EFFECTS OF AGE ON SERUM HORMONE

CONCENTRATIONS AND INTRAMUSCULAR

PROTEOLYTIC SIGNALING BEFORE AND AFTER
A SINGLE BOUT OF RESISTANCE TRAINING
VINCENT J. DALBO,1 MICHAEL D. ROBERTS,2 SCOTT E. HASSELL,3 RYAN D. BROWN,4
3,4
AND CHAD M. KERKSICK
1
Institute of Health and Social Science Research, School of Medicine and Applied Sciences, Central Queensland University,
Rockhampton, Australia; 2Department of Biomedical Science, University of Missouri-Columbia, Columbia, Missouri; 3Applied
Biochemistry and Molecular Physiology Laboratory, Health and Exercise Science Department, University of Oklahoma,
Norman, Oklahoma; and 4Endocrinology and Diabetes Section, Department of Pediatrics, University of Oklahoma Health
Sciences Center, Oklahoma City, Oklahoma

ABSTRACT groups (p , 0.05), whereas older men experienced significant

Dalbo, VJ, Roberts, MD, Hassell, SE, Brown, RD, and Kerksick, CM.
Effects of age on serum hormone concentrations and intra-
muscular proteolytic signaling before and after a single bout
of resistance training. J Strength Cond Res 25(1): 1–9,
2011—This study examined mRNA expression patterns for
atrogin-1 and muscle ring finger-1 (MuRF-1) before and
24 hours after a resistance training bout. Furthermore, basal,
5-minute and 24-hour postexercise serum concentrations of
cortisol and insulin like growth factor-1 (IGF-1) and the relation-
ships between these hormones and the genetic expression
patterns of atrogin-1 and MuRF-1 were examined. Younger and
older men completed a resistance exercise bout consisting of
3 3 10 repetitions at 80% of their predetermined 1 repetition
maximum for Smith squat, leg press and leg extension. Muscle
biopsies from the vastus lateralis were obtained before and
24 hours after exercise. Basal and postexercise gene expres-
sion differences between age groups were analyzed using the
Mann–Whitney U test, whereas separate 2 3 3 repeated
elevations in IGF-1 24 hours postexercise (p , 0.05). At base-
line, MuRF-1 gene expression was significantly greater in older
men (p = 0.03), whereas no age-related differences were found
for atrogin-1 (p = 0.24). Fold change in atrogin-1 and MuRF-1
24 hours postexercise revealed no significant differences
between younger and older men. Differential baseline expres-
sion of MuRF-1 may suggest a regulatory attempt by the aging
transcriptome to accommodate changes necessary for homeo-
static maintenance. An enhanced understanding of molecular
and genetic level adaptations can aid researchers in developing
optimal therapeutic and exercise interventions to mitigate
decrements in force, power, and loss of muscle mass seen in
aging and many clinical populations.
KEY WORDS cortisol, IGF-1, atrogenes, exercise, aging, skeletal
muscle, proteolysis, atrophy
INTRODUCTION

measures analyses of variance were performed to analyze
changes in hormone concentrations. Spearman’s correlations
were performed to examine relationships between gene expres-
sion patterns and hormone concentrations. Serum cortisol was
significantly greater in younger men before and 24 hours after
exercise (p , 0.05), whereas serum IGF-1 was significantly
greater in younger men at all time points (p , 0.001). Exercise
significantly increased cortisol 5 minutes after exercise in both
Address correspondence to Dr. Chad M. Kerksick, chad_kerksick@ou.
edu.
25(1)/1–9
Journal of Strength and Conditioning Research
Ó 2011 National Strength and Conditioning Association
H
ormones are through to be responsible for
regulating skeletal muscle adaptations by prim-
ing the body for muscle protein synthesis with
the secretion of anabolic hormones or muscle
protein catabolism with the secretion of catabolic hormones
(25). With aging, serum concentrations of anabolic hormones
and growth factors decline (7,9,12–14,22) along with
reductions in anabolic hormone concentrations, which are
thought to relate to documented declines in skeletal muscle
mass and strength (10,19). Specifically, insulin like growth
factor-1 (IGF-1) is significantly reduced in older compared to
in younger adults (7,13). Research examining the effects of
age on catabolic hormones is less prevalent and equivocal
(10,13) but nonetheless suggested that serum concentrations
of cortisol are linearly correlated with age in women (10).

