Experimental procedures are described in text. FIG.
compared with control muscles (Fig. 3), rather than a change in the catalytic activity of the enzyme (22, 34). That the enhancement of glutamine synthetase activity is not a consequence of increased transcription is shown by the data of Fig. 4, which depicts Northern-blot analysis of the level of glutamine synthetase mRNA in denervated (24 h) and control plantaris muscles. This experiment revealed no increase in the level of glutamine
The experiments described above were designed to investigate the mechanism of the increase in glutamine synthetase activity after denervation (15, 16) (Fig. 1). Our data reveal increased glutamine synthetase activity and amount without a change in the glutamine synthetase mRNA level. This phenomenon is distinct from that of dexamethasone-mediated induction of this enzyme in muscle cells in culture (21, 35) and in skeletal muscles in vivo (20). Dexamethasone caused an increase in glutamine synthetase activity that was associated with an increase in the level of glutamine synthetase mRNA (20, 21) and was blocked by RU38486 (21), a glucocorticoid antagonist (14, 27). In contrast, the denervation-mediated increase in glutamine synthetase activity was not prevented by treatment of rats with RU38486 (15). This result suggested that endogenous glucocorticoids are not involved in the denervation-mediated increase in glutamine synthetase activity and indicated a different mechanism. The present data confirm that conclusion and provide new insights into the mechanism. Immunotitration (Fig. 3) revealed an increased number of glutamine synthetase molecules in denervated muscles. However, synthesis of glutamine synthetase at the transcriptional level in denervated muscle is ruled out by Northern-blot analysis of glutamine synthetase mRNA, which was not increased in denervated muscle (Fig. 4).
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.157) on January 17, 2019.
E760
MUSCLE A
GS-1
GLUTAMINE
B
GS-24
h
C
h
GS-7
d
time after denervation
den
control
den
D
tubulin-24
h
FIG. 4. Northern-blot analysis of glutamine synthetase mRNA in denervated (den) and control (ctl) plantaris muscles. Analyses were performed at 1 h (A), 24 h (B), or 7 days (C) after denervation. Separate Northern blots were done at each time point. Autoradiograms (A-C) reveal a major band of -3 kb. D: ,& tubulin mRNA, 24 h postdenervation. E: ratio of glutamine synthetase mRNA/Ptubulin mRNA 24 h after denervation, as determined by densitometry. Data are means f SE of 4 determinations.
ctl
In addition, the effects of denervation and dexamethasone were additive (Fig. 5). Finally, differences in the mechanisms of action of dexamethasone and denervation may be provided by the time-course of the denervation effect (Fig. 1). Enzyme activity was increased dramatically within 1 h of nerve section, in contrast to the lag period noted after dexamethasone treatment of muscle cells in culture (21). A soluble activator would not seem to be present, based upon the mixing experiment (see above) and by the increase in I’,,, (Fig. 2). We hypothesize that the increase in glutamine synthetase activity after denervation may be a result of decreased degradation of the enzyme. The cause of this phenomenon might be related to glutamine levels in denervated and disused vs. control muscles. It has been
EJ
SYNTHETASE
dex dex + den
FIG. 5. Effect of denervation and dexamethasone on glutamine synthetase activity of rat plantaris muscles, 5 days postdenervation. den, denervated; dex, dexamethasone (5 mg. kg-‘.day-I). Data are means f SE of 4 determinations. Experimental procedures are described in text.
shown that removal of glutamine from the culture medium causes a large increase in glutamine synthetase activity in Lg muscle cells in culture (35). A similar phenomenon has been found in other cell types, and it has been suggested that glutamine depletion causes the enzyme to assume a more stable configuration, resulting in an increased enzyme concentration (2, 3, 7, 8, 17, 23, 36). Changes in cellular location or associations of subunits are possible contributing factors to such a phenomenon [reviewed by Rechsteiner et al. (30)]. Albina et al. (1) found a reciprocal relationship between muscle glutamine concentration and glutamine synthetase activity in vivo. It may be relevant to this hypothesis that Jaspers et al. (12) reported a decreased concentration of glutamine in rat soleus muscles undergoing denervation. However, whether the turnover of glutamine synthetase in skeletal muscle in vivo is sufficiently rapid to be compatible with this hypothesis is not known. An alternative hypothesis is that the increase in glutamine synthetase activity after denervation is a result of posttranslational covalent modification of the enzyme. In this regard, mammalian glutamine synthetase has been shown to be inactivated by ADP-ribosylation (24), although the physiological significance of this modification is not known. Finally, it is possible that altered enzyme level with no change in mRNA level could result from a change in the translational efficiency of an unaltered amount of glutamine synthetase mRNA (11, 25, 38). Translational control of muscle protein synthesis has been demonstrated for the catabolic effects of glucocorticoids (28). A possible contaminant of these results might be enzyme activity in infiltrating cells, such as lymphocytes
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.157) on January 17, 2019.
