A study examined the effects of an acute bout of lower-body resistance exercise on inflammatory gene expression in skeletal muscle in 24 post-menopausal women. Muscle biopsies obtained before and 3 hours after exercise showed significant increases in mRNA content for several inflammatory genes including TNF, IL1, IL6, IL8, and others. Blood samples obtained before and up to 48 hours after exercise showed no significant changes in circulating levels of inflammatory markers. The results indicate that resistance exercise can up-regulate transcription of inflammatory mediators locally within skeletal muscle.

1. Eur J Appl Physiol (2009) 107:463–471
DOI 10.1007/s00421-009-1145-z
O R I G I N A L A R T I CL E
Resistance exercise-induced changes of inXammatory
gene expression within human skeletal muscle
Thomas W. Buford · Matthew B. Cooke ·
Darryn S. Willoughby
Accepted: 24 July 2009 / Published online: 11 August 2009
© Springer-Verlag 2009
Abstract Aberrant local inXammatory signaling within obtained from the vastus lateralis of the dominant leg at
skeletal muscle is now considered a contributing factor to baseline and 3 h following exercise. SigniWcant (p < 0.05)
the development of sarcopenia. Recent evidence indicates up-regulation in mRNA content was observed for TNF ,
that chronic resistance training contributes to the control of IL1 , IL6, IL8, SOCS2, COX2, SAA1, SAA2, IKKB, cfos,
locally derived inXammation via adaptations to repeated, and junB. Muscle mRNA content was not signiWcantly
acute increases in pro-inXammatory mRNA within muscle. altered at the 0.05 level for IL2, IL5, IL10, or IL12 (p35).
However, only a limited number of gene transcripts related Venous blood samples were also obtained at baseline as
to the inXammatory process have been examined in the lit- well as at 3, 24, and 48 h post-exercise. Serum was ana-
erature. The present study utilized an acute bout to examine lyzed for circulating TNF , IL1 , IL6, IL8, COX2, and
the eVects of resistance exercise on several inXammatory- SAA with no signiWcant changes observed. These results
related genes in 24 physically active, post-menopausal indicate that resistance exercise is capable of up-regulating
women not currently undergoing hormone replacement transcription of numerous inXammatory mediators within
therapy. Following a standard warm-up, participants com- skeletal muscle, and these appear to be worthy of future
pleted a lower-body resistance exercise bout consisting of 3 examination in chronic studies.
sets of 10 repetitions on machine squat, leg press, and leg
extension exercises (80% intensity). Muscle biopsies were Keywords InXammation · Cytokines · Resistance training ·
mRNA · Sarcopenia · Menopause
T. W. Buford
Division of Medicine, Department of Aging and Geriatric
Research, Institute on Aging, University of Florida, Introduction
Gainesville, FL, USA
M. B. Cooke · D. S. Willoughby Skeletal muscle mass and strength are important factors in
Exercise and Biochemical Nutrition Laboratory, maintaining independence and quality of life in the elderly.
Baylor University, Waco, TX, USA More than 20 years ago, Lexell et al. (1988) noted that
there is a nearly 40% loss of muscle mass of people in their
M. B. Cooke · D. S. Willoughby
Exercise, Nutrition, and Resistance Training Research Unit, 1980s compared to when they were in their 1920s. As the
Baylor University, Waco, TX, USA world population grows ever older, the loss of skeletal mus-
cle function impacts ever-increasing numbers of people and
D. S. Willoughby
their ability to carry out daily tasks such as climbing stairs,
Baylor Biomedical Science Institute,
Baylor University, Waco, TX, USA rising from the toilet, or carrying groceries. Recent esti-
mates indicate that approximately 45% of older Americans
D. S. Willoughby (&) are sarcopenic (Melton et al. 2000) while approximately
Exercise and Biochemical Nutrition Laboratory,
20% are functionally disabled (Manton and Gu. 2001). In
Department of Health, Human Performance, and Recreation,
Baylor University, Waco, TX 76798, USA addition to the individual loss of functional capabilities, the
e-mail: Darryn_Willoughby@baylor.edu economic impact of sarcopenia is also dramatic, as it has
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2. 464 Eur J Appl Physiol (2009) 107:463–471
been estimated that the direct healthcare costs of sarcopenia related to the inXammatory process (several of which have
in the US were approximately $18 billion at the turn of the yet to be investigated within skeletal muscle) in response to
century (Janssen et al. 2004). Thus, interest in mechanisms acute resistance exercise. Thus, the purpose of this study
that underlie and interventions that could alleviate age- was to test the hypothesis that a single bout of resistance
related muscle dysfunction is signiWcant. exercise bout would signiWcantly alter the muscle mRNA
In comparison to a number of other disease states, evi- content of a more complex set of inXammatory-related
dence indicating the contribution of inXammation to sarco- genes than has previously been described.
