DOMESTIC ANIMAL ENDOCRINOLOGY

Vol. 7(2):149-164, 1990

EFFECTS OF CIMATEROL ON MUSCLE PROTEIN METABOLISM AND ITS ACTIONS IN HYPOTHYROID AND HYPERTHYROID RATS 1'= N.E. Forsberg3 and N.B. Wehr Department of Animal Science Oregon State University, Corvallis, OR 97331-6702 ReceivedJanuary9, 1989 ABSTRACT Objectives were to examine mechanisms underlying anabolic actions of cimaterol in skeletal muscle and to evaluate cimaterol's actions in hypothyroid and hyperthyroid rats. In the first study growing rats were fed either a control diet or a diet containing cimaterol for 10 days. In a second study sbam-thyroidectomized and thyroidectomized (Tx) rats were assigned to one of 5 treatments: control (sbam-Tx), Tx, Tx supplemented with cimaterol, Tx injected with triiodothyronine (T3), and Tx rats injected with T 3 and supplemented with cimaterol. Effects of treatments on growth, muscle weights and urinary N~.methylhistidine (NMH) excretion were evaluated in both trials. Muscle was also collected for determinations of DNA, RNA, protein and activities of several proteolytic enzymes. Cimaterol caused muscle hypertrophy and increased urinary NMH excretion. Hence, anabolic actions of cimaterol may resuh from an increase in myofibrillar protein synthesis which exceeds changes in myofibrillar protein degradation. Urinary NMH excretion was reduced by thyroidectomy and increased in hyperthyroid rats and both hypothyroidism and hyperthyroidism were characterized by myopathy. Cimaterol increased muscle weights in hypothyroid but not in hyperthyroid rats. Therefore, cimaterol's anabolic properties are thyroid hormone-independent and antagonized by excess thyroid hormone. Anabolic actions of cimaterol in hypothyroid rat muscle were attributed to an action on protein synthesis because urinary NMH excretion was not affected by cimaterol but muscle RNA concentration was increased. Activities of cathepsins B, D and L and neutral proteinase were dose-related to thyroid status, however, were unrelated to cimaterol-dependent perturbations in NMH excretion. Control of muscle protein balance by thyroid hormones may involve regulation of these enzymes; however, control of muscle protein degradation by cimaterol is likely directed towards other proteolytic mechanisms or to mechanisms which alter susceptibility of myofibrillar proteins to degradation. INTRODUCTION H y p e r t h y r o i d i s m and h y p o t h y r o i d i s m are o f t e n c h a r a c t e r i z e d b y m u s c l e afro. p h y and w e a k n e s s ( I , 2); h o w e v e r , t h e i r etiologies differ. H y p o t h y r o i d m y o p a t h y is associated w i t h a large r e d u c t i o n in myofibrillar p r o t e i n synthesis (MPS; 24) and a small r e d u c t i o n in myofibrillar p r o t e i n d e g r a d a t i o n (MPD; 2-4) w h e r e a s h y p e r t h y r o i d m y o p a t h y is c h a r a c t e r i z e d by a c c e l e r a t e d MPD and little change or a slight increase in MPS (5-7). Administration of b e t a - a d r e n e r g i c agonists to m a m m a l s a n d birds increases m u s c l e g r o w t h ( 8 - I 2). Mechanisms r e s p o n s i b l e have b e e n a t t r i b u t e d to changes in b o t h MPS and MPD. Ability o f b e t a - a d r e n e r g i c agonists to stimulate m u s c l e g r o w t h suggests that, in addition to their a p p l i c a t i o n as a n a b o l i c agents in l i v e s t o c k p r o d u c t i o n , t h e s e c o m p o u n d s m a y have utility in treating m y o p a t h i c disorders. Therefore, o b j e c t i v e s of this s t u d y w e r e 1) to e x a m i n e m e c h a n i s m s b y w h i c h c i m a t e r o l e n h a n c e s skeletal m u s c l e growth, 2) to e x a m i n e effects Copyright © 1990 by DOMENDO, iNC.

