Effects of Clenbuterol
on Skeletal Muscle Mass, Body Composition, From Surgical Stress in Senescent Rats
and Recovery
William J. Carter, An 0. Dang, Fred H. Faas, and Mary E. Lynch Aging decreases skeletal muscle mass and strength, which may be exacerbated
by age-related diseases. There is a need for therapeutic agents to prevent or restore loss of skeletal muscle in elderly subjects with muscle wasting disorders. Clenbuterol, a &adrenergic agonist, dramatically increases skeletal muscle mass in young animals and partially prevents or restores muscle loss in experimental models of muscle wasting. However, the protein anabolic and fat catabolic effects of clenbuterol have not been studied in senescent animals. To determine whether this drug has potential for preventing or repairing muscle loss in elderly subjects, we have examined its effects in young and old rats. Clenbuterol was administered by implanted osmotic minipumps to Fischer-344 rats ages 3,12, and 23 months, at a dose of 1.5 mg/kg/24 h for 3 weeks. The weights of five hindlimb muscles and carcass protein and fat content were determined. Clenbuterol treatment increased the weight of skeletal muscles 22% to 39% in 3-month-old rats, 19% to 35% in 12-month-old rats, and 22% to 25% in 23-month-old animals. Likewise, clenbuterol increased carcass protein content 19% in 3.month-old rats, 16% in 12-month-old rats, and 24% in 23.month-old animals. Conversely, the drug reduced carcass fat content 36% in 3.month-old rats, 32% in 12.month-old rats, and 38% in 23-month-old rats. Therefore, clenbuterol had similar anabolic and catabolic effects in all age groups. In addition, clenbuterol stimulated recovery of skeletal muscle protein lost following pump implantation in senescent rats. These findings suggest that clenbuterol or similar drugs may prove useful in stimulating skeletal muscle hypertrophy in elderly subjects with muscle wasting disorders. Copyright 0 1991 by W.B. Saunders Company
S
INCE AGING decreases skeletal muscle mass and reserve strength,‘,’ elderly subjects are particularly vulnerable to the effects of diseases that reduce skeletal muscle mass and strength below that required for independence in daily activities.3 There is a clear need for therapeutic approaches to prevent or restore the loss of skeletal muscle due to injury or disease and associated processes such as surgery, immobilization, and malnutrition. Recently, certain B,-adrenergic agonists, notably clenbuterol, were found to dramatically increase skeletal muscle mass in young mammals-’ and to reduce or restore muscle loss in animal models of muscle wasting, including denervation, loss of weight bearing, genetic muscular dystrophy, bacterial endotoxin injection, and 50% reduction in dietary intake.8-‘2 This capacity to prevent or repair muscle wasting suggests a potential clinical role for clenbuterol or similar drugs in increasing muscle mass in disadvantaged elderly subjects. However, these agents have catabolic as well as anabolic effects. Clenbuterol decreases carcass fat content, reduces the fraction of dietary energy retained as carcass energy and increases nonshivering thermogenesis.4.7 Because aging might reduce the anabolic effects of these agents and accentuate their catabolic effects, the present studies have compared the effects of clenbuterol in young and old rats. Not only did clenbuterol have similar protein anabolic and fat catabolic effects in young and old rats, it also facilitated the recovery of skeletal muscle lost following surgical stress in old rats.
MATERIALS AND METHODS Materials
Male Fischer-344 rats aged 3,12, and 23 months were purchased from the National Institute on Aging, Bethesda, MD, and Alzet osmotic minipumps, Model 2ML2, from Alza, Palo Alto, CA. Clenbuterol hydrochloride was generously supplied by Boehringer Ingelheim Animal Health, St Joseph, MO. All other chemicals used were reagent grade. Metabolism,
Vol40,
No 8 (August), 1991:
pp 855-860
Treatment of ExperimentalAnimals. All 23-month-old rats were observed and weighed daily for 7 to 10 days after receipt from the vendor. All animals (* 10%) that ate poorly and lost weight during this time were discarded on the assumption that they had a specific disease and would not be appropriate models to study effects of normal aging. Alzet osmotic minipumps were loaded with clenbuterol dissolved in water at concentrations required to deliver a dose of 1.50 mgikg/24 h. The drug solution was sterilized by filtration before pump loading. The pumps were implanted subcutaneously in the interscapular area using anesthesia induced by intramuscular injection of 5.0 mg ketamine and 0.1 mg acepromazine/lOO g. The anesthetic dose was reduced 50% in 23-month-old rats, because the reduced dose produced adequate anesthesia and allowed more rapid recovery. Pump implantation and drug treatment caused no mortality in young or old animals. Control animals received sham operations without pump implantation. During the 21-day clenbuterol infusions, rats were housed individually, weighed daily, and fed Purina Rat Chow ad lib (Ralston Purina, St Louis, MO). The amount of chow consumed was weighed on a weekly basis. At the end of the experiment, rats were killed by decapitation. The gastrocnemius, soleus, plantaris, extensor digitorum longus (EDL). and tibialis anterior muscles were rapidly dissected from both hindlimbs, weighed on an electronic balance, and frozen at -80°C until processed. Likewise, the heart and kidneys were removed, the cardiac chambers were opened, and both organs were rinsed in ice-cold 0.9% NaCI, followed by blotting, weighing, and freezing. Following removal of the visceral organs and tail, the skinned carcass was weighed, wrapped in aluminum foil, and frozen at - 20°C.
From the Veterans Administration Medical Center: Little Rock, AR; and the Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR. Supported by Veterans Administration Research Funds, Project No. 430-66-8721-0002. Address reprint requests to William J. Carter, MD, John L. McClellan Memorial Veterans Hospital (IllG-JLM}, 4300 W 7th St, Little Rock, AR 72205. Copyright 0 1991 by W.B. Saunders Company 0026-049519114008-0015$03.00l0 855
856
CARTER ETAL
60.0
Methods To prepare the carcass for analysis, it was heated in an autoclave for 3 hours at 1 bar of pressure and 120°C to soften tissues including bones. It was then homogenized in water using a Waring blender (model 700G purchased from Baxter. Scientific Products Division, Grand Prairie, TX), and brought to a volume of 500 mL. For carcass protein analysis, nitrogen in an aliquot of the homogenate was converted to ammonium sulfate by a micro-Kjeldahl procedure” and measured calorimetrically using a phenolhypochlorite method.14 Carcass protein content was estimated by multiplying carcass nitrogen content by 6.25. For muscle protein assay, muscles were homogenized in 10 mL/g of 0.05 mol/L
-30.0
lo-mL aliquot of the carcass homogenate was extracted by shaking in a uniphasic solvent mixture containing chloroform, methanol and water in a 1:2:0.8 ratio by volume.” The composition of the solvent mixture was then adjusted to 2:2:1.8 to produce a two-phase system. The upper layer was discarded and the lower chloroform layer was evaporated to dryness under a stream of nitrogen to recover carcass fat. Carcass water content was determined by drying a 10.mL aliquot of the carcass homogenate to constant weight in a 60°C oven.lh RESULTS
Food Consumption
During the Experimental Period
Clenbuterol infusion had no effect on total food consumption during the 21-day experimental period in any age group (Table 1). However, drug treatment reduced food consumption during the first week following pump implantation by 11% in 3-month, 29% in 12-month, and 48% in 23-monthold animals (Table 1). Treated animals overcame this initial deficit by consuming more chow during the final 2 weeks. Body Weights of Rats Before Clenbuterol Infusion Before osmotic pump implantation and drug treatment, control and experimental animals in each age group were closely matched for body weight, as follows (mean ? SEM, N = 6): 3-month age group, control 305 + 8 g, treated 301 2 9; 12-month age group, control 422 * 9 g, treated 425 ? 11; and 23-month age group, control 434 ? 8 g, treated 431 * 14. Rat Body Weight Changes During Clenbuterol Infusion Figures l-3 show cumulative body weight changes for control and experimental animals during the 21-day treatment period. Body weights just before osmotic pump implantation or sham operation were used as baseline
2
4
6
6
10
12
14
16
16
20
22
TREATMENTPERIOD (days) Fig 1. Cumulative weight change in 3-month-old rats during the U-day treatment period. Each data point represents the mean ? SEM of six animals. Control, 0; clenbuterol, 0. *P < .05, **P < .Ol when compared with the corresponding control by Student’s t test.