penia has steadily been accumulating over the last few
years. In fact, a number of studies have now reported links
between aberrant inXammatory/cytokine signaling and the Materials and methods
development of sarcopenia (Haddad et al. 2005; Hamada
et al. 2005; RoubenoV 2007; Visser et al. 2002). SpeciW- Participants
cally, inXammatory dysfunction may contribute to sarcope-
nia through multiple mechanisms including induction of the The present study employed 24 recreationally active
caspases (Dirks and Leeuwenburgh 2006), activation of (consistent, structured exercise at least 3£/week) female
nuclear factor kappa B (NFkB) and subsequent atrophy- subjects with an average (§SD) age of 54.54 (3.89) year,
related gene expression (Mourkioti and Rosenthal 2005), or height of 159.67 (5.22) cm, and body mass of 72.43
via the impairment of skeletal muscle growth/repair mecha- (15.58) kg. Participants were required to attain written phy-
nisms such as the growth hormone (GH)-insulin-like sician consent before being allowed to participate. Post-
growth factor 1 (IGF1) axis (Frost and Lang 2005; Haddad menopausal status was veriWed by physician conWrmation
et al. 2005). of amenorrhea concurrent with elevated serum levels of fol-
In conjunction with the consumption of adequate pro- licle stimulating hormone (FSH) for at least the previous
tein, resistance training provides a safe, cost-eVective 12 months (World Health Organization 1981; Rannevik
method of preserving muscle mass and strength in older et al. 2008). Participants with contraindications to exercise
individuals. Additionally, resistance training has been as outlined by the American College of Sports Medicine
shown to provide anti-inXammatory beneWt to skeletal mus- were not allowed to participate. In addition, participants
cle (Greiwe et al. 2001). It now appears possible, if not who were on estrogen replacement therapy were ineligible
likely, that these adaptations are due to local changes in the to participate. All eligible participants signed university-
muscle rather than systemic changes. Skeletal muscle has approved informed consent documents and approval was
come to be viewed as an endocrine organ actively involved granted by the Institutional Review Board for Human
in inXammation due to its ability to produce cytokines such Subjects. Additionally, all experimental procedures involved
as interleukin 6 (IL6) and tumor necrosis factor alpha in the study conformed to the ethical consideration of the
(TNF ) (Plomgaard et al. 2005; Steensberg et al. 2000). Helsinki Code. Investigators explained the purpose of the
Further, more recent studies have indicated that exercise study, the protocol to be followed, and the experimental
regimens including resistance training improve muscle procedures to be used prior to allowing prospective partici-
strength (Bruunsgaard et al. 2004) and reduce inXammatory pants to enter the study.
mRNA expression (Lambert et al. 2008) without altering
plasma levels of IL6 or TNF . Yet the number and precise Experimental procedures
role of endogenously produced inXammatory mediators
within skeletal muscle has been incompletely delineated During the familiarization session, lower-body maximum
and needs further investigation given the development of strength [one repetition maximum (1-RM)] was determined
low-grade inXammation in aged skeletal muscle. for machine squat, leg press, and leg extension exercises.
Although the information gained from acute exercise During the exercise testing session, participants underwent
studies is typically less clinically relevant than that a resistance exercise bout consisting of 3 sets of 10 repeti-
obtained from training studies; these investigations can pro- tions at 80% of 1RM on each of the three exercises. Exer-
vide a wide range of valuable information concerning bio- cise order was consistent for all participants and between
logical targets in training studies. In order to gain a more the familiarization to the exercise bout. Prior to beginning
comprehensive understanding of how resistance training the bout, participants underwent a warm-up consisting of
provides anti-inXammatory beneWt to the muscle, we con- 5 min cycling on a Monarch (Model 818E, Varberg, Sweden)
ducted the present investigation to gain pilot data regarding cycle ergometer (50 rpm) and 2 sets of 10 repetitions (50%
the transcriptional response of a number of inXammatory 1RM) on the leg press.
