Limited resistance of hypertrophied skeletal muscle to glucocorticoids

0022-4731/86$3.00+ 0.00 Pergamon Journals Ltd

J. s&voidBiochem.Vol. 24, No. 6, pp. 117~1183, 1986 Printed in Great Britain.

LIMITED RESISTANCE OF HYPERTROPHIED SKELETAL MUSCLE TO GLUCOCORTICOIDS R. C. HICKsoNt, T. M. GALASSI, J. A. CAPACCIO and R. T. CHATTERTONJR* Department of Physical Education, University of Ihinois at Chicago, Chicago, IL 60680 and *Department of Obstetrics and Gynecology, Northwestern University Medical School, Chicago, IL 6061 I, U.S.A. (Received 3 April 1985) Summary-Male hypophysectomized rats were initially assigned to a control or an overloaded group that underwent compensatory hypertrophy of plantaris muscles to steady-state levels following removal of synergistic musculature. Plantaris muscle mass of overloaded animals was higher than that of controls by 38% (391 k 8 vs 284 + 7 mg) and glucocorticoid cytosol specific binding concentrations, using [3H]triamcinolone acetonide (TA) as the labeled steroid, was also signi&antly higher in hypertrophied muscles (83.3 + 3.9 fmol . mg protein-‘) than in control muscles (56.3 & 3.9 fmol . mg protein-‘). Cortisone acetate (CA) was then administered daily su~utaneously in high, 100 mg; inte~~iate, IO mg; or low, l.Omg . kg-’ body wt doses. Groups of rats were killed after l/4, 2 days and 7 days. Absolute muscle mass losses after 7 days of CA treatment were approx 80 mg with high doses and 60 mg with intermediate doses in both hypcrtrophied and control muscles. The low CA dose did not produce atrophy. The absolute depletion of [‘HI TA binding activity with CA treatment was always greater in hypertrophied muscles of high and intermediate dose treated than those of their respective controls, but TA binding capacities remained higher in hypertrophied muscles than in controls at almost all time points in all treatment groups, Unlike previous findings in which the simultaneous initiation of overload prevented glucocorticoid induced muscle wasting, no resistance to the effect of CA treatment was observed when treatment was begun after hypertrophy had occurred.

INTRODUCTION

EXPERIMENTAL

A number of studies have shown that it is possible to

Animal care and experimental treatments

inhibit

Male Sprague-Dawley rats were hypophysectomixed at 72 days of age by Hormone Assay, Chicago, Ill. Further treatments began when the animals reached 80 days of age. They were housed individually and provided a diet of Purina rat chow and water ad Zibitum. Compensatory hypertrophy of plantaris muscles was induced in approximately half of the animals by surgical removal of soleus and most of the gastrocnemius muscles using procedures described previously [7]. The remaining animals were assigned as controls. At least 30 days were allowed for the muscles of the overloaded animals to reach a steadystate of enlargement before continuing further experimentation [8,9]. For glucocorticoid treatment both overloaded and control animals were divided into 1 of 4 groups that received subcutaneous injections of either a vehicle (1% aqueous carboxymethyl cellulose), or cortisone acetate (CA) in the following doses: 100 mg ’ kg-’ body weight (b.wt) (high), 10 mg . kg-’ b.wt (intermediate), and 1.Omg . kg-’ b.wt (low). Animals from each of these groups were killed after a single injection, 6 h later, or after additional single daily injections for 2 or 7 days. Both the administered steroid and 7-day duration point were used for comparison with previous work, in which the effectiveness of the onset of overload in resisting glucocorticoid actions were studied IS]. To confirm specific observations in

or retard

the muscle

atrophying

process

in-

duced by glucocorticoids through increasing the contractile activity of skeletal muscle [l-6]. In particular, it has been shown that almost all of the expected muscle mass loss from glucocorticoid treatment can be prevented by imposing a compensatory overload simultaneously with steroid treatment [5]. This finding has raised the additional question of whether skeletal muscles that have enlarged to higher steady state levels through overload are also capable of withstanding the glucocorticoid effects. Therefore, the main purpose of this investigation was to determine whether overloaded-hy~rtrophied muscles are as resistant to glucocorticoids as overloaded muscles undergoing enlargement. Furthermore, since higher glucocorticoid cytosol binding concentrations were previously observed in muscles spared from atrophy than in atrophied muscles [5], it was postulated that similar glucocorticoid binding differences would be observed if hypertrophied muscles resisted the atrophying effects of glu~~orticoids. The dose-response relationship of administered hormone in relation to muscle mass and glucocorticoid cytosol binding was also investigated.

tAddress for correspondence: Department of Physical Education, University of Illinois at Chicago, Box 4348, Chicago, IL 60680, U.S.A.