measures. Since clenbuterol-treated rats had osmotic pump implantation and control animals had sham operations. the weight of the loaded pump was subtracted from the body weight of each treated animal at all points following implantation. Pump weights were 8.01 -+ 0.064 g (mean +- SEM, N = 18) before implantation. Since these pumps imbibe tissue fluid as the drug solution is expelled. weight change was minimal during the infusion period. Figure 1 shows body weight changes in 3-month-old rats during drug infusion. Sham operated controls lost an average of 8 g during the initial 2 days, recovered to baseline at 6 days and slowly gained weight for the remainder of the 21-day treatment period. In contrast, clenbuteroltreated rats had a larger initial weight loss. averaging 18 g, recovered to baseline at 6 days, and thereafter gained weight more rapidly than controls. Figure 2 shows body weight changes in I?-month-old rats, Sham-operated controls initially lost an average of 15 g during days 2 to 5. recovered to baseline at day 13, and remained stable for the remainder of the treatment period. In contrast, clenbuteroltreated rats had a larger initial weight loss, averaging 22 g, but surpassed the weight gain of control rats by day 9. From day 9 until the end of the experimental period, clenbuteroltreated rats gained weight more rapidly than controls and exceeded both control weight gain and the baseline weight. Figure 3 shows body weight changes in 23-month-old rats. Sham-operated controls slowly lost an average of 28 g during the initial 15 days and reached a plateau with no
Table 1. Weekly and Total Chow Consumption (g) in 3-, 12-, and 23-Month-Old-Rats
~_~_
3.Month Control
Clenbuterol (n = 61
Istwk
117 t- 10
104 +- 8*
14
23.Month
12.Month
(n = 6)
Interval
I 0
potassium phosphate buffer, pH 7.0, and an aliquot used for nitrogen determination as above. For carcass fat determination, a
Control (n = 6)
105 2 6
Clenbuterol
Control
Cienbuteroi
(n = 6)
In = 6)
,n = 6)
75 t 3t
90.4 ? 5
47.2 2 8t
2nd wk
125?
123 * 14
129 t 5
133 t 3
111 t3
3rd wk
122 + 3
135 f 4*
135 t 9
151+4
109 -r 4
100 + 15 143 i 8t
Total
364 + 19
361 -t 13
369 -t 19
360 5 8
311 *9
290 + 14
NOTE. As described in the Methods section, animals in each age group were given a 21-day clenbuterol infusion at 1.5 mglkgi24 h. Values are means + SEM, with six animals in each treatment category and refer to the weight of chow consumed in the designated interval. *P < .05, tP < .Ol when compared with the corresponding control of the same age by Student’s f test.
CLENBUTEROL
857
EFFECTS IN OLD RATS
-3o.oc
I 2
0
I 4
I 6
1 6
10
I 12
1 14
I 16
I 16
I 20
9 22
TREATMENTPERIOD (days) Fig 2. Cumulative weight change in l&month-old rats during the 21-day treatment period. Each data point represents the mean k SEM of six animals. Control, 0; clenbuterol, 0. V < .05, l*P < .Ol when compared with the corresponding control by Student’s t test.
tendency to return to baseline weight. In contrast, clenbuterol-treated rats lost more weight during the initial 2 to 5 days, but gained weight from 5 to 17 days, reaching a plateau that exceeded control values and approached baseline. A common pattern of response to operative procedures and drug treatment emerged in all age groups. Although the initial weight loss was greater in clenbuteroltreated animals, they subsequently gained more weight and surpassed control animals. The weight loss following sham operation was particularly pronounced and prolonged in senescent rats. Clenbuterol. stimulation caused the body weight of senescent rats to recover to baseline following implantation surgery, while controls remained below baseline. Effect of Clenbuterol Infusion on Skeletal Muscle Weight and Muscle to Body Weight Ratios
As illustrated in Table 2, clenbuterol infusion increased the wet weight of slow-twitch, fast-twitch, and mixed-fiber muscles in all age groups. Clenbuterol increased the weight of the slow-twitch soleus 26% in 3-month, 25% in 12month, and 25% in 23-month-old rats. Likewise, the drug 5.0
0.0
l
1
-5.0 2 g
-15.0 -10.0
;
-20.0
e & P
-25.0
6,
Q-P-g
0
1
.’
-30.0
22 -35.0
_.,j-i’i
P\,-0 I
I
71.’
l’O--0 1
I \
-4o.o-! 0
I 4
I 6
I 8
I 10
I 12
I 14
I 16
I 18
Effect of Clenbuterol Infusion on Muscle Protein Content
Clenbuterol infusion increased muscle protein content in proportion to wet weight. In 3-month-old rats, clenbuterol treatment increased gastrocnemius protein content 27%, from 304 * 16 mg to 385 2 11 mg (mean t SEM, N = 6). In contrast, drug treatment did not alter the muscle protein to wet weight ratio, which was 200 ? 4 mg/g in control rats and 204 ? 2 in treated ones. In 23-month-old rats, clenbuterol treatment increased gastrocnemius protein content 26%, from 274 2 5 mg to 344 ? 15 mg. Again, drug treatment did not influence the muscle protein to wet weight ratio, which was 212 ? 3 mg/g in control rats and 214 * 3 in treated ones.
1
I I 2
increased the weight of the fast-twitch EDL 39% in 3-month, 35% in lZmonth, and 25% in 23-month-old animals. Clenbuterol treatment increased the weights of the remaining gastrocnemius, plantaris, and tibialis anterior 22% to 28% in 3-month, 19% to 22% in 12-month, and 22% to 26% in 23-month-old animals. The drug increased muscle weights more than body weights, as indicated by increased muscle to body weight ratios at all ages (Table 2). Clenbuterol increased the soleus muscle to body weight ratio 17% in 3-month, 14% in 12-month, and 18% in 23-month-old rats. Likewise, the drug increased the EDL muscle to body weight ratio 31% in 3-month, 27% in 12-month, and 19% in 23-month-old rats. Clenbuterol treatment increased the muscle to body weight ratios of the gastrocnemius, plantaris, and tibialis anterior 13% to 19% in 3-month, 11% to 20% in 12-month, and 17% to 20% in 23-month-old animals. Therefore, clenbuterol treatment increased both the weights and muscle to body weight ratios of all hindlimb muscles examined at all ages. In Table 2, clenbuterol effects on muscle weights and muscle to body weight ratios were evaluated using multiple Student’s t tests. Since multiple individual comparisons may increase the type I error rate, a two-way ANOVA was performed using PC SAS, release 6.03, and differences between means were evaluated by Duncan’s multiple range test.” When the clenbuterol effect on muscle weights and muscle to body weight ratios was evaluated using combined data from all age groups, it reached or exceeded the 5% level for both parameters in all muscles studied. This analysis supports the validity of the apparent clenbuterolinduced increase in muscle weights and muscle to body weight ratios. When the effect of age was evaluated using combined data from treated and control animals, muscle weights and muscle to body weight ratios were uniformly lower in 23-month versus 12-month-old rats at the 5% level or beyond. This analysis supports the use of senescent rats as a model to study age-related muscle loss.
I 20
I 22
TREATMENTPERIOD (days) Fig 3. Cumulative weight change in 23-month-old rats during the Zl-day treatment period. Each data point represents the mean ‘_ SEM of six animals. Control, 0; clenbuterol, 0. lP < .05, l*P < .Ol when compared with the corresponding control by Student’s t test.