149

0739-7240/90/$3.00

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FORSBERG AND WEHR

of thyroid diseases on muscle protein metabolism, 3) to evaluate effects of cimaterol on muscle growth and protein metabolism in hypothyroidism and hyperthyroidism and 4) to evaluate the interactions between cimaterol and thyroid status in control of muscle growth. MATERIALS AND METHODS Experiment 1. Objectives of this study were to evaluate mechanisms by which cimaterol stimulates muscle growth. Sixteen male Sprague-Dawley rats weighing approximately 218 g were obtained from Charles River Breeding Laboratories (Wilmington, MA) and were assigned randomly to one of two dietary treatments (Table 1) consisting of either a cimaterol-free diet (control) or a diet containing cimaterol (25 mg/kg; American Cyanamid Co., Princeton, N.J.). Animals were housed in individual stainless steel cages for 10 days with feed and water available ad libitum. Feed consumption and body weights were determined daily over a 10 day experimental period. Water consumption and total urine excretion during 24 hr periods were determined beginning on days 4 and 8 of the study. Urine was collected in . 5 ml of 4 N hydrochloric acid to prevent microbial utilization of its components. Aliquots of urine were stored at -20 C until further analysis. A 12 hr:12 hr light:dark cycle was used. On the tenth day of the study animals were anesthetized with halothane and euthanized by exsanguination. A mixture of hind-limb muscles, which consisted of biceps femoris, caudofemoralis, gluteus maximus, semimembranosus and semitendinosus, was removed from each animal, weighed and quickly frozen between blocks of dry ice. Tissues were wrapped in plastic and stored at -80 C until further analysis. Experiment 2. Objectives of this study were to examine effects of hypothyroidism and hyperthyroidism on muscle growth and to evaluate cimaterol's ability to c o u n t e r myopathies associated with these conditions. Forty-five male Sprague Dawley rats (36 thyroidectomized (Tx) and 9 that had received sham thyroidectomies) were obtained from Charles River Breeding Laboratories (Wilmington, MA). Of the thyroidectomized group, 35 were in good health. Six days following surgery and after maintenance for 1 day on control diets (Table 1), rats were assigned to one of five groups. Nine sham-Tx rats weighing 227 -+ 3 g were assigned to a control treatment and were given control diet. The remaining 35 Tx rats were assigned randomly to one of four treatments which included 1) Tx rats given control diet, 2) Tx rats given the cimaterol diet, 3) hyperthyroid rats (Tx rats receiving twice daily subcutaneous injections of 50 I~g of triiodothyronine (T3; Sigma Chemical Co., St. Louis, MO) dissolved in .5 ml of sterile saline at 0800 and 2000 hr given control diet, and 4) Tx rats receiving T 3 injections and the cimaterol diet. Nine rats were allotted to each treatment e x c e p t for the latter group which was assigned eight rats. All animals not receiving T3 received twice daily injections o f . 5 ml vehicle. Animals were housed in individual stainless steel metabolic cages which allowed for determination of feed and water consumption and urine excretion. A 13 h r : l l hr light:dark cycle was used to allow for the twice daily injections 12 hr apart. Water, which was s uppl em e nt e d with .45% calcium chloride, and feed were available ad libitum. Animals were maintained on these treatments for 10 days after which they were euthanized and tissues collected as described previously. DNA, RNA, protein, NMH and creatinine determinations. Total left and right hindlimb muscle and liver were separately homogenized in 4 volumes of

CIMATEROL AND THYROID STATUS

151

distilled water and frozen at .80 C until analyzed. DNA content of muscle samples was determined according to the methods of LaBarca and Paigan (13). RNA content of tissues was determined according to the method of Munro and Fleck (14). DNA and RNA contents were expressed both as a concentration in 'muscle excised from the hind limb and as total DNA present in this complex of muscle. Protein concentrations were determined using the method of Bradford (15). Determinations of urine NMH were conducted according to the method of Forsberg and Liu (16). Creatinine levels in urine were determined using Sigma Kit 555-A (Sigma; St. Louis, MO). A lO-fold dilution of urine was needed for sample absorbance to develop in the linear range of this assay. Enzyme Assays. Frozen muscle samples were thawed and minced with scissors, then homogenized twice (one.half speed) at 20 sec each in a Polytron. A 30 second cooling period on ice between homogenization steps was used. Activities of cathepsins B, D and L and neutral proteinase were determined in these homogenates. In each case samples were processed in triplicate with duplicate zero time controls. All assays were conducted for a period of time during which linearity of reactions prevailed. Cathepsin B (EC 3.4.22.1) activity (17) was determined by diluting 40 $tl of a 10% (w/v) hind limb muscle homogenate with buffer such that its final concentration consisted of 155 mM K2HPO4, 4.5 mM citric acid, 4 mM ethylenediaminetetraacetic acid at pH 6.0. To this, 250 !11 of 2 mM dithiothreitol, 170 ~tl water and 40 ~tl of CBZ-alanyl-arginyl-arginyl-MNA (Enzymes Systems Products; Livermore, CA) were added such that final substrate concentration was 5 mM. Samples were incubated for 2 hr at 37 C and reaction stopped by addition of 2 ml 1 N HCI. Samples were centrifuged at 1000 g for 10 min and the fluorescence of [3-naphthylamine (MNA) within the supematant (excitation 292 nm, emmission 410 nm) determined using an SLM Aminco Model II fluorocolorimeter. The blank consisted of the addition of HCI immediately following addition of tissue homogenate. Cathepsin D (EC 3.4.23.5) activity was determined by the method of Takahashi and Tang (18) using bovine hemoglobin as a substrate. Incubation time was 20 rain. Cathepsin L (EC 3.4.22.15) activity during 30 rain incubations was determined according to the method of Barrett and Kirschke (19) using azocasein as a substrate. Neutral proteinase activity was determined by the method of Kar and Pearson (20). Tissue homogenates (.1 ml) were combined with .9 ml of buffer which consisted of Tris-HCl (50 raM; pH 7.5) and Z-glycyl-glycyl-arginyl-NNap (.2 raM; Bachem Fine Chemicals, Torrance, CA) and incubated at 37 C for 3 hr. The release of MNA was examined fluorometrically (excitation 292 nm, emission 410 nm). All enzyme activity data were expressed on a gram protein basis. Statistical Analyses. In the first study differences between the two experimental treatments were examined with the use of a Student's t-test (21). Data from the second study were analyzed as a completely randomized design by use of analysis of variance. Differences among individual means were tested for significance by use of Student Neuman Keul's multiple range test. A significance level of 5 percent was used for all comparisons (21). RESULTS E x p e r i m e n t 1. Cimaterol increased (P < .05) rate of gain by 38% (Table 2). This was accompanied by a 14% increase (P < .05) in feed intake. Feed intake was depressed by cimaterol during the first three days of the study (data

