Effects of chronic β2 adrenergic agonist clenbuterol administration on 3-hydroxyacyl-CoA dehydrogenase activity in rat skeletal muscles

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(1)Title. Effects of chronic β2 adrenergic agonist clenbuterol administrat ion on 3-hydroxyacyl-CoA dehydrogenase activity in rat skeletal m uscles. Author(s). 鈴木, 淳一. Citation. 冬季スポーツ研究, 3(1): 13-18. Issue Date. 2001-03. URL. http://s-ir.sap.hokkyodai.ac.jp/dspace/handle/123456789/6785. Rights. Hokkaido University of Education.

(2) Effects of chronic R, adrenergic agonist clenbuterol administration on 3hydroxyacyl-CoA dehydrogenase activity in rat skeletal muscles Junichi SUZUKI. Laboratory of Sports Physiology, Research and Education Centerfur Winter Sports, Ho kkaido University of Education, 002-8502 Japan Abstract The effects of chronic clenbuterol administration on O-oxidative enzyme activity in skeletal muscles were studied in young (10-week-old) male Wistar rats. Rats of the treated groups were fed a diet containing 2 m$Kg clenbutero! hydrochloride continuously for 1 week, followed by a intermittent protocol (2-day-on / Zday-ofF) for following 25 days. Clenbuterol treatment increased body weight and weishis of the heart and hindlimb muscles. The protein concentration (mg!g wet wt) showed no significant difference after the treatment in all skeletal muscle examined. The clenbuterol treatment significantly increased the protein content (mgJwhole muscle) by 18%, 37% and 37%, respectively, in soleus, plantaris and gastricnemius muscles (P<0.05). Afier the treatment, the activity of 3-hydroxyacyl-CoA dehydrogenase was significantly decreased by 53% and 33%, respectively, in plantaris and gastrocnemius muscles (P<Q.05). In SOL,however, the activity did not change after the treatment. These results suggest that clenbuterol decreased B-oxidative enzyme activity in predominantly fast-twitch muscles, The muscle hypertrophy without improving oxidative capacity may reduce the endurance performance in skeletal muscles.. Key words:. 3-hydroxyacyl-CoA dehydrogenase; clenbuterol; skeletal muscle; total pro-. tein content. The 8,-agonicts are widely used as 'bronchodilators for the prevention and treatment of symptoms of exercise-induced asthma. As well as hypertrophic effects on skeletal and cardiac 5.7,9>.~2,~51 , the B-agonists affect lipid metaboIism '), Reduction of body fat and increased energy expenditure are among the most pronounced physiological effects of chronic FJagonist treatment '[". Decreased body fat may be a consequence of increased fat synthesis in adipose tissue and liver, or a combination of both. The ability of adrenaline to modulate lipid metabolism directly in fiver and adipose tissue has been demonstrated in many animal species 4*'". Binding of l3-agonists to adipose tissue adrenergic receptors activates in turn adenylate cyclase, cyclic AMP levels, the protein kinase cascade, and Ieads to the activation of the hormone sensitive Eipase and triacylglycerol hydrolysis. The. effects of a-agonists on lipid metabolism have been extensively investigated in the laboratory rodent species It is well documented that f3agonists can stimulate lipolysis in rat adipocytes and inhibit fatty acid synthesis in hepatocytes and adipocytes 8.'0! It is therefore possible that D-agonists can stimulate lipid metabolism in skeletal muscles. However, the effects of Ragonists on Iipid metabolism in skeletal muscle have not yet been determined, In this study, therefore, we investigated the activity of 3-hydroxyacyl-CoA dehydrogenase (I-IAD; EC 1.1.1.35). the key enzyme of mitochondrial R-oxidation, after long-term clenbuterol treatment in the hindlimb skeletal muscles.. ""',. Methods.

