Beruflich Dokumente
Kultur Dokumente
Adams
J Appl Physiol 93:394-403, 2002. First published Feb 8, 2002; doi:10.1152/japplphysiol.01153.2001
This article cites 60 articles, 29 of which you can access free at:
http://jap.physiology.org/cgi/content/full/93/1/394#BIBL
This article has been cited by 35 other HighWire hosted articles, the first 5 are:
Skeletal muscle growth in young rats is inhibited by chronic exposure to IL-6 but preserved
by concurrent voluntary endurance exercise
P. W. Bodell, E. Kodesh, F. Haddad, F. P. Zaldivar, D. M. Cooper and G. R. Adams
J Appl Physiol, February 1, 2009; 106 (2): 443-453.
[Abstract] [Full Text] [PDF]
Gene expression profiling of skeletal muscle in exercise-trained and sedentary rats with
inborn high and low VO2max
Updated information and services including high-resolution figures, can be found at:
http://jap.physiology.org/cgi/content/full/93/1/394
Additional material and information about Journal of Applied Physiology can be found at:
http://www.the-aps.org/publications/jappl
Journal of Applied Physiology publishes original papers that deal with diverse areas of research in applied physiology, especially
those papers emphasizing adaptive and integrative mechanisms. It is published 12 times a year (monthly) by the American
Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2005 by the American Physiological Society.
ISSN: 8750-7587, ESSN: 1522-1601. Visit our website at http://www.the-aps.org/.
J Appl Physiol 93: 394–403, 2002.
First published February 8, 2002; 10.1152/japplphysiol.01153.2001.
highlighted topics
Exercise Effects on Muscle Insulin Signaling and Action
Selected Contribution: Acute cellular and molecular
responses to resistance exercise
FADIA HADDAD AND GREGORY R. ADAMS
Department of Physiology and Biophysics, University of California, Irvine, Irvine, California 92697
Received 21 November 2001; accepted in final form 5 February 2002
Haddad, Fadia, and Gregory R. Adams. Selected Con- grams in humans. Clearly, alterations in the types and
tribution: Acute cellular and molecular responses to resistance amounts of cellular proteins present in myofibers must
Address for reprint requests and other correspondence: G. R. The costs of publication of this article were defrayed in part by the
Adams, Univ. of California Irvine, Dept. of Physiology and Biophys- payment of page charges. The article must therefore be hereby
ics, 346-D Medical Sciences 1, Irvine, CA 92697-4560 (E-mail: marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
gradams@uci.edu). solely to indicate this fact.
394 8750-7587/02 $5.00 Copyright © 2002 the American Physiological Society http://www.jap.org
CELLULAR RESPONSE TO RESISTANCE EXERCISE 395
possible to fully understand the regulatory components vivarium cages and allowed food and water ad libitium. All
that control and coordinate adaptation. treatment protocols were approved by the institutional ani-
On the basis of both in vitro and in vivo studies, it mal research committee.
has become clear that two primary processes are in- Unilateral muscle exercise protocol. Rats were anesthetized
with ketamine and acepromazine (40 and 2 mg/kg, respective-
volved with the compensatory hypertrophy response in ly), and a small incision was made along the posterior aspect of
mammalian skeletal muscles. The first and most obvi- the right lower limb to allow the placement of fine wire plati-
ous would be anabolic processes necessary for the ac- num electrodes adjacent to the sciatic nerve. The rats were then
cretion of protein to support myofiber enlargement. positioned in a specially built training platform described pre-
The second process involves the proliferation of satel- viously (11). The right leg was positioned in a foot plate at-
lite cells, which appears to be necessary to provide tached to an isometric force transducer interfaced with a com-
additional myonuclei to the enlarging myofibers (5, 45, puter. The electrodes were connected to a Grass S-48 stimulator
46, 48 –50, 54). In each case, the cellular and molecular via a stimulus isolation unit. The voltage (6–9 V), stimulation
mechanisms underlying these processes have been ex- frequency (35–40 Hz), and foot position (e.g., ankle ⬃85–90°)
tensively characterized in a number of cell types, par- were adjusted to produce maximal isometric tension. For exer-
cise bouts, the stimulation parameters were 3 maximal isomet-
ticularly in cell culture settings. In particular, the ric contractions per minute, 4-s duty cycle and 16 s of rest, for 30
steps associated with the initiation of cellular prolifer- min. The timing of the contractions was controlled by the
ation are generally well known (e.g., can be found in interfaced computer. Force output was monitored on the com-
cell biology textbooks). puter screen during each contraction. After the exercise bout,
The principles of resistance training implicitly as- the electrodes were withdrawn and the incision was closed with
sume that multiple bouts of exercise, imposed within a single wound clip.