152

FORSBERG AND WEHR TABLE 1. COMPOSITION OF RAT DIETS. Ingredient

Percentage as Fed

G r o u n d alfalfa Ground corn G r o u n d barley Soybean m e a l (41% c r u d e p r o t e i n ) Ground corncobs a Bentonite Feathermeal Dried w h e y Vitamin, m i n e r a l p r e m i x b Tricalcium phosphate

10 27.09 30 12 5.21 2.5 2 10 .5 .7 100

•In t h e c i m a t e r o l - - c o n t a i n i n g diet, c i m a t e r o l was u s e d at 4 8 0 m g / k g in a g r o u n d corn cob meal carrier to a c h i e v e a final c i m a t e r o l c o n c e n t r a t i o n o f 25 p p m . bVitamin a n d m i n e r a l p r e m i x at .5 p e r c e n t o f dietary dry m a t t e r r e s u l t e d in t h e f o l l o w i n g c o n c e n t r a t i o n s o f v i t a m i n s a n d minerals: c a r o t e n e , 2 m g / k g ; v i t a m i n A, 15 IU/g; v i t a m i n D, 4.41 IU/g; a l p h a - - t o c o p h e r o l , 35 m g / k g ; t h i a m i n e , 14 m g / k g ; riboflavin, 6.5 m g / k g ; niacin, 60 rag/ kg; p a n t o t h e n i c acid, 18 m g / k g ; choline, 1 7 8 0 m g / k g ; p y r i d o x i n e , 8.13 m g / k g ; folic acid, 1.6 m g / k g ; biotin, . 15 m g / k g ; v i t a m i n B,2, 30 gtg/kg a n d m e n a d i o n e s o d i u m bisulfate, 2.8 m g / k g .

not shown); however, intake increased after this time with cimaterol-treated rats consuming more than control-fed rats on the fourth day of the study and thereafter. Feed per gain ratio was improved (P < .05) by 17% by dietary cimaterol. Cimaterol also increased water consumption and urine volume (P < .05, Table 2). Cimaterol exerted its characteristic growth-promoting actions on skeletal muscle (Table 3). Muscle weights, whether expressed on an absolute weight basis or as a proportion of body weight, were significantly (P < .05) increased by cimaterol within the 10 day experimental period. Muscle DNA concentration was reduced (P < .05) by cimaterol; however, total DNA in muscle tissue was unaffected. RNA concentration, an estimate of protein synthetic capacity in tissues (22, 23) was not affected by cimaterol (Table 3); however, total RNA in muscle was significantly increased (P < .05). Muscle protein concentrations were not affected by cimaterol (P > .05; Table 3). Effects of cimaterol on urinary creatinine and NMH excretion and on activities of several proteolytic enzymes are shown in Table 4. Cimaterol did not significantly affect creatinine excretion (P > .05) although there was a tendency for creatinine excretion to increase in cimaterol-fed animals. Cimaterol increased (P < .05) NMH excretion on days 4 and 8 of the study irrespective TABLE 2. EFFECT OF DIETARY CIMATEROL ON tkVERAGE DAILY GAIN, FEED INTAKE, FEED PER G~aN RATIO, WATER INTAKE AND URINE VOLUME IN GROWING RATS*'b'¢.

Dieta¢ T T r e a t m e n t Control 8 Initial w e i g h t (g) Final w e i g h t (g) Feed intake ( g ' d - 0 Average daily gain ( g ' d - t ) Feed/gain (g.g-l) W a t e r intake ( g ' d -1) day 4 day 8 Urine volume (ml'd-') day 4 day8

217.3 285.4 24.7 6.8 3.6

-+ 5.8 ± 7.1' -+ .81" -+ .7' ± .4"

Cimaterol

-~

218.8 312.5 28.2 9.4 3.0

+ 5.3 ± 4.5 b _+ .78 b + .3 ~ -~ .1 b

36.5 + 5.5" 3 6 . 8 ± 3.9"

45.0 ± 6.5 b 51.6 _+ 7.1 b

9.4 -+ 2 . 6 ' 11.1 __- 3 . 2 '

13.8 -+ 4 . 8 ~ 17.1 -~ 3.3 b

• Values are m e a n s -~ SEM. b Values in t h e s a m e r o w w i t h differing s u p e r s c r i p t s differ significantly (p < .05) " Total w a t e r i n t a k e s and u r i n e v o l u m e s w e r e d e t e r m i n e d on t h e f o u r t h a n d e i g h t h days of t h e study.

CIMATEROL AND THYROID STATUS

153

oF Dlln'ARY CIMA'I~ROL ON MUSCLE WEIGHTS, DNA, RNA AND PP.OTEIN CotcrEN'rs, AND R N A / D N A RATIO IN GROWING RATS'.

