Effect of endurance training on skeletal muscle myokine expression in obese men: identification of apelin as a novel myokine.
ABSTRACT IntroductionIt has been suggested that metabolic benefits of physical exercise could be mediated by myokines. We examined here the effect of exercise training on skeletal muscle expression of a panel of myokines in humans. Pathways regulating myokine expression were investigated in human myotubes.Methods
Eleven obese non diabetic male subjects were enrolled in an 8-week endurance training program. Insulin sensitivity was assessed by oral glucose tolerance test. Subcutaneous adipose tissue and Vastus Lateralis muscle biopsies were collected before and after training. RNAs were prepared from adipose tissue and skeletal muscle. Primary culture of myoblasts was established.ResultsAs expected, exercise training improved aerobic capacity and decreased fat mass. No significant change in interleukin 6, fibroblast growth factor 21, myostatin or irisin mRNA level was found in muscle after training. A 2-fold increase in apelin mRNA level was found in muscle but not in adipose tissue. No change in circulating myokine and adipokine plasma levels was observed in the resting state in response to training. Interestingly, apelin was significantly expressed and secreted in primary human myotubes. Apelin gene expression was up-regulated by cyclic AMP and calcium unlike the other myokines investigated. Importantly, muscle apelin mRNA levels were positively related to whole-body insulin sensitivity.Conclusion
Collectively, our data show that exercise training up-regulates muscle apelin expression in obese subjects. Apelin expression is induced by exercise signalling pathways and secreted in vitro in human primary myotubes, and may behave as a novel exercise-regulated myokine with autocrine/paracrine action.International Journal of Obesity accepted article preview online, 27 August 2013. doi:10.1038/ijo.2013.158.
- SourceAvailable from: Joseph V Moxon[Show abstract] [Hide abstract]
ABSTRACT: BACKGROUND: Peripheral artery disease (PAD) is associated with impaired mobility and a high rate of mortality. The aim of this systematic review was to investigate whether reduced lower extremity performance was associated with an increased incidence of cardiovascular and all-cause mortality in people with PAD. METHODS AND RESULTS: A systematic search of the MEDLINE, EMBASE, SCOPUS, Web of Science, and Cochrane Library databases was conducted. Studies assessing the association between measures of lower extremity performance and cardiovascular or all-cause mortality in PAD patients were included. A meta-analysis was conducted combining data from commonly assessed performance tests. The 10 identified studies assessed lower extremity performance by strength tests, treadmill walking performance, 6-minute walk, walking velocity, and walking impairment questionnaire (WIQ). A meta-analysis revealed that shorter maximum walking distance was associated with increased 5-year cardiovascular (unadjusted RR=2.54, 95% CI 1.86 to 3.47, P<10(-5), n=1577, fixed effects) and all-cause mortality (unadjusted RR=2.23 95% CI 1.85 to 2.69, P<10(-5), n=1710, fixed effects). Slower 4-metre walking velocity, a lower WIQ stair-climbing score, and poor hip extension, knee flexion, and plantar flexion strength were also associated with increased mortality. No significant associations were found for hip flexion strength, WIQ distance score, or WIQ speed score with mortality. CONCLUSIONS: A number of lower extremity performance measures are prognostic markers for mortality in PAD and may be useful clinical tools for identifying patients at higher risk of death. Further studies are needed to determine whether interventions that improve measures of lower extremity performance reduce mortality.J Am Heart Assoc. 01/2014; 3(4).