Effect of Clenbuterol Infusion on Weights of Cardiac Ventrkles and Kidneys
As shown in Table 2, clenbuterol infusion did not increase the weight of the combined cardiac ventricles in 3-month-old rats, but caused a 15% increase in 12-monthold rats and an 18% increase in 23-month-old rats. On the other hand, clenbuterol treatment did not influence com-
a58
CARTER
Table 2. Skeletal Muscle, Cardiac Ventricle, and Kidney Weights and Muscle/Organ of 3-, 12-, and 23-Month-Old 3.Month CClntrd
MUSCki
23.Month
Control
Clenbuterol
Organ
(n = 6)
(n = 6)
(n = 6)
In = 6)
Gastroc
2.98 + 0.1
3.74 + 0.1t
3.24 + .oa
4.04 _f 0.1t
(9.15 2 0.2)
(10.7 ? 0.1jt
(7.65 & 0.1)
0.24 + .Ol
Soleus
0.30 + .01t
(0.72 + .Ol)
Plant EDL Tib ant Cvent
Kidneys
(0.85 2 .03)t
Clenbuterol
in = 6)
(a.90 + 0.2)t
In = 61
2.60 ? .05
3.26 .: .08t
(6.40 + 0.1)
17.67 I O.l)t
0.28 2 .Ol
0.34 i- .01t
0.24 2 .07
0.30 - .01t
(0.66 * .02)
(0.75 + .02)X
(0.59 +- .02)
(0.70 : .04)’
0.62 t .02
0.80 k .02t
0.72 + .02
0.91 _t .03t
0.61 i .02
(2.28 t .02)t
(1.69 & .04)
(2.01 t .06)t
(1.50 z .03)
0.76 2 04t (1.79 I
06)t
0.28 z .oi
0.40 2 .01t
0.34 + .Ol
0.46 2 .Olt
0.30 t .Ol
(0.87 -c .oi)
(1.14 2 .02)t
(0.79 * .Ol)
(1 .oo r .03jt
(0.74 -t ,011
1.14 + .03
1.38 -+ .04t
1.34 * .Ol
1.60 -c .06t
1.18 + .02
1.44 + 04t
(3.51 t ,081
(3.98 + .06)t
(3.39 t .08)t
0.37 1 .01t (0.88 ?
03jt
(3.16 2 ,031
(3.52 r O.l)*
(2.90 + .04)
0.78 + .02
0.83 k .03
0.91 2 .Ol
1.04 i .02t
0.94 2 .Ol
1.10 -’ .03t
(2.38 + .03)
(2.40 k .04)
(2.14 i .Ol)
(2.30 t .02)t
(2.31 ? ,031
(2.59 i .041t
2.04 + .05
2.16 + .05
(5.86 2 .06)t
in the Methods
(g), whereas values in parentheses Gastroc,
2.59 + .05
2.57 t .05
2.75 t .07
2.66 t .06
(6.11 r .09)
(5.66 + .Ol)t
(6.78 -+ 0.1)
(6.27 t O.l)t
section, animals in each age group were given a 21-day clenbuterol
means ? SEM, with six animals in each treatment Abbreviations:
Control
(1.91 ‘- .04)
(6.64 2 .06) NOTE. As described
to Body Weight Ratios
Rats (g and mg/g) 12.Month
Clenbuterol
ETAL
category. Values not in parentheses
refer to the designated
gastrocnemius;
combined
Plant, plantaris,
muscle/organ
EDL, extensor
infusion
at 1.5 mg/kg/24
refer to wet weight of the designated
combined
h. Values are muscle/organ
to body weight ratio (mgig).
digitorum
longus:
TIE ant, tibialis
anterior;
C vent, combined
cardiac
ventricles. *P < .05, tP < .Ol when compared
with the corresponding
control of the same age by Student’s t test.
bined kidney weights in any age group (Table 2). In fact, drug treatment reduced the kidney to body weight ratio 12% in 3-month-old rats, 7% in 12-month-old rats, and 8% in 23-month-old rats. This suggests that clenbuterol did not increase visceral protein stores while increasing somatic protein stores.
the increased protein and decreased fat content. drug treatment increased carcass water content and percentage carcass weight due to water in all age groups (Table 3). Therefore, clenbuterol infusion caused a substantial increase in carcass protein and decrease in carcass fat content that was similar in all age groups.
Effect of Clenbuterol Infusion on Carcass Composition
DISCUSSION
As shown in Table 3, clenbuterol increased carcass protein content 19% in 3-month, 16% in 12-month. and 24% in 23-month-old animals. Likewise, drug treatment increased the percentage of carcass weight due to protein 10% in 3-month, and 13% in 23-month-old rats. In contrast, clenbuterol treatment reduced carcass fat content 36% in 3-month, 32% in 12-month, and 38% in 23-month-old rats (Table 3). Likewise, drug treatment reduced the percentage of carcass weight due to fat 41% in 3-month, 41% in 12-month, and 44% in 23-month-old rats. As expected from
The major objective of this study was to determine if clenbuterol treatment would stimulate skeletal muscle growth in senescent rats with a declining muscle mass as it does in young animals with an increasing muscle mass. As illustrated in Tables 2 and 3. clenbuterol did indeed stimulate growth of skeletal muscles and carcass protein content to a similar degree in young and old animals. Furthermore, this agent reduced carcass fat content to a similar degree in young and old rats. These proteinenhancing and fat-reducing effects of clenbuterol are simi-
Table 3. Carcass Composition of 3-, 12-, and 23-Month-Old
23.Month
12.Month
3.Month Control
Rats (g and % carcass weight)
Clenbuterol
Control
Clenbuterol
Control
Clenbuterol
(n = 6)
(n = 6)
(n = 6)
(II = 6)
in = 61
Protein
29.5 c 1.3
35.2 t l.Ot
39.6 ? 1.2
46.1 t 1.5t
35.4 2 0.7
(17.9 c 0.5) 110 r 3.3
(19.7 * 0.5)X
(la.3 k 0.3)
(18.6 i- 0.5)
(17.6 e 0.2)
Water
126 ?z 5.1*
137 + 2.4
171 + 5.5t
126 f 2.6
151 t 5.4t
(70.4 * 0.4)t
(63.5 i 0.8)
(68.9 + 0.6)-t
(62.9 i 0.4)
(68.4 t 0.9)t
Fat
(66.6 2 0.5) 16.6 c 1.8
10.6 ? 0.7t
29.9 k 1.8
20.3 -t 1.5t
30.3 2 1.3
18.9 - 3.0t
(5.9 k 0.4p
(13.8 k 0.7)
Component
(10.0 c 0.8)
NOTE. As described
in the Methods
section, animals in each age group were given a 21.day clenbuterol
means ? SEM, with six animals in each treatment whereas values in parentheses
category.
refer to the percentage
lP < .05, tP 4 .Ol when compared
(8.2 -t 0.5)t
Values not in parentheses
infusion
refer to carcass content
of carcass weight due to the designated
with the corresponding
(15.1 t 0.6)
component
control of the same age by Student’s f test.
