Anabolic steroids increase exercise tolerance
Tetsuro
Tamaki1,
Shuichi
Uchiyama4,
Yoshiyasu
Uchiyama3,
Akira
Akatsuka2,
Roland R.
Roy5, and
V. Reggie
Edgerton5,6
1 Division of Human Structure and Function, Department of
Physiology, 2 Laboratory for Structure and Function Research,
and 3 Department of Orthopedics, Tokai University School of
Medicine, Kanagawa 259-1193; 4 Tokai University School
of Physical Education, Kanagawa 259-1292, Japan; and
5 Brain Research Institute and 6 Department of
Physiological Science, University of California Los Angeles, Los
Angeles, California 90095
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ABSTRACT |
The influence of
an anabolic androgenic steroid (AAS) on thymidine and amino acid uptake
in rat hindlimb skeletal muscles during 14 days after a single
exhaustive bout of weight lifting was determined. Adult male rats were
divided randomly into Control or Steroid groups. Nandrolone decanoate
was administered to the Steroid group 1 wk before the exercise bout.
[3H]thymidine and [14C]leucine labeling
were used to determine the serial changes in cellular mitotic activity,
amino acid uptake, and myosin synthesis. Serum creatine kinase (CK)
activity, used as a measure of muscle damage, increased 30 and 60 min
after exercise in both groups. The total amount of weight lifted was
higher, whereas CK levels were lower in Steroid than in Control rats.
[3H]thymidine uptake peaked 2 days after exercise in both
groups and was 90% higher in Control than in Steroid rats, reflecting a higher level of muscle damage. [14C]leucine uptake was
~80% higher at rest and recovered 33% faster postexercise in
Steroid than in Control rats. In a separate group of rats, the in situ
isometric mechanical properties of the plantaris muscle were
determined. The only significant difference was a higher fatigue
resistance in the Steroid compared with the Control group. Combined,
these results indicate that AAS treatment 1) ameliorates CK
efflux and the uptake of [3H]thymidine and enhances the
rate of protein synthesis during recovery after a bout of weight
lifting, all being consistent with there being less muscle damage, and
2) enhances in vivo work capacity and the in situ fatigue
resistance of a primary plantarflexor muscle.
anabolic androgenic steroid; nandrolone decanoate; serum creatine
kinase; muscle fiber damage; mitotic activity
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INTRODUCTION |
THE ADMINISTRATION OF
ANABOLIC androgenic steroids (AAS) increases skeletal muscle mass
(hypertrophy) and protein synthesis (9), and these
responses are enhanced when AAS is given in combination with resistance
exercise (11). Skeletal muscle fibers are multinucleated,
and hypertrophy is accompanied by an increase in the number of
myonuclei, thereby maintaining a relatively constant myonuclear
domain, i.e., myonuclei per cytoplasmic volume (6, 12, 14,
21). A primary source of new myonuclei appears to be from the
activation, proliferation, and incorporation of satellite cells, in
that inactivation of satellite cells via irradiation prevents
hypertrophy in functionally overloaded muscles (19, 20).
However, there are no reports examining the relationship between
protein synthesis and mitotic activity in extensor and flexor muscles
after AAS treatment with and without exercise. In addition, a
"membrane stabilizing effect" of AAS agents that diminishes the
rise in serum creatine kinase (CK) efflux after muscle damage has been
suggested (22, 23). Resistance exercise, such as weight
lifting, appropriately induces muscle hypertrophy and is commonly
associated with muscle damage and increased levels of serum CK in
humans (5, 15, 30).
Recently, we (24, 25, 26) have reported a
morphological and biochemical myogenic response associated with muscle
damage and regeneration in the plantarflexor muscles after a single
exhaustive session of weight lifting in previously nontrained adult
rats. The severity of weight lifting-induced muscle damage was
associated with the level of increase in serum CK activity after the
exercise bout (24, 25). In addition, we found
[3H]thymidine and [14C]leucine labeling in
vivo to be useful methods to detect the mitotic activity of
proliferating cells and amino acid uptake in the muscles after the
exercise session (24, 26). For example, after
activation of satellite cells, other stem cells, and/or fibroblasts,
thymidine uptake in the nuclei of these cells is essential for DNA
duplication and cell proliferation, and amino acid uptake is necessary
for the differentiation of these cells. Similarly, elevations in amino
acid uptake and protein synthesis are necessary for increasing the
cytoplasm in hypertrophying muscle fibers.
