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Influence of clenbuterol on the protein digestibility and on nitrogen balance in rats
Camp. Biochem. Physiol. Vol. lOlA, No. 3, pp. 619-623, 1992
0300-9629/92$5.00+ 0.00
Printed in Great Britain
0
INFLUENCE DIGESTIBILITY
1992 Pergamon
Press plc
OF CLENBUTEROL ON THE PROTEIN AND ON NITROGEN BALANCE IN RATS
FRANCISCAPER&LLAMAS and SALVADORZAMORA* Department of Physiology and Pharmacology, University of Murcia, 30100, Murcia, Spain. Telephone: 968-833000 (Received 25 June 1991)
Abstract-l. The influences of a j-agonist, clenbuterol, on the fecal and urinary nitrogen excretion, the protein digestibility and the nitrogen balance and retention in female Wistar rats of three different weights (70, 150 and 200 g) have been studied. 2. The addition of clenbuterol to the diet did not alter the fecal nitrogen excretion, nor did it change the digestibility coefficient of the protein, irrespective of the age/weight of the animals and the amount of the B-agonist used. 3. Clenbuterol decreased the urinary nitrogen excretion, especially in older/heavier animals and fed diets with the same dose of the agonist. 4. Nitrogen balance and retention, which, as expected, decrease as the age/weight of the animals decreases, increased significantly with the use of clenbuterol, especially in older/heavier animals. 5. From the results of this study, it can be concluded that, for the range of weight studied, the effect of this B-agonist does not show on the absorption of proteins at the digestive level; at a metabolic level, however, it does affect the nitrogen retention although, again, to a greater extent in older/heavier animals.
INTRODUCTION It has been shown that P-adrenergic
agonists (jagonists) induce significant changes in the energetic, glycidic, lipidic, and proteic metabolisms, and, as a result, changes in the body composition in different species (Li and Jefferson, 1977; Emery et al., 1984; Hamby et al., 1985; Hanrahan, 1987; Williams, 1987; McRae et al., 1988; Yang and McElligott, 1989; Buttery and Dawson, 1990). The most significant effects shown in animals treated with various j-agonists are an increase in the muscle mass (Baker et al., 1984; Dalrymple et al., 1984a, b, c; Berne et al., 1985; Hamby et al., 1985; Beermann et al., 1985a, b; Kim et al., 1987; PCrezLlamas and Zamora, 1991; Pkrez-Llamas et al., 1991) and a net reduction of body-fat content (Blum et al., 1982; Fain and Garcia-Sainz, 1983; Muir et ul., 1985; Alderson et al., 1988; Coleman et al., 1988; Mersmann et al., 1989), as well as an improvement in feed efficiency (Jones et al., 1985; Moser et al., 1986; Bohorov et al., 1987; Pbrez-Llamas and Zamora, 1991). In the last few years, considerable progress has been made in the study of the mechanisms of these compounds capable of improving carcass quality, and recent research has proposed different direct and indirect action mechanisms (Maltin et al., 1987; Lafontan et al., 1988; Higgins et al., 1988; Zeman et al., 1988; Maltin et al., 1989; Claeys et al., 1989; McLennan and Edwards, 1989; Yang and McElligott, 1989). In previous studies we have shown that both the quality of the administered diet (in regard to the amount and quality of the protein), and the age of *To whom all correspondence
should be addressed.
the animals are two capital factors to consider in the study of the influence of p-agonists on body growth, and even more so, on the increase in muscle mass (PBrez-Llamas and Zamora, 1991; Ptrez-Llamas et al., 1991). In the present study we have designed a series of experiments to determine whether the increase in the amount of muscle proteins and the improvement in feed efficiency found in animals treated with clenbuterol can be accounted for by an increase in the absorption of the dietary protein and/or by a higher nitrogen retention, induced by the b-agonist. The possible influence of age on these digestive and metabolic processes has also been studied. MATERIAIS AND METHODS Animals and diets
Female Wistar rats of three different weights were used (60-70, 14&150 and 190-2OOg) in nine lots, each consisting of 10 individuals. The animals were kept in individual metabolism cages permitting the separate collection of urine and faeces, in a room with controlled photoPeriod (12 hr light: 12 hr darkness), a temperature of 23 + 2°C and constant relative humidity (60%). Their diet in all the experiments was a standard rat feed of the following composition in dry substance (humidity 8.8%): proteins 26.2%, fat 4.2%, fiber 3.1%, total minerals 6.6% and nitrogen free extract (NFE) 59.9%.
The B-agonist used was clenbuterol (I-(4-amino-3,5dichlorophenyl)-2-ter-butylaminoethanol) (Itesba, S.A., Barcelona) added to the diet in powder form in different doses: 0, 30 and 50mg/kg. Experimental
design
Three experiments, corresponding to the three initial weights were designed. Each experiment consists of three 619
FRANCISCAPEI&Z-LLAMASand SALVAWRZAMORA
250 . B :: 200 . F 8 f -150. &S 2 >lOO. e s50
o-
70
assays, corresponding to the dose of b-agonist used. The experiments lasted 10 days, divided into two periods of 3 days (in which the animals adapted to the medium/ environment and diet) and 7 days, the principal part of the experiment, when the rats were fed the normal diet with the agonist added. Calculations The following calculations were determined:
1. Fecal nitrogen excretion, expressed as mg nitrogen per g ingested dry matter. 2. Apparent digestibility coefficient of the protein (ADC): 100.