mediators within skeletal muscle. We thus proposed to Prior to each testing session, total body mass (kg) was
investigate the transcription of a number of other genes determined on a calibrated electronic scale with a precision
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3. Eur J Appl Physiol (2009) 107:463–471 465
of §0.02 kg (Detecto, Webb City, MO, USA). In addition, the needle to collect a muscle piece. The biopsy needle was
participants underwent assessment of heart rate and blood removed from the pilot hole and the muscle specimen was
pressure upon arrival at each session as well as following removed from the biopsy needle using a sterile scalpel, sep-
the exercise bout. Heart rate was determined by palpation arated from connective and/or adipose tissues, and immedi-
of the radial artery using standard procedures, and blood ately frozen in liquid nitrogen and stored at ¡80°C for
pressure was assessed by auscultation of the brachial artery further analyses. This whole biopsy procedure was repeated
using a mercurial sphygmomanometer using standard clini- two to three times in order to obtain suYcient muscle tissue
cal procedures. (»15 mg). This muscle collection approach was used as an
Venous blood samples were obtained from the antecubital attempt to minimize scarring, invasiveness, and discomfort
vein into a 10-mL collection tube using a standard Vacu- attributed to traditional needle muscle biopsy procedures.
tainer apparatus at baseline as well as 3, 24, and 48 h post- Hayot et al. (2005) previously reported similar gene expres-
exercise. Blood samples were then centrifuged at room sion via this technique as compared to the traditional Berg-
temperature at 2,000 rpm for 15 min. The serum was strom technique while participants reported less pain and
removed and frozen at ¡80°C for later analysis. Serum was discomfort. Additionally, the small sample size and the
analyzed for serum amyloid A (SAA) using a DADE method of extraction limit the content of connective and/or
Dimension RXL clinical chemistry analyzer (Dade-Dehring, adipose contained within the sample, and removal of these
Inc, Newark, DE, USA). The analyzer was calibrated daily tissues is simpliWed compared to the Bergstrom technique.
using liquid assay multiqual (Bio-Rad, Hercules, CA, Total cellular RNA was extracted from biopsy samples
USA), and two levels of quality control with known con- with a monophasic solution of phenol and guanidine isothi-
centrations were performed. In addition, serum tumor ocyanate contained within the TRI-reagent (Sigma Chemi-
necrosis factor alpha TNF , IL1 , IL6, IL8, and cyclooxy- cal Co., St. Louis, MO, USA). Aliquots of total RNA were
genase 2 (COX2) were assessed using commercially made then separated with agarose gel electrophoresis and moni-
enzyme-linked immunoabsorbent assay (ELISA) kits tored under an ultraviolet light (Chemi-Doc XRS, Bio-Rad,
(Invitrogen Corp., Carlsbad, CA, USA; EMD Chemicals Hercules, CA, USA) to verify RNA integrity and absence
Inc., San Diego, CA, USA). Concentrations of each target of RNA degradation, indicated by prominent 28 and 18 s
protein were determined using the colorimetric method at ribosomal RNA bands (data not shown), as well as an
an optical density of 450 nm with a microplate reader OD260/OD280 ratio of approximately 2.0. The RNA samples
(Wallac Victor 1420, Perkin Elmer, Boston, MA, USA). were stored at ¡80°C until later analysis (Willoughby
Standard curves were generated using commercially avail- 2004).
able microplate reader-compatible statistical software Two microgram of total skeletal muscle RNA were
(MicroWin 2000, Microtek Laborsysteme GmbH, Overath, reverse-transcribed to synthesize cDNA using the iSscript
Germany). cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA).
Fine needle aspiration muscle biopsies were performed A reverse transcription reaction mixture [2 g of cellular
at baseline and 3 h post-exercise. 3 h post-exercise was RNA, 5£ reverse transcription buVer (20 mM Tris–HCL,
chosen as the post-exercise biopsy as most contraction- pH 8.3; 50 mM KCl; 2.5 mM MgCl2; 100 g of bovine
induced mRNA increases occur between 3 and 12 h post- serum albumin/ml), a dNTP mixture containing 0.2 mM
exercise (Bickel et al. 2005; Pilegaard et al. 2000; Yang each of dATP, dCTP, dGTP, and dTTP, 0.8 M MgCl2,
et al. 2005) and due to the previously demonstrated spike in 0.5 g/ l of oligo(dT) 15 primer, and 25 / g of MMLV
skeletal muscle mRNA expression of TNF , IL6, and IL8 RNAase H+ reverse transcriptase enzyme (Bio-Rad, Hercu-
between 2 and 4 h post-resistance exercise (Louis et al. les, CA, USA)] was incubated at 25°C for 5 min, 42°C for
2007). Participants were instructed to refrain from exercise 30 min, heated to 85°C for 10 min, and then quick-chilled
for at least 48 h prior to the initial biopsy. Muscle was on ice. The cDNA concentration was determined by using
extracted from the lateral portion of the vastus lateralis an OD260 equivalent to 50 g/ l and the starting cDNA
midway between the patella and iliac crest of the dominant template concentration was standardized by adjusting all
leg using a TRU-CORE® 1 Automatic Reusable Biopsy samples to 200 ng prior to ampliWcation (Willoughby
Instrument (Angiotech, Medical Device Technologies, 2004).
