Camp. Biochem.
Physiol.
$5.00+ 0.00 0300-9629/92
Vol. IOZA,No. I, PP. 141-145,1992
0
Printedin Great Britain
1992 Pergamon Press plc
EFFECT
OF CLENBUTEROL ON SKELETAL MUSCLE ATROPHY IN MICE INDUCED BY THE GLUCOCORTICOID DEXAMETHASONE
E. T. AGBENYEGA* and A. C. WmEHAMt# tDepartment of Physiological Sciences, School of Biological Sciences, Medical School, University of Manchester, Manchester Ml3 9PT, U.K. Telephone: 061-275-5372; Fax: (061) 275-5600 (Received 9 September 1991) Abstract-l. The ability of clenbuterol to antagonize the catabolic effect of the glucocorticoid dexamethasone on the skeletal muscles, soleus, gastrocnemius and extensor digitorum longus was studied in mice. 2. Daily injections of 5 mg dexamethasone/kg body weight over 10 days caused a significant (20%) loss of muscle weight and protein content in fast twitch but not in slow twitch muscles. 3. Inclusion of clenbuterol (4mg/kg) in the diet for the period of dexamethasone treatment partly prevented glucocorticoid-induced muscle atrophy, and increasing the concentration of clenbuterol to 8 mg/kg diet totally prevented glucocorticoid-induced protein loss in all muscles.
INTRODUCTION Excessive amounts of glucocorticoids, whether produced endogenously as in Cushing’s disease, or administered exogenously in the treatment of a variety of medical conditions (Mayer and Rosen, 1977; Bayliss, 1984), are associated with the suppression of growth in the young (Blodgett et al., 1956; Loeb, 1976) and whole body atrophy in older animals (Loeb, 1976). Skeletal muscle is the tissue most affected by the catabolic action of glucocorticoids (Afifi et al., 1968; Kelly and Goldspink, 1982). Some /?,-adrenergic agonists stimulate growth and muscle protein deposition in rodents (Emery et al., 1984; Sainz and Wolff, 1988) and in farm animals (Ricks et al., 1984; Bohorov et al., 1987). The most potent of these is clenbuterol. Clenbuterol has been reported to both stimulate protein synthesis (Emery et al., 1984) and inhibit proteolysis (Garber et al., 1976; Li and Jefferson, 1977), resulting in an increase in muscle mass. In normal young rat muscle, the anabolic action of clenbuterol is expressed as fibre hypertrophy (Maltin et al., 1986) and has been attributed to a reduction in proteolysis (Reeds et al., 1986). The objective of the present study was to determine to what extent administration of clenbuterol in the diet could reduce the degree of atrophy of mouse fast and slow twitch skeletal muscle induced by daily injections of the glucocorticoid dexamethasone. MATERIALSAND
METHODS
Twenty-eight 4-5-week-old male C57 BL/6J mice were used. Animals were fed on a powdered diet (CRM LABSURE). In all experiments the &-agonist clenbuterol was administered orally, mixed with the powdered diet. *Current address: Department of Physiology, School of Medical Sciences, University of Science and Technology, Kumasi, Ghana. $To whom all correspondence should be addressed.
Control animals were fed on the normal diet. All animals were allowed free access to food and water. Muscle atrophy was induced in C57 mice by daily subcutaneous injections of dexamethasone, 5 mg/kg body weight, a dose previously demonstrated to induce muscle atrophy in rats (Kelly and Goldspink, 1982). The vehicle used in dexamethasone injections was normal saline. Four age and weight matched groups were used and given one of the following treatments: Group 1, saline injections + control diet; Group 2, dexamethasone injections + control diet; Group 3, dexamethasone injections + 4 mg/kg clenbuterol diet; Group 4, dexamethasone injections + 8 mg/kg clenbuterol diet. The animals were weighed daily. Muscle growth or atrophy was determined by comparing muscle wet weight after 10 days treatment with that of control animals. To eliminate any change in wet weight that might have been due to electrolyte and/or water retention, total muscle protein was also measured. Assay of muscles for protein content was by the Biorad dye binding method (Bio-Rad. Watford. U.K.) after solubilization in 0.3 M NaOH, with bovine serum albumin (BSA) as standard. Before analysis, muscles were stored frozen at - 18°C. Significance of differences between means were determined using the Student’s t-test for unpaired values (two-tailed probabilities). I
RESULTS
Effect of clenbuterol on dexamethasone-induced body weight
loss of
The growth of mice in the four groups, measured as change in body weight, is shown in Fig. 1. The average daily increase in mean body weight of the control mice was approximately 2%. A fall in body weight of 3.5% occurred in the first 24 hr of dexamethasone treatment alone. The mean body weight attained after 24 hr was stable for a further 6 days, followed by a daily increase of 1%. A bigger fall in mean body weight of 7% occurred in the first 24 hr of the dexamethasone group treated with 4mg/kg diet of clenbuterol. Increasing the dose of clenbuterol to 8 mg/kg caused an even greater fall of 12% after 24 hr. These decreases in body weight did not 141
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AGBENYEGA and A. C. WAREHAM
continue after the first 24 hr in the clenbuterol treated groups but were immediately followed by 1 to 2% daily increases. The final body weight after 10 days of treatment with dexamethasone + clenbuterol was significantly less (P c 0.01) than in the saline treated controls.
