The influence of testosterone on the alkaline proteolytic activity in rat skeletal muscle

479

Biochimica et Biophysica Aeta, 641 (1980) 479--486 © Elsevier/North-Holland Biomedical Press

BBA 29361

THE I N F L U E N C E OF T E S T O S T E R O N E ON THE ALKALINE P R O T E O L Y T I C ACTIVITY IN RAT SKELETAL MUSCLE

BURKHARDT DAHLMANN, BIRGIT MAI and HANS REINAUER Diabetes Forsehungsinstitut, Biochemical Department, Universitiit Diisseldorf, Auf'm Hennekamp 65, D-4000 Diisseldorf (F.R.G.)

(Received March 17th, 1980) Key words: Skeletal muscle; Alkaline proteinase; Testosterone; (Rat skeletal muscle)

Summary The effects of t e s t e c t o m y and subsequent administration of testosterone propionate on the activity of the alkaline proteinases in rat skeletal muscle were investigated. Castration of the mature rat was followed b y a short-term delay in protein accretion in skeletal muscle tissue as measured by the protein/ DNA ratio and was paralleled b y a 2--3-fold increase in specific activity of the alkaline proteinase(s). This increase of proteolytic activity was equally significant when expressed relative to/~g DNA. Although the gain in b o d y weight was significantly lower in the castrated rats, nevertheless the protein/DNA ratio in muscle after 6 weeks approximated the values of sham-operated control rats w i t h o u t normalization of the proteolytic activity. Treatment of the castrated rats with testosterone propoinate resulted in restoring normal levels of previously elevated levels of alkaline proteolytic activity in muscle tissue. The normalization of enzyme activity as well as protein accretion in muscle was dose-dependent. Treatment of the rats with a low dose (0.1 mg/day) of testosterone propionate failed to restore the proteolyric activity, b u t led to a small increase of the protein/DNA ratio as well as to a progressive increase in b o d y weight. These data indicate a regulatory role of testosterone in the adaptive behaviour o f the alkaline proteolytic system in rat skeletal muscle.

Introduction Protein turnover in skeletal muscle is k n o w n to be under the control of several hormones [ 1--4]. Among these, testosterone has been shown to have an anabolic effect in skeletal muscle [5,6]. This effect has been illustrated in

480 studies where castration of rats was followed by a decreased growth rate of all skeletal muscles, as well as reduced b o d y growth [7]. Restoration of these parameters to normal levels was achieved by testosterone administration [8,9]. In parallel, protein synthesis in skeletal muscle is diminished after castration and normalized b y testosterone treatment of the animals [10]. These studies suggest that testosterone elicits its primary regulatory effect by an overall stimulation o f protein synthesis [ 11 ]. While the protein anabolic effects of the hormone are established fairly well, very little information exists as to its influence on the rate of protein degradation in muscle tissue. There is good evidence that intracellular protein degradation is of physiological importance in controlling muscle growth [12]. Thus, after castration o f rats, the fiber width in muscle decreases, which is probably due to the loss of contractile proteins. Both phenomena are reversed by a treatm e n t of the rats with testosterone [13--17]. These data indicate that, in the absence of testosterone, protein degradation in muscle -- as opposed to protein synthesis -- is n o t retarded. However, the question arises whether the breakdown machinery continues to perform at a constant activity level or, moreover, whether it is enhanced in the absence of testosterone. Recently, we described an alkaline proteolytic activity which participates in the degradation of muscle proteins during a protein catabolic situation, such as diabetes mellitus [18]. Since it would be of interest to obtain some more direct information on whether testosterone influences the protein degradative system in skeletal muscle, we have, in the present investigation, studied the effects of testectomy and subsequent testosterone administration on the alkaline proteolytic activity. Materials and Methods Male rats of Wistar HaN strain of 180--200 g b o d y weight were obtained from Winkelmann, Paderborn, F.R.G. The animals were fed standard diet (Sniff, Intermast, Soest, F.R.G.) and water ad libitum. All chemicals used were of the highest purity available and were purchased from Merck AG, Darmstadt, F.R.G., unless otherwise stated. [~4C]Haemoglobin was prepared as described by R o t h et al. [19] using bovine haemoglobin (Sigma, Mtinchen, F.R.G.) and K~4CNO (specific activity 50--60 mCi/mmol) from Amersham Buchler, Braunschweig, F.R.G. Azocasein and deoxyribonucleic acid from calf thymus were from Serva, Heidelberg, F.R.G. Testosterone propionate (Testoviron) was from Schering AG, Berlin, F.R.G. The radioimmunoassay kit for testosterone was purchased from Serono, Pharmazeutische Pr~iparate GmbH, Freiburg, F.R.G. Castration and hormonal treatment

Rats were castrated under pentobarbital anesthesia. Sham-operated controls got the same anesthesia, but a scrotal incision only. The efficiency of surgery was assessed b y measuring the serum testosterone levels of the animals before and after castration or sham-operation, respectively. Blood samples were taken from the retro-orbital plexus [20] and serum testosterone levels were determined by radioim.munoassay. Testosterone propionate was dissolved in sesame oil and the hormone solution was injected subcutaneously.