(n = 61
44.0 t 2.4t (19.9 i 0.6)t
(8.4 t l.l)t
at 1.5 mglkgi24
of the designated
(% carcass weight).
h. Values are
component
(g),
CLENBUTEROL
859
EFFECTS IN OLD RATS
lar to previous observations in young rats.4.6.7Therefore, age did not attenuate the anabolic effects of this &-agonist or accentuate its catabolic effects. The failure of clenbuterol to increase kidney weight and the reduced kidney to body weight ratio in treated animals are consistent with previous observations that the anabolic effect of clenbuterol is limited to skeletal and cardiac muscle.6 In all age groups, body weights had a biphasic response to clenbuterol treatment with an early enhanced weight loss and a later enhanced weight gain (Figs l-3). The reason for the increased early weight loss in rats receiving clenbuterol appears to be reduced food intake during the first week of treatment (Table 1). Previous studies using a similar dose of clenbuterol administered orally or by twice daily subcutaneous injection did not show this early reduction in food intake.4.b This suggests that optimization of the route of administration or dosage may minimize the initial anorexic effect. Following the initial lag period, clenbuterol-treated rats gained weight more rapidly than controls and achieved a greater cumulative weight gain (Figs l-3). Evidence that the clenbuterol-induced weight gain reflects deposition of skeletal muscle protein comes from increased skeletal muscle weights and the large increase in carcass protein and decrease in carcass fat observed in treated rats. As calculated from values shown in Table 2, clenbuterol increased the carcass protein to fat ratio 86% in 3-month-old rats, 72% in 12-month-old rats, 99% in 23-month-old animals. Further evidence that clenbuterol-induced weight gain represents skeletal muscle protein accretion is provided by a recent study.‘* Increased skeletal muscle protein content, as estimated from carcass creatine content, could entirely account for the clenbuterol induced increase in carcass protein content. The capacity of clenbuterol to stimulate recovery of skeletal muscle mass following surgical stress in senescent rats may have important therapeutic implications. Since 23-month-old rats showed a reduction in skeletal muscle weights and muscle to body weight ratios when compared with 12-month-old rats (Table 2) they may serve as a model for muscle loss in human aging. In the clinical setting, normal aging is associated with declining muscle mass and functional reserve.‘.’ This expected age-related decline is often accelerated by disease and associated factors such as surgical stress, immobilization, and malnutrition.3 As a result, muscle strength declines below the threshold required for independent performance of daily activities such as ambulation and bathing. Perhaps clenbuterol or related
drugs will prove useful in preventing or reversing agerelated declines in muscle mass and strength. Clenbuterol treatment has prevented or reversed skeletal muscle wasting in several animal models. Clenbuterol partially prevented denervation atrophy when treatment began at the time of denervation and partially restored muscle mass when treatment was delayed following denervation.“~” Clenbuterol reduced muscle atrophy by 50% following denervation and increased the isometric force generated by denervated muscles.” This drug has also reduced muscle atrophy following hindlimb suspension4 Likewise, clenbuterol treatment increased skeletal muscle mass and the carcass protein to fat ratio in mice with genetic muscular dystrophy to a greater degree than in normal controls.“’ In a recent studies, clenbuterol treatment either prevented loss or accelerated recovery of skeletal muscle mass following bacterial endotoxin injection’ and 50% dietary restriction.‘* The capacity of clenbuterol to prevent or repair muscle loss in these models of muscle wasting also suggests a therapeutic role for it or similar drugs in disadvantaged patients with muscle wasting. The energy wasting and cardiac stimulating effects of clenbuterol may limit its utility in elderly subjects because marginal nutrition and heart disease are frequent in this age group. A recent study indicates that the skeletal muscle anabolic effects of this agent can be separated from its fat catabolic and cardiac effects.‘” The nonspecific l3-antagonist, propranolol, and the specific P,-antagonist, atenolol, when fed at doses of 200 and 20 mg,kg diet, did not disturb the anabolic effect of clenbuterol on carcass protein content and enhanced its stimulation of hindlimb muscle protein content. On the other hand, these doses of the p-antagonists blocked clenbuterol-induced cardiac hypertrophy and increased energy expenditure and partially blocked its antilipogenic action. It should be noted that a parenteral dose of propranolol sufficient to block the muscle cyclic adenosine monophosphate (CAMP) response to clenbuterol also blocked the skeletal muscle anabolic effect.” These studies indicate that the p-receptor binding affinity or other features of the skeletal muscle response differ from that of the heart and adipose tissue, since clenbuterol effects on the latter tissues are more readily blocked by P-antagonists. This difference in responsivity of skeletal muscle and other target tissues suggests that new anabolic &-agonists with fewer catabolic effects may be developed and provides a pharmacologic strategy for decreasing the catabolic effects of clenbuterol.
REFERENCES 1. Cohn SH, Vartsky D, Yasumura S, et al: Compartmental body analysis based on total-body nitrogen, potassium and calcium. Am J Physiol239:E524-E530,1980
Geriatric Medicine and Gerontology (ed 2). New York, NY, McGraw-Hill, 1990,pp 319-330 4. Emery PW, Rothwell NJ, Stock MJ, et al: Chronic effects of
2. Larsson L, Grimby G, Karlsson J: Muscle strength and speed of movement in relation to age and muscle morphology. J Appl Physiol46:451-456.1979 3. Brummel-Smith K: Rehabilitation of the geriatric patient, in Hazard WR, Andres R, Bierman EL, et al (eds): Principles of
&-adrenergic agonists on body composition and protein synthesis in the rat. Biosci Rep 4:83-91, 1984 5. MacRae JC, Skene PA, Connell A, et al: The action of the B-agonist clenbuterol on protein-energy metabolism in fattening wether lambs. Br J Nutr 59:457-465, 1988
860
6. Reeds PJ, Hay SM, Dorwood PM, et al: Stimulation of muscle growth by clenbuterol: Lack of effect on muscle protein biosynthesis. Br J Nutr 56:249-258, 1986 7. Yang YT, McElligott MA: Multiple actions of p-adrenergic agonists on skeletal muscle and adipose tissue. Biochem J 261:1-10, 1989 8. Babij P, Booth FW: Alpha actin and cytochrome c mRNAs in atrophied adult rat skeletal muscle. Am J Physiol 254:C657-C660, 1988 9. Choo JJ, Horan MA, Little RA. et al: Wasting associated with endotoxemia in the rat: Modification by the /I$-adrenoceptor agonist clenbuterol. Biosci Rep 9:615-621, 1989 10. Rothwell NJ, Stock MJ: Modification of body composition by clenbuterol in normal and dystrophic mice. Biosci Rep 5:755760,1985 11. Zeman RJ, Ludemann R, Etlinger JD: Clenbuterol, a &agonist, retards atrophy in denervated muscles. Am J Physiol 252:El%E155,1987 12. Choo JJ, Horan MA, Rothwell NJ: Effects of the & adrenoceptor agonist, clenbuterol, on muscle atrophy due to food deprivation in the rat. Metabolism 39:647-650,647-650, 1990 13. Munro HN, Fleck A: Analysis of tissue and body fluids for nitrogenous constituents, in Munro HN (ed): Mammalian Protein Metabolism. ~013. San Diego, CA, Academic, 1969, pp 424-438
CARTER
ET AL
14. Mann LT: Spectrophotometric determination ot nitrogen total micro-Kjeldahl digests. Anal Chem 35:2179-21X?. 1963
in
15. Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purification. Can J Biochem Physiol37:91 l-917. 1959 16. Burgi U, Burgi-Saville ME, Ziegler F. et al: Food intake. body and heart composition. and heart rate in T, plus atenololtreated rats. Am J Physiol256:E459-E466. 1989 17. SAS Institute: SASiSTAT User’s Guide, tion. Gary, NC, SAS Institute. 1988, pp 125-154
Release
6.113 Edi-
18. Reeds PJ. Hay SM. Dotwood PM. et al: The effect of P-agonists and antagonists on muscle growth and body composition of young rats. Comp Biochem Physiol89(7:337-341.1988 19. Maltin CA. Reeds PJ. Deiday MI. et al: lnhibition and reversal of denervation-induced atrophy by the P-agonist growth promoter, clenbuterol. Biosci Rep 6:81 I-818. 1986 20. Maltin CA. Hay SM. Delday MI. et al: Evidence that the hypertrophic action of clenbuterol on denervated rat muscle is not propranolol sensitive. Br J Pharmacol Y6:X17-X22. IYX9 21. MacLennan PA. Edwards RHT: Effect of clrnhuterol and propranolol on muscle mass. Evidence that clenbuterol stimulates p-adrenoceptors to induce hypertrophy. Biochem J 264573-579. 1989