On the basis of these findings, our primary hypothesis was that AAS
would enhance the uptake of both thymidine and amino acids and thus the
adaptive potential selectively in plantarflexor muscles after a single
bout of weight lifting. There also is evidence that AAS treatment may
directly improve the endurance capacity of skeletal muscles. For
example, improved submaximal running capacity of rats (28)
and an improved fatigue resistance of rat skeletal muscles tested via
electrical stimulation (7) have been reported after AAS
treatment. Thus a second hypothesis was that AAS treatment would
enhance the work tolerance of the animal based on a weight-lifting
regimen performed in vivo (25) and on the in situ
mechanical properties of a primary plantarflexor, i.e., the plantaris muscle.
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MATERIALS AND METHODS |
Experimental groups.
Specific pathogen- and virus antigen-free Wistar male rats (14-20
wk old; 380-520 g body wt; n = 90) were divided
randomly into two groups: a Control (n = 53) and an
AAS-treated (Steroid, n = 37) group. One dose of
nandrolone decanoate (deca-Durabolin; Organon, 3.75 mg/kg body wt) was
administered intramuscularly in the gluteus medius muscle 1 wk before
the single exercise session (see Exercise protocol). This
drug is a long-acting steroid ester that is hydrolyzed slowly to give a
constant tissue level of steroid for >4 wk. Exercised (see
Exercise protocol) and nonexercised subgroups were studied,
and the nonexercised subgroups were used to obtain resting values. In
addition, a subsample (n = 7) of the rats from the
Control group used in the testing of the mechanical properties of the
plantaris muscle (see In situ contractile
properties) were administered an oil vehicle
intramuscularly at the same time as the AAS treatment. The animals were
housed in standard cages and were provided food and water ad
libitum. The room temperature was kept at 23 ± 1°C,
and a 12:12-h light-dark cycle was maintained throughout the
experiment. All experimental procedures were conducted in accordance
with the Japanese Physiological Society Guide for the Care and Use of
Laboratory Animals as approved by the Tokai University School of
Medicine Committee on Animal Care and Use and followed the American
Physiological Society Animal Care Guidelines.
Exercise protocol.
The hindlimbs of all exercised rats were trained for one exhaustive
session of weight lifting as described in detail elsewhere (24-26). This exercise was performed preferentially
by the plantarflexors, with minimal usage of the dorsiflexors. The
exercise session involved multiple sets of 10 repetitions (lifts) per
set with ~1 min rest between each set. The first set of lifts was
with a 500-g load. In the subsequent sets, an additional 500-g load was
added until the rat could not complete 10 repetitions. The load then
was adjusted in 100-g increments and/or decrements until the maximum
load at which 10 repetitions could be completed, i.e., the
10-repetition maximum (10RM), was determined. The 10RM was repeated
until the rat could not complete the set, and then the load was
decreased by 500 g. This procedure was followed until the rat
failed to complete three consecutive sets even when the weight was
being reduced. The total time of the exercise bout was ~30-40
min in both groups. The 10RM (g), number of sets, and total amount of load lifted (number of lifts × load lifted by different groups in
absolute values expressed as kg) were recorded. A subsample of rats
(n = 6 randomly selected rats from each group) also was tested for maximum weight-lifting capacity (1RM in kg).
Measurement of serum creatine kinase activity.
Blood samples (0.2 ml) were obtained from the caudal vein before and 30 and 60 min after the exercise session in both the Control
(n = 46) and Steroid (n = 33) groups.
Serum creatine kinase (CK) activity was measured using a standard kit
(Monotest CK-NAC, Boehringer Mannheim, Mannheim, Germany) and was used
to estimate exercise-induced muscle damage. The activities were
expressed in international units (IU)/ml.
Analyses of mitotic activity and amino acid uptake.
Our previous data indicate that in vivo [3H]thymidine and
[14C]leucine labeling are useful methods to detect the
mitotic activity of satellite (and/or other stem) cells and amino acid
uptake in muscles after the exercise session (24, 26).
