SciELO - Scientific Electronic Library Online

 
vol.41 issue12Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutansTopographic distribution of the tibial somatosensory evoked potential using coherence author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand

Journal

Article

Indicators

Related links

Share


Brazilian Journal of Medical and Biological Research

Print version ISSN 0100-879XOn-line version ISSN 1414-431X

Braz J Med Biol Res vol.41 no.12 Ribeirão Preto Dec. 2008

https://doi.org/10.1590/S0100-879X2008001200003 

Braz J Med Biol Res, December 2008, Volume 41(12) 1054-1058

The effect of a low dose of clenbuterol on rat soleus muscle submitted to joint immobilization

K.M. Cancelliero1, J.L.Q. Durigan1, R.P. Vieira2, C.A. Silva3 and M.L.O. Polacow3

1Departamento de Fisioterapia, Universidade Federal de São Carlos, São Carlos, SP, Brasil
2Departamento de Fisioterapia (LIM 34) e Patologia (LIM 05), Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brasil
3Departamento de Fisioterapia, Universidade Metodista de Piracicaba, Piracicaba, SP, Brasil

Abstract
Introduction
Material and Methods
Results
Discussion
References
Correspondence and Footnotes


Abstract

The aim of the present study was to evaluate the effect of joint immobilization on morphometric parameters and glycogen content of soleus muscle treated with clenbuterol. Male Wistar (3-4 months old) rats were divided into 4 groups (N = 6 for each group): control, clenbuterol, immobilized, and immobilized treated with clenbuterol. Immobilization was performed with acrylic resin orthoses and 10 µg/kg body weight clenbuterol was administered subcutaneously for 7 days. The following parameters were measured the next day on soleus muscle: weight, glycogen content, cross-sectional area, and connective tissue content. The clenbuterol group showed an increase in glycogen (81.6%, 0.38 ± 0.09 vs 0.69 ± 0.06 mg/100 g; P < 0.05) without alteration in weight, cross-sectional area or connective tissue compared with the control group. The immobilized group showed a reduction in muscle weight (34.2%, 123.5 ± 5.3 vs 81.3 ± 4.6 mg; P < 0.05), glycogen content (31.6%, 0.38 ± 0.09 vs 0.26 ± 0.05 mg/100 mg; P < 0.05) and cross-sectional area (44.1%, 2574.9 ± 560.2 vs 1438.1 ± 352.2 µm2; P < 0.05) and an increase in connective tissue (216.5%, 8.82 ± 3.55 vs 27.92 ± 5.36%; P < 0.05). However, the immobilized + clenbuterol group showed an increase in weight (15.9%; 81.3 ± 4.6 vs 94.2 ± 4.3 mg; P < 0.05), glycogen content (92.3%, 0.26 ± 0.05 vs 0.50 ± 0.17 mg/100 mg; P < 0.05), and cross-sectional area (19.9%, 1438.1 ± 352.2 vs 1724.8 ± 365.5 µm2; P < 0.05) and a reduction in connective tissue (52.2%, 27.92 ± 5.36 vs 13.34 ± 6.86%; P < 0.05). Statistical analysis was performed using Kolmogorov-Smirnov and homoscedasticity tests. For the muscle weight and muscle glycogen content, two-way ANOVA and the Tukey test were used. For the cross-sectional area and connective tissue content, Kruskal-Wallis and Tukey tests were used. This study emphasizes the importance of anabolic pharmacological protection during immobilization to minimize skeletal muscle alterations resulting from disuse.

Key words: Immobilization; Clenbuterol; Connective tissue; Glycogen content; Morphometry; Rat soleus muscle


Introduction

Skeletal muscle function depends of many factors, such as intact proprioceptive activity and innervation, mechanical load, and joint mobility (1-3). In many adverse situations, such as injury of skeletal muscle, bone and ligaments, immobilization is the most common method of treatment (1-3). However, one of its most preeminent effects is muscular atrophy (1-3). Several experimental models have been proposed to identify the events initiated by muscle disuse and the onset of precursors of atrophy, which include denervation, tenotomy, prolonged bed rest, hindlimb suspension, or unilateral immobilization (4). Muscular disuse induces insulin resistance and a catabolic state in human skeletal muscles (5) associated with a decrease in GLUT4 transporters (6). Although it is frequently necessary, immobilization is accompanied by side effects, such as muscular cell atrophy, intramuscular fibrosis, loss of muscle extensibility, and limitations in joint motion (7). Several studies have demonstrated that concomitant with muscular hypotrophy, great modifications occur in skeletal muscle homeostasis, affecting myofibrillar protein synthesis, contractile dynamics as well as the effectiveness of metabolic signals, that can lead to a reduction in glycogen reserves, force and fatigue resistance (4,8,9).

