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1 Faculty of Pharmaceutical Sciences, University of Kanazawa, Kakuma, Kanazawa 920-1192; and 2 Department of Life Sciences, University of Tokyo, Komaba, Tokyo 153-8902, Japan
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We hypothesized that a shift in muscle
fiber type induced by clenbuterol would change monocarboxylate
transporter 1 (MCT1) content and activity of lactate dehydrogenase
(LDH) and isoform pattern and shift myosin heavy chain (MHC) pattern in
soleus (Sol) and extensor digitorum longus (EDL) of male rats.
In the clenbuterol-administered rats (2.0 mg · kg
1 · day1
subcutaneously for 4 wk), the ratio of muscle weight to body weight
increased in the Sol (P < 0.05) and the EDL
(P < 0.01). Clenbuterol induced the appearance of fast
MHC2D and decreased slow MHC1 in Sol (13%) but
had no effect on EDL. The MHC pattern of Sol changed from slow to fast
type. Clenbuterol increased LDH-specific activity (P < 0.01) and the ratio of the muscle-type isozyme of LDH to the heart type
(P < 0.05) in Sol. The LDH total activity of the EDL
muscle was also increased (P < 0.05). Furthermore, MCT1 content significantly (P < 0.05) decreased in
both Sol and EDL (27 and 52%, respectively). This study suggests that
clenbuterol might mediate the shift of MHC from slow to fast type and
the changes in the regulation of lactate metabolism. Novel to this study is the observation that clenbuterol decreases MCT1 content in the
hindlimb muscles and that the decrease in MCT1 is not muscle-type specific. It may suggest that the genetic expressions of individual factors involving slow-type MHC, heart-type isozyme of LDH, and MCT1
are associated with one another but are regulated independently.
lactate dehydrogenase; myosin heavy chain; 
2-agonist; transition
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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CLENBUTEROL IS ONE OF
THE selective 2-adrenergic-receptor agonists and
is mainly used as a bronchodilator and tocolytic agent to relax the
uterus. It also has potent anabolic properties (2, 9, 14,
35); thus its usage as a drug for doping is banned by many
athletic committees (33). However, there are still unclear effects on lipid metabolism, thermogenesis, and hyperglycemia (25, 43). The higher distribution of
2-receptors in slow-twitch muscle (i.e., the
increased intracellular cAMP via the receptor) is one of the plausible
explanations of the specific effect of clenbuterol on the slow-type
muscles, including the transition of myosin types from slow to fast and
the metabolic shift from oxidative to anaerobic glycolysis (22,
39). Lactate dehydrogenase (LDH) plays a pivotal role in
anaerobic glycolysis (11). The ratio between the
muscle-type isozyme (LDH-M), which has a higher Km to pyruvate, and the heart type (LDH-H),
which has a lower Km to pyruvate, is an
important index of the glycogen and lactate metabolism of skeletal
muscle (37). Increased lactate or LDH-M activity in the
soleus (Sol) muscle was reported, and the increase in glycolysis was
suggested to be due to the administration of clenbuterol (26,
40). Lactate not only is an end product of glycolysis and
glycogenolysis but is also a substrate for oxidation and
gluconeogenesis (6, 18, 19). Several recent studies have
shown that movement of lactate across the sarcolemma depends, in part,
on a monocarboxylate transporter (MCT) (6, 8, 17, 18, 31).
It has been reported that there are eight isoforms of MCT (5, 20,
23, 34). MCT1 is present in rats mainly in oxidative red muscles
and in the heart (1, 31). Recently, Brooks et al.
(6-8) showed that MCT1 was located in the sarcolemmal membrane as well as the mitochondria. They showed that MCT4 was also
located in the sarcolemmal membrane (15). In cell-cell interaction, the uptake of lactate in red muscles and in the heart is
increased when MCT1 increases after endurance training. Therefore, MCT1
is highly related to the oxidative capacities of skeletal muscles and
their capacity to take up lactate extracellularly (3, 4, 17,
33) and intracellularly (7, 8). It has been
suggested that the higher distribution of LDH-H subunits is related to
MCT1 distribution in skeletal muscles (27, 28). In
slow-twitch muscles, the shift of metabolic property from oxidative to
glycolytic with clenbuterol is demonstrated by increased
phosphofructokinase activity and decreased citrate synthase activity
(14), as well as decreased ratio of LDH-H to LDH-M
(40). However, a change in MCT1 has not been reported. In
the present study, we hypothesized that these shifts of metabolic and
contractile properties by clenbuterol are muscle-type specific.
