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J Appl Physiol 99: 71-80, 2005. First published March 17, 2005; doi:10.1152/japplphysiol.00448.2004
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Clenbuterol induces muscle-specific attenuation of atrophy through effects on the ubiquitin-proteasome pathway

Tossaporn Yimlamai,1 Stephen L. Dodd,1 Stephen E. Borst,1,2 and Sooyeon Park1

1Department of Applied Physiology and Kinesiology, University of Florida, Gainesville; and 2Geriatric, Research, Education, and Clinical Center, Malcom Randall Veterans’ Affairs Medical Center, Gainesville, Florida

Submitted 28 April 2004 ; accepted in final form 15 March 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ubiquitin-proteasome pathway is primarily responsible for myofibrillar protein degradation during hindlimb unweighting (HU). {beta}-Adrenergic agonists such as clenbuterol (CB) induce muscle hypertrophy and attenuate muscle atrophy due to disuse or inactivity. However, the molecular mechanism by which CB exerts these effects remains poorly understood. The aims of this study were to investigate whether CB attenuates HU-induced muscle atrophy through an inhibition of the ubiquitin-proteasome pathway and whether insulin-like growth factor I (IGF-I) mediates this inhibition. Rats were randomized to the following groups: weight-bearing control, 14-day CB-treated, 14-day HU, and CB + HU. HU-induced atrophy was associated with increased proteolysis and upregulation of components of the ubiquitin-proteasome pathway (ubiquitin conjugates, ubiquitin conjugating enzyme E2-14kDa, and 20S proteasome activity). Upregulation of the ubiquitin proteasome occurred in all muscles tested but was more pronounced in muscles composed primarily of slow-twitch fibers (soleus) than in fast-twitch muscles (plantaris and tibialis anterior). Although CB induced hypertrophy in all muscles, CB attenuated the HU-induced atrophy and reduced ubiquitin conjugates only in the fast plantaris and tibialis anterior and not in the slow soleus muscle. CB did not elevate IGF-I protein content in either of the muscles examined. These results suggest that CB induces hypertrophy and alleviates HU-induced atrophy, particularly in the fast muscles, at least in part through a muscle-specific inhibition of the ubiquitin-proteasome pathway and that these effects are not mediated by the local production of IGF-I in skeletal muscle.

hindlimb unweighting; protein degradation; insulin-like growth factor 1

HINDLIMB UNWEIGHTING (HU) is often used to model the effects of disuse and microgravity on skeletal muscle (32). HU causes severe muscle atrophy, especially in muscles composed primarily of slow fibers (18, 34), and the precise mechanism causing the atrophy remains unclear. HU causes muscle atrophy mainly by increasing the rate of protein degradation, although a decrease in protein synthesis also occurs (8, 45). Skeletal muscle contains three major pathways of protein degradation: a lysosomal pathway, a calcium-dependent pathway, and an ATP-dependent ubiquitin-proteasome pathway. Of the three, the ubiquitin-proteasome pathway is responsible for the bulk of muscle proteolysis, including the major contractile proteins actin and myosin (4, 24, 40). It is important that this system also degrades damaged proteins after oxidative stress (13) since HU has been shown to increase oxidative stress and to decrease antioxidant capacity in skeletal muscle (27, 28).

The importance of the ubiquitin-proteasome pathway in HU-induced muscle wasting has been clearly demonstrated (14, 23, 44). Taillandier et al. (44) reported that the proteasome system accounts for most of the enhanced protein degradation seen in unweighted rat soleus (Sol) muscle, whereas the lysosomal and Ca2+-dependent pathway were responsible for only a small fraction (15–20%) of total protein degradation. These changes are accompanied by increased mRNAs for polyubiquitin, E2-14kDa ubiquitin conjugating enzyme, and subunits of the proteasome. These observations are supported by a recent study demonstrating similar increases in mRNAs for components of the ubiquitin-proteasome pathway in muscle from animals subjected to spaceflight (23). However, it is not yet clear whether elevated mRNA translates into elevated protein levels. Moreover, there is now substantial evidence that activation of the ubiquitin-proteasome pathway is muscle fiber-type specific in conditions such as hyperinsulinemia (27), burn injury (17), and muscle transformation (43). One purpose of our study was to determine whether HU-induced activation of the proteasome is muscle specific.