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1180 some instances, separate animals ment duration for 14 days.

R. C. received

HICKSON er

the treat-

Radioimmunoassay of serum cortisol The animals were sacrificed by decapitation within 30 s of removal from their cages. Blood was collected from the severed vessels, allowed to coagulate, centrifuged at 1OOOg for 20 min, and serum stored at -20°C until assay. Anticortisol antiserum was obtained from Radioassay Systems Laboratories, Carson, Calif. Cortisol was extracted directly from serum with ethyl ether and assayed according to procedures described previously from this laboratory [7]. Preparation of cytosol Following exsanguination, the plantaris muscles were rapidly removed, kept on ice, trimmed of adjacent musculature, fat, and connective tissue, weighed, and placed in pre-cooled polyethylene (50 ml) tubes. The samples were kept on ice, minced with scissors, and homogenized in 5 vol of TE buffer (0.05 M Tris, 0.0015 M EDTA, pH 7.4) containing 1.0 mM dithiothreitol (added fresh) with two 30 s passes at setting 6 using a Polytron (Brinkman Instruments). The homogenate was centrifuged at 30,000 g at 4°C using a Sorvall (RC-SB) centrifuge. After centrifugation, the supernatant fluid, referred to a cytosol, was removed for the steroid binding assay. The 30,OOOg and 100,000 g supernatant fractions gave identical binding values. Using immunocytochemical procedures, Antakly and Eisen[lO] have recently demonstrated the existence of glucocorticoid receptors in cytoplasm as well as in nuclei of a number of target tissues. This observation is in contrast with two other studiesIll, 121 that indicated that most of cellular estrogen receptors are found in the nucleoplasm of several types of target tissues. Thus, glucocorticoid hormone-receptor binding appears consistent with the well-established model of steroid action as being initiated in the cytoplasm of cells [cf. 131. Assay of [‘HI triamcinolone acetonide binding The glucocorticoid receptor in rat muscle is highly specific for triamcinolone acetonide (TA), dexamethasone, and corticosterone, and to a smaller extent, progesterone, but has little or no affinity for androgens or estrogens [S, 141. Triamcinolone acetonide and dexamethasone are known to bind with high affinity to the same receptor site in skeletal muscle [ 151. Their binding cannot be attributed to the presence of corticosteroid-binding globulin (CBG) in muscle cytosol as they do not bind to CBG [cf. IS]. On the other hand, TA has little affinity for the androgen receptor in muscle [5,15]. TA binding to progesterone receptor sites is also unlikely since progesterone receptors have not been detected in skeletal muscle [7, 16, 171. Steroid binding procedures were carried out according to the methodology developed by Ho-Kim

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et al.[l8]. For the present purposes, single point analyses were used to compare binding differences between the respective groups. Muscle cytosols were incubated with a saturating concentration (16 mM) of [6,7-‘Hltriamcinolone acetonide, 9-fluoro- I I,21 dihydroxy- 16,17,21 -tetrahydroxy-pregnaI ,4-diene3,20-dione cyclic 16, I ‘I-acetal, (New England Nuclear, Boston, Mass.), for approx 18 h at 4 C. Total and nonspecific binding were determined following dextran-coated charcoal treatment [I 81. Specific binding was determined by the difference between [3H]triamcinolone acetonide bound in the absence and presence of a ISO-fold excess of unlabeled triamcinolone acetonide. Protein was determined by the Folin-phenol method [19] using ovalbumin as standard. In some groups as needed to obtain sufficient tissues, the muscles of several animals were combined for assay. Statistical procedures The data were analyzed using analysis of variance. When appropriate, mean comparisons following significant F-ratio’s were evaluated with the Duncan’s New Multiple Range Test. Statistical significance was set at the 0.05 level. RESULTS

