One session of exercise or endurance training does not influence serum levels of Irisin in rats

Abstract

Irisin induces the browning of adipose tissue. The goal of this study was to investigate the influence of acute exercise in untrained and trained rats and endurance training on FNDC5 mRNA and irisin levels in white and red skeletal muscle and serum. Rats (n=60) were randomly divided into two groups: untrained and trained (subjected to 6-week endurance training with increasing load). Subgroups of rats from each group were sacrificed before (controls), immediately after, or 3 hours following acute exercise with the same work load. Muscle samples (red and white) and serum were collected. FNDC5 mRNA was evaluated using RT-PCR. Irisin levels were measured using an immunoenzymatic method. Muscle FNDC5 mRNA decreased immediately after acute exercise compared with baseline levels, but not in red muscle in trained rats. Atrend toward a return to baseline appeared 3 hours after the exercise, but only in white muscle in untrained group. Irisin protein levels increased after acute exercise in red muscle 3 hours post-exercise compared with samples taken immediately after exercise, and decreased 3 hours post-exercise compared to pre-exercise level in white muscles. FNDC5 mRNA did not change following training, whereas irisin protein levels increased in red muscle and decreased in white muscle. Serum irisin levels remained unchanged following acute exercise and training. We concluded that changes in irisin mRNA and protein levels in rat muscle after acute exercise are limited and depend on training status and the muscle type. Irisin serum levels remained stable after acute exercise or endurance training.

Full text

INTRODUCTION
Recently, skeletal muscle was identified as an endocrine organ
that produces and releases a wide range of cytokines and other
peptides. These cytokines and peptides are collectively named
myokines. Myokines exert their effects in autocrine, paracrine,
and endocrine manners and play an important role in driving
adaptive changes to physical exercise. Myokines provide the basis
for understanding cross-talk between exercising muscles and other
organs, such as adipose tissue, liver, heart and vessels, and brain
and could explain the beneficial effects of physical activity in
preventing non-communicable diseases (1-3). Physical inactivity
is associated with higher BMI and greater risk for excessive
accumulation of fat in the central part of the body (4).
Recently, a new myokine, irisin was discovered, which is a
downstream product of the transcriptional co-activator, PGC-1a
(peroxisome proliferator-activated receptor-gcoactivator-1a).
PGC-1ais increased in muscles by physical exercise and
mediates many adaptive changes related to energy metabolism,
for instance the expression of mitochondrial proteins encoded in
both nuclear and mitochondrial genes. It stimulates also the
expression of several skeletal muscle genes, including FNDC5
(fibronectin type III domain containing 5). The FNDC5 gene
encodes a type I membrane protein that is subsequently
proteolytically processed to form irisin. Irisin is secreted from
muscles into the blood and induces browning of adipose tissue
and UCP-1 (uncoupling protein 1) expression, and improves
systemic metabolism by increasing energy expenditure (5-8).
Irisin serum levels are positively correlated with body mass
index, body weight, fat mass, and free fat mass (9).
Because of the short time since the discovery of irisin, there
are very few experimental studies regarding the influence of
physical exercise on FNDC5 expression in muscles and irisin
protein levels in muscle and serum, and the results are
controversial. The studies of Bostrom et al.(6) showed that
FNDC5 mRNA is increased in muscles and irisin protein is
increased in plasma following 3 weeks of training in mice, and
after 10 weeks of training in humans, but Timmons et al.(10)
confirmed the increase in muscle FNDC5 mRNA only in the
case of highly active elderly human subjects.
The goal of the present study was to investigate the influence
of a single session of acute exercise in untrained rats compared
with trained rats, and prolonged endurance training on FNDC5
mRNA and irisin protein levels in white and red muscle and
serum. Additionally, to determine whether endurance training,
similar to that recommended for people, induces irisin in the
signalling pathway, and subsequently directly induces the
browning of adipose tissue.
JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2014, 65, 3, 449-454
www.jpp.krakow.pl
B. CZARKOWSKA-PACZEK1,M. ZENDZIAN-PIOTROWSKA2,K. GALA3,M. SOBOL1,L. PACZEK3
ONE SESSION OF EXERCISE OR ENDURANCE TRAINING DOES NOT INFLUENCE
SERUM LEVELS OF IRISIN IN RATS
1Department of Biophysics and Human Physiology, Medical University of Warsaw, Warsaw, Poland;
2Department of Physiology, Medical University of Bialystok, Bialystok, Poland;
3Department of Immunology, Transplantology and Internal Diseases, Medical University of Warsaw, Warsaw, Poland
Irisin induces the browning of adipose tissue. The goal of this study was to investigate the influence of acute exercise
in untrained and trained rats and endurance training on FNDC5 mRNA and irisin levels in white and red skeletal muscle
and serum. Rats (n=60) were randomly divided into two groups: untrained and trained (subjected to 6-week endurance
training with increasing load). Subgroups of rats from each group were sacrificed before (controls), immediately after,
or 3 hours following acute exercise with the same work load. Muscle samples (red and white) and serum were collected.
FNDC5 mRNA was evaluated using RT-PCR. Irisin levels were measured using an immunoenzymatic method. Muscle
FNDC5 mRNA decreased immediately after acute exercise compared with baseline levels, but not in red muscle in
trained rats. A trend toward a return to baseline appeared 3 hours after the exercise, but only in white muscle in untrained
group. Irisin protein levels increased after acute exercise in red muscle 3 hours post-exercise compared with samples
taken immediately after exercise, and decreased 3 hours post-exercise compared to pre-exercise level in white muscles.
FNDC5 mRNA did not change following training, whereas irisin protein levels increased in red muscle and decreased
in white muscle. Serum irisin levels remained unchanged following acute exercise and training. We concluded that
changes in irisin mRNA and protein levels in rat muscle after acute exercise are limited and depend on training status
and the muscle type. Irisin serum levels remained stable after acute exercise or endurance training.
Key words: exercise, irisin, endurance training, browning of fat, energy expenditure, fibronectin type III domain containing 5,
red skeletal muscle

