87

Effect of work-load exercise on the compensatory muscle
hypertrophy in the castrated rat
Shigeru Yamada
Depa ment of Sports Sciences, College of Arts and Sciences, the University of Tokyo,
3-8-l Komaba, Meguro-ku, TokYo 153

Summary
Effects of workload exercise on skeletal muscles of the castrated rat were compared with those ofnormal rats. Castration has little effect on the development of compensatory muscte hyPertrophy andthe accumulation of albumin in the hypertrophied muscle.

Key llotds: Albumin, Skeletal muscle, Compensatory hypertrophy, Testosterone

IntroductionWe reported that the tenotomy-induced hyPertrophy in the rat skeletal muscle accompanies asignificant increase in the leyel of albumin in muscle (1). Study employing the albumin-deficient rat(NAR) subsequently demonstrated that the work-induced muscle hype rophy, which is constantlyobserved with normal rat, fails to take Place in the work-loaded skeletal muscle of this mutant rat (2).
However, since NAR is known to exhibit lowered inborn levels of testosterone and growth hormone(3), it has been argued that the inability of NAR to develop the work'induced muscle hypertrophy
might be accounted for by the lowered hormone levels intrinsic to NAR.
Present study has been undertaken to asertain whether the serum level of testosterone affects
the compensatory of muscle hypertrophy, and changes in the weight and protein composition of thework-loaded skeletal muscles were compared betweefl castrated and normal rats.

Materials and Methods

Animals
Sprague-Dawley rats were obtained from the stock of CLEA Japan, Kanagawa.

Tenotomy

Rats of the same developmental stage were divided into control and experimental groups byweight-matching. Tenotomy and sham-operation of the hind legs were performed in the same manneras described previously (1). The plantaris and the soleus muscles in both opelated and contralaterallimbs were removed for examination 7 days after operation.
Castration
Rats of approximately 250 g body weight were castrated after anesthesia with an intrapedtoneal

injection of sodium pentobarbital (50 mg/kg body weight). Sham-operated control received the sameanesthesia, but only scrotal incision.

Assay of testosterone

Blood samples were collected from rats l5-20 min afte! anesthesia and were centdfuged to separate
plasma. Testosterone in the plasma was determined by use of a doubte-antibody radioimmunoassay kit
supplied by EIKEN, Tokyo (2).
Preparation of antibody

64KDa protein (rat albumin) was purified from whole skeletal muscle and anti-64KDa protein
antibody was prepared in rabbit as described previously (l ).
Polyacrylamide gel electrophoresis
Polyacrylamide gel electrophoresis was performed as described by l,aemmli (5) on luro acrylamide
slab gels. Total muscle proteins were dissolved in a sample buffer, containing 25 mM Tris-Hcl,2% sDS,
pH 6.5 and heated in boiling water for 2 min. Electrophoresis was performed at 25 mAconstantcurrent

until the bromophenol blue tracking dye moved to the end of the gel. After electrophoresis, proteins
were stained with Coomassie blue (l ).
Transfer of proteins onto nitrocellulose and immunoblolting

The methods of rowbin et at. (6) and Legocki and verma (7) were adopted with some modifica-tions. After electrophoresis, gels were equilibrated with 40 mM glycine containing 5 mM Tris-base(blotting buffer) for 30 min. The protein in the gel were then electrophoretically transferedto nitrocellulose membranes by transverse application of 125 V for l hr. The nitrocellulose membrane
successively treated with 0.03 M borate buffer containing 0. l5 M Nacl, I @o casein,azide, pH 8.0 (masking solution) for 30 min, then masking solution containing

wasand 0. l% sodium

0.5% specific antibody

and' o.02% sDS for 90 min. After washing the membrane ten times with o.o3 M borate

buffer containg

0.15 M Nacl pH8.0 (washing solution), the membrane was treated with masking solution
0.02% sDs and 0.5% goat antirabbit IgG conjugated with peroxidase for 90 min andcontaining

washed with tenchanges of washing solution. The immunocomplex was located by staining for peroxidase

activity with

4-chloro- l-naphthol and hydrogen peroxide as described by Hawke et al. (g).