INC., Gainsville, FL, USA). BrieXy, 1.0 ml of 1.0% Lido- The mRNA sequences of human skeletal muscle pub-
caine HCl was injected subcutaneously prior to making a lished in the NCBI Entrez Nucleotide database (http://
small pilot hole with a 23 gauge sterile needle. The biopsy www.ncdi.nlm.hih.gov) were used to construct oligonu-
needle was placed into the biopsy device and the inserted cleotide PCR primers using Beacon Designer software
into the previously made pilot hole (a depth of about (Bio-Rad, Hercules, CA, USA). Table 1 shows the sense
5–10 mm). A muscle sample was obtained by the activation sequence, anti-sequence, amplicon size, and NCBI acces-
of a trigger button, which unloaded the spring and activated sion number for each gene assessed in the study. The sense
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4. 466 Eur J Appl Physiol (2009) 107:463–471
Table 1 mRNA sequences of primers used in the real-time PCR procedure
Primer name NCBI accession Sense sequence (5 ! 3 ) Anti-sense sequence (5 ! 3 ) Amplicon
number size (bp)
Beta-actin NM_001101 ATC GTG CGT GAC ATT AAG GTC ATC ACC ATT GGC AAT 102
IKKB NM_001556 AAC CAG CAT CCA GAT TGA C GCC ATC ATC CGT TCT ACC 156
Cox-2 M90100 TCC CTG AGC ATC TAC GGT TTG CAT CGC ATA CTC TGT TGT GTT CC 108
JunB NM_002229 TCT CTC TAC ACG ACT ACA AAC GAC AAT CAG GCG TTC CAG 197
TNF- NM_000594 CAG CAA GGA CAG CAG AGG AGT ATG TGA GAG GAA GAG AAC C 139
SAA1 M23698 CTC CTT GGT CCT GGG TGT C TTG TCT GAG CCG ATG TAA TTG G 126
SAA2 NM_030754 GGC AGG AGT GGC AGA GAC C TGA GAG CAG AGT GAA GAG GAA GC 83
cfos V01512 CGG CAG GAT GGA AGA GAC GCA GCA GTG AAG AGA AAC G 128
IL-1 NM_000576 TGA TGG CTT ATT ACA GTG GCA ATG GTA GTG GTG GTC GGA GAT TCG 140
IL-2 NM_000586 CAA GAA TCC CAA ACT CAC CAG CGT TGA TAT TGC TGA TTA AGT CC 179
IL-4 M13982 AGT TGA CCG TAA CAG ACA TC GAG CCG TTT CAG GAA TCG 182
IL-5 J03478 GGA TGT GGA ACC TGT AAC AAC TCA GTC TTT CTA ATG GG 192
IL-6 NM_000600 GGT CCA GTT GCC TTC TCC TGT CAA TTC GTT CTG AAG AGG 136
IL-8 NM_000584 AGA GAC AGC AGA GCA CAC GTT CTT TAG CAC TCC TTG GC 174
IL-10 NM_000572 GAA CCA AGA CCC AGA CAT C CAT TCT TCA CCT GCT CCA C 137
IL-12 AF101062 GCA GCC TCC TCC TTG TGG GGG AAC ATT CCT GGG TCT GG 94
and anti-sense primers were synthesized commercially (TAE) buVer to verify positive ampliWcation and the gel
(Integrated DNA Technologies, Coralville, IA, USA). stained with ethidium bromide (present in the TAE buVer at
-actin was used as an internal control standard for each 1 g/ml) and illuminated with UV transillumination
reaction due to its consideration as a constitutively (Chemi-Doc XRS, Bio-Rad, Hercules, CA, USA). The rela-
expressed “housekeeping gene,” and the fact that it has tive expression of mRNA was assessed by determining the
been shown to be an appropriate external reference stan- ratio between the CT values of each target mRNA and the
dard in real-time PCR in human skeletal muscle following CT values for -actin for each muscle sample obtained at
acute exercise (Mahoney et al. 2004). each timepoint (Willoughby et al. 2007). Test–retest reli-
Based on previous guidelines, 200 ng of cDNA were ability of performing this procedure of mRNA expression
added to each of the 25 l PCR reactions using iQ SYBR on samples in this laboratory has demonstrated low mean
Green Supermix (Bio-Rad, Hercules, CA, USA). SpeciW- coeYcients of variation and high reliability (1.6%, intra-
cally, each PCR reaction contained the following mixtures: class r = 0.95).
