1 Unité de Physiologie Générale des Muscles, and 2 Unité de Diabétologie et Nutrition, Université Catholique de Louvain, 1200 Brussels, Belgium
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ABSTRACT |
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Clenbuterol induces hypertrophy and a slow-to-fast phenotype change in skeletal muscle, but the signaling mechanisms remain unclear. We hypothesized that clenbuterol could act via local expression of insulin-like growth factor I (IGF-I). Administration of clenbuterol to 3-mo-old female Wistar rats resulted in a 10 and 13% increase of soleus muscle mass after 3 and 9 days, respectively, reaching 16% after 4 wk. When associated with triiodothyronine, clenbuterol induced a dramatic slow-to-fast phenotype change. In parallel, clenbuterol administration induced in soleus muscle a fivefold increase in IGF-I mRNA levels associated with an eightfold increase in IGF-binding protein (IGFBP)-4 and a fivefold increase of IGFBP-5 mRNA levels on day 3. This increased IGF-I gene expression was associated with an increase in muscle IGF-I content, already detected on day 1 and persisting until day 5 without increase in serum IGF-I concentrations. These data show that muscle hypertrophy induced by clenbuterol is associated with a local increase in muscle IGF-I content. They suggest that clenbuterol-induced muscle hypertrophy could be mediated by local production of IGF-I.
insulin-like growth factor; insulin-like growth
factor-binding proteins; 2-adrenoceptor agonists; hypertrophy; fiber type
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INTRODUCTION |
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2-ADRENOCEPTOR
AGONISTS such as clenbuterol and cimaterol are known to exert
anabolic effects on skeletal muscle. Long-term administration also
induces a change of phenotype from slow to fast fiber type (7,
17, 22, 23). The mechanism of these effects is unclear. One
hypothesis to explain the increase in skeletal muscle mass induced by
clenbuterol involves the major anabolic hormones such as insulin,
growth hormone, and testosterone. However, muscle hypertrophy in
response to
2-agonists is observed even in severely
diabetic insulin-deficient as well as in hypophysectomized or castrated
rats (5, 20). These observations plead, therefore, against
the role of these classical anabolic hormones in the muscle anabolic
action of clenbuterol. The possibility that
2-agonists might locally stimulate the production of growth factors by skeletal muscle has not been yet investigated. This hypothesis is,
however, suggested by the recent demonstration that clenbuterol
could protect cerebral tissue against ischemic damage by
inducing local expression of nerve growth factor, basic fibroblast
growth factor, and transforming growth factor-
1 (8, 9,
11). The induction of local expression of growth factors in
skeletal muscle by
2-adrenoceptor agonists might thus
mediate the anabolic effects of these drugs. In skeletal muscle, it is
well established that overloading and stretch result in local
production of insulin-like growth factor (IGF) I, which acts in an
autocrine and paracrine manner to induce skeletal muscle hypertrophy
(1). In view of these data, and because it has been shown
that clenbuterol did not increase the serum concentration of IGF-I
(21), we hypothesize that local changes in IGF-I produced by skeletal muscle could be the signaling mechanisms by which clenbuterol exerts its anabolic effect. In this paper, we investigated whether the hypertrophy of the rat soleus muscle by clenbuterol is associated with an increase in IGF-I expression.
In a previous study, we showed that changes in muscle loading induced specific changes not only of IGF-I, but also of IGF-binding protein-4 and -5 (IGFBP-4 and -5) associated with phenotype changes, suggesting a possible role of these binding proteins in the changes of fiber types (3). On the other hand, thyroid hormones are known to induce a fast muscle phenotype, and triiodothyronine (T3) has been shown to act synergistically with clenbuterol to cause a profound slow-to-fast phenotype change (15). Therefore, the effects of these drugs on IGFBP-4 and IGFBP-5 expression in soleus muscle were also investigated simultaneously with the study of IGF-I expression.
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MATERIALS AND METHODS |
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Animals.