Plantaris muscle development and response to glucocorticoid treatment. Vehicle-treated overloaded muscle mass averaged 391 f 8 mg; whereas, vehicle-treated control muscle mass averaged 284 f 7 mg (P < 0.01). This 40% muscle enlargement is consistent with our previous work using this experimental model [S, 71. For subsequent data presentation in Figs 1-3, the terminology “hypertrophied” versus “control” are used. High doses (100 mg . kgg’ body weight (b.wt)) of cortisone acetate resulted in a marked reduction of almost 80 mg in plantaris muscle mass following 7 days of treatment in both hypertrophied and control muscles (Fig. 1). The curves show that most of the atrophy (60 mg) occurred between 2-7 days. In addition, the mass of the hypertrophied group averaged 304 &- 10 mg at 7 days, which was not significantly different from that in the vehicle-treated controls (284 + 7 mg). To determine whether additional CAtreatment would produce further atrophy, separate groups were administered CA for 14 days. The hypertrophied muscles averaged about the same as on 7 days (302 f 8). However, comparisons with controls were not possible as they were unable to survive this treatment duration. As shown in Fig. 1, the intermediate dose (10 mg kg-’ b.wt) of cortisone acetate resulted in about 60 mg losses of plantaris muscle mass in both groups. All of the decline occurred between the 2nd and 7th day of injections. With low dose (l.Omg kg-’ b.wt) treatment, no atrophy was observed at any time point in either

Hypertrophied

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skeletal muscle and atrophy resistance

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cortisol levels were between 5-10 mg/ml by 6 h and remained in this range with subsequent injections (Fig. 2). Both hypertrophied and control groups exhibited similar serum responses to the injected hormone. [3H]Triamcinolone acetonide cytosol binding (TA )

Fig. 1. Plantaris muscle weight responses to high (100 mg kg-’ b.wt), intermediate (10 mg kg-’ b.wt), and low (1.0 mg . kg-’ b.wt) doses of cortisone acetate. The arrows, which are located at the junction of the ordinate and abscissa of each graph, point to the values for the vehicletreated groups.

group with the exception of a small decline at the 7-day point in the hypertrophied muscles (Fig. 1).

Additional treatment of this low dose for 14 days failed to substantiate an effect as plantaris muscle mass averaged 399 + 17 mg. Serum cortisol Both vehicle-treated groups had similar but barely detectable levels of serum cortisol that averaged less than 1.0 ng/ml. In Fig. 2, the responses to cortisone acetate administration are shown in both groups. With high doses serum cortisol increased to 45-65 ng/ml after 6 h in both groups and continued to increase progressively throughout the 7-day period (Fig. 2). Serum cortisol increased to 20 ng/ml after 6 h and was only slightly higher thereafter in the intermediate dose group. In low-dose treated groups,

Mean [3H]TA specific binding was 83.3 + 3.9 fmol . mg protein-’ in the plantaris muscles of the vehicle-treated hypertrophied group as compared to 56.3 &-3.9 fmol mg protein-’ in those of the vehicletreated controls (P < 0.01). The increase in glucocorticoid cytosol binding of hypertrophied muscles is in agreement with the results of our previous study [5]. High doses of cortisone acetate depleted [3H]TA binding by more than 50% after 6 h. With further treatment, [3H]TA binding was further reduced and the observed differences between hypertrophied and control muscles were almost eliminated by 7 days of treatment. A single injection of the intermediate dose depleted [3H]TA cytosol binding levels at 6 h to the same extent as observed for the high dose. Repeated injections produced no additional changes in binding concentrations, and there were no differences in binding at 2 or 7 days between groups. Administration of low doses reduced [‘HITA specific binding by 15-20% after 6 h with little further decrement observed at additional time points. In both absolute and relative terms, [3H]TA binding differences at 7 days between hypertrophied and control muscles were still maintained at the same magnitude as found in the respective vehicle-treated groups (Fig. 3).

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Fig. 3. ~HJTr~amcinolone acetonide cytosol binding after single and repeated daily injections of high, intermediate, and low doses of cortisone acetate in hypertrophied and control plantaris muscles. The arrows, which are located at the junction of the ordinate and abscissa of each graph, point to the values for the vehicle-treated groups.