on Skeletal Muscle Mass, Body Composition, From Surgical Stress in Senescent Rats
and Recovery
William J. Carter, An 0. Dang, Fred H. Faas, and Mary E. Lynch Aging decreases skeletal muscle mass and strength, which may be exacerbated
by age-related diseases. There is a need for therapeutic agents to prevent or restore loss of skeletal muscle in elderly subjects with muscle wasting disorders. Clenbuterol, a &adrenergic agonist, dramatically increases skeletal muscle mass in young animals and partially prevents or restores muscle loss in experimental models of muscle wasting. However, the protein anabolic and fat catabolic effects of clenbuterol have not been studied in senescent animals. To determine whether this drug has potential for preventing or repairing muscle loss in elderly subjects, we have examined its effects in young and old rats. Clenbuterol was administered by implanted osmotic minipumps to Fischer-344 rats ages 3,12, and 23 months, at a dose of 1.5 mg/kg/24 h for 3 weeks. The weights of five hindlimb muscles and carcass protein and fat content were determined. Clenbuterol treatment increased the weight of skeletal muscles 22% to 39% in 3-month-old rats, 19% to 35% in 12-month-old rats, and 22% to 25% in 23-month-old animals. Likewise, clenbuterol increased carcass protein content 19% in 3.month-old rats, 16% in 12-month-old rats, and 24% in 23.month-old animals. Conversely, the drug reduced carcass fat content 36% in 3.month-old rats, 32% in 12.month-old rats, and 38% in 23-month-old rats. Therefore, clenbuterol had similar anabolic and catabolic effects in all age groups. In addition, clenbuterol stimulated recovery of skeletal muscle protein lost following pump implantation in senescent rats. These findings suggest that clenbuterol or similar drugs may prove useful in stimulating skeletal muscle hypertrophy in elderly subjects with muscle wasting disorders. Copyright 0 1991 by W.B. Saunders Company
S
INCE AGING decreases skeletal muscle mass and reserve strength,‘,’ elderly subjects are particularly vulnerable to the effects of diseases that reduce skeletal muscle mass and strength below that required for independence in daily activities.3 There is a clear need for therapeutic approaches to prevent or restore the loss of skeletal muscle due to injury or disease and associated processes such as surgery, immobilization, and malnutrition. Recently, certain B,-adrenergic agonists, notably clenbuterol, were found to dramatically increase skeletal muscle mass in young mammals-’ and to reduce or restore muscle loss in animal models of muscle wasting, including denervation, loss of weight bearing, genetic muscular dystrophy, bacterial endotoxin injection, and 50% reduction in dietary intake.8-‘2 This capacity to prevent or repair muscle wasting suggests a potential clinical role for clenbuterol or similar drugs in increasing muscle mass in disadvantaged elderly subjects. However, these agents have catabolic as well as anabolic effects. Clenbuterol decreases carcass fat content, reduces the fraction of dietary energy retained as carcass energy and increases nonshivering thermogenesis.4.7 Because aging might reduce the anabolic effects of these agents and accentuate their catabolic effects, the present studies have compared the effects of clenbuterol in young and old rats. Not only did clenbuterol have similar protein anabolic and fat catabolic effects in young and old rats, it also facilitated the recovery of skeletal muscle lost following surgical stress in old rats.
MATERIALS AND METHODS Materials
Male Fischer-344 rats aged 3,12, and 23 months were purchased from the National Institute on Aging, Bethesda, MD, and Alzet osmotic minipumps, Model 2ML2, from Alza, Palo Alto, CA. Clenbuterol hydrochloride was generously supplied by Boehringer Ingelheim Animal Health, St Joseph, MO. All other chemicals used were reagent grade. Metabolism,
Vol40,
No 8 (August), 1991:
pp 855-860
Treatment of ExperimentalAnimals. All 23-month-old rats were observed and weighed daily for 7 to 10 days after receipt from the vendor. All animals (* 10%) that ate poorly and lost weight during this time were discarded on the assumption that they had a specific disease and would not be appropriate models to study effects of normal aging. Alzet osmotic minipumps were loaded with clenbuterol dissolved in water at concentrations required to deliver a dose of 1.50 mgikg/24 h. The drug solution was sterilized by filtration before pump loading. The pumps were implanted subcutaneously in the interscapular area using anesthesia induced by intramuscular injection of 5.0 mg ketamine and 0.1 mg acepromazine/lOO g. The anesthetic dose was reduced 50% in 23-month-old rats, because the reduced dose produced adequate anesthesia and allowed more rapid recovery. Pump implantation and drug treatment caused no mortality in young or old animals. Control animals received sham operations without pump implantation. During the 21-day clenbuterol infusions, rats were housed individually, weighed daily, and fed Purina Rat Chow ad lib (Ralston Purina, St Louis, MO). The amount of chow consumed was weighed on a weekly basis. At the end of the experiment, rats were killed by decapitation. The gastrocnemius, soleus, plantaris, extensor digitorum longus (EDL). and tibialis anterior muscles were rapidly dissected from both hindlimbs, weighed on an electronic balance, and frozen at -80°C until processed. Likewise, the heart and kidneys were removed, the cardiac chambers were opened, and both organs were rinsed in ice-cold 0.9% NaCI, followed by blotting, weighing, and freezing. Following removal of the visceral organs and tail, the skinned carcass was weighed, wrapped in aluminum foil, and frozen at - 20°C.
From the Veterans Administration Medical Center: Little Rock, AR; and the Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR. Supported by Veterans Administration Research Funds, Project No. 430-66-8721-0002. Address reprint requests to William J. Carter, MD, John L. McClellan Memorial Veterans Hospital (IllG-JLM}, 4300 W 7th St, Little Rock, AR 72205. Copyright 0 1991 by W.B. Saunders Company 0026-049519114008-0015$03.00l0 855
856
CARTER ETAL
60.0
Methods To prepare the carcass for analysis, it was heated in an autoclave for 3 hours at 1 bar of pressure and 120°C to soften tissues including bones. It was then homogenized in water using a Waring blender (model 700G purchased from Baxter. Scientific Products Division, Grand Prairie, TX), and brought to a volume of 500 mL. For carcass protein analysis, nitrogen in an aliquot of the homogenate was converted to ammonium sulfate by a micro-Kjeldahl procedure” and measured calorimetrically using a phenolhypochlorite method.14 Carcass protein content was estimated by multiplying carcass nitrogen content by 6.25. For muscle protein assay, muscles were homogenized in 10 mL/g of 0.05 mol/L
-30.0
lo-mL aliquot of the carcass homogenate was extracted by shaking in a uniphasic solvent mixture containing chloroform, methanol and water in a 1:2:0.8 ratio by volume.” The composition of the solvent mixture was then adjusted to 2:2:1.8 to produce a two-phase system. The upper layer was discarded and the lower chloroform layer was evaporated to dryness under a stream of nitrogen to recover carcass fat. Carcass water content was determined by drying a 10.mL aliquot of the carcass homogenate to constant weight in a 60°C oven.lh RESULTS
Food Consumption
During the Experimental Period
Clenbuterol infusion had no effect on total food consumption during the 21-day experimental period in any age group (Table 1). However, drug treatment reduced food consumption during the first week following pump implantation by 11% in 3-month, 29% in 12-month, and 48% in 23-monthold animals (Table 1). Treated animals overcame this initial deficit by consuming more chow during the final 2 weeks. Body Weights of Rats Before Clenbuterol Infusion Before osmotic pump implantation and drug treatment, control and experimental animals in each age group were closely matched for body weight, as follows (mean ? SEM, N = 6): 3-month age group, control 305 + 8 g, treated 301 2 9; 12-month age group, control 422 * 9 g, treated 425 ? 11; and 23-month age group, control 434 ? 8 g, treated 431 * 14. Rat Body Weight Changes During Clenbuterol Infusion Figures l-3 show cumulative body weight changes for control and experimental animals during the 21-day treatment period. Body weights just before osmotic pump implantation or sham operation were used as baseline
2
4
6
6
10
12
14
16
16
20
22
TREATMENTPERIOD (days) Fig 1. Cumulative weight change in 3-month-old rats during the U-day treatment period. Each data point represents the mean ? SEM of six animals. Control, 0; clenbuterol, 0. *P < .05, **P < .Ol when compared with the corresponding control by Student’s t test.