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Effect of dexamethasone on skeletal muscle weights
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90.
The fast twitch extensor digitorum longus (EDL) and gastrocnemius (GASTROC) and slow twitch soleus (SOL) muscles of the hind limb were investigated for signs of atrophy after 10 days of treatment with dexamethasone. When compared to the saline injected controls, the dexamethasone treated EDL weighed 20% less (P < 0.01) (Fig. 2) and GASTROC, 18% less (P < 0.01) (Fig. 3). The small (12%) apparent loss of weight in SOL was not statistically significant (P > 0.05) (Fig. 4). A corresponding fall in the protein content of all the muscles was observed. This was 21% (P < 0.05) in EDL, 24% (P < 0.01) in GASTROC and 9% (P > 0.05) in SOL.
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Effect of clenbuterol on dexamethasone induced muscle atrophy
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The extent of glucocorticoid induced muscle atrophy was significantly reduced in groups of mice fed clenbuterol. The difference in muscle weight was 13% (P < 0.05) and 19% (P < 0.05) in EDL (Fig. 2) and GASTROC (Fig. 3) respectively, in the group fed 4 mg/kg clenbuterol, when compared to treatment
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Fig. 2. Weight and protein content of EDL muscle in clenbuterol and dexamethasone treated male C57 BL/6J mice. The wet weight and protein content of EDL muscle from four groups of male C57 mice injected with normal saline or with dexamethasone over a period of 10 days. Details of treatment as for Fig. 1. The number of animals in each group was seven. *Statistically significantly (see text) different from the control. #Statistically significantly (see text) different from dexamethasone treatment alone.
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Fig. 1. Growth curve of clenbuterol and dexamethasone treated male C57 BL/6J mice. The mean daily body weight of four groups of male C57 mice injected with normal saline or with dexamethasone over a period of 10 days. The saline injected group was fed on the control diet. The other three groups labelled 0, 4 and 8 mg/kg were injected with a daily subcutaneous dose of 5 mg/kg of dexamethasone. The group labelled 0 mg/kg was fed the control diet and the groups labelled 4 and 8 mg/kg were fed on diets containing clenbuterol in the respective weights per kg. Number of animals in each group was seven, each point represents the mean daily body weight for each group and bars the SEM.
with dexamethasone alone. A higher dose of clenbuterol (8 mg/kg) resulted in a bigger difference in muscle weight of 20% (P c 0.01) for EDL (Fig. 2) and 22% (P < 0.01) for GASTROC (Fig. 3). In both types of muscle there were corresponding changes in protein content (Figs 2 and 3). The mean wet weight of SOL in the dexamethasone treated group fed with clenbuterol (4 mg/kg) was 20% (P < 0.05) greater than in the dexamethasone alone treated group and no different from the control group. A higher dose of clenbuterol (8 mg/kg) further increased the weight difference to 29% (P < 0.01) and the difference in protein content to 40% (P < 0.01) when compared to dexamethasone treatment alone. This concentration of clenbuterol caused hypertrophy as indicated by a 29% increase in protein content above that of the controls. Hence, in all muscles studied the higher dose of clenbuterol completely prevented the atrophy induced by dexamethasone treatment. DISCUSSlON
Daily injections of dexamethasone caused a persistent reduction in the rate of body growth of mice.
Glucocorticoid induced muscle atrophy Inclusion of clenbuterol in the diet of dexamethasone treated mice caused a transient loss of body weight, probably resulting from a reduced food intake. Whilst this was not measured here, clenbuterol added to the diet of rats has been shown by others (Reeds et al., 1986; Choo et al., 1990) to result in transient reductions in food intake. Interestingly, subcutaneous injections of clenbuterol have not been shown to reduce food intake in rats, mice or calves (Emery et cd., 1984; Rothwell and Stock, 1985; Williams et al., 1987). Failure of the clenbuterol
treated mice to attain the same body weights as control mice by the end of the study was probably partly due to a failure to compensate for the weight loss of day one. Clenbuterol was able to completely prevent the dexamethasone-induced atrophy of fast twitch muscles and to induce hypertrophy of slow twitch muscles, despite dexamethasone treatment, from hind limbs of freely moving mice. Glucocorticoids have previously been used to induce skeletal muscle atro-
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Fig. 4. Weight and protein content of soleus muscle in clenbuterol and dexamethasone treated male C57 BL/6J mice. The wet weight and protein content of soleus muscle in male C57 mice injected with normal saline or with dexamethasone over a period of 10 days. Details of treatment as for Fig. 1. The number of animals in each group was seven. Columns represents the mean of each group and the bars represent the SEM. *Statistically significantly (see text) different from dexamethasone treatment alone.