481

Tissue preparation Rats were killed by a blow on the head and the muscle tissue of the upper thigh (hind limb) was excised, freed from fat and connective tissue, frozen in liquid nitrogen and then pulverized in a mortar. 1 g of muscle powder was diluted 10-fold (w/v} with 0.05 M Tris-HC1, pH 7.4, containing 1 M KC1 and 0.2% (w/v) Triton X-100, and homogenized using an MSE Homogenizer (60 s, 14 000 rev./min). This mixture was centrifuged at 3000 X g for 10 min at 4°C. The s u p e r n a t a n t fraction was decanted and stored at 0°C. The sediment was resuspended by homogenization followed by centrifugation as described above. The supernatant fractions of the two centrifugation steps were pooled. The supernate was then diluted with 0.05 M Tris-HC1/1 M KC1/0.2% Triton X-100, pH 7.4, to obtain a protein concentration of 7 mg/ml, and further diluted with 0.25 M Tris-HC1/1 M KC1/0.2% Triton X-100, pH 9.0, to arrive at a final protein concentration of 5 mg/ml and a pH of 9.0. The protein concentration was tested using the biuret method. Measurement o f pro teoly tic activity 0.1 ml of & 6% (w/v) aqueous solution of [14C]haemoglobin was incubated with 0.2 ml muscle extract (5 mg protein/ml) and 0.2 ml 0.05 M Tris-HC1/1 M KC1/0.2% Triton X-100, pH 9.0. The mixture was incubated for 60 min at 37°C and the reaction was stopped by the addition of 0.1 ml of a 50% (w/v) solution of trichloroacetic acid. Blanks contained Tris-HC1 buffer instead of muscle extract. The precipitated proteins were spun d o w n (11 000 X g for 5 min) and the radioactivity in the supernatant fractions was measured using a liquid scintillation counter. The proteolytic acitivity is given as the a m o u n t of radioactive (cpm) peptides released into the trichloroacetic acid-soluble fraction after a 1 h incubation period. Hydrolysis of [14C]haemoglobin was linear and proportional to the a m o u n t of muscle extract within the range used in all experiments. The enzymatic hydrolysis of azocasein was performed as described elsewhere [21]. Measurement o f DNA The DNA c o n t e n t was determined in an aliquot of 0.5 ml of the muscle homogenate with 3,5-diaminobenzoic acid hydrochloride in a fluorescence test as described by Afting and Elce [22], b u t omitting the pronase digestion step. Calf t h y m u s DNA was used as the standard. Statistical analysis The statistical significance of differences between the mean values of the data obtained for the animals of various groups was tested by Student's t test. Pro tocol o f experiments Experiment 1 : 8 8 rats were randomly divided into t w o groups: group C, 48 rats were castrated; group N, 40 rats were sham-operated to serve as controls. At the times indicates (Table I; Fig. 1) eight animals of each group were killed and skeletal muscle tissue was excised. Experiment 2 : 1 0 0 rats were castrated and each group of eight rats was killed 1 or 2 weeks after the surgical treatment, respectively. 1 week later (3 weeks after the castration) the remaining 84 animals were divided into four

482 TABLE I EFFECT OF CASTRATION MUSCLE TISSUE

ON

BODY

WEIGHT

AND

PROTEIN/DNA

RATIO

IN

SKELETAL

R a t s o f 1 8 0 - - 2 0 0 g b o d y w e i g h t w e r e c a s t r a t e d , and killed at t h e t i m e s g i v e n in the first c o l u m n . T h e increase in b o d y w e i g h t ( d i f f e r e n c e b e t w e e n b o d y w e i g h t at d a y o f c a s t r a t i o n and at day o f killing the a n i m a l ) a n d p r o t e i n / D N A ratio per g m u s c l e tissue are given as m e a n values + S . D . ; P values are given for c o m p a r i s o n o f C vs. N. N, s h a m - o p e r a t e d rats; C, c a s t r a t e d rats; n.s., n o t s i g n i f i c a n t (P > 0 . 0 2 ) . T i m e after castration (week)