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ABSTRACT: Recent findings regarding the response of fibronectin type III domain-containing protein 5 (Fndc5) and irisin to exercise are partly controversial. While the 25 kDa form of Fndc5 can be observed in muscle and serum of different species, the ~12 kDa irisin band was not detectable up to now. The present study aimed to clarify whether irisin exists in its theoretical size of ~12 kDa in mice and if it is affected by exercise. Male mice were randomly assigned to a sedentary control group (CO), a group with free access to running wheels (RW), and a treadmill group (TM). Blood and leg muscles were collected to investigate the regulatory cascade including peroxisome proliferator-activated receptor gamma co-activator 1-alpha (Ppargc1a) and Fndc5. In western blot analysis, antibodies were used capable of differentiation between full-length Fndc5 and irisin. This enabled us to demonstrate that irisin exists in muscle and serum of mice independent of exercise and that it is increased immediately after acute exercise. Different transcripts of Ppargc1a mRNA, but not Fndc5 mRNA, were up-regulated in the TM group. Furthermore, neither Fndc5 (25 kDa) nor Ppargc1a protein was elevated in muscle tissue. The Ppargc1a-Fndc5/irisin pathway did not clearly respond to mild exercise in the RW group. Our results provide evidence for the existence of irisin and for its immediate response to acute exercise in mice.International journal of biological sciences 01/2014; 10(3):338-349. · 3.17 Impact Factor
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ABSTRACT: Context: The myokine irisin may increase energy expenditure and affect metabolism. Objective: To elucidate predictors of irisin and study whether circulating irisin may have day-night rhythm in humans. Design: Observational, cross-sectional study with an additional 24h prospective observational arm (Day-night rhythm sub-study) and two prospective interventional arms (Mixed meal sub-study and Exercise sub-study). Setting: The Hellenic Military School of Medicine, Thessaloniki, Greece. Patients and Interventions: One hundred twenty-two healthy, young individuals were subjected to anthropometric and body composition measurements and their eating and exercise behavior profiles were assessed with validated questionnaires. Sub-groups were subjected to day night rhythm, standardized meal ingestion and 30-min aerobic exercise studies. Main outcome measures: Circulating irisin levels. Results: Irisin levels were lower in males than females (p=0.02) after adjustment for lean body mass (LBM), which was its major determinant. Irisin levels followed a day-night rhythm (p<0.001) with peak at 9 pm. Irisin levels were increased at the end of exercise (84.1 ± 10.0 vs. 105.8 ± 14.3 ng/mL; p<0.001). Irisin levels were not affected by intake of a standardized meal and were not associated with caloric intake or diet quality. Conclusions: In healthy, young individuals circulating irisin displays a day-night rhythm, is correlated with lean body mass and increases acutely after exercise.The Journal of clinical endocrinology and metabolism. 06/2014;
Effect of endurance training on skeletal muscle myokine
expression in obese men: identification of apelin as a novel
A Besse-Patin1,2, E Montastier1,2,3, C Vinel2,4, I Castan-Laurell2,4, K Louche1,2, C Dray2,4, D Daviaud2,4, L Mir1,2, M-A Marques1,2,
C Thalamas1,2,5, P Valet2,3, D Langin1,2,3, C Moro1,2,6and N Viguerie1,2,6
BACKGROUND: It has been suggested that the metabolic benefits of physical exercise could be mediated by myokines.
We examined here the effect of exercise training on skeletal muscle expression of a panel of myokines in humans. Pathways
regulating myokine expression were investigated in human myotubes.
METHODS: Eleven obese non-diabetic male subjects were enrolled in an 8-week endurance training program. Insulin sensitivity
was assessed by an oral glucose tolerance test. Subcutaneous adipose tissue and Vastus lateralis muscle biopsy samples were
collected before and after training. RNAs were prepared from adipose tissue and skeletal muscle. Primary culture of myoblasts was
RESULTS: As expected, exercise training improved aerobic capacity and decreased fat mass. No significant change in interleukin 6,
fibroblast growth factor 21, myostatin (MSTN) or irisin mRNA level was found in muscle after training. A twofold increase in apelin
mRNA level was found in muscle but not in adipose tissue. No change in circulating myokine and adipokine plasma levels was
observed in the resting state in response to training. Interestingly, apelin was significantly expressed and secreted in primary
human myotubes. Apelin gene expression was upregulated by cyclic AMP and calcium, unlike the other myokines investigated.