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Fig. 3. Weight and protein content of gastrocnemius muscle in clenbuterol and dexamethasone treated male C57 BL/6J mice. The wet weight and protein content of gastrocnemius muscle from four groups of male C57 mice injected with normal saline or with dexamethasone over a period of 10 days. Details of treatment as for Fig. 1. The number of animals in each group was seven. Columns represents the mean of each group and the bars represent the SEM. *Statistically significantly (see text) different from the control. #Statistically significantly (see text) different from dexamethasone treatment alone.
phy in laboratory animals (Shoji, 1975; Mayer et al., 1976; Kelly et al., 1986). The reduced body weight and muscle weight of C57 mice resulting from dexamethasone treatment in this study agree with these findings. The reduction in muscle weight was greater in fast twitch EDL and gastrocnemius than in slow twitch soleus. A greater effect of dexamethasone on fast compared to slow twitch muscles has been described by others (Kelly and Goldspink, 1982; Kelly et al., 1986; Robinson and Clamann, 1988). Most glucocorticoids possess some mineralocorticoid activity which could lead to retention of water and electrolytes by muscle. Although dexamethasone has little mineralocorticoid activity, reductions in the total muscle protein levels are a more reliable indication of muscle atrophy. In all cases the protein contents of muscles reflected changes in wet weights. Muscle atrophy could result from an increased rate of proteolysis or a decreased rate of protein synthesis. Interestingly, glucocorticoids have been shown to both increase proteolysis (Goldberg, 1969; Kayali et al., 1987; Seene et al., 1988) and to decrease protein synthesis (Kelly and Goldspink, 1982; Garlick et al., 1987). Which might be the more significant action is not clear, although Odera and Millward (1982)
144
E. T. AGBENYEGA and A. C. WAREHAM
concluded that the major effect of glucocorticoids on skeletal muscle was the reduction of protein synthesis, with a lesser effect of stimulation of protein degradation. The mechanism(s) by which clenbuterol promotes muscle growth is also unclear, but cannot be ascribed to alterations in circulating levels of the major anabolic hormones such as insulin or growth hormone (Emery et al., 1984). It was still effective in promoting muscle growth in orchidectomized and adrenalectomized rats (Rothwell and Stock, 1988). The muscle growth promoting effect of clenbuterol has been suggested to result from a reduction in proteolysis by some workers (Reeds et al., 1986; Bohorov et al., 1987; Williams et al., 1987) and from an increase in protein synthesis by others (Emery et al., 1984; Maltin et al., 1987; Pell et al., 1987). Hence, the antagonistic effects of clenbuterol and dexamethasone on skeletal muscle growth could be due to opposing effects on both synthesis and degradation of muscle proteins. Muscle wasting occurs in many diseased states, often as a result of disuse. Physical injury leads to a negative nitrogen balance (Cuthbertson, 1930) and net breakdown of muscle proteins (Frayn et al., 1980; Tischler and Fagan, 1980). Hence bone fracture (Stoner, 1976; Frayn et al., 1980) and denervation (Goldspink, 1976; Goldspink et al., 1983) are both potent stimuli of muscle protein breakdown. Glucocorticoids may be involved in the muscle wasting associated with disuse. Elevated levels of plasma glucocorticoids have been reported after limb casting (Seider et al., 1982) and hind limb suspension (Popovic et al., 1982). On the other hand, Jasper and Tischler (1986) reported that disuse atrophy of muscle still occurred in adrenalectomized rats. Other workers have, however, provided evidence to support the involvement of glucocorticoids in the process of disuse atrophy of skeletal muscles. Numbers of glucocorticoid receptor sites have been shown to increase in situations leading to atrophy, including joint immobilization (Dubois and Almon, 1980), denervation (Dubois and Almon, 1981) and limb casting (McGrath and Goldspink, 1978). With the demonstration that the effects of glucocorticoids on muscle are mediated via specific receptor sites (Snochowski et nl., 1980) it seems probable that glucocorticoids play a significant role in the process of muscle atrophy. Interestingly, clenbuterol has been shown to be particularly effective in reducing muscle atrophy associated with denervation (Maltin et al., 1987; Zeman et al., 1987; Maltin et al., 1989; Agbenyega and Wareham, 1990) and in increasing muscle mass in dystrophic mice (Rothwell and Stock, 1985). However, in the case of muscle wasting associated with femur fracture, clenbuterol was ineffective, except under conditions of food restriction (Choo et al., 1990). It may be significant that there is an increase in the number of glucocorticoid receptors in denervated muscle (Dubois and Almon, 1981). However, Nicholson et al. (1984) found that atrophy of denervated muscle preceded the increase in glucocorticoid receptor density. In addition, normal soleus muscle, which was least affected by glucocorticoid treatment in the present experiments, has been reported to have
a higher glucocorticoid receptor density than EDL (Shoji and Pennington, 1977). It is unlikely that there is a simple relationship between receptor density and glucocorticoid induced atrophy. The prevention of the catabolic effect of dexamethasone by clenbuterol may indicate a possible use for &agonists in reversing the wasting effect on skeletal muscle during clinical use of these drugs.