Increase in b o d y w e i g h t N (g)

0 3 6 10 14

C (g)

0 104 107 161 242

0 69 82 120 169

+- 1 6 _+ 5 6 _+ 21 +- 3 6

+ 7 + 22 _+ 1 4 -+ 2 4

Level of significance (P value)

<0.005 n.s. <0.005 ~0.005

Protein/DNA N (mg//~g)

C (mg/~g)

0.31 0.41 0.46 0.42 0.48

0.31 0.41 0.41 0.52

+- 0 . 0 3 _+ 0 . 0 9 -+ 0 . 0 7 + 0.07 + 0.05

-+- 0 . 0 3 -+ 0 . 0 7 -+ 0 . 0 9 -+ 0 . 0 4

Level of significance (P value)

<0.02 n.s. n.s. n.s.

groups, each consisting of 21 rats. The animals of each group were treated as follows: group Cs, 0.1 ml o f sesame oil/day; group Ctl, 0.1 mg o f testosterone propionate/day; group Cts, 0.5 mg of testosterone propionate/day; group Ct25, 2.5 mg of testosterone propionate/day. At weekly intervals seven animals of each group were killed and skeletal muscle tissue was excised. Results

Effect of castration Before surgery, the testosterone level in the rats was 2 . 8 -+ 1 . 0 ng/ml serum. This concentration was increased to more than 4 ng/ml serum in the shamoperated rats, whereas no serum testosterone was detectable in the castrated

1500

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500

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3 6 I0 Time after castration ( w e e k s )

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14

Fig. 1. T h e e f f e c t o f c a s t r a t i o n o n the alkaline p r o t e o l y t i c a c t i v i t y Ln s k e l e t a l m u s c l e o f rats. o . . . . . . o N, , c o n t r o l rats; • -- C, c a s t r a t e d rats. [ 1 4 C ] H a e m o g i o b l n w a s u s e d as substrate. M e a n s -+S.E. are indic a t e d . P v a l u e s are g i v e n f o r c o m p a r i s o n o f C and N at t h e t i m e s i n d i c a t e d .

483

animals. In these animals the gain in body weight was retarded when compared with control rats (Table I). As shown in Table I, the protein/DNA ratio in muscle tissue of shamoperated rats increased during the experimental period. This was due to an accretion of protein from about 100 mg at the beginning of the experiment to 125 mg/g muscle tissue after 14 weeks. In contrast, castration of the rats was followed by a decrease in muscle protein (77 mg/g muscle tissue at 3 weeks after castration). Since DNA content in muscle tissue of sham-operated and castrated rats was identical (255 + 10 #gig muscle tissue), it follows that the gain in protein/DNA ratio was delayed in the castrated rats (Table I). In addition, there was a remarkable effect of castration on the alkaline proteinases of skeletal muscle, with a 2--3-fold increase in specific activity, which persisted during the experimental period. This significant change was observed independent of whether haemoglobin or azocasein were used as substrate (Figs. 1 and 3). Essentially the same difference between castrated and control rats was obtained when the proteolytic activity was expressed relative to the DNA content of the muscle tissue (data not shown}. The effect o f testosterone administration to castrated rats In the second experiment we found that administration to castrated rats of testosterone propionate led to a dose-dependent increase of the weight of ventral prostate-seminal vesicles as well as to an increase in gain of body weight. In agreement with earlier findings [9], prolonged (10--14 days} administration of high doses of testosterone propionate (0.5 and 2.5 mg/animal} again decreased the gain in body weight and only injections of 0.1 mg testosterone propionate/ day resulted in a continuous growth. ~ T