Importantly, changes in muscle apelin mRNA levels were positively related to whole-body insulin sensitivity improvement.
CONCLUSION: Collectively, our data show that exercise training upregulates muscle apelin expression in obese subjects. Apelin
expression is induced by exercise signaling pathways and secreted in vitro in human primary myotubes, and may behave as a novel
exercise-regulated myokine with autocrine/paracrine action.
International Journal of Obesity advance online publication, 24 September 2013; doi:10.1038/ijo.2013.158
Keywords: exercise; skeletal muscle cells; apelin; myokines
Regular physical activity protects against numerous chronic
diseases such as obesity, type 2 diabetes and cardiovascular
diseases.1Some of the beneficial effects of regular exercise
include lower blood pressure, improved glucose homeostasis and
lipid profile, higher resting energy expenditure and reduced fat
mass. Several mechanisms underlie such benefits. It is now widely
accepted that regular exercise increases nutrient metabolism in
various tissues by regulating the expression and activity of key
metabolic control genes, leading to enhanced insulin sensitivity
and metabolic flexibility.2The skeletal muscle exhibits remarkable
metabolic adaptations to exercise, including mitochondrial
biogenesis and improved substrate metabolism.3,4How exactly
the contracting muscle mediates the metabolic and physiological
adaptations of exercise is still unclear.5It has long been
hypothesized that the muscle can produce endocrine signals
capable of mediating the health benefits of exercise.6As the
skeletal muscle is the largest organ of the body, the discovery of
several factors secreted by the contracting muscle has led to a
new field of research. These so-called myokines are secreted in
response to exercise and can regulate in an autocrine and
endocrine fashion the function of muscle and other organs.6
Skeletal muscle has been first considered as an endocrine organ
because of its ability to produce interleukin 6 (IL6) as an exercise-
released factor.7IL6 induces lipolysis and improves insulin-
stimulated glucose uptake.8Another well-documented myokine
is myostatin (MSTN). MSTN exhibits antihypertrophic effects in the
skeletal muscle and MSTN null mice are characterized by an
excessive muscle mass.9Irisin originating from the proteolytic
cleavage of fibronectin type III domain-containing protein 5
(FNDC5) and fibroblast growth factor 21 (FGF21) was recently
homeostasis in mice, and seems to behave as a thermogenic
factor involved in the browning of white subcutaneous adipose
tissue in mice.10FGF21 enhances whole-body insulin sensitivity
and thermogenesis in brown adipose tissue.12,13Although the
1Inserm, UMR1048, Obesity Research Laboratory, I2MC, Institute of Metabolic and Cardiovascular Diseases, Toulouse, France;2University of Toulouse, UMR1048, Paul Sabatier
University, Toulouse, France;3Nutrition and Clinical Biochemistry Departments, Toulouse University Hospitals, Toulouse, France;4Inserm, UMR1048, Adipolab, I2MC, Institute of
Metabolic and Cardiovascular Diseases, Toulouse, France and5Clinical Investigation Centre Inserm, CIC-9302, Department of Clinical Pharmacology, Toulouse University Hospitals,
Toulouse, France. Correspondence: Dr N Viguerie, Inserm, UMR1048, Obesity Research Laboratory, I2MC, Institute of Metabolic and Cardiovascular Diseases, CHU Rangueil,
Av J Poulhe `s, Toulouse, 31432 Cedex 4, France.
6These authors contributed equally to this work.
Received 2 April 2013; revised 4 July 2013; accepted 20 July 2013; accepted article preview online 27 August 2013
International Journal of Obesity (2013), 1–7
& 2013 Macmillan Publishers LimitedAll rights reserved 0307-0565/13
effect of exercise training on the plasma levels of these myokines
has been previously studied, inconsistent findings have been
In the present study, we investigated skeletal muscle
myokines (IL6, MSTN, Irisin, FGF21) gene expression and plasma
levels in obese individuals in response to an 8-week aerobic
exercise training intervention. To extend the knowledge on the
adaptation of muscle to training, we performed a comprehen-
sive gene expression profiling of human skeletal muscle.
