Effects of cimaterol on protein synthesis, protein degradation, amino acid transport and acetate oxidation in sheep external intercostal muscle

NUTRITION RESEARCH, Vol. 8, pp. 1287-1296, 1988 0271-5317/88 $3.00 + .00 Printed in the USA. Copyright (c) 1988 Pergamon Press plc. All rights reserved.

EFFECTS OF CIMATEROL ON PROTEIN SYNTHESIS, PROTEIN DEGRADATION, AMINO ACID TRANSPORT AND ACETATE.OXIDATION IN SHEEP EXTERNAL INTERCOSTAL MUSCLE I M.A. Wilson , M.S., C. Zhong , M.S., N.E. Forsberg , Ph.D. 2, R.H. Dalrymple +, Ph.D., C.A. Ricks +, Ph.D. Department of Animal Science, Oregon State University Corvallis, OR 97331 +American Cyanamid Co., Princeton, N.J. 08540

ABSTRACT The objectives of this study were to examine mechanisms by which cimaterol, a beta-2 adrenergic agonist, may stimulate muscle protein gain. Sixteen wether lambs were assigned to a control or cimaterol-containing diet (n = 8) and allowed ad libitum intake for 28 days. The effects of dietary cimaterol on protein synthesis, protein degradation, amino acid transport and acetate oxidation were examined in external intercostal muscle bundles isolated from each experimental animal. Additionally, the effects of dietary cimaterol on liveweight gain and of cimaterol added directly to incubation media on protein synthesis and protein degradation were evaluated. Dietary cimaterol increased average daily gains; however, it did not affect either the synthesis or degradation of muscle proteins. Addition of cimaterol directly to incubations of skeletal muscle tissue did not affect either of these processes. Dietary cimaterol increased acetate oxidation in isolated muscles and stimulated transport of amino acids via a sodium gradientdependent process. Increased acetate oxidation may reflect enhanced energetic requirements of cimaterol-treated tissues and the increase in amino acid transport detected may play an important role in cimaterol-dependent muscle hypertrophy. Key words:

Sheep, cimaterol, skeletal muscle metabolism, protein synthesis, protein degradation, amino acid transport.

ipublished as paper Experiment Station.

number

8318

of

the

Oregon

Agricultural

2To whom correspondence should be sent: N.E. Forsberg, Department of Animal Science, Oregon State Univ., Corvallis, OR 97331.

1287

IZB8

M.A. WILSON et al. INTRODUCTION

Dietary beta-adrenergic agonists stimulate muscle protein accretion in sheep (1,2), pigs ( 3 ) , beef cattle (4), and chickens (5). The mechanisms underlying these observations have been the objects of considerable study in recent years. Since these compounds remove constraints to normal muscle growth, an understanding of their mechanisms would be useful in elucidating the cellular control of muscle growth. Evidence obtained from a variety of investigations suggests that protein degradation is reduced by the administration of these compounds (6-10). Protein synthesis in treated animals has been reported to be increased (lO,11), unchanged (7a,8,9) and, in one case, reduced in rats treated with clenbuterol for extended periods of time (8). It remains unclear whether the effects of these compounds on muscle are mediated via their direct interaction with muscle cells or result from indirect release of other hormones or both. Moreover, the extent to which muscle hypertrophy results from direct or indirect actions of these compounds on muscle protein turnover versus their direct or indirect actions on other physiological processes, which may also constrain muscle growth, is not known. Although beta-adrenergic agonists increase blood supply to the hindlimb (12), increase amino acid transport into muscle (13, 14) and may spare hepatic amino acid catabolism by providing fatty acids to the liver it is not known whether these changes would occur in response to increased nutrient requirements of hypertrophying muscle or whether they would cause hypertrophy. The administration of beta-adrenergic agonists may cause muscle hypertrophy through several of the above-mentioned mechanisms. The variability in response to these compounds may be dependent upon the beta-adrenergic agonist used, the species and age of animals tested and their physiologic and nutritional status. The present study was designed to investigate the direct and indirect effects of cimaterol on protein synthesis and protein degradation and the effects of dietary cimaterol on amino acid transport and acetate oxidation in skeletal muscle of growing wether lambs.