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0.2 I

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1 2 Time after

i 3 castration

I 4 (weeks

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F i g . 2. E f f e c t o f t e s t o s t e r o n e p r o p i o n a t e a d m i n i s t r a t i o n o n t h e p r o t e i n / D N A r a t i o p e r g m u s c l e t i s s u e o f c a s t r a t e d rats. M e a n s +-S.E. a r e i n d i c a t e d . T h e p e r i o d o f t e s t o s t e r o n e ( s e s a m e off) i n j e c t i o n s is g i v e n b y t h e bar. T h e v a l u e s i n d i c a t e d b y a n a s t e r i s k a r e o f s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e (P <: 0 . 0 0 5 ) f r o m t h e Cs values. ¢ -- Cs, c a s t r a t e d r a t s t r e a t e d w i t h s e s a m e oil; o o C t l , castrated rats t r e a t e d w i t h 0.1 mg testosterone/day; ~..... -~ C t 5 , c a s t r a t e d r a t s t r e a t e d w i t h 0 . 5 m g t e s t o s t e r o n e / d a y ; [] [] Ct2 s , c a s t r a t e d r a t s t r e a t e d w i t h 2.5 m g t e s t o s t e r o n e / d a y . Fig. 3. T h e e f f e c t o f t e s t o s t e r o n e p r o p i o n a t e i n j e c t i o n s o n the a l k a l i n e P r o t e o l y t i c a c t i v i t y in s k e l e t a l m u s c l e t i s s u e o f c a s t r a t e d rats. M e a n s +S.E. a r e g i v e n . P r o t e o l y t i c a c t i v i t y w a s m e a s u r e d w i t h a z o c a s e i n as s u b s t r a t e . T h e s h a d e d a r e a o u t l i n e s t h e a c t i v i t i e s m e a s u r e d in s k e l e t a l m u s c l e t i s s u e o f s h a m - o p e r a t e d r a t s in t h e f i r s t s e t o f e x p e r i m e n t s . T h e v a l u e s o f Ct5 a n d C t 25 arc all s t ~ d f i e a n t i y d i f f e r e n t f r o m t h e Cs v a l u e s (P ~ 0 . 0 5 ) . F o r e x p l a n a t i o n o f s y m b o l s see F i g . 2.

484 In parallel, administration o f 0.1 mg testosterone propionate/day produced a slow progression in protein/DNA ratios in muscle tissue, while the injection of 0.5 or 2.5 mg testosterone propionate/day was followed b y a marked increase of this ratio (Fig. 2). These changes were due to an increased protein content and a decreased DNA content per gram muscle tissue. Restoration of the alkaline proteolytic activity was achieved by injection into the rats of both 0.5 and 2.5 mg testosterone propionate, whereas 0.1 mg testosterone was ineffective (Fig. 3). The decrease in specific activity cannot be the result of an increase in protein concentration: after 2 or 3 weeks administration to castrated rats of 0.1 mg testosterone propionate, the protein/DNA in muscle tissue was o f the same ratio (0.7--0.8 mg/pg) as in animals that had been administered 0.5 mg testosterone propionate for 1 week only (0.72 mg/#g; see Fig. 2). Finally, the proteolytic activity measured in group Ct~ was maintained at a similarly high level, as found in the control rats treated only with sesame oil (Fig. 3). Discussion In their investigation, Kochakian et al. [23] found constant absolute amounts of DNA in various muscles of guinea pigs at two different age groups. Since the protein content increased with age, it implies that the relative DNA concentration decreased. This is consistent with the increasing protein/DNA ratio measured in the control rats during our first set of experiments (Table I). Furthermore, castration of guinea pigs did n o t affect the total amount of DNA in musculus obliquus and gastrocnemius [23]. These data agree well with our own observation that the gain in protein/DNA ratio of muscle tissue was delayed only during the first 3 weeks after castration of the rats b u t then returned to control values (Table I). The retardation of the protein/DNA ratio appears to be due to an enhanced catabolic activity in protein metabolism, in that alkaline proteinase activity of rat muscle tissue increased by 100--200% while the protein content of muscle tissue was reduced by 20% after castration of the rats as compared to sham-operated animals. These data would be consistent with the increase of urea nitrogen excretion after castration of rats [24]. Despite an enhanced muscle protein catabolism, the animals were increasing in b o d y weight and the protein/DNA ratio was restored to normal (Table I). We therefore conclude that protein synthesis in castrated rats still exceeds protein breakdown. Nevertheless, as shown in the second set of experiments, testosterone propionate administration improved the protein/DNA ratio in muscle tissue (Fig. 2). Most importantly, the normalization of the alkaline proteolytic activity in muscle tissue occurred in parallel to the above effects. It is, therefore, n o t unreasonable to assume that the protein anabolic effect of testosterone is of a dual nature: it n o t only mediates the stimulation of protein synthesis b u t also represses the protein degradative system. Even in healthy (noncastrated) rats, administration of high doses of testosterone propionate has been shown to cause a decrease in alkaline proteolytic activity (Dahlmann, B., Widjaja, A. and Reinauer, H., unpublished data). Despite the clearly demonstrable effects of testosterone as presented in this study, hormonal regulation of proteolytic activity in rat skeletal muscle must

