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00005768-200202000-0001400005768_2002_34_267_kitaura_longitudinal_2article< 100_0_11_11 >Medicine and Science in Sports and Exercise© 2002 Lippincott Williams & Wilkins, Inc.Volume 34(2)February 2002pp 267-273Inhibited longitudinal growth of bones in young male rats by clenbuterol[BASIC SCIENCES: Original Investigations]KITAURA, TAKASHI; TSUNEKAWA, NAOKO; KRAEMER, WILLIAM J.Faculty of Pharmaceutical Sciences, University of Kanazawa, Kakuma, Kanazawa 920-1192, JAPAN; and The Human Performance Laboratory, The University of Connecticut, Storrs, CT 06269-1110Submitted for publication March 2001.Accepted for publication June 2001.AbstractKITAURA, T., N. TSUNEKAWA, and W. J. KRAEMER. Inhibited longitudinal growth of bones in young male rats by clenbuterol. Med. Sci. Sports Exerc., Vol. 34, No. 2, pp. 267–273, 2002.Purpose: Clenbuterol is one of the beta-2 adrenergic receptor agonists with potent anabolic properties in muscles, yet the concomitant effects on muscle and bone in young animals remain to be resolved. Therefore, the purpose of this study was to determine the effects of clenbuterol administration on muscles and bones of young rats.Methods: Twelve male Sprague-Dawley rats (9-wk-old) were randomly assigned to either a control (CON, N = 6) or clenbuterol group (CLE, N = 6). Clenbuterol of 2 mg·kg body wt·d−1 was administered subcutaneously for 4 wk. After treatment, the soleus (SOL), extensor digitorum longus (EDL), and ventricle (VENT) muscles and the femurs (FE) and tibiae (TI) bones were excised and analyzed. The bone mineral content (BMC), area, and bone mineral density (BMD) of FE and TI were measured by dual-energy x-ray absorptiometry (DXA). The longitudinal lengths of bones were measured with the Vernier calipers.Results: CLE showed smaller body weight than CON (P < 0.05) after the treatment. The muscle wet weights in CLE tended (P = 0.08) to be higher than CON in SOL (9%) and EDL (12%), but the ratio of muscle wet weight-to-body weight were higher (SOL:P < 0.05, EDL:P < 0.01) than CON. VENT of CLE showed increases in both the wet weight and the ratio (P < 0.01). FEs in CLE showed smaller values in BMC (P < 0.01), area (P < 0.01), and length (P < 0.05) than CON but not in BMD. TIs showed significant decreases (P < 0.01) in BMC, area, and length but not in BMD.Conclusion: These results indicated that clenbuterol induced the muscular hypertrophy but inhibited the longitudinal growth of bones in young male rats, which may be a serious concern in any ergogenic use.Selective beta-2 adrenergic receptor agonists, for example, clenbuterol, are often used as bronchodialators in the treatment of asthma. However, clenbuterol also has potent anabolic properties. Therefore, it has been postulated as a possible drug for muscle atrophy consequent to muscle disease or microgravity (1,35), as well as a candidate for countermeasures of osteoporosis or cancellous osteopenia (35). Although its use is banned as a drug in many athletic organizations (25,34), the details of its diverse effects remain to be elucidated, particularly the effects on the bone, which are essentially unknown.Many investigators have studied the anabolic effects of the clenbuterol on skeletal and cardiac muscles, which still remain equivocal in many cases (13,22,33). The various treatment effects may be due to the different experimental conditions like species, dose, sex, ages, and so on (6,19,21,25). Furthermore, the interrelationships among various organs are very complicated (24), and more information is needed to clarify the various operational mechanisms. It has been shown that beta receptor agonists like epinephrine, norepinephrine, or isoproterenol can increase adenylate cyclase activity or intracellular adenosine-3′, 5′-cyclic monophosphate (cAMP) contents in either developing palate or in cloned bone cell lines MC3T3-E1 (16) or UMR-106 (14,31). It has also been suggested that the cAMP concentrations are very important for the modeling or remodeling of bones (1). However, the mechanism of effects on bone growth and metabolism are not well explained.Recently, a few new phosphodiesterase-4 (PDE-4) inhibitors, which will increase intracellular cAMP concentrations, have been developed as candidates for countermeasures of osteoporosis or cancellous osteopenia (20). Clenbuterol can also increase the intracellular cAMP concentrations via the beta-2 adrenergic receptor (17,21,34). There are also a few reports that beta-2 receptors are located in the osteoblast (14,29). Zeman et