Analyses of mitotic activity and muscle amino acid uptake were
performed at rest and 3, 6, 12, and 18 h and 1, 2, 3, 4, 7, 10, and 14 days after exercise in Control (n = 53, 3-8/time point) and Steroid (n = 37, 3-6/time point) rats. [3H]thymidine
([methyl-3H]; 15.5 MBq/kg ip; specific
activity 247.9 GBq/mmol, NEN Life Science Products, Boston, MA) and
[14C]leucine ([U-14C]; 1.15 Mbq/kg ip;
specific activity 13.6 GBq/mmol, NEN Life Science Products) were
injected 1 and 3 h before sampling to label proliferating cells or
proteins that use leucine during protein synthesis, respectively. The
rats were overdosed with pentobarbital sodium (60 mg/kg ip), and the
following muscles were removed bilaterally: primary plantarflexors
[soleus (Sol), plantaris (Plt), and gastrocnemius (Gas)] and primary
dorsiflexors [tibialis anterior (TA) and extensor digitorum longus
(EDL)]. After excess connective tissue and fat were removed, each
muscle was wet weighed and homogenized in 0.02 M phosphate buffer (pH
7.4) at a 1:20 dilution at 4°C. Then, 1 ml of the homogenate from
each muscle sample was added to 5 ml of 10% trichloroacetic acid (TCA)
and mixed well. This mixture was centrifuged (2,050 g for 10 min), and the upper solution (TCA soluble) was removed. This procedure
was repeated five times, and the remaining TCA-insoluble material was
collected and dried in 70% ethanol. The dried material was treated
overnight with 1 ml of dissolving solution (Solvable, Packard
Instruments, Meriden, CT) at 45°C, and then a 10-ml liquid
scintillation cocktail (Atomlight, Packard Instruments) was added to
count radioactivity (Beckman LS4800, Fullerton, CA). The total protein
concentration in each homogenate was measured, and the radioactivity of
each sample was expressed in disintegrations per minute per milligram
of protein.
We have reported previously (24, 26) that the uptake of
thymidine and leucine into individual muscles within a rat and for an
individual muscle across rats varies widely, most likely reflecting
varying levels of recruitment of each muscle during the weight-lifting
task. However, in all cases, the pooled values for the plantarflexors
(Sol, Plt, and Gas) had a higher amino acid uptake than the pooled
values for the dorsiflexors (TA and EDL). Thus, to minimize the impact
of the intra- and intermuscle variability on the effects of exercise on
thymidine and leucine uptake, the difference in the uptake between the
plantarflexor and dorsiflexor muscles in both legs of each rat is
reported along with the absolute values.
Analysis of myosin synthesis.
The determination of myosin synthesis was performed at the same time
points and for the same groups as for the analysis of mitotic activity
and amino acid uptake. Myosin was extracted with 0.6 M KCl solution (50 ml) from a 1-ml homogenate for 15 min at 4°C and filtered with three
sheets of gauze. The myosin-extracted KCl solution was diluted with
cool, distilled water (1:20), which resulted in the reappearance of
myosin deposits. The diluted solution was passed through an omnipore
nondissolving membrane filter (10-µm aperture and 47-mm diameter;
Nihon Millipore, Yonezawa, Japan). The membrane containing the deposits
was dried, cut into several pieces, and soaked in a dissolving solution
overnight at 45°C. The radioactivity was counted using the same
procedures employed for the mitotic activity and protein synthesis
analyses. Values were expressed in disintegrations per minute per
milligram of protein.
In situ contractile properties.
The in situ isometric mechanical properties of the Plt muscle were
determined under urethane anesthesia (800 mg/kg ip) 1 wk after the
single AAS or oil vehicle treatment. Twenty-seven rats (Control,
n = 11; Vehicle, n = 7; Steroid,
n = 9) were studied for this portion of the study. The
body (rectal) temperature was maintained at ~36 ± 1°C using a
heating pad, and atropine sulfate (0.05 mg/kg sc) was administered to
avoid parasympathetic secretory hyperfunction. A tracheal tube was
inserted. The jugular vein was cannulated, and warm Ringer's solution
containing 5% glucose was administered intravenously, as necessary,
i.e., based on changes in heart rate (monitored via electrocardiogram)
and breathing rate and depth.