MATERIALS AND METHODS
All procedures in the study were approved by the Ethical
Committee of the Medical University in Bialystok and were
performed according to EU regulations governing the treatment
of laboratory animals.
This study used 60 male Wistar rats. The rats had access to
water ad libitum,were fed Labofeed B, and were maintained in
a12/12 h light/dark cycle. The experimental protocol was as
previously described (11-13).
During the first 5 successive days of the experiment, the rats
were subjected to exercise adaptation, which consisted of 10 min
of treadmill running at 15 m/min per day. Following adaptation,
rats were randomly assigned to two groups: untrained (UT,
n=30) and trained (T, n=30). Rats from the T group were
subjected to prolonged endurance training consisting of
treadmill running 5 days a week for 6 weeks. During the first
week, the exercise time was increased by 10 min each day,
starting from running 10 min/per day at a speed of 1200 m/h.
During subsequent weeks of training, running time was a
constant 60 min/day. During week 2, the running speed was
1500 m/h, and then it was increased to 1680 m/h during weeks
3–6. No additional stimulus was applied to enhance running.
The UT group remained at rest during the training period.
Twenty-four hours after the last training session, each group (UT
and T) was randomly divided into three subgroups. Ten rats from
each subgroup (UTpre, n=10 and Tpre, n=10) were sacrificed
under anaesthesia (intraperitoneal chloral hydrate, 1 ml/100 mg
body mass). The remaining rats in each UT and T subgroup were
subjected to an acute exercise session consisting of 60 min of
treadmill running at 1680 m/h. Relative effort was higher in the UT
group compared with the T group. The rats from the two subgroups
subjected to acute exercise were sacrificed either immediately
following the acute exercise (UT0h, n=10 and T0h, n=10) or 3
hours after the acute exercise (UT3h, n=10 and T3h, n=10).
Immediately after sacrifice, samples from the gastrocnemius (red
and white portions) muscle were collected according to the
described redistribution of specific fibres (14) from all rats in each
group and stored at –80°C for subsequent analyses.
The mean body mass of rats in the UT group on the day of
acute exercise was 271±11.6 g and 283.17±24.67 g in the T group.
mRNA isolation
Approximately 50 mg of skeletal muscle tissue (red and
white portions of the gastrocnemius) was homogenized in a
TissueLyser bead mixer (Qiagen, Germany) at a frequency of 25
Hz for 5 minutes. Total mRNA isolation was performed using an
EZ1 RNA Universal Tissue Kit and Biorobot EZ1 (Qiagen,
Germany) according to the manufacturer’s instructions. Total
RNA concentrations were measured at 260 nm using NanoDrop
spectrophotometry (ND-1000 Spectrophotometer, NanoDrop
Technologies, Inc). Samples were then frozen and stored at
–80°C for further analyses.
Reverse transcription
Reverse transcription of total mRNA into cDNA was
performed using the Thermomixer Comfort (Eppendorf,
Germany) with the SuperScript III First-Strand Synthesis
450
Fig.1.Relative skeletal muscle
(gastrocnemius, red) FNDC5
mRNA and irisin protein levels
(mean ±S.D.) after an acute bout of
exercise in untrained (UT) and
trained (T) rats.
Analyses were performed in
samples collected from each group
prior to an acute bout of exercise
(UTpre, Tpre), just after the
cessation of exercise (UT0h, T0h),
and 3 hours post exercise (UT-3h,
T3h). * Statistically significant
difference (p<0.05).