TABL~ 3. E ~ c r s

Dietary Treatment Control Muscle w e i g h t -

g

3.68 1.30 1.53 5.63 1.33 4.89 203

--g'lOOg BW -t D N A - - m g ' g -~ - - t o t a l nag RNArag-g-~

-- total rag Protein - r a g . g - ,

± .09" + .03" + .14 = ± .15 ± .15 ± .32" ± 14

Cimaterol 4.54 1.45 1.33 6.04 1.45 6.58 221

± 12 b ± .02 b + .12 b + .24 ± .19 ± .43 b ± 17

• Values are means _ SEM. Values in the same r o w w i t h differing superscripts differ significantly (p < ,05).

of whether NMH was expressed in absolute, per 100 g body weight or on creatinine bases. Because muscle size increased 23%, yet NMH increased 200 to 270%, the increase in NMH excretion was only partially the result of increased muscle mass. Activities of cathepsins B, D and L and neutral proteinase in muscle were unaffected (P > .05) by cimaterol treatment. E x p e r i m e n t 2. The combination ofT3 injection with dietary cimaterol proved to be stressful. On the ninth day of the study one rat assigned to this treatment died and on the final day of the study two more rats died. Implications of these effects are discussed later. All analyses of tissues from this group are therefore based on five animals. No other deaths occurred; hence, analyses associated with other treatments consisted of nine replicates each. Effects of thyroid hormone status and of dietary cimaterol on rat growth, feed and water intake and urine volume are shown in Table 5. Initial weights of sham-thyroidectomized animals were higher (P < .05) than thyroidectomized rats because of the time required for recovery, shipping and adaptation following surgery. Thyroidectomy reduced feed intake (P < .05) by 34% and caused a 79% reduction in gain (P < .05). Administration of T3 to thyroidectomized animals enhanced (P < .05) intake but further reduced (P < .05) gain (Table 5). Cimaterol did not affect (P > .05) feed intake by Tx or hyperthyroid rats but improved (P < .05) growth rate of Tx but not of hyperthyroid rats. Water intake was influenced by thyroid status. Water intake was reduced (P < .05) in Tx animals and increased (P < .05) in hyperthyroid rats. Cimaterol TABLE 4 . EFFECT OF DIETARY CIMATEROL ON URINARY CREATININE AND N%MKI'HYLHISTIDINE EXCR~'TION AND ON ACTlVI'l'lE$ OF SEVERAL PROTEOLY'rIc ENZYM~ IN MUSCL~ OF GROWING RATSt.

Dietary Treatment Control Creatinine ( g r a o l e s • d - t ) -

day 4 - day 8 Urinary NMH (nraoles - d - ' ) - - d a y 4 --day 8 (nranleNMH • 100 gBW-' " d -t) -- day4

-- day 8 (nraole N M H • g r a o l e c r e a t i n i n e - ' ) Carhepsin B b Cathepsin D " Cathepsin La - Neutral proteinase b - -

-

" Values are means ± SEM. Values (p < .05) b C a t h e p s i n B and neutral proteinase peptides • rng protein -~ • r a i n - ' . " C a t h e p s i n D activity is e x p r e s s e d d C a t h e p s i n L activity is expressed

-- day 4 -- day 8

2.77 2.45 417 393 174 147 151 160 2.00 .394 .071 5.32

± .29 ± .43 -+ 1 0 5 = -+ 1 3 8 " -+ 4 0 a -+ 3 8 " ± 31" ± 39" ± .10 ± .123 ± .016 +1.08

Cimaterol 3.73 3.24 834 1070 338 368 224 330 2.32 .339 .083 5.20

± .56 ± .42 ± 151 b ± 101 b + 60 b ± 48 b ± 39 b ± 47 ~ ± .08 ± .090 ± .011 ± .32

in the same r o w with differing superscripts differ significantly activity are e x p r e s s e d as n m o l e s of MNA released from synthetic as Abs2,o • g p r o t e i n - ' as A b s 3 ~ • g p r o t e i n - '

• rain-'. • rain-'.

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FORSBERG AND WEHR

TABLE 5. EFFECT OF THYROID STATUS AND CIMATEROL ON AVERAGE DAILY GAIN, FEED INTAKE, WATER INTAKE AND URINE VOLUME IN GROWING RATS~, Experimental Sham

n

9

Initial weight (g) 227 Final weight (g) 300 Feed intake b (g'd-') 26.8 Average daily gain (g'd-')

Tx + cimaterol

9

*" 3 ' _+ 5 •

213 228

208 249

.6"

1 7 . 6 +-

.5 b

7.3 +

.2'

1.5 +

.4 b

4.1

-+ 2 . 1 b m 2.8 b

28.7 27.2

-+ 4 . 0 ~ ~- 2 . 4 "

27.8 25.7

9

_+ 5 b -+ 5 c

19.3 ~

T x -~ T~ + cimaterol

T x + T~

9

_ 3b ± 4b

+-

Water intake (gd-') day4 35.9 day8 33.1 Urine volume day 4 day8

"Ix

Treatment

210 194

8

x 3D _+ 34

212 204

.3 bc

20.9

+-_ 1 . 0 ".*

.4 c

-1.6

_+

_* 2 . 0 b _+ 2 . 1 ~

45.0 59.0

+ 2.2 c -+ 3 . 0 ~

48.1 58.3

1 5 . 4 _~ 1 . 6 • 1 5 . 4 -+ 1 . 5 •

22.1 22.4

+ 1.6 b _+ 1 . 8 ~

1 7 . 1 • 1.0 • 2 2 . 8 -~ 3 . 0 b

_

.3 d

22.6

2 5b ± 74

-.83

+_ -+

.9 d .50 d

-~ 3 . 6 ~ -+ 6 . 3 ~

(ml'dl~) .2 _+ .7 ~ 1 7 . 0 m 1.0"