MUSCLE
GLUTAMINE
and macrophages. However, this appears unlikely because neither lymphocytes nor macrophages appear to possess a significant capacity for glutamine synthesis (1, 4). Furthermore, morphological examination of denervated and control muscles 1 h postdenervation did not reveal cellular infiltration (results not shown). We thank Drs. B. H. Sohmer and S. E. Poduslo for helpful comments, B. Pasko for preparation of the typescript, and M. A. Wennes and H. Smith for expert technical assistance. The antiglutamine synthetase antiserum was the generous gift of Dr. R. E. Miller. Dr. R. H. Wilson generously provided the Chinese hamster glutamine synthetase gene. This work was supported by National Institute of Child Health and Human Development Grant HD-16596 and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-41022. Present addresses: M. Konagaya and Y. Konagaya, Dept. of Neurology, Nara Medical University, 840, Shijo-cho, Kashihara, Nara 634, ?Japan; J. W. Thomas, CNS Research, Searle Research and Development, Monsanto, St. Louis, MO 63198. Address for reprint requests: S. R. Max, Dept. of Neurology, University of Maryland School of Medicine, Baltimore, MD 21201. Received
18 July
1988; accepted
in final
form
19 December
1989.
REFERENCES 1. ALBINA, J. E., W. HENRY, P. A. KING, J. SHEARER, B. MASTROFRANCESCO, L. GOLDSTEIN, AND M. D. CALDWELL. Glutamine metabolism in rat skeletal muscle wounded with X-carageenan. Am. J. Physiol. 252 (Endocrinol. Metab. 15): E49-E56, 1987. 2. ARAD, G., A. FREIKOPF, AND R. G. KULKA. Glutamine-stimulated modification and degradation of glutamine synthetase in hepatoma tissue culture cells. CeLL 8: 95-101, 1976. 3. ARAD, G., AND R. G. KULKA. Effects of glutamine, methionine sulfon and dexamethasone on rates of synthesis of glutamine synthetase in cultured hepatoma cells. Biochim. Biophys. Acta 544: 153-162,1978. 4. ARDAWI, M. S. M., AND E. A. NEWSHOLME. Metabolism in lymphocytes and its importance in the immune response. Essays Biochem. 21: l-44,1985. 5. CHANG, T. W., AND A. L. GOLDBERG. The metabolic fates of amino acids and the formation of glutamine in skeletal muscle. J. Biol. Chem. 253: 3685-3695,1978. 6. CHIRGWIN, J. M., A. E. PRZYBYLA, R. J. MACDONALD, AND W. J. RUTTER. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299,197l. 7. CROOK, R. B., AND G. M. TOMKINS. Effect of glutamine on the degradation of glutamine synthetase in hepatoma tissue culture cells. Biochem. J. 176: 47-52, 1978. 8. FREIKOPF, A., AND R. G. KULKA. Involvement of protein synthesis in the post translational control of glutamine synthetase activity of cultured hepatoma cells. Biochem. Biophys. Res. Commun. 72: 1195-1200,1976. 9. GOLDBERG, A. C., AND T. W. CHANG. Regulation and significance of amino acid metabolism in skeletal muscle. Federation Proc. 37: 2301-2307,1978. 10. GOLDSTEIN, L. A. Interorgan glutamine relationships. Federation Proc. 45: 2176-2179,1986. 11. HUNT, T. False starts in translational control of gene expression. Nature Lond. 316: 580-581,1985. 12. JASPERS, S. R., E. J. HENRIKSEN, S. SATURAGE, AND M. E. TISCHLER. Effects of stretching and disuse on amino acids in muscles of rat hind limbs. Metabolism 38: 303-310, 1989. 13. KOMINZ, D. R., A. HOUGH, P. SYMONDS, AND K. LAKI. The amino acid composition of actin, myosin, tropomyosin and the meromyosins. Arch. Biochem. Biophys. 50: 148-159, 1954. 14. KONAGAYA, M., P. A. BERNARD, AND S. R. MAX. Blockade of glucocorticoid receptor binding and inhibition of dexamethasoneinduced muscle atrophy in the rat by RU38486, a potent glucocorticoid antagonist. Endocrinology 119: 375-380, 1986. 15. KONAGAYA, M., Y. KONAGAYA, AND S. R. MAX. Evaluation of the endogenous glucocorticoid hvnothesis of denervation atronhv. J.