Furthermore, we hypothesized a decrease in MCT1 in the slow-twitch
muscle type, based on the evidence that 2-receptor
density has been shown to be correlated with the oxidative potential,
the percentage of slow-type fibers (22), and the
coexpression of MCT1 and LDH-H (28). Therefore, we
evaluated clenbuterol's effects on the relationship between the
metabolic changes and the shift of myosin types.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal care and experimental protocols.
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.
Animal care and use protocols were in accordance with the Guiding
Principles for the Care and Use of Animals approved by the Council of
the Physiological Society of Japan. Two animals were housed per cage
(25 × 40 × 20 cm), and, at 9 wk of age, animal pairs were
randomly assigned to a saline (Con, n = 6) or
clenbuterol (Cleb, n = 6) group. Clenbuterol (2 mg · kg body wt1 · day1;
Sigma Chemical) was administered subcutaneously once daily for 4 wk.
Con rats were injected with 0.5 ml/kg body wt of normal saline once
daily for 4 wk.
Tissue sampling.
At the end of the 4 wk of treatment, the animals were killed with an
intraperitoneal injection of 50 mg/kg pentobarbital sodium and
decapitated. The Sol and extensor digitorum longus (EDL) muscles were
quickly removed and weighed. Muscle samples from the left hindlimb were
used to determine LDH activity and LDH isozymes, and samples from the
right hindlimb were immediately frozen in isopentane cooled by liquid
nitrogen for the analysis of myosin heavy chain (MHC) isoforms and MCT1
and then stored at 
80°C until the assay. Protein concentration was
determined by using the bicinchoninic acid protein assay kit (Pierce,
Rockford, IL) for subsequent analyses.
LDH analysis. For the analysis of metabolic property, the tissues were homogenized for 10 s in a 1:50 (wt/vol) dilution of LDH extraction buffer containing 50 mM Tris · HCl, 0.3 M sucrose, 0.1 M KCl, 1 mM EDTA, 5 mM MgCl2, and 0.5 mM phenylmethylsulfonyl fluoride at pH 7.4 with a polytron homogenizer. Homogenates were then centrifuged at 4°C for 5 min at 600 g. The resulting supernatant produced an enzyme sample that was decanted and assayed for LDH activity and LDH isozymes (13, 42). The LDH activity was measured spectrophotometrically by using a Monotest LDH kit from Boehringer (Mannheim, Germany). The reaction mixture was composed of 50 mM phosphate buffer (pH 7.5), 0.6 mM sodium pyruvate, and 0.18 mM NADH. After preincubation at 30°C for ~10 min, an enzyme sample was added to the reaction mixture, and the reaction was carried out at 30°C, measuring the average decrease in absorbance of NADH at 365 nm. The extinction coefficient of 9,118 was used to convert the absorbance change to the molar concentration of the product formed, and the values obtained were converted to those of 25°C using conversion factor 0.75. One unit of enzyme activity was defined as 1 mmol of NADH reduced per minute at 25°C.
Aliquots of the enzyme sample were also used for LDH isozyme analysis electrophoretically by using the method of Diets and Lubrano (13). The gels were composed of 6.5% acrylamide, 0.17% N,N'-methylene-bis-acrylamide (bis-acrylamide), 375 mM Tris · HCl (pH 8.9), 0.05% ammonium persulfate (APS), and 0.05% N,N,N',N'-tetramethylenediamine (TEMED) and were 45 ×85 ×1 mm. Each lane was loaded with ~5 µg of protein, and the gels were run in a vertical-slab gel system (TS-35, Wakamori, Tokyo, Japan) at 20 mA/gel (constant current) in a cold room at 4°C for 60 min. After electrophoresis, the gels were stained enzymatically in a solution containing 0.1 M sodium lactate, 10 mM NaCl, 0.5 mM MgCl2, 1 mg/ml NAD, 0.25 mg/ml nitro blue tetrazolium, 0.025 mg/ml phenazine methosulfate, and sodium phosphate buffer, pH 7.4.MHC analysis. MHC composition was assessed by using SDS-PAGE procedures essentially as described by Talmadge and Roy (38). Crude myofibrillar extracts containing MHCs were obtained from samples of ~100 mg, pulverized in liquid nitrogen, treated with the denaturing solution (10% glycerol, 2% SDS, 5% 2-mercaptoethanol, 62.5 mM Tris · HCl, pH 6.8) for SDS-PAGE in a glass homogenizer, and heated for 5 min in boiling water.