3. Urinary nitrogen excretion, expressed as mg nitrogen per 100 average body weight. 4. Nitrogen balance (NB): NB=p
I-F-U
x 100.
I
5. Nitrogen retention (NR): I-F-U
Average weight
x 100.
Abbreviations used: I = ingested nitrogen; F = fecal nitrogen; U = Urinary nitrogen. Nitrogen was determined by Kjedhal’s method. 100
I 150
BO
Fig. 1. Effect of clenbuterol on fecal nitrogen excretion, the doses used: Omg/kg (white column), 30mgjkg (black column) and 5Omg/kg (striped column). The data are expressed as means (N = lo), SEM shown by vertical bars. There were no significant differences with respect to the control (white column).
ADC=
%$70
(g)
BODY WEIGHT
NR=
.
IOY WEIGHT (g)
Fig. 3. Effect of clenbuterol on urinary nitrogen excretion, the doses used: 0 mg/kg (white column, 30 mg/kg (black column) and 5Omg/kg- (striped column). The data are exnressed as means (N = 10). SEM shown by vertical bars. Significant differences with’ respect to the control (white column) were: *P < 0.05 and **P < 0.001. Statistical analysis The data are expressed as means f SEM. Tests for significance were carried out by use of Student’s r-test. P values of less than 0.05 were considered significant. RESULTS The following histograms represent the influence of clenbuterol (0, 30 and 50 mg/kg) on the parameters studied, in relation to the weight of the rats (70, 150 and 200 g). Each bar represents the average value of 10 data + SEM. Figure 1 shows the effect of clenbuterol on nitrogen fecal excretion, expressed in relation to food intake. None of the assays carried out showed any effects of the /?-agonist on the elimination of nitrogen in the stool, irrespective of animal age. Figure 2 represents the effect of clenbuterol on the digestibility coefficient of the protein. This value shows a great constancy between those animals treated and the control lots, which again suggests that the B-agonist has no effects on the absorption of the dietary protein. Figure 3 shows the influence of clenbuterol on urinary nitrogen excretion, related to the average weight of the animals. The b-agonist decreased the elimination of urinary nitrogen in all the lots studied.
r
40 20 I
o-
70
150 BODY WEIGHT
200
(g)
Fig. 2. Effect of clenbuterol on apparent digestibility coelI&ient of the protein (ADC), the doses used: 0 n&kg (white column). 30 ma/ka (black column) and 50 mg/kg (striped columdj. The dataam expressed as’means (N =-io>; SEM shown by vertical bars. There were no significant differences with respect to the control (white column).
70
2w
150 BODY WEIGHT
(g)
Fig. 4. Effect of clenbuterol on nitrogen balance (mg N/mg N consumed), the doses used: Omgjkg (white column), 30 mg/kg (black column) and 50 mg/kg (striped column). The data are expressed as means (N = lo), SEM shown by vertical bars. Significant differences with respect to the control (white column) were: *P < 0.05 and +*P < 0.001.
Clenbuterol and dietary protein utilization
g
200
% F50 5
100
:: ij
50
0
70
150 BODY WEIGHT
200
(9)
Fig. 5. Effect of clenbuterol on nitrogen retention (mg N/100 g body weight), the doses used: Omg/kg (white column), 30 mg/kg (black column) and 50 mg/kg (striped column). The data are expressed as means (N = lo), SEM shown by vertical bars. Significant differences with respect to the control (white column) were: *P < 0.05 and **p < 0.001. However, the decreasing of this parameter is statistically significant (P < 0.05) only in the older/heavier lots. Figures 4 and 5 represent the effect of clenbuterol on nitrogen balance (Fig. 4) and on nitrogen retention (Fig. 5). These figures show the positive effect of the /?-agonist on both parameters. When the results are expressed as a function of the ingested nitrogen (Fig. 4), the influence of age on these processes becomes apparent. The effect of clenbuterol is noticeable only in the heavier lots but, in leaving the influence of age aside, and expressing the results as a function of the average weight (Fig. 5), the B-agonist significantly increases (P < 0.001) the nitrogen retention in all the lots studied. DISCUSSION
The fecal nitrogen excretion slightly increases as the age of the animals increases, but this increase becomes statistically significant (P < 0.05) only in comparing the lots with the extreme body weights (70 and 200 g). This fact is shown in Fig. 2, where the apparent digestibility coefficient (ADC) is determined. These results show that proteic absorption at the intestinal level decreases, though very slightly, as the age/weight of the animals increases. The addition of clenbuterol to the diet had no effect on the nitrogen fecal excretion nor did it change the ADC, irrespective of the body weight of the animals and of the doses of p-agonist used. These results coincide with other researches made on goats (Williams et al., 1987). In our experimental conditions the results confirm that, though its pharmacological actions might suggest the opposite (Beth and Uvnas-Moberg, 1986; Lafontan et al., 1988), this P-agonist does not alter the dietary protein absorption at the digestive level in any way. From the above it can be concluded that the positive increases in body and muscle growth that both a number of authors (Baker et al., 1984; Dalrymple et al., 1984a, b, c; Kim et al., 1987; Yang and McElligott, 1989; Buttery and Dawson, 1990) and we, in similar experimental conditions (WrezLlamas and Zamora, 1991), have found after the