[10£ PCR buVer, 0.2 M dNTP mixture, 2.0 M of a cock-
tail containing both the sense and anti-sense RNA oligonu- Statistical analyses
cleotide primers, 2 mM MgCL2, 1.0 / l of hot-start iTaq
DNA polymerase, SYBR Green I dye, and nuclease-free Data were analyzed initially for normality and homogeneity
dH2O]. Each PCR reaction was ampliWed using real-time of variances. The ratios of the mRNA of target genes to the
quantitative polymerase chaine reaction (PCR) (iCycler IQ mRNA of the housekeeping gene ( -actin) were then ana-
Real-Time PCR Detection System, Bio-Rad, Hercules, CA, lyzed then analyzed using a single-factor multivariate analy-
USA). The ampliWcation proWle was run for 40-cycles sis of variance (MANOVA) with repeated measures, with
employing a denaturation step at 95°C for 30 s, primer univariate tests on each dependent variable conducted as fol-
annealing at 58°C for 30 s, and extension at 72°C for 30 s. low-up tests to the MANOVA. Blood data were analyzed
Fluorescence was measured after each cycle, resulting from using individual ANOVAs. SigniWcance was set at P < 0.05
the incorporation of SYBR green dye into the ampliWed (two-tailed) throughout. Statistical analyses were performed
PCR product. The speciWcity of the PCR was demonstrated using the SPSS 16.0 for Windows software package.
with absolute negative controls using separate PCR reac-
tions containing no cDNA template or primers, and a single
gene product was conWrmed using DNA melt curve analy- Results
sis. Additionally, to assess positive ampliWcation of mRNA,
aliquots (20 l) of the PCR reaction mixtures were electro- No signiWcant changes were observed for serum levels of
phoresed in 1.5% agarose gels in 1£ Tris-Acetate-EDTA TNF , IL1 , IL6, IL8, COX2, or SAA for 48 h following
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5. Eur J Appl Physiol (2009) 107:463–471 467
Table 2 Circulating serum levels of TNF- , IL-1 , IL-6, IL-8, COX2, and SAA at baseline as well as 3, 24, and 48 h post-exercise
Protein Pre-exercise 3 h post-exercise 24 h post-exercise 48 h post-exercise p value
TNF- (pg/mL) 10.77 § 6.98 9.24 § 8.49 12.20 § 8.16 12.90 § 8.44 0.408
IL-1 (pg/mL) 4.59 § 3.04 4.15 § 2.68 6.33 § 4.47 6.37 § 5.22 0.117
IL-6 (pg/mL) 3.93 § 4.39 2.60 § 3.19 3.28 § 3.32 3.83 § 3.82 0.585
IL-8 (pg/mL) 4.76 § 3.34 3.75 § 3.29 5.02 § 4.19 4.75 § 3.47 0.623
COX2 (ng/mL) 1.367 § 1.220 0.981 § 1.332 1.872 § 2.176 1.622 § 1.978 0.327
SAA (ng/mL) 412.25 § 283.64 421.08 § 247.58 588.46 § 290.63 532.00 § 292.74 0.084
Fig. 1 mRNA expression of TNF IL1 , IL6, and IL8 at baseline and Fig. 2 mRNA expression of IL2, IL5, IL10, and IL12 at baseline and
3 h post-exercise relative to -actin from muscle biopsies extracted 3 h post-exercise relative to -actin from muscle biopsies extracted
from the vastus lateralis. *SigniWcantly diVerent from baseline mRNA from the vastus lateralis. No signiWcant changes were observed,
expression at the p < 0.05 level. *9SigniWcantly diVerent from baseline although a weak trend for an increase in SAA was observed
mRNA expression at the p < 0.005 level
exercise; although a weak trend was observed for circulat- Discussion
ing SAA (p = 0.084) (Table 2). Meanwhile, at 3 h post-
exercise, elevations in skeletal muscle mRNA expression As the world’s population ages, the loss of skeletal muscle
of 11 genes of interest were observed in comparison to mass and function in older individuals has become a signiW-
-actin. Several of these were signiWcantly upregulated at cant societal problem. Resistance training provides a
