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The Journal of Clinical Endocrinology & Metabolism
Copyright © 2000 by The Endocrine Society
Vol. 85, No. 1
Printed in U.S.A.
Androstenedione Does Not Stimulate Muscle Protein
Anabolism in Young Healthy Men*
BLAKE B. RASMUSSEN, ELENA VOLPI, DENNIS C. GORE,
ROBERT R. WOLFE
AND
Departments of Surgery (B.B.R., D.C.G., R.R.W.) and Internal Medicine (E.V.), University of Texas
Medical Branch, and the Metabolism Department, Shriners Hospital (B.B.R., E.V., R.R.W.), Galveston,
Texas 77550
2
H5]phenylalanine, blood sampling from femoral artery and vein, and
muscle biopsies. Plasma testosterone, androstenedione, LH, and estradiol concentrations were determined by RIA.
After ingestion of oral androstenedione, plasma testosterone and
LH concentrations did not change from basal, whereas plasma androstenedione and estradiol concentrations were significantly increased (P , 0.05). Compared to a control group, androstenedione did
not affect muscle protein synthesis and breakdown, or phenylalanine
net balance across the leg.
We conclude that oral androstenedione does not increase plasma
testosterone concentrations and has no anabolic effect on muscle
protein metabolism in young eugonadal men. (J Clin Endocrinol
Metab 85: 55–59, 2000)
ABSTRACT
Androstenedione is the immediate precursor of testosterone. Androstenedione intake has been speculated to increase plasma testosterone levels and muscle anabolism. Thus, androstenedione supplements have become widely popular in the sport community to improve
performance. This study was designed to determine whether 5 days
of oral androstenedione (100 mg/day) supplementation increases skeletal muscle anabolism.
Six healthy young men were studied before the treatment period and
after 5 days of oral androstenedione supplementation. Muscle protein
turnover parameters were compared to those of a control group studied
twice as well and receiving no treatment. We measured muscle protein
kinetics using a three-compartment model involving infusion of L-[ring-
A
that even if androstenedione does not stimulate testosterone
release, it may directly stimulate muscle protein synthesis.
The purpose of this study was to assess 1) whether 5 days
of oral androstenedione supplementation increases plasma
testosterone concentration, and 2) whether oral androstenedione has an anabolic effect on skeletal muscle.
NDROSTENEDIONE is the immediate precursor to
testosterone during steroid biosynthesis (1). Recently,
it has been speculated that exogenous androstenedione is
converted to testosterone and thus may have an anabolic
effect on skeletal muscle. Numerous studies have shown that
testosterone increases muscle size (2– 6) by increasing muscle
protein synthesis (4, 7–9) in healthy and hypogonadic men,
and that it increases maximum voluntary muscle strength in
eugonadal men (3). Because of these speculations, but without any published reports on the effects of exogenous androstenedione on muscle protein synthesis, androstenedione
has, on the one hand, been banned from use by the Olympic
Committee and many professional sports organizations and,
on the other hand, become a widely popular, commercially
available, oral supplement. However, a 1966 study from Horton and Tait (10) evaluated the pharmacokinetics of androstenedione and showed that if androstenedione was given
through the gastrointestinal route, only about 2% was converted to testosterone. On the other hand, androstenedione
is a steroid possessing an androgenic activity of 10 –20%
relative to the activity of testosterone (11). Thus, it is possible
Materials and Methods
Study subjects
Six healthy male volunteers (age, 32 6 4 yr; height, 179 6 2 cm;
weight, 77 6 6 kg; body mass index, 23.9 6 1.6 kg/m2) were studied in
the postabsorptive state before and after androstenedione administration. The leg volume, estimated using an anthropometric approach (12),
was 12.3 6 1.6 L. Six healthy volunteers (three women and three men)
were also studied twice in the postabsorptive state and used as a control
group for determinations of muscle protein kinetics (age, 31 6 3 yr;
height, 168 6 3 cm; weight, 67 6 5 kg; body mass index, 23.7 6 1.2 kg/m2;
leg volume, 9.5 6 0.5 L). All subjects gave informed, written consent
before participating in the study, which was approved by the institutional review board of the University of Texas Medical Branch
(Galveston, TX). Each subject was screened for determination of health
status at the General Clinical Research Center at the University of Texas
Medical Branch. Subjects were recreationally active, although none was
involved in consistent resistance exercise training. None of the subjects
was taking any form of medication, creatine, amino acid supplements,
or anabolic steroids or was on an excessive protein diet.