an appropriate time frame, must stimulate and rein- An isometric training protocol was chosen to minimize the
The 18S primers were mixed with competimers at an opti- bodies used detected changes in phosphorylation of residue
mized ratio that could range from 1:4 to 1:10, depending on Thr389 of S6K1 and Thr183 and Tyr185 of rat ERK2. In each
the abundance of the target mRNA. Inclusion of 18S compe- case, phosphorylation at these sites is critical for increased
timers was necessary to bring down the 18S signal, which activity in vivo (40, 57). Muscle samples were extracted by
allows its linear amplification to the same range as the homogenization in seven volumes of ice-cold buffer A [50 mM
coamplified target mRNA (Ambion, relative RT-PCR kit pro- Tris 䡠 HCl, pH 7.8, 2 mM potassium phosphate, 2 mM EDTA,
tocol). 2 mM EGTA, 50 mM -glycerophosphate, 10% glycerol, 1%
For each specific target mRNA, the RT-PCR reactions were Triton X-100, 1 mM dithiothreitol, 3 mM benzamidine, 1 mM
carried out under identical conditions by using the same sodium orthovanadate, 10 M leupeptin, 5 g/ml aprotinin,
reagent premix for all the samples to be compared in the 200 g/ml soybean trypsin inhibitor, and 1 mM 4-(2-amino-
study. To validate the consistency of the analysis procedures, ethyl)-benzenesulfonyl fluoride HCl] by use of a motor-driven
at least one representative from each group was included in glass pestle. The homogenate was immediately centrifuged
each RT-PCR run. at 12,000 g for 30 min at 4°C. The supernatant was immedi-
One microliter of each RT reaction (0- to 10-fold dilution
ately saved in aliquots at ⫺80°C for subsequent use in im-
depending on target mRNA abundance) was used for the
munoblotting. The supernatant protein concentration was
PCR amplification. The PCR reactions were carried out in the
determined by using the Bio-Rad protein assay with BSA as
presence of 2 mM MgCl2 by using standard PCR buffer
(GIBCO), 0.2 mM dNTP, 1 M specific primer set, 0.5 M standard. Approximately 50 g of supernatant proteins were
18S primer/competimer mix, and 0.75 unit Taq DNA poly- subjected to SDS-PAGE [12.5% T, according to standard
merase (GIBCO) in 25 l of total volume. Amplifications were protocol (32)] and then electrophoretically transferred to a
carried out in a Stratagene Robocycler with an initial dena- polyvinylidene difluoride membrane (Immobilon-P) with the
turing step of 3 min at 96°C, followed by 25 cycles of 1 min at use of 10% methanol, 1 mM orthovanadate, 25 mM Tris, and
96°C, 1 min at 55°C (55–60°C depending on primers), 1 min 193 mM glycine, pH 8.3. Phospho-ERK1/2 and phospho-S6K1
Fig. 3. Increased mechano-growth factor (MGF) and insulin-like growth factor (IGF)-I mRNA after resistance
exercise. Representative PCR gel scans demonstrate the observed changes in MGF and IGF-I mRNA in the
nonstimulated contralateral muscles (C) and in muscles at 16 (T16), 24 (T24), and 40 h (T40) after 2 bouts of
exercise with a 48-h rest interval.
tiation of satellite cells or myogenic precursor cells (2, on the basis of observations from previously efficacious
21, 27, 33, 47, 51, 62). Myogenin mRNA increased in all training programs. As a result, a number of differing
muscles after resistance exercise (Fig. 8, C and D). The permutations of frequency, intensity, and duration
absolute magnitude of this increase was similar after may be applied, some of which may prove effective in
both the one- and two-bout protocols. However, two simulating desirable adaptations. However, regardless
bouts of exercise and in particular those involving 24 of the protocol employed, the effectiveness of a training
and 48 h of rest would appear to have resulted in this program will not be evident until an appreciable period
parameter being elevated for a greater proportion of of time has elapsed, allowing for the completion of a
the 40-h period over which data were collected. relatively large number of training sessions. This reli-
The cdk inhibitor p21 inhibits progression through ance on end-point measures, i.e., overt measures of
the cell cycle and initiates the process of differentia- hypertrophy, precludes understanding of how each of
tion. We previously reported that cdk inhibitor p21 the three parameters and more specifically the individ-
Fig. 5. Increased IGF binding protein 4 (IGFBP4) and IGF-I receptor (IGFR1) mRNA after resistance exercise.