The stacking gel consisted of 4% acrylamide, 0.08% bis-acrylamide, 30% glycerol, 70 mM Tris · HCl (pH 6.8), 4 mM EDTA, 0.4% SDS, 0.1% APS, and 0.05% TEMED. The separating gel was composed of 8% acrylamide, 0.16% bis-acrylamide, 30% glycerol, 0.4% SDS, 200 mM Tris · HCl, 100 mM glycine, 0.1% APS, and 0.05% TEMED (pH 8.7). The running buffer in the upper chamber contained 100 mM Tris · HCl, 150 mM glycine, and 0.1% SDS (pH 8.3). The running buffer in the lower chamber contained 50 mM Tris · HCl, 0.075 M glycine, and 0.05% SDS. Each lane was loaded with ~500 ng/lane of protein, and the gels (106 × 100 × 1 mm) were run in an electrophoretic chamber (KS-8012, Marysol, Tokyo, Japan) at 70 V (constant voltage) in a cold room at 4°C for 22 h. After electrophoresis, the individual MHC isoforms were measured after they were shown using a silver-stain technique (30). The MHC isoforms were identified based on data reported in the literature (36) and on digitized densitometric data sets with the use of these procedures (order of migration: fast-type 2A, fast-type 2D, fast-type 2B, and slow-type 1 MHC isoforms). The LDH isozymes or MHC isoforms were photographed, scanned with a digital imaging system, and quantified using computerized densitometry (NIH Image 1.61 analysis software).MCT1 analysis. Polyclonal antibody directed against COOH terminus of MCT1 (N'-CPQQNSSGDPAEEESPV-C') (23) was produced by immunizing New Zealand White rabbits with a synthetic peptide corresponding to amino acids 478-494 of MCT1 (Sawady Technology, Tokyo, Japan). This antibody was raised and affinity purified as described elsewhere (32). The polyclonal antibody yielded a single band on a Western blot that corresponded to a molecular weight of ~43,000, consistent with the molecular weight reported for MCT1 (1, 17). It was confirmed that the antibody cross-reacted with MCT1 from rat and Chinese hamster, but not human, in a preliminary experiment. Sample preparation for Western blotting was done according to McCullagh et al. (28). Briefly, muscles were homogenized in 210 mM sucrose, 2 mM EGTA, 40 mM NaCl, 30 mM HEPES, 5 mM EDTA, and 2 mM phenylmethylsulfonyl fluoride, pH 7.4. After homogenization, 1.17 M KCl and 58.3 mM tetrasodium pyrophosphate were added. After ultracentrifugation at 230,000 g for 75 min at 4°C, the supernatant fluid was discarded, and the pellet was washed and resuspended in 10 mM Tris · HCl and 1 mM EDTA, pH 7.4, solution. Then 16% of SDS solution was added. Samples were centrifuged at 1,100 g, and the supernatant was used for protein assay and Western blotting detection of MCTs. Western blotting of MCT1 was done according to McCullagh et al. (27) and Wilson et al. (41). Samples were applied to 12% SDS-polyacrylamide gels. Proteins were transferred from the gel to membrane (Hybond-C extra, Amersham). Membranes were blocked by 20 mM Tris · HCl, 137 mM NaCl, 0.1% Tween 20, and 10% nonfat dry milk, pH 7.5, solution. Membranes were then incubated with antibody solution for 1 h. After being washed in 20 mM Tris · HCl, 137 mM NaCl, and 0.1% Tween 20, pH 7.5, membranes were incubated with donkey anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:3,000; Bio-Rad) in 20 mM Tris · HCl, 137 mM NaCl, and 0.1% Tween 20, pH 7.5. MCT1 was detected using an enhanced chemiluminescence detection method (Amersham) by exposing the membrane to film (Fuji, Tokyo, Japan). Band densities of MCT1 were obtained by scanning the film and using the NIH Image 1.61 analysis software.