Received June 4, 1999. Revision received July 28, 1999. Rerevision
received October 7, 1999. Accepted October 14, 1999.
Address all correspondence and requests for reprints to: Robert R.
Wolfe, Ph.D., Shriners Hospital, Metabolism Department, 815 Market
Street, Galveston, Texas 77550. E-mail: [email protected].
* This work was supported by NIH Grant DK-33952 and Shriners
Grant 8490. It was conducted at the General Clinical Research Center at
the University of Texas Medical Branch (Galveston, TX), funded by
Grant M01-RR-00073 from the National Center for Research Resources,
NIH, USPHS. This study was presented in part at the Experimental
Biology Meeting, April 1999, Washington, D.C.
Protocol
Each subject was studied twice. In the subjects given androstenedione, the first study was performed to acquire baseline data, and the
second study was performed after 5 days (5–7 days after the initial study)
of androstenedione administration (Fig. 1a). The six subjects included in
the control group were also studied twice. The control group was used
to assess the variability of muscle protein kinetics within the same
subject on two different occasions. The subjects did not engage in phys-
55
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RASMUSSEN ET AL.
because there is no known psychological component to the rate of
muscle protein turnover. The second study for the treatment group was
initiated on day 5 of oral androstenedione administration. It was identical to the first study, outlined above. After basal blood samples were
taken, the subjects were administered the morning dose of androstenedione, and the tracer infusion was started. Blood samples for model kinetics and hormone concentrations and muscle biopsies were taken as
described for the initial study. A time course for androstenedione and
testosterone concentrations after the administration of oral androstenedione was measured in one representative subject. The second
study for the control group was also identical to the first infusion
protocol, described above.
Analysis
FIG. 1. A, The study design for the treatment group included two
studies separated by 5 days of oral androstenedione administration.
The control group did not receive androstenedione. B, The infusion
protocol used before and after androstenedione treatment and for the
control group.
ical exercise the day before being studied. They were asked to maintain
their normal dietary patterns, and they kept a notebook detailing their
food intake and physical activity. The night before each study the subjects were admitted at the General Clinical Research Center of the University of Texas Medical Branch. After 2200 h, the subjects were allowed
only water ad libitum. After an overnight fast, a Teflon catheter was
placed in a forearm vein for isotope infusion, another catheter was
placed in a wrist vein of the opposite arm for arterialized blood sampling, and femoral arterial and venous catheters were placed. A blood
sample was drawn for background phenylalanine enrichment and androstenedione, testosterone, estradiol, and LH determinations (Fig. 1b).
A primed (2 mmol/kg), continuous infusion (0.05 mmol/kgzmin) of
l-[ring-2H5]phenylalanine (98% enriched; Cambridge Isotope Laboratories, Woburn, MA) was then started and maintained throughout the
study. After 2 h of infusion, an initial muscle biopsy was taken from the
vastus lateralis, approximately 20 cm above the knee, using a 5-mm
Bergström biopsy needle (Stille, Stockholm, Sweden). To measure leg
blood flow, an indocyanine green (ICG) infusion was started (0.5 mg/
min) in the femoral artery 4 h after the start of the isotope infusion and
continued for 30 min. Blood samples were collected at 10-min intervals
from the femoral vein and the heated wrist vein. Subsequently, the ICG
infusion was stopped, and four arterial and venous blood samples were
collected every 10 min for analysis of phenylalanine enrichment. Additional blood was collected at the beginning and end of the fifth hour
of the study to measure hormone concentrations. At the end of the study
a muscle biopsy needle was inserted into the leg in the opposite direction
from the previous biopsy, and a second biopsy was taken.