SYNTHETASE
E761
Neurol. Sci. 79: 1179-1183, 1987. KONAGAYA, M., Y. KONAGAYA, AND S. R. MAX. Neural control of glutamine synthetase activity in rat skeletal muscle. Sot. Neurosci. Abstr. 12: 1107, 1987. 17. KULKA, R. G., AND H. COHEN. Regulation of glutamine synthetase activity of hepatoma tissue culture cells by glutamine and dexamethasone. J. Biol. Chem. 248: 6738-6743,1973. 18. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275,1951. 19. MANIATIS, T., E. F. FRITSCH, AND J. SAMBROOK. Molecular Cloning. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982, p. 190. 20. MAX, S. R., J. MILL, K. MEAROW, M. KONAGAYA, Y. KONAGAYA, J. W. THOMAS, C. BANNER, AND L. VITKOVI~. Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles. Am. J. Physiol. 255 (Endocrinol. Metab. 18): E397-E402, 1988. 21. MAX, S. R., J. W. THOMAS, C. BANNER, L. VITKOVIC, M. KONAGAYA, AND Y. KONAGAYA. Glucocorticoid-receptor mediated induction of glutamine synthetase in skeletal muscle cells in vitro. Endocrinology 120: 1179-1183,1987. 22. MILLER, R. E., AND D. A. CARRINO. Dibutyryl cyclic AMP decreases glutamine synthetase in cultured 3T3-L1 adipocytes. J. Biol. Chem. 255: 5490-5500,198O. 23. MILMAN, G., L. A. PORTNOFF, AND D. C. TIEMEIER. Immunochemical evidence for glutamine-mediated degradation of glutamine synthetase in cultured Chinese hamster cells. J. Biol. Chem. 250: 1393-1399,1975. 24. Moss, J., P. A. WATKINS, S. J. STANLEY, M. R. PURNELL, AND W. R. KIDWELL. Inactivation of glutamine synthetase by an NAD:arginine ADP-ribosyltransferase. J. Biol. Chem. 259: 5lOO5104, 1984. 25. OCHOA, S. Regulation of protein synthesis initiation in eukaryotes. Arch. Biochem. Biophys. 223: 325-349,1983. 26. ODESSEY, R., E. A. KHAIRALLAH, AND A. L. GOLDBERG. Origin and possible significance of alanine production by muscle. J. Biol. Chem. 249: 7623-7629,1974. 27. PHILIBERT, D. RU38486: an original multifaceted antihormone in vivo. In: Adrenal Steroid Antagonism, edited by M. K. Agarwal. Berlin: Gruyter, 1984, p. l-25. 28. RANNELS, S. R., D. E. RANNELS, A. E. PEGG, AND L. S. JEFFERSON. Glucocorticoid effects on peptide-chain initiation in skeletal muscle and heart. Am. J. Physiol. 235 (Endocrinol. Metab. Gastrointest. Physiol. 4): E134-E139, 1978. 29. RAVE, N., R. CRKVENJAKOV, AND H. BOEDTKER. Identification of procollagen mRNAs transferred to diazobenzylmethyl paper from formaldehyde agarose gels. Nucleic Acids Res. 6: 3559-3567, 1979. 30. RECHSTEINER, M., S. ROGERS, AND K. ROTE. Protein structure and intracellular stability. Trends Biochem. Sci. 12: 390-394, 1987. 31. RENNIE, M. J., H. S. HUNDAL, P. BABIJ, P. MACLENNON, P. M. TAYLOR, P. W. WATT, M. M. JEPSON, AND D. J. MILLWARD. Characteristics of a glutamine carrier in skeletal muscle have important consequences for nitrogen loss in injury, infection, and chronic disease. Lancet 2: 1008-1012, 1986. 32. RUDERMAN, N. B., AND M. BERGER. The formation of glutamine and alanine in skeletal muscle. J. Biol. Chem. 249: 5500-5506,1974. 33. SARKAR, P., AND S. CHAUDHURY. Messenger RNA for glutamine synthetase. Molec. Cell. Biochem. 53/54: 233-244, 1983. 34. SHIMKE, R. T. Methods for analysis of enzyme synthesis and degradation in animal tissues. Methods Enzymol. 40E: 241-266, 1975. 35. SMITH, R. J., S. LARSON, S. E. STRED, AND R. DURSCHLAG. Regulation of glutamine synthetase and glutaminase activities in cultured skeletal muscle cells. J. Cell Physiol. 120: 197-203, 1984. 36. TIEMEIER, D. C., AND G. MILMAN. Regulation of glutamine synthetase in cultured Chinese hamster cells. Induction and repression by glutamine. J. Biol. Chem. 247: 5722-5727, 1972. an energy source for 37. TILDON, J. T., AND H. R. ZIELKE. Glutamine: mammalian tissues. In: Glutamine and Glutamate in Mammals, edited by E. Kvamme. New York: CRS, 1988, p. 167-182. 38. WALDEN, W. E., AND R. E. THACH. Translational control of gene expression in a normal fibroblast. Characterization of a subclass of mRNAs with unusual kinetic properties. Biochemistry 25: 20332041,1986.