(3) Suzuki J. This study was approved by the Animal Care and Use Committee of Hokkaido University of Education and performed in accordance with the "Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences" of the Physiological Society o f Japan. Animals Sixteen young (9 week-old) male Wistar rats were purchased from Clea Japan Inc. (Tokyo, Japan). After the rats were fed for 7 days to allow adaptation to the new environment, rats of each age group were randomly divided into control (YC; n=8) and cIenbuterol fed (YCL; n=8) groups. All rats were housed under conditions of control temperature (24i1 "C) and a reIative humidity of about 50%. Lighting (7:OO19:OO) was controlled automatically. All rats were given tap water ad libitum. Control groups were fed a standard commercial laboratory chow (MF-type, Oriental East Co., Tokyo, Japan). The clenbuteral fed groups were fed a powdered diet (MF-type, Oriental East Co.) containing 2 mg kg- 1 clenbuterol hydrochloride (C-5423, Sigma Co., St. Louis, MO, USA) continuously for 1 week, followed by a intermittent protocoI (2-day-on / 2-day-off) for following 25 days. The intermittent administration could prevent an attenuation of the response occurred while continuous treatment '". The average clenbuterol intake, estimated from food intake, was 116 pg JKglday. Because the rats were housed two or three per cage, the food intake per individual rat was estimated from the food intake per cage.. Tissuepreparation Under pentobarbital anaesthesia (50 rnglkg i. p.), soleus (SOL), plantaris (PL) and gastrocnemius (GAS) muscles were rapidly excised, washed in cold saline, made free of surrounding connective tissue, weighed and frozen in liquid nitrogen. Then the hearts were excised, and whole hearts and leR ventricles (LV) were. weighed. The muscle samples were stored at -80 "C until biochemical analyses.. Sample preparation Muscle hornogenates (5% (W/V)) were obtained from around 50 mg of frozen tissue homogenized for two interrupted 15-s bursts with Polytron homogenizer (set at 15,000 rpm) in ice-cold buffer (lOmM Tris-HC1, pH 7.0; 175mM KCI, 1OmM glutathione (reduced from); 2mM EDTA). After centrifugation at 600 g for 10 min at 0 OC, the supernatant was divided into aliquotes for enzyme assay and protein assay. The aliquotes for protein assay were stored at -80 "C. HAD enzyme nctiviQ defermittathn Immediately after the sample preparation, HAD activity was determined using a spectsophotometric assay as previously described ". A volume of 100 pL supernatant was added to 890 pL of reagents ( I 00 mM triethanolarnine-MCI. pH 7.0; 5 m M EDTA; 0.45 mM NADH). The reaction was initiated by adding 10 pL of 10 mM acetoacetyI-CoA (SIGMA, A- 1625)- The measurements were carried out at 340 nm on a spectrophotometer (U-200 1 , Hitachi Co., Tokyo, Japan) at 25 "C. Table 1. Efec~sof clenhrrrerol feding on hn* orEan weiffl~(s. YC (n=X). and. YCL (n=R). Initial hody weight (g3. 294.3 f4.9. 298.0 14.9. Final hody weigh1 (g) Organ wcights (mg) Whole heart. 374.9 L8.9. 4 14.0 f6.6*. 886.3 f 17.9. 1050.7 f20.3*. I,elt ventricle. 633.6 f20.8. 725.6 & ] 7 . 7 *. Solcus Ganrocncmius Plantaris Valucs arc means group at P 4 . 0 5. 170.2kg.1. 194.5415.3. 1789.4 3 3 . 4. 2386.0 f60.OC. 413. I f 16.7. 566.4 f 17.8*. * SE. *. significant1 y di1Ttrt.n~liorn control.