measures. Since clenbuterol-treated rats had osmotic pump implantation and control animals had sham operations. the weight of the loaded pump was subtracted from the body weight of each treated animal at all points following implantation. Pump weights were 8.01 -+ 0.064 g (mean +- SEM, N = 18) before implantation. Since these pumps imbibe tissue fluid as the drug solution is expelled. weight change was minimal during the infusion period. Figure 1 shows body weight changes in 3-month-old rats during drug infusion. Sham operated controls lost an average of 8 g during the initial 2 days, recovered to baseline at 6 days and slowly gained weight for the remainder of the 21-day treatment period. In contrast, clenbuteroltreated rats had a larger initial weight loss. averaging 18 g, recovered to baseline at 6 days, and thereafter gained weight more rapidly than controls. Figure 2 shows body weight changes in I?-month-old rats, Sham-operated controls initially lost an average of 15 g during days 2 to 5. recovered to baseline at day 13, and remained stable for the remainder of the treatment period. In contrast, clenbuteroltreated rats had a larger initial weight loss, averaging 22 g, but surpassed the weight gain of control rats by day 9. From day 9 until the end of the experimental period, clenbuteroltreated rats gained weight more rapidly than controls and exceeded both control weight gain and the baseline weight. Figure 3 shows body weight changes in 23-month-old rats. Sham-operated controls slowly lost an average of 28 g during the initial 15 days and reached a plateau with no
Table 1. Weekly and Total Chow Consumption (g) in 3-, 12-, and 23-Month-Old-Rats
~_~_
3.Month Control
Clenbuterol (n = 61
Istwk
117 t- 10
104 +- 8*
14
23.Month
12.Month
(n = 6)
Interval
I 0
potassium phosphate buffer, pH 7.0, and an aliquot used for nitrogen determination as above. For carcass fat determination, a
Control (n = 6)
105 2 6
Clenbuterol
Control
Cienbuteroi
(n = 6)
In = 6)
,n = 6)
75 t 3t
90.4 ? 5
47.2 2 8t
2nd wk
125?
123 * 14
129 t 5
133 t 3
111 t3
3rd wk
122 + 3
135 f 4*
135 t 9
151+4
109 -r 4
100 + 15 143 i 8t
Total
364 + 19
361 -t 13
369 -t 19
360 5 8
311 *9
290 + 14
NOTE. As described in the Methods section, animals in each age group were given a 21-day clenbuterol infusion at 1.5 mglkgi24 h. Values are means + SEM, with six animals in each treatment category and refer to the weight of chow consumed in the designated interval. *P < .05, tP < .Ol when compared with the corresponding control of the same age by Student’s f test.
CLENBUTEROL
857
EFFECTS IN OLD RATS
-3o.oc
I 2
0
I 4
I 6
1 6
10
I 12
1 14
I 16
I 16
I 20
9 22
TREATMENTPERIOD (days) Fig 2. Cumulative weight change in l&month-old rats during the 21-day treatment period. Each data point represents the mean k SEM of six animals. Control, 0; clenbuterol, 0. V < .05, l*P < .Ol when compared with the corresponding control by Student’s t test.
tendency to return to baseline weight. In contrast, clenbuterol-treated rats lost more weight during the initial 2 to 5 days, but gained weight from 5 to 17 days, reaching a plateau that exceeded control values and approached baseline. A common pattern of response to operative procedures and drug treatment emerged in all age groups. Although the initial weight loss was greater in clenbuteroltreated animals, they subsequently gained more weight and surpassed control animals. The weight loss following sham operation was particularly pronounced and prolonged in senescent rats. Clenbuterol. stimulation caused the body weight of senescent rats to recover to baseline following implantation surgery, while controls remained below baseline. Effect of Clenbuterol Infusion on Skeletal Muscle Weight and Muscle to Body Weight Ratios
As illustrated in Table 2, clenbuterol infusion increased the wet weight of slow-twitch, fast-twitch, and mixed-fiber muscles in all age groups. Clenbuterol increased the weight of the slow-twitch soleus 26% in 3-month, 25% in 12month, and 25% in 23-month-old rats. Likewise, the drug 5.0
0.0
l
1
-5.0 2 g
-15.0 -10.0
;
-20.0
e & P
-25.0
6,
Q-P-g
0
1
.’
-30.0
22 -35.0
_.,j-i’i
P\,-0 I
I
71.’
l’O--0 1
I \
-4o.o-! 0
I 4
I 6
I 8
I 10
I 12
I 14
I 16
I 18
Effect of Clenbuterol Infusion on Muscle Protein Content
Clenbuterol infusion increased muscle protein content in proportion to wet weight. In 3-month-old rats, clenbuterol treatment increased gastrocnemius protein content 27%, from 304 * 16 mg to 385 2 11 mg (mean t SEM, N = 6). In contrast, drug treatment did not alter the muscle protein to wet weight ratio, which was 200 ? 4 mg/g in control rats and 204 ? 2 in treated ones. In 23-month-old rats, clenbuterol treatment increased gastrocnemius protein content 26%, from 274 2 5 mg to 344 ? 15 mg. Again, drug treatment did not influence the muscle protein to wet weight ratio, which was 212 ? 3 mg/g in control rats and 214 * 3 in treated ones.
1
I I 2
increased the weight of the fast-twitch EDL 39% in 3-month, 35% in lZmonth, and 25% in 23-month-old animals. Clenbuterol treatment increased the weights of the remaining gastrocnemius, plantaris, and tibialis anterior 22% to 28% in 3-month, 19% to 22% in 12-month, and 22% to 26% in 23-month-old animals. The drug increased muscle weights more than body weights, as indicated by increased muscle to body weight ratios at all ages (Table 2). Clenbuterol increased the soleus muscle to body weight ratio 17% in 3-month, 14% in 12-month, and 18% in 23-month-old rats. Likewise, the drug increased the EDL muscle to body weight ratio 31% in 3-month, 27% in 12-month, and 19% in 23-month-old rats. Clenbuterol treatment increased the muscle to body weight ratios of the gastrocnemius, plantaris, and tibialis anterior 13% to 19% in 3-month, 11% to 20% in 12-month, and 17% to 20% in 23-month-old animals. Therefore, clenbuterol treatment increased both the weights and muscle to body weight ratios of all hindlimb muscles examined at all ages. In Table 2, clenbuterol effects on muscle weights and muscle to body weight ratios were evaluated using multiple Student’s t tests. Since multiple individual comparisons may increase the type I error rate, a two-way ANOVA was performed using PC SAS, release 6.03, and differences between means were evaluated by Duncan’s multiple range test.” When the clenbuterol effect on muscle weights and muscle to body weight ratios was evaluated using combined data from all age groups, it reached or exceeded the 5% level for both parameters in all muscles studied. This analysis supports the validity of the apparent clenbuterolinduced increase in muscle weights and muscle to body weight ratios. When the effect of age was evaluated using combined data from treated and control animals, muscle weights and muscle to body weight ratios were uniformly lower in 23-month versus 12-month-old rats at the 5% level or beyond. This analysis supports the use of senescent rats as a model to study age-related muscle loss.
I 20
I 22
TREATMENTPERIOD (days) Fig 3. Cumulative weight change in 23-month-old rats during the Zl-day treatment period. Each data point represents the mean ‘_ SEM of six animals. Control, 0; clenbuterol, 0. lP < .05, l*P < .Ol when compared with the corresponding control by Student’s t test.
