Download Mechanical load increases muscle IGF

Am J Physiol Endocrinol Metab
280: E383–E390, 2001.
Mechanical load increases muscle IGF-I and androgen
receptor mRNA concentrations in humans
MARCAS M. BAMMAN,1,2 JAMES R. SHIPP,1 JIE JIANG,4
BARBARA A. GOWER,2.3 GARY R. HUNTER,1,3 ASHLEY GOODMAN,1
CHARLES L. MCLAFFERTY, JR.,1 AND RANDALL J. URBAN4
Departments of 1Human Studies, 2Physiology and Biophysics, and 3Nutrition Sciences,
University of Alabama at Birmingham, Birmingham, Alabama 35294; and 4Department of
Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77550
Received 20 January 2000; accepted in final form 25 October 2000
involves alternating
concentric (CON) and eccentric (ECC) muscle actions
against a constant external load, the magnitude of
which is limited by the individual’s CON strength. The
hypertrophic response after this biphasic training is
well documented (17, 23); however, the mechanism(s)
by which resistance training induces myofiber hypertrophy is as yet unclear. High-resistance ECC muscle
actions induce more sarcolemma damage than CON
muscle actions, as evidenced by greater elevations in
serum creatine kinase, myoglobin, and troponin I (32).
Furthermore, ECC loading leads to more severe and
more prolonged myofibrillar disruption, soreness, and
force deficit (15). Whether these events are causally
related to the growth response remains unknown; however, myofiber hypertrophy is blunted if the ECC phase
is omitted from conventional resistance training (17).
Hather et al. (17) previously found that CON/ECC
training induces hypertrophy of both type I and type II
myofibers, whereas hypertrophy is absent in type I
fibers and blunted in type II fibers after CON training
(matched with CON/ECC for total work). ECC loading
has also been shown to elevate muscle protein synthesis for a prolonged period in animals (35). It appears
that ECC loading, which is submaximal only during
conventional resistance exercise [ECC strength is 20–
50% greater than CON (6)], is important for maximizing hypertrophy.
Although no physiological consequences have been
demonstrated, transient elevations in serum anabolic factors such as growth hormone (GH) and testosterone have been documented after a bout of conventional resistance exercise (24), and increased
serum growth factors have also been noted across
several weeks of resistance training (23). This has
led to the speculation that one or more of these
factors may somehow stimulate resistance exerciseinduced myofibrillar protein synthesis. However, because the hypertrophic response is specific to the
loaded muscle(s), activation by a systemic hormone
would require load-mediated modulation of the hormone’s efficacy in the exercised muscle. Load-mediated modulation of receptor expression or binding
affinity in the muscle might explain localization of
the growth response with elevated serum anabolic
Address for reprint requests and other correspondence: Marcas M.
Bamman, Muscle Research Laboratory, EB207, 901 South 13th St.,
The Univ. of Alabama at Birmingham, Birmingham, AL 35294-1250
([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.
insulin-like growth factor I; muscle hypertrophy; resistance
exercise; creatine kinase; concentric; eccentric
CONVENTIONAL RESISTANCE TRAINING
http://www.ajpendo.org
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society
E383
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Bamman, Marcas M., James R. Shipp, Jie Jiang,
Barbara A. Gower, Gary R. Hunter, Ashley Goodman,
Charles L. McLafferty, Jr., and Randall J. Urban. Mechanical load increases muscle IGF-I and androgen receptor
mRNA concentrations in humans. Am J Physiol Endocrinol
Metab 280: E383–E390, 2001.—The mechanism(s) of loadinduced muscle hypertrophy is as yet unclear, but increasing
evidence suggests a role for locally expressed insulin-like
growth factor I (IGF-I). We investigated the effects of concentric (CON) vs. eccentric (ECC) loading on muscle IGF-I
mRNA concentration. We hypothesized a greater IGF-I response after ECC compared with CON. Ten healthy subjects
(24.4 ⫾ 0.7 yr, 174.5 ⫾ 2.6 cm, 70.9 ⫾ 4.3 kg) completed eight
sets of eight CON or ECC squats separated by 6–10 days.