MATERIALS AND METHODS

Sixteen wether lambs (Suffolk cross whiteface) weighing an average of 37.3 kg were alloted to two experimental treatments. Eight animals were randomly assigned to a control diet (Table I) and eight animals were assigned to a cimaterol-containing diet (5 ppm). The diets contained 15.4% crude protein and 12.3 MJ M.E./kg. Animals were housed in group pens and allowed ad libitum consumption of both water and feed for a period of 28 days. Animals were weighed at the start and at the completion of the study and pen intakes of feed and feed refusals were recorded daily. Surgical biopsies Following the 28 day feeding period biopsies of the external intercostal muscle (5-6 cm in length) were taken from the lambs according to the methods of Wijayasinghe et al. (15) and transported to the laboratory in a Krebs-Henseleit bicarbonate (KHB) buffer containing I0 mM glucose and 5 mM acetate (pH 7.4, 22 ~ C., 95% 02:5% C02). Biopsies were attached to a stainless steel frame, with

SHEEP MUSCLEMETABOLISM

1289

TABLE 1 Composition of Experimental Diets I

Ingredient

Corn Soybean meal Ground orchardgrass hay Ground corn cobs Alfalfa meal Cane molasses Salt (NaCI) Limestone Vitamin and mineral premix

Percent dry matter

56 lO 13 2 15 2.5 .5 .75 .25

Calculated feed contents: Crude protein Acid detergent fiber Calcium Phosphorous

15.4 12.4 .65 .32

1-The cimaterol-containing diet was identical to the above except that cimaterol (5 ppm) was included using ground corn cobs as a vehicle. Cimaterol was provided by American Cyanamid Co., Princeton, N.J. Both diets were pelleted and were formulated to meet the requirements for growing lambs (26). 2-Provided per kg diet: Vitamin A palmitate, llO00 IU; vitamin D, 2650 IU; vitamin E acetate, 22 IU; Zn, 125 mg; Fe, 125 mg; Mn, 62.5 mg; Cu, lO mg; I, 1.25 mg; Co, 0.5 mg; Se, 0.I mg.

the use of a cyanoacrylate adhesive (Krazy Glue, Chicago, IL), immersed in the transport buffer and individual muscle bundles (15 - 25 mg), with tendons attached, were removed using microdissection equipment (Fine Science Tools, Belmont, CA) under a stereozoom microscope. Larger numbers of bundles were prepared than reported by Wijayasinghe et al. (15) by taking two biopsies (5-10 g) from each animal at staggered intervals, by dividing each biopsy into two smaller samples and by employing two individuals in the dissection of each muscle sample. Individual muscle bundles were attached to a plastic frame with the use of small surgical clips (Fine Science Tools, Belmont, CA) at their approximate resting length and incubated as described below. Viability of individual muscle preparations was determined by assessing initial and final ATP concentrations with two additional muscle bundles taken from each muscle biopsy. Samples of muscle were immediately frozen in liquid nitrogen prior to assay and ATP concentrations were determined using a kit from Sigma Chemical Co. (St. Louis, MO; catalogue 366-UV). Recovery of ATP was determined by the addition of known quantities of ATP to assay tubes. In all cases ATP concentrations from freshly isolated and incubated muscle were greater than 2.5 ~moles/g tissue.

1290

M.A. WILSON et al.