Three-month-old female Wistar rats were randomly split into four groups
of 16 animals. The animals of the first group (Clenb) were
supplemented with clenbuterol (Sigma, St. Louis, MO) in their drinking water (10 parts/million) (15). Rats of the second
group (T3) were given subcutaneous injections of
3,3',5-triiodo-L-thyronine (300 µg/kg body wt) every
48 h for 4 wk. Rats of the third group (Clenb/T3)
received both clenbuterol and T3, and the fourth group (Ctrl) served as a control. Animals of Ctrl and Clenb groups were given
subcutaneous injections of T3 vehicle. After 3, 9, and 28 days of treatment, rats were decapitated, and soleus muscles were dissected, weighed, frozen in liquid nitrogen, and kept at 70°C. The blood of each animal was collected, and the serum was recovered by
centrifugation for storage at
20°C. In a second experiment, 16 rats
(4 in each time point group) were treated only with clenbuterol at the
same dose during 1, 2, 3, and 5 days, and a group of 20 rats served as control.
Electrophoretic analysis of myosin heavy chains. Soleus muscles of the 28-day experiment were used to determine the proportions of different myosin heavy chains (MHCs). Frozen muscles were pulverized and extracted in Guba-Straub solution as described previously (13). MHCs were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) including 33% (wt/vol) glycerol in both separating (7% polyacrylamide) and stacking gels (3% polyacrylamide). Identification of the bands was based on their electrophoretic mobilities. Their proportions were quantified by densitometry. All measurements were made in duplicate for each muscle. The results are expressed as percentage of total MHCs.
RNA isolation and Northern blot hybridization. Total RNA was isolated from soleus muscle by the TRIPUR reagent (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. Each sample of RNA (10 µg) was denatured in formaldehyde-MOPS and subjected to electrophoresis on 1.2% agarose gel. Homogeneity of RNA loading was assessed by ultraviolet transillumination of the gel after staining with ethidium bromide. The RNA was transferred to nylon membranes (Hybond, Amersham, Buckinghamshire, UK) by vacuum blotting (Vacugene, Pharmacia, Uppsala, Sweden). The amounts of IGF-I, IGFBP-4, and IGFBP-5 mRNA were determined by hybridization with specific riboprobes. The 194-bp AvaII-HinfI rat IGF-I exon 4 complementary DNA fragment was inserted into the plasmid vector Bluescript (pBSM13+) and linearized with EcoRI. A 221-bp rat IGFBP-4 gene fragment was ligated into the plasmid vector Bluescript (pBS SK+) and linearized with BamHI. A 656-bp rat IGFBP-5 gene fragment was ligated into the plasmid vector Bluescript (pBS SK+) and linearized with EcoRI. The specific riboprobes were generated from linearized plasmids with uridine 5'-[32P]triphosphate using T7 or T3 RNA polymerases. The mRNA levels were quantified by densitometric scanning of the hybridization signal (LKB Ultroscan XL laser densitometry; LKB, Bromma, Sweden) using the software Gel Scan (Pharmacia). All sizes/classes of IGF-I mRNA transcripts were pooled together. The mRNA levels were normalized by assigning an arbitrary value of 100% to the mRNA level observed at the beginning of the experiment (day 0).
RIA of IGF-I.
Serum samples for IGF-I were extracted by C18 Sep-Pak
(Waters, Milford, MA) chromatography. Serum IGF-I concentration was measured by RIA, following a method described previously
(19). Muscle extraction was performed with acetic acid.
Ice-cold 1 M acetic acid (1 ml) was added to 100 mg of powdered muscle.
The mixture was vortexed vigorously and allowed to stand on ice for 2 h. After centrifugation at 20,000 g for 30 min at
4°C, the supernatant was transferred to a polypropylene tube, and the
pellet was reextracted. Both supernatants were combined and desiccated
in a Speed-Vac concentrator. The material was kept at 20°C and
reconstituted in 1 ml of assay buffer 24 h before RIA. The
recovery of recombinant IGF-I added to the tissue powder-acetic acid
mixture was 42.5 ± 3.5% (mean ± SD; n = 4). The intra- and interassay coefficients of variation were,
respectively, 4.2 and 4.6%. The results are expressed as nanograms of
IGF-I per gram of tissue and are not corrected for recovery.