Clenbuterol is a β2-adrenoceptor agonist used as a bronchodilator for treating lung diseases (10). It also has an anabolic muscular effect and lipolytic activity (11). Low doses of clenbuterol (10 µg/kg) minimize muscular atrophy in rats, which is partially associated with denervation (12). Other studies on rats also reported that low doses of clenbuterol can cause anabolic effects without myocyte death (13,14). Clenbuterol also presents anabolic effects on the skeletal muscle of non-immobilized mice (15).

Therefore, the objective of the present study was to evaluate the effect of low doses of clenbuterol on morphometric and metabolic parameters of rat soleus muscles submitted to joint immobilization for 7 days.


Material and Methods

All experimental procedures were approved by the Ethics Committee for Experimental Research of the University Federal de São Carlos (#03/06).

Male Wistar rats (3-4 months old; 250-300 g) were maintained under controlled conditions of temperature and humidity, with free access to food and water. The animals were divided into 4 groups (N = 6 for each group): control, non-immobilized clenbuterol, immobilized, and immobilized treated with clenbuterol for 7 days.

The rats were anesthetized (intraperitoneal injection of sodium pentobarbital, 50 mg/kg body weight) and the left hindlimb was immobilized in neutral ankle position with acrylic resin orthoses (8). Clenbuterol (10 µg/kg body weight) was administered subcutaneously daily at 9:00 am (6). After 7 days, the animals were killed and the soleus muscle was isolated and weighed by analytic scale and divided for analysis of glycogen content, cross-sectional area and connective tissue content.

The muscle extremities were used for analysis of glycogen content. Samples were digested in KOH, the glycogen was precipitated with ethanol, centrifuged and submitted to acid hydrolysis in the presence of phenol and measured according to Lo et al. (16).

The soleus belly was processed in paraffin and 5 non-serial 7-µm thick cross-sections were obtained and stained with hematoxylin-eosin for morphometric analysis. Image analysis system was carried out with the Image Pro-plus 4.0 software (Media Cybernects, USA), a digital camera (JVC, USA) coupled to a microscope (Zeiss, Germany), and connected to a computer. All images were captured at 100X magnification and analyzed as described (8). Cross-sectional area measurements were made on 375 myofibers from 5 fields on each of the 5 muscle sections. A planimetry system was used for intramuscular connective tissue analysis, by the method of Weibel (17) and the density (%) was calculated.

The statistical analysis was performed by Kolmogorov-Smirnov and homoscedasticity tests. For the muscle weight and muscle glycogen content, two-way ANOVA (factors: immobilization x treatment) and the Tukey test were used. For the cross-sectional area and connective tissue content, Kruskal-Wallis and Tukey tests were used (P < 0.05).


Results

The clenbuterol-treated group presented higher soleus glycogen (81.6%, P < 0.05) without alterations in weight, cross-sectional area and connective tissue compared with the control group (Table 1, Figure 1A and B).

Rats immobilized for 7 days had 34.2% less soleus muscle weight and 31.6% less glycogen content (Table 1) compared with the control group (P < 0.05). The cross-sectional area of soleus muscle was 44.1% less and there was 216.5% more connective tissue (from 8.82 ± 3.55 to 27.92 ± 5.36%) compared with the control group (P < 0.05, Table 1, Figure 1C).

When the immobilized group was compared with the immobilized + clenbuterol group, it is clear that the clenbuterol-treated rats had higher muscle weight (15.9%, from 81.3 ± 4.6 to 94.2 ± 4.3 mg), and glycogen content (92.3%, from 0.26 ± 0.05 to 0.50 ± 0.17 mg/100 mg; P < 0.05; Table 1).

When the clenbuterol group was compared with the immobilized + clenbuterol group, greater cross-sectional area (19.9%, P < 0.05) and less connective tissue (52.2%, P < 0.05) were observed with clenbuterol (Table 1; Figure 1B and D). Thus, although the clenbuterol treatment increased the cross-sectional area in immobilized animals, it did not revert completely the decrease caused by immobilization, with a significant difference between these groups (P < 0.05). Clenbuterol treatment in the immobilized group inhibited connective tissue accumulation compared with non-clenbuterol-treated animals (P < 0.05, Figure 1D); however, it did not inhibit completely the connective tissue accumulation at control levels.