Incubations Protein synthesis was determined by incubating individual muscle bundles for 2 hours in 25 ml Erlenmeyer flasks, fitted with serum stoppers, (37 ~ C.; 95% 0o:5% COo; lO0 oscillations per min) containing 3 ml KHB buffer with 1 mM acetate, 2 mM~glucose, .i U/ml bovine insulin (Sigma Chemical Co., 1~t. Louis, MO), all L-amino acids including .128 mM tyrosine and .3 uCi/ml U -~ C tyrosine (New England Nuclear (NEN), Boston, MA). Preliminary studies indicated that rates of protein synthesis were constant throughout this time. Following incubation the muscle bundles were homogenized and the protein precipitated with 3 percent trichloroacetic acid (TCA; w/v). Cell protein was obtained by centrifugation followed by washing twice. The pellet was subsequently solubilized in 1 ml Protosol (NEN; Boston, MA) and the radioactivity associated with protein determined by liquid scintillation counting. Protein degradation was determined by incubating muscle bundles for 4 hours in 4 ml of KHB buffer (37 ~ C., 95% 02:5% COo; i00 oscillations per min) containing 1 mM acetate, 2 mM glucose and .l~U/ml bovine insulin. Cycloheximide (0.5mM) was included in the buffer to minimize recycling of released tyrosine. In preliminary studies rates of protein degradation were constant during a 4 hour incubation period. Protein degradation was estimated by the release of free tyrosine into the TCA-soluble supernatant during the incubation. Tyrosine release was determined fluorometrically (16). Appropriate initial and final tissue free tyrosine values were determined to correct for net tyrosine release. These values did not change appreciably during the course of the incubation. The effects of dietary cimaterol on protein synthesis and degradation were compared in control versus cimaterol-fed animals. The possibility that cimaterol may directly affect protein synthesis or protein degradation in muscle tissue was evaluated by incubating additional muscle bundles, taken from the same sheep, in the presence of O, I, i0 or I00 ~M cimaterol. The effects of medium cimaterol on protein degradation were investigated in each of the first group of animals biopsied (i.e., 4 control-fed and 4 cimaterol-fed; mean weight 43.0 kg at biopsy, SEM 1.6) and the effects of medium cimaterol on protein synthesis were evaluated in each of the second group of animals biopsied (4 control and 4 cimaterol-fed; mean weight 50.5 kg at biopsy, SEM; 1.7). Cimaterol-HCl, provided by American Cyanamid Co. Princeton, N.J., was included in incubation media (pH 7.4) dissolved in KHB. Incubation conditions were identical to those described above. In all cases the effects of variable concentrations of cimaterol and the effects of dietary versus medium cimaterol were evaluated using triplicate observations in each animal. Effects of dietary cimaterol on muscle acetate oxidation were examined by incubating muscle bundles taken from control and cimaterol-fed animals, in duplicate, in KHB buffer1~ontaining 1 mM acetate, 2 mM glucose, .i U/ml bovine insulin and .05 pCi/ml I - ~ C acetate (NEN, Boston, MA). Conversion of acetate to carbon dioxide was determined (17). Amino acid transRort Transport of aminoisobutyric acid (AIB; .2 mM) was determined in both sodium-adequate media (Na-KHB), which consisted of KHB buffer containing 1 mM potassium acetate and 2 mM glucose, and sodium-free media (choline-KHB; choline chloride and choline bicarbonate replaced their respective salts in Na-KHB) to determine the effects of cimaterol on sodium gradient-dependent and independent transport of AIB. Additionally the transport of .2 mM AIB in the presence of 25 mM N-methylaminoisobutyric acid (MeAIB; a model analogue transported specifically by transport system A; 18) in Na-KHB was used to determine the effects of dietary cimaterol on the proportion of AIB transport