621
treatment with different /I-agonists cannot be accounted for by any improvement in the use of the dietary protein induced by clenbuterol. Urinary nitrogen excretion in the control lots (Fig. 3) shows no clear tendency in relation to the age of the animals, this possibly being due to the type of diet used; our intention in administering a proteinrich (26.2%) diet was not the study of the nitrogen balance in the control lots, but the study of the influence of clenbuterol on the excess of amino acids ingested and absorbed. Since the diet has a high level of proteins and high digestibility coefficient, the level of amino acids absorbed is considerably high in absolute terms. Thus, our research is aimed to study how clenbuterol can improve the body and muscle growth. The results as to nitrogen balance and retention in the control lots (Figs 4 and 5) are as expected for the range of weights studied. The maximum values of both indices were obtained in the youngest animals, that is, in those with higher growth rates. A statistically significant decrease (P < 0.001) in relation to the age of the animals is also observed. This suggests that as the animals are older, their growth rate becomes slower and the metabolic activity in this range of age gradually decreases. Treatment with clenbuterol decreased the urinary nitrogen excretion (Fig. 3), though not significantly in all the assays carried out. These changes once again confirm that this b-agonist does alter the proteic metabolism at the biosynthetic and/or proteolytic level, as has already been mentioned (Deschaies et al., 1981; Hamby et al., 1985; Reeds et al., 1986; Bohorov et al., 1987; Hovel1 et al., 1987; Challiss et uf., 1988; McLennan and Edwards, 1989). Administration of the /?-agonist in our experimental conditions significantly improves nitrogen balance (Fig. 4) in the older/heavier animals (150 and 200 g), but the influence of the drug is much more marked on the nitrogen retention/average weight ratio (Fig. 5), which increases (P < 0.001) even in the younger animals, showing a higher metabolic rate. Given that (A) the expression of these results shows the influence of the animals’ weight/age in Fig. 4 (balance), and of the intake in Fig. 5 (retention), and (B) the results obtained by either of the two procedures are similar but not identical, it could be inferred that clenbuterol presents a double effect. Firstly, on the intake, which is slightly increased by the agonist and also on the improvement of the metabolic use of the protein, and as result, on the general decreasing in the urinary nitrogen excretion. This two-fold effect accounts for the significant increase (P < 0.001) in the nitrogen retention, even in the younger animals, a fact which cannot be observed in studying the balance, since in this case the possible influences of the p-agonist on the intake are not taken into account. The results obtained in this study may clearly account for the positive effect of clenbuterol on body growth and, what is more important, on muscle mass growth, in accordance with our previous findings and reports (Perez-Llamas and Zamora, 1991; PerezLlamas et al., 1991). These effects would be a consequence of the positive effect of the /?-agonist on nitrogen retention.
622
FRANCISCAPEG&-LLAMASand SALVADC~R ZAMORA
Acknowledgements-The authors wish to thank Professor M. Canteras Jordana for his help in the statistical analysis. REFERENCES Alderson E. M., Scaife J. R. and Galbraith H. (1988) The effect of oestradiol-17jI and cimaterol 8iven alone or in combination on lipid metabolism and c-AMP concentrations in tissues of male castrate lambs. Proc. Nutr. Sot. 47, 109A. Baker P. K., Dahymple R. H., Ingle D. L. and Ricks C. A. (1984) Use of a /J-adrenergic agonist to alter muscle and fat deposition in lambs. J. Anim. Sci. 59, 1256-1261. Beth K. and U~ass-Mo~rg K. (1986) B-Adrenergic agonists inhibit gastric acid and pepsin secretion through somatostatin release in dogs. Stand. J. Gustroenterol. 21, 531-536. Beermann D. H., Fishell V. K., Hogue D. E., Ricks C. A. and Dalrymple R. H. (1985a) Effects of the repartitioning aaent. cimaterol (CL 263. 780) on skeletal muscle fiber tipe and fiber hypkrtrophy in lambs. J. Anim. Sci. 61,254 (Abstr. 116). Beermann D. H., Hogue D. E., Dalrymple R. H. and Ricks C. A. (1985b) Effects of the repartitioning agent cimaterol (CL263, 780) and tishmeai on skeletamuscle and fat accretion in lambs. J. Anim. Sci. 61, 255 (Abstr. 117). Beme R. S., Novakofski J. and Bechtel P. J. (1985) Effects of the &-agonist clenbuterol on body and tissue weights in four strains of rats. J. A&m. Sci. 61, 256 (Abstr. 