Testosterone administration to older men improves muscle
function: molecular and physiological mechanisms
Arny A.
Ferrando1,
Melinda
Sheffield-Moore1,
Catherine W.
Yeckel1,
Charles
Gilkison2,
Jie
Jiang2,
Alison
Achacosa3,
Steven A.
Lieberman2,
Kevin
Tipton1,
Robert R.
Wolfe1, and
Randall J.
Urban2
Departments of 1 Surgery, 2 Internal Medicine, and
3 Physical Therapy, The University of Texas Medical Branch,
Galveston, Texas 77550
 |
ABSTRACT |
We investigated the effects
of 6 mo of near-physiological testosterone administration to older men
on skeletal muscle function and muscle protein metabolism. Twelve older
men (
60 yr) with serum total testosterone concentrations <17 nmol/l
(480 ng/dl) were randomly assigned in double-blind manner to receive
either placebo (n = 5) or testosterone enanthate (TE;
n = 7) injections. Weekly intramuscular injections were
given for the 1st mo to establish increased blood testosterone
concentrations at 1 mo and then changed to biweekly injections until
the 6-mo time point. TE doses were adjusted to maintain nadir serum
testosterone concentrations between 17 and 28 nmol/l. Lean body mass
(LBM), muscle volume, prostate size, and urinary flow were measured at
baseline and at 6 mo. Protein expression of androgen receptor (AR) and
insulin-like growth factor I, along with muscle strength and muscle
protein metabolism, were measured at baseline and at 1 and 6 mo of
treatment. Hematological parameters were followed monthly throughout
the study. Older men receiving testosterone increased total and leg LBM, muscle volume, and leg and arm muscle strength after 6 mo. LBM
accretion resulted from an increase in muscle protein net balance, due
to a decrease in muscle protein breakdown. TE treatment increased
expression of AR protein at 1 mo, but expression returned to pre-TE
treatment levels by 6 mo. IGF-I protein expression increased at 1 mo
and remained increased throughout TE administration. We conclude that
physiological and near-physiological increases of testosterone in older
men will increase muscle protein anabolism and muscle strength.
aging; muscle strength; lean body mass; insulin-like growth factor
I
 |
INTRODUCTION |
MOST AGING MEN
SHOW A REDUCTION in circulating serum testosterone concentrations
(16, 22). This reduction in serum testosterone concentration is a core physiological event in what is termed andropause. Andropause can be clinically characterized by decreased potency and libido, increased fatigability, and decreased muscle strength (13, 24). A significant decrease in serum total
testosterone occurs as early as ages 50-59 (16). This
decrease in testosterone production is associated with the loss of lean
body mass (LBM) and muscle strength. When men are made hypogonadal with
a gonadotropin-releasing hormone analog (14), LBM and
muscle strength are lost. Once weakened, older individuals are prone to
falls that prevent an independent living status and diminish the
quality of life. As the population of older Americans grows, the need
to develop therapies to counteract the aging-induced loss in skeletal
muscle mass and function becomes critically important.
Previously we demonstrated that testosterone administration primes
skeletal muscle for growth by increasing net protein synthesis in the
fasted state (10, 18). The logical extrapolation of a
continued increase in net protein synthesis is an increase in lean body
mass and strength. Bhasin et al. (2) demonstrated that
supraphysiological doses of testosterone can induce increases in muscle
size and strength in younger men without concomitant exercise. This
relationship holds true in relatively hypogonadal populations, where
the increase of circulating testosterone increases muscle protein
synthesis (23), LBM (3, 20), and muscle strength (3, 23). In an earlier study (23),
we demonstrated that 1 mo of testosterone administration increased
muscle anabolism and strength in six older men. We also demonstrated
that the increase in muscle anabolism was associated with an increase
in the expression of intramuscular mRNA for insulin-like growth factor
I (IGF-I) (23). Because IGF-I has also been demonstrated
to be a potent anabolic hormone (11), the relationship
between testosterone administration and IGF-I levels was investigated
in the present study.