SHEEP MUSCLEMETABOLISM

1291

which was mediated by system A-independent mechanisms (18). The transport of cycloleucine (c-leu) in choline-KHB was examined to determine the effects of dietary cimaterol on Na gradient-independent transport. Incubations were conducted in sealed 25 ml Erlenmeyer flasks containing 3 ml transport medium in a shaking water bath (37 ~ C.; 95% 02:5% CO?; I00 oscillations per minute). Sheep muscle bundles incubated for 2 ~r in this manner concentrated AIB intracellularly to approximately four-fold extracellular concentrations (N.E. Forsberg, unpublished observations). Tissues were incubated for 30 minutes, during which time linear uptake of amino acid analogues was evident, (N.E. Forsberg, unpublished data) after which they were rinsed with ice-cold analogue-free Na-KHB or choline-KHB. The extracellular analogue component, ~ e d e d for the calculation of actual transport, was determined with the use of "C-inulin (.2 ~Ci/ml; NEN; Boston, MA) in replicate flasks. Inulin space was not influenced by the extracellular analogue concentration or the presence or absence of sodium in incubation media. Carbon-14 labeled amino acid analogues were obtained from NEN (Boston, MA) and were added to incubation media at .2 Ci/ml. All observations were conducted in duplicate in each animal. Statistical analyses The synthesis, examined cimaterol analysis effects.

effects of dietary cimaterol on average daily gains, protein protein degradation, acetate oxidation and amino acid transport were with the use of a two-tailed t-test (19). The effects of medium on protein synthesis and degradation were examined using two-way of variance with dietary and medium cimaterol examined as main The level of significance adopted for all comparisons was 5 percent.

RESULTS

Average daily gains of cimaterol-fed animals (.378 + .026 kg/day) during the 28 day feeding trial were significantly greater (p < .O5) compared to gains of control-fed animals (.301 • .030 kg/day). Since animals were group-fed, individual feed intake data were not gathered; however, pen intake data indicated that the cimaterol did not affect intake and that improved efficiency of liveweight gain accounted for the increased rate of growth. The effects of dietary cimaterol on rates of protein synthesis, protein degradation and acetate oxidation in external intercostal muscle tissue are shown in Table 2. Dietary cimaterol did not affect protein synthesis or protein degradation. Muscle acetate oxidation was significantly increased (p < .05) by the presence of cimaterol in the diet (Table 2). The effects of medium cimaterol on protein synthesis in the first eight animals biopsied and on protein degradation in the second eight animals biopsied are shown in Table 3. Medium cimaterol did not affect protein synthesis or protein degradation. The transport of amino acid analogues under a variety of conditions was examined to determine whether dietary cimaterol induced changes in activities of amino acid transport systems (Table 4). Transport of AIB was increased by dietary cimaterol when muscle samples were incubated in Na-KHB but not when incubated in choline-KHB. Aminoisobutyric acid transport in the presence of MeAIB, was not affected by cimaterol. Cycloleucine transport was not affected by cimaterol.