SUMMARY
The /I-adrenergic agonist, clenbuterol, has been shown to be effective in increasing lean body mass in many different species. In particular, it has been effective in preventing muscle wasting normally associated with denervation. Such adrenergic agonists could have potential clinical use in the treatment of muscle wasting conditions associated with old age, immobility and disease. Glucocorticoid treatment is known to be associated with suppression of growth and skeletal muscle wasting. We have designed a series of experiments to study the ability of clenbuterol to oppose the catabolic effect of the glucocorticoid dexamethasone on skeletal muscle of mice. Daily injections of dexamethasone (5 mg/kg body weight) for 10 days led to a 20% loss (P < 0.01) of muscle weight of fast twitch muscles. An apparent 12% loss in weight of slow twitch muscle did not reach the level of significance. Inclusion of clenbuterol in the diet at 4 and at 8 mg/kg for the period of dexamethasone treatment was effective in opposing this catabolic action on muscle, the higher concentration totally preventing any muscle wasting. Changes in total muscle protein paralleled changes in muscle wet weights. Hence, it can be concluded that the changes in muscle mass induced by dexamethasone and by clenbuterol were not due to changes in muscle water content.
REFERENCES Afifi A. K., Bergmann R. A. and Harvey J. 0. (1968) Steroid myopathy: clinical, histological and cytological observations. Johns Hopkins Med. J. 123, 158-173. Agbenyega E. T. and Wareham A. C. (1990) Effect of clenbuterol on normal and denervated muscle growth and contractility. Muscle Nerve 13, 199-203. Bayliss R. I. S. (1984) Corticosteroid and corticotrophin treatment in non-endocrine disease. In Oxford Textbook of Medicine (Edited by Weatherall D. J., Ledingham J. G. G. and Warrel D. A.). Oxford University Press, Oxford. Blodgett F. M., Burgin L., Iezzoni D., Gribetz D. and Talbot N. D. (1956) Effect of prolonged cortisone therapy on statural growth skeletal muscle maturation and metabolic status of children. N. Engl. J. Med. 254, 636641. Bohorov O., Buttery P. J., Correia J. H. R. D. and Soar J. B. (1987) The effect of the /3-adrenergic agonist clenbuterol on implantation with oestradiol plus trebolone acetate on protein metabolism in whether lambs. Br. J. Nutr. 57, 99-107. Choo J. J., Horan M. A., Little R. A., Rothwell N. J. and Wareham A. C. (1990) Anabolic &agonist clenbuterol fails to modify muscle atrophy due to femur fracture. Circulatory Shock 32, 16171.
Glucocorticoid
indul ted muscle atrophy
Cuthbertson D. P. (1930) The disturbance of metabolism produced by bony and non-bony injury, with notes on certain abnormal conditions of bone. Biochem. J. 24, 1244-1263. Dubois D. C. and Almon R. R. (1980) Disuse atrophy of skeletal muscle is associated with an increase in number of glucocorticoid receptors. Endocrinology 107, 1649-1651. Dubois D. C. and Almon R. R. (1981) A possible role for glucocorticoids in denervation atrophy. Muscle Nerve 4, 370-373. Emery P. W., Rothwell N. J., Stock M. J. and Winter P. O’D. (I 984) Chronic effects of the P,-adrenergic agonist on body composition and protein synthesis in the rat. Biosci. Rep. 4, 83-91. Frayn K. N., Little R. A. and Threfall C. J. (1980) Protein metabolism after unilateral femoral fracture in the rat, and comparison with sham operation. Br. J. exp. Path. 61, 474478. Garber A. J., Karl I. E. and Kipnis D. M. (1976) Alanine and glutamine synthesis and release from skeletal muscle IV. /I-Adrenergic inhibition of amino acid release. J. biol. Chem. 252, 851-857. Garlick P. J., Grant I. and Glennie R. T. (1987) Short-term effects of corticosterone treatment on muscle protein synthesis in relation to the response to feeding. Biochem. J. 248, 43942. Goldberg A. L. (1969) Protein turnover in skeletal muscle. II: Effect of denervation and cortisone on protein in skeletal muscle. J. biol. Chem. 244, 3223-3229‘. Goldsnink D. F. (1976) The effects of denervation on protein turnover of rat skeletal muscle. Biochem. J. 156, 71-80. Goldspink D. F., Garlick P. J. and McNurlan M. A. (1983) Protein turnover measured in vivo and in vitro in muscle undergoing compensatory growth and subsequent denervation hypertrophy. Biochem. J. 210, 89-98. Jasper S. R. and Tischler M. E. (1986) Role of glucocorticoids in the rat leg muscle to reduced activity. Muscle Nerve 9, 554-561. Kayali A. G., Young V. R. and Goodman M. N. (1987) Sensitivity of myofibrillar proteins to glucocorticoid induced atrophy. Am. J. Physiol. 