Statistical analyses. Differences between groups were determined using one-way ANOVA. Post hoc differences were determined with Scheffé's test and unpaired t-test where appropriate. Significance was evaluated in all statistics at P < 0.05 and/or P < 0.01. Data are reported as means ± SD.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The effect of clenbuterol treatment on body weight and muscle wet
weight of rats is shown in Table 1. The
Cleb group showed significantly lower body weight than the Con group
(P < 0.05). The muscle wet weights of the Cleb group
were higher than those of the Con group (P > 0.05;
Table 1) in the Sol (9%) and EDL (12%), and the ratio of muscle wet
weight to body weight was significantly higher (Sol: P < 0.05; EDL: P < 0.01) in the Cleb group than in the
Con group. Therefore, the skeletal muscles tended to be hypertrophied as a result of clenbuterol treatment.
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The MHC isoform distribution is shown in Table
2. In the Sol, which consisted of only
MHC1 and MHC2A, the percentage of
MHC1 decreased (P < 0.01) with
clenbuterol. Furthermore, MHC2A did not change, but
MHC2D increased (P < 0.01) in the Sol.
This suggests that the transition of MHCs from slow to fast might have
occurred within the muscle.
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In the EDL, the percentage of fast-type MHC2B and MHC2D tended to increase, and both MHC1 and MHC2A decreased, but these differences were not significant.
The LDH activity is shown in Table 3.
Both the LDH-specific activity of Sol (P < 0.01) and
the total activity were higher in Cleb rats than in Con
(P < 0.05). In EDL, the total activity was
significantly higher than in Con (P < 0.05).
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The LDH isozyme distribution is summarized in Table
4. In Sol, the distribution pattern of
LDH isozymes shifted from LDH-H predominant to LDH-M predominant, and
the ratio of LDH-M to LDH-H increased significantly with clenbuterol,
but the isozymes of EDL were not affected.
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Figure 1 shows the MCT1 contents in Sol
and EDL. MCT1 contents of the Cleb group were significantly lower
(P < 0.05) than those of the Con group in both the Sol
and the EDL muscles (Sol Cont vs. Cleb: 17.80 ± 3.91 vs.
12.98 ± 3.23%; EDL Cont vs. Cleb: 8.12 ± 3.47 vs.
3.92 ± 1.93% of rat heart standard). This means that clenbuterol
induced the reduction of MCT1 expression in both Sol (27%) and EDL
(52%).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The present study tested the hypothesis that the
2-agonist clenbuterol would produce muscle-type-specific
differential results (i.e., a slow-to-fast shift in the MHC content in
the slow-twitch Sol muscle, an oxidative-to-glycolytic metabolic shift
in the LDH isoforms and the MCT1 content, but no changes in fast-twitch EDL muscle). A novel and significant finding is the demonstration that
clenbuterol administration decreases MCT1 content in both Sol and EDL
muscles and the decrease of MCT1 is not muscle-type specific (Fig. 1).
Our data (Tables 2-4) clearly support our hypothesis in the Sol
muscle but argues against it in the fast-twitch EDL muscle. These data
suggest that the genetic expressions of individual factors involving
slow-type MHC, LDH-H, and MCT1 are associated with one another but are
regulated independently.
Previous studies show that clenbuterol administration induces a
transition from slow to fast MHC phenotypes in the slow-twitch Sol
muscle (10, 14) but no change in MHCs in fast-twitch
plantaris and gastrocnemius muscles (14). In Sol muscle,
our results (Table 2) support these findings, and the magnitude of the
decrease in MHC1 (13%) was a little larger than that of
the decrease (about 8%) in MHC1 obtained in a prior
study by Dodd et al. (14). This difference might be due to
the longer treatment of clenbuterol for 4 wk in the present study than
their treatment for 2 wk. In fast-twitch EDL muscle, we also showed
that there was no change in MHCs. The present study (Table 2) shows
that the expression of fast-type MHCs is induced in the rat Sol by
clenbuterol treatment. The MHC isoform profile shows a partial
transformation toward a "fast" phenotype with a marked increase in
MHC2D and a corresponding relative decrease in slow-type
MHC1 (Table 2). This shift and the enlargement of muscle
(Table 1) may account for the increased contraction velocity and
tension development in these muscles reported by others
(14). They were explained by the relationship of
clenbuterol to MyoD, which regulates fast MHC synthesis, or myogenin to
slow MHC (12, 29).
MCT1 has a close relationship to lactate metabolism, and there is a
significant relationship between MCT1 content and the percent muscle
fiber type (slow oxidative + fast oxidative glycolytic) (3,
4, 27, 28). Therefore, as our data (Tables 2-4) show, there
must be some influence on lactate metabolism and MCT1 content when the
percentage of muscle-fiber-type changes (28, 31). Although
this interactive relationship between the metabolic changes and
muscle-fiber composition is not established, it might be explained in
part by a genetic regulation via cAMP. In our study, the relationship between the expression of MCT1 and the genetic regulation by cAMP was
not tested; however, a strong inverse relationship can be expected.