(4) 3-hydmx) acyI-CoA dchydrogenase activity after clenbuterol treatment. SOL. GAS. Figure 1 IJll'cct ~Tclenhuzcroltreatment on the total protein conccntratlon in solcus (SOL). plantaris (PC) and gastrocnc~nius(GAS) fnusclcs.. Totnl protein defermination AII protein concentrations were determined in duplicate by the bicinchoninic acid (BCA) assay (Pierce Chemical Co., IL, USA) using BSA as a standard. SInti.~ficnl Anabrsis All results are expressed as meaneSE. Using the Kolrnogrov-Smirnoff test, we first tested the distribution of all parameters to detennine whether it was compatible with a norma! distribution. In the present study, all data sets showed a normal distribution. Unpaired Student's t test was used for parametric two sample comparison. Differences were considered to be statistically significant at P<0.05.. Results. Table 1 shows body and organ weights. At the end of the treatment, body weights of treated groups were significantly greater than those of control groups (P4.05). The clenbuterol treat-. SOL. 1'1,. GAS. Fi~vr2 e 1311'cct of clcnbutcrol lrcatmcnt on the total protcin contcnt in soleus (Sol,), plan~aris(I'L) and gastrocncmlus {GAS) musclcs. *. Slgni ticanlly dilkrcnt from YC (Pc0.05 j.. men! significantly increased the weights of whole heart, the LV and all skeletal muscles examined (P<0.05).Except for the SOL, the weights of all muscles were significantly greater in treated group than in the control g o u p (P<0.05$. The protein concentration (rnglg wet wt) showed no significant difference after the clenbuterol treatment in all skeletal rnuscIe examined (Fig. 1). The clenbuterol treatment significantly increased the protein content (mdwhole muscle) by 18%, 37% and 37%, respectively, in SOL, PL and GAS (Pc0.05, Fig. 2). The clenbuterol treatment significantly decreased HAD activity by 53% and 37%, respectively, in PL and GAS (W0.05; Fig. 3). In SOL, however, the activity did not change after the treatment.. Discussion. The results of this study show that denbuterol feeding for 32 days decreased 3-.

(5) Suzuki J. Sol,. PI,. GAS. Figure 3 El't'ict oi'clcnbutcrol trcntlncnt on 3-hydrosyncyl-CoA dchydropcnasc activity in solcus (SOL), plantnris (PL) nnd gas~rucnzmius(GAS) muscIes. *, Signilicanlly diff'eren~from YC (1'<0.05).. hydroxyacyl-CoA dehydrogenase (HAD) activity in fast- but not in slow-twitch muscles. As reported by previous investigat~rs5.7-".".'5' , clenbuterol treatment caused hypertrophy in fastand a lesser extent in slow-twitch muscles identified by muscle weights and total protein content in the present study. In the present study, the experimental groups were fed a diet containing 2 mg / kgIood clenbuterol. Cartana & Stock (1 995) ') observed that the administration of a diet containing the same amount of clenbuterol fbr 3 days caused a significant increase in the weights of hindlimb muscles in male Wistar rats. When clenbuterol is administered, a marked response is obtained within 1-2 weeks ' . 5 * ' " ' . After several weeks of continuous treatment, the response is attenuated, presumably by a downregulation of thc adrenoreceptors IR'. In the present study, the experimental groups were fed the clenbuterol diet continuously for 1 week, followed by a intermittent protocol (2-day-on / 2-day -off) fer. following 25 days. The intermittent administration could prevent an attenuation of the response occurred w-hile continuous treatment "'. The major finding of this study was that the activity of 3-hydroxyacy 1-CoA dehydrogenase, the key enzyme of mitochondria1 B-oxidation, was significantly decreased after clenbuterol treatment (P<0.05; Fig. 3). This change was observed in PL and GAS muscles but not in the SOL. In our previous study, the succinate dehydrogenase (SDH) activity of type IEa and Ilb fibers was significantly decreased after 32-day intermittent clenbuterol treatment 17'. I n type I fiber and left ventricle, however, the SDW activity did not change after the treatment 17). These reductions in rnitochondrial oxidative enzyme activity after clenbuterol treatment may be secondary to selectively increased myodibrillar protein. No change in the activity of oxidative enzymes in slow-twitch muscles suggests that mitochondria1 volume was increased in proportion to rnyofibrjllar protein during chronic clenbuterol treatment. The transformation of slow to fast-twitch fibers may also contribute to the reduction of oxidative enzyme activity. In our previous study, the composition of type IIa and IIb fibers was significantly increased in the SOL and PL, respectively, in both young and middle-aged rats after 32-day clenbuterol treatment "'. It has been reported that the transformation of type IIa to type IIb fibers was observed in extensor digitorurn longus muscle (EDL), but not in SOL, after 2 1-day clenbuterol treatment I>). Zeman et al. (1988) found that, in SOL and to a lesser extent in EDL, the transformation of slow to fast-twitch fibers was induced by clenbuterol treatment for 8 and 12 weeks. The anabolic effects of clenbuterol on slowand fast-twitch fibers are equivocal ':'-'n.'h.'9'. The present results show that, after clenbuterol treatment, the totat protein content was significantIy increased in all skeletal muscles examined (Fig. 2). The degree of increase, however,. "'.