After the first study, each subject in the treatment group was given
androstenedione (Ultimate Nutrition, Farmington, CT; 50 mg/capsule;
minimum purity, 99.5%) for 5 days at a dosage of 100 mg/day, which
is double that suggested by the manufacturer. Androstenedione was
taken orally each morning with breakfast. We confirmed that the capsules given to the subjects contained 50 mg pure androstenedione by gas
chromatography/mass spectrometry (GC/MS) analysis. The control
group did not take oral androstenedione. A placebo was not given,
Blood phenylalanine enrichments and concentrations were determined by GC/MS (model 5973, Hewlett-Packard Co., Palo Alto, CA)
after purification of the amino acids (13) and derivatization to tertbutyldimethylsilyl derivative. Isotopic enrichments are expressed as the
tracer to tracee ratio (13).
Free muscle intracellular phenylalanine enrichments were measured
by GC/MS after extraction and purification as previously described (13).
The enrichments of protein-bound phenylalanine were measured after
hydrolysis of the extracted muscle proteins (13) using the external standard curve approach (14).
Leg blood flow was determined from blood samples collected during
the continuous infusion of ICG (15, 16). Sera from blood samples were
analyzed in a spectrophotometer with absorbance set at l 5 805 nm. The
coefficient of variation of each ICG measurement (intrasubject) was less
than 5%.
Plasma androstenedione, testosterone, LH, and estradiol concentrations were determined by RIA (Diagnostic Products, Los Angeles, CA).
According to the manufacturer, the cross-reactivity of androstenedione
on the estradiol assay is not detectable.
Calculations
The three-compartment model of leg muscle amino acid kinetics used in
this study has been described previously (17). Use of this model allowed
us to determine the rate of utilization of phenylalanine for muscle protein
synthesis and its intracellular appearance from muscle protein breakdown.
The model assumptions are addressed in Refs. 17 and 18. The parameters
of the three-compartment model of leg amino acid kinetics used to determine the phenylalanine kinetics are defined as follows (Fig. 2): phenylalanine entry into leg: Fin 5 CA 3 BF (1); phenylalanine exit from leg: Fout 5
CV 3 BF (2); net balance across the leg: NB 5 (CA 2 Cv) 3 BF (3); muscle
inward transport: FM,A 5 {[(EM 2 EV)/(EA 2 EM)] 3 CV 1 CA} 3 BF (4);
muscle outward transport: FV,M 5 {[(EM 2 EV)/(EA 2 EM)] 3 CV 1 CV} 3
BF (5); A-V shunting: FV,A 5 Fin 2 FM,A (6); protein breakdown: FM,O 5 FM,A
3 [(EA/EM) 2 1] (7); and protein synthesis: FO,M 5 FM,O 1 NB (8). Components of the kinetic parameters are defined as follows: CA and CV,
concentrations of phenylalanine in the artery and vein, respectively; BF, leg
blood flow; and EA, EV, and EM, enrichments (tracer/tracee) of phenylalanine in the femoral artery and vein, and intracellular muscle, respectively.
The muscle protein fractional synthetic rate (FSR) was calculated using the
precursor-product model previously described (19, 20).
Statistical analysis
Data are expressed as the mean 6 sem. Pre-post differences in hormone concentrations in the androstenedione group were assessed using
the two-tailed paired t test. Differences in muscle protein kinetics between control and androstenedione were analyzed using ANOVA for
repeated measures. Pairwise multiple comparisons were carried out
using the t test with Bonferroni’s inequalities. Differences were considered significant at P , 0.05.