McCullagh et al. (27) showed that MCT1 content is inversely correlated with LDH total activity. The
2-agonist clenbuterol increases an intracellular cAMP,
which regulates the expression of cAMP response element (CRE) and CRE
binding protein in the nucleus. This CRE or CRE binding protein
regulates LDH-M expression (16, 24). These data suggest
that the increased cAMP stimulated by clenbuterol directly induced an
increase in LDH-M in skeletal muscles (i.e., the increased LDH activity
and the decreased ratio of LDH-H to LDH-M). Our data (Tables 3 and 4)
in Sol muscle support this notion. The smaller distribution of the
2-receptor in fast-twitch muscle (22) may
explain the effect of clenbuterol on LDH in EDL but may not explain the
significant decrease of MCT1 in EDL. Although the tendency of increased
LDH-specific activity in EDL was not significant (P = 0.08), the increased LDH total activity may be explained by the
increased muscle mass (Table 1). Therefore, the different response to
clenbuterol in LDH and MCT1 remains to be explained. One plausible
explanation is that the turnover rate of MCT1 might be faster than that
of LDH. On the other hand, the genetic regulation in MCT1 expression
might be different from LDH-H or LDH-M. As it is unknown whether MCT1 expression is inhibited by cAMP, the transcriptional effect of clenbuterol on MCT1 expression awaits further characterization.
MCT1 plays a role in the muscle's capacity to exchange lactate among cells, as well as to take up and oxidize lactate in cells with high mitochondrial density (15). Slow-twitch muscles have more MCT1 in sarcolemmal and mitochondrial membranes than do fast-twitch muscles (7, 15) and may be able to oxidize more lactate than can fast-twitch muscle due to the higher LDH-H percent. As Brooks et al. (7, 8, 15) showed, the important localization of MCT1 in mitochondrial membranes and the presence of LDH-M in mitochondria should also be studied to explain the difference between Sol muscle and EDL in metabolic adaptation. The speculation that EDL has a higher distribution of LDH-M in the mitochondria-like liver may explain the lesser effects of clenbuterol on LDH and MCT1 in EDL than in Sol, which is speculated to have higher distribution of LDH-H in the mitochondria. Future investigations need to examine MCT4 as a possible candidate for intra- and extracellular lactate shuttles mechanism in the muscle (7, 15, 23). Furthermore, we should consider the effect of lypolysis, which is increased by clenbuterol (43). The MCT1 in the mitochondria can transport pyruvate along with ketone bodies, resulting in the conversion to acetyl-CoA (20). Increased acetyl-CoA inhibits pyruvate dehydrogenase activity in the mitochondria by end-product inhibition. This would be followed by an increase in the production of lactate due to the pyruvate dehydrogenase inhibition (21). This sustained specific condition produced by clenbuterol may induce the downregulation of MCT1 and the upregulation of LDH-M. Thus the higher distribution of MCT1 in the mitochondria may be able to explain the faster increase of LDH-M in the Sol compared with the EDL (Tables 3 and 4).
In conclusion, the present study shows that clenbuterol increases Sol and EDL muscle mass-to-body weight ratio, induces the appearance of the fast-type MHC2D isoforms, and decreases the slow-type MHC1 in Sol muscle. Finally, because clenbuterol depressed MCT1 expression in both Sol and EDL muscles, it appears that changes in MCT1 are not muscle-type specific like LDH isozymes or MHC isoforms. The present study is the first to demonstrate that clenbuterol administration decreases MCT1 concentrations in EDL as well as in Sol skeletal muscles.
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ACKNOWLEDGEMENTS |
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We thank Dr. William J. Kraemer, Dr. William Fink, Dr. Bruce Craig, and Mary Ann for helpful advice and assistance during the final reading of the manuscript.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Kitaura, Faculty of Pharmaceutical Sciences, Univ. of Kanazawa, Kakuma, Kanazawa 920-1192, Japan (E-mail: kitaura{at}dbs.p.kanazawa-u.ac.jp).
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 5 October 2000; accepted in final form 12 February 2001.
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TOP
ABSTRACT
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METHODS
RESULTS
DISCUSSION
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