485 be considered more complex. For example, treatment of rats with adrenal glucocorticoids markedly increases the alkaline proteolytic activity in rat skeletal muscle [25,26]. Since, in contrast to the anabolic action of androgens, the hormonal action of glucocorticoids in muscle is strongly catabolic, the enhanced alkaline proteolytic activity in muscle after castration appears to be a consequence of the one-sided action of glucocortocoids after testosterone depletion. This notion is supported by the fact that androgens and glucocorticoids compete for the same site in muscle cytosol [27--29]. This would also explain the increased alkaline proteolytic activity in muscle of diabetic rats [18,25,30]. In these animals, serum as well as testicular testosterone levels are substantially lower than in metabolically healthy animals [ 31]. In conclusion, new evidence has been presented supporting the view that the alkaline proteolytic activity in rat skeletal muscle displays adaptive properties to muscle wasting conditions. Acknowledgements The authors gratefully acknowledge the technical assistance of Ms. Gabriele Neubert and wish to thank Dr. Dieter Kuschak for his help with the testosterone determinations. This work was supported by the Bundesministerium fiir Jugend, Familie und Gesundheit, Bonn, F.R.G., and by the Ministerium fiir Wissenschaft und Forschung des Landes Nordrhein-Westfalen, Diisseldorf, F.R.G. References 1 Kochaklan, C.D. and Stettner, C.E. (1948) Am. J. Physiol. 155, 255--261 2 Flaim, K.E., Li, J.B. and Jefferson, L.S. (1978) Am. J. Physiol. 235, E231--E236 3 Cabin, G.F. Jr., Aoki, T.T. and Marliss, E.B. (1972) in Handbook of Physiology, Endocrinology, Sect. 7, Vol. I, Ch. 36, pp. 563--577, Am. Physiol. Sot., Washington, DC 4 Goldberg, A.L. (1969) J. Biol. Chem. 244, 3223--3229 5 Kochakian, C.D. (1966) in The Physiology and Biochemistry of Muscle as a Food (Briskey, E.J., Cassens, R.G. and Trautman, J.C., eds.), Ch. 7, pp. 81--112, The University of Wisconsin Press, Madison 6 Kochakian, C.D. (19'/6) in AnaboHc-Androgenic Steroids (Kochakian, C.D., ed.), Ch. IV A, pp. 211-228, Springer-Verlag, Berlin 7 Kochakian, C.D., Tillotson, C. and Endahl, G.L. (1956) Endocrinology 52, 226--231 8 Scow, R.C. (1952) Endocrinology 51, 42---51 9 Kochakian, C.D. and Endahl, B.R. (1959) Proc. Soc. Exp. Biol. Med. 100, 520--522 I 0 Breuer, C.B. and Florlni, J.R. (1965) Biochemistry 4, 1544--1550 11 Florini, J.R. (1970) Biochemistry 9, 909---912 12 Goldberg, A.L., Howell, E.M., Li, J.B., Matte1, S.B. and Prouty, W.F. (1974) Fed. Proc. 33, 1112-1120 13 Scow, R.C. and Hagan, S.N. (1955) Am. J. Physiol. 180, 31--36 14 Gori, Z., PeUegrino, C. and PoUera, M. (1967) Exp. Molec. Pathol. 6, 172--198 15 Gori, Z., Pellegrlno, C. and Pollera, M. (1969) Exp. Molec. Pathol. 10, 199--218 16 Bel,gamini, E., Bombara, O. and Pellegrlno, C. (1965) Biochem. J. 97, 11P 17 Bergamini, E., Bomba~a, G. and Pene~rino, C. (1969) Biochim. Blophys. Acta 177, 220--234 18 Dahlmann, B., Schroeter, C., Herbertz, L. and Ralnauer, H. (19"/9) Bio'ehem. Med. 21, 33--39 19 Roth, J.S., Losty, T. and Wierbickl (1971) Anal. Biochem. 42, 214---221 20 N~ller, H.G. (1955) KILn. Wschr. 3S, 770---771 21 Dahlmann, B. and Reinauer, H. (1976) Biochem. J. 171, 803--810 22 Afting, E.G. and Elce, J.S. (1978) Anal. Biochem. 86, 90--99 23 Kochakian, C.D., Hill, J. and Harrison, D.G. (1964) Endocrinology 74, 635---642 24 Dohm, G.L. and Louis, T.M. (1978) Proc. Soc. Exp. Biol. Med. 158, 622--625 25 Mayer, M., Amin, R. and Shafrir, E. (1974) Arch. Biochem. Biophys. 161, 20---25

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