Effect of Clenbuterol Infusion on Weights of Cardiac Ventrkles and Kidneys
As shown in Table 2, clenbuterol infusion did not increase the weight of the combined cardiac ventricles in 3-month-old rats, but caused a 15% increase in 12-monthold rats and an 18% increase in 23-month-old rats. On the other hand, clenbuterol treatment did not influence com-
a58
CARTER
Table 2. Skeletal Muscle, Cardiac Ventricle, and Kidney Weights and Muscle/Organ of 3-, 12-, and 23-Month-Old 3.Month CClntrd
MUSCki
23.Month
Control
Clenbuterol
Organ
(n = 6)
(n = 6)
(n = 6)
In = 6)
Gastroc
2.98 + 0.1
3.74 + 0.1t
3.24 + .oa
4.04 _f 0.1t
(9.15 2 0.2)
(10.7 ? 0.1jt
(7.65 & 0.1)
0.24 + .Ol
Soleus
0.30 + .01t
(0.72 + .Ol)
Plant EDL Tib ant Cvent
Kidneys
(0.85 2 .03)t
Clenbuterol
in = 6)
(a.90 + 0.2)t
In = 61
2.60 ? .05
3.26 .: .08t
(6.40 + 0.1)
17.67 I O.l)t
0.28 2 .Ol
0.34 i- .01t
0.24 2 .07
0.30 - .01t
(0.66 * .02)
(0.75 + .02)X
(0.59 +- .02)
(0.70 : .04)’
0.62 t .02
0.80 k .02t
0.72 + .02
0.91 _t .03t
0.61 i .02
(2.28 t .02)t
(1.69 & .04)
(2.01 t .06)t
(1.50 z .03)
0.76 2 04t (1.79 I
06)t
0.28 z .oi
0.40 2 .01t
0.34 + .Ol
0.46 2 .Olt
0.30 t .Ol
(0.87 -c .oi)
(1.14 2 .02)t
(0.79 * .Ol)
(1 .oo r .03jt
(0.74 -t ,011
1.14 + .03
1.38 -+ .04t
1.34 * .Ol
1.60 -c .06t
1.18 + .02
1.44 + 04t
(3.51 t ,081
(3.98 + .06)t
(3.39 t .08)t
0.37 1 .01t (0.88 ?
03jt
(3.16 2 ,031
(3.52 r O.l)*
(2.90 + .04)
0.78 + .02
0.83 k .03
0.91 2 .Ol
1.04 i .02t
0.94 2 .Ol
1.10 -’ .03t
(2.38 + .03)
(2.40 k .04)
(2.14 i .Ol)
(2.30 t .02)t
(2.31 ? ,031
(2.59 i .041t
2.04 + .05
2.16 + .05
(5.86 2 .06)t
in the Methods
(g), whereas values in parentheses Gastroc,
2.59 + .05
2.57 t .05
2.75 t .07
2.66 t .06
(6.11 r .09)
(5.66 + .Ol)t
(6.78 -+ 0.1)
(6.27 t O.l)t
section, animals in each age group were given a 21-day clenbuterol
means ? SEM, with six animals in each treatment Abbreviations:
Control
(1.91 ‘- .04)
(6.64 2 .06) NOTE. As described
to Body Weight Ratios
Rats (g and mg/g) 12.Month
Clenbuterol
ETAL
category. Values not in parentheses
refer to the designated
gastrocnemius;
combined
Plant, plantaris,
muscle/organ
EDL, extensor
infusion
at 1.5 mg/kg/24
refer to wet weight of the designated
combined
h. Values are muscle/organ
to body weight ratio (mgig).
digitorum
longus:
TIE ant, tibialis
anterior;
C vent, combined
cardiac
ventricles. *P < .05, tP < .Ol when compared
with the corresponding
control of the same age by Student’s t test.
bined kidney weights in any age group (Table 2). In fact, drug treatment reduced the kidney to body weight ratio 12% in 3-month-old rats, 7% in 12-month-old rats, and 8% in 23-month-old rats. This suggests that clenbuterol did not increase visceral protein stores while increasing somatic protein stores.
the increased protein and decreased fat content. drug treatment increased carcass water content and percentage carcass weight due to water in all age groups (Table 3). Therefore, clenbuterol infusion caused a substantial increase in carcass protein and decrease in carcass fat content that was similar in all age groups.
Effect of Clenbuterol Infusion on Carcass Composition
DISCUSSION
As shown in Table 3, clenbuterol increased carcass protein content 19% in 3-month, 16% in 12-month. and 24% in 23-month-old animals. Likewise, drug treatment increased the percentage of carcass weight due to protein 10% in 3-month, and 13% in 23-month-old rats. In contrast, clenbuterol treatment reduced carcass fat content 36% in 3-month, 32% in 12-month, and 38% in 23-month-old rats (Table 3). Likewise, drug treatment reduced the percentage of carcass weight due to fat 41% in 3-month, 41% in 12-month, and 44% in 23-month-old rats. As expected from
The major objective of this study was to determine if clenbuterol treatment would stimulate skeletal muscle growth in senescent rats with a declining muscle mass as it does in young animals with an increasing muscle mass. As illustrated in Tables 2 and 3. clenbuterol did indeed stimulate growth of skeletal muscles and carcass protein content to a similar degree in young and old animals. Furthermore, this agent reduced carcass fat content to a similar degree in young and old rats. These proteinenhancing and fat-reducing effects of clenbuterol are simi-
Table 3. Carcass Composition of 3-, 12-, and 23-Month-Old
23.Month
12.Month
3.Month Control
Rats (g and % carcass weight)
Clenbuterol
Control
Clenbuterol
Control
Clenbuterol
(n = 6)
(n = 6)
(n = 6)
(II = 6)
in = 61
Protein
29.5 c 1.3
35.2 t l.Ot
39.6 ? 1.2
46.1 t 1.5t
35.4 2 0.7
(17.9 c 0.5) 110 r 3.3
(19.7 * 0.5)X
(la.3 k 0.3)
(18.6 i- 0.5)
(17.6 e 0.2)
Water
126 ?z 5.1*
137 + 2.4
171 + 5.5t
126 f 2.6
151 t 5.4t
(70.4 * 0.4)t
(63.5 i 0.8)
(68.9 + 0.6)-t
(62.9 i 0.4)
(68.4 t 0.9)t
Fat
(66.6 2 0.5) 16.6 c 1.8
10.6 ? 0.7t
29.9 k 1.8
20.3 -t 1.5t
30.3 2 1.3
18.9 - 3.0t
(5.9 k 0.4p
(13.8 k 0.7)
Component
(10.0 c 0.8)
NOTE. As described
in the Methods
section, animals in each age group were given a 21.day clenbuterol
means ? SEM, with six animals in each treatment whereas values in parentheses
category.
refer to the percentage
lP < .05, tP 4 .Ol when compared
(8.2 -t 0.5)t
Values not in parentheses
infusion
refer to carcass content
of carcass weight due to the designated
with the corresponding
(15.1 t 0.6)
component
control of the same age by Student’s f test.