the p < 0.005 level, including IL1 (f = 27.47, p < 0.001), simple, cost-eVective, and feasible alternative method for
IL6 (f = 32.39, p < 0.001), IL8 (f = 12.33, p = 0.001), preserving/improving skeletal muscle mass in older indi-
COX2 (f = 9.57, p = 0.003), cfos (f = 33.63, p < 0.001) and viduals, and part of this beneWt appears to be via alterations
junB (f = 28.50, p < 0.001) (Figs. 1, 3, and 4). Further, in skeletal muscle inXammatory signaling. Although sarco-
mRNA of TNF (f = 7.90, p = 0.007), IKKB (f = 4.14, penia aVects both genders, women with sarcopenia have
p = 0.048), suppressor of cytokine signaling 2 [(SOCS2) been reported to have lower functional capacities than com-
(f = 6.48, p = 0.014)], SAA1 (f = 6.77, p = 0.012), and plimentary males (Bassey et al. 1992; Frontera et al. 1991).
SAA2 (f = 4.94, p = 0.031) were signiWcantly elevated at While opposing evidence exists (Gower and Nyman 2000;
the p < 0.05 level (Figs. 1, 3, and 4). No signiWcant changes TaaVe et al. 1995), it appears that the loss of endogenous
were observed for expression of genes IL2 (f = 1.69, estrogens signiWcantly contributes to this functional decline
p = 0.200), IL5 (f = 0.773, p = 0.384), IL10 (f = 0.067, (Douchi et al. 1998). One mechanism by which the loss of
p = 0.796), or IL12 (f = 2.94, p = 0.093) (Fig. 2). estrogen has been reported to contribute to sarcopenia is
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6. 468 Eur J Appl Physiol (2009) 107:463–471
through the increase in pro-inXammatory cytokines as
estrogen withdrawal has been shown to increase these pro-
teins (Greeves et al. 1999; Phillips et al. 1993). Primarily
because of the eVects of menopause on bone mass, many
women are prescribed hormone replacement therapy (HRT)
in an attempt to minimize the detrimental eVects of estro-
gen withdrawal. Yet some are now becoming worried about
associated side eVects of HRT such as increased risk of
breast cancer (Girasole et al. 1999; Kramer et al. 2004). For
these women, resistance training provides a safe alternative
that can provide beneWt to both skeletal muscle and bone.
For these reasons, we chose post-menopausal women as our
study population for examining the eVects of resistance
exercise on inXammatory mRNA expression within muscle.
As previously stated, previous investigations have sug-
gested that skeletal muscle is capable of inducing changes
in skeletal muscle mRNA expression independent of the
circulation. Lambert and colleagues (2008) proposed that
the changes in cytokine gene expression within muscle are
derived from the muscle itself due to the unresponsiveness
Fig. 3 mRNA expression of IKKB, cFos, and JunB at baseline and 3 h of circulating levels to exercise. Further, Akerstrom et al.
post-exercise relative to -actin from muscle biopsies extracted from
(2005) previously observed this response following acute
the vastus lateralis. *SigniWcantly diVerent from baseline mRNA
expression at the p < 0.05 level. *9SigniWcantly diVerent from baseline knee extensor exercise, as IL8 mRNA and protein expres-
mRNA expression at the p < 0.005 level sion were increased within muscle while circulating levels
remained unchanged. Our resistance training protocol was
thus chosen in an attempt to produce mRNA alterations
without inducing a systemic inXammatory response. Rippy
and Marsden (2006). Choosing such a limited protocol left
some risk for not observing signiWcance due to intensity
insuYciency; however, only four genes (IL2, IL4, IL10,
IL12) were not signiWcantly altered and none of these four
have previously been reported to be altered by exercise.