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 Exercise and Age Effects of Proteolytic Expression
Moreover, Kraemer et al. (13) found younger (29.8 6 5.3
years) men to possess more favorable hormonal profiles for
IGF-1 and cortisol after a 10-week training program both at
rest and after resistance exercise when compared to older
(62.0 6 3.2 years) men.
Additionally, the Kraemer et al. (13) investigation found
younger adults to experience significantly more skeletal
muscle growth after training, a result that may be explained
by increased protein synthesis or decreased rates of skeletal
muscle catabolism in younger compared to older adults. In
this regard Yarasheski et al. (33) found younger adults to have
significantly greater rates of mixed muscle protein synthesis
than older adults before training, but found no significant
differences between the groups after 2 weeks of resistance
training. However, older adults could have experienced
a greater increase in nonfunctional components of skeletal
muscle compared to younger adults, and in a follow-up
investigation Welle et al. (30) found younger adults to have
significantly greater rates of myofibrillar protein synthesis
compared to older adults before and after 9 weeks of
resistance training. As a result, age-related differences in the
functional components of skeletal muscle protein synthesis
seems to play a role in the blunted hypertrophy response
seen in older compared to younger men after resistance
training. Age-related differences in proteolytic activity could
also partially explain the blunted hypertrophy response in
older adults and is a research topic currently garnering
attention.
The ubiquitin–proteasome pathway (UPP) is primarily
responsible for skeletal muscle protein degradation and
increased pathway activity is associated with increased
expression of 2 muscle-specific ubiquitin ligases (E3 ligages):
atrogin-1 and muscle ring finger-1 (MuRF-1) (2,23). In
addition, IGF-1 (23,27) and glucocorticoids (1,5,20,27) have
been found to influence UPP activity. Insulin like growth
factor-1 stimulates muscle protein synthesis via activation of
the phosphatidylinositol 3-kinase (PI3K)–Akt pathway,
which has been found to reduce skeletal muscle atrophy
by preventing the upregulation of atrogin-1 and MuRF-1
mRNA and protein expression (27). In this respect, initiation
of Akt pathway activity (via IGF-1 secretion) can phosphor-
ylate FOXO transcription factors (3) whereby they trans-
locate from the nucleus leading to an inhibition of
transcriptional processes linked to atrophy (28). More
recently, Stitt et al. (27) found that although active FOXO1
is not able to induce transcription of atrophy related genes,
the inactivation of the FOXO transcription factor is a required
step in the blockade of glucocorticoid induced transcriptional
alterations suggesting the prevention of skeletal muscle
atrophy may be triggered by IGF-1 signaling via the PI3K
and Akt pathway. In support of these findings (27), Sacheck
et al. (23) reported that exposure of C2C12 myoblasts to
IGF-1 suppressed the transcription of atrogin-1 and other
atrogenes responsible for myofibrillar protein degradation.
Similarly, glucocorticoid exposure is linked with significant
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increases in atrogin-1 (2,27) and MuRF-1 (2,5,27) expression
suggesting an upregulation of UPP activity. Furthermore and
in an attempt to demonstrate the importance of atrogin-1
and MuRF-1 in skeletal muscle proteolysis, when atrogin2/2
and MuRF2/2 mice were compared to wild-type littermates,
the null mice were found to experience less skeletal muscle
atrophy after muscle denervation (2).
With age, there is a decrease in the serum concentrations of
anabolic hormones such as IGF-1 (7,13) and a possible
increase in catabolic hormone concentrations such as cortisol
(10) at rest. In this respect, Kraemer et al. (13) found a less
favorable anabolic and catabolic response after resistance
exercise when older men were compared to younger men.
Moreover, age-related skeletal muscle adaptations include
a decrease in skeletal muscle fiber diameter (15,16,34,35),
which may be attributed to decreased physical activity and
possibly decreases in satellite cell number and activation (8).
However, these changes could also be influenced by age-
related differences in transcriptional activity of skeletal
muscle proteolysis, an area that remains relatively un-
explored. As a result, it was hypothesized that in response
to a single bout of heavy resistance training, older men would
have less favorable hormone concentrations when compared
to younger men. As a result of these anticipated hormonal
responses, it was further hypothesized that older men would
have a higher baseline mRNA expression of atrogin-1 and
MuRF-1 suggesting an upregulation of UPP activity.
Therefore, the purpose of this investigation was to examine
baseline and 24-hour postexercise mRNA expression of
atrogin-1 and MuRF-1 in younger and in older men.
METHODS
Experimental Approach to the Problem
To investigate age-related differences in acute changes in serum
hormones and intramuscular expression of proteolytic genes, 1
group of younger and 1 group of older men completed the study
design in a paired approach. After baseline blood and muscle
sampling, participants were familiarized to equipment and
completed 1-repetition maximum (1RM) for each exercise.
Later, all participants completed a standardized workout
consisting of 3 sets of 10 repetitions at 80% 1RM. This
combination of sets, repetitions, and intensity was chosen for
its ability to stimulate acute changes in the measured hormones
and initiate an optimal amount of motor unit and subsequently
muscle fiber involvement. Five minutes after completion of the
exercise and 24 hours after completion of the exercise bout,
a second muscle sample was collected. Blood was analyzed for
concentration changes in serum hormones, whereas muscle
samples were analyzed for changes in intramuscular genes of
interest.
Subjects
Thirteen younger (age: 21 6 1 years, weight: 83.9 6 5.2 kg,
BMI: 26.6 6 1.8; [mean 6 SEM]) and 9 older (68 6 1 years,
82.5 6 2.7 kg, 26.6 6 1.8 kg
m22) apparently healthy men
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(i.e., 1 workout per week) for