Previous studies of testosterone administration in older men used a
standard clinical dosing paradigm (3, 15, 21). Although
this dosing is clinically feasible and convenient, it does not account
for individual response to hormone administration. We have previously
noted that a given dosage of testosterone administration results in
widely varied blood concentrations (23). Although group
means often reveal significant increases in testosterone, individual
variation may mask a consistency in outcomes. For example, Bhasin et
al. (3) and Tenover (21) each used a standard
clinical replacement dose in elderly men for up to 3 mo. However,
Bhasin et al. demonstrated an increase in muscle strength, whereas
Tenover did not. Individual response can be resolved in part by using supraphysiological doses (2); however, these doses may be
associated with the potential for increased side effects such as
altered lipid profiles (12) or hemodynamic profiles
(15). In the present study, we endeavored to adjust
individual testosterone concentrations to remain within the mid- to
high physiological range. We reasoned that remaining within or near
physiological testosterone concentrations would diminish potential side
effects while allowing the investigation of testosterone's anabolic
effects. We hypothesized that increases in testosterone within or near
the physiological range would also stimulate muscle anabolism and
increase muscle strength in older men much like previous studies where
supplementation resulted in supraphysiological concentrations (2,
15). To accomplish this, we carefully adjusted individual nadir
hormone concentrations to remain within the physiological range
throughout the 6-mo study. This dosing paradigm permits the
investigation of the efficacy of long-term testosterone administration
at or near physiological concentrations in older men.
 |
METHODS |
Subjects.
Twelve healthy, older male subjects were randomly assigned in
double-blind fashion to receive either testosterone enanthate (TE) or
placebo for 6 mo. Seven subjects [68 ± 3 (SE) yr; 91 ± 5 kg] were randomized to receive TE, whereas five subjects (67 ± 3 yr; 99 ± 7 kg) received a placebo consisting of sesame seed oil.
The study was approved by the Institutional Review Board at The
University of Texas Medical Branch (UTMB). Informed consent was
obtained after the study was explained to each individual. Subjects
were selected on the basis of the following inclusion criteria:
1) prostate-specific antigen (PSA)
4.0 µg/l
(6), 2) serum total testosterone
17 nmol/l
(480 ng/dl), 3) serum low-density lipoprotein (LDL)
200
ng/dl (7), 4) completion of a Bruce treadmill
exercise test without significant findings of cardiovascular disease,
and 5) no medical illnesses causing disability. The serum testosterone cutoff was chosen because it has been shown that 85% of
healthy older men (age 60-98 yr) have serum testosterone concentrations <17 nmol/l but still in the low-normal range of >10
nmol/l (1). Exclusion criteria included a history of
prostate cancer and severe coronary artery disease (due to the possible hypertrophic and atherogenic effects of testosterone), knee replacement (for reasons of strength determination), or use of a blood
anticoagulant, e.g., Coumadin (for fear of excessive bleeding during
biopsy and catheterization procedures). Because we wanted to determine
the outcomes of testosterone without the confounding effects of
exercise (2), we excluded subjects engaged in regular
training (defined as 30 min of aerobic or resistance training activity
2 days/wk). These exclusion/inclusion criteria were similar to those
of previously published studies by our group and others (21,
23).
Experimental protocol.
The studies were performed at the General Clinical Research Center
(GCRC) at UTMB. Subjects were studied at baseline, after 1 mo, and
after 6 mo of treatment. Each GCRC admission consisted of ~3 days. On
day 1, subjects were admitted in the afternoon and underwent
Cybex II isokinetic dynamometer testing for muscular endurance.
Subjects followed a standardized protocol that included 15 min of
pretest stretching. Muscular endurance was defined as the total work
performed for 20 repetitions at 240°/s. On the morning of
day 2, subjects were weighed in hospital gowns, resting (recumbent) blood pressure was taken, and blood was drawn from the
fasted subjects for hematological measures. Subjects were then taken
for magnetic resonance imaging (MRI) of the lower body. Leg muscle
volume was determined by analysis of images collected by MRI (GE Signa
1.5-Tesla whole body imager; General Electric, Milwaukee, WI) as
previously described (9). Image data files generated at
the MRI facility were analyzed for appendicular total and muscle
volumes using NIH Image software (NIH Image public domain analysis
package). Muscle volume (cm3) was computed as the addition
of individual slice areas multiplied by the slice thickness (10 mm).