A second purpose of our study was to determine whether clenbuterol (CB) inhibits HU-induced activation of the ubiquitin-proteasome pathway and whether such inhibition occurs in a muscle-specific manner. CB is a {beta}2-adrenergic agonist with growth-promoting properties that cause increases in muscle mass (50) and reduction in muscle atrophy attributable to muscular dystrophy (21), denervation (51, 52), and hindlimb suspension (15, 46). The precise mechanism of CB action remains unclear, although a reduction in muscle protein degradation has been proposed (33, 39). The few studies examining the effects of {beta}2-agonists on the protease activity are contradictory (see Ref. 30 for review). To our knowledge, there is little information regarding the effect of {beta}2-agonists on the ubiquitin-proteasome pathway.

A mediator of the action of CB may be IGF-I. Although IGF-I is known to play a pivotal role in myogenesis and postnatal skeletal muscle growth (1, 19) through its stimulation of protein synthesis (18), it is not known whether IGF-I is a mediator of CB-induced muscle hypertrophy. Recently, it has been shown that CB administration to rats caused a transient increase in muscle IGF-I protein content (5) and that IGF-I inhibits protein degradation both in vivo and in vitro, possibly by suppressing the upregulation of several components of the ubiquitin-proteasome pathway (10, 17, 22, 48). Furthermore, Li et al. (29) have recently shown that IGF-I can protect cultured myotubes by inhibiting dexamethasone-induced upregulation of the proteasome. Thus a third purpose of our study was to determine whether muscle IGF-I is altered by CB, HU, or a combination of CB and HU.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and experimental design.   Adult, female Sprague-Dawley rats, weighing ~200 g, were obtained from Harlan Sprague Dawley (Indianapolis, IN). Females were chosen for study to avoid the confounding influences of androgens while studying effects on muscle mass. Animals were housed at 20–22°C and maintained on a 12-h light-dark cycle. They were given standard food and water ad libitum and handled daily to reduce contact stress during a 7-day acclimation period. Animals were then assigned randomly to one of four groups: control (C; n = 8), CB-treated (n = 9), HU (n = 9), and CB combined with HU (HU-CB; n = 9). The number of animals per experimental group was determined by power analysis. Animals in C and CB groups were housed individually in conventional rat cages. The animals in HU and HU-CB groups were housed separately in standard rabbit cages to provide increased area for movement. At the end of treatment, animals were killed and muscle tissue removed under pentobarbital anesthesia. These experiments were approved by the Institutional Animal Care and Use Committee and conformed to the Guiding Principles in the Care and Use of Animals of the American Physiological Society.

HU.   According to the protocol of Roer and Dillaman (37), tails were wrapped with orthopedic tape, connected to a swivel, and suspended for 14 days. We chose 14 days of suspension because it has been shown that the rate of protein degradation is maximal at this time (15). The rats were suspended from the top of a cage with hindlimbs 1–2 cm above the floor, and they had a 360° range of motion by use of the forelimbs. This arrangement prevents the animal from climbing up the side of the cage and hanging from the top.

CB treatment.   CB-treated animals were supplemented with CB (30 mg/l, Sigma, St. Louis, MO) in their drinking water for 14 days, and water intake was recorded every other day. The average daily intake of CB was 0.9 mg/day during the entire experimental period. This dose has been shown to promote maximal growth of several types of muscles (39). CB was replaced every 3 days to prevent oxidation. Administration of CB in the drinking water avoids unnecessary handling of the animals and is as effective as injection (10, 42).