System for RT-PCR (Invitrogen, USA) according to the
manufacturer’s instructions.
Real-time PCR to quantify FNDC5 in mRNA
Detection of mRNA was performed using an ABI-Prism
7500 Sequence Detection System (Applied Biosystems, USA).
Specific primers and probes and TaqMan Universal Master Mix
for rat FNDC5 were purchased from Applied Biosystems. The
relative amount of specific mRNA was normalized to rat
GAPDH. The relative expression of mRNA was calculated using
the 2-DDCt method (15).
Protein quantization in skeletal muscle and serum
The evaluation of irisin concentrations in serum and in tissue
homogenates was performed using enzyme-linked
immunosorbent assay (ELISA). Each sample of skeletal muscle
was homogenized in a TissueLyser bead mixer (Qiagen,USA)
and centrifuged (10,000 rpm for 10 minutes, 4°C). The
supernatant was collected and frozen at –80°C until analyses.
Total protein concentration was measured at 562 nm on a Bio-
Tek Power Wave XS spectrophotometer (Bio-Tek Instruments,
USA) using bicinchoninic acid (BCA) Protein Assay Reagent
(Pierce, Holland), according to the manufacturer’s instructions.
Tissue and serum irisin protein concentrations were measured
using an Irisin/FNDC-5 ELISA kit (Phoenix Pharmaceuticals,
INC, USA). The results are presented as the ratio of irisin
concentration/protein concentration.
Statistical analyses
Results are provided as the mean ±S.D. and as relative fold
changes. Differences in mRNA levels (for statistics, DCTwas
used) and protein levels between groups were analysed using
Kruskal-Wallis non-parametric ANOVA followed by post hoc
Duncan’s Test. Pre- and post-training values of mRNA and
protein levels in skeletal muscle tissue were compared using the
Student’s ttest. p<0.05 was considered to be statistically
significant.
RESULTS
The relative changes in FNDC5 mRNA and irisin protein
levels in red skeletal muscle (gastrocnemius, red portion) after
an acute bout of exercise in UT and T rats are shown in Fig.1.
In UT rats, there was a significant decrease in FNDC5 mRNA in
the UT0h and UT3h groups compared with the UTpre group
(p=0.0003 and p=0.001, respectively). In T rats, FNDC5 mRNA
levels were similar between groups between time points. Irisin
protein levels decreased, but not significantly, in the UT0h group
compared with the UTpre group, and there was a significant
increase in the UT3h group compared with the UT0h group
(p=0.002). In T rats, irisin protein levels were similar between
time points.
The relative changes in FNDC5 mRNA and irisin protein
levels in white skeletal muscle (gastrocnemius, white portion)
after an acute bout of exercise in UT and T rats are shown in Fig.
451
Fig.2.Relative skeletal muscle
(gastrocnemius, white) FNDC5
mRNA and irisin protein levels
(mean ±S.D.) after an acute bout of
exercise in untrained (UT) and
trained (T) rats.
Analyses were performed in
samples collected from each group
prior to an acute bout of exercise
(UTpre, Tpre), just after the
cessation of exercise (UT0h, T0h),
and 3 hours post exercise (UT-3h,
T3h). * Statistically significant
difference (p<0.05).