Results
Ingestion of androstenedione in the treatment group
caused the plasma concentration of androstenedione to increase approximately 3-fold when measured 4 h after inges-
ANDROSTENEDIONE DOES NOT PROMOTE ANABOLISM
57
FIG. 3. Time course of androstenedione and testosterone concentrations after ingestion of 100 mg androstenedione in one representative
subject.
FIG. 2. Three-compartment model of leg amino acid kinetics. Free
amino acid pools in femoral artery (A), femoral vein (V), and muscle
(M) are connected by arrows, indicating unidirectional amino acid
flow between each compartment. Abbreviations are identified in the
text.
TABLE 1. Plasma androstenedione, testosterone, estradiol, and
LH concentrations in normal men before and after 5 days of
treatment with oral androstenedione
Hormone
Androstenedione (nmol/L)
Basal
Last hour of infusion study
Testosterone (nmol/L)
Basal
Last hour of infusion study
LH (IU/L)
Basal
Last hour of infusion study
Estradiol (nmol/L)
Basal
Last hour of infusion study
Pretreatment
Oral androstenedione
7.5 6 0.7
7.0 6 0.8
12.8 6 0.8
37.2 6 10.7a
21.0 6 2.6
24.5 6 4.7
19.9 6 1.1
24.5 6 3.3
1.95 6 0.30
1.51 6 0.38
1.85 6 0.22
1.73 6 0.22
0.10 6 0.01
0.10 6 0.01
0.12 6 0.02
0.17 6 0.02a
Data are expressed as the mean 6
a
P , 0.05 vs. pretreatment.
SEM.
androstenedione (P 5 0.070, pre vs. post within the androstenedione group; P 5 0.521, pre vs. post within the control
group). Muscle protein synthesis tended to be higher with
time in both groups (P 5 0.051). Although the synthesis was
slightly elevated in the androstenedione group, the change
was not different from that observed in the control group, nor
was the change as large in magnitude as the increase in
breakdown. Net muscle balance of phenylalanine across the
leg, a measure of net protein deposition, did not change over
time in either group. However, net balance tended to be more
negative in the androstenedione group (time by treatment
interaction; P 5 0.093), indicating an increase in net protein
catabolism rather than an improvement in anabolism as predicted (Table 3).
Muscle protein synthesis was measured by the precursorproduct method (FSR) in all of the subjects in the androstenedione group and in three subjects in the control group
(Fig. 4). Consistent with the data from the three-compartment model, no differences were found between groups, and
no time effect was seen in either the treatment group (0.074 6
0.015%/h vs. 0.077 6 0.023%/h) or the control group (0.056 6
0.017%/h vs. 0.057 6 0.008%/h).
Discussion
tion (P , 0.05). Nonetheless, plasma testosterone and LH
concentrations did not change (P 5 NS). On the other hand,
androstenedione ingestion significantly increased the estradiol concentration (P , 0.05; Table 1). The time courses for
androstenedione and testosterone concentrations after androstenedione ingestion for one representative subject are
shown in Fig. 3.
Blood flow, phenylalanine enrichments, and concentrations were not significantly different between the control and
treatment groups and were unaffected by time (Table 2).
The muscle amino acid kinetic data are reported in Table
3. Phenylalanine transport into and out of the leg did not
change with time in both groups, with no differences between the groups. There was a trend for an overall increase
in muscle protein breakdown with time (time effect, P 5
0.086), which was entirely attributable to the group receiving
To our knowledge this is the first report of the response of
muscle protein metabolism to androstenedione supplementation in human subjects. The data show that 5 days of oral
androstenedione supplementation do not stimulate muscle
protein anabolism or increase plasma testosterone concentrations. Rather, oral androstenedione is aromatized to estradiol and, probably reduced and conjugated for excretion
by the liver (10, 11). As the plasma androstenedione concentration increased more than 3-fold, our data also indicate
that androstenedione has no direct anabolic effect on skeletal
muscle.