Preparation of myofibrillar and cytosolic fractions.   A portion (~100 mg) of frozen muscle was minced and homogenized in 10 volumes of ice-cold buffer (50 mM Tris·HCl, pH 8.0, 1 mM EGTA, 1 mM EDTA, 10 µM E-64, 1 µM pepstatin A, 1 mM PMSF, 10% glycerol) using a Kontes ground glass homogenizer. Myofibrillar proteins were pelleted by centrifugation at 1,500 g for 10 min at 4°C. Pellets were washed three times in the same buffer containing 1% Triton X-100 and resuspended in 8 M urea, 50 mM Tris·HCl, pH 7.5. Cytosolic fractions were prepared by centrifugation of homogenates at 10,000 g for 10 min and 100,000 g for 1 h at 4°C. Supernatants were stored at –80°C.

Protein degradation.   Sol muscle and tendon were removed, mounted on plastic supports at resting length, and placed in Krebs-Henseleit buffer, pH 7.4, 10 mM glucose, 0.2 mM valine, 0.17 mM leucine, 0.1 mM isoleucine, and saturated with 95% O2-5% CO2. After incubation for 1 h at 37°C in a shaking water bath to allow for metabolic steady state, the muscle was transferred to fresh buffer containing 0.5 mM cycloheximide and incubated for an additional 2 h. This concentration of cycloheximide inhibits protein synthesis by 95% without effecting protein degradation (43). Protein degradation was then estimated by measuring tyrosine release into the media as previously described (47). Briefly, after centrifugation (1,000 g, 4°C, 10 min), 750 µl of the supernatant was added to 750 µl of 5% trichloroacetic acid, 750 µl of 1% nitrosonaphthol (wt/vol), and 750 µl of 20% nitric acid containing 2.5% of NaN2 (wt/vol), followed by incubation at 55°C for 30 min and addition of 7.5 ml of dichloroethane. After centrifugation as above, tyrosine in the medium was measured by spectrofluorometry at excitation and emission wavelength of 450 and 550 nm, respectively, and expressed as nanomoles per gram of muscle per 2 h. Measurement was not performed in tibialis anterior (TA) and plantaris (PA) because these muscles are too thick to allow for full oxygenation.

Protein determination.   Protein content of homogenates and of myofibrillar and cytosolic fractions was determined in duplicate by the Lowry method using the Coomassie Plus Protein Assay Reagent Kit (Pierce, Rockford, IL) with bovine serum albumin as a standard.

Proteasome activity assay.   The trypsin-like and chymotrypsin-like activities of the proteasome were measured using 100 µM Boc-LRR-7-amido-4-methyl-coumarin (AMC) and 100 µM Suc-LLVY-AMC, respectively, as described by Stein et al. (41) with some modifications. Enzymatic activity was determined in triplicate by incubating 10 µg of protein (cytosolic fraction) with 50 mM Tris·HCl, pH 8, 5 mM MgCl2, and 1 mM DTT, in 200 µl ± 10 µM lactacystin (Boston Biochem). Proteolytic activity was monitored continuously by release of the fluorescent group AMC with a temperature-controlled microplate fluorometric reader (SpectraMax-GeminiXS, Molecular Devices) during 30 min at 37°C using an excitation wavelength of 380 nm and an emission wavelength of 460 nm. The standard curve was established using a free AMC. The difference between peptidase activities in the reaction medium, with and without proteasome inhibitor, was calculated, and the data was expressed as nanomoles per minute per micrograms of protein. Lactacystin caused a significant suppression (>90%) of the total trypsin and chymotrypsin activities if the cytosolic activity was based on proteasome function.