This led to the identification of apelin (APLN) as a novel
factor upregulated by exercise training. Apelin expression,
secretion andregulation by
pathways were next investigated in vitro in human primary
SUBJECTS AND METHODS
Eleven sedentary obese male volunteers who had stable weight during the
previous 3 months were recruited at the Toulouse Clinical Investigation
Centre (Table 1). The subjects were on their usual diet before the study and
ate a weight-maintaining diet consisting of 35% fat, 16% protein and 49%
carbohydrates 2 days before the experiment. None were previously
enrolled in an endurance activity training. They were asked to maintain
their dietary habits during the study and to refrain from vigorous
physical activity 48h before each clinical investigation day. Dietary
intake was assessed by a dietician from a 3-day weighed food record,
including 2 week days and 1 weekend day, the week before the first
investigation day. Dietary records were assessed at baseline and during
the last week of the program. Nutrient intake was calculated using
PRoFIL software v6.7 (Audit Conseil en Informatique Me ´dicale, St
Doulchard, Bourges, France) with the CIQUAL French food composition
database for diet composition.
The study was performed according to the latest version of the Declaration
of Helsinki and the Current International Conference on Harmonization
(ICH) guidelines. It was approved by the Ethics Committee of Toulouse
University Hospitals and all subjects gave written informed consent. The
study is registered in Clinical Trials NCT01083329 and EudraCT 2009-
Anthropometric and clinical parameters
Anthropometric parameters, blood samples, adipose tissue and skeletal
muscle biopsy samples were analyzed during a 2-day investigation, 1 week
apart, before and after the training program as follows. On day 1, after an
overnight fast, maximal oxygen consumption (VO2max) was measured on
a braked bicycle ergometer as described in de Glisezinski et al.19On day 2,
after an overnight fast, blood samples were drawn and percutaneous
biopsy samples of the Vastus lateralis muscle and of the abdominal
subcutaneous adipose tissue were obtained as previously described.20,21
Ninety minutes after the end of biopsy sampling, an oral glucose tolerance
test (OGTT) with 75g glucose load was performed at 30-min intervals
(times 30, 60, 90, 120). Body composition was assessed by dual-energy
X-ray absorptiometry performed with a total body scanner (DPX, Software
3.6, Lunar Radiation Corp., Madison, WI, USA). Blood glucose was assayed
using the glucose oxidase technique (Biome ´rieux, Paris, France). Plasma
nonesterified fatty acids were assayed with an enzymatic method (Wako
kit, Unipath, Dardilly, France). Serum insulin was measured by using a
bi-insulin IRMA kit (Bertin Pharma, Montigny le Bretonneux, France). Plasma
FGF21 and apelin were quantified with the Human FGF-21 Quantikine
ELISA Kit (R&D Systems Europe, Lille, France) and the human EIA apelin-12
kit (Phoenix Pharmaceuticals, Belmont, CA, USA), respectively. Retinol
binding protein 4 (RBP4) was analyzed by immuno-nephelemetry on a BN
ProSpec (Siemens HealthCare Diagnostics, Cergy-Pontoise, France). Other
parameters were determined using standard clinical biochemistry
methods. As it is a good reflection of the insulin sensitivity measured by
an euglycemic insulin clamp, the Matsuda insulin sensitivity index (ISI-M)
derived from oral glucose tolerance test is widely used in clinical and
epidemiological research.22ISI-M was calculated as: 10000 per square root
of [(fasting glucose?fasting insulin)?(mean glucose?mean insulin
during oral glucose tolerance test)].