DISCUSSION

We had previously found that the simultaneous initiation of both compensatory overload and glucocorticoids prevented essentially all of the steroidinduced atrophy caused by glucocorticoids alone [5]. This observation provided the stimulus for testing the hypothesis that muscles which have enlarged to higher steady-state weight by compensatory overload are also capable of resisting the atrophy. The present findings clearly indicate that hypertrophied skeletal muscles were unable to counteract the atrophy. In fact, the high dose (100mg. kg-’ b.wt) abolished almost all of the muscle mass increases within the 7 day experimental period. The implementation of an intermediate-dose group which was 10% of the high dose, was still based on expectations that the hypertrophied muscles could withstand some degree of

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glucocorticoid action. But the same pattern ofmuscie atrophy was observed in both groups, although the overall rate of decline was somewhat less than that with the high dose. One common connection the present findings have with those obtained when overload and gfucocorticoids were imposed simultaneously I.51is that the overload was effective in counteracting the atrophy only when the muscle mass approaches a “normal” range. This was evidenced by the fact that no further decline from the hypertrophied state was observed from 7 to 14 days of glucocorti~oid treatment even though non-hypertrophied muscles decreased to lower weights in the presence of glucocorticoids. In the low dose-treated groups, where no atrophy occurred, [‘HITA binding in plantaris muscles was reduced by about the same amount in both hypertrophied and control groups. While the rate of TA binding depletion was somewhat similar between groups receiving high and intermediate doses, the absolute depletion was greater in the muscles of the hypertrophied group, Since the TA binding levels were initially higher in the muscles of the vehicletreated hypertrophied group than those in the corresponding controls, the possibility exists that the effect of the overload was diminished by this greater response potential of the muscle to administered hormone. Nevertheless, some caution is required in this interpretation since nuclear-bound receptors were not determined and whether the total number of receptors per cell were increased is not known. With the surgical overload model, hypertrophied muscles increase their fiber area in all cell types [20]. Thus, the observed 50% or more[5] increases in glucocorticoid cytosol binding capacity in the enlarged muscles expressed per mg cytosol protein, would not initially appear to result from marked changes in cell proportions. In contrast, the atrophy associated with excess glucocorticoids occurs initially in the white muscle regions while the red fiber types remain resistant to atrophy [l, 31. In this case, the number of sites per mg of cytosol protein would increase. This observation is based on recent unpublished studies in this laboratory that have shown the red fiber types contain 2-3 times as many binding sites as the white fibers. Nevertheless, additional investigation of homogeneous regions of cell types or even single fiber analyses are needed to resolve whether [‘H]TA binding changes in hypertrophied or atrophied muscles are related to altered numbers of receptor sites per cell or to redistribution of cell proportions. Glucocorticoids are known to depress both protein and RNA synthesis [21-231. However. compensatory overload causes muscle enlargement by stimuiatin~ protein and RNA synthesis [24,25]. Thus, a specific mechanism for sparing muscle from atrophy exists. Nevertheless, it appears that the rates of muscle protein synthesis in enlarged muscles are not sufficient to maintain muscle mass at the higher

Hypertrophied

skeletal muscle and atrophy resistance

steady-state levels and simultaneously override the glucocorticoid effects. Our laboratory is in the process of examining these possibilities by determining the separate and combined influence of overload and glucocorticoids on protein synthesis and breakdown. From the foregoing data, we conclude that skeletal muscles, hypertrophied to steady-state levels by compensatory overload, are not able to counteract the catabolic effects of glucocorticoids sufficiently to maintain the hypertrophied condition, but overload is able to prevent muscle loss to weights below those of the normal hypertrophied condition. Acknowledgements-This research was supported by NIH grant AM 26408 and Research Career Development Award KO4AMOllOO to R. C. Hickson. The technical assistance of Mr Gregory Andrews and secretarial assistance of Mrs Mary Ann Fritsch are greatly appreciated. REFERENCES

1. Goldberg

2.

3. 4.

5.

A. L. and Goodman H. M.: Relationship between cortisone and muscle work in determining muscle size. J. Physiol. 200 (1969) 667-675. Gardiner P. F., Hibl B., Simpson D. R., Roy R. R. and Edgerton V. R.: Effects of a mild weight-lifting program on the progress of glucocorticoid-induced atrophy in rat hindlimb muscles.-P@gers Arch. 385 (1980) 147-153. Hickson R. C. and Davis J. R.: Partial prevention of glucocorticoid-induced muscle atrophy by endurance training. Am. J. Physiol. 241 (1981) E226-E232. Scene T. and Viru A.: The catabolic effects of alucocoticoids on different types of skeletal muscle fib& and its dependence upon muscle activity and interaction with anabolic steroids. J. steroid Eiochem. 16 (1982) . , 349-352. Kurowski T. T., Chatterton R. T. Jr and Hickson R. C.: Countereffects of compensatory overload and glucocorticoids in skeletal muscle: androgen and glucocorticoid cytosol receptor binding. J. steroid Biochem. 21 (1984) 137-145.