119). Blum J. W., Froehli D. and Kunz P. (1982) Effects of catecholamines on piasma free fatty acids in fed and fasted cattle. Endocrinology 110, 452-456. Bohorov O., Buttery P. J., Correia J. H. D. and Soar J. B. (1987) The effect of the /I,-adrenergic agonist clenbuterol or implantation with oestradiol plus trenbolone acetate on protein metabolism in wether lambs. Er. f. Nutr. 57, 99-107. Buttery P. J. and Dawson J. M. (1990) Growth promotion in farm animals. Proc. Nutr. Sot. 49, 459466. Challis5 R. A., Leighton B., Wilson S., Thurlby P. L. and Arch J. R. S. (1988) An investigation of the /I-adrenoceptor that mediates metabolic responses to the novel agonist BRL 28410 in rat soleus muscle. Eiochem. Pharmat. 37,947-950. Claeys M. C., Mulvaney D. R., McCarthy F. D., Gore M. T., Marple D. N. and Sartin J. L. (1989) Skeletal muscle protein synthesis and growth hormone accretion in young lambs treated with clenbuterol. J. Anim. Sci. 67, 2245-2254. Coleman M. E., Ekeren P. A. and Smith S. B. (1988) Lipid synthesis and adipocyte growth in adipose tissue from sheep chronically fed a fl-adrenergic agent. J. Anim. Sci. 66, 372-378. Dalrymple R. H., Baker P. K., Doscher M. E., Ingb D. L., Pankavich J. A. and Ricks C. A. (1984a) Effect of the repartitioning agent CL 263,780 on muscle and fat accretion in finishing swine. J. Anim. Sci. 59,212 (Abstr. 164). Dalrymple R. H., Baker P. K., Gingher P. E., Ingle D. L., Pensack J. M. and Ricks C. A. (1984b) A repartitioning agent to improve performance and carcass composition of broilers. Puultrv Sci. 63. 23762383. Dahymple R. H, Baker P. K. and Ricks C. A. (1984~) Repartitioning agents to improve performance and body composition. In Proceedings of Georgia Nutrition Conference for the Feed Industry, pp. 111-l 18. University of Georgia, Georgia, U.S.A. Deschaies Y., Willemot 3. and Leblac J. (1981) Protein synthesis, amino acid uptake and pools during isoproterenol-induced hypertrophy of the rat heart and tibialis muscle. Can. J. Physiol. Pharmnc. 59, 113-121.
Emery P. W., Rothwell N. J., Stock M. J. and Winter P. D. (1984) Chronic effects of 8,-adreneric auonists on body imposition and protein s&he& c&e&t. Biosci. Rep. 4, 83-91. Fain J. N. and Garcia-Sainz J. A. (1983) Adrenergic regulation of adipocyte metabolism. J. Lipid Res. 24, 945-966. Hamby P. L., Stouffer J. R. and Smith S. B. (1985) Muscle metabolism and carcass traits in lambs fed diets containing a /?-agonist. J. An&n. Sci. 61, 246 (Abstr. 95). Hanrahan J. P. (1987) &to Agonbts and their Effects on Animal Growth and Carcass Composition. Elsevier Applied Science, London. Higgins J. A., Lass&t Y. V., Bardsley R. G. and Buttery P. J. (1988) The relation between dietary restriction or clenbuterol (a selective /?-agonist) treatment on muscle growth and calpain proteinase (EC 3.4.22.17) and calpastatin activities in lambs. Br. J. Nutr. 60, 645652. Hovell F. D. D., Kiie D. J., Reeds P. J. and Beermann D. H. (1987) The effect of /I,-agonists on the endogenous nitrogen loss of sheep. Pro;. iktr. Sot. 47, 13A.Jones R. W.. Easter R. A.. McKcith F. K.. Dah-vrnnle R. H., Maddock H. M. and Bechtel P. J. (1985) Ef$xt-of fi-adrenergic agonist cimaterol (CL 263, 780) on the growth and carcass characteristics of tinishing swine. J. Anim. Sci. 61, 905-913. Kim Y. S., Lee Y. B. and Dalrymple R. H. (1987) Effect of the repartitioning agent eimaterol on growth, carcass and skeletal muscle characteristics in lambs. J. Anim. Sci. 65, 1392-1399. Lafontan M., Berlan M. and Prud’hon M. (1988) Les agonistes ~~-adr~ner~q~. Mecanismes d’action: lipomobilisation et anabolisme. Repr. Nutr. Dbvelopp. 28, 61-84. Li J. B. and Jefferson L. S. (1977) Effect of isoproterenol on amino acid levels and protein turnover in skeletal muscle. Am. J. Phvsiol. 232. E243-E249. Maltin C. A., Hay S. M., Delday M. I., Reeds P. J. and Palmer R. M. (1989) Evidence that the hypertrophic action of clenbuterol on denervated rat muscle is not propranolol-sensitive. Br. J. Pharmac. 96, 817-822. Maltin C. A., Hay S. M., Delday M. I., Smith F. G., Lobley G. E. and Reeds P. 1. (1987) Ci~bu~rol, a beta-agonist, induces gMJWthin innervated and denervated rat soleus muscle via apparently different mechanisms. Biosci. Rep. 7, 525-532. Mersmann H. J., Hu C. Y., Pond W. G., Rule D. C., Novakofski J. E. and Smith S. B. (1989) Growth and adipose tissue metabolism in young pigs ftd cimaterol with adequate and low dietary protein. J. Anim. Sci. 64, 13841394. McLennan P. A. and Edwards R. H. T. (1989) Effects of clenbuterol and propranolol on muscle mass. Evidence that clenbuterol stimulates muscle adrenoceptors to induce hypertrophy. Riochem. J. 264, 573-579. McRae J. C., Skene P. A., Connell A., Buchan V. and Lobley G. E. (1988) The action on the b-agonist clenbuterol on protein and energy metabolism in fattening wether lambs. Br. J. Nutr. 59, 457-465. Moser R. L., Dah-ymple R. H., Cornelius S. G., Pettigrew J. E. and Allen C. E. (1986) E&et of eimaterol (CL 263, 780) as a ~pa~itioning agent in the diet for finishing pigs. J. Anim. Sci. 62,21-26. Muir L. A., Wien S., Duquette P. F. and Olson G. (1985) Effect of the beta-adrenergic agonist L-640-033 on lipid metabolism, growth and carcass characteristics of female broiler chickens. J. A&n. Sci. 61, 263 (Abstr. 138). Wren-Llamas F., Sastre J. F. and Zamora S. (1991) Influence of dietary protein level on growth: effect of clenbuterol. Comp. Biochem. PhysioI. %A, 671475.