Immunoblotting analysis.   20S proteasome {alpha}- and {beta}-subunits, 14-kDa E2, and conjugated ubiquitin were measured by Western analysis. Cytosolic (for 20S proteasome and 14-kDa E2) and myofibrillar fractions (for ubiquitin conjugates) were subjected to 4–12% SDS-PAGE. Ponceau staining (Pierce) was used to ensure equal loading of protein. Samples from all experimental groups were electrophoresed on the same gel. Samples were transferred to nitrocellulose (0.45 µm, Millipore, Bedford, MA), and membranes were blocked overnight at 4°C in TBS-Tween 20 (20 mM Tris·HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) containing 5% nonfat dry milk, followed by incubation for 2 h at room temperature with one of the following primary antibodies: 1:1,000 mouse anti-20S proteasome {alpha}- and {beta}-subunits (Calbiochem, CA), 1:100 mouse anti-ubiquitin (Santa Cruz Biotechnology), or 1:1,000 rabbit anti-E2-14kDa (BostonBiochem, MA). Antibodies against 20S proteasome {alpha}- and {beta}-subunits have been reported by the manufacturer to cross-react. The membranes were then washed in TBS-Tween 20 buffer for 3 x 10 min and incubated for 1 h at room temperature with secondary antibodies (anti-mouse or rabbit IgG conjugated to horseradish peroxidase, 1:5,000, Amersham, UK). Bands were detected by ECL (Amersham Pharmacia Biotech), and band intensity was measured using Kodak ID Image Analysis Software (Eastman Kodak Scientific Imaging Systems). As previously reported, the 20S proteasome {alpha}- and {beta}-subunits each produced a single major band due to the fact that each of the primary antibodies used reacts with a core subunit and not a specific subunit of {alpha}- and {beta}-subunits (27). The high molecular weight ubiquitin-conjugated proteins were assessed as the sum of bands with molecular weight of >65. The density of bands was analyzed three times (with the variation < 10%), and the average value was reported.

Skeletal muscle IGF-I content.   Muscle IGF-I was measured by RIA as we have previously described (38). Briefly, IGF-I was extracted by the method of Fan et al. (16). Muscle samples (200 mg) were homogenized in 1 ml of 0.5 N HC1 at 4°C and centrifuged at 13,500 g for 10 min. Supernatants were purified using Sepak C18 Plus minicolumns (Waters, Milford, MA), eluted with methanol, dried, and suspended in RIA buffer containing 10 mM disodium EDTA, 30 mM phosphate, 0.02% sodium azide, 0.02% protamine sulfate, 0.25% bovine serum albumin, pH = 7.5. RIA was performed using [125I]-[des MET-0] human IGF-I 1–70 (Bachem, Torrance, CA) that was radiolabeled by the iodogen method. IGF-I antiserum IUB2-495 was obtained through the national Hormone and Pituitary Program, National Institute of Diabetes, Digestive, and Kidney Diseases. The standard curve employed rat IGF-I (GroPep, Adelaide, Australia). Bound and free 125I were separated by incubation with goat anti-rabbit gamma globulin (Pel Freez Biologicals, Rogers, AR) and normal rabbit serum, followed by centrifugation at 4,000 g for 30 min. Data were analyzed using PRISM software.

Statistical analysis.   All values are reported as means ± SE. Unless otherwise stated, a two-way ANOVA followed by a Newman-Keuls test post hoc was used for statistical analysis. Statistical significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of HU and CB on body and muscle weights.   There were no significant differences in initial body weight among groups. At the end of the experiment, body weight was significantly increased by 8.5% in C animals and by 19 and 6.8% in CB and HU-CB animals, respectively, whereas the body weight of HU animals was decreased by 5.2% compared with the initial body weight (Table 1). Sol, PA, and TA muscle weights were significantly reduced (46.3, 24.2, and 11.8%, respectively) after 14 days of HU compared with C (Table 1). CB treatment increased the weight of all three muscles 35.5% in TA, 28.3% in PA, and 17.4% in Sol. CB attenuated (P < 0.05) the loss of muscle weight associated with unweighting in PA and TA but not in Sol. HU-induced loss of muscle mass in PA and TA was –24.2 and –11.8%, respectively, without CB, compared with –9.1 and 13.1% with 14 days of CB treatment. HU significantly reduced the ratio of muscle weight-to-body weight in Sol and PA but not in TA. CB increased the same ratio in PA and TA but not in Sol. CB treatment of HU animals significantly increased the ratio of muscle weight-to-body weight in TA but not in PA or Sol.