6. Hickson R. C., Kurowski T. T., Capaccio 3. A. and Chatterton R. T. Jr: Androgen cytosol binding in exercise-induced sparing of muscle atrophy. Am. J. Physiol. (1984) E597-E602. 7. Hickson R. C., Galassi T. M., Kurowski T. T., Daniels D. G. and Chatterton R. T. Jr: Skeletal muscle cytosol [3H] methyltrienolone receptor binding and serum androgens: effects of hypertrophy and hormonal state. J. steroid Biochem. 19 (1983) 1705-1712.

8. Ianuzzo C. D. and Chen V.: Metabolic character of hypertrophied rat muscle. J. appl. Physiol. 46 (1979) 738-742.

9. Armstrong R. B., Marum P., Tullson P. and Saubert C. W. IV: Acute hypertrophic response of skeletal muscle to removal of synergists. J. appl. Physiol. 46 (1979) 835-842.

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10. Antakly T. and Eisen H. J.: Immunocytochemical localization of glucocorticoid receptor in target cells. Endocrinology 115 (1984) 1984-l 989. II. King W. J. and Green G. L.: Monoclonal antibodies localize oestrogen receptor in nuclei of target cells. Nature 307 (1984) 745-747. 12. Welshons W. V., Lieberman M. E. and Gorski J.: Nuclear localization of unoccupied oestrogen receptors. Nature 387 (1984) 747-749. l3 King W. J.: Cytoplasmic receptors for steroid ’ hormones-characteristics and assay. In Biochemistry of Mammalian Reproduction (Edited by L. J. D. Zanefeld and R. T. Chatterton). Wiley-Interscience, New York (1982) pp. 455481. 14. Snochowski M., Dahlberg E. and Gustafsson J.-A.: Characterization and quantification of the androgen and glucocorticoid receptors in cytosol from rat skeletal muscle. Eur. J. Biochem. 111 (1980) 6033616. 15. Mayer M., Kaiser N., Milholland R. J. and Rosen F.: The binding of dexamethasone and triamcinolone acetonide to glucocorticoid receptors in rat skeletal muscle. J. biol. Chem. 249 (1974) 52365240. 16. Dahlberg E., Snochowski M. and Gustafsson J.-A.: Regulation of the androgen and glucocorticoid receptors in rat and mouse skeletal muscle cytosol. Endocrinology 108 (1981) 1431-1440. 17. Ho-Kim M. A., Tremblay R. R. and Dube J. Y.: Binding of methyltrienolne to glucocorticoid receptors in rat muscle cytosol. Endocrinology 109 (1981) 1418-1423.

18. Ho-Kim M. A., Tremblay R. R. and Dube J. Y.: Determination of occupied cytoplasmic glucocorticoid receptor sites by an exchange assay in rat muscles. J. steroid Biochem. 18 (1983) 179-184.

19. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J.: J. biol. Chem. 193 (1951) 265-275. 20. Ianuzzo C. D., Gollnick P. D. and Armstrong R. B.: Compensatory adaptation of skeletal muscle fiber types to a long-term functional overload. Life Sci. 19 (1976) 1517-1524. 21, Bullock G. R., Carter E. E., Elliot P., Peters R. F., Simpson P. and White A. M.: Relative changes in the function of muscle ribosomes and mitochondria during the early phase of steroid-induced catabolism. Biochem. J. 127 (1972) 881-892.

22. Shoji S. and Pennington R. J. T.: The effect of cortisone on protein breakdown and synthesis in rat skeletal muscle. Molec cell. Endocr. 6 (1977) 159-169. 23 Rannels D. E., Rannels S. R., Li J. B., Pegg A. E., Morgan H. E. and Jefferson L. S.: Effects of glucocorticoids on peptide chain initiation in heart and skeletal muscle. In Advances in Mvocardiolonv (Edited by M. Tajuddin, P. K. Das, M. Tahg and N. g Dhalla). University Park Press, Baltimore, Maryland, Vol. 1 (1980) pp. 493-501. 24 Goldberg A. L.: Protein synthesis during work-induced growth of skeletal muscle. J. cell Biol. 36 (1968) 653658.

25. Sohel B. E. and Kaufman S.: Enhanced RNA polymerase activity in skeletal muscle undergoing hypertrophy. Archs biochem. Biophys. 137 (1970) 469476.