Clenbuterol and dietar y protein utilization Wrez-Llamas F. and Zamora S (1991) The influence of clenbuterol on growth in rats. Camp. Biochem. Physiol. WA, 241-244. Reeds P. J., Hay S. M., Donvood P. M. and Palmer R. M. (1986) Stimulation of muscle growth by clenbuterol: lack of effect on muscle protein biosynthesis. Br. J. NW. 56, 249-258. Williams P. E. V. (1987) The use of j-agonists as a means of altering body composition in livestock species. Nutr. Abs. Rev. B57, 453464.
Williams P. E. V., Pagliani L., Innes G. M., Pennie K.,
623
Harris C. I. and Garthwaite P. (1987) Effects of a j-agonist (clenbuterol) on growth, carcass composition, protein and energy metabolism of veal calves. Br. J. Nutr. 57, 417428.
Yang Y. T. and McElligott M. A. (1989) Multiple actions of /?-adrenergic agonists on skeletal muscle and adipose tissue. Bioch&. J! Ml, I-10. Zeman R. J., Ludemann R., Easton T. G. and Etlinger J. D. (1988) Slow to fast alterations in skeletal muscle fibers caused by clenbuterol, a &-receptor agonist. Am. J. Physiol. 254, E726-E732.
0300-9629/92$5.00+ 0.00
Printed in Great Britain
0
INFLUENCE DIGESTIBILITY
1992 Pergamon
Press plc
OF CLENBUTEROL ON THE PROTEIN AND ON NITROGEN BALANCE IN RATS
FRANCISCAPER&LLAMAS and SALVADORZAMORA* Department of Physiology and Pharmacology, University of Murcia, 30100, Murcia, Spain. Telephone: 968-833000 (Received 25 June 1991)
Abstract-l. The influences of a j-agonist, clenbuterol, on the fecal and urinary nitrogen excretion, the protein digestibility and the nitrogen balance and retention in female Wistar rats of three different weights (70, 150 and 200 g) have been studied. 2. The addition of clenbuterol to the diet did not alter the fecal nitrogen excretion, nor did it change the digestibility coefficient of the protein, irrespective of the age/weight of the animals and the amount of the B-agonist used. 3. Clenbuterol decreased the urinary nitrogen excretion, especially in older/heavier animals and fed diets with the same dose of the agonist. 4. Nitrogen balance and retention, which, as expected, decrease as the age/weight of the animals decreases, increased significantly with the use of clenbuterol, especially in older/heavier animals. 5. From the results of this study, it can be concluded that, for the range of weight studied, the effect of this B-agonist does not show on the absorption of proteins at the digestive level; at a metabolic level, however, it does affect the nitrogen retention although, again, to a greater extent in older/heavier animals.
INTRODUCTION It has been shown that P-adrenergic
agonists (jagonists) induce significant changes in the energetic, glycidic, lipidic, and proteic metabolisms, and, as a result, changes in the body composition in different species (Li and Jefferson, 1977; Emery et al., 1984; Hamby et al., 1985; Hanrahan, 1987; Williams, 1987; McRae et al., 1988; Yang and McElligott, 1989; Buttery and Dawson, 1990). The most significant effects shown in animals treated with various j-agonists are an increase in the muscle mass (Baker et al., 1984; Dalrymple et al., 1984a, b, c; Berne et al., 1985; Hamby et al., 1985; Beermann et al., 1985a, b; Kim et al., 1987; PCrezLlamas and Zamora, 1991; Pkrez-Llamas et al., 1991) and a net reduction of body-fat content (Blum et al., 1982; Fain and Garcia-Sainz, 1983; Muir et ul., 1985; Alderson et al., 1988; Coleman et al., 1988; Mersmann et al., 1989), as well as an improvement in feed efficiency (Jones et al., 1985; Moser et al., 1986; Bohorov et al., 1987; Pbrez-Llamas and Zamora, 1991). In the last few years, considerable progress has been made in the study of the mechanisms of these compounds capable of improving carcass quality, and recent research has proposed different direct and indirect action mechanisms (Maltin et al., 1987; Lafontan et al., 1988; Higgins et al., 1988; Zeman et al., 1988; Maltin et al., 1989; Claeys et al., 1989; McLennan and Edwards, 1989; Yang and McElligott, 1989). In previous studies we have shown that both the quality of the administered diet (in regard to the amount and quality of the protein), and the age of *To whom all correspondence
should be addressed.
