Download Anabolic effects of testosterone are preserved during inhibition of 5

Am J Physiol Endocrinol Metab 293: E507–E514, 2007.
First published May 8, 2007; doi:10.1152/ajpendo.00130.2007.
Anabolic effects of testosterone are preserved during inhibition
of 5␣-reductase
Stephen E. Borst,1,2,3 Christine F. Conover,1 Christy S. Carter,1,3 Chris M. Gregory,4
Emanuele Marzetti,3 Christiaan Leeuwenburgh,3 Krista Vandenborne,4 and Thomas J. Wronski5
1
Geriatric Research, Education, and Clinical Center, Veterans Affairs Medical Center;
Departments of 2Applied Physiology and Kinesiology, 3Aging and Geriatric Research,
4
Physical Therapy, and 5Physiological Sciences, University of Florida, Gainesville, Florida
Submitted 26 February 2007; accepted in final form 30 April 2007
aged 60 or older are hypogonadal, as
defined by a serum total testosterone concentration of 300 ng/dl
(10.4 nM) or less (32). In older men, a low circulating testosterone concentration is associated with a decreased sense of
well-being, with loss of muscle mass and strength and with loss
of bone mineral density and increased risk of fractures (41). In
recent years there have been many trials of testosterone replacement as a strategy for combating sarcopenia in older men.
When administered to hypogonadal men at replacement doses
of 35–100 mg/wk, testosterone improves mood and cognition
and increases the hematocrit (32) but produces little increase in
bone mineral density (38). Although some studies report that
testosterone replacement produces a substantial increase in
lower body strength (20), most have found either no change in
strength or increases that are small compared with what is
obtainable through resistance exercise training (8, 15, 27, 36).
Recently, Bhasin et al. (5) have shown that treating older men
with very high doses of testosterone, up to 600 mg/wk, produces a remarkable increase in strength. However, in this same
study, 25% of those receiving the highest doses dropped out
due to serious adverse events such as polycythemia, edema,
and urinary retention. In addition to concerns over short-term
adverse effects, there are unanswered concerns that higher
doses of testosterone might accelerate underlying early-stage
prostate cancer (25). To date, studies of testosterone replacement in older men have not reported an increased incidence of
prostate cancer (25). However, the number of subjects studied
and the duration of treatment are not sufficient to rule out the
possibility. Such concerns have been rekindled by publication
of a 40-yr longitudinal study from the Baltimore Longitudinal
Study on Aging showing a positive correlation between
life-long blood testosterone levels and the risk of prostate
cancer (29). In addition, autopsy data from men over 60 who
died of all causes revealed that 42% carry early-stage
prostate cancer (28).
5␣-Reductase is the enzyme responsible for the conversion
of testosterone to dihydrotestosterone (DHT). A review of the
literature by Gormley (22) indicates that significant expression
of 5␣-reductase occurs in prostate but not in muscle or bone.
This finding led us to hypothesize that administering the
combination of a high dose of testosterone and a 5␣-reductase
inhibitor might produce anabolic effects on muscle and bone
without producing prostate enlargement. There are two isoforms of 5␣-reductase, type I and type II. In humans, benign
prostatic hypertrophy (BPH) requires 5␣-reductase type II, as
BPH is inhibited both by finasteride, an inhibitor of type I, and
by dutasteride, an inhibitor of type I and II (40). Because we
were not sure which isoform mediates prostate enlargement in
rats, we selected an inhibitor capable of blocking both isoforms. In the rat, both finasteride and MK-434 block both
isoforms of 5␣-reductase. MK-434 was chosen for the present
study because it has a relatively high potency in the rat (16, 17,
31, 34, 35).
We have published a pilot study in rats (9) demonstrating
that MK-434 blocks prostate enlargement caused by administration of a supraphysiological dose of testosterone for 28 days
Address for reprint requests and other correspondence: Stephen Borst, VA
Medical Center, GRECC-182, 1601 SW Archer Rd., Gainesville, FL 326081197 ([email protected]).
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.
dihydrotestosterone; prostate; body composition; bone resorption
APPROXIMATELY 40% OF MEN
http://www.ajpendo.org
E507
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on June 17, 2017
Borst SE, Conover CF, Carter CS, Gregory CM, Marzetti E,
Leeuwenburgh C, Vandenborne K, Wronski TJ. Anabolic effects
of testosterone are preserved during inhibition of 5␣-reductase. Am J
Physiol Endocrinol Metab 293: E507–E514, 2007. First published
May 8, 2007; doi:10.1152/ajpendo.00130.2007.—At replacement doses,
testosterone produces only modest increases in muscle strength and
bone mineral density in older hypogonadal men. Although higher
doses of testosterone are more anabolic, there is concern over increased adverse effects, notably prostate enlargement. We tested a
novel strategy for obtaining robust anabolic effects without prostate
enlargement. Orchiectomized (ORX) male rats were treated for 56
days with 1.0 mg testosterone/day, with and without 0.75 mg/day of
the 5␣-reductase inhibitor MK-434. Testosterone administration elevated the prostate dihydrotestosterone concentration and caused prostate enlargement. Both effects were inhibited by MK-434. ORX
produced a catabolic state manifested in reduced food intake, blunted
weight gain, reduced hemoglobin concentration, decreased kidney
mass, and increased bone resorption, and in the proximal tibia there
was both decreased cancellous bone volume and a decreased number
of trabeculae. In soleus and extensor digitorum longus muscles, ORX
reduced both the percentage of type I muscle fibers and the crosssectional area of type 1 and 2 fibers. Testosterone administration
caused a number of anabolic effects, including increases in food
intake, hemoglobin concentration, and grip strength, and reversed the
catabolic effects of ORX on bone. Testosterone administration also
partially reversed ORX-induced changes in muscle fibers. In contrast
to the prostate effects of testosterone, the effects on muscle, bone, and
hemoglobin concentration were not blocked by MK-434. Our study
demonstrates that the effects of testosterone on muscle and bone can
be separated from the prostate effects and provides a testable strategy
for combating sarcopenia and osteopenia in older hypogonadal men.