IGF-I, IGF binding protein-4 (IGFBP-4), and androgen receptor (AR) mRNA concentrations were determined in vastus
lateralis muscle by RT-PCR before and 48 h after ECC and
CON. Serum total testosterone (TT) and IGF-I were measured serially across 48 h, and serum creatine kinase activity
(CK), isometric maximum voluntary contraction (MVC), and
soreness were determined at 48 h. IGF-I mRNA concentration increased 62% and IGFBP-4 mRNA concentration decreased 57% after ECC (P ⬍ 0.05). Changes after CON were
similar but not significant (P ⫽ 0.06–0.12). AR mRNA concentration increased (P ⬍ 0.05) after ECC (63%) and CON
(102%). Serum TT and IGF-I showed little change. MVC fell
10% and CK rose 183% after ECC (P ⬍ 0.05). Perceived
soreness was higher (P ⬍ 0.01) after ECC compared with
CON. Results indicate that a single bout of mechanical loading in humans alters activity of the muscle IGF-I system, and
the enhanced response to ECC suggests that IGF-I may
somehow modulate tissue regeneration after mechanical
damage.
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MECHANICAL LOAD AND MUSCLE IGF-I IN HUMANS
concentrations of IGF-I and IGFBP-4. We hypothesized
that the local IGF-I response after a single bout of
resistance exercise would be greater when heavy ECC
muscle actions were performed as opposed to heavy
CON muscle actions. Levels of IGF-I and IGFBP-4
mRNA in vastus lateralis muscle samples were determined before and 48 h after ECC and CON. We also
measured muscle androgen receptor (AR) mRNA concentration and serum levels of testosterone and IGF-I
to investigate possible interactions between serum anabolic factors and local tissue responses.
METHODS
Subjects. Ten healthy subjects (7 men, 3 women) ⬎19 yr of
age participated in this study. Each subject was screened for
health history by means of a health status questionnaire and
for level of activity by use of the Baecke Questionnaire of
Habitual Physical Activity. Exclusion criteria included current lower-body resistance training or previous history of
diagnosed condition or illness that would endanger the subject during strenuous resistance exercise. All subjects were
given an oral and written briefing of the study before signing
informed consent forms. The study was approved by the
Institutional Review Board and was conducted in the General Clinical Research Center (GCRC) at the University of
Alabama at Birmingham.
Familiarization sessions. Each subject completed three
familiarization sessions on a Smith squat machine (York
Barbell, Wright Exercise Equipment, Birmingham, AL) to
learn proper execution of the exercise and to become familiar
with heavy loads. The third familiarization was completed
ⱖ5 days before the first exercise session in an effort to
prevent residual effects of the familiarization routine. In
familiarization 1, subjects warmed up with a light weight
and performed one set of 10 biphasic (CON/ECC) repetitions
with a comfortable submaximal load. In familiarization 2,
which was 3 days later, subjects warmed up, completed a
one-repetition maximum (1RM) strength test, and concluded
the session with three sets of 10 repetitions at 60% of 1RM.
Approximately 3–4 days later, subjects returned for the final
familiarization, which consisted of a warm-up, a 1RM test,
three sets of 10 repetitions at 70% of that day’s 1RM, and two
sets of 8 repetitions at 80% of 1RM.
Strength testing. As mentioned in the preceding section,
each subject performed a 1RM squat strength test during the
second and third familiarizations. After a sufficient warm-up
period, sets of one repetition were executed with increasing
load until two failed attempts occurred at a given weight.
1RM was recorded as the highest weight successfully lifted.
Each attempt was separated by 2 min.
Isometric maximum voluntary contraction (MVC) strength
was assessed unilaterally during knee extension. MVC was
determined at a knee angle of 1.91 rad (110°) using a calibrated force transducer (Omega) interfaced with a desktop
computer. Force output was recorded at 100 Hz, and the
system provided visual numeric feedback for both subject
and investigator. During each test, three MVCs (6 s duration)
were performed separated by 1- to 2-min rest periods. The
mean of the two highest peak forces (kg) obtained across the
three trials was used for analysis. Subjects were instructed to
contract as hard as possible and were verbally encouraged
throughout each trial. MVC was assessed before the familiarization sessions and was repeated 48 h post-CON and
post-ECC.
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hormones. Whether ECC loading enhances local
mechanism(s) in humans is largely unknown.
Increasing evidence indicates that modulation of
muscle protein turnover is tightly regulated by a number of locally expressed tissue growth factors (1, 12,
13). Insulin-like growth factor I (IGF-I) is known to
stimulate myoblast proliferation and differentiation in
vitro (13) as well as muscle protein synthesis (22) and,
as such, has received increasing attention in studies of
muscle hypertrophy. Local expression of IGF-I in skeletal muscle appears to be load sensitive and acts independently of any change in serum GH or IGF-I (1).