the animals are two capital factors to consider in the study of the influence of p-agonists on body growth, and even more so, on the increase in muscle mass (PBrez-Llamas and Zamora, 1991; Ptrez-Llamas et al., 1991). In the present study we have designed a series of experiments to determine whether the increase in the amount of muscle proteins and the improvement in feed efficiency found in animals treated with clenbuterol can be accounted for by an increase in the absorption of the dietary protein and/or by a higher nitrogen retention, induced by the b-agonist. The possible influence of age on these digestive and metabolic processes has also been studied. MATERIAIS AND METHODS Animals and diets
Female Wistar rats of three different weights were used (60-70, 14&150 and 190-2OOg) in nine lots, each consisting of 10 individuals. The animals were kept in individual metabolism cages permitting the separate collection of urine and faeces, in a room with controlled photoPeriod (12 hr light: 12 hr darkness), a temperature of 23 + 2°C and constant relative humidity (60%). Their diet in all the experiments was a standard rat feed of the following composition in dry substance (humidity 8.8%): proteins 26.2%, fat 4.2%, fiber 3.1%, total minerals 6.6% and nitrogen free extract (NFE) 59.9%.
The B-agonist used was clenbuterol (I-(4-amino-3,5dichlorophenyl)-2-ter-butylaminoethanol) (Itesba, S.A., Barcelona) added to the diet in powder form in different doses: 0, 30 and 50mg/kg. Experimental
design
Three experiments, corresponding to the three initial weights were designed. Each experiment consists of three 619
FRANCISCAPEI&Z-LLAMASand SALVAWRZAMORA
250 . B :: 200 . F 8 f -150. &S 2 >lOO. e s50
o-
70
assays, corresponding to the dose of b-agonist used. The experiments lasted 10 days, divided into two periods of 3 days (in which the animals adapted to the medium/ environment and diet) and 7 days, the principal part of the experiment, when the rats were fed the normal diet with the agonist added. Calculations The following calculations were determined:
1. Fecal nitrogen excretion, expressed as mg nitrogen per g ingested dry matter. 2. Apparent digestibility coefficient of the protein (ADC): 100.
3. Urinary nitrogen excretion, expressed as mg nitrogen per 100 average body weight. 4. Nitrogen balance (NB): NB=p
I-F-U
x 100.
I
5. Nitrogen retention (NR): I-F-U
Average weight
x 100.
Abbreviations used: I = ingested nitrogen; F = fecal nitrogen; U = Urinary nitrogen. Nitrogen was determined by Kjedhal’s method. 100
I 150
BO
Fig. 1. Effect of clenbuterol on fecal nitrogen excretion, the doses used: Omg/kg (white column), 30mgjkg (black column) and 5Omg/kg (striped column). The data are expressed as means (N = lo), SEM shown by vertical bars. There were no significant differences with respect to the control (white column).
ADC=
%$70
(g)
BODY WEIGHT
NR=
.
IOY WEIGHT (g)
Fig. 3. Effect of clenbuterol on urinary nitrogen excretion, the doses used: 0 mg/kg (white column, 30 mg/kg (black column) and 5Omg/kg- (striped column). The data are exnressed as means (N = 10). SEM shown by vertical bars. Significant differences with’ respect to the control (white column) were: *P < 0.05 and **P < 0.001. Statistical analysis The data are expressed as means f SEM. Tests for significance were carried out by use of Student’s r-test. P values of less than 0.05 were considered significant. RESULTS The following histograms represent the influence of clenbuterol (0, 30 and 50 mg/kg) on the parameters studied, in relation to the weight of the rats (70, 150 and 200 g). Each bar represents the average value of 10 data + SEM. Figure 1 shows the effect of clenbuterol on nitrogen fecal excretion, expressed in relation to food intake. None of the assays carried out showed any effects of the /?-agonist on the elimination of nitrogen in the stool, irrespective of animal age. Figure 2 represents the effect of clenbuterol on the digestibility coefficient of the protein. This value shows a great constancy between those animals treated and the control lots, which again suggests that the B-agonist has no effects on the absorption of the dietary protein. Figure 3 shows the influence of clenbuterol on urinary nitrogen excretion, related to the average weight of the animals. The b-agonist decreased the elimination of urinary nitrogen in all the lots studied.
r
40 20 I
o-
70
150 BODY WEIGHT
200
(g)
Fig. 2. Effect of clenbuterol on apparent digestibility coelI&ient of the protein (ADC), the doses used: 0 n&kg (white column). 30 ma/ka (black column) and 50 mg/kg (striped columdj. The dataam expressed as’means (N =-io>; SEM shown by vertical bars. There were no significant differences with respect to the control (white column).
70
2w
150 BODY WEIGHT
(g)
Fig. 4. Effect of clenbuterol on nitrogen balance (mg N/mg N consumed), the doses used: Omgjkg (white column), 30 mg/kg (black column) and 50 mg/kg (striped column). The data are expressed as means (N = lo), SEM shown by vertical bars. Significant differences with respect to the control (white column) were: *P < 0.05 and +*P < 0.001.