The rat was placed in a prone position on a custom-made operation table
that allowed stabilization of the head and limbs with surgical tape. A
midsagittal incision was made extending from the popliteal area to the
base of the calcaneus to expose the posterior aspect of the lower
hindlimb. The right Plt muscle was exposed and freed from surrounding
tissues, care being taken to avoid any interference with the normal
blood and nerve supplies. The distal tendon of the Plt muscle was cut
and attached to a transducer (TB-611T, Nihon Kohden, Tokyo, Japan)
connected to an amplifier (AP-621G, Nihon Kohden). The surrounding skin
was used to form a mineral oil bath maintained at 35 ± 1°C
using radiant heat to prevent tissue drying and to minimize electrical
interference. The sciatic nerve (~15 mm long) was dissected
carefully, exposed under the gluteus medius muscle, and then immersed
in a small bath of mineral oil. A bipolar silver electrode (Ag/Ag,
distance between the two electrodes fixed at 2 mm) was placed under the sciatic nerve ~15 mm proximal to the branching of the tibial and peroneal nerves. The surface electromyographic (EMG) signals during electrical stimulation of the Plt muscle were recorded using a bipolar
surface electrode (Ag/Ag, 1.0-mm diameter and distance between the two
electrodes fixed at 2 mm) placed at the midbelly of the muscle. The
surgical preparation was completed within 30 min, and measurement of
the contractile properties was started ~60 min after anesthesia induction.
Recording procedures.
Whole muscle maximum isometric twitches were elicited using 1.0-ms
duration single pulses (0.5 Hz) with the voltage set ~2-3 times
above the threshold for a maximum response (0.8-1.2 V). Optimum
muscle length was determined from the maximum twitch length-tension relationship, and all contractile measures were taken at this muscle
length. Time to peak tension, one-half relaxation time (1/2RT), and the
time between stimulation and the appearance of an EMG signal
(conduction and transmission time) were measured for 10 consecutive
twitches at 1 Hz and averaged. The maximum tetanic tension produced at
stimulation frequencies of 80, 100, 120, and 140 Hz and a train
duration of 500 ms were determined. One minute of rest was allowed
between each tetanus, and the highest tension recorded was considered
the maximum tetanic tension. Measurements of the twitch and tetanic
properties were completed within 10 min. After a 5-min rest period, the
muscle was stimulated continuously at 12 Hz. The fatigue test was
considered to be the time that elapsed between the initial twitch to a
50% fall in twitch. After the fatigue test, the rats were given 0.5 ml
of Ringer's solution and a 10-min rest. Subsequently, the same
contractile measurements were obtained from the contralateral Plt
muscle. The mechanical properties are reported as the average of the
two muscles from each animal.
All mechanical and electrical measurements were recorded on a
Linearcorder (Mark VII, WR3101, Graphtec, Tokyo, Japan) and an FM tape
recorder (PC208A, SONY Magnescale, Tokyo) and then stored on a
digital audio tape (DAT, DT120RN). The measurements also were recorded
with a microcomputer (Quadra 840-AV, Apple Japan, Tokyo) and analyzed
using MacADIOS II and SuperScorp II (GW Instruments, Somerville, MA).
The analog-to-digital sampling rate was set at 5 kHz.
Statistical analyses.
All data are expressed as means ± SE. Differences in body mass,
muscle mass, record of exercise task, and [3H]thymidine
and [14C]leucine uptake levels between Control and
Steroid groups were determined using Student's t-tests.
Analysis of variance (ANOVA) was used to determine overall differences,
and Duncan's post hoc analyses for individual group differences were
used for the pre- and serial postexercise data for
[3H]thymidine, [14C]leucine, and myosin
fractional uptake and for the comparison of the mechanical properties
among the three groups. Standard regression analysis, Pearson Product
correlation procedures, and Fisher's correlation-coefficient table
were used to determine the relationship between
[3H]thymidine uptake and CK activity. Differences were
considered statistically significant at P
0.05.
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RESULTS |
Body and muscle mass.
The mean final body mass and mass of each muscle studied (TA, EDL, Sol,
Plt, and Gas) were similar for the two groups (Table 1).
Exercise capacity.
The weight-lifting performance during the single exercise session was
significantly better in the Steroid than in the Control group (Table
2). The total amount of weight lifted,
the total number of sets, 10RM, and number of complete sets at 10RM
were 47, 12, 22, and 81% higher in the Steroid than in the Control group, respectively. In addition, there was no difference in the 1RM
between the subsamples of rats tested in the Control and Steroid groups, i.e., 4.0 ± 0.3 and 4.1 ± 0.1 kg, respectively.
Serum CK activity.
The mean preexercise serum CK levels were similar in the Steroid and
Control groups (Fig. 1). These levels
were increased significantly in both groups 30 and 60 min after the
exercise session. The postexercise values were significantly lower in
the Steroid than in the Control group at both time points.