“0
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.157) on January 17, 2019.
synthetase
activity
BO FENG, MASAAKI KONAGAYA, YOKO KONAGAYA, JOHN W. THOMAS, CARL BANNER, JOHN MILL, AND STEPHEN R. MAX Department of Neurology and Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201; Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore 21228; and Laboratory of Molecular Biology, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892
FENG, Bo, MASAAKI KONAGAYA, YOKO KONAGAYA, JOHN W. THOMAS, CARL BANNER, JOHN MILL, AND STEPHEN R. MAX. Neural control of glutamine synthetase activity in rat skeletal muscles. Am. J. Physiol. 258 (Endocrinol. Metab. 21): E757-E761, 1990.-The mechanism of glutamine synthetase induction in rat skeletal muscle after denervation or limb immobilization was investigated. Adult male rats were subjected to midthigh section of the sciatic nerve. At 1, 2, and 5 h and 1, 2, and 7 days after denervation, rats were killed and denervated, and contralateral control soleus and plantaris muscles were excised, weighed, homogenized, and assayed for glutamine synthetase. Glutamine synthetase activity increased -twofold 1 h after denervation in both muscles. By 7 days postdenervation enzyme activity had increased to three times the control level in plantaris muscle and to four times the control level in soleus muscle. Increased enzyme activity after nerve section was associated with increased maximum velocity with no change in apparent Michaelis constant. Immunotitration with an antiglutamine synthetase antibody suggested that denervation caused an increase in the number of glutamine synthetase molecules in muscle. However, Northern-blot analysis revealed no increase in the steady-state level of glutamine synthetase mRNA after denervation. A mixing experiment failed to yield evidence for the presence of a soluble factor involved in regulating the activity of glutamine synthetase in denervated muscle. A combination of denervation and dexamethasone injections resulted in additive increases in glutamine synthetase. Thus the mechanism underlying increased glutamine synthetase after denervation appears to be posttranscriptional and is distinct from that of the glucocorticoidmediated glutamine synthetase induction previously described by us.
muscle; denervation
SKELETAL MUSCLE PRODUCES and releases glutamine, which serves as a substrate for the energy metabolism of a number of cells and tissues, including intestine, lymphocytes, kidney, and, possibly, brain (4, 10, 37). The release of glutamine by muscle is increased under conditions of stress, such as fasting and trauma (10). However, the quantity of glutamine contained in muscle proteins is insufficient to account for all the glutamine released by muscle (5, 13). Therefore, glutamine is synthesized de novo in muscle (9, 26). Although the exact
pathway is undefined, glutamine synthetase appears to catalyze the final step (32). Glutamine synthetase expression in skeletal muscle is subject to a number of effecters, notably glucocorticoids (20, 21, 35). In this regard, we recently showed that dexamethasone-mediated induction of glutamine synthetase activity is associated with an increase in the level of glutamine synthetase mRNA in intact skeletal muscles (20) or in L6 skeletal muscle cells in culture (21). Because glucocorticoids cause pronounced muscle atrophy, we postulated that induction of glutamine synthetase may play a role in the progression of muscular atrophy from other causes. Indeed, Rennie et al. (31) recently hypothesized that glutamine may control the rate of muscle protein turnover. Because denervation also causes muscle atrophy, we assessed the effects of nerve section on glutamine synthetase activity. Indeed, denervation causes a significant increase in glutamine synthetase activity in rat skeletal muscles (15, 16). We now report the results of investigations into the mechanism of this increase. MATERIALS
AND METHODS
Adult male rats of the crl:CD(SD)BR strain (Charles River, Wilmington, MA) weighing 236 t 40 (SD) g were used. They were fed Purina Laboratory Chow (no. 5001) and water ad libitum and maintained on a schedule of 12-h light:12-h darkness. Denervation was carried out under ether anesthesia by midthigh section of the sciatic nerve. At 1, 2, and 5 h and 1,2, and 7 days after denervation rats were anesthetized with ether and then decap‘itated. Experimental (i.e., denervated) and contralateral control soleus and plantaris muscles were removed, dissected free of fat and connective tissue, and weighed. They were minced finely with scissors in ice-cold homogenization buffer (0.25 M sucrose, 0.2 mM EDTA, pH 7.4, and 2 mM 2-mercaptoethanol). The minced suspensions were homogenized with a polytron homogenizer (Brinkman Instruments). Homogenates were prepared and centrifuged, and the cytosol (30,000 g supernate) was assayed for glutamine synthetase activity as described by Smith et al. (35), except that the glutamate concentration in our assays was 5 mM (higher concentrations of glutamate resulted E757
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.157) on January 17, 2019.