When the control group was compared with immobilized + clenbuterol (10 µg/kg), no difference in glycogen content was found; however, there was a decrease of 23.7% (P < 0.05) in weight, this value being lower than that of the immobilized group. The cross-sectional area and connective tissue also presented significant differences (P < 0.05, Figure 1A and D).


Figure 1. Soleus muscle fibers stained with hematoxylin and eosin. A, Control group. B, Clenbuterol treatment for 7 days. C, Immobilized in ankle neutral position for 7 days. A reduction of muscle fiber area (asterisk) and an increase of intramuscular connective tissue (arrow) can be seen compared with the control group. D, Immobilized and clenbuterol treatment for 7 days. An increase of muscle fiber area (asterisk) and a decrease of intramuscular connective tissue (arrow) can be seen compared with the immobilized group. Original magnification: 100X.

[View larger version of this image (209 K JPG file)]


Table 1. Effect of clenbuterol on atrophy caused by immobilization of soleus muscle in the rat.

[View larger version of this table (88 K JPG file)]


Discussion

As far as we know, this is the first study that analyzed the effects of a low dose of clenbuterol in a disuse muscle model. Data analysis revealed that clenbuterol prevents muscle loss in soleus muscle, including muscle weight, muscle glycogen content and cross-sectional area of fibers, which could be beneficial in cachexia and disuse conditions.

Selective agonists of β-adrenoceptors, such as clenbuterol, may counter hypotrophy by disuse because of their anabolic effects. However, the mechanisms by which β-adrenergic agents induce anabolism are still not fully understood (18,19). Fitton et al. (6) reported that low doses of clenbuterol have a protective effect on denervated muscle before its reinnervation, reducing protein loss and muscular fiber atrophy and partially preserving contractile performance.

The results of the present study demonstrated the anabolic effects of low doses of clenbuterol under conditions of disuse, i.e., prevented loss of soleus weight and cross-sectional area. This correlates with the Canu et al. study (20), in which the anabolic action of clenbuterol was observed in both normal and atrophied muscles. Pellegrino et al. (21) also observed increased cross-sectional area of mouse soleus muscle in the presence of clenbuterol.

Akatsu et al. (18) reported that regulatory mechanisms for skeletal muscle hypertrophy by clenbuterol are unclear; however, they suggest that transforming growth factor β could be involved. Matsumoto et al. (22) suggested that one hypothesis to explain the skeletal muscle hypertrophy induced by clenbuterol is the stimulated production of peptide growth factors such as insulin-like growth factors. However, these studies differ in regard to doses, including low and large doses. Chen and Alway (13) observed that the administration of clenbuterol at 10 µg/kg body weight reduced atrophy in the slow muscle of senescent rats. Burniston et al. (14) observed that 1 mg/kg clenbuterol increased the protein content associated with myofiber hypertrophy.

Despite glycogen utilization, the immobilized group presented glycogen reduction. Bevan (23) showed that insulin acts in different ways, and that there is participation by subunit PI3-K (phosphatidylinositol 3-kinase), which is responsible for several mechanisms including glycogen synthesis. Muscular disuse induces insulin resistance and a catabolic state in human skeletal muscles (5). In rat hindlimbs immobilized for 7 days, Hirose et al. (24) observed a reduction in intracellular signaling stimulated by insulin and in glucose uptake, suggesting deficit in PI3-K activation, which could explain the results of decrease glycogen in this study; this being one of the mechanisms activated by PI3-K subunit. In the present study, glycogen was increased in clenbuterol and immobilized + clenbuterol groups, which could be explained by clenbuterol causing an increase in tissue glucose uptake, possibly because of the permissive effect of insulin action (25,26). Pan et al. (26) observed that the increased glucose uptake stimulated by clenbuterol was parallel to the increase in glycogen reserves. According to Hunt et al. (27), the possibility of increased insulin sensitivity in the skeletal muscle after clenbuterol treatment could be due to the reduced influence of epinephrine on insulin action as a result of down-regulation of β-adrenergic receptors.

The connective tissue area density values were lower in the immobilized + clenbuterol group compared with the immobilized group. Because clenbuterol has an anabolic action, an increase in protein synthesis of the extracellular matrix was also expected (28). As this was not observed, it may be that differences in rate of renewal among intramuscular proteins and extracellular matrix might have influenced the results. Because collagen proteins have a slower rate of renewal than muscular tissue (29), clenbuterol did not increase the cross-sectional area (19.9%) as intensely as it decreased the connective tissue (52.2%) in the immobilized + clenbuterol group. This finding is related to a study by Jiang et al. (30), in which the authors observed that clenbuterol (60 µg for more than 3 months) minimized collagen proliferation in denervated skeletal muscle of humans. An important fact in the present study is that the clenbuterol treatment for 7 days led to a decrease in connective tissue using a dose six times smaller than the dose used by Jiang et al. (30).