Statistical analysis. Results are expressed as means ± SE. Differences between control and treated muscles were assessed by analysis of variance (ANOVA) followed by a Newman-Keuls test. Statistical significance was set at P < 0.05.
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RESULTS |
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Soleus muscle mass.
Before the possible role of local IGF-I in the anabolic effects of
clenbuterol on skeletal muscle was examined, the hypertrophic response
in soleus muscle was evaluated. As shown in Table
1, administration of clenbuterol to rats
for 3 days induced a significant 10% increase (P < 0.01) of muscle mass (100.8 ± 2.5 mg for Clenb vs. 91.2 ± 1.9 mg for Ctrl). The hypertrophy reached 16% after 4 wk of
clenbuterol supplementation. In contrast to the anabolic effect of
clenbuterol, T3 induced a 10% decrease (P = 0.09) of muscle mass after 4 wk of treatment. Combining the two drugs
resulted in a 12% increase (P = 0.09) of muscle mass
after 4 wk.
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MHC isoforms.
The effects of long-term administration of clenbuterol on muscle
phenotype were assessed by the determination of MHC proportions. Four
weeks of clenbuterol administration induced a trend toward a faster
phenotype, characterized by a decrease of the proportion of MHC1 (from
97.5 ± 1.6 to 93.5 ± 2.7%), an increase of that of
MHC2a (from 2.5 ± 1.6 to 5 ± 1.2%), and the appearance of
MHC2x (1.5%) (Fig. 1). These changes in
MHC proportions were only minor and nonsignificant. T3
treatment induced a slow-to-fast phenotype change with significant
alterations of the proportion of each MHC. Soleus muscles of
T3-treated animals were thus composed of 83.5 ± 2.2%
of MHC1, 12.5 ± 1.4% of MHC2a, and 4 ± 1% of MHC2x. When
administered together, these two drugs acted synergistically and
induced a dramatic slow-to-fast phenotype change: the soleus muscles of
Clenb/T3-treated rats presented the following MHC
proportions: 48.9 ± 6% of MHC1, 30.9 ± 4.6% of MHC2a,
17.8 ± 1.2% of MHC2x, and 2.4 ± 0.1% of MHC2b (Fig. 1).
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IGF-I, IGFBP-4, and IGFBP-5 gene expression in soleus muscle.
Clenbuterol for 3 days significantly increased (5-fold;
P < 0.05) IGF-I mRNA levels in the soleus muscle, as
shown in Fig. 2. In contrast,
T3 alone did not change the gene expression of IGF-I.
However, in muscles of animals receiving clenbuterol and T3
together, the increase of IGF-I mRNA levels was slightly more marked
(7.5-fold) than after clenbuterol alone. The increase of muscle IGF-I
mRNA levels induced by clenbuterol (or clenbuterol + T3) after 3 days disappeared after 9 days of treatment.
Clenbuterol alone or combined with T3 also increased
IGFBP-4 and IGFBP-5 mRNA levels. The time pattern of these changes was
similar to that of IGF-I mRNA. Levels of IGFBP-4 and IGFBP-5 mRNA
increased significantly after 3 days of administration and returned to
control values after 9 days. T3 alone induced a small but
significant (P < 0.05) increase of IGFBP-4 mRNA levels
after 3 and 9 days but had no effect on muscle IGFBP-5 gene expression.
After 28 days of clenbuterol administration, mRNA levels of IGF-I,
IGFBP-4, and IGFBP-5 were, respectively, 56, 70, and 40% higher than
the levels in control muscles. Neither T3 alone nor in
association with clenbuterol had an effect on IGF-I, IGFBP-4, and
IGFBP-5 mRNA levels after 28 days of administration (data not shown).
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Muscle IGF-I peptide content.
Concentrations of IGF-I peptide in muscle were determined by RIA after
3 and 9 days of clenbuterol administration. As shown in Fig.
3, 3 days of treatment resulted in a
threefold increase of IGF-I peptide (P < 0.05). The
levels of muscle IGF-I peptide later decreased to a value that was not
significantly different from the control at 9 days. Clenbuterol thus
induces parallel changes of IGF-I mRNA and peptide in rat soleus
muscle.