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  		Table 1. Effects of 14 days of HU and CB on body weight muscle weight, muscle weight to body weight ratio (MW/BW), and myofibrillar and total protein contents

 
Effects of HU and CB on total and myofibrillar protein content.   HU significantly reduced total and myofibrillar protein content of all three muscles (P < 0.05; Table 1). This reduction was greater in Sol (53%) than in PA (29.9%) or TA (20%). CB significantly increased total and myofibrillar protein in all three muscles (P < 0.05). Total and myofibrillar protein were increased by 45.2 and 49.1%, respectively, in TA, by 30.2 and 30.3% in PA, and by 33.3 and 30.1% in Sol. CB prevented the HU-induced reduction in total and myofibrillar protein contents in PA and TA but not in Sol.

Effects of HU and CB on protein degradation rate.   The rate of protein degradation in Sol was estimated in vitro by measuring release of tyrosine into the medium. The Sol is small enough to allow for adequate oxygenation so that the release of tyrosine is not greater than normal. HU caused a 69% increase in protein degradation in the Sol (Fig. 1). CB treatment failed to attenuate the increase.



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  		Fig. 1. Effects of 14 days of hindlimb unweighting (HU) and clenbuterol treatment (CB) on protein degradation rates as measured by the rate of tyrosine release from the soleus muscle. Experimental groups are control (Con), HU, CB, and CB plus HU (CB + HU). Values are means ± SE; n = 5–6. *P < 0.05 vs. Con.

 
Effects of HU and CB on components of the ubiquitin-proteasome pathway.   We examined the association of HU-induced muscle atrophy with the activation of individual components of the ubiquitin-proteasome pathway in fast- and slow-twitch muscles. In all muscles, HU induced a significant increase in high molecular weight ubiquitin conjugates (P < 0.05; Fig. 2). High molecular weight ubiquitin conjugates were increased by 255% in Sol, 157% in PA, and 135% in TA muscle. CB significantly reduced ubiquitin conjugates in PA and TA but not in Sol. In addition, CB completely prevented the HU-induced increased accumulation of ubiquitin conjugates in the PA and TA but not in Sol.



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  		Fig. 2. Effects of 14 days of HU and CB on the level of high-molecular weight (HMW) ubiquitin (Ub) conjugates in soleus (A), plantaris (B), and tibialis anterior (C) muscles. Values are means ± SE arbitrary units of optical density, expressed as % of control; n = 6–8. *P < 0.05 vs. Con. #P < 0.05 vs. HU.

 
HU caused increases in protein expression of the ubiquitin-conjugating enzyme 14-kDa E2 that were significant in PA (20%; Fig. 3B) and TA (31%; Fig. 3C) but not in Sol (20%; Fig. 3A). CB treatment induced a significant increase in the expression level of 14-kDa E2 in the TA (P < 0.05) but not in PA or Sol. The expression level of 14-kDa E2 protein was increased in CB-HU compared with C in TA but not in Sol or PA.



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  		Fig. 3. Effects of 14 days of HU and CB on the level of Ub-conjugating enzyme E2-14kDa in soleus (A), plantaris (B), and tibialis anterior (C) muscles. Values are means ± SE arbitrary units of optical density, expressed as % of control; n = 6–8. *P < 0.05 vs. Con.

 
Chymotrypsin-like and trypsin-like activities of the 20S proteasome were determined in crude muscle extracts as described in METHODS. HU significantly increased both chymotrypsin-like (34%) and trypsin-like (32%) activity of 20S proteasome in TA (P < 0.05) but not in PA and Sol (Figs. 4 and 5). CB had no effect on the activity of 20S proteasome in any of the three muscles examined. However, CB treatment of HU animals effectively suppressed the HU-induced increased activity level of chymotrypsin-like activity in both PA and TA muscles. The level of trypsin-like activity of HU animals was also significantly reduced in the TA but not in the PA or Sol after CB treatment.