16.1 ~ 2.2 • 1 7 . 6 - 1.6"

V a l u e s a r e m e a n s _+ SEM. V a l u e s i n t h e s a m e r o w w h i c h d o n o t s h a r e c o m m o n

superscripts differ

(p < .05). b V a l u e s a r e m e a n s o f i n t a k e c a l c u l a t e d f r o m t o t a l i n t a k e d u r i n g t h e 10 d a y s t u d y .

did not affect (P > .05) water intake (Table 5). Urine v o l u m e was increased in h y p e r t h y r o i d rats (P < .05) but was unaffected b y t h y r o i d e c t o m y or cimaterol (P > .05). Effects of thyroid status and of cimaterol on m u s c l e weights and m u s c l e DNA, RNA and p r o t e i n contents are s h o w n in Table 6. Effects o f treatments on m u s c l e weights w e r e d e p e n d e n t u p o n w h e t h e r m u s c l e weights w e r e e x p r e s s e d on an absolute w e i g h t basis or as a p r o p o r t i o n of b o d y weight. W h e n m u s c l e weights w e r e e x p r e s s e d on an absolute w e i g h t basis, b o t h t h y r o i d e c t o m y and h y p e r t h y r o i d i s m r e d u c e d (P < .05) muscle weight. A r e d u c t i o n (P < .05) in m u s c l e w e i g h t was seen o n l y in the h y p e r t h y r o i d g r o u p w h e n weights w e r e expressed as a p r o p o r t i o n o f b o d y weight. Irrespective o f the m o d e of expression, cimaterol e n h a n c e d (P < .05) m u s c l e weights of "Ix b u t not o f h y p e r t h y r o i d rats (Table 6). In c o m p a r i s o n to sham.Tx rats DNA c o n c e n t r a t i o n was increased (P < .05) in h y p e r t h y r o i d but not in Tx rat muscle (Table 6). Total DNA p r e s e n t in m u s c l e was r e d u c e d (P < .05) in b o t h Tx and h y p e r t h y r o i d rats. Cimaterol did not affect (P > .05) e i t h e r DNA c o n c e n t r a t i o n or c o n t e n t in m u s c l e tissue in e i t h e r Tx or h y p e r t h y r o i d rats. RNA c o n c e n t r a t i o n , an estimate o f p r o t e i n synthetic capacity (22, 23) was r e d u c e d (P < .05) in Tx animals and increased (P < .05) in h y p e r t h y r o i d animals. Cimaterol further increased (P < .05) RNA c o n c e n t r a t i o n in Tx but not in h y p e r t h y r o i d rats (Table 6). Muscle p r o t e i n concentrations w e r e not affected (P > .05) by thyroid h o r m o n e status or cimaterol (Table 6). Effects o f treatments on urinary creatinine and NMH e x c r e t i o n and on activities o f various p r o t e o l y t i c enzymes in m u s c l e are s h o w n in Table 7. On day 4, c r e a t i n i n e levels w e r e not affected b y any o f the treatments; however, by day 8 o f the study "Ix, h y p e r t h y r o i d and h y p e r t h y r o i d plus cimaterol treatments h a d elevated (P < .05) urinary creatinine relative to sham-Tx rats. Effects o f treatments o n urinary NMH w e r e d e p e n d e n t u p o n m o d e o f expressing NMH data and u p o n day o f treatment. NMH e x c r e t i o n , e x p r e s s e d in absolute quantities p e r day, was r e d u c e d (P < .05) in Tx rats on b o t h days 4 and 8 o f the study (Table 7) w h e n c o m p a r e d to sham-Tx rats. On day 4, T 3 injection increased NMH e x c r e t i o n (P < .05) w h e n c o m p a r e d to Tx rats but not w h e n c o m p a r e d to sham-Tx rats. On day 8, T 3 injection increased (P
.05) NMH excretion in hypothyroid rats but increased (P < .05) NMH excretion in hyperthyroid rats. To normalize NMH excretion between animal groups which may vary in body or muscle weights other investigators have expressed NMH either as a proportion of body weight (5, 8) or as a function of urinary creatinine (24). Expression of NMH per unit body mass yielded similar results to absolute NMH excretion patterns except for the observation that on day 4 T 3 treatment increased (P < .05) NMH excretion in hyperthyroid rats compared to sham. Tx rats. Urinary NMH was clearly dose-dependent to thyroid status. With expression of NMH on a urine creatinine basis, results were different compared to expression of data uncorrected for muscle mass. On day 4, a dose response of NMH/creatinine to thyroid status was evident; however, by day 8 T 3 did not further increase NMH/creatinine compared to sham-Tx rats. Cimaterol increased (P < .05) NMH/creatinine in Tx rats on day 8 and in hyperthyroid rats on both days 4 and 8 (Table 7). Significant effects of treatments on cathepsins B, D and L and neutral proteinase were detected. All enzymes increased in activity in a dose-responsive manner as thyroid hormone status progressed from hypothyroid (Tx) to euthyroid (sham-Tx) to hyperthyroid. Administration of cimaterol to Tx animals increased (P < .05) activities of cathepsins B and L and neutral proteinase (Table 7). However, in hyperthyroid animals there was a tendency for cimaterol to have the opposite effect. Cimaterol reduced (P < .05) activities of cathepsins B and D in these animals. DISCUSSION E x p e r i m e n t 1. Beta-adrenergic agonists increase muscle growth in many species (8-12). Our data are similar to these reports. Not only was muscle size increased but muscle weight, expressed as a proportion of total body weight, was increased by cimaterol. Therefore cimaterol induced fundamental changes in composition of growth. Mode of action of beta-agonists in muscle remains controversial. Some have attributed muscle growth stimulation to an increase in muscle protein synthesis (25-27) and others attribute hypertrophy to a reduction in muscle protein degradation (8, 27-31). In this study cimaterol enhanced urinary NMI-I excretion. Although caution must be used in interpreting urine NMH data, since there exist extra-myofibrillar sources of NMH (34), our data suggest that cimaterol increased myofibrillar protein degradation. The possibility that acute exposure of muscle to catecholamine-like compounds enhances myofibrillar protein degradation is supported by the recent observation that treatment of isolated rat epitrochlearis muscle with epinephrine increased release of NMH into incubation media (32). If c .hanges in urinary NMH reflect changes in myofibrillar protein degradation, an increase in net muscle protein synthesis in excess of the effect on protein degradation would be required for muscle hypertrophy to occur. Protein synthesis per unit muscle mass may be increased either through an increase in ribosome concentration or through an increase in protein synthesis per unit ribosome content (22, 23). We did not assess the latter possibility; however, others who have assessed effects of clenbuterol on rat muscle (30, 31) reported no change in protein synthesis per unit RNA. In our study and