Furthermore, Yang et al. (37) recently identified two
isoforms of IGF-I in skeletal muscle, one of which
appears to be regulated exclusively by mechanical load.
Some have proposed the name mechanogrowth factor
(MGF) for this mechanically sensitive isoform (16).
This local IGF-I (or MGF) is thought to induce myofiber
hypertrophy by autocrine (i.e., direct stimulation of
myofibrillar protein synthesis) and/or paracrine (i.e.,
satellite cell proliferation, differentiation, and fusion)
action (1).
The efficacy of muscle IGF-I is dependent not only on
its expression but also on its availability, which is
regulated by a family of six IGF binding proteins (BPs)
and by the abundance of the type 1 IGF receptor. For
example, in muscle, IGFBP-4 has a high affinity for
IGF-I and thus inhibits its myogenic effects, whereas
IGFBP-5 may facilitate (13) or inhibit (21) IGF-I-stimulated differentiation under certain conditions. Additionally, IGFBP-1 has been shown to inhibit IGF-Istimulated protein synthesis (14).
The increase of serum IGF-I with exogenous administration of GH or IGF-I does not appear to stimulate
myofiber hypertrophy in the absence of mechanical
load (4), nor does systemic GH treatment enhance the
hypertrophic effect of resistance training (33, 38). However, induction of exogenous IGF-I directly into skeletal muscle does increase muscle mass (2), suggesting
that any stimulus causing an increase in muscle IGF-I
availability may lead to muscle growth. Urban et al.
(34) have recently shown that exogenous testosterone
administration in hypogonadal older men increases
muscle strength and protein synthesis and is associated with increased muscle IGF-I mRNA concentration
with a concomitant reduction in IGFBP-4 mRNA. Muscle IGF-I mRNA content is also increased in muscle
after heavy exercise in humans and after stretch in
animals (37). Taken together, these findings lead to the
attractive speculation that mechanical load associated
with resistance exercise may increase muscle IGF-I
availability, which in turn may cause increased myofibrillar protein synthesis, satellite cell activation, and
consequent myofiber hypertrophy. Because ECC loading appears to enhance the hypertrophic response to
resistance training (17) and because stretch tension
increases muscle IGF-I mRNA (37), it also seems plausible that ECC loading may enhance the local IGF-I
response.
In this study we investigated the separate effects of
CON and ECC resistance exercise on muscle mRNA
MECHANICAL LOAD AND MUSCLE IGF-I IN HUMANS
side dominant. Each subject rated muscle soreness on a 0–10
scale that included corresponding descriptions of the levels of
discomfort he/she was experiencing, with 0 ⫽ no soreness
and 10 ⫽ unbearably sore and difficult to move.
Muscle biopsy procedure. The vastus lateralis muscle of the
right leg was sampled by percutaneous needle biopsy as we
described previously (5). Each subject was biopsied on three
occasions: 48 h before beginning the familiarizations, 48 h
post-CON, and 48 h post-ECC. Tissue samples were snap
frozen in liquid nitrogen and stored at ⫺80°C until analysis.
Total RNA isolation and qualitative RT-PCR. RT-PCR was
performed as described previously (30). Total RNA was isolated from muscle biopsy specimens (50–100 mg) using RNAzol B (Tel-Test, Friendswood, TX). Total RNA (0.5–1.0 ␮g)
was then converted to cDNA by means of a reverse transcription kit (Promega, Madison, WI). Five microliters of cDNA
solution were then subjected to PCR in the presence of the
appropriate primers (Table 1). PCR began with 1 cycle (2
min) at 94°C followed by amplification cycles consisting of 1
min at 94°C, 1 min at 55°C, and 1 min at 72°C. The optimum
number of amplification cycles was predetermined in control
experiments and consisted of 26 cycles for AR and 25 cycles
for both IGF-I and IGFBP-4.
The products of the PCR were run on Southern gel, and
amplified DNA products were sized by DNA ladder. Within
subjects, samples from the three time points were run in
adjacent lanes. Southern blots were then made and hybridized to oligonucleotides of the DNA fragment (Table 1). Band
densities on the Southern blots were quantified by densitometry. For standardization, optical density of each band was
corrected for background, and the band density for glyceraldehyde phosphate dehydrogenase was used as the adjusting
factor.