After breakfast, subjects were taken to the UTMB Field House for
one-repetition maximum (1RM) determinations for bicep curl, tricep
extension, leg extension, and leg curl on specific equipment (Cybex)
designed for each movement. Subjects were initially familiarized on the
equipment after screening and selection. For 1RM testing, subjects
first warmed up on a stationary bike set at 30 W for 10 min. The
determination of 1RM was accomplished by increasing the load on each
machine until successful completion of the movement was no longer
possible. The heaviest load lifted was considered the 1RM. At
approximately noon, subjects received dual-energy X-ray absorptiometry
(DEXA) to determine LBM and fat mass. Body mass components were
determined with regional analysis software as previously described
(8). Finally, subjects were referred to the Department of
Urology at UTMB for prostate ultrasound and urine flow measurements.
Prostate volume was measured by transrectal ultrasound, and urinary
flow rate measures were made using a Life-Tech uroflowmeter (Life Tech, Houston, TX).
On day 3, subjects received a stable isotope infusion to
determine skeletal muscle protein metabolism. Muscle protein net balance and fractional synthesis rate (FSR) of skeletal muscle were
determined by infusion of the stable isotope
[2H3]ketoisocaproic acid, arteriovenous
sampling, and muscle biopsies as previously described
(10). Briefly, skeletal muscle FSR was calculated from the
determination of the rate of tracer incorporation into the protein and
the enrichment of the intracellular pool as the precursor
where Ep1 and Ep2 are the enrichments of
the protein-bound [2H3]leucine (from
transamination of [2H3]ketoisocaproic acid)
from the biopsies at 2 and 5 h of isotope infusion; EM
represents the average intracellular
[2H3]leucine enrichment over the time of
incorporation; and t is the time in minutes. The factors 60 and 100 are required to express FSR in percent per hour. Each biopsy
was divided to be used for both Western blot and isotopic enrichment analyses.
After the isotope infusion study on day 3, subjects were
given injections and discharged. Subjects returned every week for fasted blood draw and injections for the first 4 wk and then every 2 wk
for the remainder of the study. Serum total testosterone concentrations
were measured on each occasion and adjusted to between 17 and 28 nmol/l
(500 and 800 ng/dl; based on the concentration for the visit before
each injection) to approximate concentrations found in young men. The
aforementioned measurements were made at baseline and at 1 and 6 mo.
However, at 1 mo, the MRI, DEXA, and urology measures were omitted. We
designed the TE dosing paradigm for weekly injections for the 1st mo so
that we could adjust TE doses and establish increased testosterone
concentrations by the first measurements that were done at 1 mo. This
paradigm was reproduced from our initial study (23). A
biweekly injection paradigm would not have allowed TE dose adjustment
before the assessments at 1 mo.
Clinical measures.
Measurement of clinical parameters (see Table 2) such as testosterone
(DPC, Los Angeles, CA), estradiol (DPC), blood lipids (Vitros 250 Chemistry System, Johnson & Johnson, Arlington, TX), PSA, liver
function tests (Vitros 250), and hematocrit (Couter Onyx, Beckman
Coulter, Brea, CA) were done on a monthly basis by a UTMB clinical
laboratory. Subjects were also monitored monthly for breast tenderness
and the presence of gynecomastia by history and physical examination.
Serum testosterone concentrations were determined by the clinical
laboratory, so that adjustments in TE doses could be made on the basis
of the previous serum testosterone concentration.
Western blot analysis.