1292

M.A. WILSON et al. DISCUSSION

External intercostal muscle of sheep consists of 34% Type I and 66% Type II fibers (Y.B. Lee, personal communication). Beta-adrenergic agonists cause hypertrophy of both Type I and II fibres (20). Hence, the external intercostal muscle of cimaterol-fed sheep should provide an adequate model to study the effects of these compounds on muscle metabolism. Although we did not study the effects of these compounds on muscle hypertrophy in this study, we did observe hypertrophy (p < .05) of semitendinosus, biceps femoris and gastrocnemius muscles of sheep fed I and I0 ppm cimaterol in a concurrent study. Although direct effects of beta-adrenergic agonists on muscle protein synthesis have not been reported there is evidence that they they increase messenger RNA concentrations of specific myofibrillar proteins (21). Whether or not these changes result in an increase in myofibrillar protein synthesis is not known. However, our data suggest that the presence of cimaterol in the diet did not increase muscle growth by increasing muscle protein synthesis. The methods used in this study assessed the synthesis of all muscle proteins simultaneously. It is possible that the synthesis of specific myofibrillar proteins is influenced by beta-adrenergic agonists and that the rapid turnover of nonmyofibrillar proteins obscured this effect. Determination of the conversion of tyrosine to specific myofibrillar proteins is needed to assess this possibility. Using in vivo infusion techniques other investigators (8,9) have determined that beta-adrenergic agonists enhance muscle growth by reducing protein degradation. Methods used in these studies involved direct determination of fractional rates of muscle protein synthesis and muscle protein accretion and indirect calculation of fractional rates of protein degradation. We attempted to assess the effects of these compounds on muscle protein degradation directly by using biopsied muscle and determined that cimaterol, whether provided in the diet or incubation media, did not influence muscle protein degradation. Several reasons for this disagreement with previous reports (8,9) may exist. It is possible that protein degradation was not affected in animals treated chronically with cimaterol, as in this study, or that artifactual increases in protein degradation of isolated muscle, a characteristic of isolated sheep skeletal muscle (Forsberg et al., unpublished observations), mask cimaterol's effects. Alternatively, the effects of cimaterol on protein degradation may have been lost during the 45 minute transport and processing time required for preparation of tissues for incubation. Amino acid transport represents a barrier to the incorporation of amino acids into protein; however, the significance of the individual transport mechanisms and the regulation of these processes to muscle growth is not known. Several investigators have shown that administration of catecholamines and their analogues to rodents increased amino acid transport activity in muscle tissue (13, 14, 22) whereas the direct administration of these compounds to muscle tissue caused a reduction in transport (13). These observations suggest an indirect effect of cathecholamines on transport. We investigated the effects of dietary cimaterol on rates of transport of AIB and c-Leu under a variety of experimental conditions in which, if alterations in transport activity were observed, determination of individual transport agencies affected by cimaterol could be determined. Dietary cimaterol stimulated transport of AIB when muscle samples were incubated in Na-KHB but not when incubated in choline-KHB. This implies that cimaterol stimulated the activity of a sodium gradient-dependent transport agency. When MeAIB (25 mM), a model substrate for transport system A, was added to Na-KHB the effect of cimaterol on AIB transport was not evident. Hence, it is believed that dietary cimaterol

SHEEP MUSCLEMETABOLISM

1293

stimulated the activity of a sodium gradient-dependent system A-like transport agency. In a previous study (7b) we determined that activity of System A in sheep skeletal muscle is either repressed or non-existent. It is possible that chronic administration of cimaterol derepressed system A activity.

TABLE 2 Effect of Dietary Cimaterol on Protein Synthesis, Protein Degradation and Acetate Oxidation in Sheep External Intercostal Muscle '

Dietary cimaterol (ppm)

0 Parameter assessed Protein synthesis 3 4 Protein degradatiRn Acetate oxidation-

5

mean

SEM

mean

SEM

.131 .314 4.46 a

.022 .054 .34

.125 .31 ~ 5.65

.014 .039 .57

1Amino acids provided in incubation (mM) included L-alanine, .382; L-arginine, .184; L-asparagine, .250; L-aspartic acid, .0023; L-cystine, .049; L-glutamic acid, .048; L-glutamine, .lO0; glycine, .200; L-histidine, .063; L-isoleucine, .lOl; L-leucine, .203; L-lysine, .397; L-methionine, .064; L-phenylalanine, .083; L-proline, .371; L-serine, .107; L-threonine, .269; L-tryptophan, .082; L-tyrosine, .128; L-valine, .228. 2values with (p<.05). 3

different

superscripts

in

the

same

row differ significantly

nmoles tyrosine converted to protein per mg tissue per 2 h.

4nmoles tyrosine released from muscle protein per mg tissue per 4 h. 5nmoles acetate converted to carbon dioxide per mg tissue per 2 h.

The significance of increased activity of a system A-like transport agency to protein synthesis and degradation in skeletal muscle is not known. If intracellular concentrations of amino acids were normally limiting rates of protein synthesis, increased transport could stimulate protein synthesis and protein accretion. An increase in amino acid transport coupled to increased blood flow to muscle (12) could play an important permissive role in cimateroldependent muscle hypertrophy. Transport of c-leu, a model neutral non-polar amino acid analogue (23), in choline-KHB reflects the combined activities of sodium gradient-independent transport mechanisms. Most of this activity in other tissues is represented by transport system L (18, 24). We did not find evidence for an alteration in sodium gradient-independent transport activity. This may be expected as sodium gradient-independent transport mechanisms are not as intensely regulated as system A (18).