TABLE 1. Primer sequences used for the genes of interest.*
Primer sequence
Gene (forward and reverse)
Atrogin-1 5’-ATG TGC GTG TAT CGG ATG G-3#
5’-AAG GCA GGT CAG TGA AGC-3’
MuRF-1 5’-GCC TTC TTC GCC TTC TCC-3’
5’-AGC TCA TAC AGA CTC AGT TCC-3’
b-Actin (HKG)† 5’-AGT CAT CAT CTG GCT CTG G-3’
5’-AAT GGA GGC TTG AGG TAG G-3’
*HKG = housekeeping gene; MuRF-1 = muscle RING (really interesting and novel gene)
finger-1.
†b-Actin was used as an HKG.
at least 1 year; (c) have not
ingested or were currently con-
Accession
suming creatine, beta-hydrox-
number y-beta-
methyl-butyrate (HMB), thermo-
NM_058229 genic aids or other nutritional
supplements (excluding multi-
NM_002931
vitamins) for an 8-week period
NM_001101 before beginning the study; (d)
be classified as low risk accord-
ing to American College of
Sports Medicine criteria with
no medical contraindications to
resistance exercise.

Familiarization Session (Visit 1,

volunteered to participate in this study. Subjects were
informed as to the experimental procedures and signed
informed consent statements and medical history forms in
adherence with the human subjects’ guidelines of the
University of Oklahoma Health Sciences Center before any
data collection.
Procedures
Entrance Criteria. Before participation, subjects had to (a) sign
statements stating that they had no current or past use of
anabolic steroids, human growth hormone, or other phar-
maceutical drugs that affect muscle mass; (b) had not
participated in a structured resistance training program
Day 0). Eligible subjects re-
ported to the laboratory between 0600 and 0900. After
anthropometric measurements (i.e., height, weight, and body
composition via 3-site skinfolds), subjects completed 1RM
tests for the Smith squat, bilateral leg press, and bilateral leg
extension exercises. During the Smith squat (Cybex In-
ternational Inc., Medway, MA, USA) exercise, 2 warm-up sets
of 10 repetitions at ;50% 1RM were completed before 3–5
progressive 1RM attempts with 3 minutes of rest given
between attempts. Squat depth was monitored by having
participants eccentrically descend until their hips touched
a 44-cm box, which typically required all participants to attain
a 90° knee bend at the bottom portion of the exercise. Subjects
were required to maintain good lifting form (i.e., heels in

TABLE 2. Demographics, strength, lifting volume, and dietary attributes.*†

Variable Younger (n = 13) Older (n = 9) Significance

Demographics
Age (y) 21 6 1 68 6 1 p , 0.001
Body mass (kg) 83.9 6 5.2 82.5 6 2.7 p = 0.83
BMI (kg
m22) 26.5 6 1.8 26.6 6 0.9 p = 0.97
Strength and volume
Squat 1RM (kg) 117 6 13 76 6 18 p = 0.051
Leg press 1RM (kg) 258 6 28 202 6 34 p = 0.18
Leg extension (kg) 97 6 6‡ 55 6 6 p , 0.001
Lifting volume (kg)§ 10,232 6 993‡ 7,062 6 477 p = 0.02
Dietary intake
Calories (kcal
kg21
d21) 27.5 6 2.4 24.3 6 1.9 p = 0.178
Protein (g
kg21
d21) 1.2 6 0.2 0.9 6 0.1 p = 0.08
Carbohydrate (g
kg21
d21) 3.2 6 0.4 3.2 6 0.3 p = 0.61
Fat (g
kg21
d21) 1.1 6 0.1 0.9 6 0.1 p = 0.07

*1RM = 1 repetition maximum; BMI = body mass index.

†Values are expressed as means 6 SE.
‡Young . old.
§Denotes lifting volume during visit 2.