Serum IGF-I and total testosterone. Total IGF-I and total
testosterone were determined in serum samples withdrawn
before exercise and at 0.5, 1, 2, 4, 8, 24, and 48 h after CON
and ECC bouts. Total IGF-I was determined by immunoradiometric assay (Diagnostic Systems Laboratories,) using
125
I. Total testosterone was determined by solid-phase radioimmunoassay (Diagnostic Products, Los Angeles, CA) using
125
I. All samples within subjects for a given hormone were
assayed in random order during a single run. Mean intraassay coefficients of variation (CVs) ranged from 2 to 7%, and
mean interassay CVs ranged from 3 to 10%.
Serum creatine kinase. Total creatine kinase (CK) activity
was determined in serum samples immediately before exercise and at 24 and 48 h after CON and ECC. CK assays of all
samples were performed in random order by means of a
Table 1. PCR primers and hybridization oligonucleotides used
IGF-I
AR
IGFBP-4
GAP, 206 bp
GAP, 473 bp
sense (1 ␮mol/l)
antisense (1 ␮mol/l)
oligonucleotide
sense (2 ␮mol/l)
antisense (2 ␮mol/l)
oligonucleotide
sense (1 ␮mol/l)
antisense (1 ␮mol/l)
oligonucleotide
sense (0.04 ␮mol/l)
antisense (0.04 ␮mol/l)
oligonucleotide
sense (0.02 ␮mol/l)
antisense (0.02 ␮mol/l)
oligonucleotide
5⬘-AAATCAGCAGTCTTGGAACC-3⬘
5⬘-CTTCTGGGTCTTGGGCATGT-3⬘
5⬘-CAGGGGCTTTTATTTCAACAAG-3⬘
5⬘-GATGCTCTACTTCGCCCCTGA-3⬘
5⬘-CCCAGCAAATAGAATTCCATGAC-3⬘
5⬘-CTGGGTGTGGAAATAGATG-3⬘
5⬘-CCATCCAGGAAAGCCTGCA-3⬘
5⬘-TGGAAGTTGCCGTTGCGGT-3⬘
5⬘-CAGGTGCCTGCAGAAGCA-3⬘
5⬘-GGAGTCAACGGATTTGGT-3⬘
5⬘-GTGATGGGATTTCCATTGAT-3⬘
5⬘-TCAGCCTTGACGGTGCCATG-3⬘
5⬘-GGTATCGTGGAAGGACTCAT-3⬘
5⬘-TCCACCACCCTGTTGCTGTA-3⬘
5⬘-GTGGGTGTCGCTGTTGAAGT-3⬘
IGF-I, insulin-like growth factor I; AR, androgen receptor; GAP, glyceraldehyde phosphate dehydrogenase.
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GCRC procedures. Each subject had two exercise days; one
day, ECC-only resistance exercise was performed, and the
other day, CON-only resistance exercise was performed.
CON and ECC bouts were separated by 6–10 days, and order
was randomly assigned. For CON, subjects performed eight
sets of eight repetitions with resistance for the first set
established at 85% of the 1RM achieved in the third familiarization. For ECC, subjects also performed eight sets of
eight repetitions, but the resistance for the first set was
established at 110% of the 1RM. The resistance was set at
these levels to maintain relative workloads (because ECC
strength is 20–50% greater than CON) and thus maximize
motor unit recruitment in both CON and ECC modes. Resistance was decreased if a subject began to fatigue to the point
that proper form, repetition number, and/or pace could not be
maintained. If a subject reached volitional fatigue after six
repetitions or fewer, the load was reduced 5–10 kg for the
next set. Volitional fatigue during CON was defined as an
inability to ascend without assistance. During ECC, fatigue
was defined as a controlled descent faster than the 2-s minimum. Within subjects, a minimum squat depth was defined
during familiarization sessions as a point in the range of
motion at which the subject’s femur was horizontal (parallel
with the floor). This minimum descent was used as a criterion
for successful performance during both 1RM testing and the
CON and ECC exercise bouts.
On each of the two exercise days, subjects reported to the
GCRC in a fasted state between 0700 and 0800, and a
catheter was placed in an antecubital vein shortly after
arrival. The subjects lay quietly for 30–40 min before a
baseline blood draw. This was immediately followed by the
prescribed exercise bout. Subjects remained in the GCRC for
8 h after exercise for serial blood sampling and dietary
control. Postexercise blood samples (5 ml) were withdrawn at
0.5, 1, 2, 4, and 8 h. A mixed diet was prescribed by the GCRC
dietetics staff. The diet was designed to be isocaloric based on
the subject’s body weight, and the same three meals were
given on each of the two exercise days. The three meals were
given at the following times: 1) just after the 1-h postexercise
blood draw, 2) 3 h postexercise, and 3) 6 h postexercise.