1294

M.A. WILSON et al. TABLE 3 Effects of Medium and Dietary Cimaterol on Protein Synthesi~ and Protein Degradation in Sheep External Intercostal Muscle ~.

Medium cimaterol (~M)

0

n

Protein synthesis 2 ^ Protein degradation ~

mean

8 8

.103 .323

1

SEM

.013 .045

mean

.095 .375

I0

SEM

i00

mean

.022 .043

SEM

.099 .334

.020 .058

mean

SEM

.092 .316

.019 .048

Ivalues are means • SEM of 8 animals per treatment. 2nmoles of tyrosine converted to protein per mg tissue per 2 hr. 3nmoles of tyrosine released from protein per mg tissue per 4 hr.

TABLE 4 Effects of Dietary Cimaterol on Transport of Amino Ac~d2Analogues in Sheep External Intercostal Muscle Tissue. '

Dietary treatment

Label

Inhibitor

Labeled analogue

mM

Inhibitory analogue

AIB AIB AIB

.2 .2 .2

...... ...... MeAIB

c-leu c-leu

.2 25

...... ......

Control

mM

25

Medium

Na-KHB choline-KHB Na-KHB choline-KHB choline-KHB

Ivalues are means of 8 animals • SEM. _~nd are transported per g tissue per 30 min 2values

(p<.05).

in

the

same

row

with

differing

Cimaterol

Mean

SEM

Mean

SEM

.109 a .044 .lO0

.019 .003 .010

.135 b .050 .103

.006 .004 .015

.040 2.00

expressed

.003 .36

.041 2.21

as p moles

.005 .43

analogue

superscripts differ significantly

SHEEP MUSCLE METABOLISM

1295

A recent study reported that clenbuterol increased heat production in sheep (25). This implies that energetic requirements of animals fed betaadrenergic agonists may be elevated. If energy requirements of muscle tissue are increased by the administration of beta-adrenergic agonists we expected that substrate oxidation would be increased to meet the enhanced maintenance energy requirement. Accordingly, we evaluated the oxidation of acetate, a substrate oxidized by ruminant muscle tissue (15), in control and cimaterol-fed animals and determined that acetate oxidation was increased by cimaterol. This implies that the energetic requirements of muscle may be increased by cimaterol; however, direct measurements of oxygen consumption are needed to confirm this hypothesis.

ACKNOWLEDGEMENT

We are grateful to Dr. Yu Bang Lee of the University of California at Davis for conducting muscle fibre typing on sheep external intercostal muscle.

REFERENCES

I.

Baker P K, Dalrymple R H, Ingle D L, Ricks C A. Use of a beta-adrenergic agent to alter muscle and fat deposition in lambs. J Anim Sci 1984; 59:1256-61.

2.

Beermann D H, Hogue D E, Fishell V K, Dalrymple R H, Ricks C A. Effects of cimaterol and fishmeal on performance, carcass characteristics and skeletal muscle growth in lambs. J Anim Sci 1986; 62:370-80.

3.

Ricks C A, Baker P K, Dalrymple R H, Doescher M E, Ingle D L. clenbuterol to alter muscle and fat accretion in swine. Fed Proc 43: Supp. 1., 857.

4.

Ricks C A, Dalrymple R H, Baker P K, Ingle D L. Use of a beta-agonist to alter fat and muscle deposition in steers. J Anim Sci 1984b; 59:1247-55.

5.

Dalrymple R H, Baker P K, Gingher P E, Ingle D L, Pensack J M, Ricks C A. A repartitioning agent to improve performance and carcass composition in broilers. Poultry Sci 1984; 63:2376-83.

6.

Li J B, Jefferson L S. Effect of isoproterenol on amino acid levels protein turnover in skeletal muscle. Am J Physiol 1977; 232:E243-49.