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 Exercise and Age Effects of Proteolytic Expression

press 1RM was completed us-

ing a standard
leg press (Cybex International
Inc.). Subjects were positioned
on their back at a 45° angle
whereby the eccentric portion
of the lift positioned their thighs
approximately 1–2 in. from their
torso and their knees at an angle
approximately equal to a 90°
knee flexion angle. Sled position
and foot placement was stan-
dardized between testing ses-
sions, and all subjects were
required to maintain good lift-
ing form (i.e., hands at their
sides with lower back flat on the
back pad). Finally, a 1RM de-
termination was performed us-
ing a bilateral leg extension
Figure 1. Serum insulin-like growth factor-1 (IGF-1) (A) and cortisol (B) concentrations (in nanomoles) at rest and
machine (Cybex International
after exercise; all values are expressed as mean 6 SE; ‡Young . old (p , 0.05); †increase from respective pre-
exercise value (p , 0.05). Inc.) whereby the participants’
legs began at a 90° angle and
extended to an approximate
170° or 180° knee angle to finish
contact with the floor, no bouncing the hips off of the box, no the lift. During all 1RM tests, subjects were advised to employ
resting the hips on the box between descending and the aforementioned lifting criteria and were verbally encour-
ascending the weight) during all lifts. A successful 1RM aged by the experimenters. After the familiarization session,
determination was assessed when participants could not participants were told to record a 3-day dietary log before
perform a successful eccentric–concentric repetition or could their second visit. Detailed instructions were also provided
no longer maintain good lifting form as determined by the in regards to estimating portion sizes and logging food
experimenter. Once Smith squat 1RM was determined, preparation (i.e., logging grilled vs. fried foods, etc.).
subjects were given a 3-minute rest before following similar Furthermore, participants were instructed to maintain their
procedures to determine the 1RM with the leg press. Leg normal caloric intake throughout the duration of the study.

Figure 2. Baseline expression of the genes of interest (GOIs) at rest by comparing raw CT values (A) and the fold change in the expression of the GOI 24 hours
after exercise (B) (mean 6 SE). Note that the x-axis in (A) is expressed as log10, and the dashed line in (B) represents baseline expression (i.e., onefold).
‡Significant difference between groups (young . old, p = 0.03). MuRF1 = muscle RING (really interesting and novel gene) finger 1.

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TABLE 3. Postexercise time course response for atrogin-1 and MuRF-1 mRNA expression.*

Protocol and biopsy Atrogin-1 MuRF-1

Investigation Population time points expression expression

Jones et al. (11) 9 UT men Leg casted for 2 wks
Pre and postcast
(18–30 y) biopsies obtained
Postcast resistance
exercise bout performed
and a 24-h post-ex
biopsy obtained
Yang et al. (32) 8 UT men Exercise: 3 3 10 bilateral
(23 y) knee extensions at 65%
of 1RM
Muscle biopsies
obtained before, 4-h and
24-h post-ex
Coffey et al. (6) 7 RT (31 y) Exercise: 8 3 5 maximal
and 8 ET effort leg extensions
(29 y) men Muscle biopsies obtained
before and 3 hours post-ex
Churchley et al. (4) 7 RT men Both legs examined; one
(30 y) leg glycogen depleted
After glycogen depletion
men returned to the
laboratory for pre- and 3-h
post-ex biopsies
Exercise: 8 3 5 leg extensions
at 80% of 1 RM for each leg
Louis et al. (17) 6 RT adults Exercise: 3 3 10 bilateral
(25 y) knee extensions at 70%
of 1 RM
Muscle biopsies obtained
before and 0, 1, 2, 4, 8, 12,
and 24 h post-ex
Raue et al. (21) 6 Older (85 y) Exercise: 3 3 10 knee
and 8 younger extensions at 70% of 1 RM
(23 y) UT Muscle biopsies obtained
women before and 4 h post-ex
Y From post-cast Y From post-cast
to 24-h post-ex to 24-h postex
biopsy (p , 0.05) biopsy (p , 0.05)

In MHC I fibers, Y In MHC I fibers, [
at 24-h vs. 4-h at both post-ex
post-ex (p , 0.05) time points
(p , 0.05)
No change in MHC In MHC IIa fibers, [
IIa fibers at both post-ex time
points (p , 0.05)
No change 3 h Not examined
post-ex
No change in No change in
glycogen-depleted glycogen-depleted
leg leg
Y in control leg 3-h No change in
post-ex (p , 0.05) control leg
Y 8 and 12-h post- [ 1, 2, and 4 h
ex (p , 0.05) post-ex (p , 0.05)
No change in YW [ 4 h post-ex in YW
and OW (p , 0.05)
[ 4 h post-ex in
OW (p , 0.05)

*UT = untrained; RT = resistance-trained; ET = endurance-trained; post-ex = post-exercise; YW = young women; OW = old women.