Subjects returned to the GCRC in a fasted state at 24 h
postexercise for a blood draw and at 48 h postexercise for a
blood draw and muscle biopsy. Also at 48 h, subjects rated
perceived soreness on an 11-point scale and completed the
MVC strength test to assess functional damage within the
muscle. Because these tests immediately followed the muscle
biopsy (right thigh), MVC and soreness were determined in
the contralateral left limb. Nine of ten subjects were right-
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MECHANICAL LOAD AND MUSCLE IGF-I IN HUMANS
RESULTS
Subject characteristics are shown in Table 2. The
mean repetitions and mean loads for CON and ECC
were computed from each subject’s average across
eight sets. As described in METHODS, the target repetition number was eight per set. Every effort was made
to achieve an equivalent number of repetitions on CON
and ECC days; thus individual loads were adjusted
slightly for each set based on performance in the previous set (see METHODS for details). As shown in Table 2,
the method of load adjustment resulted in nearly identical numbers of repetitions during CON and ECC.
Moreover, the average loads for CON (82%) and ECC
(102%) approximated the target loads of 85 and 110%
1RM for CON and ECC, respectively. ECC loading
elicits a blunted metabolic response and activates
fewer motor units than CON at the same absolute
workload (3, 28). Loads were 20–30% higher during
ECC loading in an effort to match relative intensity
Table 3. Indexes of muscle soreness and/or damage
MVC,* kg
Soreness, ordinal
scale, 0–10
Creatine kinase,
U/l
CON
ECC
Baseline
48 h PostCON
48 h Post-ECC
72.4 ⫾ 4.6
77.6 ⫾ 4.2
65.3 ⫾ 3.9†‡
0 (0–3)
6 (2–8)‡
Baseline
24 h
postexercise
48 h
postexercise
345.2 ⫾ 67.0
224.6 ⫾ 47.0
326.0 ⫾ 71.5
449.4 ⫾ 89.0
264.6 ⫾ 65.1
636.3 ⫾ 244.6†
Ordinal soreness data are shown as median (range). All other
values are means ⫾ SE. MVC, maximum voluntary isometric knee
extension contraction. Baseline MVC was tested only once (before
any exercise). * Main time effect, P ⬍ 0.05; † different from baseline,
P ⬍ 0.05; ‡ different from 48 h post-CON, P ⬍ 0.01.
and thus maximize motor unit activity in both modes of
exercise.
Measurements used to assess relative muscle damage after CON and ECC are presented in Table 3.
Isometric MVC was unchanged 48 h after CON but was
depressed 10% 48 h after ECC (P ⬍ 0.05). Total CK was
not different from baseline 24 and 48 h after CON. CK
activity was elevated 183% 48 h after ECC (P ⬍ 0.05).
The average rating on the 11-point soreness scale was
significantly higher (P ⬍ 0.01) 48 h after ECC compared with CON.
RT-PCR results for IGF-I, IGFBP-4, and androgen
receptor mRNAs are shown in Fig. 1. Repeated-measures ANOVA revealed significant time effects (P ⬍
0.05) for IGFBP-4 and AR mRNA concentrations but
not for IGF-I mRNA concentration (P ⫽ 0.21). Results
for IGF-I mRNA concentration were highly variable
between subjects and across time. A priori planned
comparisons between the baseline value and 48 h postCON and between baseline and 48 h post-ECC for
IGF-I mRNA concentration showed a 62% increase
Table 2. Descriptive characteristics of all subjects
Age, yr
Height, cm
Weight, kg
1RM squat, kg
Average repetitions per set
CON
ECC
Average load per set, kg
CON
ECC
24.4 ⫾ 0.7
174.5 ⫾ 2.6
70.9 ⫾ 4.3
136.1 ⫾ 10.3
7.9 ⫾ 0.1
7.7 ⫾ 0.1
111.6 ⫾ 7.6
139.2 ⫾ 11.8
Values are means ⫾ SE; n ⫽ 10 subjects. 1RM, one-repetition
maximum; CON, concentric exercise; ECC, eccentric exercise.
Fig. 1. Concentrations of mRNA for insulin-like growth factor
(IGF) I, IGF binding protein-4 (IGFBP-4), and androgen receptor
(AR) at baseline and 48 h after concentric (CON) and eccentric (ECC)
loading. Values are means ⫾ SE. *Different from baseline, P ⬍ 0.05.