Use of 1984a;

and

7a. Forsberg N E, Merrill G F. Effects of cimaterol on protein synthesis and degradation in monolayer cultures of rat and mouse myoblasts. J Anim Sci 1986; 63:Supp. l, 222. 7b. Forsberg N E, Kaneps A J, Riebold J W. Neutral amino acid transport in sheep external intercostal muscle. Can J Anim Sci 1988 (In Press). 8.

Reeds P J, Hay S M, Dorwood P M, Palmer R M. Stimulation of muscle growth by clenbuterol: Lack of an effect on muscle protein biosynthesis. Br J Nutr 1986; 56:249-58.

9.

Bohorov O, Buttery P J, Correia J H R D, Soar J B. The effects of the beta-2 adrenergic agonist clenbuterol or implantation with estradiol plus trenbolone acetate on protein metabolism in wether lambs. Br J Nutr 1987; 57:99-107.

1296

M.A. WILSON et al.

lO. Zeman R J, Ludemann R, Etlinger J D. Clenbuterol, a beta-2 agonist, retards atrophy in denervated muscles. Am J Physiol 1987; 252:E152-55. Ii. Emery P W, Rothwell N J, Stock M J, Winter P D. Chronic effects of beta-2 adrenergic agonists on body composition and protein synthesis in the rat. Biosci Rep 1984; 4:83-91. 12. Eisemann J H, Huntington G B. Effect of clenbuterol on blood flow and oxygen use in two tissue beds of steers. Fed. Proc. 1987; 46:Suppl. I, 1177. 13

Sanders R B, Jacob W H. Action of epinephrine on amino acid uptake rat diaphragm in vitro and in vivo. Pharmacol. 1972; 7:327-48.

into

14. Deschaies Y, Willemot J, LeBlanc J. Protein synthesis, amino acid uptake, and pools during isoproterenol-induced hypertrophy of the rat heart and tibialis muscle. Can J Physiol Pharmacol 1981; 59:113-21. 15. Wijayasinghe M S, Milligan L P, Thompson J R. The preparation and in vitro viability of isolated external intercostal muscle fiber bundles from sheep. Can J Anim Sci 1984; 64:785-89. 16. Waalkes T P, Udenfriend S. A fluorometric method for the estimation of tyrosine in plasma and tissues. J Lab Clin Med 1957; 50:733-36. 17. Forsberg N E, Smith N E, Baldwin R L. Roles of acetate and its interactions with glucose and lactate in cow mammary tissue. J Dairy Sci 1984; 67:2247-54. 18. Shotwell M A, Kilberg M S, Oxender D L. The regulation of neutral amino acid transport in mammalian cells. Biochim. Biophys. Acta 1983; 737:26784. 19. Steel R G D, Torrie J H. Principles and Procedures of Statistics, McGraw-Hill Book Co. New York.

1980.

20. Kim Y S, Lee Y B, Ashmore C R, Dalrymple R H. Effect of cimaterol on skeletal muscle characteristics of lambs. J Anim Sci 1987; 65:Supp. l, 278. 21. Smith S B, Garcia D K, Davis S K, Patton M A, Anderson D B. Specific gene expression in longissimus muscle of steers fed ractopamine. J Anim Sci 1987; 65:Supp l, 278. 22. Nutting D F. Anabolic effects of catecholamines in diaphragm muscle from hypophysectomized rats. Endocrinol 1982; 110:307-17. 23. LeCam A, Freychet P. Neutral amino acid transport. Characterization of the A and L systems in isolated rat hepatocytes. J Biol Chem 1977; 252:148-56. 24. Shotwell M A, Jayme D W, Kilberg M S, Oxender D L. Neutral amino acid transport in Chinese hamster ovary cells. J Biol Chem 1981; 256:5422-27. 25. Brockway J M, MacRae J C, Williams P E V. Side effects of clenbuterol as a repartitioning agent. Vet Record 1987; 381-83. 26. National Research Council. Nutrient Requirements for Sheep, 1985. National Academy Press. Washington, D.C. Accepted for publication May 31, 1988.

6th Edition.