2.In UT rats, FNDC5 mRNA levels decreased in the UT0h
group compared with the UTpre group (p=0.03). In the UT3h
group, FNDC5 mRNA was increased in comparison with levels
in the UT0h group (p=0.01). In T rats, there was a significant
decrease in FNDC5 mRNA in the T0h group compared with the
Tpre group (p=0.0007). The trend toward increased levels
appeared 3 hours post exercise; however, this increase was not
significantly different compared with the earlier time points.
The irisin protein levels were decreased in the UT3h group
compared with the UTpre group (p=0.02). In T rats, the irisin
protein levels were similar between time points.
The relative changes in FNDC5 mRNA and irisin protein
levels in red and white skeletal muscle following prolonged
endurance training in T rats compared with UT rats are shown in
Fig.3.FNDC5 mRNA did not change after prolonged training in
red or white muscle. Irisin protein levels increased in red muscle
(p=0.03) and decreased in white skeletal muscle (p=0.02).
Serum irisin levels did not change after an acute bout of
exercise in either UT or T rats and after prolonged training (Fig.4).
DISCUSSION
The most surprising effect of our experiment was the stable
irisin serum levels after acute bouts of exercise in untrained and
trained animals and after prolonged training. These findings are
in contrast with the results reported by Bostron et al.(6);
however, their results referred to humans and mice, and the
results may not be transferable between species. Our results are
in line with the recently published data obtained by Fain et al.
(16), who reported that prolonged training do not result in
increase of FNDC5 mRNA and protein in skeletal muscle and
irisin level in serum in normal pigs. Huh et al.(17) reported an
increase in irisin serum levels after an acute sprint in untrained
humans and unchanged irisin levels after the same exercise in
trained humans. The level of irisin increased when the level of
ATP dropped, but remained unchanged when the level of ATP
also remained unchanged. They proposed the hypothesis that
when ATP concentrations in muscle are decreased, irisin
production is upregulated. Thus, irisin could contribute to
restoration of ATP homeostasis, but return to baseline levels
soon after this restoration is completed. Based on Huh’s results
and conclusions, we hypothesize that the mode of energy
metabolism could explain the lack of increase in irisin serum
levels in our study. Running at the speeds used in our
experiment, the exercise was moderate for the rats so ATP
depletion was low and primarily red muscle fibres were recruited
(18). Myosin heavy chain composition of muscle fibres has an
impact on the oxygen cost of exercise. (19). This could explain
why we did not observe any immediate changes in irisin protein
452
Fig.3.Relative changes in skeletal
muscle (gastrocnemius, red, panel
A, and gastrocnemius, white, panel
B) FNDC5 mRNA and irisin protein
levels (mean ±S.D.) after prolonged
endurance training (T vs. UT). *
Statistically significant difference
(p<0.05).

levels after an acute bout of exercise in red or white muscle in
either group. However, other regulatory mechanism could be
involved to induce an increase in irisin protein level in red
muscle in the UT group 3 hours after the exercise compared with
the time point immediately after exercising. Otherwise, in the
UT group in white muscle, there was a decrease in irisin protein
level 3 hours after an acute bout of exercise compare to pre-
exercise level. Similar results were found after prolonged
training as the increase in irisin protein levels were observed
only in red muscle; in white muscle, a decrease in irisin protein
levels was observed. It seems that small changes observed 3
hours after an acute bout of exercise occurred during the training
period. The aforementioned increases in irisin protein levels in
muscles seem too small to induce an increase in irisin serum
levels after acute bouts of exercise or prolonged training.
In our experiment, FNDC5 mRNA expression was
decreased right after an acute exercise session in red and white
muscles in trained and untrained rats, however non significantly
in trained group in red muscle. A trend towards a return to
baseline levels was observed 3 hours following cessation of
exercise, but only in white muscle in untrained group. Bostrom
at al.(6) reported that acute exercise did not change FNDC5
mRNA levels in mice; however, they did not describe the
parameters of the exercise and time points of samples taken in
relation to the cessation of exercise. They observed increased
FNDC5 mRNA following prolonged training in mice and
humans. In our experiment, FNDC5 mRNA levels did not
change after endurance training in red or white muscles.
Timmons et al.found an increase in FNDC5 mRNA levels in
humans, but only in active elderly subjects. They did not confirm
Bostrom’s results with regard to young active subjects after an
aerobic exercise, and found no relationship between the FNDC5
mRNA in muscle and diabetes status in humans (10).
Changes in irisin protein levels following training in red or
white muscle did not follow the changes inFNDC5 mRNA levels
in the muscles. The possible reason for this could be the different
proteolytic activities in muscle that are responsible for
translating irisin protein from the product of the FNDC5 gene, or
possible post-translational changes. In mammalian cells, the
correlation coefficient between mRNA and protein levels is less
than 0.5 (20).
In conclusion, serum level of irisin was stable after acute
exercise or prolonged training and changes in irisin mRNA and
protein levels after acute exercise and prolonged training in the
muscles of rats are limited and depend on the training status and
muscle type.
Abbreviations: PGC-1a,peroxisome proliferator-activated
receptor-gcoactivator-1a;FNDC5, fibronectin type III domain
containing 5; UCP-1, uncoupling protein 1
Acknowlwdgements:These studies were partially funded by
the Medical University of Bialystok (grant no: 123-18810) and
by Medical University of Warsaw (grant no:
NZME/DYW/10/12).
Conflict of author’s: None declared.
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Received: August 8, 2013
Accepted: May 16, 2014
Author’s address: Dr. Bozena Czarkowska-Paczek,
Department of Biophysics and Human Physiology, Medical
University of Warsaw, 5 Chalubinskiego Street, 02-004 Warsaw,
Poland
E-mail: dom_paczek@o2.pl
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