al. (35) showed that clenbuterol reduced net bone loss in denervated hindlimbs. However, the effects of clenbuterol on bone are not understood as well as in muscles. Therefore, we hypothesized that clenbuterol may increase the growth of bones as well as muscles, and we examined the effects of clenbuterol on muscle weights, bone size, bone mineral content (BMC), and bone mineral density (BMD) in young (9- to 13-wk-old) male Sprague-Dawley rats.METHODSAnimal care and drug treatment.Twelve male Sprague-Dawley rats (8-wk-old) were fed rat chow and water ad libitum and were maintained on a 12:12-h light-dark photoperiod. At the beginning, their body weights ranged from 225 to 240 g. Two animals were housed per cage (25 × 40 × 20 cm). At 9 wk of age, they were randomly assigned to either a treatment with saline (group CON, N = 6) or clenbuterol (group CLE, N = 6) for 4 wk. Clenbuterol (Sigma Chemical, St. Louis, MO) 2 mg·kg body weight·d−1 was administered subcutaneously once daily. Control rats were injected with 0.5 mL·kg−1 body weight normal saline once daily. The experimental protocol was approved by the Kanazawa University Animal Care and Use Committee. The policies of Kanazawa University regarding the care and use of laboratory animals are consistent with the policies endorsed by the American College of Sports Medicine.Muscle and bone treatments.At the end of the experimental period, all rats were weighed, anesthetized, and sacrificed with an intraperitoneal injection of 50 mg·kg−1 pentobarbital sodium and decapitation. The soleus (SOL), extensor digitorum longus (EDL), and ventricle (VENT) muscles were quickly removed and weighed. In skeletal muscles, the average values of the right and left were used for data analyses. Then the right and left bones of both femurs (FE) and tibiae (TI) were excised and cleared of fat and connective tissue.The longitudinal lengths of the bones were measured with a stainless steel Vernier calipers (Mitutoyo, Kawasaki, Japan). The BMC, area, and BMD of FE and TI were measured by dual-energy x-ray absorptiometry (DXA) using the Aloka DCS-600R for small animals (Aloka, Tokyo, Japan) with Small Subject software (version 8.5, Aloka). Scanning speed was 10 mm·s−1, with resolution set at 0.2 × 1 mm. Coefficients of variation were determined from five repeat scans on two tibiae over several days, with repositioning for each scan. The average coefficient of variation was 3.2% for BMC and 3.1% for BMD. The minimum limits of measure for this apparatus were 25 mg for BMC and 22 mg·cm−2 for BMD. The five-segmented analysis was done automatically on the long axis of the FE and TI by using the software (details in Fig. 7). To delete the effect of the bone size, the relative data of BMC and area from each bone in segmental analysis were shown into %BMC and %area. The data from thin fibula, which is continual to TI, was negligible (<1%) but deleted from the data of TI with the software. The average values of right and left bones were used for analyses in this investigation. FIGURE 7— Schematic diagram of five evenly divided segments of the femur and the tibia of rat. The segments were named from proximal to distal like F1–F5 or T1–T5.Statistical analysis.The data were analyzed using a one-way ANOVA. Post hoc differences were determined with Scheffe’s test for the values from DXA and unpaired t-test for the values including body weight, muscle wet weight, and bone length for high accuracy. By using nQuery software (Statistical Solutions, Saugus, MA), the statistical power for the N size used range from 0.72 to 0.80. Data are reported as the mean ± SD. Significance was defined as P ≤ 0.05.RESULTSBody weights and muscle weights.Body weight of control rats increased normally during the experimental period, but CLE showed a rapid decrease for 2 or 3 d after injection of the drug but recovered quickly (Fig. 1). About 2 wk later, CLE demonstrated increases in body weight which were greater than CON. However, after 3 wk of the clenbuterol treatment, the growth rate of body weight was inhibited. FIGURE 1— Changes of average body weights of male rats. Values are means ± SD Filled symbols: control group (CON) of six rats;open symbols: clenbuterol-treated group (CLE) of six rats. Data are means of each group;bars are standard deviations. CON was linked with a solid line. CLE was linked with a dashed line. Arrow shows a start of the daily injections for 4 wk.The body weight and muscle wet weight after clenbuterol treatment for 4 wk are shown in Table 1. Finally, CLE showed a significant smaller body weight than CON (P < 0.05). The muscle wet weights in CLE were a little higher than CON (P > 0.05;Table 1) in the SOL (9%) and EDL (12%), but the ratios of muscle wet weight-to-body weight were significantly higher (SOL:P < 0.05, EDL:P < 0.01) than CON. This indicates that the skeletal muscles tended to be hypertrophied. The VENT of CLE group showed significantly an increased wet weight (14%, P < 0.01) and ratio (20%, P < 0.01). Therefore, the cardiac hypertrophy was clearly evident a being induced by clenbuterol treatment. Table 1. Body weights and muscle weights.Values are means ± SD;N, no. of observations.SOL, soleus; EDL, extensor digitorum longus.