Protein was isolated from muscle biopsy samples by slicing frozen
muscle in very small pieces with a clean razor blade and thawing the
tissue in lysis buffer (150 mM NaCl, 10 mM Tris, 1% Triton X-100, 1%
Na deoxycholate, 0.1% SDS, 5 mM EDTA) containing protease inhibitors
(1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 10 µg/ml
aprotinin, 50 µg/ml leupeptin, 1 µg/ml pepstatin A) at a
concentration of 3 ml of ice cold lysis buffer per gram of tissue. The
tissue was homogenized with a Dounce homogenizer (4°C) and
centrifuged at 15,000 g for 20 min, and the supernatant was
removed and centrifuged again to result in total cell lysate. The
androgen receptor (AR) antibody (Santa Cruz Biotechnology, Santa Cruz,
CA) was incubated with 80 mg of cell lysate run on standard
SDS-PAGE gel with a working solution concentration range of
1:15-20. The IGF-I antibody (Santa Cruz Biotechnology) was incubated with 40 mg of cell lysate run on standard SDS-PAGE gel with a working solution concentration of 1:100. The actin
"housekeeping" antibody (Sigma) was used with a working solution
concentration range of 1:100-200. This anti-actin antibody is a
broad-based antibody that recognizes an epitope located on the
NH2-terminal region of actin and demonstrates a broad
reactivity among multiple actin isoforms in various species. The
housekeeping antibody was used to correct the results for protein
loading of the gel. Western analysis allows the direct measurement of
protein expression in the muscle biopsy samples.
Statistical analysis.
Comparison of 1- and 6-mo measures to baseline values was accomplished
by 2-way repeated-measures ANOVA with Dunnett's multiple comparison
test. Comparison of clinical outcome values over the 6-mo study period
was accomplished by ANOVA with Dunnett's multiple comparison test.
Where 1-mo measures were omitted, a paired t-test was used
to statistically compare 6-mo and baseline values. Statistical significance was P
0.05. Data are presented as means
±SE.
 |
RESULTS |
Clinical outcomes.
Figure 1 shows the mean testosterone
profiles of each group at 2-wk intervals over the 6-mo study period.
Table 1 shows the individual testosterone
concentrations for each of the seven subjects who received TE and the
dose adjustment made for each individual. None were clinically
hypogonadal at the beginning of this study. TE injections were adjusted
by an independent clinician to maintain levels within the normal range
(17-28 nmol/l). As can be seen in Table 1, the serum testosterone
concentrations and the doses of TE administered were variable from
individual to individual. Following such a paradigm, especially with
the use of intramuscular injections, the older men were exposed to
serum testosterone concentrations at various times during the 6-mo
study that were above the physiological range. Therefore, this study
assesses a mix between physiological and near-physiological
administration. However, serum testosterone concentrations were greater
in the treatment group at all time points after baseline
(P < 0.05). Serum testosterone did not change in the
placebo group. Table 2 delineates subject
characteristics and laboratory values over the 6-mo study period.
Treatment subjects remained normotensive, and liver function tests,
blood lipid profiles, and PSA were unchanged. Estradiol increased upon
treatment and, for the most part, remained elevated throughout the 6-mo
period without causing breast tenderness or gynecomastia by report
or examination. Hematocrit was elevated after 4 mo of TE and remained elevated until the end of the study.

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Fig. 1.
Serum testosterone profiles throughout treatment. Values
are means ± SE. Testosterone treatment group values were
significantly higher at all time points after baseline. Arrows indicate
study time points.
|
|
Prostate volume was not significantly increased with TE administration.
Prostate volume in the treatment group was 44 ± 15 ml at
baseline, whereas the placebo group was 41 ± 8 ml. Six-month values were 47 ± 13 and 35 ± 7 ml, respectively, for the
treatment and placebo groups. Urinary flow rate also did not change
over time or as a result of treatment. Baseline flow rate was 8.3 ± 1.5 and 8.9 ± 1.3 ml/s, whereas 6-mo values were 7.5 ± 1.4 and 8.7 ± 1.6 ml/s for the treatment and placebo groups, respectively.
Western blot analysis.
TE administration significantly increased skeletal muscle AR protein
expression at 1 mo (P < 0.05), but AR returned to
baseline levels at 6 mo. Figure 2 shows a
representative autoradiogram of a Western blot for skeletal muscle AR
from a subject receiving testosterone and a graph of the densitometry
data from the treatment group. There was no correlation between the
serum testosterone concentration at 1 mo and the change of AR
expression from baseline to 1 mo for individuals. IGF-I protein
expression in skeletal muscle increased at 1 mo and remained elevated
at 6 mo (P < 0.05; Fig.
3). AR and IGF-I protein expression did
not change in the placebo group (data not shown).

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Fig. 2.