E508
5␣-REDUCTASE AND TESTOSTERONE ANABOLIC EFFECT
without blocking testosterone-induced muscle hypertrophy or
suppression of a urine maker of bone resorption. The present
study was undertaken to determine whether MK-434 could
prevent testosterone-induced prostate enlargement over a
longer period and to determine the effects of testosterone and
MK-434 on bone morphology, muscle fiber type and size,
strength, and muscle apoptosis.
MATERIALS AND METHODS
AJP-Endocrinol Metab • VOL
293 • AUGUST 2007 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on June 17, 2017
Animals and experimental design. Barrier-raised and viral pathogen-free Fischer 344 male rats aged 3 mo were obtained from Charles
River Laboratories (Wilmington, MA). Experimental procedures conformed to the Institute of Laboratory Animal Resources Guide to the
Care and Use of Experimental Animals and were approved by the
Institutional Animal Care and Use Committee at the Gainesville
Veterans Affairs Medical Center. Rats were fed a diet of Purina
Rodent chow, no. 5001 (Purina Mills, St. Louis, MO), containing 3.3
kcal/g and distributed at 58.9% carbohydrate, 12.4% fat, and 28.7%
protein. Closed orchiectomy (ORX) was performed under isoflurane
anesthesia on 32 rats and involved removal of testes, epididymis, and
epididymal fat. Sham surgery was performed on eight rats. In the
same procedure, 30-day drug pellets (Innovative Research of America, Sarasota, FL) were implanted subcutaneously to deliver 1.0 mg
testosterone/day vs. placebo and 0.75 mg MK-434/day vs. placebo.
This dose of testosterone is supraphysiological in rats, causing marked
prostate enlargement and elevation of the prostate DHT concentration
in our previous study (9). Similarly, we have shown that this dose of
MK-434 is sufficient to markedly inhibit testosterone-induced increases in prostate mass and DHT concentration (9). Rats were treated
for 56 days under the following experimental conditions: Sham, ORX,
ORX ⫹ T (testosterone), ORX ⫹ MK, and ORX ⫹ T ⫹ MK. For 2
days after surgery, rats received a nutritional supplement of Jello-O
with added fat (STAT VME; J. A. Webster, Alachua, FL), added
protein (Challenge Isolated Soy Protein 95 powder; General Nutrition
Centers), and Pediasure (Abbott Laboratories). Instructions for preparation are available online at http://www.researchtraining.org/
moduletext.asp?intModuleID⫽602. After 28 days, a second surgery
was performed to replace drug pellets. MK-434 was a gift from Merck
(Rahway, NJ).
Forelimb grip strength was measured, as we have previously
reported (13, 39), using an automated grip strength meter (Columbus
Instruments, Columbus, OH). The experimenter grasped the rat by the
tail and suspended it above a grip ring. After ⬃3 s, the animal was
gently lowered toward the grip ring and allowed to grasp the ring with
its forepaws. The experimenter then quickly lowered the body to a
horizontal position and tugged the tail until its grasp of the ring was
broken. The mean force in grams was determined with a computerized
electronic pull strain gauge fitted directly to the grasping ring and was
divided by body mass. Average measurements from three successful
trials were taken as the final outcome. Successful trials were defined
as those in which the animal grasped the ring with both forepaws and
pulled the ring without jerking. The experimenter was blinded as to
which treatment group each rat belonged to.
Urine collection and analysis of deoxypyridinoline and creatinine
clearance. Deoxypyridinoline (Dpd) is a degradation product of type
I collagen and a specific marker for bone resorption. Rats were housed
in metabolic cages for 24-h urine collection. Dpd was measured using
a Pyrilinks-D EIA kit with a sensitivity of 1.1 nmol/l and an interassay
coefficient of variation (CV) of 4% (Quidel, Santa Clara, CA).
Because the concentration of urine solutes is altered by water excretion, Dpd was normalized to urine creatinine and reported as nanomolars Dpd per minimolars creatinine. Creatinine was measured using
a colorimetric assay kit with an interassay CV of 2% (Sigma Chemical). After collection of 24-h urine, blood was collected by tail tip
amputation for analysis of serum creatinine. Creatinine clearance was
calculated from the serum and urine creatinine values and expressed
as milliliters per minute.
Death and hemoglobin determination. Rats were euthanized under
pentobarbital anesthesia. Blood was sampled by tail tip amputation
and the hemoglobin concentration measured using a Hemoport H2
hemoglobin analyzer (Stanbio Laboratory, Boerne, TX). Soleus
(SOL) and extensor digitorum longus (EDL) muscles were mounted
in embedding medium and frozen in isopentane that was cooled by
liquid N2. Muscles and other tissues were stored frozen at ⫺80°C.
Bone morphology. Tibiae were stripped of musculature and placed
in 10% phosphate-buffered formalin (pH 7.4) for 24 h before transfer
to 70% ethanol and dehydration in increasing concentrations of
ethanol. Bones were then embedded undecalcified in modified methyl
methacrylate (3) and sectioned longitudinally at 4-␮m thickness with
Leica/Jung 2050 or 2165 microtomes. The bone sections were stained
by the Von Kossa method with a tetrachrome counterstain. Cancellous
bone structural variables were measured in these sections with the
Osteometrics (Atlanta, GA) system. The sample area within the
proximal tibial metaphysis began 1 mm distal to the growth plate
to exclude the primary spongiosa. The following bone variables
were measured or calculated (21): cancellous bone volume (%),
trabecular width (␮m), trabecular number (no./mm), and trabecular
separation (␮m).
Muscle fiber type. The right SOL and EDL muscles were stored at
⫺80°C prior to analysis. Serial sections (10 ␮m) were cut from the
central portion of each muscle, and immunohistochemical reactions
were performed against anti-myosin heavy chain antibodies (BA-D5,
SC-71, BF-F3, and BF-35) and incubated at 4°C overnight. Sections
were subsequently incubated in rhodamine-conjugated anti-rabbit IgG
and FITC-conjugated anti-mouse IgG (Nordic Immunological Laboratories) and were classified as either type I, IIa, IIx, or IIb. Additional
sections were stained with hematoxylin and eosin and matched to
those used to identify fiber type. Stained cross sections were photographed at ⫻10 with a Leica fluorescence microscope and a digital
camera. The relative proportion of fibers of each type as well as
measures of individual fiber cross-sectional area (CSA) were determined in each sample. Fiber CSA values were determined on a
Macintosh computer using the public domain National Institutes of
Health (NIH) Image (version 1.62) program (developed at NIH and
available on the Internet at http://rsb.info.nih.gov/nih-image/). Fiber
type and fiber CSA analyses were performed on an average of 488 and
266 fibers in the SOL and EDL, respectively.