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also in other studies (30, 31, 33) beta-adrenergic agonists did not affect muscle ribosome concentration either. However, total RNA content of muscle was increased by beta-agonists in each of these studies. Therefore, protein synthesis per unit muscle mass may remain unchanged in beta-agonist-treated animals but net protein synthesis in individual muscles may be increased. To determine a mechanism by which cimaterol increased urinary" NMH excretion we examined activities of several proteolytic enzymes in skeletal muscle. Cathepsins have been implicated in digestion of both non-myofibriIlar proteins and in secondary digestion of myofibrillar proteins (35-37) and neutral proteinases have been implicated in human muscle degenerative conditions (20). Activities of these enzymes were not influenced by cimaterol. Activities of calcium.dependent proteinases (CDPs), which may initiate myofibrillar protein degradation (38), were not examined in this study. In a recent study with rabbits (8), long-term exposure to cimaterol reduced urinary" NMH excretion and reduced activities of CDPs and calpastatin but did not affect activities of cathepsins B or D or neutral proteinase. Therefore, it is possible that regulation of muscle protein degradation by cimaterol is directed towards CDPs. In previous studies with sheep (39), cimaterol reduced muscle cathepsin B activity. Others (40) reported that clenbuterol increased muscle cathepsin B activity and did not affect cathepsin D activity in rat skeletal muscle. Reason(s) for these inconsistencies are not known; however, differences in duration of agonist administration, animal maturity, species, muscle fiber type and betaagonist used may all influence the nature of the anabolic response in muscle. Conflicting reports of the ability of beta-agonists to both reduce (8, 27, 30) and to stimulate muscle protein degradation (this study; 32) may be reconciled through investigation of the temporality of their effects. Acute exposure of isolated rat muscle to epinephrine increases NMH release (32), whereas longterm treatment of rabbits with cimaterol (21-28 days) reduces urinary NMH excretion (8). An interesting effect of cimaterol was its stimulation of water intake and urine output. The difference between intake and excretion, approximating water balance, suggests greater retention of water in cimaterol-fed versus control-fed rats. A portion of water retained would have been associated with cimaterol-dependent hypertrophy; however, the balance must have been lost through evaporation and respiration. Brock'way et al. (41) reported that clenbuterol enhanced heat production in sheep. Therefore it is possible that beta agonists increase need for water intake for thermoregulatory purposes. The reduction in DNA concentration in cimaterol-treated animals is likely due to the dilution of existing DNA. The observation that total DNA in muscle was unaffected by cimaterol treatment is supportive of this conclusion. E x p e r i m e n t 2. Creatinine excretion has been used as an index of muscle mass and used to normalize NMH excretion data (24). Urinary creatinine excretion was enhanced in both Tx and hyperthyroid rats despite reductions in muscle and body weights associated with these treatments. Creatinine excretion in thyroid imbalances therefore is more heavily influenced by thyroid status than by muscle mass. For these reasons we have relied upon NMH excretion per unit body mass to provide an estimate of MPD. Thyroidectomy and T3 administration profoundly altered growth and muscle protein metabolism. The reduction in muscle weight of Tx rats was associated