E758
MUSCLE
GLUTAMINE
in no increase in enzyme activity). Protein was determined by the procedure of Lowry et al. (18) by use of crystalline bovine serum albumin as standard. For mRNA analyses, muscles were frozen and pulverized in liquid nitrogen in a mortar and pestle that had been treated with diethyl pyrocarbonate (19). The powders were stored at -70°C. Total cellular RNA was isolated from the frozen muscle powders by means of the guanidine isothiocyanate procedure of Chirgwin et al. (6). Equal amounts of RNA, as determined both spectrophotometrically and through ethidium bromide staining, were fractionated by electrophoresis through denaturing agarose gels containing formaldehyde (29)) transferred to nitrocellulose filters, and hybridized with a radioactive cDNA probe (20). In some experiments (1 h and 7 days postdenervation) a radioactive RNA probe was used (21). The ,&tubulin probe was as described (20). Northern autoradiograms were quantified using an LKB laser densitometer. Immunotitration of glutamine synthetase from denervated and control soleus muscles was performed by the method of Shimke (34) by use of a rabbit antiglutamine synthetase antiserum prepared with purified rat liver glutamine synthetase as antigen (22). The incubation conditions of Miller and Carrino (22) were used. After immunoprecipitation, glutamine synthetase activity was assayed in the supernate. Where indicated (experiment of Fig. 5), dexamethasone was injected for 5 days (5 rng- kg-‘. day-l SC) into normal rats or into rats with a denervated plantaris muscle. Statistical analysis was carried out with paired t tests @-tailed).
SYNTHETASE
M. plantaris
M. soleus
I
1
I
I
.
1
2
5
24
48
Hours
after
,
168
Denervation
1. Effect of denervation on glutamine synthetase specific activity (expressed as percent control) in rat soleus and plantaris muscles (M). Data are means -I- SE of 5 determinations. Glutamine synthetase activity in denervated muscles was significantly different from that in contralateral control muscles in every case, P < 0.001. Experimental procedures are described in text. FIG.
RESULTS
Denervation of soleus and plantaris muscles caused a significant increase in the activity of glutamine synthetase (Fig. 1). A twofold increase was apparent as early as 1 h after nerve section; by 7 days after denervation, glutamine synthetase activity was increased threefold in plantaris muscle and fourfold in soleus (Fig. 1). Increased glutamine synthetase activity in plantaris muscles was associated with an increase in maximum velocity ( Vmax)(48 h denervated = 21.9 nmol . h-l. mg protein-‘; control = 10.49 nmol .h-l. mg protein-‘), with no change in apparent Michaelis constant (K,; 48 h denervated = 1.48 x low3 M; control = 1.9 x 10m3M) (Fig. 2). The increase in Vmaxsuggested an increase in the amount of enzyme, possibly via enhanced synthesis. Indeed, immunotitration experiments by use of an antiglutamine synthetase antiserum showed the equivalence point for the enzyme to be the same from denervated (24 h) and control muscles (Fig. 3A), and that more antibody is required to precipitate glutamine synthetase in denervated than in control muscles (Fig. 3B). These results suggest a twofold increase in the number of glutamine synthetase molecules in 24-h denervated vs. control muscles, in excellent correspondence to the increase in enzyme activity (Fig. 1). Thus denervated muscles appear to contain an increased number of enzyme molecules
1 /
-2
V
0
2
4 6 8 1 /[glutamate] ( 109 FIG. 2. Effect of glutamate concentration on glutamine synthetase activity of denervated (48 h) and control rat plantaris muscles: Lineweaver-Burk plot. Experimental procedures are described in text. CTL, control; DEN, denervation.
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.157) on January 17, 2019.
MUSCLE
0
ctl
l
den
GLUTAMINE
0 0
E759
SYNTHETASE
synthetase mRNA after denervation. A similar result was obtained with Northern blots prepared 1 h or 7 days after denervation (Fig. 4). Stripping and rehybridization of the 24-h Northern blot with a radioactive probe for ,& tubulin revealed no difference between denervated and control muscles (Fig. 4). Therefore the glutamine synthetase mRNA results are not caused by unequal loading of the gels. The half-life of glutamine synthetase mRNA has been estimated to be 6-8 h (33). Therefore, it is unlikely that our determinations would have missed a transient change in mRNA level. To compare further the mechanism of the denervationmediated increase in glutamine synthetase with that of dexamethasone (20, 21), rats with denervated or unoperated muscles were given dexamethasone (5 mg kg-‘. day-l SC) for 5 days. The dexamethasone-treated-plusdenervated muscles were compared with denervated muscles and with muscles from rats receiving dexamethasone alone. The effects of dexamethasone and denervation were additive, indicating that different mechanisms are involved (Fig. 5). A mixing experiment was performed to investigate the possible presence of soluble factors influencing enzyme activity. Mixing equal portions of supernatant fractions from denervated (24 h) and control soleus muscles resulted in a specific activity equal to the average of the activities when these fractions were assayed separately (control, 13.3 nmol . h-l. mg protein-‘; denervated, 45.0 nmol . h-l . mg protein-l; control + denervated, 26.4 nmol . h-’ . mg protein-‘). Thus denervated muscles do not appear to contain a soluble activator molecule; conversely, control muscles do not appear to contain a soluble inhibitory molecule. l
600
total
activity
(cpm)
.B
v
denervalion
DISCUSSION
mtibody
added
(pi)
3. Immunotitration of glutamine synthetase 24 h postdenervation. A: increasing amounts of cytosol were added to tubes containing fixed amount of antiserum. B: increasing amounts of antiserum were added to tubes containing fixed amount of cytosol. Glutamine synthetase activity was assayed in supernate after immunoprecipitation. Separate groups of muscles were used for these experiments. Experimental procedures are described in text. FIG.