252, E621-E626. Kelly F. J. and Goldspink D. F. (1982) The differing response of four muscle types to dexamethasone treatment in the rat. Blochem. J. 208, 147-152. Kelly F. J., McGrath J. A., Goldspink D. F. and Cullen M. J. (1986) Morphological/biochemical study on the action of corticosteroids on rat skeletal muscle. Muscle Nerve 9, I-10. Li J. B. and Jefferson L. S. (1977) Effects of isoproterenol on amino acid levels and protein turnover in skeletal muscles. Am. J. Physiol. 232, E243-E249. Loeb J. N. (1976) Corticosteroids and growth. N. Engl. J. Med. 295, 547-55 1. Mahin C. A., Delday M. I. and Reeds P. J. (1986) The effect of the growth promoting drug, clenbuterol, on fibre frequency and area in the hind limb muscles from young male rats. Biosci. Rep. 6, 293-299. Maltin C. A., Hay S. M., Delday M. I., Lobley G. E. and Reeds P. J. (1989) The action of the /?-agonist clenbuterol on protein metabolism in innervated and denervated phasic muscles. Biochem. J. 261, 965-971, Maltin C. A., Hay S. M., Delday M. I., Smith F. G., Lobley G. E. and Reeds P. J. (1987) Clenbuterol, a /?-agonist, induces growth in innervated and denervated rat soleus muscles via apparently different mechanisms. Biosci. Rep. 7, 525-531. Mayer M. and Rosen F. (1977) Interaction of glucocorticoids and androaens with skeletal muscle. Metabolism 26. 937-962. Mayer M., Shafiq E., Kaiser N., Milholland R. J. and Rosen F. (1976) Interaction of glucocorticoid hormones with rat
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skeletal muscle: catabolic effects and hormone binding. Metabolism 25, 157-167. McGrath J. A. and Goldspink D. F. (1978) The effect of cortisone on the protein turnover of the soleus muscle after immobilization. Biochem. Sot. Trans. 6, 1017-1019. Nicholson W. F., Watson P. A. and Booth F. W. (1984) Level of blood-bourne factors and cytosol glucocorticoid receptors during the initiation of muscle atrophy in rodent hindlimbs. PfGgers Arch. 401, 321-323. Odera B. R. and Millward D. J. (1982) Effect of corticosterone treatment on muscle protein turnover in adrenalectomized rats and diabetic rats maintained on insulin. Biochem. J. 204, 663472. Pell J. M., Bates P. C., Elcock C., Lane S. E. and Simmonds A. D. (1987) Growth hormone and clenbuterol: action and interaction of muscle growth, protein turnover and serum IGF 1 concentration in dwarf mice. J. Endow. 115, suppl. 68A. Popovic V., Popovic P. and Honeycut C. (1982) Hormonal changes in anti orthostatic rats. Physiologist 25, 577-578. Reeds P. J., Hay S. M., Donvard P. M. and Palmer R. M. (1986) Stimulation of muscle growth by clenbuterol: lack of effect on protein biosynthesis. Br. J. Nutr. 56, 249-256. Ricks C. A., Dalrymple R. H., Baker P. K. and Ingle D. L. (1984) Use of a p-agonist to alter fat and muscle deposition in steers. J. Anim. Sci. 59, 1247-1255. Robinson A. J. and Clamann H. P. (1988) Effects of glucocorticoids on motor units in cat hindlimb muscles. Muscle Nerve 11, 703-713. Rothwell N. J. and Stock M. J. (1985) Modification of body composition by clenbuterol in normal and dystrophic (mdx) mice. Biosci. Rep. 5, 755-760. Rothwell N. J. and Stock M. J. (1988) Increased bodyweight gain and body protein in castrated and adrenalectomised rats treated with clenbuterol. Br. J. Nutr. 60, 355-360. Sainz R. D. and Wolff J. E. (1988) Effects of the B-agonist, cimaterol, on growth, body composition and energy expenditure in rats. Br. J. Nutr. 60. 85-90. Seenk T., Umnova M ., Alex K. and Pehme A. (1988) Effects of glucocorticoids on contractile apparatus of skeletal muscle. J. Steroid Biochem. 29, 313-317. Seider M. J., Nicholson W. F. and Booth F. W. (1982) Insulin resistance for glucose metabolism in diused soleus muscle of mice. Am. J. Physiol. 242, E12-E18. Shoji S. (1975) Experimental study of steroid myopathy. Adv. Neurol. Sci. 19, 157-167. Shoji S. and Pennington R. J. T. (1977) Binding of dexamethasone and cortisol to receptors in rat extensor digitorum longus and soleus muscle. Exp. Neurol. 57, 342-348. Snochowski M. E., Dahlberg E. and Gustafsson J. A. (1980) Characteristics and quantification of the androgen and glucocorticoid receptors in the cytosol from rat skeletal muscle. Eur. J. Biochem. 111, 603406. Stoner H. B. (1976) Causative factors and efferent stimuli involved in the metabolic response to injury. In Metabolism and the Responses IO Injury (Edited by Wilkinson A. W. and Cuthbertson D. P.), pp. 204212. Pitman Medical, Tunbridge Wells. Tischler M. E. and Fagan J. M. (1980) Response to trauma of protein, amino acid, and carbohydrate metabolism in injured and uninjured rat skeletal muscles. Metabolism 32, 853-868. Williams P. E. V., Pagliani L., Innes G. M., Pennie K., Harris G. and Garthwaite P. (1987) Effects of B-agonist (clenbuterol) on the growth, carcass composition, protein and energy metabolism in veal calves. Br. J. Nutr. 57, 417428. Zeman R. J., Ludemann R. and Etlinger J. D. (1987) Clenbuterol a P,-agonist retards atrophy in denervated muscles. Am. J. Physiol. 252, E152-E155.
Physiol.