Food records were subsequently analyzed for average energy
and macronutrient intake using ESHA Food Processor,
Version 10.2 (Salem, OR, USA).
Training Day (Visit 2, Day 7). Participants reported to the
laboratory between 0600 and 0900 hours after a 12-hour fast.
Before the exercise bout, participants donated 15 mL of venous
blood from the median cubital vein of their right arm using
standard phlebotomy techniques and a 15- to 30-mg muscle
specimen from the vastus lateralis of their dominant leg. Briefly,
1.5 ml of 1.0% Lidocaine HCl was injected subcutaneously
10 minutes before making a pilot incision with an 18 G
hypodermic needle. Muscle specimens were extracted by
repeatedly inserting a 16 G biopsy needle (Medical Devices
Technologies Inc, Gainesville, FL, USA) in the same collection
site 3–5 times. All visible fat and connective tissue from the
specimen was removed before being frozen in liquid nitrogen,
and all muscle samples were subsequently stored at 280°C
until later analysis. All blood samples were centrifuged for 15
minutes at 3,500 rpm, and the serum supernatant was
transferred into 2 microcentrifuge tubes and stored at
220°C for subsequent metabolite analyses.
After the preliminary muscle biopsy and blood collection
procedures, participants cycled at 60 rpm at 60 W for 5
minutes. Participants then completed 1 warm-up set of the
Smith machine squat exercise at 50% of their previously
obtained 1RM. After a 3-minute rest period, each participant

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 Exercise and Age Effects of Proteolytic Expression
completed a resistance exercise bout consisting of 3 sets of
10 repetitions at 80% of their 1RM for Smith squats, leg press
and leg extensions. A 3 minute rest period was given between
each set and between exercises. Five minutes after the
workout, participants donated 15 mL of venous blood from
the previous collection site. Participants were then instructed
to return 24 hours later for a follow-up muscle collection
whereby participants donated a second muscle sample and
third blood sample from approximately the same regions
using identical collection and specimen storage procedures.
Biochemical Analyses
Hormonal Analyses. Commercial enzyme immunoassays (Di-
agnostic Systems Laboratories, Webster, TX, USA) were used
to determine serum cortisol and free IGF-1 before, 5 minutes
post, and 24 hours postexercise. All old and young
participants were grouped on 1 microtiter plate at each time
point (i.e., 1 plate was devoted to old participants, etc.) to
avoid interassay variability within subjects. All assays were
performed according to the provided manufacturer guidelines
using a wavelength of 450 nm against a known standard
curve. Serum concentrations of all samples were determined
in duplicate with a Model 680 microplate reader (Bio-Rad Inc,
Hercules, CA, USA). The average interassay and intraassay
CV values for cortisol and IGF-1 were under 10%.
Total RNA Isolation. Approximately 10–12 mg of muscle was
homogenized using the TRI-reagent method (Sigma Chem-
ical Co., St. Louis, MO, USA). All muscle samples were
preweighed, placed into an autoclaved microcentrifuge tube,
and homogenized using a micropestle and 500 mL of
TRI-reagent per 25 mg tissue. After homogenization,
approximately 100 mL of chloroform was added to each
sample, the samples were vortexed for 15 seconds and
allowed to stand at room temperature for 10 minutes. The
samples were then centrifuged at 12,000 rpm at 4°C for 15
minutes. This homogenization procedure yields 3 phases
including an upper aqueous phase containing total RNA, an
interphase containing DNA, and an organic phase containing
myofibrillar protein. The upper aqueous phase was trans-
ferred into a new autoclaved microcentrifuge tube. Approx-