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Synchron LX System (Beckman Coulter, Fullerton, CA) following manufacturer’s instructions. By use of this system,
the within- and between-run CVs for serum in our laboratory
are 2.1 and 1.2%, respectively.
Data analysis. Results are reported as means ⫾ SE. Muscle biopsy and isometric strength performance data were
analyzed by one-way repeated-measures ANOVA with three
time points, defined as preexercise baseline, 48 h post-CON,
and 48 h post-ECC. A priori planned comparisons were analyzed between the baseline value and each of the two 48-h
postexercise values.
Serum hormone and CK data were analyzed using a new
baseline blood draw for each exercise day. The new baseline
was established to prevent any residual effect of previous
exercise on the postexercise values after the second exercise
session. Hormone levels were evaluated by repeated-measures ANOVA with two repeat factors: 1) mode (CON and
ECC) and 2) eight time points (baseline and 0.5, 1, 2, 4, 8, 24,
and 48 h postexercise). Total testosterone data for three
female subjects were excluded from the testosterone analysis, because several values were below the detectable range of
the assay. CK levels were tested by repeated-measures
ANOVA in similar fashion but with three time points (baseline, 24 h postexercise, and 48 h postexercise). The least
squares difference (LSD) test was employed for post hoc
testing. Scores on the subjective soreness rating 48 h after
CON and ECC were compared using the Wilcoxon matched
pairs test. Significance for all tests was accepted at P ⬍ 0.05.
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MECHANICAL LOAD AND MUSCLE IGF-I IN HUMANS
after CON (P ⫽ 0.005). IGF-I was elevated 48 h after
CON (P ⫽ 0.006), but no changes were found after ECC
loading.
DISCUSSION
after ECC (P ⬍ 0.05) with a similar trend after CON
(P ⫽ 0.12). Planned comparisons identified a 57% reduction in IGFBP-4 mRNA after ECC (P ⬍ 0.01) and a
strong trend toward decreased IGFBP-4 after CON
(P ⫽ 0.06). AR mRNA concentration was elevated (P ⬍
0.05) 102 and 63% after CON and ECC, respectively.
Southern blot results for IGF-I, IGFBP-4, and AR
mRNAs are shown in Fig. 2.
Serum levels of total testosterone and IGF-I are
shown in Table 4. Because testosterone was not within
the detectable range for several samples among the
three women, testosterone data are presented only for
men. Main time effects were found for testosterone
after both CON (P ⬍ 0.001) and ECC (P ⫽ 0.026)
loading. After both exercise bouts, total testosterone
levels tended to fall. Testosterone was depressed after
CON at 1, 2, 4, and 8 h after exercise (P ⬍ 0.05). After
ECC, testosterone was lower than baseline for all time
points beyond 0.5 h postexercise (P ⬍ 0.05) except at
24 h. A time effect was noted for serum IGF-I (n ⫽ 10)
Table 4. Serum hormone levels before and after CON and ECC loading
Testosterone* (ng/dl)
CON†
ECC†
IGF-I (ng/ml)
CON†
ECC
Baseline
0.5 h
1h
2h
4h
8h
24 h
48 h
621 ⫾ 63
589 ⫾ 60
555 ⫾ 62
580 ⫾ 77
504 ⫾ 50‡
486 ⫾ 65‡
417 ⫾ 45‡
447 ⫾ 63‡
416 ⫾ 44‡
444 ⫾ 46‡
451 ⫾ 48‡
451 ⫾ 36‡
586 ⫾ 67
496 ⫾ 73
542 ⫾ 40
472 ⫾ 46‡
349 ⫾ 37
412 ⫾ 70
317 ⫾ 22
361 ⫾ 43
345 ⫾ 36
371 ⫾ 45
358 ⫾ 32
361 ⫾ 47
352 ⫾ 44
350 ⫾ 34
372 ⫾ 49
373 ⫾ 35
385 ⫾ 35
379 ⫾ 41
416 ⫾ 49‡
386 ⫾ 34
Values are means ⫾ SE. * Testosterone data for men only (n ⫽ 7). † Main time effect, P ⬍ 0.05; ‡ different from baseline, P ⬍ 0.05.
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Fig. 2. Sample Southern blot results for IGF-I, IGFBP-4, and AR
mRNAs before loading (PRE), 48 h after concentric loading (CON),
and 48 h after eccentric loading (ECC). Samples from each subject
were run in adjacent lanes as depicted here. A: ECC performed 6–10
days before CON; B and C: CON performed 6–10 days before ECC.