In contrast, in the study of Patiyal and Katoch (31), high doses of clenbuterol (2 mg/kg) caused collagen content proliferation in the mouse gastrocnemius due to the anabolic effect on protein of clenbuterol in the skeletal muscle. In the Burniston study (14), there was myocyte death and collagen increase in myocardium (100 µg/kg or 1 mg/kg). Along these lines, collagen proliferation seems to be related to the dose administered since the present study demonstrated that low doses of clenbuterol for a short period induced connective tissue reduction in soleus muscle during 7 days of joint immobilization.

In conclusion, we found that 10 µg/kg clenbuterol, sc, for a short period of time was efficient in minimizing reduction of muscle weight, fiber area and glycogen content during joint immobilization. The extrapolation of our results to clinical practice would suggest that low doses of clenbuterol could be useful during muscle disuse periods, i.e., in joint immobilization before rehabilitation.


References

1. Appell HJ. Morphology of immobilized skeletal muscle and the effects of a pre- and postimmobilization training program. Int J Sports Med 1986; 7: 6-12.         [ Links ]

2. Degens H. Age-related skeletal muscle dysfunction: causes and mechanisms. J Musculoskelet Neuronal Interact 2007; 7: 246-252.         [ Links ]

3. Mattiello-Sverzut AC, Carvalho LC, Cornachione A, Nagashima M, Neder L, Shimano AC. Morphological effects of electrical stimulation and intermittent muscle stretch after immobilization in soleus muscle. Histol Histopathol 2006; 21: 957-964.         [ Links ]

4. Reardon KA, Davis J, Kapsa RM, Choong P, Byrne E. Myostatin, insulin-like growth factor-1, and leukemia inhibitory factor mRNAs are upregulated in chronic human disuse muscle atrophy. Muscle Nerve 2001; 24: 893-899.         [ Links ]

5. Ferrando AA, Lane HW, Stuart CA, Davis-Street J, Wolfe RR. Prolonged bed rest decreases skeletal muscle and whole body protein synthesis. Am J Physiol 1996; 270: E627-E633.         [ Links ]

6. Fitton AR, Berry MS, McGregor AD. Preservation of denervated muscle form and function by clenbuterol in a rat model of peripheral nerve injury. J Hand Surg 2001; 26: 335-346.         [ Links ]

7. Kannus P, Jozsa L, Jarvinen TL, Kvist M, Vieno T, Jarvinen TA, et al. Free mobilization and low- to high-intensity exercise in immobilization-induced muscle atrophy. J Appl Physiol 1998; 84: 1418-1424.         [ Links ]

8. da Silva CA, Guirro RR, Polacow ML, Cancelliero KM, Durigan JL. Rat hindlimb joint immobilization with acrylic resin orthoses. Braz J Med Biol Res 2006; 39: 979-985.         [ Links ]

9. Jozsa L, Kannus P, Thoring J, Reffy A, Jarvinen M, Kvist M. The effect of tenotomy and immobilisation on intramuscular connective tissue. A morphometric and microscopic study in rat calf muscles. J Bone Joint Surg Br 1990; 72: 293-297.         [ Links ]

10. Ng GY, Ohlsson A. Bronchodilators for the prevention and treatment of chronic lung disease in preterm infants. Cochrane Database Syst Rev 2001; CD003214.         [ Links ]

11. Duncan ND, Williams DA, Lynch GS. Deleterious effects of chronic clenbuterol treatment on endurance and sprint exercise performance in rats. Clin Sci 2000; 98: 339-347.         [ Links ]

12. Maltin CA, Delday MI, Hay SM, Baillie AG. Denervation increases clenbuterol sensitivity in muscle from young rats. Muscle Nerve 1992; 15: 188-192.         [ Links ]

13. Chen KD, Alway SE. A physiological level of clenbuterol does not prevent atrophy or loss of force in skeletal muscle of old rats. J Appl Physiol 2000; 89: 606-612.         [ Links ]

14. Burniston JG, Clark WA, Tan LB, Goldspink DF. Dose-dependent separation of the hypertrophic and myotoxic effects of the beta(2)-adrenergic receptor agonist clenbuterol in rat striated muscles. Muscle Nerve 2006; 33: 655-663.         [ Links ]