compared with control muscles (Fig. 3), rather than a change in the catalytic activity of the enzyme (22, 34). That the enhancement of glutamine synthetase activity is not a consequence of increased transcription is shown by the data of Fig. 4, which depicts Northern-blot analysis of the level of glutamine synthetase mRNA in denervated (24 h) and control plantaris muscles. This experiment revealed no increase in the level of glutamine
The experiments described above were designed to investigate the mechanism of the increase in glutamine synthetase activity after denervation (15, 16) (Fig. 1). Our data reveal increased glutamine synthetase activity and amount without a change in the glutamine synthetase mRNA level. This phenomenon is distinct from that of dexamethasone-mediated induction of this enzyme in muscle cells in culture (21, 35) and in skeletal muscles in vivo (20). Dexamethasone caused an increase in glutamine synthetase activity that was associated with an increase in the level of glutamine synthetase mRNA (20, 21) and was blocked by RU38486 (21), a glucocorticoid antagonist (14, 27). In contrast, the denervation-mediated increase in glutamine synthetase activity was not prevented by treatment of rats with RU38486 (15). This result suggested that endogenous glucocorticoids are not involved in the denervation-mediated increase in glutamine synthetase activity and indicated a different mechanism. The present data confirm that conclusion and provide new insights into the mechanism. Immunotitration (Fig. 3) revealed an increased number of glutamine synthetase molecules in denervated muscles. However, synthesis of glutamine synthetase at the transcriptional level in denervated muscle is ruled out by Northern-blot analysis of glutamine synthetase mRNA, which was not increased in denervated muscle (Fig. 4).
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.157) on January 17, 2019.
E760
MUSCLE A
GS-1
GLUTAMINE
B
GS-24
h
C
h
GS-7
d
time after denervation
den
control
den
D
tubulin-24
h
FIG. 4. Northern-blot analysis of glutamine synthetase mRNA in denervated (den) and control (ctl) plantaris muscles. Analyses were performed at 1 h (A), 24 h (B), or 7 days (C) after denervation. Separate Northern blots were done at each time point. Autoradiograms (A-C) reveal a major band of -3 kb. D: ,& tubulin mRNA, 24 h postdenervation. E: ratio of glutamine synthetase mRNA/Ptubulin mRNA 24 h after denervation, as determined by densitometry. Data are means f SE of 4 determinations.
ctl
In addition, the effects of denervation and dexamethasone were additive (Fig. 5). Finally, differences in the mechanisms of action of dexamethasone and denervation may be provided by the time-course of the denervation effect (Fig. 1). Enzyme activity was increased dramatically within 1 h of nerve section, in contrast to the lag period noted after dexamethasone treatment of muscle cells in culture (21). A soluble activator would not seem to be present, based upon the mixing experiment (see above) and by the increase in I’,,, (Fig. 2). We hypothesize that the increase in glutamine synthetase activity after denervation may be a result of decreased degradation of the enzyme. The cause of this phenomenon might be related to glutamine levels in denervated and disused vs. control muscles. It has been
EJ
SYNTHETASE
dex dex + den
FIG. 5. Effect of denervation and dexamethasone on glutamine synthetase activity of rat plantaris muscles, 5 days postdenervation. den, denervated; dex, dexamethasone (5 mg. kg-‘.day-I). Data are means f SE of 4 determinations. Experimental procedures are described in text.
shown that removal of glutamine from the culture medium causes a large increase in glutamine synthetase activity in Lg muscle cells in culture (35). A similar phenomenon has been found in other cell types, and it has been suggested that glutamine depletion causes the enzyme to assume a more stable configuration, resulting in an increased enzyme concentration (2, 3, 7, 8, 17, 23, 36). Changes in cellular location or associations of subunits are possible contributing factors to such a phenomenon [reviewed by Rechsteiner et al. (30)]. Albina et al. (1) found a reciprocal relationship between muscle glutamine concentration and glutamine synthetase activity in vivo. It may be relevant to this hypothesis that Jaspers et al. (12) reported a decreased concentration of glutamine in rat soleus muscles undergoing denervation. However, whether the turnover of glutamine synthetase in skeletal muscle in vivo is sufficiently rapid to be compatible with this hypothesis is not known. An alternative hypothesis is that the increase in glutamine synthetase activity after denervation is a result of posttranslational covalent modification of the enzyme. In this regard, mammalian glutamine synthetase has been shown to be inactivated by ADP-ribosylation (24), although the physiological significance of this modification is not known. Finally, it is possible that altered enzyme level with no change in mRNA level could result from a change in the translational efficiency of an unaltered amount of glutamine synthetase mRNA (11, 25, 38). Translational control of muscle protein synthesis has been demonstrated for the catabolic effects of glucocorticoids (28). A possible contaminant of these results might be enzyme activity in infiltrating cells, such as lymphocytes
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.157) on January 17, 2019.