Prostate DHT was extracted by the method of Theobald et al. (42).
Briefly, homogenates were extracted twice with 10 volumes of anhydrous ethyl ether. Organic phases were dried under N2 and reconstituted in assay buffer for the commercial DHT radioimmunoassay kit
obtained from Diagnostic Systems Laboratories (Webster, TX). The
assay has a sensitivity of 4 pg/ml and an interassay CV of 4.5%.
Skeletal muscle apoptosis was assessed by quantifying DNA fragmentation in biceps muscle, using a cell death ELISA kit (Roche
Molecular Biochemicals). This assay allows for the quantification of
cytosolic mono- and oligonucleosomes (180 base pair nucleotides or
multiples). Briefly, samples were minced on ice in 5 volumes of
isolation buffer (0.21 M mannitol, 0.07 M sucrose, 0.005 M HEPES,
0.001 M EDTA, and 0.2% fatty acid-free bovine serum albumin, pH
7.4), homogenized on ice in a Potter-Elvehjem glass-to-glass homogenizer, and centrifuged for 10 min at 1,000 g at 4°C. The resulting
supernatant was collected and centrifuged again for 20 min at 14,000
g at 4°C. The supernatant (cytosolic fraction) was used for the assay.
Results are reported as arbitrary optical density units/milligram protein. Protein concentrations were determined by Bradford assay.
Statistics. Statistical analyses were performed using Prism software
(GraphPad Software, San Diego, CA). Unless otherwise noted, data
were analyzed by one-way ANOVA with treatment as a factor (Sham,
ORX, ORX ⫹ T, ORX ⫹ MK, ORX ⫹ T ⫹ MK). Post hoc analyses
of differences between individual experimental groups were made
using the Newman-Keuls multiple comparison test. Body mass and
E509
5␣-REDUCTASE AND TESTOSTERONE ANABOLIC EFFECT
Dpd data were analyzed by two-factor repeated-measures ANOVA,
with time and treatment as independent variables. Apoptosis data were
analyzed by Kruskal-Wallis testing because of nonhomogeneous variance. Significance was set at P ⬍ 0.05. Values are reported as
means ⫾ SE.
RESULTS
Body mass and food intake. Over the course of 56 days, food
intake was reduced by 13.5% in ORX rats compared with
Sham (P ⬍ 0.001; see Fig. 1). The reduction in food intake was
⬃60% inhibited in ORX ⫹ T rats (P ⬍ 0.001) and fully
inhibited in ORX ⫹ T ⫹ MK rats (P ⬍ 0.05; see Table 1).
Despite this, ORX ⫹ T and ORX ⫹ T ⫹ MK rats did not gain
substantially more weight than ORX rats, especially toward the
end of the treatment period. In orchiectomized rats, the 56-day
increase in body mass was inhibited by 32% compared with
Sham (P ⬍ 0.002 at all time points). In ORX ⫹ T and ORX ⫹
T ⫹ MK rats, the ORX-induced decrease in weight gain was
inhibited briefly following initial surgery for ORX and pellet
implantation (P ⬍ 0.0001 at 3 and 7 days, P ⫽ 0.012 at 14
days) and again following surgery at 28 days to replace drug
pellets (P ⫽ 0.0057).
Body composition and tissue weights. In orchiectomized rats
we observed a small, 6% decrease in the combined wet weights
of five skeletal muscles compared with Sham (plantaris, SOL,
Fig. 2. Effects of ORX, T, and MK-434 on blood hemoglobin concentration. *Significantly increased compared with ORX. Values are means ⫾
SE; n ⫽ 40.
tibialis anterior, EDL, and biceps humerus, P ⬍ 0.05; see Table
1). Muscle mass in ORX ⫹ T and ORX ⫹ T ⫹ MK rats was
not different than ORX. None of the treatment groups showed
a change in adiposity. The latter was assessed as the wet weight
of three adipose depots removed at death: inguinal subcutaneous fat, retroperitoneal depot of visceral fat, and mesenteric
visceral fat. In ORX rats, kidney mass was decreased 22%
compared with Sham (P ⬍ 0.001; see Table 1). The decrease
was prevented in the ORX ⫹ T and ORX ⫹ T ⫹ MK groups
(P ⬍ 0.001). Changes in kidney mass were not associated with
corresponding changes in creatinine clearance, which was
0.801 ⫾ 0.112 ml/min for Sham, 0.592 ⫾ 0.030 for ORX,
0.622 ⫾ 0.026 for ORX ⫹ T, 0.621 ⫾ 0.036 for ORX ⫹ MK,
and 0.937 ⫾ 0.065 for ORX ⫹ MK ⫹ T.
Hemoglobin. Blood hemoglobin concentration was not significantly different in ORX vs. Sham rats (see Fig. 2). However, hemoglobin was elevated 8.2% in ORX ⫹ T rats compared with ORX (P ⬍ 0.05), and the effect of testosterone was
not blocked by MK-434 (P ⬍ 0.05).
Prostate. Prostate mass was decreased 88% in ORX rats
compared with Sham (P ⬍ 0.001; see Fig. 3) and was increased
12-fold in ORX ⫹ T compared with ORX. (P ⬍ 0.001).