(n = 61
44.0 t 2.4t (19.9 i 0.6)t
(8.4 t l.l)t
at 1.5 mglkgi24
of the designated
(% carcass weight).
h. Values are
component
(g),
CLENBUTEROL
859
EFFECTS IN OLD RATS
lar to previous observations in young rats.4.6.7Therefore, age did not attenuate the anabolic effects of this &-agonist or accentuate its catabolic effects. The failure of clenbuterol to increase kidney weight and the reduced kidney to body weight ratio in treated animals are consistent with previous observations that the anabolic effect of clenbuterol is limited to skeletal and cardiac muscle.6 In all age groups, body weights had a biphasic response to clenbuterol treatment with an early enhanced weight loss and a later enhanced weight gain (Figs l-3). The reason for the increased early weight loss in rats receiving clenbuterol appears to be reduced food intake during the first week of treatment (Table 1). Previous studies using a similar dose of clenbuterol administered orally or by twice daily subcutaneous injection did not show this early reduction in food intake.4.b This suggests that optimization of the route of administration or dosage may minimize the initial anorexic effect. Following the initial lag period, clenbuterol-treated rats gained weight more rapidly than controls and achieved a greater cumulative weight gain (Figs l-3). Evidence that the clenbuterol-induced weight gain reflects deposition of skeletal muscle protein comes from increased skeletal muscle weights and the large increase in carcass protein and decrease in carcass fat observed in treated rats. As calculated from values shown in Table 2, clenbuterol increased the carcass protein to fat ratio 86% in 3-month-old rats, 72% in 12-month-old rats, 99% in 23-month-old animals. Further evidence that clenbuterol-induced weight gain represents skeletal muscle protein accretion is provided by a recent study.‘* Increased skeletal muscle protein content, as estimated from carcass creatine content, could entirely account for the clenbuterol induced increase in carcass protein content. The capacity of clenbuterol to stimulate recovery of skeletal muscle mass following surgical stress in senescent rats may have important therapeutic implications. Since 23-month-old rats showed a reduction in skeletal muscle weights and muscle to body weight ratios when compared with 12-month-old rats (Table 2) they may serve as a model for muscle loss in human aging. In the clinical setting, normal aging is associated with declining muscle mass and functional reserve.‘.’ This expected age-related decline is often accelerated by disease and associated factors such as surgical stress, immobilization, and malnutrition.3 As a result, muscle strength declines below the threshold required for independent performance of daily activities such as ambulation and bathing. Perhaps clenbuterol or related
drugs will prove useful in preventing or reversing agerelated declines in muscle mass and strength. Clenbuterol treatment has prevented or reversed skeletal muscle wasting in several animal models. Clenbuterol partially prevented denervation atrophy when treatment began at the time of denervation and partially restored muscle mass when treatment was delayed following denervation.“~” Clenbuterol reduced muscle atrophy by 50% following denervation and increased the isometric force generated by denervated muscles.” This drug has also reduced muscle atrophy following hindlimb suspension4 Likewise, clenbuterol treatment increased skeletal muscle mass and the carcass protein to fat ratio in mice with genetic muscular dystrophy to a greater degree than in normal controls.“’ In a recent studies, clenbuterol treatment either prevented loss or accelerated recovery of skeletal muscle mass following bacterial endotoxin injection’ and 50% dietary restriction.‘* The capacity of clenbuterol to prevent or repair muscle loss in these models of muscle wasting also suggests a therapeutic role for it or similar drugs in disadvantaged patients with muscle wasting. The energy wasting and cardiac stimulating effects of clenbuterol may limit its utility in elderly subjects because marginal nutrition and heart disease are frequent in this age group. A recent study indicates that the skeletal muscle anabolic effects of this agent can be separated from its fat catabolic and cardiac effects.‘” The nonspecific l3-antagonist, propranolol, and the specific P,-antagonist, atenolol, when fed at doses of 200 and 20 mg,kg diet, did not disturb the anabolic effect of clenbuterol on carcass protein content and enhanced its stimulation of hindlimb muscle protein content. On the other hand, these doses of the p-antagonists blocked clenbuterol-induced cardiac hypertrophy and increased energy expenditure and partially blocked its antilipogenic action. It should be noted that a parenteral dose of propranolol sufficient to block the muscle cyclic adenosine monophosphate (CAMP) response to clenbuterol also blocked the skeletal muscle anabolic effect.” These studies indicate that the p-receptor binding affinity or other features of the skeletal muscle response differ from that of the heart and adipose tissue, since clenbuterol effects on the latter tissues are more readily blocked by P-antagonists. This difference in responsivity of skeletal muscle and other target tissues suggests that new anabolic &-agonists with fewer catabolic effects may be developed and provides a pharmacologic strategy for decreasing the catabolic effects of clenbuterol.
REFERENCES 1. Cohn SH, Vartsky D, Yasumura S, et al: Compartmental body analysis based on total-body nitrogen, potassium and calcium. Am J Physiol239:E524-E530,1980
Geriatric Medicine and Gerontology (ed 2). New York, NY, McGraw-Hill, 1990,pp 319-330 4. Emery PW, Rothwell NJ, Stock MJ, et al: Chronic effects of
2. Larsson L, Grimby G, Karlsson J: Muscle strength and speed of movement in relation to age and muscle morphology. J Appl Physiol46:451-456.1979 3. Brummel-Smith K: Rehabilitation of the geriatric patient, in Hazard WR, Andres R, Bierman EL, et al (eds): Principles of
&-adrenergic agonists on body composition and protein synthesis in the rat. Biosci Rep 4:83-91, 1984 5. MacRae JC, Skene PA, Connell A, et al: The action of the B-agonist clenbuterol on protein-energy metabolism in fattening wether lambs. Br J Nutr 59:457-465, 1988
860
6. Reeds PJ, Hay SM, Dorwood PM, et al: Stimulation of muscle growth by clenbuterol: Lack of effect on muscle protein biosynthesis. Br J Nutr 56:249-258, 1986 7. Yang YT, McElligott MA: Multiple actions of p-adrenergic agonists on skeletal muscle and adipose tissue. Biochem J 261:1-10, 1989 8. Babij P, Booth FW: Alpha actin and cytochrome c mRNAs in atrophied adult rat skeletal muscle. Am J Physiol 254:C657-C660, 1988 9. Choo JJ, Horan MA, Little RA. et al: Wasting associated with endotoxemia in the rat: Modification by the /I$-adrenoceptor agonist clenbuterol. Biosci Rep 9:615-621, 1989 10. Rothwell NJ, Stock MJ: Modification of body composition by clenbuterol in normal and dystrophic mice. Biosci Rep 5:755760,1985 11. Zeman RJ, Ludemann R, Etlinger JD: Clenbuterol, a &agonist, retards atrophy in denervated muscles. Am J Physiol 252:El%E155,1987 12. Choo JJ, Horan MA, Rothwell NJ: Effects of the & adrenoceptor agonist, clenbuterol, on muscle atrophy due to food deprivation in the rat. Metabolism 39:647-650,647-650, 1990 13. Munro HN, Fleck A: Analysis of tissue and body fluids for nitrogenous constituents, in Munro HN (ed): Mammalian Protein Metabolism. ~013. San Diego, CA, Academic, 1969, pp 424-438
CARTER
ET AL
14. Mann LT: Spectrophotometric determination ot nitrogen total micro-Kjeldahl digests. Anal Chem 35:2179-21X?. 1963
in
15. Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purification. Can J Biochem Physiol37:91 l-917. 1959 16. Burgi U, Burgi-Saville ME, Ziegler F. et al: Food intake. body and heart composition. and heart rate in T, plus atenololtreated rats. Am J Physiol256:E459-E466. 1989 17. SAS Institute: SASiSTAT User’s Guide, tion. Gary, NC, SAS Institute. 1988, pp 125-154
Release
6.113 Edi-
18. Reeds PJ. Hay SM. Dotwood PM. et al: The effect of P-agonists and antagonists on muscle growth and body composition of young rats. Comp Biochem Physiol89(7:337-341.1988 19. Maltin CA. Reeds PJ. Deiday MI. et al: lnhibition and reversal of denervation-induced atrophy by the P-agonist growth promoter, clenbuterol. Biosci Rep 6:81 I-818. 1986 20. Maltin CA. Hay SM. Delday MI. et al: Evidence that the hypertrophic action of clenbuterol on denervated rat muscle is not propranolol sensitive. Br J Pharmacol Y6:X17-X22. IYX9 21. MacLennan PA. Edwards RHT: Effect of clrnhuterol and propranolol on muscle mass. Evidence that clenbuterol stimulates p-adrenoceptors to induce hypertrophy. Biochem J 264573-579. 1989