Clenbuterol and dietary protein utilization
g
200
% F50 5
100
:: ij
50
0
70
150 BODY WEIGHT
200
(9)
Fig. 5. Effect of clenbuterol on nitrogen retention (mg N/100 g body weight), the doses used: Omg/kg (white column), 30 mg/kg (black column) and 50 mg/kg (striped column). The data are expressed as means (N = lo), SEM shown by vertical bars. Significant differences with respect to the control (white column) were: *P < 0.05 and **p < 0.001. However, the decreasing of this parameter is statistically significant (P < 0.05) only in the older/heavier lots. Figures 4 and 5 represent the effect of clenbuterol on nitrogen balance (Fig. 4) and on nitrogen retention (Fig. 5). These figures show the positive effect of the /?-agonist on both parameters. When the results are expressed as a function of the ingested nitrogen (Fig. 4), the influence of age on these processes becomes apparent. The effect of clenbuterol is noticeable only in the heavier lots but, in leaving the influence of age aside, and expressing the results as a function of the average weight (Fig. 5), the B-agonist significantly increases (P < 0.001) the nitrogen retention in all the lots studied. DISCUSSION
The fecal nitrogen excretion slightly increases as the age of the animals increases, but this increase becomes statistically significant (P < 0.05) only in comparing the lots with the extreme body weights (70 and 200 g). This fact is shown in Fig. 2, where the apparent digestibility coefficient (ADC) is determined. These results show that proteic absorption at the intestinal level decreases, though very slightly, as the age/weight of the animals increases. The addition of clenbuterol to the diet had no effect on the nitrogen fecal excretion nor did it change the ADC, irrespective of the body weight of the animals and of the doses of p-agonist used. These results coincide with other researches made on goats (Williams et al., 1987). In our experimental conditions the results confirm that, though its pharmacological actions might suggest the opposite (Beth and Uvnas-Moberg, 1986; Lafontan et al., 1988), this P-agonist does not alter the dietary protein absorption at the digestive level in any way. From the above it can be concluded that the positive increases in body and muscle growth that both a number of authors (Baker et al., 1984; Dalrymple et al., 1984a, b, c; Kim et al., 1987; Yang and McElligott, 1989; Buttery and Dawson, 1990) and we, in similar experimental conditions (WrezLlamas and Zamora, 1991), have found after the
621
treatment with different /I-agonists cannot be accounted for by any improvement in the use of the dietary protein induced by clenbuterol. Urinary nitrogen excretion in the control lots (Fig. 3) shows no clear tendency in relation to the age of the animals, this possibly being due to the type of diet used; our intention in administering a proteinrich (26.2%) diet was not the study of the nitrogen balance in the control lots, but the study of the influence of clenbuterol on the excess of amino acids ingested and absorbed. Since the diet has a high level of proteins and high digestibility coefficient, the level of amino acids absorbed is considerably high in absolute terms. Thus, our research is aimed to study how clenbuterol can improve the body and muscle growth. The results as to nitrogen balance and retention in the control lots (Figs 4 and 5) are as expected for the range of weights studied. The maximum values of both indices were obtained in the youngest animals, that is, in those with higher growth rates. A statistically significant decrease (P < 0.001) in relation to the age of the animals is also observed. This suggests that as the animals are older, their growth rate becomes slower and the metabolic activity in this range of age gradually decreases. Treatment with clenbuterol decreased the urinary nitrogen excretion (Fig. 3), though not significantly in all the assays carried out. These changes once again confirm that this b-agonist does alter the proteic metabolism at the biosynthetic and/or proteolytic level, as has already been mentioned (Deschaies et al., 1981; Hamby et al., 1985; Reeds et al., 1986; Bohorov et al., 1987; Hovel1 et al., 1987; Challiss et uf., 1988; McLennan and Edwards, 1989). Administration of the /?-agonist in our experimental conditions significantly improves nitrogen balance (Fig. 4) in the older/heavier animals (150 and 200 g), but the influence of the drug is much more marked on the nitrogen retention/average weight ratio (Fig. 5), which increases (P < 0.001) even in the younger animals, showing a higher metabolic rate. Given that (A) the expression of these results shows the influence of the animals’ weight/age in Fig. 4 (balance), and of the intake in Fig. 5 (retention), and (B) the results obtained by either of the two procedures are similar but not identical, it could be inferred that clenbuterol presents a double effect. Firstly, on the intake, which is slightly increased by the agonist and also on the improvement of the metabolic use of the protein, and as result, on the general decreasing in the urinary nitrogen excretion. This two-fold effect accounts for the significant increase (P < 0.001) in the nitrogen retention, even in the younger animals, a fact which cannot be observed in studying the balance, since in this case the possible influences of the p-agonist on the intake are not taken into account. The results obtained in this study may clearly account for the positive effect of clenbuterol on body growth and, what is more important, on muscle mass growth, in accordance with our previous findings and reports (Perez-Llamas and Zamora, 1991; PerezLlamas et al., 1991). These effects would be a consequence of the positive effect of the /?-agonist on nitrogen retention.