Exercise training program
The exercise training program was performed at the Centre de Ressources
d’Expertise et de Performance Sportives (CREPS) of Toulouse. The 45–60-
min exercise sessions consisted mainly of cycling and running, 5 times a
week, for 8 weeks. Subjects exercised 3 times per week under supervision
during the first 4 weeks and 2 times per week during the last 4 weeks. They
exercised on their own during other sessions. All daily sessions consisted of
at least a 20-min warm-up at 35% VO2max followed by progressively
increasing exercise intensity (up to 85% VO2max) and duration (up to 1h)
throughout the training program. The subjects exercised at a target heart
rate corresponding to 35–85% of their VO2max. Heart rate was monitored
with a Suunto T3 Cardiometer (MSE, Strasbourg, France). Compliance with
training was good and the percentage of sessions completed was greater
than 85% at the end of the study. Subjects were instructed to keep their
usual dietary habits during the study. Adherence to the training program
was self-reported. At the end of the 8-week training program they were
investigated 48–72h after the last exercise bout.
For determination of VO2max, gas exchanges were measured as previously
described.23Breath-by-breath measurements were taken at rest and
throughout the exercise to assess air flow, and O2and CO2concentrations
in expired gases using a computerized ergospirometer (Ultima PFX,
Medical Graphics, St. Paul, MN, USA). Oxygen concentration was analyzed
by a zirconium cell and CO2concentration by an infrared analyzer. Certified
calibration gases were used to calibrate the analyzers every day before the
beginning of the assay. The VO2max exercise trial occurred in a ventilated
room to ensure a constant room temperature and hygrometry from the
calibration just before the trial.
DNA microarray and reverse transcription-quantitative PCR
Total RNA from frozen biopsies was prepared as previously described.20,24
Based on the concentration (Nanodro ND-1000 Spectrophotometer,
weeks of exercise training: (a) anthropometric and clinical parameters;
(b) circulating adipokines and myokines
Changes in bio-clinical characteristics before and after 8
Before trainingAfter training
Body weight (kg)
Fat free mass (kg)
Fat mass (kg)
Food intake (kcal/day)
Fasting insulin (mUIl?1)
Fasting glucose (mM)
VO2max/fat free mass
Abbreviations: BMI, body mass index; ISI-Matsuda, insulin sensitivity
Matsuda index. Data are mean±s.d. (n¼11). P-value is extracted from
A Besse-Patin et al
International Journal of Obesity (2013) 1–7
& 2013 Macmillan Publishers Limited
Labtech, Jebel Jeloud, Tunisia) and quality (Experion, Bio-Rad, Marnes-la-
Coquette, France) check, 9 subjects had enough high quality total RNA
available for gene expression study. For reverse transcription-quantitative PCR,
500ng of total RNA was used for first-strand cDNA synthesis using random
hexamers and poly(dT) according to the Multiscribe reverse transcriptase kit
(High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Foster
City, CA, USA). TaqMan Assays (Applied Biosystems) were used with 18S RNA
(Taqman Control Assays) for gene expression normalization. Microarray
experiments were performed using Agilent 4?44k oligonucleotide arrays as
described in Viguerie et al.25Hybridization quality check resulted in the
analysis of microarray data from eight subjects. Microarray data have been
deposited in NCBI’s Gene Expression Omnibus and are accessible through
GEO Series accession number GSE40551.
Satellite cells were isolated from fresh V lateralis biopsy samples obtained
before training and cultured as previously described.26On day 4 of
differentiation, the cells were treated as indicated with ionomycin, insulin,
GW7647, GW0742 (Sigma-Aldrich, Courtaboeuf, France) or forskolin
(Calbiochem Corp., Darmstadt, Germany). After 24h, the medium was
collected to measure secreted factors and cells were harvested for mRNA
extraction. Samples were stored at ?801C.