MUSCLE
GLUTAMINE
and macrophages. However, this appears unlikely because neither lymphocytes nor macrophages appear to possess a significant capacity for glutamine synthesis (1, 4). Furthermore, morphological examination of denervated and control muscles 1 h postdenervation did not reveal cellular infiltration (results not shown). We thank Drs. B. H. Sohmer and S. E. Poduslo for helpful comments, B. Pasko for preparation of the typescript, and M. A. Wennes and H. Smith for expert technical assistance. The antiglutamine synthetase antiserum was the generous gift of Dr. R. E. Miller. Dr. R. H. Wilson generously provided the Chinese hamster glutamine synthetase gene. This work was supported by National Institute of Child Health and Human Development Grant HD-16596 and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-41022. Present addresses: M. Konagaya and Y. Konagaya, Dept. of Neurology, Nara Medical University, 840, Shijo-cho, Kashihara, Nara 634, ?Japan; J. W. Thomas, CNS Research, Searle Research and Development, Monsanto, St. Louis, MO 63198. Address for reprint requests: S. R. Max, Dept. of Neurology, University of Maryland School of Medicine, Baltimore, MD 21201. Received
18 July
1988; accepted
in final
form
19 December
1989.
REFERENCES 1. ALBINA, J. E., W. HENRY, P. A. KING, J. SHEARER, B. MASTROFRANCESCO, L. GOLDSTEIN, AND M. D. CALDWELL. Glutamine metabolism in rat skeletal muscle wounded with X-carageenan. Am. J. Physiol. 252 (Endocrinol. Metab. 15): E49-E56, 1987. 2. ARAD, G., A. FREIKOPF, AND R. G. KULKA. Glutamine-stimulated modification and degradation of glutamine synthetase in hepatoma tissue culture cells. CeLL 8: 95-101, 1976. 3. ARAD, G., AND R. G. KULKA. Effects of glutamine, methionine sulfon and dexamethasone on rates of synthesis of glutamine synthetase in cultured hepatoma cells. Biochim. Biophys. Acta 544: 153-162,1978. 4. ARDAWI, M. S. M., AND E. A. NEWSHOLME. Metabolism in lymphocytes and its importance in the immune response. Essays Biochem. 21: l-44,1985. 5. CHANG, T. W., AND A. L. GOLDBERG. The metabolic fates of amino acids and the formation of glutamine in skeletal muscle. J. Biol. Chem. 253: 3685-3695,1978. 6. CHIRGWIN, J. M., A. E. PRZYBYLA, R. J. MACDONALD, AND W. J. RUTTER. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299,197l. 7. CROOK, R. B., AND G. M. TOMKINS. Effect of glutamine on the degradation of glutamine synthetase in hepatoma tissue culture cells. Biochem. J. 176: 47-52, 1978. 8. FREIKOPF, A., AND R. G. KULKA. Involvement of protein synthesis in the post translational control of glutamine synthetase activity of cultured hepatoma cells. Biochem. Biophys. Res. Commun. 72: 1195-1200,1976. 9. GOLDBERG, A. C., AND T. W. CHANG. Regulation and significance of amino acid metabolism in skeletal muscle. Federation Proc. 37: 2301-2307,1978. 10. GOLDSTEIN, L. A. Interorgan glutamine relationships. Federation Proc. 45: 2176-2179,1986. 11. HUNT, T. False starts in translational control of gene expression. Nature Lond. 316: 580-581,1985. 12. JASPERS, S. R., E. J. HENRIKSEN, S. SATURAGE, AND M. E. TISCHLER. Effects of stretching and disuse on amino acids in muscles of rat hind limbs. Metabolism 38: 303-310, 1989. 13. KOMINZ, D. R., A. HOUGH, P. SYMONDS, AND K. LAKI. The amino acid composition of actin, myosin, tropomyosin and the meromyosins. Arch. Biochem. Biophys. 50: 148-159, 1954. 14. KONAGAYA, M., P. A. BERNARD, AND S. R. MAX. Blockade of glucocorticoid receptor binding and inhibition of dexamethasoneinduced muscle atrophy in the rat by RU38486, a potent glucocorticoid antagonist. Endocrinology 119: 375-380, 1986. 15. KONAGAYA, M., Y. KONAGAYA, AND S. R. MAX. Evaluation of the endogenous glucocorticoid hvnothesis of denervation atronhv. J.