$5.00+ 0.00 0300-9629/92
Vol. IOZA,No. I, PP. 141-145,1992
0
Printedin Great Britain
1992 Pergamon Press plc
EFFECT
OF CLENBUTEROL ON SKELETAL MUSCLE ATROPHY IN MICE INDUCED BY THE GLUCOCORTICOID DEXAMETHASONE
E. T. AGBENYEGA* and A. C. WmEHAMt# tDepartment of Physiological Sciences, School of Biological Sciences, Medical School, University of Manchester, Manchester Ml3 9PT, U.K. Telephone: 061-275-5372; Fax: (061) 275-5600 (Received 9 September 1991) Abstract-l. The ability of clenbuterol to antagonize the catabolic effect of the glucocorticoid dexamethasone on the skeletal muscles, soleus, gastrocnemius and extensor digitorum longus was studied in mice. 2. Daily injections of 5 mg dexamethasone/kg body weight over 10 days caused a significant (20%) loss of muscle weight and protein content in fast twitch but not in slow twitch muscles. 3. Inclusion of clenbuterol (4mg/kg) in the diet for the period of dexamethasone treatment partly prevented glucocorticoid-induced muscle atrophy, and increasing the concentration of clenbuterol to 8 mg/kg diet totally prevented glucocorticoid-induced protein loss in all muscles.
INTRODUCTION Excessive amounts of glucocorticoids, whether produced endogenously as in Cushing’s disease, or administered exogenously in the treatment of a variety of medical conditions (Mayer and Rosen, 1977; Bayliss, 1984), are associated with the suppression of growth in the young (Blodgett et al., 1956; Loeb, 1976) and whole body atrophy in older animals (Loeb, 1976). Skeletal muscle is the tissue most affected by the catabolic action of glucocorticoids (Afifi et al., 1968; Kelly and Goldspink, 1982). Some /?,-adrenergic agonists stimulate growth and muscle protein deposition in rodents (Emery et al., 1984; Sainz and Wolff, 1988) and in farm animals (Ricks et al., 1984; Bohorov et al., 1987). The most potent of these is clenbuterol. Clenbuterol has been reported to both stimulate protein synthesis (Emery et al., 1984) and inhibit proteolysis (Garber et al., 1976; Li and Jefferson, 1977), resulting in an increase in muscle mass. In normal young rat muscle, the anabolic action of clenbuterol is expressed as fibre hypertrophy (Maltin et al., 1986) and has been attributed to a reduction in proteolysis (Reeds et al., 1986). The objective of the present study was to determine to what extent administration of clenbuterol in the diet could reduce the degree of atrophy of mouse fast and slow twitch skeletal muscle induced by daily injections of the glucocorticoid dexamethasone. MATERIALSAND
METHODS
Twenty-eight 4-5-week-old male C57 BL/6J mice were used. Animals were fed on a powdered diet (CRM LABSURE). In all experiments the &-agonist clenbuterol was administered orally, mixed with the powdered diet. *Current address: Department of Physiology, School of Medical Sciences, University of Science and Technology, Kumasi, Ghana. $To whom all correspondence should be addressed.
Control animals were fed on the normal diet. All animals were allowed free access to food and water. Muscle atrophy was induced in C57 mice by daily subcutaneous injections of dexamethasone, 5 mg/kg body weight, a dose previously demonstrated to induce muscle atrophy in rats (Kelly and Goldspink, 1982). The vehicle used in dexamethasone injections was normal saline. Four age and weight matched groups were used and given one of the following treatments: Group 1, saline injections + control diet; Group 2, dexamethasone injections + control diet; Group 3, dexamethasone injections + 4 mg/kg clenbuterol diet; Group 4, dexamethasone injections + 8 mg/kg clenbuterol diet. The animals were weighed daily. Muscle growth or atrophy was determined by comparing muscle wet weight after 10 days treatment with that of control animals. To eliminate any change in wet weight that might have been due to electrolyte and/or water retention, total muscle protein was also measured. Assay of muscles for protein content was by the Biorad dye binding method (Bio-Rad. Watford. U.K.) after solubilization in 0.3 M NaOH, with bovine serum albumin (BSA) as standard. Before analysis, muscles were stored frozen at - 18°C. Significance of differences between means were determined using the Student’s t-test for unpaired values (two-tailed probabilities). I
RESULTS
Effect of clenbuterol on dexamethasone-induced body weight
loss of
The growth of mice in the four groups, measured as change in body weight, is shown in Fig. 1. The average daily increase in mean body weight of the control mice was approximately 2%. A fall in body weight of 3.5% occurred in the first 24 hr of dexamethasone treatment alone. The mean body weight attained after 24 hr was stable for a further 6 days, followed by a daily increase of 1%. A bigger fall in mean body weight of 7% occurred in the first 24 hr of the dexamethasone group treated with 4mg/kg diet of clenbuterol. Increasing the dose of clenbuterol to 8 mg/kg caused an even greater fall of 12% after 24 hr. These decreases in body weight did not 141
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AGBENYEGA and A. C. WAREHAM
continue after the first 24 hr in the clenbuterol treated groups but were immediately followed by 1 to 2% daily increases. The final body weight after 10 days of treatment with dexamethasone + clenbuterol was significantly less (P c 0.01) than in the saline treated controls.
IJ Salins
Effect of dexamethasone on skeletal muscle weights
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90.