imately 250 mL of 100% isopropanol per 500 mL of TRI
reagent was used to precipitate the RNA from the aqueous
phase. The RNA pellet was exposed to subsequent ethanol
washes and finally dissolved in 30 mL of RNase-free water
with repeated pipetting and vortexing. This procedure has
been shown to yield undegraded RNA, free of DNA and
proteins as indicated by prominent 28S and 18S ribosomal
RNA bands, and an OD260/OD280 ratio of approximately 1.4,
a value consistent with previous work by Mahoney et al. (16)
The diluted RNA samples were stored at 280°C until later
analyses.
Total mRNA Determination and Reverse Transcription. Total
RNA concentration was determined using a high Sensitivity
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RNA analysis kit with the Experion Automated Electropho-
resis platform (Bio-Rad). This method separates and
quantitates RNA ranging from 50 to 6,000 nucleotides in
length using a laser-excitable RNA stain and RNA ladder
provided by the manufacturer. The preparation of reagents
and the RNA ladder was performed according to the
manufacturer’s instructions. Furthermore, all RNA samples
and the RNA ladder were thawed on ice during the assay to
preserve RNA integrity. The average CV for total RNA was 5.8%.
The standardized solutions of total cellular RNA were
reverse transcribed to synthesize complimentary DNA
(cDNA). In short, a reverse transcription buffer, a deoxynu-
cleotide triphoshphates mixture, MgCl2, RNase inhibitor, an
oligo(dT)15 primer, 10 mL of nuclease-free H2O and 1
U
mL21 MMLV reverse transcriptase enzyme [Bio-Rad])
were incubated at 42°C for 40 minutes, heated to 85°C for 5
minutes then quick chilled on ice yielding the cDNA product.
Starting cDNA template concentrations were diluted in
nuclease-free H2O and quantified using spectrophotometric
procedures (1 OD unit = 50 mg
mL21) and standardized to
200 ng
mL21 before polymerase chain reaction (PCR)
amplification. The standardized cDNA solutions were frozen
at 280°C until real-time PCR was performed.
Real-Time Real-Time Polymerase Chain Reaction. Antisense and
sense oligonucleotide primer pairs were constructed using
commercially available Beacon Designer software (Bio-Rad)
and commercially synthesized (Integrated DNA Technolo-
gies, Coralville, IA, USA). b-Actin was used as an internal
reference gene for detecting relative change in the quantity of
target mRNA because of its consideration as a constitutively
expressed housekeeping gene after resistance training (16).
The primer sequences for atrogin-1, MuRF-1, and b-actin are
listed in Table 1. Four hundred nanograms of cDNA was
added to each of the PCR reactions for atrogin-1, MuRF-1,
and b-actin. Each 25 mL PCR reaction mixture contained the
following mixtures: 2 mL of cDNA template added along
with 12.5 mL of 23 SYBR Green Supermix (Bio-Rad) (100
mM KCl mixture, 40 mM Tris–HCl, 0.4 mM of each dNTP,
50 U
mL21 of iTaq DNA polymerase, 6.0 mM MgCl2, SYBR
Green I, 20 nM flourescein), 1.5 mL of sense and antisense
primers, and 7.5 mL nuclease-free dH2O. The PCR reactions
were amplified with a thermal cycler (Bio-Rad, Hercules)
whereby the amplification sequence involved an initial
3-minute denaturation step to activate the Taq polymerase
followed by a 40-cycle period with a denaturation step at
95°C for 10 seconds and primer annealing and extension step
at 55°C for 30 seconds. Emitted fluorescence from SYBR
green was measured after each cycle and was used to quantify
gene expression. All CT values were assessed in the linear
portion of amplification, and a DNA melt curve analysis was
performed after amplification to assure that the single gene
products were amplified in the absence of primer–dimers.
Agarose gel electrophoresis using 25-mL aliquots of the finalized
PCR reaction mixtures was performed in 1.5% agarose gels