Representative PCR gel scans demonstrate the observed changes in IGFBP4 and IGFR1 mRNA in the nonstimu-
lated contralateral muscles (C) and in muscles at 16 (T16), 24 (T24), and 40 h (T40) after 2 bouts of exercise with
a 48-h rest interval.
and molecular markers chosen had been previously and Esser (39) reported increased ERK phosphoryla-
identified as being responsive to increased loading in a tion at earlier time points after resistance exercise.
variety of in vitro or in vivo models (e.g., Refs. 2, 15, 39). Two bouts of resistance exercise, particularly with a
Cellular Signaling Responses 48-h rest interval, appear to prolong the elevation of
ERK2 phosphorylation (Fig. 2D).
The activation of S6K1 has a relatively modest, pos- Together, these results demonstrate that intracellu-
itive impact on translation in general, but more impor- lar signaling pathways, which have been reported to be
tantly it increases the translation of specific mRNAs associated with hypertrophy, are responsive to acute
that encode components of the translational apparatus bouts of increased loading. These responses appear to
itself (26, 55). The contribution of this regulatory step exhibit temporal and/or quantitative summation that
in supporting anabolic responses would therefore be of is sensitive to the duration of the rest interval between
Fig. 7. Increased cyclin D1 and myogenin mRNA after resistance exercise. Representative PCR gel scans
demonstrate the observed changes in cyclin D1 and myogenin mRNA in the nonstimulated contralateral muscles
(C) and in muscles at 16 (T16), 24 (T24), and 40 h (T40) after 2 bouts of exercise with a 48-h rest interval.
pronounced when the rest intervals were longer (Fig. 4, As noted above, an increase in myogenin expression
B and D). Resistance exercise also induced significant is generally thought to reflect the differentiation of
increases in the mRNAs for components of the IGF-I satellite cells and/or myogenic precursor cells in intact
system such as IGFBP4 and the primary IGF-I recep- mammalian skeletal muscle. In the present study, both
tor (Fig. 6). IGF-I actions in skeletal muscles are re- the single- and dual-exercise-bout protocols resulted in
ported to include stimulation of the proliferation and significant increases in myogenin mRNA. As we had
differentiation of satellite cells as well as insulin-like reported previously (4), the increases seen in myogenin
anabolic effects (41). In the present study, the IGF-I- were paralleled by, and highly correlated with, those in
related changes appear to be consistent with cellular cdk inhibitor p21 in this study. This result would
responses that, if sustained, would be expected to con- support the idea that myogenin expression was occur-
tribute to the development of hypertrophic adaptations. ring in muscle lineage cells that were preparing to
Markers of Cellular Proliferation and Differentiation withdraw from the cell cycle and to begin differenti-
were not completely resolved at 40 h postexercise. vals. The methods used in this study should be useful
However, in the cases of the phosphorylation of S6K1 in the study of regulatory mechanisms as well as the
and the increase in cyclin D1 expression, the single- evaluation of exercise training parameters.
exercise-bout responses did not reach statistical signif-
icance. With the possible exception of IGFBP4 and the The authors thank Angi Qin, Ming Zeng, and Sam McCue for
technical assistance.
IGFR1, the imposition of a second bout of exercise, at This work was supported by National Institute of Arthritis and
time points when the response to the initial bout was Musculoskeletal and Skin Diseases Grant AR-45594.
ongoing, appears to have substantially increased the
magnitude and/or duration of the increase in mRNAs. REFERENCES
In most cases, the 48-h rest interval resulted in a
substantially greater increase and/or prolongation of 1. Adams GR and Haddad F. The relationships between IGF-1,
DNA content, and protein accumulation during skeletal muscle
the observed response. Twenty-four hours of rest were hypertrophy. J Appl Physiol 81: 2509–2516, 1996.
also fairly effective, whereas the 8-h interval was, in 2. Adams GR, Haddad F, and Baldwin KM. Time course of
most cases, clearly inferior to the longer rest times in changes in markers of myogenesis in overloaded rat skeletal
terms of stimulating increases in signaling or mRNA. muscles. J Appl Physiol 87: 1705–1712, 1999.