Our failure to demonstrate an effect of androstenedione on
either testosterone concentrations or muscle protein synthesis is probably not due to the study design. For example, we
have shown that 5 days after an im injection of 200 mg
testosterone enanthate in young men whose activity level
and physical characteristics were similar to those of the vol-
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RASMUSSEN ET AL.
TABLE 2. Leg blood flow, phenylalanine concentrations, and enrichments in normal men before and after 5 days of treatment with oral
androstenedione
Control
Blood flow (mL/min z 100 mL leg vol)
Concentrations (nmol/mL)
Femoral artery
Femoral vein
Enrichments (%)
Femoral artery
Femoral vein
Muscle
Data are expressed as the mean 6
Androstenedione
P value (by ANOVA)
Pre
Post
Pre
Post
Effect of
time
Effect of
treatment
Interaction
3.9 6 0.4
3.1 6 0.3
3.1 6 0.4
3.5 6 0.1
0.390
0.660
0.066
55 6 3
62 6 4
55 6 3
61 6 3
48 6 3
52 6 3
49 6 3
55 6 3
0.716
0.661
0.097
0.072
0.641
0.328
7.05 6 0.53
5.51 6 0.33
4.12 6 0.43
7.45 6 0.28
5.52 6 0.24
4.09 6 0.56
7.38 6 0.28
5.81 6 0.21
4.43 6 0.21
7.20 6 0.21
5.19 6 0.16
4.46 6 0.32
0.747
0.202
0.991
0.912
0.962
0.514
0.380
0.188
0.911
SEM.
Control subjects did not receive androstenedione.
TABLE 3. Leg muscle phenylalanine kinetics in normal men before and after 5 days of treatment with oral androstenedione
Control
Phenylalanine inflow (Fin)
Phenylalanine outflow (Fout)
Inward transport (FM,A)
Outward transport (FV,M)
A-V shunting (FV,A)
Protein breakdown (FM,O)
Protein synthesis (FO,M)
Net balance (NB)
Androstenedione
P values (by ANOVA)
Pre
Post
Pre
Post
Effect of
time
Effect of
treatment
Interaction
212 6 24
238 6 26
112 6 29
138 6 29
101 6 23
69 6 10
44 6 9
225 6 7
165 6 9
183 6 10
97 6 14
115 6 16
68 6 12
78 6 11
59 6 12
218 6 2
151 6 22
164 6 23
75 6 12
88 6 13
76 6 16
48 6 7
35 6 5
213 6 2
170 6 9
189 6 9
130 6 19
150 6 20
39 6 22
74 6 6a
55 6 6
219 6 2
0.334
0.353
0.230
0.197
0.041
0.086
0.051
0.964
0.187
0.141
0.946
0.768
0.251
0.180
0.496
0.142
0.045
0.026
0.045
0.015
0.896
0.356
0.800
0.093
Data are expressed as nanomoles per min/100 mL leg vol (mean 6
a
P 5 0.070 for time effect within the androstenedione group.
FIG. 4. Muscle FSRs of control and androstenedione treatment
groups. Subjects were studied twice in the postabsorptive, resting
state. pre, Initial study; post, second study (5 days after ingestion of
100 mg/day androstenedione for the androstenedione treatment
group). No differences were detected between the control group and
the treatment group or between pre and post values.
unteers of the present study, plasma testosterone was significantly elevated, and muscle protein synthesis increased
2-fold (7). We have also detected an anabolic effect on muscle
protein synthesis when a synthetic anabolic hormone (oxandrolone) was given orally for 5 days, even though testosterone plasma concentrations significantly declined, and oxandrolone concentrations increased only to approximately 2
ng/dL. (21). To further substantiate the validity of our study
SEM).
design, we have included a control group, showing that
muscle protein synthesis is not affected by prior determination of the same value.