Meanwhile, we were successful in mirroring previous stud-
ies by signiWcantly altering muscular mRNA expression
without aVecting circulating protein levels of six represen-
tative markers. The lack of a signiWcant change in circulat-
ing levels of representative markers within the serum
observed in the present study appears to indicate that the
source of the mRNA increases is not due to peripheral
blood mononuclear cells. While adipocytes and hepatocytes
also produce a number of these mRNA, it appears highly
unlikely that these cells contributed to the mRNA increases
given the fact that the protocol utilized was focused on con-
traction-induced changes and involved little metabolic per-
turbation. These data taken together with previous evidence
lead us to believe that muscle is the source of the additional
mRNA and thus suggest several inXammatory mediators
within skeletal muscle worthy of further research in con-
junction with chronic exercise training. Additional evi-
Fig. 4 mRNA expression of COX2, SOCS2, SAA1, and SAA2 at dence is necessary, however, to conWrm this hypothesis.
baseline and 3 h post-exercise relative to -actin from muscle biopsies
extracted from the vastus lateralis. *SigniWcantly diVerent from base-
In response to acute resistance exercise, we observed
line mRNA expression at the p < 0.05 level. *9SigniWcantly diVerent signiWcant (p < 0.05) changes in mRNA content of 11
from baseline mRNA expression at the p < 0.005 level genes pertinent to tissue inXammation. These data agree
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7. Eur J Appl Physiol (2009) 107:463–471 469
with previous evidence showing transcriptional up-regula- indicate the same in response to resistance exercise,
tion of several of these genes in response to resistance exer- although one study has reported otherwise (Puntschart et al.
cise (Louis et al. 2007; Akerstrom et al. 2005; Hamada 1998).
et al. 2005). Little or no evidence exists for the presence of As a whole, the eVects of exercise on these genes is
several of these mRNA within skeletal muscle, nor for their interesting given the traditional paradigm that the direc-
regulation by exercise. Most notable among these is the tional change of the mRNA for exercise-induced genes is
expression of two isoforms of SAA. SAA is a generic term typically thought to occur in the same direction as the
for a family of cytokine-induced acute-phase proteins pri- encoded protein with long-term adaptation being accom-
marily produced by the liver. Its functions in inXammation plished by each short-term bout leading to a change in the
include the capacity to induce chemotaxis, cell adhesion change in the steady state of that protein (Durham et al.
and migration (Akerstrom et al. 2005) and the ability to act 2004). The inXammatory genes, however, appear to be
as an extracellular matrix adhesion protein (Badolato et al. down-regulated in response to repeated, transient increases,
1995). While extra-hepatic expression has been reported similar to recent Wndings concerning ROS/antioxidant
(Hershkoviz et al. 1997), no information exists concerning interactions with insulin signaling (CoVey and Hawley
SAA within skeletal muscle of mammals. Here, we 2007).
observed signiWcant increases in both SAA1 and SAA2 The exact mechanisms that underlie the seemingly
mRNA expression following the exercise session. SAA “inverse” adaptations are yet to be delineated; however,
production is known to be responsive to cytokines such as they appear to be necessary components of the muscle
IL6 and TNF ; however, it appears that its upregulation repair process following mechanical stretch. While chronic
may be contraction-responsive as circulating levels of these muscle inXammation induces catabolism, short-term
cytokines were not altered and muscle translation of the inXammation is a necessary part of muscle repair. Recently,
cytokines could not have occurred by 3 h post-exercise. Cheng and colleagues (2008) reported that damaged skele-
Additionally, we observed signiWcant increases in an tal muscle expressed both mRNA and protein of interferon-
isoform of suppressor of cytokine signaling (SOCS2), a (IFN ) and that IFN null mice showed signiWcantly
signaling molecule for the NFkB cascade, and family mem- impaired muscle healing compared to normal mice. These
bers of the activator protein 1 (AP1) transcription factor. authors concluded that endogenous IFN promotes muscle
Some evidence exists for the eVects of exercise on these healing in part by stimulating the formation of new muscle
mRNA; however, this evidence is far from conclusive Wbers. Additional evidence has also shown that COX2 is
(Kovacevic et al. 2008). SOCS proteins are negative regu- necessary for proper muscle regeneration following muscle
lators of cytokine signaling that are typically expressed at injury (Cheng et al. 2008). It seems plausible that this
low levels in un-stimulated cells, but respond rapidly upon explanation applies to all of the examined inXammatory
cytokine stimulation. They are also associated with growth mediators and that skeletal muscle plays an extremely
hormone signaling in skeletal muscle. Given the known important role in control of inXammation and in its own
stimulation of cytokines by exercise, it seems intuitive repair. While the mechanism of chronic down-regulation is
that expression would increase. However, Haddad and unknown, it is possible that this adaptation is moderated by
colleagues (2005) reported decreases in skeletal muscle the action of muscle-speciWc IGF1 (mIGF1), as a recent
SOCS2 mRNA in response to exercise in young rats with transgenic model has shown that mIGF1 expression signiW-
no changes in old rats. It is curious how the Haddad group cantly accelerates the regenerative process of injured
observed no signiWcant increases in SOCS2 mRNA, while muscle and down-regulates pro-inXammatory cytokines
the changes observed in this study were quite robust. It is (Bondesen et al. 2004, 2006). Further, mIGF1 enhanced
likely that the either the exercise mode (isometric vs. iso- connective tissue remodeling and limited muscle Wbrosis,
tonic) or relative intensity are behind this discrepancy; each positive eVects of chronic exercise. Given that mIGF1
although the possibility of a species-speciWc response does has been shown to be robustly increased by resistance train-
exist. Meanwhile, NFkB and AP1 are redox-sensitive tran- ing (Pelosi et al. 2007), we propose here a model in which
scription factors that regulate expression of numerous acute exercise resistance stimulates muscle inXammation
inXammation-related genes. In addition, NFkB plays a early in the repair process, followed by a signal from
well-documented role in muscle atrophy signaling mIGF1 to shut down transcription.