Results

Fig. l, shows the serum testosterone concentrations of castrated and control rats 7 days aftersurgery. It is evident that testosterone concentrations in the serum of castrated rats declines to much

lower levels than that in the control animals.
To study the effect of work-load exercise on the muscle hypertroPhy in castrated rat, the soleus
and plantaris muscles of one limb were work'loaded by tenotomy of the synergistic gastrocnemius
muscle. The contralateral limb which received only sharn-operation served as control. During the week

following tenotomy, the work-loaded muscles of the tenotomized limb of castrated rat exhibited
statistically significant changes in wet weight as compared to those of control muscles. By contrast, weroutinely observed 30-5Ma weight gain in the work-loaded muscles of normal rats when the synergisticmuscle was tenotomized (l).

To verify whether the work-load exercise brings about qualitative or quantitative change in theprotein components of the skeletal muscles of castrated rat, total protein from the work-loaded andthe control muscle were analyzed by means of SDS polyacrylamide gel electrophoresis and the muscle
albumin levels were compared between the hypertrophied and control muscles by means of Westem-blotting. As shown in Fig. l, the amount of albumin significantly increased in the work-loaded muscleof the testosterone-depleted animal (l). Although weights of the work-loaded planta s muscles are
somewhat greater in castrated rats than in control animals, differences between two groups are
statistically insignificant (Fig. 3). Therefore, it appears that the serum level of testosterone has little
effect on the work-induced muscle hypertrophy.

Discussion
As evidenced previously, compensatory hypertrophy in rat skeletal muscle accompanies an increasein muscle concentration of albumin (l ). Studies on the physiological role of albumin in comPensatory
muscle hypertrophy would be greatly facilitated by the availability ofa rat strain defective in albuminbiosynthesis (9, lO). In the present experiments, tenotomy was performed on the skeletal muscle ofcastrated rats and changes in weight and protein composition of the workioaded synelgistic muscles
were compared with those of normal rats. In contrast to the case with normal rats, we found that
castrated rat succeed to develop significant muscle hypertrophy in response to the work-load training.

The result strongly suggests positive involvement of albumin in the work-induced enlargement ofskeletal muscle, though mechanisms by which albumin participates in the muscle hypertrophy remain
to be elucidated.
Among biochemical parameters which may influence the rate of muscle hypertroPhy, serum levelsof testosterone is known to exert pronounced effects (l l, 12, 13, 14,15,16,17, 18, l9). Testosterone
primarily regulates the DNA dependent RNA synthesis in target tissues, and facilitates the synthesis ofmale specific proteins (20,21,22). In addition, this steroid is also known to play an important role inmuscie hypertrophy. Although the responses of different tissues to treatment with this hormonesis
are not uniform and the mechanisrns by which these homones affect muscle mass and muscle Protein
composition remain only poorly understood, the accumulated facts raise the possibility that theobserved inability of the NAR skeletal muscle to respond to work load training might be the con'sequence of tlle lowered inborn levels of testosterone and growth hormone in this mutant rat (10).

︵一﹄ ∞目︶ 暉０● ﹄一●Ｏｏ●ｏＯ Ｃ Ｏ︼ｏ一め０一∽０い Ｅ ●︼０∽・¨NCFi& l. Effect of castration on the se m testosterone concentation. Blooal was withdrawn fiom theabdomina.l aorta at 15-20 min after pentobarbital injection and testostetone concentration wasdetemited by the radio-immunoassay as described in ..Materials and Methods',. N and C
indicate the testosterone concentration in the setum ofnotmal and castrated rats, respectively.

¨島¨ｐ澪 ｏ コ ち ３Ｓ︼ ︻ｓ︐NC
Fi& 3. Effect of tenotomy and training on weight of the plantaris muscle in normal and castrated rats.
N and C indicate the muscle weights of the normal Iat gioup anal the castrated rat group,respectively. Bars indicate the relative changes in wet weight of the plantais muscle in normal
and castnted rat ? days after operation. Normar and castrated rats were subjected to trai[ingfor 7 days after tenotomy. The weights of the plantaris muscles of normal and castmted rats
were detetmined.