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  		Fig. 4. Effects of 14 days of HU and CB on the chymotrypsin-like activity of proteasome in soleus (A), plantaris (B), and tibialis anterior (C) muscles. Values are means ± SE; n = 6–8. *P < 0.05 vs. Con. #P < 0.05 vs. HU.

 


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  		Fig. 5. Effects of 14 days of HU and CB on the trypsin-like activity of proteasome in soleus (A), plantaris (B), and tibialis anterior (C) muscles. Values are means ± SE; n = 6–8. *P < 0.05 vs. Con. #P < 0.05 vs. HU.

 
In all muscles, no treatments caused significant changes in expression level of the catalytic {beta}-subunit of 20S proteasome (data not shown). Similarly, in PA and TA muscles, treatments did not effect the level of the noncatalytic {alpha}-subunit of 20S proteasome (Fig. 6). However, in Sol, HU and CB-HU caused a significant decrease in the {alpha}-subunit of 20S proteasome (P < 0.05), whereas CB had no effect.



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  		Fig. 6. Effects of 14 days of HU and CB on 20S proteasome {alpha}-subunit content in soleus (A), plantaris (B), and tibialis anterior (C) muscles. Values are means ± SE arbitrary units of optical density, expressed as % of control; n = 6–8. *P < 0.05 vs. Con.

 
Effects of HU and CB on skeletal muscle IGF-I content.   To further examine the mechanism of CB-induced hypertrophy or attenuation of HU-induced atrophy, IGF-I protein content was measured in two fast-twitch muscles that showed changes in muscle mass (PA and TA). HU causes a significant reduction in IGF-I content in PA, but the reduction in TA was not significant (P > 0.05). This effect was not reversed by CB (Fig. 7). Surprisingly, CB treatment produced a significant decline in the level of IGF-I protein in PA, although not in TA.



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  		Fig. 7. Effects of 14 days of HU and CB on muscle IGF-1 content in plantaris (A) and tibialis anterior (B) muscles. Values are means ± SE; n = 5–6. *P < 0.05 vs. Con.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Overview of major findings.   In summary, HU-induced muscle atrophy was greatest in the predominantly slow-twitch Sol and was associated with increased protein expression of several components of the ubiquitin-proteasome proteolytic pathway. CB induced muscle hypertrophy and attenuated HU-induced atrophy. The attenuation of atrophy may have occurred through an inhibition of the ubiquitin-proteasome system in a muscle type-specific fashion. The effects of CB did not appear to be mediated by increases in muscle content IGF-I protein.

HU effects on muscle weight and protein contents.   The finding that HU causes a decrease in muscle mass (greater in slow muscle than in fast) and muscle protein is congruent with other studies (23, 25, 26). The mechanism(s) responsible for the decrease in total and myofibrillar protein in HU muscle are not clearly understood but are thought to be due to a combination of decreased protein synthesis and increased protein degradation. Our finding that protein degradation is increased by ~60% in the unweighted Sol is in agreement with the findings of other laboratories (25, 44). Of interest is our finding that CB treatment does not attenuate this increase. Because the rate of protein synthesis was not measured in the present study, the relative contribution of decreased protein synthesis to HU-induced muscle atrophy could not be quantified. Nonetheless, our findings strongly support the hypotheses that HU-induced muscle atrophy is due, in large part, to an increased rate of protein degradation.

CB effects on muscle weight and protein contents.   Our findings that CB induced hypertrophy and a marked increase in both total and myofibrillar protein are in agreement with previous findings (51). Because CB preferentially affects fast fibers, it is surprising that it also had a significant effect on the predominantly slow Sol muscle. The finding that CB treatment attenuates HU-induced muscle atrophy in the fast-twitch PA and TA muscles, but not in the slow Sol, is also consistent with those of others (42, 46, 51). Protein accretion in response to {beta}2-agonists may be due either to increased protein synthesis or to reduced protein degradation, but recent evidence suggests that protein degradation may be the predominant effect (6, 33, 35).