GAP, glyceraldehyde phosphate dehydrogenase.
Based on previous work linking muscle hypertrophy
with local availability of IGF-I (2) or with ECC loading
(17), the primary objective of this study was to determine whether ECC loading was associated with increased availability of local IGF-I in the working muscles. We noted a high degree of variability in IGF-I
mRNA concentrations between subjects and across
time. As a result, no main time effect was found in the
ANOVA model. However, mean IGF-I mRNA levels
trended upward 48 h after both CON and ECC loading,
with the increase after ECC being significant as determined by planned comparison. To our knowledge, this
is the first evidence in humans of muscle IGF-I transcriptional modulation after a single bout of heavy
resistance exercise. Using immunohistochemistry, two
recent investigations report increased IGF-I staining
in human muscle after several bouts of high-intensity
exercise (18, 31). Hellsten et al. (18) found an increased
number of IGF-I immunoreactive capillaries and satellite cells in vastus lateralis muscle after 7 days of
intense military training including 150 km of terrain
marching with a 30-kg overload (i.e., gear). The increased IGF-I staining was accompanied by a sixfold
increase in serum CK activity. Singh et al. (31) found a
marked 491% increase in IGF-I staining in vastus
lateralis of frail elderly after 10 wk of leg resistance
training. Greater IGF-I staining at baseline was predictive of the magnitude of muscle hypertrophy (r ⫽
0.70). It should be noted that both of these exercise
protocols included both CON and ECC loading. IGF-I
immunoreactivity within myofibers has also been
shown to rise 4 days after 5 days of successive electrically stimulated ECC loading in the rat tibialis anterior (36). Others have reported increases in muscle
IGF-I peptide with no concomitant increase in IGF-I
gene expression after 5 days of endurance training in
rats (11).
The optimum time point for an acute postexercise
elevation in muscle IGF-I content is unknown. Increased myofiber IGF-I mRNA has been detected by in
situ hybridization immediately after 6 days of continuous stretch in rabbits (37). We elected to biopsy the
vastus lateralis muscle 48 h after the loading bouts to
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MECHANICAL LOAD AND MUSCLE IGF-I IN HUMANS
Based on the CK and soreness data after ECC loading, we suggest that myofibrillar disruption and/or
sarcolemma damage may play a role in activating the
muscle IGF-I system. We have previously shown an
increase in serum levels of acidic fibroblast growth
factor (aFGF) after resistance exercise with an ECC
component (7). The FGF response paralleled an increase in serum CK activity. FGFs are powerful proliferative agents in myoblast cultures (12) and have been
shown to induce myotube hypertrophy (8). Additionally, FGFs are known to be released directly from
mechanically wounded muscle cells (8). Because we
report increased IGF-I mRNA after 48 h and others
have shown increased IGF-I peptide after 4–6 days (31,
36), it is possible that the IGF-I response is activated
by an earlier local release of FGF or some other factor(s). Based on these data, we suggest that IGF-I
activity is somehow linked to mechanisms involved in
tissue regeneration after mechanical damage. One
problem with this hypothesis, however, is the fact that
ECC exercise-induced muscle damage does not necessarily result in hypertrophy. Marked muscle damage
follows unaccustomed endurance exercise with a large
ECC component (e.g., downhill running) (32); however,
endurance exercise is not a potent hypertrophic stimulus (23). The ECC damage associated with high-tension contractions during resistance exercise is somehow uniquely different. This is an attractive area for
further study.
Another possible mechanism for increasing muscle
IGF-I availability is via androgen activation. Increased
muscle protein synthesis after testosterone therapy in
older men has been shown to be associated with increased IGF-I mRNA concentration and a concomitant
reduction in IGFBP-4 mRNA concentration (34). Furthermore, inducing androgen deficiency in young men
decreases muscle IGF-I mRNA concentration and
causes muscle atrophy (26). These data suggest that
IGF-I action in muscle is secondary to androgen activity. In the seven men in the present study, serum total
testosterone was not increased but fell after both CON
and ECC loading. The testosterone response to resistance exercise is highly variable (24). The fall in serum
testosterone across time in the present study most
likely reflects diurnal variation, as testosterone levels
are highest in the morning and decrease into the
evening hours. In support of this, testosterone values
were not different from the initial morning levels 24 h
after both exercise bouts. However, we found a substantial increase in AR mRNA concentration after both
CON and ECC loading. To our knowledge, this is the
first report of such an increase in humans after an
acute exercise bout. AR content has been shown to
increase in type II muscle after resistance training in
rats (10). Also in rats, gastrocnemius hypertrophy by
electrical stimulation has been associated with an increased number of muscle ARs (20). Our data indicate
both AR and IGF-I mRNA concentrations are upregulated by heavy mechanical load. Although the mechanism is not clear, muscle androgen and IGF-I activities
may be related.