Gaussian distribution and homoscedasticity of data were tested with
corrected Kolmogorov–Smirnov and Levene tests, respectively. Microarray
data analyses were performed as described in Viguerie et al.25Mann–
Whitney or Wilcoxon tests were used in non-parametric simple compar-
isons. Kruskal–Wallis and Dunn’s post tests were performed in multiple
comparisons and non-parametric data analyses. Spearman correlation
analysis was used to assess the correlation between variables in non-
parametric univariate analysis and P-values adjusted for multiple
comparisons using the Benjamini–Hochberg procedure. To test for an
independent association between insulin resistance and myokines, multi-
ple linear regression models were computed using hierarchical regression
in which changes (D) in fat mass were entered in a first block, D VO2max,
D plasma adipokines and myokines, and D myokine mRNA levels were
introduced in the model using a stepwise procedure in a second block and
D APLN mRNA level was introduced in the subsequent block. Statistical
analyses were performed with GraphPad Prism software (GraphPad
Software, La Jolla, CA, USA) and SPSS Statistics 17.0 software (SPSS,
Chicago, IL, USA). Threshold for statistical significance was Po0.05.
Anthropometric and clinical characteristics before and after
The 11 obese male volunteers were aged 35.4±1.5 years. As
expected, exercise training increased whole-body aerobic capacity
(VO2max) by about 7%, slightly reduced fat mass (mean fat mass
loss 0.8±0.4kg) and tended to increase fat-free mass (mean fat-
free mass gain 1.2±0.6kg) (Table 1a). Changes in body
composition occurred despite no change in food intake. No
significant changes in ISI-M and fasting plasma glucose were
observed, whereas fasting plasma insulin tended to decrease in
response to training (Table 1a).
Effect of endurance training on skeletal muscle myokine gene
We investigated the mRNA levels of IL6, FGF21, MSTN and FNDC5
in the human skeletal muscle of obese individuals pre- and post-
exercise intervention. Endurance training did not significantly
change gene expression of the four candidate myokines
Apelin mRNA level is increased in the skeletal muscle from obese
individuals after endurance training
Analysis of the human skeletal muscle transcriptome before and
after endurance training led to the identification of APLN as the
most upregulated transcript encoding a known protein. APLN
encodes apelin, a peptide that was so far known as an
adipokine.27Reverse transcription-quantitative PCR confirmed a
twofold increase in APLN after endurance training and displayed
no change in its receptor APJ (Figure 2). A positive correlation
between changes in muscle APLN mRNA levels and changes in ISI-
M was found (r¼0.81, P¼0.008) in response to exercise training
(Figure 3). A significant negative correlation with changes in
fasting plasma insulin was also found (r¼–0.70, P¼0.036). In a
stepwise regression analysis, the best predictive model of change
in ISI-M included changes in plasma RBP4 (b¼ ?0.723), skeletal
muscle APLN mRNA (b¼0.241) and fat mass (b¼–0.203). This
model explained 89% of the variability in ISI-M (P¼0.008).
weeks of endurance training using reverse transcription-quantitative PCR (RT-qPCR) normalized to 18S. Data are mean±s.e.m. (n¼9).
Effect of endurance training on myokine mRNA level in the human skeletal muscle. mRNA levels were measured before and after 8
A Besse-Patin et al
& 2013 Macmillan Publishers LimitedInternational Journal of Obesity (2013) 1–7
Regulation of myokine mRNA level and secretion in human
We next investigated the regulation of APLN and other candidate
myokines in vitro in human primary myotubes established from V
lateralis muscle. Myotubes were treated with drugs, mimicking the
activation of exercise signaling pathways and enhancing calcium
and cyclic adenosine monophosphate (cAMP) intracellular levels,
ionomycin (a calcium ionophore) and forskolin (an adenylyl
cyclase activator), respectively (Figure 4). Ionomycin treatment
induced a 1.6-fold increase in APLN and 2-fold decrease in FNDC5
expression. Ionomycin also slightly increased MSTN by 30% and
decreased IL6 by 30%. Forskolin treatment decreased IL6, FGF21,
FNDC5 and MSTN by 90%, 50%, 50% and 35%, respectively,
whereas APLN increased by 3.3-fold. We also showed that
activation of peroxisome proliferator-activated receptors (PPAR) -
a and -d signaling as well as insulin treatment had no effect on
APLN gene expression (Supplementary Figure 1). Of interest,
FGF21 and apelin were detected in the culture medium at low
levels when compared with plasma values (Figure 5 and Table 1B).