MK-434 alone had no effect on prostate mass, but prostate
mass was reduced 66% in ORX ⫹ T ⫹ MK compared with
ORX ⫹ T (P ⬍ 0.001). ORX caused an 81% reduction in the
prostate DHT concentration (1,333 ⫾ 822 pg/tissue in Sham
Table 1. Effects of ORX, T, and MK-434 on food intake and body composition
Sham
ORX
ORX ⫹ T
ORX ⫹ MK
ORX ⫹ MK ⫹ T
Food intake, g/day
Muscle, g
IWAT, g
RPWAT, g
MWAT, g
Kidney, g
17.50⫾0.20
15.13⫾0.20*
16.61⫾0.34#
14.98⫾0.17
17.69⫾0.27#
1.291⫾0.022
1.214⫾0.017*
1.131⫾0.025
1.210⫾0.025
1.186⫾0.022
3.14⫾0.11
3.80⫾0.18
3.25⫾0.12
3.69⫾0.24
3.68⫾0.20
1.80⫾0.10
2.11⫾0.16
1.74⫾0.10
2.09⫾0.10
1.91⫾0.10
4.32⫾0.20
4.83⫾0.26
4.90⫾0.22
4.63⫾0.25
5.78⫾0.32
1.013⫾0.024
0.785⫾0.018*
1.088⫾0.028#
0.807⫾0.013*
1.075⫾0.017#
Values are means ⫾ SE; n ⫽ 40. ORX, orchiectomy; T, testosterone; IWAT, inguinal subcutaneous depot of white adipose tissue; RPWAT, retroperitoneal
depot of visceral white adipose tissue; MWAT, mesenteric depot of white adipose tissue. At death, we measured the combined wet weights of 5 skeletal muscles
(muscle), the IWAT, the RPWAT, and the MWAT. *P ⬍ 0.05 vs. Sham; #P ⬍ 0.05 vs. ORX.
AJP-Endocrinol Metab • VOL
293 • AUGUST 2007 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on June 17, 2017
Fig. 1. Effects of orchiectomy (ORX), testosterone (T), and MK-434 on body
mass and food intake. ORX reduced food intake and weight gain over the
course of 56 days. T prevented the reduction in food intake and prevented the
reduction in weight gain only during the first 14 days after surgery. *Significant effect of ORX vs. Sham; #significant effect of T vs. ORX. MK-434 had
no effect on food intake or weight gain. Some error bars are contained within
the width of the symbol. Values are means ⫾ SE; n ⫽ 40.
E510
5␣-REDUCTASE AND TESTOSTERONE ANABOLIC EFFECT
vs. 257 ⫾ 21.7 in ORX, P ⬍ 0.001). The prostate DHT
concentration was 4.2-fold higher in ORX ⫹ T (1,077.1 ⫾ 273
pg/tissue, P ⫽ 0.001) compared with ORX, and that increase
was reduced by 44% in ORX ⫹ T ⫹ MK rats (611 ⫾ 117
pg/tissue, P ⬍ 0.05). The prostate DHT concentration was low
in ORX rats and was only 19% lower in ORX ⫹ MK rats
(209.4 ⫾ 13.4, P ⬍ 0.05).
Physical performance. Grip strength was not significantly
changed in ORX rats compared with Sham (P ⫽ 0.084; see
Fig. 4). Compared with ORX, grip strength was significantly
Fig. 5. Effects of ORX, T, and MK-434 on bone resorption. Urine deoxypyridinoline (Dpd) was measured as marker of bone resorption and was normalized to urine creatinine. *Dpd is increased with ORX; #Dpd is suppressed by
T; ‡Dpd is decreased over time. Values are means ⫾ SE; n ⫽ 40.
elevated in ORX ⫹ T, ORX ⫹ MK, and ORX ⫹ T ⫹ MK rats
(P ⬍ 0.05).
Bone. Sham animals, aged 3 mo at the beginning of the
experiment, displayed a 50% decrease in bone resorption over
56 days (P ⬍ 0.0001; see Fig. 5). Compared with sham, ORX
rats displayed a 25% increase in Dpd/creatinine at 28 days
(P ⬍ 0.05) and an 80% increase at 56 days (P ⬍ 0.05).
Compared with ORX, Dpd/creatinine was reduced in ORX ⫹
T and ORX ⫹ T ⫹ MK rats (P ⬍ 0.05) and was not different
from Sham. In proximal tibiae, ORX rats had a 28.4% decrease
in cancellous bone volume compared with Sham (P ⬍ 0.05;
see table 2). Compared with ORX, cancellous bone volume
was substantially increased in ORX ⫹ T and ORX ⫹ T ⫹ MK
rats (P ⬍ 0.05). Compared with Sham, ORX rats displayed a
33% decrease in the number of bone trabeculae (P ⬍ 0.001).
This decrease was prevented in ORX ⫹ T and ORX ⫹ T ⫹
MK rats (P ⬍ 0.05). Finally, ORX rats displayed a 61%
increase in trabecular separation compared with Sham (P ⬍
0.005). This increase was also prevented in ORX ⫹ T and
ORX ⫹ T ⫹ MK rats (P ⬍ 0.05). The effect of ORX on bone
resorption was robust enough to cause a 6% decrease in the wet
weight of tibiae (0.620 ⫾ 0.007 g for Sham vs. 0.586 ⫾ 0.006
g for ORX, P ⬍ 0.05). This decrease was significantly preTable 2. Structural properties of tibial cancellous bone
Sham
ORX
ORX ⫹ T
ORX ⫹ MK
ORX ⫹ MK ⫹ T
Fig. 4. Effects of ORX, T, and MK-434 on grip strength. *Significantly
increased compared with ORX. Values are means ⫾ SE; n ⫽ 40.
AJP-Endocrinol Metab • VOL
Bone
Volume, %
Trabecular
Width, ␮m
Trabecular
No., no./mm
Trabecular
Separation, ␮m
14.8⫾1.61
10.6⫾0.834*
14.7⫾1.36#
10.3⫾1.36
20.1⫾1.85#
43.6⫾2.42
43.3⫾2.09
45.2⫾2.05
48.1⫾2.63
50.9⫾2.62
4.00⫾0.284
2.67⫾0.24*
3.90⫾0.361#
2.50⫾0.252
4.70⫾0.315#
219.6⫾17.6
354.2⫾32.7*
236.5⫾30.4#
391.8⫾49.8
177.0⫾16.2#
Values are means ⫾ SE; n ⫽ 40. Effects of ORX, T, and MK-434 on
morphology of cancellous bone in proximal tibia. *Significant effect of ORX;
#significant effect of T.
293 • AUGUST 2007 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on June 17, 2017
Fig. 3. Effects of ORX, T, and MK-434 on prostate mass. *Significantly
decreased compared with Sham; #significantly increased compared with ORX;
‡significantly decreased compared with ORX ⫹ T. Values are means ⫾
SE; n ⫽ 40.
5␣-REDUCTASE AND TESTOSTERONE ANABOLIC EFFECT
E511
vented in ORX ⫹ T rats (P ⬍ 0.05). Tibia weights were
0.593 ⫾ 0.011 g for ORX ⫹ T, 0.575 ⫾ 0.016 g for ORX ⫹
MK, and 0.575 ⫾ 0.005 g for ORX ⫹ MK ⫹ T.
Muscle fiber type. In SOL muscle, the mean CSA of all
myofibers was reduced by 16.1% in ORX rats compared with
Sham (see Fig. 6, P ⬍ 0.001), with reductions of 12.1 and
23.6% in the CSA of type I and type IIa fibers, respectively.
The change in mean CSA was prevented in ORX ⫹ T and in
ORX ⫹ T ⫹ MK rats (P ⬍ 0.01). Also in SOL muscle, the
fraction of type I fibers was reduced by 8% in ORX rats
compared with Sham (P ⬍ 0.001; see Fig. 7). This change was
prevented in ORX ⫹ T and in ORX ⫹ T ⫹ MK rats (P ⬍
0.001).
In EDL muscle, the CSA of all muscle fibers was reduced by
17.7% in ORX rats compared with Sham (P ⬍ 0.001; see Fig.
6), with reductions of 26.6 and 20.6% in the CSA of type I and
IIb fibers, respectively. These changes were partially prevented
in ORX ⫹ T and in ORX ⫹ T ⫹ MK rats (P ⬍ 0.05). Also in
EDL muscle, the percentage of type I fibers was reduced by
57% in ORX rats compared with Sham (P ⬍ 0.01; see Fig. 7).
Fig. 8. Effects of ORX, T, and MK-434 on skeletal muscle apoptosis. DNA
fragmentation in biceps humerus muscle was assessed as described in
MATERIALS AND METHODS. Values are means ⫾ SE; n ⫽ 39. OD, optical
density.
Again, these changes were partially prevented in ORX ⫹ T and
in ORX ⫹ T ⫹ MK rats (P ⬍ 0.05).
Muscle apoptosis. Assessment of apoptosis in biceps humerus muscle yielded variable results (see Fig. 8), and because
the variance was not homogeneous, Kruskal-Wallis statistical
analysis was performed. Apoptosis may have been higher in
ORX rats compared with control, but the change was not
statistically significant. Compared with ORX, apoptosis may
have been lower in ORX ⫹ T and ORX ⫹ MK rats, but again
the change was not statistically significant.
DISCUSSION
The present study confirms our previous report (9) and the
report of Brown et al. (11) demonstrating an ORX-induced
catabolic state in rats. Our main new findings are 1) that
administration of a high dose of testosterone prevented those
Fig. 7. Effects of ORX, T, and MK-434 on the fraction
of type I muscle fiber. Muscle fiber analysis was performed as described in MATERIALS AND METHODS in
soleus and EDL muscles. *Significant effect of ORX;
#significant effect of T. Values are means ⫾ SE; n ⫽ 40.
AJP-Endocrinol Metab • VOL
293 • AUGUST 2007 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on June 17, 2017
Fig. 6. Effects of ORX, T, and MK-434 on type I and type II muscle fiber
cross-sectional area (CSA). Muscle fiber analysis was performed as described
in MATERIALS AND METHODS in soleus and extensor digitorum longus (EDL)
muscles. *Significant effect of ORX; #significant effect of T. Values are
means ⫾ SE; n ⫽ 40.
E512
5␣-REDUCTASE AND TESTOSTERONE ANABOLIC EFFECT
AJP-Endocrinol Metab • VOL
spinal cord injury, suggesting that testosterone may increase
muscle endurance as well as strength. In further support of this
concept is the report of Van Zyl et al. (44), who found that
treatment with testosterone increases exercise treadmill performance in rats.
Phillips and Leeuwenburgh (30) and Dirks and Leeuwenburgh (18) have shown that apoptosis is markedly increased in
type II muscle fibers with aging male rats. We hypothesized,
since aging is associated with a decrease in sex steroid hormones, that ORX might result in an increase in DNA fragmentation and that testosterone might reverse that increase. In the
present study, the level of DNA fragmentation observed was
far below what we have previously observed in older animals.
Our method may not be sensitive enough to detect a small
increase in apoptosis occurring in young animals following
ORX. Although Sinha-Hikim et al. (37) have shown that
significant activation of muscle satellite cells occurs mainly at
higher doses of testosterone, inhibition of apoptosis may be
another mechanism by which long-term testosterone replacement might prevent progression of sarcopenia.
As a marker of bone resorption, we measured urine excretion of Dpd, a covalent cross-linker of bone collagen fibers. We
observed a progressive decrease in bone resorption in intact,
sham-operated animals that were aged 3 mo at the beginning of
the study and 5 mo at the end. This finding is in agreement with
previous reports (33) showing that skeletal maturation is associated with a decrease in bone turnover. At 28 and 56 days
following surgery, Dpd increased progressively in ORX rats
compared with sham. Our findings are in agreement with those
of Erben et al. (19), who reported that ORX of rats results in a
high-turnover osteopenia, with a large increase in bone resorption and a small reflex increase in bone formation. In ORX rats,
increased bone resorption over the course of 56 days resulted
in dramatic decreases in the volume of cancellous bone and in
the number of trabeculae, with no change in trabecular width
and an increase in trabecular separation. Probably the most
robust findings of this study are that testosterone administration
completely prevented these changes and that MK-434 did not
inhibit the effect of testosterone.
We found that testosterone also has a robust trophic effect on
the kidney, one that was not blocked by MK-434. The kidney
expresses 5␣-reductase type I (22), and MK-434 inhibits both
type I and II 5␣-reductase in the rat. However, our data show
that the effects of testosterone on the kidney are direct and do
not require conversion to DHT. We found that changes in
kidney mass were not associated with changes in creatinine
clearance, a measure of the glomerular filtration rate (GFR).
However, this result is expected because GFR is not maximal
in healthy young animals (14, 24). Future studies will aim to
determine whether testosterone supports maximal kidney filtration rates and maintains GFR in aging.
In the present study, we found that 56 days of testosterone
treatment in young ORX Fisher 344 rats caused a 12-fold
prostate enlargement that was 66% inhibited by MK-434.
Previously, we (9) reported that 28 days of testosterone treatment in mature ORX Brown Norway rats caused a fivefold
prostate enlargement that was nearly completely blocked by
MK-434. Several factors may contribute to the lesser degree to
which MK-434 blocked testosterone-induced prostate in the
present study. Those factors include strain and age differences
293 • AUGUST 2007 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on June 17, 2017
catabolic changes and caused prostate enlargement and 2) that
coadministration of the 5␣-reductase inhibitor MK-434 substantially inhibited prostate enlargement while allowing most
of the anabolic effects of testosterone to occur. To produce
consistent prostate enlargement, we selected a supraphysiological dose of 1.0 mg testosterone/day. This dose is the
equivalent of 1,200 mg/wk for a person weighing 70 kg. In
comparison, human replacement doses are in the range of
35–100 mg/wk (10, 15, 27, 36), and the highest dose administered to humans is 600 mg/wk (5).
ORX-induced catabolic effects included reduced food intake
and body weight, reduced prostate and kidney mass, decreased
cross-sectional area of type I and type IIb muscle fibers,
increased bone resorption, and loss of cancellous bone. Testosterone caused marked prostate enlargement as well as the
following anabolic effects: increased food intake, increased
kidney mass, increased blood concentration of hemoglobin,
increased strength and partial prevention of muscle fiber
changes, and decreased bone resorption and prevention of bone
loss. In general, MK-434 substantially blocked testosteroneinduced prostate enlargement but did not block the anabolic
effects of testosterone.
5␣-Reductase type II is expressed in androgenic tissues
(e.g., prostate, seminal vesicles) and also in other tissues,
where it may play a role in undesirable effects of testosterone
(22). 5␣-Reductase type II is also expressed in hair follicles,
where it plays a role in male pattern baldness and increased
body hair, and in sebaceous glands, where it plays a role in
acne. In tissues expressing 5␣-reductase, DHT is the primary
androgen. However, 5␣-reductase is not expressed in appreciable quantities in muscle or bone (22). Although to our
knowledge sex steroid concentrations have not been measured
in these tissues, testosterone is presumably the primary androgen. These considerations led us to the hypothesis that a
5␣-reductase inhibitor might block testosterone-induced prostate enlargement without blocking anabolic effects on muscle
or bone.
Brown et al. (11) reported that ORX in Sprague-Dawley rats
caused a decrease in the peak tetanic tension of several muscles
without any loss of muscle mass. Our findings were for the
most part consistent with theirs. We found that ORX reduced
the percentage and size of type I fibers in SOL and EDL
muscles. Reductions were also noted in the size of type IIa
fibers in SOL and in the size of type IIb fibers in EDL. ORX
may have reduced grip strength, although the effect was not
statistically significant. We found that ORX caused only a very
small decrease in muscle mass. It is well established (1, 43)
that, over the long term, low serum testosterone is associated
with both reduced muscle mass and strength. Taken together,
these findings suggest that loss of testosterone produces a loss
of muscle quality that precedes a measurable loss in muscle
mass.
As expected, testosterone caused an increase in grip
strength, and the effect was not inhibited by MK-434. The
ORX-induced changes in the number and size of muscle fibers
were at least partially prevented by testosterone. Notably, the
effects of testosterone were not inhibited by MK-434. The
effects of testosterone on fiber type composition and fiber size
are consistent with the known effects of testosterone on
strength and power (6, 46). We (23) have previously reported
that testosterone attenuates the loss of type I fibers following
5␣-REDUCTASE AND TESTOSTERONE ANABOLIC EFFECT
AJP-Endocrinol Metab • VOL
GRANTS
This work was supported by a Veterans Administration Merit Award to
S. E. Borst.
REFERENCES
1. Abassi A, Drinka PJ, Mattson DE, Rudman D. Low circulating levels
of insulin-like growth factor and testosterone in chronically institutionalized men. J Am Geriatr Soc 48: 975–981, 1993.
2. Azzolina B, Ellsworth K, Andersson S, Geissler W, Bull HG, Harris
GS. Inhibition of rat alpha-reductases by finasteride: evidence for isozyme
differences in the mechanism of inhibition. J Steroid Biochem Mol Biol
61: 55– 64, 1997.
3. Baron R. Processing of undecalcified bone specimens for bone histomorphometry. In: Bone Histomorphometry, edited by Recker RR. Boca Raton,
FL: CRC Press, 1983, p. 13–35.
4. Behre HM, Bohmeyer J, Nieschlag E. Prostate volume in testosteronetreated and untreated hypogonadal men in comparison to age-matched
normal controls. Clin Endocrinol (Oxf) 40: 341–349, 1994.
5. Bhasin S, Woodhouse L, Casaburi R, Singh AB, Mac RP, Lee M,
Yarasheski KE, Sinha-Hikim I, Dzekov C, Dzekov J, Magliano L,
Storer TW. Older men are as responsive as young men to the anabolic
effects of graded doses of testosterone on the skeletal muscle. J Clin
Endocrinol Metab 90: 678 – 688, 2005.
6. Bhasin S, Woodhouse L, Storer TW. Proof of the effect of testosterone
on skeletal muscle. J Endocrinol 170: 27–38, 2001.
7. Blazer DG. Testosterone and Aging: Clinical Research Directions (Online). Institute of Medicine. http://www.iom.edu/CMS/3740/4862/16398/
16440. aspx [2003].
8. Borst SE. Interventions for sarcopenia and muscle weakness in older
people. Age & Aging 33: 538 –555, 2004.
9. Borst SE, Lee JH, Conover CF. Inhibition of 5␣-reductase blocks
prostate effects of testosterone without blocking anabolic effects. Am J
Physiol Endocrinol Metab 288: E222–E227, 2005.
10. Brill KT, Weltman AL, Gentili A, Patrie JT, Fryburg DA, Hanks JB,
Urban RJ, Veldhuis JD. Single and combined effects of growth hormone
and testosterone administration on measures of body composition, physical performance, mood, sexual function, bone turnover, and muscle gene
expression in healthy older men. J Clin Endocrinol Metab 87: 5649 –5657,
2002.