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.6 on August 12, 2017
make the sample time coincide with previous reports of
1) increased mixed muscle protein synthesis after an
acute bout of heavy resistance exercise (29) and 2)
drops in isometric force and increases in myofibrillar
disruption after ECC loading (19). Although a direct
link is not apparent, our data indicate that important
changes in the local IGF-I system occur during the
acute phase of tissue repair and regeneration after
mechanical loading. Singh et al. (31) and Yan et al. (36)
studied muscle 4–6 days after exercise and found large
increases in immunoreactivity for IGF-I peptide, suggesting that the posttranscriptional effect continues for
at least a few days.
The biological activity of IGF-I is regulated by a
family of six IGFBPs in serum and in extravascular
tissues. In skeletal muscle, the myogenic effects of
IGF-I are inhibited by IGFBP-4, which appears to
exert a strong affinity for IGF-I, thereby reducing the
level of free IGF-I (9, 34). In cultured myoblasts, both
proliferation and differentiation are inhibited by overexpression of IGFBP-4 (9). We report a 56% drop in
IGFBP-4 mRNA after ECC loading and a 31% (nonsignificant) decrease after CON. Additional components of
the IGF-I system not measured in this study (including
other IGFBPs and the type 1 IGF receptor) can certainly modulate the availability of IGF-I in muscle.
Because, however, IGFBP-4 has been consistently
shown to inhibit IGF-I action in muscle cells, we suggest that the decrease in IGFBP-4 mRNA concentration coupled with the increase in IGF-I mRNA found in
this study would promote an increase in IGF-I availability within the muscle after mechanical loading.
These changes occurred in the absence of any increase
in serum IGF-I. This is not surprising. Several others
have reported that muscle IGF-I activity is independent of changes in serum IGF-I (1). Further, this is not
the first report of unchanging serum IGF-I after a
single bout of heavy resistance exercise (25).
Although evidence linking local IGF-I availability to
growth potentiation in muscle is mounting (1), the
mechanism by which mechanical load modulates IGF-I
expression is not clear. In his comprehensive review,
Adams (1) proposes that this load-sensitive IGF-I in
muscle works by paracrine or autocrine action to induce satellite cell proliferation and differentiation, followed by fusion of differentiated myoblasts to hypertrophying myofibers. The process of myoblast fusion is
thought to maintain myonuclear domain size and thus
the capacity for muscle protein synthesis. Indeed, myonuclear domain is maintained during mechanical loadinduced myofiber hypertrophy (27). The model proposed by Adams (1) is well supported; however, the
direct link between high-intensity mechanical load and
muscle IGF-I modulation requires further investigation. Goldspink (16) has proposed a mechanotransduction mechanism involving cytoskeletal proteins during
stretch. By this proposed mechanism, stretch tension
on the basement membrane (e.g., laminin) physically
activates intracellular signaling via a membranebound signaling molecule on the plasmalemma.
MECHANICAL LOAD AND MUSCLE IGF-I IN HUMANS
We report for the first time changes in muscle
mRNAs associated with tissue growth and repair after
a single bout of resistance exercise in humans. The
novel approach of studying high-intensity CON and
ECC loading separately enabled us to test the influence
of ECC action on the IGF-I and AR responses to mechanical load. The results indicate that high-intensity
lengthening and shortening contractions both induce
muscle IGF-I and AR gene transcription. Moreover, the
enhanced IGF-I activation after ECC loading supports
the concept that IGF-I is somehow involved in tissue
regeneration after mechanical load-induced damage.
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We thank the participants for their tolerance and tireless effort.
We thank Leo Wright of Wright Exercise Equipment in Birmingham,
Alabama, for providing the exercise equipment used in this study.
This work was funded by a University of Alabama at Birmingham
Provost’s Faculty Development Award (M. M. Bamman), National
Institutes of Health GCRC Grant M01-RR-00032, and National Institutes of Health 1 R01-AG/AR-11000.
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