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Fig. 1.
Mean serum creatine kinase (CK) activity at rest and 30 and 60 min after an exhaustive resistive bout of weight lifting. Bars
are SE. A significant increase in CK leakage was observed 30 and 60 min
after exercise in both groups. However, the values were significantly
lower in the Steroid than in the Control group. IU, international unit.
Rest, preexercise. P 0.05 (Control vs. Steroid);
*P 0.05 (rest vs. postexercise in Control group);
P 0.05 (rest vs. postexercise in Steroid
group).
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Thymidine and amino acid uptake rates.
Compared with the Control group, both the plantarflexor (Sol, Plt, and
Gas) and dorsiflexor (TA and EDL) musculature of the Steroid group had
significantly lower uptakes of [3H]thymidine (
16% in
the dorsiflexors and
26% in the plantarflexors) and higher uptakes
of [14C]leucine (+64% in the dorsiflexors and +90% in
the plantarflexors) in the resting state (Fig.
2, A and B). The
mean uptake values of both amino acids also were significantly higher
in the plantarflexors than in the dorsiflexors in both groups.

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Fig. 2.
Mean uptake of [3H]thymidine (A)
and [14C]leucine (B) in the plantarflexors and
dorsiflexors in the resting state. Bars are SE. Uptakes of both
[3H]thymidine and [14C]leucine were
significantly higher in plantarflexors than in dorsiflexors in both
groups. Uptake of [3H]thymidine was significantly lower
and uptake of [14C]leucine significantly higher in
Steroid than in Control for both plantarflexors and dorsiflexors.
Plantarflexors: soleus, plantaris, and gastrocnemius; dorsiflexors:
tibialis anterior and extensor digitorum longus. r, rest values for
nonexercised control rats; dpm, disintegrations per minute.
P 0.05 (Control vs. Steroid); §P 0.05 (plantarflexors vs. dorsiflexors).
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Compared with resting levels, the absolute [3H]thymidine
uptake in the plantarflexors was significantly higher after 1-3
days in Control and after 2 days in Steroid rats (Fig.
3, A and B). In
contrast, there were no significant changes in the dorsiflexor muscles
of either group during the 2-wk postexercise period. A significant
increase in [14C]leucine uptake was observed at 10 days
after exercise in the plantarflexors of Control rats (Fig.
4A). No significant changes were observed in the dorsiflexors of the Control group or in
either muscle group of the Steroid rats (Fig. 4, A and
B).

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Fig. 3.
Absolute change in uptake of [3H]thymidine
in plantarflexors and dorsiflexors during the 2 wk after a single
exhaustive bout of weight lifting in Control (A) and Steroid
(B) groups. Values are means ± SE. d, Day.
*P 0.05, (rest vs. postexercise in Control group);
*P 0.05 (rest vs. postexercise in Steroid
group). Note that the overall mean uptakes of
[3H]thymidine for the dorsiflexor muscles after the
exercise bout were 35.4 ± 2.5 and 27.2 ± 1.5 for Control
and Steroid, respectively. These values were not significantly
different from resting values.
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Fig. 4.
Absolute change in uptake of [14C]leucine
in the plantarflexors and the dorsiflexors during the 2 wk after a
single exhaustive bout of weight lifting in Control (A) and
Steroid (B) groups. Values are means ± SE.
*P 0.05 (rest vs. postexercise in Control group). Note
that the overall mean uptakes of [14C]leucine for the
dorsiflexor muscles after the exercise bout were 127.4 ± 5.8 and
178.8 ± 5.4 for Control and Steroid, respectively. These values
were not significantly different from resting values.
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Because there was no exercise effect on the overall mean uptakes of
these thymidine and amino acids for the dorsiflexor muscles of either
group, we then normalized the exercise effects of the plantarflexors to
the dorsiflexor muscles (Fig. 5). In
effect, this procedure minimized the intra-animal variability. The
relative uptake of [3H]thymidine increased significantly
1-3 days after exercise and showed a peak at 2 days after exercise
in both groups (Fig. 5A). The peak value at 2 days after
exercise, however, was significantly lower in the Steroid than in the
Control group. There was a tendency for the [14C]leucine
uptake in the Control group to be elevated compared with rest values
from 1 to 10 days after exercise, with significant peaks at 3 and 10 days postexercise (Fig. 5B). In the Steroid group, however,
there was a tendency for the [14C]leucine uptake to be
higher than rest values from 6 h to 3 days after exercise, with a
significant peak at 1 day postexercise. Significant differences in the
[14C]leucine uptake levels between the Control and
Steroid groups were observed at rest, at 6 and 12 h, and at 1 and
10 days after exercise. Interestingly, the uptake levels of
[14C]leucine at 3 h after exercise tended to be
lower than rest levels in both groups (P
0.05 in the
Steroid group).