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with reduced urinary NMH. This suggests that myofibrillar protein degradation is reduced in hypothyroidism, an observation supported by previous studies (2-4), and that a large reduction in muscle protein synthesis is needed to account for muscle growth attenuation. Reduced protein synthetic capacity (RNA/g) and reduced total RNA of thyroidectomized rat muscle indicates that low ribosome content could account for reduced muscle growth (22, 23). Reduced RNA content of skeletal muscle of thyroidectomized rats has been reported previously (2.4) and represented an early adaptation of muscle to experimentally-induced hypothyroidism (4). A reduction in muscle weight caused by excess T~ has been observed by previous investigators (6,7) and, in this study, was associated with a 70% increase in urinary NMH excretion on day 8. These data and data from other studies (2, 6, 7) indicate that hyperthyroid myopathy is caused by enhanced myofibrillar proteolysis. It is possible that protein synthesis per unit muscle mass in hyperthyroid muscle was increased, since RNA concentration was increased by 38 percent; however, net protein synthesis may have been reduced since total muscle RNA was reduced by T 3. Cimaterol increased weight gain and muscle weights of Tx but not of hyperthyroid rats. Therefore growth-promoting actions of cimaterol in muscle are thyroid hormone-independent and antagonized by excess T3. Because cimaterol-dependem muscle hypertrohy in Tx animals was not associated with changes in urinary NMH, it is likely that its anabolic effects were directed towards protein synthesis. The cimaterol-clependent increase in RNA concentration and in total RNA content of muscle taken from Tx rats is supportive of this hypothesis and indicates that these effects may also be thyroid hormoneindependent and antagonized by excess T 3. The inability of cimaterol to increase RNA concentration in euthyroid (experiment I) and in hyperthyroid muscle may be due to antagonistic actions of endogenous and supplemental thyroid hormones, respectively. The ability of both thyroid hormone and cimaterol to regulate muscle ribosome concentration is apparent in Tx rats. Endocrine regulation of ribosome synthesis and degradation has been reported (22, 23). Further studies which examine mechanisms by which ribosome turnover is regulated by these hormones is needed to provide a better understanding of endocrine control of muscle growth. Cimaterol did not improve gain or muscle growth in hyperthyroid rats. Instead it exerted effects which stressed the rats. Three deaths occurred in this treatment group and there was further enhancement of urinary NMI-I excretion by cimaterol on the eighth day of the study. The reason why the latter effect did not reduce muscle weight is unclear; however, it is possible that this effect developed late in the study (i.e., changes in urinary NMH excretion caused by cimaterol treatment of hyperthyroid animals were apparent on day 8 but not on day 4) and sufficient time had not elapsed for measurable changes in muscle weight to result. Alternatively, cimaternl-dependent changes in urinary NMH may have arised from changes in degradation of extra-myofibrillar NMH pools. Changes in urinary NMH excretion caused by variable thyroid status were accompanied with corresponding changes in activities of the cathepsins and neutral proteinase. Similar effects of variable thyroid status on activities

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of cathepsins B and D have been reported (42). This implies that control of muscle protein degradation by thyroid hormones may involve these enzymes. In Tx rats, cimaterol did not affect urinary NMH but increased muscle cathepsins B and L and neutral proteinase activities. In hyperthyroid rats, cimaterol enhanced urinary NMH, but reduced cathepsins B, D and L activities. Therefore, cimaterol may either increase or reduce proteinase activities, depending on thyroid status, but its regulation of these enzymes does not provide insight to mechanisms by which urinary NMH excretion is regulated by betaagonists. Therefore, it is unlikely that regulation of activities of cathepsins or neutral proteinase is a primary mechanism by which cimaterol influences rat muscle growth. Degradation of cellular proteins involves not only the proteolytic system but also mechanisms which selectively target proteins for various degradative pathways. For example, ubiquitination of proteins targets proteins for degradation via an ATP-dependent proteolytic mechanism (43), and modification of protein conformation may reveal PEST or KFERQ sequences which target proteins for degradation (44, 45). Hence, lack of change in activity of an enzyme does not necessarily indicate the lack of involvement of that enzyme in degradation of a specific protein. Cimaterol enhanced urinary NMH excretion in euthyroid (Experiment 1) and hyperthyroid rats but not in Tx rats. Therefore, control of urinary NMH excretion by cimaterol may be thyroid hormone-dependent. Perhaps thyroid hormones potentiate the actions of cimaterol on this process. Cimaterol did not affect water intake in Tx or hyperthyroid rats; however, thyroid hormone enhanced water intake. Similar effects of excess thyroid hormone on water intake have been reported (46). Since heat production is enhanced in hyperthyroidism (47) it is likely that water intake is increased for thermoregulatory purposes. Reductions in DNA content in muscles taken from Tx and hyperthyroid rats indicate reductions in muscle cellularity, caused either by reduced satellite cell contribution of DNA to existing muscle cells or reduced development of other cell types associated with muscle occurred. Similar effects of thyroidectomy on muscle DNA content have been reported and were attributed not to a lack of thyroid hormone but instead to reduced growth hormone concentration resulting from thyroid hormone deficiency (4). Previous work in our laboratory (8) indicated the importance of protein degradation in cimaterol-dependent control of muscle growth; however, in the present study alterations in myofibrillar protein synthesis may have accounted for cimaterol's short-term anabolic effects. Although we did not assess protein synthesis, the cimaterol-dependent increase in NMH excretion in experiment 1 implies that a large stimulation of net protein synthesis would be required for muscle hypertrophy to occur. Also, the ability of cimaterol to increase growth of hypothyroid muscle was likely dependent upon an effect on protein synthesis since NMH excretion in Tx rats was unaffected by cimaterol. Effects of beta-adrenergic agonists on muscle metabolism are influenced by other hormones. Sharpe et al. (48) reported glucocorticoid dependency for growth-promoting actions of clenbuterol in rats. This study demonstrates that thyroid status influences responses in muscle to cimaterol. Further work on the bases for these interactions is needed and will likely reveal important mechanisms underlying the endocrine control of postnatal muscle growth.