Androgen receptor (AR) protein expression in skeletal
muscle during 6 mo of testosterone administration in older men.
Top: representative Western blot from one of the 7 subjects
assessed for protein expression of AR by use of standard Western
analysis. Actin was used as an internal control for protein loading.
Bottom: means ± SE from the 7 subjects that received
testosterone. Five subjects who received placebo demonstrated no change
throughout the study in AR expression (data not shown). Data are
expressed as arbitrary units calculated as the ratio of the band
densities of AR over the band densities of actin. *Statistical
significance was determined by ANOVA, P 0.05.
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Fig. 3.
Insulin-like growth factor I (IGF-I) protein expression
in skeletal muscle during 6 mo of testosterone administration in older
men. Top: representative Western blot from one of 7 subjects
assessed for expression of IGF-I by use of standard Western analysis.
Bottom: mean data from the 7 subjects receiving testosterone
administration. Five subjects who received placebo demonstrated no
change throughout the study in IGF-I expression (data not shown). Data
were derived as described in Fig. 2.
|
|
Physiological outcomes.
The net balance of muscle protein was less negative in the fasted state
in the treatment group throughout TE administration (Fig.
4; P < 0.05), but still
less than zero. In other words, treatment subjects were less catabolic
when fasting than those in the placebo group. The more favorable net
balance was due to a decrease in fasting protein breakdown, as
fractional synthetic rate of muscle protein remained constant
throughout (0.071 ± 0.02 to 0.084 ± 0.013 to 0.062 ± 0.016%/h at baseline and 1 and 6 mo, respectively).

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Fig. 4.
Fasting net phenylalanine balance across the leg.
Phenylalanine net balance describes the net balance between muscle
protein synthesis and breakdown. *Significantly less negative than the
placebo group and baseline testosterone, by ANOVA, P < 0.05.
|
|
The resultant improvement in net protein balance led to an increase in
LBM. Table 3 outlines the changes in LBM
and muscle strength over the 6-mo study period. The treatment group
demonstrated increases in total and leg LBM, whereas the percentage of
total body fat diminished. Leg muscle volume by MRI was also increased significantly after 6 mo of TE administration. All 1RM strength scores
increased in the treatment group after 6 mo of TE. Muscular endurance,
as tested by an isokinetic dynamometer, did not increase at 1 or 6 mo.
 |
DISCUSSION |
This study demonstrates that testosterone increases within or near
the physiological range can produce increases in muscle anabolism, LBM,
and muscle strength similar to supraphysiological administration. We
monitored serum testosterone concentrations and adjusted the dose of TE
to maintain testosterone concentrations in older men in ranges
comparable with those of younger men. During the 6 mo of TE
administration, some subjects experienced testosterone concentrations
that exceeded the physiological; however, testosterone concentrations
were consistently maintained above baseline values. The older men in
this study demonstrated an increase in LBM that was comparable to that
achieved with a standard replacement regimen that resulted in higher
testosterone concentrations (5). We also found that,
similar to younger men (2), testosterone will increase
muscle anabolism and strength in older men. The strength increases of
the older men in this study were greater than those demonstrated with
standard replacement paradigms (15, 21) or with
testosterone patch administration over 36 mo (20). Our data suggest that a standard paradigm of testosterone administration that does not include individual dose adjustment may not always achieve
desired outcomes if the subjects have not received adequate testosterone to stimulate metabolic changes in muscle. Because we
studied only a small number of subjects, we cannot draw any conclusions
regarding the risk-to-benefit ratio of testosterone administration in
older men. However, we found no significant side effects in our small
group other than an increase in hematocrit. Our data indicate that
testosterone can improve muscle strength in older men when careful
dosing ensures sustained blood testosterone increases. Our first study
demonstrated that short-term administration with standard replacement
dosages resulted in LBM and strength increases (23). The
present study indicates that these LBM and strength increases can be
maintained over 6 mo with careful dose adjustments that ensure
primarily physiological testosterone levels. This study also
demonstrates that the muscle's response to testosterone changes over
the 6-mo period of administration, indicating that alternative
paradigms of testosterone administration (i.e., cyclic administration)
can be of physiological benefit.