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Early changes of IGF-I, IGFBP-4, and IGFBP-5 gene expression.
To investigate the possibility of rapid changes in gene expression
induced by clenbuterol, the effect of this drug on muscle IGF-I,
IGFBP-4, and IGFBP-5 mRNA was studied on days 1, 2, 3, and 5. As shown in Fig.
4, clenbuterol induced a progressive
increase of IGF-I mRNA levels, which reached a value significantly
different from that of control on day 3 (P < 0.001). The IGF-I mRNA level then decreased back to control values
on day 5. Like IGF-I mRNA, IGFBP-4 mRNA levels increased
progressively from day 1 to reach a peak on day 3 (P < 0.001). From day 1 onward, the
increase in IGFBP-4 mRNA levels was significantly different from
control values (P < 0.01). Significant changes in
IGFBP-4 mRNA levels thus preceded those in IGF-I mRNA. In contrast to
the effects on IGF-I and IGFBP-4, clenbuterol induced first a
significant decrease of IGFBP-5 mRNA levels on day 1 (P < 0.01), followed by a progressive increase reaching a peak value on day 3 (P < 0.001).
From day 3, IGFBP-4 and IGFBP-5 mRNA, like IGF-I mRNA,
decreased and reached control values on day 5.
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Early changes in muscle IGF-I peptide.
As shown in Fig. 5, clenbuterol
administration to rats induced a significant increase of muscle IGF-I
contents starting on day 1. The elevated value of IGF-I
peptide reached a peak on day 3 and persisted until
day 5. Thus changes in muscle IGF-I peptide preceded those
observed at mRNA levels and persisted at least until day 5,
a time point when IGF-I mRNA levels had returned to control values.
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Effects of clenbuterol on circulating IGF-I. As shown by Fig. 5, IGF-I levels in serum of the rats receiving clenbuterol were lower than levels in control animals. The decline in serum IGF-I was more marked on day 1 of clenbuterol administration. On day 2, serum IGF-I rose to control values and then declined during the following days (days 3 and 5).
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DISCUSSION |
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The present study shows for the first time that clenbuterol activates IGF-I expression in skeletal muscle at both mRNA and peptide levels. Because IGF-I can induce muscle hypertrophy, our data suggest that the hypertrophic effect of clenbuterol might be mediated by the overexpression of muscle IGF-I.
The hypertrophic effect of IGF-I on skeletal muscle is well established. In vitro, IGF-I not only activates myoblast proliferation and differentiation but also induces hypertrophy of resulting myotubes. In vivo, overexpression or infusion of IGF-I in skeletal muscle also results in muscle hypertrophy (2, 6). Moreover, it has been demonstrated that muscle hypertrophy induced by stretching or overloading occurred via local expression of IGF-I, which exerts its effects in an autocrine and a paracrine manner (1). Recently, it has been shown that IGF-I induced muscle hypertrophy via a calcium calcineurin pathway (16, 18). Because clenbuterol induced in skeletal muscle an early overexpression of IGF-I, which preceded the increase in muscle mass and persisted at least for 5 days, IGF-I appears to be a link between clenbuterol and muscle hypertrophy.
The early rise in muscle IGF-I peptide on day 1 induced by clenbuterol preceded the significant increase of IGF-I mRNA levels, which occurred on day 3. This suggests that the early change in IGF-I peptide did not result from accelerated IGF-I gene expression. Similar data were reported by Eliakim et al. (10), who observed an increase of IGF-I peptide in skeletal muscle of rats submitted to exercise without changes of muscle IGF-I mRNA or plasma IGF-I peptide. Because clenbuterol has been shown to induce skeletal muscle protein synthesis by increasing translation of mRNA at the early time of its administration (12), this mechanism might be involved in the rapid increase of IGF-I peptide in response to clenbuterol.