(1 mg
mL21) using 13 Tris–Boric acid–ethylenediaminetetra-
acetic acid buffer and illuminated with a UV transilluminator
(Chemi-Doc XRS, Bio-Rad) to verify positive amplification of
target mRNA (data not shown). All assays were performed in
duplicate, and the CV values for the CT values of atrogin-1 and
MuRF-1 were both under 10%.
Baseline and Postexercise Quantification of Real-Time RT-PCR.
Baseline gene expression data were analyzed using the 22DCT
(DCT = CT for sample with lowest average 2 CT for sample
of interest) and is expressed in arbitrary units. Although this
method does not normalize expression with a housekeeping
gene, it does allow for the comparison of baseline levels of
all genes. Postexercise fold change in gene expression
was analyzed using the Livak method (i.e., 22DDCT as-
suming 100% primer efficiency where DDCT = [CT gene
of interest 2 CT b-actin]post-exercise 2 [CT gene of interest
2 CT b-actin]pre-exercise). Hence, a onefold change indicates
no change in gene expression relative to baseline mRNA
levels.
Statistical Analyses
All data are reported as mean 6 SEM, and all statistical
analyses were performed using SPSS (version 14.0, SPSS Inc.,
Chicago, IL, USA). Participant demographics and dietary
intake was compared using independent samples t-tests.
Shapiro–Wilk tests were performed on all genetic data to test
for distribution normality. When the normality assumption
was violated and traditional transformation approaches were
not feasible, Mann–Whitney U tests were used to compare
differences between age groups. Hormonal data were
compared between groups using a 2 3 3 (group 3 time
point) analysis of variance with repeated measures. Finally,
parametric and nonparametric correlations (i.e., Pearson’s
and Spearman’s r) were performed for selected dependent
variables. Significance for all statistical analyses was de-
termined using an alpha level of # 0.05.
RESULTS
Distribution Normality for Dependent Variables
Shapiro–Wilk tests revealed that baseline and postexercise
mRNA expression profiles for atrogin-1 and MuRF-1 were
not normally distributed (p , 0.05) and as a result non-
parametric approaches were used.
Demographics, Total Lifting Volume, and Dietary Intake
Baseline demographics are presented in Table 2. The young
group exhibited significantly greater values for the 1RM
Smith squat and leg extension and total lifting volume during
visit 2 (p , 0.05).
Hormonal Response to Exercise
Hormone concentrations before and after the exercise bout are
presented in Figure 1. No group 3 time interactive effects for
cortisol were revealed (p = 0.89), although there was a signi-
ficant main effect for group (p , 0.05) and time (p , 0.001).
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Within-group pairwise comparisons revealed that cortisol
significantly increased in younger (p , 0.01) and older (p ,
0.05) men at 5 minutes postexercise. Independent samples
t-tests revealed that cortisol levels were significantly greater in
younger vs. older men before (p , 0.05) and 24 hours
postexercise (p , 0.001). No group 3 time interactive effects
were found for IGF-1 concentrations (p = 0.62). There was
a significant main effect for group (p , 0.001) with separate
independent samples t-tests revealing that IGF-1 levels were
significantly greater in younger vs. older men at all time points
(p , 0.001). Changes over time tended to be different (p =
0.051) whereby within-group pairwise comparisons revealed
that IGF-1 significantly increased in older (p , 0.05) men 24
hours postexercise, whereas serum IGF-1 tended to increase in
younger men 5 minutes postexercise (p = 0.07).
Gene Expression
Basal expression patterns for atrogin-1 and MuRF-1 are
presented in Figure 2A. No differences in baseline expression
of atrogin-1 (p = 0.24) were revealed. MuRF-1 mRNA
expression were significantly greater in the older men when
compared to the younger men at baseline (p , 0.05).
Postexercise expression patterns for atrogin-1 and MuRF-1
are presented in Figure 2B. No between-group age differ-
ences (p . 0.05) 24 hours after exercise were revealed for
atrogin-1 and MuRF-1. Further, no significant within-group
changes in response to the exercise stimulus were revealed
for either gene (p . 0.05).
Correlations
Spearman’s correlations were used to examine relationships
between baseline serum concentrations of cortisol and IGF-1
and the baseline mRNA expression of atrogin-1 and MuRF-1.
No significant correlations were found between cortisol and
atrogin-1 (r = 20.070; p = 0.75) or MuRF-1 (r = 20.028; p =
0.90). Additionally, no significant correlations were found
between IGF-1 and atrogin-1 (r = 20.316; p = 0.13), but
a significant correlation was found between IGF-1 and
MuRF-1 (r = 20.424; p , 0.05).
DISCUSSION
The purpose of the current investigation was to examine
adaptations that occur in IGF-1, cortisol, and intramuscular
mRNA expression of atrogin-1 and MuRF-1 with age and in
response to an acute bout of resistance exercise. Baseline and
24-hour postexercise concentrations of cortisol were signif-
icantly higher in younger compared to older men, whereas
serum IGF-1 concentrations were significantly higher at each
time point in younger compared to older men. Moreover,
MuRF-1 mRNA expression was significantly greater in older
compared to younger men, and there was a significant
negative correlation between serum IGF-1 and MuRF-1
mRNA expression. This relationship provides further evi-
dence that IGF-1 may be able to reduce skeletal muscle
proteolysis in vivo by activating the Akt pathway and
downstream proteolytic involvement (3,28).
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 Exercise and Age Effects of Proteolytic Expression
In support of previous research and a response that is well
characterized in the literature, serum IGF-1 concentrations
were significantly greater in younger men at all time points
when compared to older men (7,13), whereas cortisol
concentrations were greater at all time points in younger
men. These findings both refute (13) and agree with
previous studies examining serum cortisol concentrations
and age (10). We hypothesized serum cortisol concen-
trations would be higher in older men because of the
progressive reduction seen in muscle in aging populations;