Forskolin and ionomycin, respectively, decreased by 33% and
increased by 2.4-fold the FGF21 concentration in the culture
medium (Figure 5a). Apelin concentration in the culture medium
was very low—about 50 times less than in plasma—and no
significant change in apelin concentration was observed com-
pared with control (Figure 5b).
Effect of endurance training on circulating levels of adipokines
and myokines in obese individuals
In agreement with the observed fat mass loss, there was a trend
for reduced plasma leptin concentrations, but no significant
change in adiponectin or RBP4 (Table 1B). Exercise training did not
change significantly the resting plasma levels of IL6, FGF21 and
apelin (Table 1B).
Endurance training is known to improve whole-body glucose
homeostasis and to reduce the risk of developing type 2
diabetes.2In this study, we identify apelin as a novel myokine
that might contribute to exercise training-mediated improvement
of whole-body insulin sensitivity in obese individuals. Interestingly,
skeletal muscle gene expression of other myokines with a role
unchanged in response to 8 weeks of exercise training in
middle-aged obese men.
Accumulating data suggest that, during and following exercise,
the skeletal muscle synthesizes and releases factors that may act
either systemically or locally within the muscle tissue to mediate
some of the metabolic and physiological adaptations of exercise.6
These secreted factors have been termed as myokines. Little is
known on the regulation by acute and chronic exercise of the few
myokines identified so far. In the present study, skeletal muscle
whole-transcriptome profiling led us to identify APLN as a novel
skeletal muscle transcript upregulated by exercise training. Apelin
was so far known as an adipocyte-secreted peptide that
modulates skeletal muscle glucose and lipid metabolism and
increases insulin sensitivity via its receptor, APJ.27No changes in
APJ mRNA levels were noted in the muscle in response to training.
In addition, mRNA levels of other known myokines such as IL6,
FGF21, MSTN and FNDC5 remained unaffected by the training
intervention in muscle (P-values40.1). Consistently, exercise
training did notsignificantly
concentrations of IL6 and FGF21. A decrease in MSTN in skeletal
muscle appears to be a hallmark of exercise training. MSTN was
previously found decreased in the muscle of old women after 12
weeks of aerobic training.28Here, despite non-significant change,
a tendency to decrease appears for MSTN. FNDC5 encodes a novel
myokine, irisin, with thermogenic potential in white adipose
tissue.29There has been a recent controversy on the regulation of
skeletal muscle FNDC5 by training in humans. In agreement with
our data, unchanged expression of muscle FNDC5 was recently
reported in response to 6 weeks of endurance cycling.30In
addition, a 12-week endurance exercise training induced no
change in serum IL6 despite a twofold decrease in its skeletal
muscle gene expression.31Conversely, it was recently shown that
a 2-week exercise program increased serum FGF21 in young
healthy women.15However, no report was made on skeletal
muscle gene or protein expression. The contribution from each
tissue to plasma level is unknown. In summary, part of the
discrepancies between our study and other studies may be
mRNA levels were measured before and after 8 weeks of endurance training using RT-qPCR normalized to 18S. Data are mean±s.e.m. (n¼9).
**Po0.01 in a Wilcoxon test.
Effect of endurance training on apelin and APJ mRNA level in the human skeletal muscle. Apelin (APLN) and apelin receptor (APJ)
mRNA levels and insulin sensitivity index during endurance training.
Spearman correlation between changes in skeletal muscle apelin
(D APLN) mRNA levels and insulin sensitivity index (D ISI Matsuda)
during an 8-week training of male volunteers (n¼9).