15. Spurlock DM, McDaneld TG, McIntyre LM. Changes in skeletal muscle gene expression following clenbuterol administration. BMC Genomics 2006; 7: 320.         [ Links ]

16. Lo S, Russell JC, Taylor AW. Determination of glycogen in small tissue samples. J Appl Physiol 1970; 28: 234-236.         [ Links ]

17. Mathieu O, Cruz-Orive LM, Hoppeler H, Weibel ER. Measuring error and sampling variation in stereology: comparison of the efficiency of various methods for planar image analysis. J Microsc 1981; 121: 75-88.         [ Links ]

18. Akutsu S, Shimada A, Yamane A. Transforming growth factor betas are upregulated in the rat masseter muscle hypertrophied by clenbuterol, a beta2 adrenergic agonist. Br J Pharmacol 2006; 147: 412-421.         [ Links ]

19. von Deutsch DA, Abukhalaf IK, Wineski LE, Silvestrov NA, Bayorh MA, Potter DE. Changes in muscle proteins and spermidine content in response to unloading and clenbuterol treatment. Can J Physiol Pharmacol 2003; 81: 28-39.         [ Links ]

20. Canu M, Stevens L, Ricart-Firinga C, Picquet F, Falempin M. Effect of the beta(2)-agonist clenbuterol on the locomotor activity of rat submitted to a 14-day period of hypodynamia-hypokinesia. Behav Brain Res 2001; 122: 103-112.         [ Links ]

21. Pellegrino MA, D'Antona G, Bortolotto S, Boschi F, Pastoris O, Bottinelli R, et al. Clenbuterol antagonizes glucocorticoid-induced atrophy and fibre type transformation in mice. Exp Physiol 2004; 89: 89-100.         [ Links ]

22. Matsumoto T, Akutsu S, Wakana N, Morito M, Shimada A, Yamane A. The expressions of insulin-like growth factors, their receptors, and binding proteins are related to the mechanism regulating masseter muscle mass in the rat. Arch Oral Biol 2006; 51: 603-611.         [ Links ]

23. Bevan P. Insulin signalling. J Cell Sci 2001; 114: 1429-1430.         [ Links ]

24. Hirose M, Kaneki M, Sugita H, Yasuhara S, Martyn JA. Immobilization depresses insulin signaling in skeletal muscle. Am J Physiol Endocrinol Metab 2000; 279: E1235-E1241.         [ Links ]

25. Torgan CE, Etgen GJ Jr, Kang HY, Ivy JL. Fiber type-specific effects of clenbuterol and exercise training on insulin-resistant muscle. J Appl Physiol 1995; 79: 163-167.         [ Links ]

26. Pan SJ, Hancock J, Ding Z, Fogt D, Lee M, Ivy JL. Effects of clenbuterol on insulin resistance in conscious obese Zucker rats. Am J Physiol Endocrinol Metab 2001; 280: E554-E561.         [ Links ]

27. Hunt DG, Ding Z, Ivy JL. Clenbuterol prevents epinephrine from antagonizing insulin-stimulated muscle glucose uptake. J Appl Physiol 2002; 92: 1285-1292.         [ Links ]

28. Kumar R, Sharma S. Remodeling of extracellular matrix protein, collagen by beta-adrenoceptor stimulation and denervation in mouse gastrocnemius muscle. J Physiol Sci 2006; 56: 87-94.         [ Links ]

29. Reeds PJ, Palmer RM, Smith RH. Protein and collagen synthesis in rat diaphragm muscle incubated in vitro: the effect of alterations in tension produced by electrical or mechanical means. Int J Biochem 1980; 11: 7-14.         [ Links ]

30. Jiang G, Gu Y, Zhang L. The effects of clenbuterol on intramuscular collagen metabolism in denervated muscle. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 1998; 12: 48-51.         [ Links ]

31. Patiyal SN, Katoch SS. Tissue specific and variable collagen proliferation in Swiss albino mice treated with clenbuterol. Physiol Res 2006; 55: 97-103.         [ Links ]


Correspondence and Footnotes

Address for correspondence: R.P. Vieira, Rua Francisco Pereira Filho, 80, Apto. 33, 12220-450 São José dos Campos, SP, Brasil. Fax: +55-12-3921-1505. E-mail: rodrelena@yahoo.com.br

R.P. Vieira is the recipient of a post-doctoral fellowship from FAPESP. Received March 4, 2008. Accepted December 1, 2008.

Creative Commons License All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution License