Download Comparison of GH, IGF-I, and testosterone with mRNA of receptors

Am J Physiol Endocrinol Metab
281: E1159–E1164, 2001.
Comparison of GH, IGF-I, and testosterone with mRNA of
receptors and myostatin in skeletal muscle in older men
TAYLOR J. MARCELL,1 S. MITCHELL HARMAN,1 RANDALL J. URBAN,3
DANIEL D. METZ,1 BUEL D. RODGERS,2 AND MARC R. BLACKMAN2
1
Intramural Research Program, National Institute on Aging, National Institutes of Health;
2
Division of Endocrinology and Metabolism, Department of Medicine, Johns Hopkins
University School of Medicine, Baltimore, Maryland 21224; and 3Department of Internal
Medicine, University of Texas Medical Branch, Galveston, Texas 77555
Received 8 September 2000; accepted in final form 20 July 2001
androgens; reverse transcription-polymerase chain reaction;
gene expression; elderly; protein metabolism
IN HEALTHY YOUNG ADULTS, under equilibrium conditions,
skeletal muscle protein synthesis and degradation are
a balanced, dynamic process with no net change occurring in skeletal muscle mass. During aging, however,
muscle tissue is gradually lost, often resulting in diminished mass and strength, a condition referred to as
sarcopenia, which contributes to frailty. This loss of
muscle results from a net imbalance between the rates
of protein synthesis and degradation (22). Protein synthesis can be stimulated by various signals, including
Address for reprint requests and other correspondence: T. J. Marcell, Kronos Longevity Research Institute, 4455 E. Camelback Rd.,
Ste. B135, Phoenix, AZ 85018 (E-mail: [email protected]).
http://www.ajpendo.org
hormones, metabolic demand, and functional overload
(10, 23). Protein breakdown is also under the influence
of these same factors, which stimulate lysosomal and
ubiquitin degradation processes. Insulin, growth hormone (GH), insulin-like growth factor I (IGF-I), and
testosterone (T) are anabolic hormones, all of which
increase muscle mass by stimulating protein synthesis
and/or inhibiting protein breakdown, whereas cortisol
is a potent stimulus to protein catabolism (6, 9, 22).
Circulating levels of these various hormones are altered by the aging process, potentially contributing to
sarcopenia (2, 5, 12, 27).
Although GH-induced IGF-I production in the liver
is the major source of circulating IGF-I and mediates
many GH metabolic effects, local IGF-I production
within target tissues, under the influence of both GH
and T (28), accounts for ⬎50% of total IGF-I production
and appears to be more important for stimulating
muscle growth and repair (10, 14, 16). How changes in
hormone levels with age affect the IGF-I pathway in
skeletal muscle remains poorly understood.
Myostatin, a recently discovered member of the
transforming growth factor (TGF)-␤ superfamily, is an
autocrine factor that is a potent inhibitor of muscle
development (20). Postnatally, myostatin is expressed
in varying levels exclusively in skeletal muscle (4, 29)
and preferentially in fast-type skeletal muscle fibers
(4). Age-related loss of skeletal muscle is associated
with a selective atrophy of the fast-type skeletal muscle fibers (17). Thus, whether the age-related decline in
anabolic hormonal status allows a progressive increase
in myostatin expression that would contribute to sarcopenia is an important question (15). However, by
what mechanism hormones interact with myostatin
remains unclear.
Therefore, in the current study, we measured plasma
levels of GH, IGF-I, T, and cortisol and, using the
reverse transcription-polymerase chain reaction (RTPCR), determined gene expression of IGF-I and myostatin in skeletal muscle biopsy samples from healthy
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0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society
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Marcell, Taylor J., S. Mitchell Harman, Randall J.
Urban, Daniel D. Metz, Buel D. Rodgers, and Marc R.
Blackman. Comparison of GH, IGF-I, and testosterone with
mRNA of receptors and myostatin in skeletal muscle in older
men. Am J Physiol Endocrinol Metab 281: E1159–E1164,
2001.—Growth hormone (GH), insulin-like growth factor I
(IGF-I), and testosterone (T) are important mediators of
muscle protein synthesis, and thus muscle mass, all of which
decline with age. We hypothesized that circulating hormones
would be related to the transcriptional levels of their respective receptors and that this expression would be negatively
related to expression of the myostatin gene. We therefore
determined content of mRNA transcripts (by RT-PCR) for
GH receptor (GHR), IGF-I, androgen receptor (AR), and myostatin in skeletal muscle biopsy samples from 27 healthy men
⬎65 yr of age. There were no significant relationships between age, lean body mass, or percent body fat and transcript
levels of GHR, IGF-I, AR, or myostatin. Moreover, there were
no significant correlations of serum GH, IGF-I, or T with
their corresponding target mRNA levels (GHR, intramuscular IGF-I, or AR) in skeletal muscle. However, GHR was
negatively correlated (r ⫽ ⫺0.60, P ⫽ 0.001) with myostatin
mRNA levels. The lack of apparent relationships of muscle
transcripts with their respective ligands in healthy older
adults suggests that age-related deficits in both GH and T
may lead to an increase in myostatin expression and a disassociation in autocrine IGF-I effects on muscle protein synthesis, both of which could contribute to age-related sarcopenia.
E1160
GHR, IGF-I, AR, AND MYOSTATIN IN ELDERLY SKELETAL MUSCLE
older men. Because circulating hormones can autoregulate their actions by effects on their own receptors
(3), we also determined mRNA levels for GH receptor
(GHR) and androgen receptor (AR). The present study
was designed to test the hypotheses that 1) circulating
anabolic hormone concentrations are significantly related to the transcriptional levels of their respective
receptors, 2) the expression of GHR, IGF-I, and AR is
up- or downregulated coordinately, and 3) the expression of anabolic hormone receptors is negatively related to expression of the myostatin gene.
SUBJECTS AND METHODS
Table 1. Subject characteristics of the
healthy older men
Age, yr
Weight, kg
BMI, kg/m2
Lean body mass, kg*
Body fat, %*
GH, ng/ml†
IGF-I, ng/ml
Testosterone, ng/dl
Cortisol, mg/dl
Mean
Range
72.7 ⫾ 0.9
78.5 ⫾ 1.7
26.7 ⫾ 0.6
51.6 ⫾ 0.8
30.0 ⫾ 0.8
0.75 ⫾ 0.10
126.0 ⫾ 8.3
428.5 ⫾ 19.1
6.6 ⫾ 0.2
66–82
58–92
22–32
42–59
22–38
0.3–2.4
63–219
252–627
4.8–9.4
Values are means ⫾ SE; n ⫽ 27 men. BMI, body mass index; GH,
growth hormone; IGF-I, insulin-like growth factor I. * Body composition determined by dual-energy X-ray absorptiometry. † Mean 12-h
(Q20 min, 0800–2000) overnight value.
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Subjects. Twenty-seven relatively healthy, ambulatory,
community-dwelling older men (Table 1) were recruited by
mass-mailed advertisement. All subjects underwent a comprehensive screening assessment that included a physical
examination, routine laboratory profiles, and a graded treadmill exercise electrocardiogram. No subject had diabetes mellitus, coronary artery disease, or untreated thyroid disease.
Study participants were nonsmokers, consumed ⬍2 oz. of
alcohol per day, and took no medications known to interfere
with the GH/IGF-I or sex steroid axes or any other outcome
measure. Because these subjects were to participate in an
intervention study examining the separate and interactive
effects of exogenously administered recombinant human GH
and/or T, subjects were included only if serum IGF-I values
were ⬎1 SD below the mean for subjects aged 20–35 yr (i.e.,
⬍230 ng/ml) and if serum total testosterone levels were ⬍470
ng/dl. Over 70% of the men screened for participation in this
study had circulating IGF-I levels below this exclusion range.
The study was approved by the combined Institutional Review Board of the Johns Hopkins Bayview Medical Center
and Intramural Research Program, National Institute on
Aging (NIA), and each subject provided written, informed
consent.
Study protocol. All subjects were admitted to the General
Clinical Research Center at the Johns Hopkins Bayview
Medical Center, where their body weight and height were
recorded and subjects underwent an assessment of body
composition for determination of percent body fat and lean
body mass (LBM) via dual-energy X-ray absorptiometry
(DEXA; Lunar Radiation, DPX-L, Madison, WI). At 1900, an
intravenous catheter was inserted into a forearm vein for
subsequent blood sampling, and heparinized (1,000 U/l) 0.9%
saline was infused slowly to prevent clotting. Blood was
sampled for GH at 20-min intervals from 2000 until the
following morning at 0800, at which time blood samples for
IGF-I and testosterone were drawn in the fasted state.
Hormone assays. Serum GH and total serum T were measured in duplicate in the Endocrine Research Laboratory of
the Intramural Research Program, NIA. The sensitivity of
the GH immunoradiometric assay was 0.05 ng/ml. Intraassay coefficients of variation (CVs) at mean GH concentrations of 2.5, 6.4, and 10.9 ng/ml were 2.0, 1.7, and 1.3%,
respectively, and interassay CVs at mean GH levels of 2.4,
6.2, and 11.2 ng/ml were 3.6, 2.7, and 4.1%, respectively.
Total T was measured by RIA (Diagnostic Products, Los
Angeles, CA) with a sensitivity of 10 ng/dl. Intra-assay CVs
at mean T concentrations of 60, 300, 597, and 998 ng/dl were
11.2, 6.7, 1.5, and 3.1%, respectively, and interassay CVs at
mean T levels of 76, 299, 707, and 1,041 ng/dl were 5.9, 3.9,
3.2, and 4.8%, respectively. Cortisol was measured by RIA
(ICN Pharmaceuticals, Diagnostics Division, Costa Mesa,
CA) with a sensitivity of 0.25 mg/dl. Intra-assay CVs at mean
cortisol concentrations of 4.0, 12.5, and 25.6 mg/dl were 4.3,
6.8, and 10.5%, respectively, and interassay CVs at mean
cortisol levels of 3.9, 10.3, and 25.7 mg/dl were 4.5, 4.5, and
5.5%, respectively. Total serum IGF-I was measured by RIA
after acid-ethanol extraction at Endocrine Sciences (Calabasas Hills, CA). Sensitivity of the IGF-I assay was 30 ng/ml,
and the intra- and interassay CVs were 5.9 and 7.3% at 289
ng/ml and 4.6 and 6.3% at 591 ng/ml.
Skeletal muscle mRNA measures by RT-PCR. Skeletal
muscle samples were obtained from the midbelly of the dominant vastus lateralis muscle, midway between its origin and
insertion, by use of the Bergstrom needle biopsy technique
(11), in the morning after the overnight blood sampling.
Visible fat and connective tissue were excised on a chilled
glass plate, and samples were placed into preweighed vials
and quick-frozen in liquid nitrogen. For RNA extraction, the
muscle sample was first pulverized in liquid nitrogen with a
mortar and pestle, followed by homogenization for 30 s at
8,000 rpm. Total RNA was then extracted using RNA
STAT-60 (Tel-Test, Friendswood, TX) and quantified by determining absorbance at 260 nm in duplicate, and the values
were averaged.
Complementary DNA (cDNA) was reverse transcribed
(RT) from 0.5 ␮g of total RNA using 1 ␮M random hexamers
and 100 U Superscript II reverse transcriptase (GIBCO-BRL,
Rockville, MD) in a final volume of 20 ␮l at 42°C for 50 min,
followed by 5 min of heat inactivation at 99°C, following the
manufacturer’s recommendation.
cDNA was amplified from the 20 ␮l of RT reaction mix in
the same tube in a final concentration of 1⫻ PCR buffer
(Perkin-Elmer, Norwalk, CT; 25 mM Tris 䡠 HCl, 50 mM KCl),
1.5 mM MgCl2, 1.0 mM deoxy-NTP, forward and reverse
primers (gene of interest, 10 pmol glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 1.5 pmol), 5 U AmpliTaq
DNA polymerase (Perkin-Elmer), and 0.0225 ␮Ci/␮l deoxy[32P]CTP (Amersham, Arlington Heights, IL) in a final volume of 50 ␮l. The linear portion of the amplification curve for
each transcript was defined and then utilized to determine
the appropriate number of PCR amplification cycles in the
RT-PCR analyses. As an example, data for the AR cycle
titration are depicted in Fig. 1. Similar curves for GHR,
IGF-I, and myostatin were also generated independently
(data not shown). PCR for AR started with an initial denaturation at 94°C for 3 min, followed by 26 cycles of denaturation at 94°C for 35 s, annealing at 58°C for 45 s, and
extension at 72°C for 80 s, with a final extension cycle at 72°C
for 7 min. GHR transcripts were similarly amplified for 26
cycles, whereas IGF-I was amplified for 28 cycles and myostatin for 30 cycles.
GHR, IGF-I, AR, AND MYOSTATIN IN ELDERLY SKELETAL MUSCLE
E1161
RESULTS
Fig. 1. Optimizing RT-PCR for androgen receptor (AR) by determining the linear phase of PCR amplification after a cycle titration. AR
was expressed in duplicate between 20 and 40 cycles. Bottom: representative gel electrophoretic pattern for AR. Similar curves were
determined for growth hormone receptor (GHR), insulin-like growth
factor I (IGF-I), and myostatin (data not shown).
The PCR products (10 ␮l from each sample tube) were then
isolated by electrophoresis using precast 6% polyacrylamideTris-borate-EDTA (TBE) gels (NOVEX, San Diego, CA) at 90
W for 90 min. Gels were subsequently dried, and incorporated 32P was determined by quantitative autoradiography
(Molecular Dynamics, Sunnyvale, CA). For final analyses,
the optical density of the transcript of interest was determined in duplicate using SigmaGel 1.05 software (SPSS,
Richmond, CA) (see Statistical analysis).
For subsequent sequencing of the GHR, IGF-I, AR, and
myostatin DNA fragments, PCR products were isolated using
a 5% polyacrylamide (1:25 bis-to-acrylamide ratio) gel with
25% glycerol. Samples were run for 3 h at 250 V in 0.5⫻ TBE
buffer. The gel was then stained with ethidium bromide, and
the bands were cut out under ultraviolet illumination. The
gel slices were then electroeluted for 30 min (Electro-Eluter
442, Bio-Rad, Hercules, CA). The samples were extracted
with phenol-chloroform (2⫻) and then ethanol precipitated.
Samples were then sequenced at the University of Texas
Medical Branch Molecular Core Laboratory with the specific
forward and reverse primers (Table 2) from the 3⬘ to 5⬘
ends by use of an ABI Prism (model 310) Sequencer (PE
Biosystems). All amplified DNA fragment sequences were
then compared to confirm identity with known sequences
(Table 2).
Statistical analysis. All data are expressed as means ⫾ SE.
Results from the GH assays were reduced using a curvefitting program, and serum concentrations were estimated
from the derived constants. Twelve-hour overnight (Q20 min,
0800–2000) GH values were measured, and mean values for
this sampling period were used for further analyses. Samples
for gene expression were reverse transcribed and then subjected to PCR from duplicate samples of total RNA. The
AJP-Endocrinol Metab • VOL
Relationships of skeletal muscle transcript levels
with age, body composition, and circulating hormones.
Subject characteristics are described in Table 1. Mean
total LBM (51.6 ⫾ 0.8 kg) and percent body fat (30.0 ⫾
0.8%), determined by DEXA, were consistent with basic good health and did not suggest frailty or morbid
obesity. To determine whether muscle transcript levels
were related to age, body composition (total weight,
body mass index, LBM, %fat) or circulating hormone
concentrations (GH, IGF-I, T, and cortisol), we performed a series of simple and multiple regression analyses. There were no significant relationships between
age or any of the measured indexes of body composition
and transcript levels of GHR, IGF-I, AR, or myostatin
(data not shown). Moreover, there were no significant
correlations of GH, circulating IGF-I, or T with their
corresponding target mRNA levels (GHR, intramuscular IGF-I, or AR) in skeletal muscle (Table 3). Furthermore, no relationships were observed between serum
GH, circulating IGF-I, T, or cortisol and skeletal muscle myostatin mRNA levels (Table 3).
Interrelationships among skeletal muscle transcript
levels. We observed no significant interrelationship between basal levels of skeletal muscle GHR and intramuscular IGF-I gene expression [r ⫽ ⫺0.16; P ⫽ nonTable 2. Oligonucleotide primer sequences for GHR,
intramuscular IGF-I, AR, and myostatin
Primer
Sequence
GHR 5⬘
GHR 3⬘
IGF-I 5⬘
IGF-I 3⬘
AR 5⬘
AR 3⬘
MSTN 5⬘
MSTN 3⬘
5⬘-AAG-GGA-TTG-ATC-CAG-ATC-TTC-TCA-AGG-3⬘
5⬘-ATC-GCT-TAG-AAG-TCT-GTC-GGT-GTC-TG-3⬘
5⬘-AGT-CTT-CCA-ACC-CAA-TTA-TTT-AAG-TGC-3⬘
5⬘-CCG-ACA-TGC-CCA-AGA-CCC-AGA-AG-3⬘
5⬘-TTG-TCC-ACC-GTG-TGT-CTT-CTT-CTG-C-3⬘
5⬘-TGC-ACT-TCC-ATC-CTT-GAG-CTT-GGC-3⬘
5⬘-ATC-ATG-CAA-AAA-CTG-CAA-CTC-3⬘
5⬘-TAG-AGG-GTA-ACG-ACA-GCA-TC-3⬘
Base
Pairs
185 bp
387 bp
225 bp
860 bp
GHR, growth hormone receptor; AR, androgen receptor; MSTN,
myostatin.
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duplicate samples were then electrophoresed, and optical
density was determined from the 32P incorporation into the
subsequent bands. The arbitrary value for optical density
was then averaged between duplicate samples. Analysis of
duplicate samples minimizes the potential variability in reverse transcription, PCR cycle efficiency, and gel loading
variations. The intra-assay variance for the entire set of
duplicate samples determined (GHR, IGF-I, AR, and myostatin) was 0.1 ⫾ 9.7% (mean ⫾ SD; n ⫽ 206 pairs). Therefore,
the mRNA data are expressed as the average optical density
(arbitrary units) of the duplicate samples measured. Because
this study was designed to focus on relationships of circulating hormone concentrations with expression of hormone receptor genes, we did not normalize our molecular data to a
“housekeeping” gene such as GAPDH, because changes in
such a housekeeper could influence the relationships studied.
Relationships between continuous variables were analyzed
using simple linear regression analysis and were expressed
as a correlation coefficient (r). Significance was set at Pⱕ
0.05.
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GHR, IGF-I, AR, AND MYOSTATIN IN ELDERLY SKELETAL MUSCLE
Table 3. Correlations between serum hormone levels and skeletal muscle gene transcripts
GHR mRNA
Serum GH, ng/ml
Serum IGF-I, ng/ml
Serum testosterone, ng/dl
Serum cortisol, mg/ml
GHR mRNA
IGF-I mRNA
AR mRNA
r
r
r
r
⫽
⫽
⫽
⫽
⫺ 0.16 (⬎ 0.4)
⫺ 0.20 (⬎ 0.3)
⫺ 0.28 (⬎ 0.1)
⫺ 0.08 (⬎ 0.7)
IGF-I mRNA
r
r
r
r
r
⫽
⫽
⫽
⫽
⫽
⫺ 0.29 (⬎ 0.1)
⫺ 0.07 (⬎ 0.7)
⫺ 0.15 (⬎ 0.4)
⫺ 0.31 (⬎ 0.1)
⫺ 0.16 (⬎ 0.4)
AR mRNA
r
r
r
r
r
r
⫽
⫽
⫽
⫽
⫽
⫽
⫺0.03 (⬎0.8)
0.19 (⬎ 0.3)
0.26 (⬎ 0.1)
0.03 (⬎ 0.9)
0.41 (0.04)
⫺0.37 (0.06)
MSTN mRNA
r
r
r
r
r
r
r
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫺0.03 (⬎0.9)
⫺0.07 (⬎0.7)
⫺0.11 (⬎0.5)
⫺0.17 (⬎0.4)
⫺0.60 (0.001)
0.16 (⬎ 0.4)
⫺0.36 (0.07)
Values ⫽ Pearson correlation coefficients (significance).
Fig. 2. Bivariate relationships of skeletal muscle transcript levels of
GHR with those of AR (A) and myostatin mRNA (B) in 27 healthy
older men.
AJP-Endocrinol Metab • VOL
not significantly related to IGF-I (r ⫽ ⫺0.16; P ⫽ NS)
mRNA levels (Table 3).
DISCUSSION
In the present study of healthy older men, we observed a significant negative relationship between
skeletal muscle myostatin and GHR gene expression,
as determined by mRNA levels. Myostatin is thought
to be a negative regulator of skeletal muscle growth,
because null mutations (31) and gene knockout experiments of myostatin (20) have resulted in significant
increases in muscle mass. Myostatin functionally decreases protein synthesis by inhibiting cell proliferation and DNA synthesis by blocking satellite cell (myoblast) activity (25, 26). In contrast, GH may exert its
stimulatory effects on muscle protein synthesis, in
part, by activating satellite cells (14). Thus the inverse
relationship between myostatin and GHR gene expression in the current study suggests that GH effects on
muscle protein synthesis (14) may, in part, occur by
inhibiting myostatin’s effects on satellite cell activation. Although the direct mechanism by which GH signaling could inhibit myostatin remains unclear, a GHresponsive element has been identified upstream of the
myostatin promoter (18, 25). Thus GH could directly
downregulate myostatin transcription. However, Kirk et
al. (13) observed no change in the expression of myostatin
levels associated with GH-induced muscle hypertrophy
in regenerating rat skeletal muscle.
Alternatively, the GH-induced expression of IGF-I
may indirectly inhibit myostatin expression. Musclederived IGF-I is associated with an upregulation of
myogenin (7), a regulatory factor that binds to myocyte
enhancer factor 2 (MEF2) start sites, and these start
sites have been identified within the myostatin promoter (13). Therefore, GH/IGF-I could modulate myostatin expression by interacting with various muscle
promoters in a competitive manner. However, we observed no relationship between the autocrine production of IGF-I and that of myostatin. Currently, we are
investigating this potential hypothesis further using a
cell culture model.
Taken together, these data suggest that GH excess
may override myostatin’s inhibition of satellite cell
activation rather than GH directly inhibiting myostatin promoter activity. Thus an increased expression of
muscle myostatin levels with age (S. Bhasin, personal
communication) may be due, in part, to the age-related
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significant (NS); Table 3]. However, we did detect a
significant direct relationship between GHR and AR
mRNA levels (r ⫽ 0.41; P ⫽ 0.04; Fig. 2), as well as a
trend indicating an inverse relationship between AR
and IGF-I mRNA levels (r ⫽ ⫺0.37; P ⫽ 0.06; Table 3),
indicating possible interactions between these anabolic
pathways.