(reviewed in (Haddad and Adams 2006). Acute aerobic We do acknowledge that several pertinent questions
exercise has been shown to up-regulate signaling of both remain unanswered and need clariWcations within future
NFkB (Kandarian and Jackman 2006) and AP1 (Ji et al. training studies. First, future investigations involving train-
2004) in skeletal muscle. Less is known concerning mRNA ing protocols should include a comparison group of
expression, but our Wndings showing increases in transcrip- younger women as well as measurements of reproductive
tion of inhibitor of kappa kinase , cfos, and junB appear to hormone status for comparison. Such a group was not
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8. 470 Eur J Appl Physiol (2009) 107:463–471
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identify molecular mediators worthy of investigation within induces calcium mobilization and chemotaxis of human mono-
cytes by activating a pertussis toxin-sensitive signaling pathway.
chronic studies, not to determine the overall eVectiveness of J Immunol 155:4004–4010
training on one particular group. Secondly, given the goal Bassey EJ, Fiatarone MA, O’Neill EF, Kelly M, Evans WJ, Lipsitz LA
of the present study and thus the chosen protocol, we can- (1992) Leg extensor power and functional performance in very
not conclude the maximal possible induction of mRNA old men and women. Clin Sci (Lond) 82:321–327
Bickel CS, Slade J, Mahoney E, Haddad F, Dudley GA, Adams GR
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of muscle mRNA production on circulating levels of these muscle to acute bouts of resistance exercise. J Appl Physiol
parameters in response to exercise. Thus, a more strenuous 98:482–488
exercise protocol would likely elicit a greater response of Bondesen BA, Mills ST, Kegley KM, Pavlath GK (2004) The COX-2
pathway is essential during early stages of skeletal muscle regen-
blood mononuclear cells and/or muscle, whereby altering eration. Am J Physiol Cell Physiol 287:C475–C483
systemic levels of protein. Finally, it must not be over- Bondesen BA, Mills ST, Pavlath GK (2006) The COX-2 pathway
looked that a limitation of the present study is the fact that regulates growth of atrophied muscle via multiple mechanisms.
we did not measure protein levels of the examined markers. Am J Physiol Cell Physiol 290:C1651–C1659
Bruunsgaard H, Bjerregaard E, Schroll M, Pedersen BK (2004) Muscle
Posttranscriptional events can eVect protein expression and strength after resistance training is inversely correlated with base-
subsequently yield diVering levels of mRNA and functional line levels of soluble tumor necrosis factor receptors in the oldest
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In conclusion, the purpose of the present was to examine tion. Sports Med 37:737–763
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Acknowledgments The authors wish to thank all participants for Sci (Lond) 97:79–84
their hard work and willingness to follow the study protocol. TB is cur- Greiwe JS, Cheng B, Rubin DC, Yarasheski KE, Semenkovich CF
rently supported by the University of Florida Institute on Aging and (2001) Resistance exercise decreases skeletal muscle tumor necro-
Claude D. Pepper Older Americans Independence Center (1 P30 sis factor alpha in frail elderly humans. FASEB J 15:475–482
AG028740). Haddad F, Adams GR (2006) Aging-sensitive cellular and molecular
mechanisms associated with skeletal muscle hypertrophy. J Appl
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