* Significantly different from control group (P < 0.05).** Significantly different from control group (P < 0.01).Figure 2 shows the relationship between muscle wet weight of EDL and the body weight. The EDL of CLE clearly showed different regression lines in growth from the CON. The similar relationship between muscle weight and body weight was also obtained in the SOL and the VENT (data not shown). FIGURE 2— Scatter plots of muscle wet weight of EDL as a function of body weight (BW) in CON (N = 6) and CLE (N = 6). Dashed lines, least squares linear regression for CLE (open squares): y = 1.859 × BW − 507.57 (r = 0.91);solid lines, least squares linear regression for CON (closed squares): y = 0.713 × BW − 72.58 (r = 0.44).Bone analyses.The bone data are shown in Table 2 and the data for DXA analyses are shown in Figure 3. Femurs in the CLE showed significantly smaller data in BMC (P < 0.01), in area (P < 0.01) and length (P < 0.05), but not in the BMD. Tibiae also showed significantly decreased values in BMC (P < 0.01), area (P < 0.01), and length (P < 0.01) but again not in BMD. Table 2. Bone analyses of young male rats treated with or without clenbuterol.Values are means ± SD;N, no. of observations.* Significantly different from control group (P < 0.05).** Significantly different from control group (P < 0.01).FIGURE 3— Sample data of the femur and the tibia with DXA. A, femur of CON; B, femur of CLE; C, tibia of CON; D, tibia of CLE. A scale bar shows 10 mm. In A and B, the dashed line is for trimming.Figure 4 showed the correlation of BMD to body weight. The BMD of both femurs (r = 0.57) and tibiae (r = 0.44) bones showed a small increase with growth, but the BMD was not significantly different between the CLE and CON groups (see Table 2). However, in Figure 5, a higher relationship between BMC and body weight in both femur (r = 0.83) and tibiae (r = 0.77) is shown. Most of CLE group located in lower range of body weight and indicated an inhibitory effect of clenbuterol on bone growth. Figure 6 showed a similar relationship with BMC in the femur (r = 0.74) and the tibia (r = 0.66). The area relationships of bones were similar with BMC and length (data not shown). FIGURE 4— Scatter plots of BMD of femurs (FE) or tibiae (TI) as a function of body weight (BW) in CON (closed symbol, N = 6), and CLE (open symbol, N = 6). FE: r = 0.57. y = 0.1561 × BW + 71.70. TI: r = 0.44. y = 0.0946 × BW + 63.72. Note that clenbuterol had no effects on BMD of both bones.FIGURE 5— Scatter plots of BMC of femurs (FE) or tibiae (TI) as a function of body weight (BW) in CON (closed symbol, N = 6) and CLE (open symbol, N = 6). FE (circle): r = 0.83. y = 0.97 × BW − 0.01. TI (square): r = 0.77. y = 0.60 × BW − 0.55. Note that clenbuterol had inhibitory effect on BMC of both bones.FIGURE 6— Scatter plots of length of femurs (FE) or tibiae (TI) as a function of body weight (BW) in CON (closed symbol, N = 6) and CLE (open symbol, N = 6). FE (circle): r = 0.74. y = 0.02116 × BW + 28.99. TI (square): r = 0.66. y = 0.02245 × BW + 32.65. Note that clenbuterol had inhibitory effect on length of both bones.Figure 7 shows the individual segments of bones. The darkened portion of fibulas was deleted from the data on the software automatically because they were below the detectable threshold. Rarely are the head portions of fibulas detectable, as shown in Figure 3, but they were very small and negligible (<1%). However, they were deleted for the data analysis of tibiae using the software program.Table 3 shows the five divided segmental data in femurs. F2 is a portion of second region from the postal end of the femur. There was a significant difference between CON and CLE treatments in BMC of F2 (P < 0.05), area of F1 and F4 (P < 0.05), and BMD of F2 (P < 0.05). However, for %BMC and %area, there were no significant differences in any of the comparative segments between CON and CLE. These data indicate that these differences might be due to the size of bone. Conversely, the significant difference of BMD in F2 (P < 0.05) was not changed by bone size theoretically. Therefore, it might be a clenbuterol treatment effect. Table 3. Segmental analysis from the femur of young male rats treated with or without clenbuterol.Values are means ± SD (N = 6).