11. Brown M, Fisher JS, Hasser EM. Gonadectomy and reduced physical
activity: effects on skeletal muscle. Arch Phys Med Rehabil 82: 93–97,
2001.
12. Carani C, Qin K, Simoni M, Faustini-Fustini M, Serpente S, Boyd J,
Korach KS, Simpson ER. Effect of testosterone and estradiol in a man
with aromatase deficiency. N Engl J Med 337: 91–95, 1997.
13. Carter CS, Cesari M, Ambrosius WT, Hu N, Diz D, Oden S, Sonntag
WE, Pahor M. Angiotensin-converting enzyme inhibition, body composition, and physical performance in aged rats. J Gerontol A Biol Sci Med
Sci 59: 416 – 423, 2004.
14. Castrop H. Mediators of tubuloglomerular feedback regulation of glomerular filtration: ATP and adenosine. Acta Physiol 189: 3–14, 2007.
15. Clague JE, Wu FC, Horan MA. Difficulties in measuring the effect of
testosterone replacement therapy on muscle function in older men. Int J
Androl 22: 261–265, 1999.
16. Cooke GM, Pothier F, Murphy BD. The effects of progesterone,
4,16-androstadien-3-one and MK-434 on the kinetics of pig testis microsomal testosterone-4-ene-5 alpha-reductase activity. J Steroid Biochem
Mol Biol 60: 353–359, 1997.
17. di Salle E, Giudici D, Biagini L, Cominato C, Briatico G, Panzeri A.
Effects of 5 alpha-reductase inhibitors on intraprostatic androgens in the
rat. J Steroid Biochem Mol Biol 53: 381–385, 1995.
18. Dirks A, Leeuwenburgh C. Apoptosis in skeletal muscle with aging.
Am J Physiol Regul Integr Comp Physiol 282: R519 –R527, 2002.
19. Erben RG, Eberle J, Stahr K, Goldberg M. Androgen deficiency
induces high turnover osteopenia in aged male rats: a sequential histomorphometry study. J Bone Miner Res 15: 1085–1098, 2000.
20. Ferrando AA, Sheffield-Moore M, Yeckel CW, Gilkison C, Jiang J,
Achacosa A, Lieberman SA, Tipton K, Wolfe RR, Urban RJ. Testosterone administration to older men improves muscle function: molecular
and physiological mechanisms. Am J Physiol Endocrinol Metab 282:
E601–E607, 2002.
293 • AUGUST 2007 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on June 17, 2017
as well as the consistency of MK-434 delivery from implanted
pellets (MK-434 was injected in the previous study). Another
intriguing possibility relates to the duration of testosterone
treatment. Although DHT is considered the chief mediator of
prostate enlargement, Winter et al. (47) have shown that
prostate enlargement in dogs is caused by administration of
DHT plus a small dose of 17␤-estradiol, but not by DHT alone,
indicating that a small amount of estrogen is required for
prostate enlargement. Because administration of androgens
inhibits endogenous testosterone secretion and because estrogen can be synthesized from testosterone, but not DHT, it is
expected that prostate estrogen concentration would be increased by testosterone administration and decreased by DHT
administration (45). In the present study, over the course of 56
days there may have been sufficient estrogen in the prostate to
allow for a small amount of growth even in the continued
presence of MK-434.
Ferrando et al. (20) administered a replacement dose of
testosterone to older hypogonadal men for 6 mo without
observing prostate enlargement. However, most studies report
an average of 20% prostate enlargement per year of testosterone replacement (4, 26, 48). There is a growing consensus that
higher-than-replacement doses of testosterone will be required
to produce substantial anabolic effects in older men. The 2003
report of the Institute of Medicine (7) questions the efficacy of
replacement doses in producing increases in strength. In addition, the question of whether testosterone treatment may accelerate underlying early-stage prostate cancer has not been
settled. For all of these reasons, protection of the prostate will
be an important consideration for the future of testosterone
therapy. Our study suggests that such protection may be
afforded with a 5␣-reductase type II inhibitor without compromising the anabolic effects of testosterone.
Loss of testosterone in older men is associated with loss of
bone mineral density (43). However, administration of testosterone at replacement doses to older, hypogonadal men has
produced only marginal increases in bone mineral density,
even in studies lasting as long as 36 mo (38). Studies of the
impact of high-dose testosterone on bone have not been performed. Our study demonstrates that substantial changes in
bone mineral can occur with high doses of testosterone. Our
finding that the effects of high-dose testosterone are not
blocked by a 5␣-reductase inhibitor is consistent with the
hypothesis that anabolic effects of testosterone in bone are
mediated by local conversion to estrogen rather than DHT.
This hypothesis was generated following the identification of
several boys who lacked functional P-450 aromatase activity
(12). These patients have an osteopenia that responds to estrogen treatment but not to testosterone.
We conclude that high-dose testosterone produces anabolic
effects in ORX rats, including effects on hemoglobin, muscle
fiber type and strength, kidney mass, and powerful effects on
bone resorption and bone morphology and also produces
marked prostate enlargement. We also conclude that coadministration of an inhibitor of 5␣-reductase can inhibit prostate
effects of testosterone without inhibiting anabolic effects. This
study provides, in an animal model, proof of principle and a
rationale for testing the effects of higher-dose testosterone and
finasteride in older hypogonadal men.
E513
E514
5␣-REDUCTASE AND TESTOSTERONE ANABOLIC EFFECT
AJP-Endocrinol Metab • VOL
37. Sinha-Hikim I, Cornford M, Gaytan H, Lee ML, Bhasin S. Effects of
testosterone supplementation on skeletal muscle fiber hypertrophy and
satellite cells in community-dwelling older men. J Clin Endocrinol Metab
91: 3024 –3033, 2006.
38. Snyder PJ, Peachey H, Hannoush P, Berlin JA, Loh L, Holmes JH,
Dlewati A, Staley J, Santanna J, Kapoor SC, Attie MF, Haddad JG Jr,
Strom BL. Effect of testosterone treatment on bone mineral density in
men over 65 years of age. J Clin Endocrinol Metab 84: 1966 –1972, 1999.