HU activates the ubiquitin-proteasome pathway of fast- and slow-twitch muscles.   The ubiquitin-proteasome system seems to be most responsible for the breakdown of intracellular proteins, including myofibrillar proteins (4, 24, 40). There are two major steps in the ubiquitin-proteasome pathway: 1) ubiquitin conjugation that involves the 14-kDa E2 and E3-{alpha} and 2) the subsequent degradation of ubiquitin-conjugates by the 20S proteasome. The finding that HU increases accumulation of ubiquitinated proteins in all muscles studied is consistent with reports in other models showing increased ubiquitinated proteins in catabolic states such as starvation, denervation (49), burn-injury (40), and HU (23).

The fact that HU did not alter the proteasome activity or expression of 20S proteasome {beta}-subunits in the Sol was unexpected, since a number of studies have reported that HU resulted in increased gene expression of several components of the ubiquitin-proteasome system, including several other subunits of the 20S proteasome (23, 44). The reason for the lack of the parallel changes between mRNA and protein is not apparent. The finding that HU markedly reduces expression of the noncatalytic {alpha}-subunit in Sol was also unexpected, especially since it was unchanged in the fast muscles. Further study is needed to determine the site(s) of differential regulation of this proteolytic pathway during unweighting.

One of the most interesting findings in this study was that HU caused an elevation of 20S proteasome activity in the fast-twitch TA that was not seen in Sol. Thus activation of the ubiquitin-proteasome system appears to be muscle-type specific. This finding is supported by several studies showing that fast-twitch muscle is more sensitive to catabolism than slow-twitch muscle in a variety of muscle-wasting conditions, including hyperinsulinemia (27) and burn injury (17). Because HU did not affect the expression of 20S proteasome {alpha}- and {beta}-subunits in fast muscles, the increased proteasome activity in the TA is likely the result of changes in the specificity of 20S proteasome. It has been shown that the 20S proteasome can be activated by a PA28 activator (12) to cause an increase in proteasome activity. Another possible explanation for our finding of increased 20S proteasome activity without increased expression may be posttranslational modifications, such as phosphorylation (9, 31). Mason et al. (31) showed that certain proteasome subunits (C8 and C9) can be phosphorylated in vivo, and dephosphorylation of these subunits results in small but significant decreases in the activity of 20S proteasome.

Interestingly, we found that PA and Sol muscles exhibited similar patterns of response with respect to the activation of the ubiquitin-proteasome system (i.e., no alterations in either the activity or expression level of proteasome subunits content). This was unexpected given that the PA contains mainly the fast-twitch fibers, whereas Sol mainly consists of slow-twitch fibers. The explanation for these similar effects may reside in the fact that, during unloading, the hindlimb assumes a plantar-flexed posture that results in shortening of PA and Sol with lengthening of TA (36). Collectively, these data suggest that the activation of the ubiquitin-proteasome system during HU is not only fiber-type specific but also muscle specific.

CB inhibits the ubiquitin-proteasome proteolysis in fast-twitch muscle.   CB is known to exert its anabolic effects largely through reduced protein degradation (6, 33, 35). Although CB caused a moderate degree of hypertrophy in Sol, it did not inhibit the activity or expression of the proteasome in Sol. This suggests that other protease systems and/or increased protein synthesis may have played the major role. However, because CB significantly reduced ubiquitin conjugates in TA and PA, it appears that CB preferentially inhibits the ubiquitin-proteasome pathway in the fast-twitch muscles. This pattern correlates with the changes seen in muscle mass with CB. Notably, because CB did not affect the activity or expression of the 20S proteasome subunits in these muscles, it seems that CB had a specific inhibitory effect on the ubiquitin conjugation. This finding is in keeping with a previous report that CB reduces expression of ubiquitin mRNAs (11). Our experimental design did not allow us to calculate the relative contributions of increased protein synthesis and decreased protein degradation to CB-induced hypertrophy of these muscles. Nevertheless, recent studies (11, 35) have shown that, in normal rats, rates of protein degradation in the fast-twitch gastrocnemius muscle were markedly reduced, whereas protein synthesis rates were not affected by CB treatment. Taken together, these findings suggest that CB exerts its hypertrophic effect, at least in part, by reducing muscle proteolysis through an inhibition of the ubiquitin-proteasome pathway in the fast-twitch PA and TA but not in the slow-twitch Sol.