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Fig. 5.
Serial changes in [3H]thymidine
(A) and [14C]leucine (B) uptake
during the 2 wk after a single exhaustive bout of weight lifting.
Uptake values were calculated as [(mean value of plantarflexors) (mean value of dorsiflexors)] in both legs and are plotted as
means ± SE. *P 0.05 (rest vs. postexercise in
Control); P 0,05 (rest vs. postexercise in
Steroid); P 0.05 (Control vs. Steroid).
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Fraction of amino acid uptake for myosin.
For the Control group, the uptake of [14C]leucine for the
myosin fraction in the plantarflexor muscles was significantly elevated from rest values at 10 days after exercise, whereas the uptake rates
were similar to rest values at all other recovery time points (Fig.
6). For the Steroid group, significant
decreases were observed at 3, 6, and 12 h and at 3 days after the
exercise bout. No other significant changes were observed throughout
the 14-day postexercise period (Fig. 6). The levels of
[14C]leucine uptake of the Steroid group, however, were
significantly higher than Control values at rest, from 3 h to 2 days, and at 14 days after exercise.

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Fig. 6.
Serial changes in absolute [14C]leucine
uptake for the contractile component (myosin) of the plantarflexor
muscles during the 2 wk after a single exhaustive bout of weight
lifting. *P 0.05, (rest vs. postexercise in Control);
P 0,05 (rest vs. postexercise in Steroid);
P 0.05 (Control vs. Steroid).
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In situ mechanical properties of the Plt muscle.
Body and muscle mass, and all mechanical properties of the Plt muscle
except for the fatigue index, were similar among the three groups
(Table 3). The Plt in the Steroid group
was more resistant to fatigue than in the other two groups. Note that
there were no differences between Control and Vehicle groups.
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DISCUSSION |
Ergogenic effects on the in vivo and in situ conditions.
The mean body and muscle weights were similar in the Control and
Steroid groups (Table 1). These results were expected, because the rats
were maintained for only 1-3 wk after the single dose of AAS and
were subjected to a single bout of exhaustive weight lifting 1 wk after
AAS treatment.
In the present study, the rats were subjected to an exhaustive
submaximal weight-lifting regimen, i.e., 10RM corresponding to
~70-80% of the maximum capacity (1RM), and the Steroid group lifted 47% (P
0.05) more total weight than the Control
group. This large difference reflects a higher number of sets and a
higher load at 10RM in the Steroid than in the Control group (Table 2). These data suggest the possibility of an improvement in the capacity of
1RM itself. However, there was no difference in the 1RM between the two
groups. Thus AAS treatment improved repetitive lifting capacity at a
relatively high load, a finding consistent with athletes using AAS
being able to withstand an enhanced work volume (weight lifting)
(11).
Suggested mechanisms for the ergogenic effects of AAS include the
possibility that these agents 1) act through the central nervous system, allowing the subjects to train harder (2)
or 2) may improve skeletal muscle function directly by
increasing protein synthesis (8, 9, 17) or membrane
stabilization (22, 23). We have made preliminary
examinations of the effects of AAS on the level of various
neurotransmitters of the central nervous system of these same rats
(unpublished observations). The level of norepinephrine and its
metabolite 4-hydroxy-3-methoxyphenylglycol appears to be higher in the
hypothalamus of Steroid than of Control rats. Thus we are hypothesizing
that this hyperadrenergic state may have resulted in an increased
cardiac output, a reduced peripheral resistance, and an enhanced muscle
blood flow, thus contributing to the enhanced endurance capacity
observed in the AAS-treated rats.
This interpretation is consistent with the in situ isometric mechanical
properties of the Plt muscle, a primary plantarflexor muscle. The
maximum isometric twitch and tetanic forces and the conduction-transmission time of the nerve/muscle were unaffected by the
AAS treatment (Table 3). These data suggest that the AAS treatment had
little direct effect on the function of the neuromuscular unit,
including the motoneuron, peripheral nerve, and muscle. However, the fatigue resistance as tested by continuous 12-Hz trains of
impulses was enhanced significantly by the AAS treatment (Table 3).