The fast twitch extensor digitorum longus (EDL) and gastrocnemius (GASTROC) and slow twitch soleus (SOL) muscles of the hind limb were investigated for signs of atrophy after 10 days of treatment with dexamethasone. When compared to the saline injected controls, the dexamethasone treated EDL weighed 20% less (P < 0.01) (Fig. 2) and GASTROC, 18% less (P < 0.01) (Fig. 3). The small (12%) apparent loss of weight in SOL was not statistically significant (P > 0.05) (Fig. 4). A corresponding fall in the protein content of all the muscles was observed. This was 21% (P < 0.05) in EDL, 24% (P < 0.01) in GASTROC and 9% (P > 0.05) in SOL.
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Fig. 2. Weight and protein content of EDL muscle in clenbuterol and dexamethasone treated male C57 BL/6J mice. The wet weight and protein content of EDL muscle from four groups of male C57 mice injected with normal saline or with dexamethasone over a period of 10 days. Details of treatment as for Fig. 1. The number of animals in each group was seven. *Statistically significantly (see text) different from the control. #Statistically significantly (see text) different from dexamethasone treatment alone.
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Fig. 1. Growth curve of clenbuterol and dexamethasone treated male C57 BL/6J mice. The mean daily body weight of four groups of male C57 mice injected with normal saline or with dexamethasone over a period of 10 days. The saline injected group was fed on the control diet. The other three groups labelled 0, 4 and 8 mg/kg were injected with a daily subcutaneous dose of 5 mg/kg of dexamethasone. The group labelled 0 mg/kg was fed the control diet and the groups labelled 4 and 8 mg/kg were fed on diets containing clenbuterol in the respective weights per kg. Number of animals in each group was seven, each point represents the mean daily body weight for each group and bars the SEM.
with dexamethasone alone. A higher dose of clenbuterol (8 mg/kg) resulted in a bigger difference in muscle weight of 20% (P c 0.01) for EDL (Fig. 2) and 22% (P < 0.01) for GASTROC (Fig. 3). In both types of muscle there were corresponding changes in protein content (Figs 2 and 3). The mean wet weight of SOL in the dexamethasone treated group fed with clenbuterol (4 mg/kg) was 20% (P < 0.05) greater than in the dexamethasone alone treated group and no different from the control group. A higher dose of clenbuterol (8 mg/kg) further increased the weight difference to 29% (P < 0.01) and the difference in protein content to 40% (P < 0.01) when compared to dexamethasone treatment alone. This concentration of clenbuterol caused hypertrophy as indicated by a 29% increase in protein content above that of the controls. Hence, in all muscles studied the higher dose of clenbuterol completely prevented the atrophy induced by dexamethasone treatment. DISCUSSlON
Daily injections of dexamethasone caused a persistent reduction in the rate of body growth of mice.
Glucocorticoid induced muscle atrophy Inclusion of clenbuterol in the diet of dexamethasone treated mice caused a transient loss of body weight, probably resulting from a reduced food intake. Whilst this was not measured here, clenbuterol added to the diet of rats has been shown by others (Reeds et al., 1986; Choo et al., 1990) to result in transient reductions in food intake. Interestingly, subcutaneous injections of clenbuterol have not been shown to reduce food intake in rats, mice or calves (Emery et cd., 1984; Rothwell and Stock, 1985; Williams et al., 1987). Failure of the clenbuterol
treated mice to attain the same body weights as control mice by the end of the study was probably partly due to a failure to compensate for the weight loss of day one. Clenbuterol was able to completely prevent the dexamethasone-induced atrophy of fast twitch muscles and to induce hypertrophy of slow twitch muscles, despite dexamethasone treatment, from hind limbs of freely moving mice. Glucocorticoids have previously been used to induce skeletal muscle atro-
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Fig. 4. Weight and protein content of soleus muscle in clenbuterol and dexamethasone treated male C57 BL/6J mice. The wet weight and protein content of soleus muscle in male C57 mice injected with normal saline or with dexamethasone over a period of 10 days. Details of treatment as for Fig. 1. The number of animals in each group was seven. Columns represents the mean of each group and the bars represent the SEM. *Statistically significantly (see text) different from dexamethasone treatment alone.
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Fig. 3. Weight and protein content of gastrocnemius muscle in clenbuterol and dexamethasone treated male C57 BL/6J mice. The wet weight and protein content of gastrocnemius muscle from four groups of male C57 mice injected with normal saline or with dexamethasone over a period of 10 days. Details of treatment as for Fig. 1. The number of animals in each group was seven. Columns represents the mean of each group and the bars represent the SEM. *Statistically significantly (see text) different from the control. #Statistically significantly (see text) different from dexamethasone treatment alone.