CB attenuates HU-induced upregulation of ubiquitin-proteasome proteolysis in fast-twitch muscle.   {beta}-Adrenergic agonists such as CB and cimaterol have been shown to increase muscle mass (50) and to reduce muscle atrophy induced by denervation (3, 52) and HU (15, 46). This study demonstrates that CB attenuates the increased activity of 20S proteasome during HU and the accumulation of ubiquitin conjugates in the myofibrillar fraction in fast-twitch muscles, with the magnitude of effect greater in the TA than the PA muscle. Moreover, it should be noted that the magnitude of these measures was below the basal control level. These data clearly indicate that CB treatment attenuates the HU-induced muscle atrophy by suppressing the activation of the ubiquitin-proteasome pathway in the fast-twitch muscle. This finding is in keeping with a previous study in which CB reduced ubiquitin mRNA in cachectic, tumor-bearing animals (11). The ubiquitinating step using 14-kDa E2 is a potentially rate-limiting step in the ubiquitin pathway. The fact that CB did not reduce 14-kDa E2 protein expression in Sol or PA, and even caused an increase in TA, suggests that 14-kDa E2 is not crucial for the activation of the ubiquitin-proteasome pathway during unweighting. This interpretation is supported by a study showing that fasting-induced muscle atrophy is unchanged in mice lacking the 14-kDa E2 gene (2).

Recently, two skeletal muscle-specific ubiquitin ligases have been identified (7, 20). Muscle RING finger 1 and muscle atrophy F-box are each thought to play a role in skeletal muscle atrophy. Both are upregulated in models of disuse such as immobilization, denervation, and HU. In addition, knockout mice lacking these proteins have significantly reduced muscle atrophy following denervation. It is not known whether CB can affect the expression of these E3 ligases. We demonstrated that CB treatment of HU animals did not inhibit protein markers for the activation of the ubiquitin-proteasome system and accelerated muscle proteolysis in Sol (Fig. 2). Taken together, these results suggest that CB may attenuate HU-induced muscle atrophy through a downregulation of the ubiquitin-proteasome proteolysis in fast-twitch muscle only.

IGF-1 as a potential mediator for {beta}2-agonist action.   Although CB binds to {beta}2 receptors and activates the synthesis of cAMP (50), it is also possible that {beta}2-agonists cause muscle hypertrophy indirectly through the production of other factors. IGF-I is an obvious candidate because it plays such a central role in myogenesis and postnatal muscle growth (19). Others have shown that HU lowers IGF-I expression in skeletal muscle (43). In the present study, we examined IGF-I in the muscles most affected by CB, TA, and PA and found that HU reduced muscle IGF-I content in PA muscle, but the decrease was not significant in the TA. Awede et al. (5) showed that CB-induced hypertrophy is associated with a transient increase in muscle IGF-I content, which was observed in Sol after 3 days of CB treatment but not after 9 days of treatment. Our findings that CB reduces IGF-I in PA muscle and had no effect on IGF-I in HU PA or TA muscles indicates that a sustained local production of IGF-I does not occur during CB-induced muscle hypertrophy. However, the possibility that a transient increase in IGF-I may play a role in CB-induced hypertrophy cannot be eliminated.

In summary, in the present study, we demonstrated that HU-induced muscle atrophy is associated with increased activity of the ubiquitin-proteasome system in both slow- and fast-twitch muscle, although to a different degree. CB treatment attenuated these effects by reducing the activation of ubiquitin-proteasome proteolysis in the predominantly fast-twitch PA and TA but not in the slow-twitch Sol. The attenuation of atrophy by CB is probably not mediated by the IGF-I peptide in skeletal muscle.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. L. Dodd, PO Box 118205, Univ. of Florida, Gainesville, FL 32611 (E-mail: sdodd{at}hhp.ufl.edu)

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.


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