39. Sonntag WE, Carter CS, Ikeno Y, Ekenstedt K, Carlson CS, Loeser
RF, Chakrabarty S, Lee S, Bennett C, Ingram R, Moore T, Ramsey
M. Adult-onset growth hormone and insulin-like growth factor I deficiency reduces neoplastic disease, modifies age-related pathology, and
increases life span. Endocrinology 146: 2920 –2932, 2005.
40. Tarter TH, Vaughan Ed. Inhibitors of 5␣-reductase in the treatment of
benign prostatic hyperplasia. Curr Pharmaceut Design 12: 775–783, 2006.
41. Tenover JL. Testosterone replacement therapy in older men. Int J Androl
22: 300 –306, 1999.
42. Theobald HM, Roman BL, Lin TM, Ohtani S, Chen SW, Peterson RE.
2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits luminal cell differentiation
and androgen responsiveness of the ventral prostate without inhibiting
prostatic 5alpha-dihydrotestosterone formation or testicular androgen production in rat offspring. Toxicol Sci 58: 324 –338, 2000.
43. van den Beld AW, de Jong FH, Grobbee DE, Pols HA, Lamberts SW.
Measures of bioavailable serum testosterone and estradiol and their relationships with muscle strength, bone density, and body composition in
elderly men. J Clin Endocrinol Metab 85: 3276 –3282, 2000.
44. Van Zyl CG, Noakes TD, Lambert MI. Anabolic-androgenic steroid
increases running endurance in rats. Med Sci Sports Exerc 27: 1385–1389,
1995.
45. Vermeulen A, Deslypere JP. Long-term transdermal dihydrotestosterone
therapy: effects on pituitary gonadal axis and plasma lipoproteins. Maturitas 7: 281–287, 1985.
46. Wang C, Swerdloff RS, Iranmanesh A, Dobs A, Snyder PJ, Cunningham G, Matsumoto AM, Weber T, Berman N; Testosterone Gel Study
Group. Transdermal testosterone gel improves sexual function, mood,
muscle strength, and body composition parameters in hypogonadal men.
J Clin Endocrinol Metab 85: 2839 –2853, 2000.
47. Winter ML, Bosland MC, Wade DR, Falvo RE, Nagamani M, Liehr
JG. Induction of benign prostatic hyperplasia in intact dogs by nearphysiological levels of 5-alpha dihydrotestosterone and 17-beta estradiol.
Prostate 26: 325–333, 1995.
48. Zitzmann M, Depenbusch M, Gromoll J, Nieschlag E. Prostate volume
and growth in testosterone-substituted hypogonadal men are dependent on
the CAG repeat polymorphism of the androgen receptor gene: a longitudinal pharmacogenetic study. J Clin Endocrinol Metab 88: 2049 –2054,
2003.
293 • AUGUST 2007 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on June 17, 2017
21. Frost HM. Bone histomorphometry: analysis of trabecular bone dynamics. In: Bone Histomorphometry, edited by Recker RR. Boca Raton, FL:
CRC Press, 1983, p. 109 –132.
22. Gormley GJ. Finasteride: a clinical review. Biomed & Pharmacother 49:
319 –324, 1995.
23. Gregory CM, Vandenborne K, Ottenweller JE, Huang HF, Dudley
GA. Effects of testosterone therapy on skeletal muscle after spinal cord
injury. Spinal Cord 41: 23–28, 2003.
24. Guyton AC, Hall JE. Textbook of Medical Physiology (9th ed.). Philadelphia, PA: WB Saunders, 1996, p. 328 –329.
25. Hajjar RR, Kaiser FE, Morley JE. Outcomes of long-term testosterone
replacement in older hypogonadal males: a retrospective analysis. J Clin
Endocrinol Metab 82: 3793–3796, 1997.
26. Jin B, Conway AJ, Handelsman DJ. Effects of androgen deficiency and
replacement on prostate zonal volumes. Clin Endocrinol (Oxf) 54: 437–
445, 2001.
27. Kenny AM, Prestwood KM, Gruman CA, Marcello KM, Raisz LG.
Effects of transdermal testosterone on bone and muscle in older men with
low bioavailable testosterone levels. J Gerontol A Biol Sci Med Sci 56:
M266 –M272, 2001.
28. Mikuz G. Pathology of prostate cancer. Adv Clin Pathol 1: 21–24, 1997.
29. Parsons JK, Carter HB, Platz EA, Wright EJ, Landis P, Metter EJ.
Serum testosterone and the risk of prostate cancer: potential implications
for testosterone therapy. Cancer Epidemiol Biomarkers Prev 14: 2257–
2260, 2005.
30. Phillips T, Leeuwenburgh C. Muscle fiber specific apoptosis and TNFalpha signaling in sarcopenia are attenuated by life-long calorie restriction.
FASEB J 19: 668 – 670, 2005.
31. Rittmaster RS, Manning AP, Wright AS, Thomas LN, Whitefield S,
Norman RW, Lazier CB, Rowden G. Evidence for atrophy and apoptosis in the ventral prostate of rats given the 5-alpha-reductase inhibitor
finasteride. Endocrinology 136: 741–748, 1995.
32. Sahni M, Borst SE, Mulligan T. Should we offer testosterone replacement therapy to men who live in nursing homes? Ann Long-Term Care 14:
27–33, 2006.
33. Sengupta S, Arshad M, Sharma S, Dubey M, Singh MM. Attainment of
peak bone mass and bone turnover rate in relation to estrous cycle,
pregnancy and lactation in colony-bred Sprague-Dawley rats: suitability
for studies on pathophysiology of bone and therapeutic measures for its
management. J Steroid Biochem Mol Biol 94: 421– 429, 2005.
34. Seo SI, Kim SW, Paick JS. The effects of androgen on penile reflex,
erectile response to electrical stimulation and penile NOS activity in the
rat. Asian J Androl 1: 169 –174, 1999.
35. Shao TC, Kong A, Marafelia P, Cunningham GR. Effects of finasteride
on the rat ventral prostate. J Androl 14: 79 – 86, 1993.
36. Sih R, Morely JE, Kaiser FE, Perry HM, Patrick P, Ross C. Testosterone replacement in older hypogonadal men: a 12 month randomized,
controlled trial. J Clin Endocrinol Metab 82: 1661–1667, 1997.