Another possibility is that clenbuterol could act on liver, the major
production site of circulating IGF-I, contributing, therefore, to
raising IGF-I levels within skeletal muscles. Interestingly, the levels
of serum IGF-I in clenbuterol-treated rats were lower than those of
control animals during the first 5 days of treatment. A similar result
has been obtained in animals treated with cimaterol, in which a 34%
decrease in plasma IGF-I levels has been reported (5). The
mechanism by which 2-agonists decrease the levels of
plasma IGF-I is not known. Nevertheless, these results plead against a
general action of clenbuterol on the liver IGF-I synthesis but rather
suggest a local stimulatory effect on skeletal muscle IGF-I, since the
anabolic effects of clenbuterol are restricted to skeletal and cardiac
muscles. Skeletal muscle cells are indeed well known to produce IGF-I
locally, which could have an autocrine or a paracrine action
(1).
2-Adrenoreceptors are expressed in
muscle cells. Together, these observations suggest that clenbuterol may
exert its anabolic action by increasing the production of IGF-I by myocytes.
In addition to changes in IGF-I expression, clenbuterol administration resulted in changes in the gene expression of IGFBP-4 and IGFBP-5 in skeletal muscle. Although the biological significance of these changes is not known, IGFBP changes could modify IGF-I bioavailability. An increased expression of IGFBPs could promote the quenching of IGF-I in skeletal muscle, increasing its concentration within the muscle. In our study, on day 1, IGFBP-4 and IGFBP-5 gene expressions were regulated in opposite ways by clenbuterol; the level of IGFBP-4 mRNA was significantly increased, whereas the IGFBP-5 mRNA level was reduced. The balance between these binding proteins at protein level could determine their contributions to the changes in muscle IGF-I peptide.
Overloading is another method for inducing muscle hypertrophy. Like clenbuterol, overloading increases muscle IGFBP-4 mRNA but, in contrast to clenbuterol, decreases IGFBP-5 mRNA levels (3). In overloaded muscles, IGF-I is thought to induce hypertrophy by exerting an anabolic effect on both myofibers and satellite cells, which proliferate, differentiate, and fuse with preexisting myofibers (1). In contrast, clenbuterol-induced hypertrophy appears to be due only to anabolic effects on preexisting myofibers (20). Thus the difference between these two models of hypertrophy could be explained by the modulation of IGF-I action by IGFBP, especially IGFBP-5. Indeed, the increased expression of IGFBP-5 in muscle of clenbuterol-treated animals could prevent the proliferation of satellite cells. This hypothesis is supported by the recent demonstration that increased secretion of IGFBP-5 by fibroblasts could inhibit the proliferation of myoblasts of Duchenne muscular dystrophy patients (14).
The changes in IGFBP-5 could also be considered in relation to muscle phenotype. On the basis of data reported in previous studies (3, 4), it has been proposed that IGFBP-5 could promote expression of the fast muscle phenotype. Increased expression of IGFBP-5 is observed in conditions inducing slow-to-fast phenotype changes such as unloading and denervation. A decreased expression is found when skeletal muscle is overloaded, a situation that promotes the expression of the slow phenotype. In the present study, the association between increased expression of IGFBP-5 and a slow-to-fast phenotype change was also observed in soleus muscle of rats fed with clenbuterol. In T3-treated rats, an increased proportion of fast myosin (MHC2) was observed, but this phenotype change was not preceded by or associated with changes in IGFBP-5 expression. These data show that, if IGFBP-5 plays any role in the determination of muscle fiber type, it does not appear as a dominant factor systematically involved in all processes inducing a change from slow-to-fast phenotype in skeletal muscle.
In conclusion, clenbuterol induces in skeletal muscle a local overexpression of IGF-I, which could result from a direct effect of the drug on muscle cells. This local overexpression of IGF-I could mediate the anabolic effects of clenbuterol on skeletal muscle. Investigation of signaling pathways leading to these effects, especially the determination of the role of calcineurin in clenbuterol-induced hypertrophy, could help to reinforce the role of IGF-I in this process.
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ACKNOWLEDGEMENTS |
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We thank Josiane Verniers for skillful technical assistance.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. Lebacq, Unité de Physiologie générale des Muscles, Université Catholique de Louvain, UCL 5540, Ave. Hippocrate 55, 1200 Brussels, Belgium (E-mail: lebacq{at}fymu.ucl.ac.be).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 19 April 2001; accepted in final form 4 September 2001.
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