622
FRANCISCAPEG&-LLAMASand SALVADC~R ZAMORA
Acknowledgements-The authors wish to thank Professor M. Canteras Jordana for his help in the statistical analysis. REFERENCES Alderson E. M., Scaife J. R. and Galbraith H. (1988) The effect of oestradiol-17jI and cimaterol 8iven alone or in combination on lipid metabolism and c-AMP concentrations in tissues of male castrate lambs. Proc. Nutr. Sot. 47, 109A. Baker P. K., Dahymple R. H., Ingle D. L. and Ricks C. A. (1984) Use of a /J-adrenergic agonist to alter muscle and fat deposition in lambs. J. Anim. Sci. 59, 1256-1261. Beth K. and U~ass-Mo~rg K. (1986) B-Adrenergic agonists inhibit gastric acid and pepsin secretion through somatostatin release in dogs. Stand. J. Gustroenterol. 21, 531-536. Beermann D. H., Fishell V. K., Hogue D. E., Ricks C. A. and Dalrymple R. H. (1985a) Effects of the repartitioning aaent. cimaterol (CL 263. 780) on skeletal muscle fiber tipe and fiber hypkrtrophy in lambs. J. Anim. Sci. 61,254 (Abstr. 116). Beermann D. H., Hogue D. E., Dalrymple R. H. and Ricks C. A. (1985b) Effects of the repartitioning agent cimaterol (CL263, 780) and tishmeai on skeletamuscle and fat accretion in lambs. J. Anim. Sci. 61, 255 (Abstr. 117). Beme R. S., Novakofski J. and Bechtel P. J. (1985) Effects of the &-agonist clenbuterol on body and tissue weights in four strains of rats. J. A&m. Sci. 61, 256 (Abstr. 119). Blum J. W., Froehli D. and Kunz P. (1982) Effects of catecholamines on piasma free fatty acids in fed and fasted cattle. Endocrinology 110, 452-456. Bohorov O., Buttery P. J., Correia J. H. D. and Soar J. B. (1987) The effect of the /I,-adrenergic agonist clenbuterol or implantation with oestradiol plus trenbolone acetate on protein metabolism in wether lambs. Er. f. Nutr. 57, 99-107. Buttery P. J. and Dawson J. M. (1990) Growth promotion in farm animals. Proc. Nutr. Sot. 49, 459466. Challis5 R. A., Leighton B., Wilson S., Thurlby P. L. and Arch J. R. S. (1988) An investigation of the /I-adrenoceptor that mediates metabolic responses to the novel agonist BRL 28410 in rat soleus muscle. Eiochem. Pharmat. 37,947-950. Claeys M. C., Mulvaney D. R., McCarthy F. D., Gore M. T., Marple D. N. and Sartin J. L. (1989) Skeletal muscle protein synthesis and growth hormone accretion in young lambs treated with clenbuterol. J. Anim. Sci. 67, 2245-2254. Coleman M. E., Ekeren P. A. and Smith S. B. (1988) Lipid synthesis and adipocyte growth in adipose tissue from sheep chronically fed a fl-adrenergic agent. J. Anim. Sci. 66, 372-378. Dalrymple R. H., Baker P. K., Doscher M. E., Ingb D. L., Pankavich J. A. and Ricks C. A. (1984a) Effect of the repartitioning agent CL 263,780 on muscle and fat accretion in finishing swine. J. Anim. Sci. 59,212 (Abstr. 164). Dalrymple R. H., Baker P. K., Gingher P. E., Ingle D. L., Pensack J. M. and Ricks C. A. (1984b) A repartitioning agent to improve performance and carcass composition of broilers. Puultrv Sci. 63. 23762383. Dahymple R. H, Baker P. K. and Ricks C. A. (1984~) Repartitioning agents to improve performance and body composition. In Proceedings of Georgia Nutrition Conference for the Feed Industry, pp. 111-l 18. University of Georgia, Georgia, U.S.A. Deschaies Y., Willemot 3. and Leblac J. (1981) Protein synthesis, amino acid uptake and pools during isoproterenol-induced hypertrophy of the rat heart and tibialis muscle. Can. J. Physiol. Pharmnc. 59, 113-121.
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Clenbuterol and dietar y protein utilization Wrez-Llamas F. and Zamora S (1991) The influence of clenbuterol on growth in rats. Camp. Biochem. Physiol. WA, 241-244. Reeds P. J., Hay S. M., Donvood P. M. and Palmer R. M. (1986) Stimulation of muscle growth by clenbuterol: lack of effect on muscle protein biosynthesis. Br. J. NW. 56, 249-258. Williams P. E. V. (1987) The use of j-agonists as a means of altering body composition in livestock species. Nutr. Abs. Rev. B57, 453464.
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Harris C. I. and Garthwaite P. (1987) Effects of a j-agonist (clenbuterol) on growth, carcass composition, protein and energy metabolism of veal calves. Br. J. Nutr. 57, 417428.
Yang Y. T. and McElligott M. A. (1989) Multiple actions of /?-adrenergic agonists on skeletal muscle and adipose tissue. Bioch&. J! Ml, I-10. Zeman R. J., Ludemann R., Easton T. G. and Etlinger J. D. (1988) Slow to fast alterations in skeletal muscle fibers caused by clenbuterol, a &-receptor agonist. Am. J. Physiol. 254, E726-E732.