Testosterone administration resulted in some noteworthy effects on AR
and IGF-I expression in skeletal muscle. AR protein expression was
increased after 1 mo of TE but had returned to pretreatment levels by 6 mo. Physiologically, it is logical that androgen would enhance its own
receptor expression as it stimulates muscle metabolism. We previously
noted an upregulation of AR expression with oxandrolone administration
(18) in young males, which also occurred concomitantly
with an increase in muscle protein synthesis. The return of AR
expression to pretreatment values after 6 mo of continuous androgen
administration indicates a steady-state adaptation to the treatment
paradigm. There is also the possibility that the AR response is nothing
more than a response to the dosing paradigm. At 1 mo, older subjects
were receiving TE weekly rather than every 2 wk, and their mean serum
testosterone concentrations were more in the supraphysiological range
than they were at 6 mo. However, this relationship is weakened by the
fact that individual testosterone concentrations at 1 mo did not
correlate with the change in AR expression from baseline to 1 mo. This
pattern of AR expression raises the possibility that cycling of
testosterone administration could produce effects on skeletal muscle
analogous to continuous administration. Such a paradigm would
be beneficial by administering significantly less testosterone for
similar anabolic outcomes, thus minimizing the possibility of side effects.
IGF-I accompanies increases in muscle mass and strength
(17). In frail elderly, progressive resistance training
that increases muscle mass and strength also increases intramuscular
IGF-I concentrations (19). Clinically, we previously
demonstrated that older men given testosterone for 1 mo increased IGF-I
transcripts in muscle while decreasing the inhibitory IGF-binding
protein (23). The present study agrees with our previous
work in that IGF-I protein expression increased at 1 mo and further
demonstrates that this increase was maintained throughout the 6 mo of
testosterone administration. This confirms that the increase in IGF-I
mRNA noted in our earlier study (23) translates into an
actual increase of IGF-I protein. A corollary to these studies found
that young men who were made hypogonadal for 10 wk by Lupron showed a
decrease in muscle strength and a decrease in intramuscular IGF-I mRNA
concentration (14). Taken together, these data indicate a
mechanistic importance of IGF-I on muscle anabolism.
Although the intracellular mechanism stimulating muscle protein
anabolism requires further clarification, it is clear that testosterone
improves net protein balance of skeletal muscle. This effect is
pronounced in the fasted state as net protein balance becomes less
negative. We have previously demonstrated (10, 18) that
one of the primary effects of testosterone (during fasting) is the
efficient reutilization of intracellular amino acids (derived from
protein breakdown) for protein synthesis. However, the present study
demonstrates that, even if breakdown is decreased, ample amino acid
precursors are present to support the initial rate of protein
synthesis. Thus testosterone administration may ameliorate the loss of
skeletal muscle nitrogen during fasting in this older population by
preventing the loss of intracellular amino acids. Not only is the
appearance of amino acids from protein breakdown reduced, but those
that are derived from protein breakdown are efficiently utilized to
maintain protein synthesis, as we have previously demonstrated
(10, 18). This retention of nitrogen during fasting, when
combined with the anabolic stimulus of a meal alone (4,
25), may lead to muscle (LBM) accretion over time and explain
the anabolic effects of chronic testosterone administration.
In summary, the present study demonstrates that careful and
near-physiological testosterone administration in older men will increase LBM and muscle strength similarly to younger men. However, further consideration should be given to the specific androgen and
length and type of administration regimen to be used in older men and
to large-scale studies initiated to determine the risk-to-benefit ratio
of testosterone administration in older men.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institutes of Health Grants
AG/AR-11000 (R. J. Urban), M01-RR-00073 (General Clinical Research
Center, University of Texas Medical Branch), GM-57295 (A. A. Ferrando), and Shriners Hospitals for Children Grant 8940 (R. R. Wolfe).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: A. A. Ferrando, Depts. of Surgery and Metabolism, Shriners Hospitals for Children, 815 Market St., Galveston, TX 77550 (E-mail:
aferrand{at}utmb.edu).
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.
10.1152/ajpendo.00362.2001
Received 25 April 2001; accepted in final form 2 November 2001.
 |
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