A

一一一■

B

灘CH

C

H

Fi& 2. SDs-polyacrylamide gel electrophoresis and Western blot analysis of the mNcle proteins in
the muscle proteins in th€ castrated rat. Normal male lats wele castnted and subjecled totenotomy and training as described in "Mate als and Methods". The plafltais muscles of thetenotomized (H) afld conttol (C) Iimbs were solubilized in SDS sample buffet. Each l2rdsolution (20 llg protein) was subjected to l07o SDs-polyacrylamide gel elechophoresis andslained for protein with Coomassie blue (A). Protein in the gpl were electrophoreticallytEnsfered to nitrocellulose filter. After the nitrocellulose lilter was successively treated byanti-rat albumin antibody and goat anti-rabbit IgG conjugated with lacloperoxidase, the labbit
tgc-goat IgG complex was located by staining for peroxidase activity with 4thloro-l_naphtholand hydrogen Peroxide (B).

Therefore, the effect of castration on compensatory muscle hypertrophy was examined in normal Iats
as respects muscle enlargement and albumin accumulation. The results show that compensatory muscle
hypertrophy is inducible in the caslrated rats as be shown in the hyPophysectomized rats. Moreover.
muscle hypertrophy observed with these organ-ectomized rats also accompanied albumin accumulationin the hypertrophied muscle. Successful induction of the compensatory muscle hypertrophy in thecastrated rats is well in agreement with the Goldberg's statement that endogenous testicular hormones
has no effect on tenotomy induced muscle hypertrophy (23).

Taken together, the prcsent results, suggest that testosterone, in not essential fol the induction ofmuscle hype rophy and that failure of the NAR muscle to exhibit work-induced hyPertrophy ispossibly due to albumin deficiency in this mutant rat.

92
ReFerencesl)Yalnada,S,Tomlno,S,lzumi,S and Aklno,M:(1984)B B A798,260‑2672)Yamada,S:(1989)東 京大学教養学部体育学紀要 23,83‑86
3)Takahatti,M,ヽヽ abayashi,K and Nagase,S:(1984)Endocmol Japan 31,185‑193
4)Utsllnomiya,T,Yamagu饉 ,S,and Kadota T:(1981)HORUMON TO RINSYOU 29(11),89‑91

5)Lcammu,uK:(1970)Nature 227,680

6)Towbh,H,S● cheLn,T and Gordon,J:(1979)PloC Nau Acad Sc1 76,4350‑4354
7) Legockl,R P and Verna,EP:(1981)Anal Biochcrn 3,385‑392

8)HawkeS,R,Niday,E and COrdon,A:(1981)J Anal Biochem l19,142‑147
9)Nagase,S,ShLnalnune,K and Shumlya,S:(1980)Jlkken Dobuts 29,33‑38
10)Esumi,H,Okui,M,Sato,S,Suglmura,T and Nagasc,S:(1980)PIoC Natl Acad Sci 77,3215‑3219

11)Goldberg,AL:(1967)Am J Pllysio1 213,1193‑1198
12)MackOVa,E V and Hnlk,P:(1976)PhysI。 l BohemOslov 25,325‑332
13)Marchettl,M,Figura,F,Candeloro,R,and FavllL,S:(1980)J Sports Mcd,20,13‑2214)Nimni,M E and Bavetta,LA:(1961)PS E B M lo6,738‑740)
15)Kochakia,C D and T■ lotson,C:(1957)Am J Physi。 1 189(3),425‑427

16)Evans,HM,Simpton,M E and Ll,CH:(1962)CIowth 12,1517)Arv■ ,A and H」 aLnarson,A:(1967)Ac● Endocrlno1 56 suppl 12218)Knobll,E:(1960)in:CrOwth ln LIvlng Systcm,ed Zatrow,MX,Basic Books,New Yo!k19) MCManus,MB,レ unb,SR,Judis,J J and Scala,J:(1975)Europ J Physlo1 34,149‑156

20)Floml,IR:(1967)Biochemisty 9),909‑912
21)0'Ma1ley,B:(1969)TranS N Y Acad Sci 31,478‑484
22)Llao,S"Lelnmgcr,KR,SagheF,D,and Barton,RW:(1965)Endocrh 77,763‑765

23)C2oldbCrg,AL,Eunger,JD,Coldsptょ ,D F and Jablccki,C:(1975)Mcd Sci Spo■s Exerclsc 7,248‑261