Although there was a trend for muscle protein synthesis
to increase, no significant differences were found between
androstenedione and control groups using two different
methods (three-pool model and precursor-product approach). On the contrary, there was a trend for muscle protein breakdown to be elevated, which was entirely attributable to androstenedione intake. The trend for an increase in
protein breakdown after androstenedione treatment may
have been the consequence of the increase in estradiol. In fact,
it has been reported that long term exposure to estrogens
decreases muscle fiber size in rats (22). Overall, the trend for
an increase in both protein breakdown and synthesis indicates that androstenedione tended to increase muscle protein
turnover. However, the increased turnover did not lead to
muscle protein anabolism, as the increase in protein breakdown exceeded the increase in protein synthesis. A similar
situation takes place in catabolic states such as sepsis or burn
injury, in which muscle protein breakdown is significantly
elevated (23, 24), and muscle protein synthesis increases as
well, although not enough to counteract the catabolic effect
of increase in breakdown. The stimulation of muscle protein
synthesis in this circumstance is probably due to the increased availability of intracellular amino acids secondary to
accelerated rate of breakdown. The trend for an increase in
muscle protein turnover may also indicate tissue remodeling
and, hence, improvement in muscle function, but a recent
study found no improvement in muscle function after an-
ANDROSTENEDIONE DOES NOT PROMOTE ANABOLISM
drostenedione supplementation (25). Regardless of the
mechanisms accountable for the response to androstenedione ingestion in the present study, the increase in protein
turnover observed did not lead to muscle protein anabolism.
The failure of androstenedione intake to raise testosterone
concentrations is somewhat surprising. A study showed that
approximately 5% of circulating androstenedione is converted
to testosterone (26). Considering that the daily production of
testosterone is about 5 mg/day (26) and that we administered
100 mg/day androstenedione, if 5% of the oral androstenedione
was converted to testosterone, we should have observed a doubling of testosterone concentrations. As this was not the case,
our results could have been due to a suppression of endogenous
testosterone secretion by androstenedione. However, the
plasma LH concentration was not suppressed by androstenedione administration, suggesting that androstenedione per se or
the derived estrogens did not inhibit the hypothalamicpituitary-Leydig cell axis. Our results are consistent with recent
data from another laboratory (25) showing that androstenedione does not increase testosterone concentrations during short
term administration (100 mg/day) or when given during resistance training (300 mg/day). In addition, the same study
reported no differences between the androstenedione supplement group and a placebo group after 8 weeks of resistance
training for the following measures: volitional muscle strength,
mean cross-sectional area of type 2 muscle fibers, and lean body
mass (25). Thus, it is likely that a significant proportion of
ingested androstenedione is reduced and conjugated by the
liver before reaching peripheral testosterone-converting tissues.
In fact, a kinetic study has shown that 5.9% of the androstenedione administered iv was converted to testosterone, whereas the
corresponding value was only 1.8% when androstenedione was
given via the gastrointestinal route (10). This difference was due
to the fact that 89% of the orally administered androstenedione
was converted to testosterone glucuronide, which was excreted
through the urinary tract (10).
In conclusion, 5 days of oral androstenedione administration at double the dosage suggested by the manufacturer
does not increase plasma testosterone concentration, nor
does it have an anabolic effect on skeletal muscle. Our inability to demonstrate the putative beneficial effects of androstenedione on skeletal muscle protein contradicts the
popular idea that androstenedione is an ergogenic aid for
athletes. To the contrary, the increase in estrogens, the possible interaction or competition with androgen receptors,
and the possible carcinogenic effect of prolonged androgen
intake make androstenedione consumption inadvisable in
healthy eugonadal men.
Acknowledgments
We thank the nursing staff at the University of Texas Medical Branch
General Clinical Research Center for assistance throughout these studies, and Zhi Ping Dong, M.S., for technical assistance.
59
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