Muscle transcript levels of the negative regulator
myostatin were inversely related to the anabolic GHR
(r ⫽ ⫺0.60; P ⫽ 0.001; Fig. 2) gene expression, and a
negative trend was observed between myostatin and
AR (r ⫽ ⫺0.36; P ⫽ 0.07) mRNA, but myostatin was
GHR, IGF-I, AR, AND MYOSTATIN IN ELDERLY SKELETAL MUSCLE
We thank Dr. Tom Wood [University of Texas Medical Branch
(UTMB) Molecular Core Laboratory] for critical comments in optimizing the RT-PCR protocol utilized and in sequencing the DNA
fragments, Dr. S. Bhasin and colleagues for supplying the primer
sets for myostatin, Dr. J. Jayme for valuable assistance in obtaining
the muscle biopsies, Carol St. Clair for performing the serum GH and
testosterone assays, Tracey A. Roy for performing the DEXA scans,
and the nursing staff of the Johns Hopkins Bayview Medical Center’s
General Clinical Research Center for expert assistance with patient
studies.
This research was supported in part by National Institutes of
Health (NIH) Research Grants T32-AG-00250–01A1 (to T. J. Marcell) and RO-1 AG-11002–5 (to M. R. Blackman); by General Clinical
Research Center Grant MO-1-RR-02719 from the National Center
AJP-Endocrinol Metab • VOL
for Research Resources, NIH, Bethesda, MD; by a Center of Excellence Grant from the ProMedica (T. J. Marcell, M. R. Blackman); and
by a Pilot Project Grant from the Sealy Center on Aging, UTMB,
Galveston, TX (T. J. Marcell).
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decrease in endogenous GH and testosterone levels,
contributing to age-related sarcopenia. Although the
functional significance of increased myostatin expression has been well documented, the mechanism by
which anabolic hormones could inhibit myostatin remains uncertain.
In keeping with the basic state of good health of
these study participants, none of the healthy older men
we studied would be considered frail or sedentary on
the basis of published values for fitness (24) or body
composition (8) for this age group. Nonetheless, aging
is associated with a progressive loss of lean body and
muscle mass and strength (2, 21), and thus these men
were relatively sarcopenic compared with published
data for younger men (17). Additionally, overnight
mean circulating GH, serum IGF-I, and T values were
similar to levels previously observed in comparably
aged men but were significantly reduced compared
with values for young adults (5, 12, 19).
Both circulating GH and T are thought to elicit their
effects on skeletal muscle protein synthesis by binding
to their receptors and increasing muscle gene transcription, including that of IGF-I (1, 28). Several lines
of evidence suggest that GH exerts effects on muscle
protein synthesis via locally produced rather than circulating IGF-I (14). Despite these mechanisms, we
could not detect relationships between circulating GH
or T and mRNA levels for their receptors, nor were
circulating GH and T related to intramuscular IGF-I
gene expression, as we had hypothesized.
As noted earlier, all of the subjects studied were old
and had relatively low hormone levels. Therefore, our
findings may reflect the limited variability of measured
GH and T within the group studied. Such relationships
may be better observed in younger persons in whom a
wider range of hormone values would be obtained.
Another possibility is that the normal relationships of
GH, IGF-I, and T to muscle gene activation are disrupted by the aging process itself. For example, in one
study of six elderly subjects, skeletal muscle IGF-I
mRNA levels were not increased after an acute GH
injection (30).
On the basis of the data in the present study, we
conclude that age-related deficits in both GH and T
may lead to an increase in myostatin expression and a
dissociation in autocrine IGF-I effects on skeletal muscle protein synthesis, both of which could contribute to
age-related sarcopenia.
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AJP-Endocrinol Metab • VOL
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EM. Dominant negative myostatin produces hypertrophy without hyperplasia in muscle. FEBS Lett 474: 71–75, 2000.
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21. Proctor DN, Balagopal P, and Nair KS. Age-related sarcopenia in humans is associated with reduced synthetic rates of
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23. Sheffield-Moore M, Wolfe RR, Gore DC, Wolf SE, Ferrer
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