This enhanced muscle fatigue resistance in the Steroid group is
consistent with the enhanced work capacity observed during the
weight-lifting task. These results also are consistent with the
reported improvement in the fatigue resistance of the rat EDL muscle to
a continuous 4-Hz stimulation train after subcutaneous injection of 1 mg of nandrolone phenylpropionate on alternate days for 5-6 wk
without a change in the mean fiber cross-sectional area
(7). Furthermore, greater submaximal running endurance has
been reported in AAS-treated rats (0.5 mg nandrolone phenylpropionate injection for 4 wk every other day), despite the observation that the
training intensity and skeletal muscle oxidative capacities were
similar in AAS- and saline-treated rats (28). These latter reports suggest that the increased muscle fatigue resistance induced by
AAS loading was not associated with a significant improvement in the
oxidative capacity of the muscle. Although the enzymatic profiles for
the muscle fibers were not determined in the present study, the
duration (1 wk) was most likely too short to have induced any changes
in enzyme levels associated with oxidative phosphorylation.
Uptake of [3H]thymidine and
[14C]leucine in the resting state.
The uptake of [3H]thymidine was lower and that of
[14C]leucine higher in the Steroid than in the Control
group for both the dorsiflexor and plantarflexor muscles in the resting
state (Fig. 2, A and B). These findings are
consistent with the reported increase in the rate of muscle protein
synthesis and muscle RNA levels with no change in DNA levels associated
with AAS treatment (8). A lower
[3H]thymidine uptake indicates reduced cell
proliferation, i.e., DNA level, in the resting muscles, suggesting that
AAS inhibits DNA replication in skeletal muscles. Increased leucine
incorporation, an indicator of increased protein synthesis rates, into
the muscles after the administration of AAS has been observed
previously in humans (9) and rats (17). It
also has been reported that AAS have a high affinity for glucocorticoid
receptors and thus may counteract the catabolic effect of high
circulating glucocorticoid concentrations resulting from training
(3, 13, 18, 29). The present study does not provide direct
evidence of the effects of AAS on muscle protein catabolism. However,
because the final muscle weights of the Steroid group were similar to
those of the Control group (Table 1), it seems reasonable to assume
that the rate of muscle protein degradation must have increased in
proportion to the increase in the rate of protein synthesis.
Serum CK activity and uptake of [3H]thymidine.
Serum CK activities were increased in both the Steroid and Control
groups 30-60 min after the exercise bout (Fig. 1). Using the same
weight-lifting model, we have shown that the degree of CK leakage is
associated with the severity of damage in the exercised muscles
(24). Weight lifting-induced muscle damage by use of this
model has been confirmed morphologically in histological sections
(24, 26). We also have reported that the intensity of
weight lifting (amount of work), the degree of muscle damage, the serum
CK activity, and the mitotic activity (uptake of
[3H]thymidine) in the exercised muscles are closely
related events in this model (24, 25).
In the present study, the serum CK activities were lower in the Steroid
than in the Control group 30-60 min after the exercise bout,
whereas the total amount of weight lifted was higher in the Steroid
group. Similarly, a diminished CK response in humans using AAS has been
reported after a single bout of heavy-resistance exercise
(4). Evidence suggests that AAS agents may have a membrane-stabilizing effect (22, 23) and that this may
blunt the rise in serum CK efflux after muscle damage. In a similar manner, vitamin E also has the potential to stabilize muscle fiber membrane (16), and the vitamin E treatment of rats
diminishes CK leakage from contraction-induced damage of the muscle
(27). However, vitamin E treatment does not ameliorate the
induction of muscle injury itself (27). In the present
study, it is highly likely that muscle damage in the Steroid group, as
reflected by the serum CK levels and [3H]thymidine uptake
rates, was minimized by AAS treatment.