3. Adams GR and McCue SA. Localized infusion of IGF-I results
It is not immediately clear why a second bout of exer- in skeletal muscle hypertrophy in rats. J Appl Physiol 84: 1716–
cise imposed 8 h after the first would be so markedly 1722, 1998.
inferior in producing an enhanced response compared 4. Adams GR, McCue SA, Zeng M, and Baldwin KM. The time
with 24 or 48 h of rest (i.e., MGF, IGF-I, myogenin, course of myosin heavy chain transitions in neonatal rats: im-
cyclin D1 mRNA). One explanation might be that some portance of innervation and thyroid state. Am J Physiol Regula-
tory Integrative Comp Physiol 276: R954–R961, 1999.
signaling mechanisms enter a refractory period after 5. Allen DL, Monke SR, Talmadge RJ, Roy RR, and Edgerton
bution: comparison of two contractile activity programs. J Appl 41. Petley T, Graff K, Jiang W, and Florini J. Variation among
Physiol 4: 2509–2516, 1993. cell types in the signaling pathways by which IGF-I stimulates
19. Diffee GM, McCue S, LaRosa A, Herrick RE, and Baldwin specific cellular responses. Horm Metab Res 31: 70–76, 1999.
KM. Interaction of various mechanical activity models in regu- 42. Quinn LS, Haugk KL, and Grabstein KH. Interleukin-15: a
lation of myosin heavy chain isoform expression. J Appl Physiol novel anabolic cytokine for skeletal muscle. Endocrinology 136:
74: 2517–2522, 1993. 3669–3672, 1995.
20. Dunn SE, Burns JL, and Michel RN. Calcineurin is required for 43. Reiss K, Meggs LG, Li P, Olivetti G, Capasso JM, and
skeletal muscle hypertrophy. J Biol Chem 274: 21908–21912, 1999. Anversa P. Upregulation of IGF1, IGF1-receptor, and late
21. Florini JR and Ewton DZ. Highly specific inhibition of IGF-I- growth related genes in ventricular myocytes acutely after in-
stimulated differentiation by an antisense oligodeoxyribonucleotide farction in rats. J Cell Physiol 158: 160–168, 1994.
to myogenin mRNA. J Biol Chem 265: 13435–13437, 1990. 44. Roberts CT, Lasky SR, Lowe WL, Seaman WT, and Le-
22. Fleck SJ and Kraemer WJ. Designing Resistance Training Roith D. Molecular cloning of rat insulin-like growth factor I
Programs. Champaign, IL: Human Kinetics, 1987. complementary deoxyribonucleic acids: differential messenger
23. Frame S and Balmain A. Integration of positive and negative ribonucleic acid processing and regulation by growth hormone in
growth signals during ras pathway activation in vivo. Curr Opin extrahepatic tissues. Mol Endocrinol 1: 243–248, 1987.
Genet Dev 10: 106–113, 2000. 45. Rosenblatt DJ and Parry DJ. Gamma irradiation prevents
24. Goldspink G. Changes in muscle mass and phenotype and the compensatory hypertrophy of overloaded mouse extensor digito-
expression of autocrine and systemic growth factors by muscle in rum longus muscle. J Appl Physiol 73: 2538–2543, 1992.
response to stretch and overload. J Anat 194: 323–334, 1999. 46. Rosenblatt JD, Yong D, and Parry DJ. Satellite cell activity
25. Haddad F, Qin AX, Zeng M, McCue SA, and Baldwin KM. is required for hypertrophy of overloaded adult rat skeletal
Effects of isometric training on skeletal myosin heavy chain muscle. Muscle Nerve 17: 608–613, 1994.
expression. J Appl Physiol 84: 2036–2041, 1998. 47. Sabourin LA and Rudnicki MA. The molecular regulation of
26. Hashemolhosseini S, Nagamine Y, Morley SJ, Desrivières myogenesis. Clin Genet 57: 16–25, 2000.
S, Mercep L, and Ferrari S. Rapamycin inhibition of the G1 to 48. Salleo A, LaSpada G, Falzea G, Denaro MG, and Cic-
S transition is mediated by effects on cyclin D1 mRNA and ciarello R. Response of satellite cells and muscle fibers to
protein stability. J Biol Chem 273: 14424–14429, 1998.