SYNTHETASE
E761
Neurol. Sci. 79: 1179-1183, 1987. KONAGAYA, M., Y. KONAGAYA, AND S. R. MAX. Neural control of glutamine synthetase activity in rat skeletal muscle. Sot. Neurosci. Abstr. 12: 1107, 1987. 17. KULKA, R. G., AND H. COHEN. Regulation of glutamine synthetase activity of hepatoma tissue culture cells by glutamine and dexamethasone. J. Biol. Chem. 248: 6738-6743,1973. 18. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275,1951. 19. MANIATIS, T., E. F. FRITSCH, AND J. SAMBROOK. Molecular Cloning. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982, p. 190. 20. MAX, S. R., J. MILL, K. MEAROW, M. KONAGAYA, Y. KONAGAYA, J. W. THOMAS, C. BANNER, AND L. VITKOVI~. Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles. Am. J. Physiol. 255 (Endocrinol. Metab. 18): E397-E402, 1988. 21. MAX, S. R., J. W. THOMAS, C. BANNER, L. VITKOVIC, M. KONAGAYA, AND Y. KONAGAYA. Glucocorticoid-receptor mediated induction of glutamine synthetase in skeletal muscle cells in vitro. Endocrinology 120: 1179-1183,1987. 22. MILLER, R. E., AND D. A. CARRINO. Dibutyryl cyclic AMP decreases glutamine synthetase in cultured 3T3-L1 adipocytes. J. Biol. Chem. 255: 5490-5500,198O. 23. MILMAN, G., L. A. PORTNOFF, AND D. C. TIEMEIER. Immunochemical evidence for glutamine-mediated degradation of glutamine synthetase in cultured Chinese hamster cells. J. Biol. Chem. 250: 1393-1399,1975. 24. Moss, J., P. A. WATKINS, S. J. STANLEY, M. R. PURNELL, AND W. R. KIDWELL. Inactivation of glutamine synthetase by an NAD:arginine ADP-ribosyltransferase. J. Biol. Chem. 259: 5lOO5104, 1984. 25. OCHOA, S. Regulation of protein synthesis initiation in eukaryotes. Arch. Biochem. Biophys. 223: 325-349,1983. 26. ODESSEY, R., E. A. KHAIRALLAH, AND A. L. GOLDBERG. Origin and possible significance of alanine production by muscle. J. Biol. Chem. 249: 7623-7629,1974. 27. PHILIBERT, D. RU38486: an original multifaceted antihormone in vivo. In: Adrenal Steroid Antagonism, edited by M. K. Agarwal. Berlin: Gruyter, 1984, p. l-25. 28. RANNELS, S. R., D. E. RANNELS, A. E. PEGG, AND L. S. JEFFERSON. Glucocorticoid effects on peptide-chain initiation in skeletal muscle and heart. Am. J. Physiol. 235 (Endocrinol. Metab. Gastrointest. Physiol. 4): E134-E139, 1978. 29. RAVE, N., R. CRKVENJAKOV, AND H. BOEDTKER. Identification of procollagen mRNAs transferred to diazobenzylmethyl paper from formaldehyde agarose gels. Nucleic Acids Res. 6: 3559-3567, 1979. 30. RECHSTEINER, M., S. ROGERS, AND K. ROTE. Protein structure and intracellular stability. Trends Biochem. Sci. 12: 390-394, 1987. 31. RENNIE, M. J., H. S. HUNDAL, P. BABIJ, P. MACLENNON, P. M. TAYLOR, P. W. WATT, M. M. JEPSON, AND D. J. MILLWARD. Characteristics of a glutamine carrier in skeletal muscle have important consequences for nitrogen loss in injury, infection, and chronic disease. Lancet 2: 1008-1012, 1986. 32. RUDERMAN, N. B., AND M. BERGER. The formation of glutamine and alanine in skeletal muscle. J. Biol. Chem. 249: 5500-5506,1974. 33. SARKAR, P., AND S. CHAUDHURY. Messenger RNA for glutamine synthetase. Molec. Cell. Biochem. 53/54: 233-244, 1983. 34. SHIMKE, R. T. Methods for analysis of enzyme synthesis and degradation in animal tissues. Methods Enzymol. 40E: 241-266, 1975. 35. SMITH, R. J., S. LARSON, S. E. STRED, AND R. DURSCHLAG. Regulation of glutamine synthetase and glutaminase activities in cultured skeletal muscle cells. J. Cell Physiol. 120: 197-203, 1984. 36. TIEMEIER, D. C., AND G. MILMAN. Regulation of glutamine synthetase in cultured Chinese hamster cells. Induction and repression by glutamine. J. Biol. Chem. 247: 5722-5727, 1972. an energy source for 37. TILDON, J. T., AND H. R. ZIELKE. Glutamine: mammalian tissues. In: Glutamine and Glutamate in Mammals, edited by E. Kvamme. New York: CRS, 1988, p. 167-182. 38. WALDEN, W. E., AND R. E. THACH. Translational control of gene expression in a normal fibroblast. Characterization of a subclass of mRNAs with unusual kinetic properties. Biochemistry 25: 20332041,1986.
“0
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.157) on January 17, 2019.