phy in laboratory animals (Shoji, 1975; Mayer et al., 1976; Kelly et al., 1986). The reduced body weight and muscle weight of C57 mice resulting from dexamethasone treatment in this study agree with these findings. The reduction in muscle weight was greater in fast twitch EDL and gastrocnemius than in slow twitch soleus. A greater effect of dexamethasone on fast compared to slow twitch muscles has been described by others (Kelly and Goldspink, 1982; Kelly et al., 1986; Robinson and Clamann, 1988). Most glucocorticoids possess some mineralocorticoid activity which could lead to retention of water and electrolytes by muscle. Although dexamethasone has little mineralocorticoid activity, reductions in the total muscle protein levels are a more reliable indication of muscle atrophy. In all cases the protein contents of muscles reflected changes in wet weights. Muscle atrophy could result from an increased rate of proteolysis or a decreased rate of protein synthesis. Interestingly, glucocorticoids have been shown to both increase proteolysis (Goldberg, 1969; Kayali et al., 1987; Seene et al., 1988) and to decrease protein synthesis (Kelly and Goldspink, 1982; Garlick et al., 1987). Which might be the more significant action is not clear, although Odera and Millward (1982)
144
E. T. AGBENYEGA and A. C. WAREHAM
concluded that the major effect of glucocorticoids on skeletal muscle was the reduction of protein synthesis, with a lesser effect of stimulation of protein degradation. The mechanism(s) by which clenbuterol promotes muscle growth is also unclear, but cannot be ascribed to alterations in circulating levels of the major anabolic hormones such as insulin or growth hormone (Emery et al., 1984). It was still effective in promoting muscle growth in orchidectomized and adrenalectomized rats (Rothwell and Stock, 1988). The muscle growth promoting effect of clenbuterol has been suggested to result from a reduction in proteolysis by some workers (Reeds et al., 1986; Bohorov et al., 1987; Williams et al., 1987) and from an increase in protein synthesis by others (Emery et al., 1984; Maltin et al., 1987; Pell et al., 1987). Hence, the antagonistic effects of clenbuterol and dexamethasone on skeletal muscle growth could be due to opposing effects on both synthesis and degradation of muscle proteins. Muscle wasting occurs in many diseased states, often as a result of disuse. Physical injury leads to a negative nitrogen balance (Cuthbertson, 1930) and net breakdown of muscle proteins (Frayn et al., 1980; Tischler and Fagan, 1980). Hence bone fracture (Stoner, 1976; Frayn et al., 1980) and denervation (Goldspink, 1976; Goldspink et al., 1983) are both potent stimuli of muscle protein breakdown. Glucocorticoids may be involved in the muscle wasting associated with disuse. Elevated levels of plasma glucocorticoids have been reported after limb casting (Seider et al., 1982) and hind limb suspension (Popovic et al., 1982). On the other hand, Jasper and Tischler (1986) reported that disuse atrophy of muscle still occurred in adrenalectomized rats. Other workers have, however, provided evidence to support the involvement of glucocorticoids in the process of disuse atrophy of skeletal muscles. Numbers of glucocorticoid receptor sites have been shown to increase in situations leading to atrophy, including joint immobilization (Dubois and Almon, 1980), denervation (Dubois and Almon, 1981) and limb casting (McGrath and Goldspink, 1978). With the demonstration that the effects of glucocorticoids on muscle are mediated via specific receptor sites (Snochowski et nl., 1980) it seems probable that glucocorticoids play a significant role in the process of muscle atrophy. Interestingly, clenbuterol has been shown to be particularly effective in reducing muscle atrophy associated with denervation (Maltin et al., 1987; Zeman et al., 1987; Maltin et al., 1989; Agbenyega and Wareham, 1990) and in increasing muscle mass in dystrophic mice (Rothwell and Stock, 1985). However, in the case of muscle wasting associated with femur fracture, clenbuterol was ineffective, except under conditions of food restriction (Choo et al., 1990). It may be significant that there is an increase in the number of glucocorticoid receptors in denervated muscle (Dubois and Almon, 1981). However, Nicholson et al. (1984) found that atrophy of denervated muscle preceded the increase in glucocorticoid receptor density. In addition, normal soleus muscle, which was least affected by glucocorticoid treatment in the present experiments, has been reported to have
a higher glucocorticoid receptor density than EDL (Shoji and Pennington, 1977). It is unlikely that there is a simple relationship between receptor density and glucocorticoid induced atrophy. The prevention of the catabolic effect of dexamethasone by clenbuterol may indicate a possible use for &agonists in reversing the wasting effect on skeletal muscle during clinical use of these drugs.
SUMMARY
The /I-adrenergic agonist, clenbuterol, has been shown to be effective in increasing lean body mass in many different species. In particular, it has been effective in preventing muscle wasting normally associated with denervation. Such adrenergic agonists could have potential clinical use in the treatment of muscle wasting conditions associated with old age, immobility and disease. Glucocorticoid treatment is known to be associated with suppression of growth and skeletal muscle wasting. We have designed a series of experiments to study the ability of clenbuterol to oppose the catabolic effect of the glucocorticoid dexamethasone on skeletal muscle of mice. Daily injections of dexamethasone (5 mg/kg body weight) for 10 days led to a 20% loss (P < 0.01) of muscle weight of fast twitch muscles. An apparent 12% loss in weight of slow twitch muscle did not reach the level of significance. Inclusion of clenbuterol in the diet at 4 and at 8 mg/kg for the period of dexamethasone treatment was effective in opposing this catabolic action on muscle, the higher concentration totally preventing any muscle wasting. Changes in total muscle protein paralleled changes in muscle wet weights. Hence, it can be concluded that the changes in muscle mass induced by dexamethasone and by clenbuterol were not due to changes in muscle water content.
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