After muscle damage, skeletal muscle fibers can regenerate, with the
satellite cells playing a primary role (1, 10). Damage of
parent fibers activates normally dormant satellite cells, which then
begin to proliferate. Peak proliferation is observed ~48 h after
muscle damage, at which time the satellite cells begin to fuse and form
multinucleated myotubes to repair the damaged portions of the parent
fibers (1, 10). Skeletal muscle fibers are long,
multinucleated cells, and there is a relatively constant amount of
cytoplasm supplied by each myonucleus, i.e., the myonuclear domain
concept (12, 14). This concept suggests that, after muscle
damage, satellite cells proliferate and provide additional myonuclei,
thus reestablishing a normal myonuclear domain size. In turn, the
mitotic activity (uptake of [3H]thymidine) at 2 days
after exercise has been associated with muscle damage. We observed a
significantly lower peak uptake of [3H]thymidine 2 days
after exercise in the Steroid group (Fig. 5A), suggesting
that AAS reduced the level of muscle disruption in the Steroid group.
This view is further supported by the following observation. A
significant correlation between CK activity (the degree of muscle
damage) 30-60 min after the exercise bout and the uptake of
[3H]thymidine (mitotic activity) 2 days after the
exercise bout was evident in both groups (Fig.
7). However, for any
[3H]thymidine uptake rate, the serum CK values for the
Steroid group were consistently lower than those of the Control group.
Together, these data suggest that AAS diminished CK leakage and
minimized muscle fiber damage after a single bout of exhaustive
resistance exercise.

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|
Fig. 7.
Relationship between serum CK activity and
[3H]thymidine uptake in the plantarflexor muscles of
Control and Steroid groups after a single exhaustive bout of weight
lifting. Abscissa: [3H]thymidine uptake 2 days after
exercise; ordinate: CK leakage 30-60 min after exercise.
Regression equations and correlations are y = 0.009x + 2.36; r = 0.95 for Control,
and y = 0.005x + 1.72;
r = 0.79 for Steroid, respectively.
|
|
Total and muscle protein synthesis.
The Control group showed a biphasic response in
[14C]leucine uptake, i.e., peaks at 3 and 10 days after
the exercise bout (Fig. 5B). We recently reported that this
first peak most likely reflects increased protein synthesis
(hypertrophy) in the primary tissues (contractile proteins, connective
tissue, revascularization, activation of satellite cells, etc.),
whereas the second peak may reflect the synthesis of the contractile
components of regenerated and/or de novo muscle fibers (hyperplasia)
(24). In the Steroid group, in contrast, there was a
tendency for an earlier increased uptake, i.e., at 6 h to 3 days
after the exercise bout, with a single peak at 1 day after the exercise
bout (Fig. 5B). Thus the increase in protein synthesis
associated with the weight-lifting exercise bout occurred more rapidly
and in a shorter time period in the AAS-treated than in the Control
rats. However, it is also clear that the cell cycle of proliferating
cells (including satellite cells) in the muscles of the Steroid group
was not affected by AAS treatment, because the peak uptake of
[3H]thymidine was observed 2 days after exercise in both
groups (Fig. 5A).
[14C]leucine uptake for myosin in the plantarflexor
muscles of the Steroid group (Fig. 6) was significantly lower at
3-12 h and 3 days after exercise than at rest, whereas the
absolute total uptake levels were similar except for the level at
3 h (Fig. 4B). Moreover, the total uptake level of
[14C]leucine was significantly higher in the Steroid than
in the Control group (Figs. 2B and 5B). Together,
these data indicate that AAS treatment enhanced protein synthesis in
the noncontractile (i.e., connective tissue and/or membrane proteins)
in addition to the contractile components of the muscle.
In conclusion, the present data indicate that AAS treatment before a
single bout of exhaustive weight-lifting exercise 1) enhances the total in vivo work capacity of the muscles, 2)
reduces the CK leakage and the uptake of [3H]thymidine of
the muscle fibers, consistent with there being less muscle fiber damage
induced by weight lifting, 3) enhances the fatigue
resistance of a primary plantarflexor muscle, and 4)
increases the protein synthesis of both the contractile and noncontractile components of the muscles. These results demonstrate an
improved adaptability of the muscle to overload and an elevated threshold at which an unusual exercise intensity can initiate a
"muscle damage syndrome."
 |
ACKNOWLEDGEMENTS |
This work was supported by a Grant-in-Aid for Scientific Research
(B-12480012) from the Ministry of Education, Science and Culture of Japan.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: T. Tamaki, Dept. of Physiology, Div. of Human Structure and Function, Tokai Univ. School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193 Japan (E-mail: tamaki{at}is.icc.u-tokai.ac.jp).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 31 July 2000; accepted in final form 14 February 2001.
 |
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