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ACKNOWLEDGEMENTS AND Fo(YrNOTES tThe authors thank Helen Chesbrough for typing this manuscript and M. Wilson for technical assistance. Submitted as paper 8739 of the Oregon Agricultural Experiment Station. ~4:imaterol was a gift of American Cyanamid Co., Princeton, N.J. 3Address correspondence and reprint requests to N.E. Forsberg. REFERENCES 1. Ramsay JD. Muscle dysfunction in hyperthyroidism. Lancet 2:931-934, 1966. 2. Brown JG, Millward DJ. Dose response of protein turnover in rat skeletal muscle to triiodothyronine treatment. Biochim Biophys Acta 757:182-190, 1983. 3. Plaim KE, Li JB, Jefferson Is. Effects of thyroxine on protein turnover in rat skeletal muscle. Am J Physiol 235:E23 I-E236, 1978. g. Brown JG, Bates PC, Holliday MA, Millward DJ. Thyroid hormones and muscle protein turnover. The effect of thyroid hormone deficiency and replacement in thyroidectomizod and hypophysectomized rats. Biochem J 194:771-782, 1981. 5. Kayali AG, Young VR, Goodman MN. Sensitivity of myofibrillar proteins to glucocorticoid-induced muscle proteolysis. Am J Physiol 252:E621-E626, 1987. 6. Miller JL, Ismail F, Waligora JK, Gevers W. Modulating influence of D,L-Propranolol on triiodothyronine-induced skeletal muscle protein degradation. Endocrinology 117:869-871, 1985. 7. Angeras U, Hasselgren PO. Protein degradation in skeletal muscle during experimental hyperthyroidism in rats and the effect of -blocking agents. Endocrinology 120:1417-1421, 1987. 8. Forsberg NE, Ilian MA, All-Bar A, Cheeke PR, Wehr NB. Effects of cimaterol on rabbit performance on myofibrillar protein degradation and on calcium.dependent proteinase and calpastatin activities in skeletal muscle. J Anita Sci 67:3313-3321, 1989. 9. Ricks CA, Dalrymple RH, Baker PK, Ingle DL. Use of beta-agonists to alter fat and muscle deposition in steers. J Anita Sci 59:1247-1255, 1984. 10. Dalrymple RH, Baker PK, Gingher PE, Ingle DL, Pensack JM, Ricks CA. A repartitioning agent to improve performance and carcass composition of broilers. Poultry Sci 63:2376-2383, 1984. 11. Beermann DH, Hogue DE, Fishell VK, Dalrymple Ri-l, Ricks CA. Effects of cimaterol and fishmeal on performance, carcass characteristics and skeletal muscle growth in lambs. J Anim Sci 62:370-380, 1986. 12. Wilson MA, Zhong C, Forsberg NE, Dalrymple RH, Ricks CA. Effects of cimaterol on protein synthesis, protein degradation, amino acid transport and acetate oxidation in sheep external intercostal muscle. Nutr Res 8:1287-1296, 1988. 13. LaBarca C, Paigen K. A simple, rapid and sensitive DNA assay procedure. Anal Biochem 102:344-352, 1980. 14. Munro HN, Fleck A. Analysis for nitrogenous constituents. Measurement of nucleic acids in tissues. In: Mammalian Protein Metaboism 3:477-479. Academic Press, N.Y., 1969. 15. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye-binding. Anal Biochem 72:248-254. 1976. 16. Forsberg NE, Liu CC. Phenylisothiocyanate derivatization of lW-methylhistidine: A method of assessing myotibrillar protein degradation. Nutr Res, 9:1269-1276, 1989. 17. Rodemarm HP, WaYman L, Goldberg AL. The stimulation of protein degradation in muscle by calcium is mediated by prostaglandin E= and does not require the calcium-activated protease. J Biol Chem 257:8716-8723, 1982. 18. Takahashi T, Tang J. Cathepsin D from porcine and bovine spleen. Meth Enzymol 80:565-569, 1981. 19. Barrett AJ, Kirschke H. Cathepsin B, cathepsin H, and cathepsin L. Meth Enzymol 80:535-565, 1981. 20. Kar NC, Pearson CM. Elevated activity of a neutral proteinase in human muscular dystrophy. Biochem Med 24:238-243, 1980. 21. Steel RGD, Torrie JH. Principles and procedures of statistics. McGraw-Hill Book Co. New York, New York, 1980.

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45. Chiang HL, Dice JF. Peptide sequences that target proteins for enhanced degradation during serum withdrawal. J Biol Chem 263:6797-6805, 1988. 46. Bello AA, Covian MR. Effect of the excess of thyroid hormone administration on water and sodium chloride intake in the rat. Physiol Behavior 43:155-157, 1988. 47. Buchmann K, Arieli A, Burger AG, Chinet AE. Triiodothyronine.induced thermogenesis: altered T3-efliciency in tissues from fed, starved and refed hypothyroid rats. Endocrinology 117:1084-1089. 1985. 48. Sharpe PM, Haynes PM, Buttery PJ. Glucocorticoid status and growth. In: Control and Manipulation of Animal Growth. P 207-222. Ed. Buttery, PJ, Lindsay, DB, Haynes, NB. Butterworths, London. 1986.