Role of IGF-I and IGFBPs in the changes of mass and phenotype induced in rat soleus muscle by clenbuterol

Bonaventure L. Awede1, Jean-Paul Thissen2, and Jean Lebacq1

1 Unité de Physiologie Générale des Muscles, and 2 Unité de Diabétologie et Nutrition, Université Catholique de Louvain, 1200 Brussels, Belgium


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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; beta 2-adrenoceptor agonists; hypertrophy; fiber type


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

beta 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 beta 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 beta 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-beta 1 (8, 9, 11). The induction of local expression of growth factors in skeletal muscle by beta 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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1.   Effects of clenbuterol, T3, and clenbuterol + T3 on soleus muscle mass

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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Fig. 1.   Proportions of myosin heavy-chain (MHC) isoforms in soleus muscles of control (Ctrl), clenbuterol (clenb), triiodothyronine (T3), and clenbuterol + T3 (Clenb/T3)-treated rats. The amount of each MHC is expressed as a percentage of total MHC. Values are given as means ± SE; n = 4/group, except Clenb/T3 on day 28, when n = 2. Significantly different from control (*P < 0.01; **P < 0.001).

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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Fig. 2.   Analysis of insulin-like growth factor (IGF) I, IGF-binding protein (IGFBP)-4, and IGFBP-5 mRNAs by Northern blotting of 10 µg of total RNA from soleus muscles of control, T3, Clenb/T3, and clenb-treated rats. A: quantification of IGF-I, IGFBP-4, and IGFBP-5 mRNA levels by densitometry expressed as a percentage of the levels on day 0. Values are given as means ± SE; n = 4/group. Significantly different (*P < 0.05) from control; **P < 0.01; ***P < 0.001. B: autoradiographs showing expression of IGF-I, IGFBP-4, and IGFBP-5 mRNAs on days 0, 3 and 9 in soleus of control and treated rats (T3, Clenb/T3, Clenb).

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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Fig. 3.   Concentration of IGF-I peptide in soleus muscles of control and clenbuterol-treated rats. Values are expressed as means ± SE; n = 4/group. Significantly different (**P < 0.01) from control group.

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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Fig. 4.   Analysis of IGF-I, IGFBP-4, and IGFBP-5 mRNAs by Northern blotting of 10 µg of total RNA from soleus muscles of control and clenbuterol-treated rats. A: quantification of IGF-I, IGFBP-4, and IGFBP-5 mRNA levels by densitometry expressed as a percentage of the levels on day 0. Values are given as means ± SE; n = 4/group. Significantly different (from control *P < 0.05; **P < 0.01; ***P < 0.001). B: autoradiographs showing expression of IGF-I, IGFBP-4, and IGFBP-5 mRNAs on days 0, 1, 2, 3 and 5 in muscles of control and clenbuterol-treated rats.

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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Fig. 5.   Concentration of IGF-I peptide (A) in soleus muscles and serum (B) of control and clenbuterol-treated rats. Values are expressed as means ± SE; n = 4/group. Significantly different from control (*P < 0.05; **P < 0.01).

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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta 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). beta 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.


    ACKNOWLEDGEMENTS

We thank Josiane Verniers for skillful technical assistance.


    FOOTNOTES

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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ABSTRACT
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RESULTS
DISCUSSION
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