however, it appears that other mechanistic involvement
should be examined.
Atrogin-1 and MuRF-1 gene and protein expression have
been used to assess skeletal muscle wasting in a number of
published investigations (2,5,24). Glucocorticoid exposure to
a cell culture (C2C12) line significantly decreased myocel-
lular diameter and increased protein expression of atrogin-1
and MuRF-1 (27) whereby IGF-1 addition increased fiber
diameter. Our in vivo findings support this data due to higher
serum IGF-1 and cortisol levels and significantly lower
mRNA expression of MuRF-1 at baseline. Furthermore, the
lack of fold-change differences between atrogin-1 and
MuRF-1 after exercise suggests that the time course of these
candidate genes may not be age dependent.
Limited research has been conducted examining the effects of
age or exercise on atrogin-1 and MuRF-1 gene expression (21)
in vivo, which underscores the need for such investigations.
However, Raue et al. have examined the effects of age and an
acute bout of resistance exercise in younger (23.4 6 1.7 years)
and older women (85.2 6 1.3 years) and found at baseline older
women expressed significantly higher mRNA levels of MuRF-1,
whereas no age-related differences were found for atrogin-1.
Both results are consistent with the findings from this study and
also suggest both genders may be responding to a resistance
training stimulus in a similar manner. In contrast, Whitman et al.
(31) found no baseline differences in the mRNA expression of
atrogin-1 and MuRF-1 gene expression between younger
(21.5 6 2.9 years) and older (72.8 6 8.3 years) adults.
Nevertheless, this study and other research models suggest an
involvement of MuRF-1 for associated changes in proteolytic
activity while results for atrogin-1 have been reported (11,21,29).
An additional consideration raised in the Raue et al. (21) article
was that the rate of muscle wasting may carry a stronger
impetus over resulting gene expression than age itself.
In the current investigation, there was a nonsignificant (p .
0.05) upregulation of atrogin-1 (younger men: 1.8 6 0.7 AU,
p . 0.05; older men: 2.9 6 1.8 AU) and MuRF-1 (younger men:
1.6 6 0.3 AU, p . 0.05; older men: 2.2 6 0.7 AU) mRNA
expression in younger and older men 24 hours after a bout of
resistance exercise. Although in contrast with other studies, the
differences in sampling time could certainly be an experimental
factor responsible for these differences; a point which is
illustrated in Table 3. To this point, an excellent investigation
was published by Louis et al. (17), which nicely illustrated the
time course changes in proteolytic gene expression.
the TM
8 Journal of Strength and Conditioning Research
Copyright © National Strength and Conditioning Association Unauthorized reproduction of this article is prohibited.
Limitations of the current investigation include not having
a measure of skeletal muscle mass because it would have
been of interest to examine if differences in serum cortisol
concentrations relative to skeletal muscle mass are present
between younger and older adults. We have also speculated
that the increased serum concentrations of IGF-1 in younger
adults activate the Akt pathway and inhibit atrogene
expression, but we did not examine any Akt pathway proteins
or FOXO transcription factor phosphorylation status as our
muscle collection procedure did not yield an adequate supply
of tissue for these assays. Additionally, our investigation
focused on 2 key atrogenes (atrogin-1 and MuRF-1) at 1 time
point (24 hours postexercise), which limits our ability to make
comparisons to the work of Raue et al. (21) or expand our
results into more acute changes in intramuscular proteolytic
adaptations. Finally, it is important to understand that
disconcordant relationships often exist between the transcrip-
tion of a gene and the translation of this gene into a functional,
viable protein. Thus, our lack of data illustrating the expression
of proteins coded by the genes we measured limits our ability
to fully interpret all aspects of this data. In this respect,
subsequent studies should be completed to examine atrogin-1
and MuRF-1 mRNA and protein expression after repeated
exercise bouts to give a clearer picture of the effects of repeated
exercise on atrogene expression. Moreover, future investiga-
tions should continue to examine more deeply the role of the
acute training variables and how manipulations in these
variables impact intramuscular changes. In this respect, it is
certainly possible that ineffective or more likely divergent
patterns of neuromuscular recruitment resulted in a muscle
sample that was a partial representation of the activated motor
units. Although this fact is apparent with all biopsy sampling
studies, the inherent difficulties with conclusions to be drawn
from our data is apparent. To this point, a recent excellent
review into this topic by Spiering et al. (26) outlined much of
this research and highlighted the fact that limited data exists
relative to proteolytic level changes.
PRACTICAL APPLICATIONS
Results from the current investigation suggest that upregulation
of MuRF-1 mRNA expression with age (21) might be
suggestive of increased UPP activity, which could be a precursor
to wasting or some adaptive outcome of the transcriptome to
regulate physiological demands. Significant muscle atrophy can
occur with detraining, rehabilitation from injury, surgery and in
older adults and clinical populations. Additional research into
the molecular control points for muscle atrophy are important
and can help elucidate modifications to resistance exercise
training to optimize results and ultimately helping fitness
professional and clinicians better understand muscle physiology
with exercise and advancing age.
ACKNOWLEDGMENTS
We would like to thank the subjects who participated in this
study and all laboratory assistants who aided with data

collection and analysis. We would also like to graciously thank
the reviewers that took the time to critique this manuscript. The
National Strength and Conditioning Foundation provided
funds for this project through a Young Investigator Grant to the
corresponding author and principle investigator (CK).
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