* Significantly different from control group (P < 0.05).The names of sections are shown in Figure 7.Table 4 shows the five-segmental data in the tibia. There were significantly decreased values for CLE treatment group in BMC of T2 (P < 0.01) and T3 (P < 0.05), in area of T1 (P < 0.05), T2 (P < 0.01), T3 (P < 0.05), and T4 (P < 0.05), in %BMC of T2 (P < 0.05), and in %area of T2 (P < 0.05). However, the T5 of CLE showed a significant increase in %BMC (P < 0.05). There were no significant differences between CON and CLE in BMD at any of the segments. Therefore, these effects might be due to the size of bone. Table 4. Segmental analysis from the tibia of young male rats treated with or without clenbuterol.Values are means ± SD (N = 6).* Significantly different from control group (P < 0.05).** Significantly different from control group (P < 0.01).The names of sections are shown in Figure 7.DISCUSSIONThe purpose of this study was to examine the hypotheses that clenbuterol would induce increases in the growth of bones as well as in muscles of young male rats. The primary findings of this study indicated significant CLE treatment effects, yet they were not similar in their adoptive responses. In the cardiac muscle, both the muscle wet weight and the ratio of wet weight to body weight significantly increased after clenbuterol treatment. In the SOL and EDL muscles, the ratio of wet weight to body weight increased significantly (see Table 1). However, the data from the DXA of the hindlimb bones showed significantly decreased BMC and area but no changes in the BMD. Our data demonstrated that the clenbuterol treatment could inhibit the longitudinal growth of bone similar to the side effects observed with the use of anabolic steroids in young individuals (11,27).As the previous studies have shown (3) the effect of beta-adrenergic agonists such as clenbuterol on muscle growth may be mediated by anorexia and the associated reduced food intake. However, in most of these cases, the body weights recover and have been shown to progress toward normal or heavier weights within a week due to the rapid increased tolerance to the drug (3,6,18). Choo et al. (7) showed that clenbuterol induced differential effects on body weights due to increases in muscle protein content and simultaneous decreases in adipose tissue mass. As our data (Fig. 1) show, the body weights of CLE tended to increase for a while until the end of the first 2 wk of the drug treatment similar to CON. The observed decreased body weights later in the treatment period might be due to other physiological mechanisms, such as increased proteolysis, increased lipid metabolism, or increased thermogenesis (7,13,34).Previous investigations have shown that clenbuterol may be useful to increase the muscle mass and help prevent the bone loss caused by the disuse of muscle and/or microgravity (1,35). In this study, we also observed similar responses to further support the hypothesis that clenbuterol induces muscle hypertrophy (see Table 1). However, the muscle hypertrophy induced by clenbuterol treatment may be explained by the direct action of the drug via the inhibition of proteolysis (4) or the increase in protein synthesis (3,17), and not by the indirect effects of growth hormone (GH), insulin-like growth factor (IGF), or testosterone (34). Also, it has been reported that the effects of clenbuterol on lipolysis and cardiac muscle hypertrophy were similar to thyroid hormone (13,33,34). Furthermore, significant changes of these hormones by clenbuterol have not been reported (3,5,13,34). In the present study, these hormones were not measured; therefore, future investigations will be needed to examine the role for such endocrine-mediated mechanisms.Generally speaking, the length of bone increases in parallel with muscle mass or body weight-for-age in young male and female humans (23). As described above (Table 1), the anabolic effect of clenbuterol on muscles is clear and has been well documented by other investigators (6,21,22,24,25). Basically, longitudinal bone growth depends not only on genetic and nutritional factors but also the regulation of some hormones including GH, IGF-1, androgen, estrogen (25,31), thyroid hormone (15,23,28), and glucocorticoid (23,32). For example, GH and IGF increase muscle mass as well as the longitudinal growth of bone (23). Interestingly, our data showed that clenbuterol inhibited the longitudinal growth of bones (see Table 2). The action of clenbuterol on CNS (2), which regulates these hormones, also must be further elucidated. Therefore, the shortened bone induced by clenbuterol might be a result of either direct or indirect mechanisms.The data on the segmental analyses (see Table 3) show that F2, the next portion of 20–40% from postal end of FE, was most affected in BMD (P < 0.05) by clenbuterol and the value of BMD was highest in FE. The causes of decreased BMD in F2, %BMC, and %area in T2 remain unknown. Whether the growth of the chondrocyte and osteoblast is inhibited or the increased osteoclast activity followed by faster epiphyseal closure is induced by clenbuterol remain to be investigated. So far, there are no reports of changes in estrogen concentrations in male animals treated with clenbuterol. Therefore, whether the cause of inhibited growth of bones is due to alterations in estrogen is unknown, but at least the faster epiphyseal closure is speculated to be involved with shortened bones (23). Furthermore, we should consider the effects of cAMP on deteriorated bones by clenbuterol stimulation. Some investigators have reported that the cAMP concentration is very important for modeling or remodeling of bones (1) and it was increased by not only a few new phosphodiesterase 4 (PDE-4) inhibitors for countermeasures of osteoporosis (20) but also clenbuterol of beta-2 adrenergic agonist (17,34). There is evidence that beta-2 receptors are located in osteoblasts (14,29). However, how the cAMP acts on the cells related with bones is unknown. Our data (see Table 3) shows that the clenbuterol also decreased partially the BMD in segment of F2. Some lysosomal enzymes, such as the cathepsins, in the cells might be stimulated by cAMP after the activation of parathyroid hormone (PTH) (12,26). This phenomenon might support the abnormality of bone growth due to the stimulated osteoblasts or osteoclasts by clenbuterol.There are a few interesting pieces of evidence indicating that the rat epiphyseal cartilage exhibited the metabolic property of aerobic glycolysis (8). The matrix vesicles from epiphyseal growth plates of young rabbits contained high lactate dehydrogenase (LDH) activity and five LDH isozymes (10). These mechanisms might be related with cell-mediated calcification (9). In our previous study of skeletal muscles, clenbuterol induced the higher LDH activity and an increase of muscle type of LDH (LDH-M) meaning higher glycolysis (30). Clenbuterol may possibly directly accelerate calcification and epiphyseal closure by elevated glycolysis via cAMP. Therefore, the mechanism of clenbuterol might be independent from those of GH, IGF-1, or other anabolic hormones. It is clear that clenbuterol inhibits the longitudinal growth of bone. These effects would be similar to the side effects observed for some anabolic steroids (11,27). Therefore, use of clenbuterol by young people should be carefully scrutinized in light of the animal data from our investigation.As Wong et al. (33) and Prather et al. (25) reported, the dose in animal experiments was large in comparison with that which has been reported in human use for clinical treatments of asthma. The higher dose increased the deposition rate of lean mass and retard adipose gain (25). This fact or the unclear side effects should be one of the reasons why athletes use the clenbuterol. We have no data on the effect of the dose in young human. Therefore, it should be studied furthermore on the human bone tissue in detail.The cause of effects by clenbuterol is very complicated and might be related with energy metabolism, sex hormones, and ages as described above. Although we had no any information on effects of clenbuterol on longitudinal growth in human bone, the present results suggest that it is necessary to discuss whether clenbuterol should be contraindicated for prepubescent and adolescent humans but not adult humans. 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Brunet-Imbault, B; Arlettaz, A; Horcajada, MN; Collomp, K; Benhamou, CL; COURTEIX, Dhttp://journals.lww.com/acsm-msse/Fulltext/2005/09000/Alteration_of_Trabecular_Bone_under_Chronic__2.7.aspx304http://pdfs.journals.lww.com/acsm-msse/2005/09000/Alteration_of_Trabecular_Bone_under_Chronic__2.00007.pdfhttp://dx.doi.org/10.1249%2f01.mss.0000177592.82507.95