(6) 3-h)dro~pacyl-CoAdehydrogenase actii.ily after clenbuterol treatment. tended to be greater in fast- than in slow-twitch. fibers. Differences in B-adrenergic receptor expressions and in the dose of R,-agonist " h a y contribute in part to the contrasting effects of the drug on fast- and slow-twitch fibers. In conclusion, we have shown that a decrease in 3-hydroxyacyl-CoA dehydrogenase activity in fast- but not in slow-twitch muscles of rats treated with clenbuterol for 32 days. The muscle hypertrophy without improving oxidative capacity may reduce the endurance performance in skeletal muscles.. '". 7.. 8.. 9. References 1. Agbenyega ET and Wareham AC (1990). 2.. 3.. 4.. 5.. 6.. Effect of cIenbuteroI on normal and denervated muscle growth and contractility. Muscle & Nerve 13, 199-203. Agbenyega ET, Morton R.H, Hatton PA and Wareham AC (1995) Effect of the TJadrenergic agonist clenbuterol on the growth of fast- and slow-twitch skeletal of the dystrophic muscle (C57BL6Jdy2JJdy2J) mouse. Comp Biochem Physiol C Pharmacal Toxic01 Endacrino1111,397-403. Bass A, Brdiczka, Eyer P, Hofer S and Pette D (1 969) Metabolic differentiation of distinct muscle types at the leveI of enzymatic organization. European J Biochem 10: 198206. Bojanic D and Naherski SR (1983) Identification and subclassification of rat adipocy te beta-adrenoceptors using (+/-)-[I 2511 cyanopindolol. EUP' J Pharmacol 93:235243. Cartana J, Segues T, Yebras M, Rothwell NJ and Stock MJ (1 994) Anabolic effects of clenbuterol after long-term treatment and withdrawal in the rat. Metaholism 43, 10861092. Cartana J and Stock MJ (1995) Effects of clenbuterol and salbutamol en tissue ru-. 10.. 11.. bidium uptake in vivo. Metaholism 44, 119125. Choo JJ, Horan MA, Little RA and Rothwell NJ (1992) Anabolic effects of clenbuterol on skeletaI muscle are mediated by B,-adrenoceptor activation. Am J Physiol 263 (Endocrinol Mriob 26), E50-E56. Emery PW, Rothwell NJ, Stock MJ and Winter PD (1984) Chronic effects of beta 2adrenergic agonists on body composition and protein synthesis in the rat. Biosci Rep 4:83-91. Hayes A and Williams DA (1994) Longterm clenbuterol administration aIters the isometric contractiIe properties of skeletal muscle from normal and dystrophindeficient mdx mice. Clin Exper Phurmacol Physiol2 1,757-765. Ingalls CP, Barnes WS and Smith SB (1 996) Interaction between clenbuterol and run training: effects on exercise performance and MLC isoform content. J Appl PhysioE80, 795-801. Lands AM, Arnold A, McAuliff JP, Luduena FP and Brown TG Jr. (1967) Differentiation of receptor systems activated by sympathomimetic amines. Nature 2 143597598.. 12. Maltin CA, Delday MI, Hay SM and BaiIlie GS (1 992) Denervation increases clenbuterol sensitivity in muscle from young rats. Muscle andiVerve 14, 188-192. 13. Martin CA, Delday MI and Reeds PJ (1986) The effect of a growth promoting drug, clenbuterol, on fibre frequency and area in hind limb muscles from young male rats. Biosci Rep 6,293-299. 14. McElligott MA, Barreto AJr and Chaung LY (1989) Effect of continuous and intermittent clenbuterol feeding on rat growth rate and muscle. Comp Biochem Physiol 92C, 135-138. 15. Moore NG, Pegg GG and Sillence MN (1994) Anabolic effects of the 13,-.

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