Correlation between changes in skeletal muscle apelin
A Besse-Patin et al
International Journal of Obesity (2013) 1–7
& 2013 Macmillan Publishers Limited
explained by the age of the subjects, their obesity status and
possibly the difference in exercise training volume and duration.
Despite non-significant change in food intake, the 8-week
training program induced a slight but significant fat-mass loss,
indicating that a slight energy deficit was achieved with exercise
training. As apelin was primarily described as an adipokine, a
described.32In type 2 diabetic individuals, 12–24-month aerobic
training programs increased plasma apelin while fat mass was
unaffected.33,34Here, the absence of changes in plasma apelin
level may be the result of compensation due to both, the increase
in muscle mass and the enhanced gene expression of skeletal
muscle APLN, while no change in APLN expression was found in
adipose tissue (data not shown). Also, mice overexpressing apelin
had no increase in plasma apelin content, whereas enhanced
metabolic function was seen in skeletal muscle.35Alternatively,
the upregulation of muscle APLN expression in the face of no
apparent changes in plasma apelin levels may suggest that apelin
is produced to act locally on skeletal muscle fibers in a similar
fashion to interleukin 8.36In addition, a positive correlation between
change in skeletal muscle APLN and increase in ISI-M was observed.
Besides a change in plasma RBP4 level, the increase in skeletal
muscle APLN appeared as a weak independent contributor of
insulin sensitivity during exercise training. This is in agreement with
at least another study.34RBP4 is an adipokine with a controversial
role in insulin resistance,37whereas apelin has been previously
shown to promote glucose uptake in skeletal muscle.38Collectively,
our data indicate that skeletal muscle apelin might have a role in
the exercise training-induced improvement of insulin sensitivity
through autocrine/paracrine effects within the skeletal muscle.
To confirm that apelin is a novel myokine, we next investigated
its expression, secretion and regulation in vitro in human primary
myotubes, besides other known myokines (IL6, MSTN, FGF21 and
was expected,as recently
FNDC5). Thus, little is known on the regulation of myokines
expression in vitro. Exercise induces muscle contraction through a
rise in intracellular cyclic adenosine monophosphate and calcium
along with other pathways.5It was previously shown that
activation of cyclic adenosine monophosphate/protein kinase A
and calcium signaling pathways in human primary myotubes
induces PGC-1a and peroxisome proliferator-activated receptors -
d gene expression, and promotes mitochondrial biogenesis.39
These pathways also favor lipid oxidation and glycogen storage. In
this study, activation of cyclic adenosine monophosphate
signaling by forskolin consistently downregulated IL6, MSTN,
FGF21 and FNDC5 expression in vitro, while it strongly induced
APLN. In the same line, activation of calcium signaling by
downregulating IL6 and FNDC5. As previous studies have shown
that both exercise and muscle contraction induce IL6 mRNA,40this
suggests that the transcriptional regulation of myokines is
complex and involvesmultiple
Interestingly, we could show that both apelin and FGF21 are
secreted in the culture medium of human primary myotubes. Of
note, FGF21 secretion paralleled its gene transcription pattern in
response to forskolin treatment. In contrast, apelin concentration
in the culture medium was very low, about 50 times less than in
plasma, and apelin secretion remained unchanged in response to
both forskolin and ionomycin treatments. This suggests that
apelin may be secreted by skeletal muscle cells, but because of its
very short half-life (o5min)41the peptide may be quickly
degraded, thus preventing a significant accumulation in the
medium overtime. A possible interaction of apelin with circulating
proteins35and instability in human plasma42was also reported.
Additionally, skeletal muscle may not contribute significantly to
circulating concentrations of plasma apelin. Furthermore, as
various apelin isoforms exist in plasma,42the possibility of a
(4mM), ionomycin (0.5mM) or vehicle (control) for 24h. Myokine mRNA level was measured using RT-qPCR normalized to 18S. Data are
presented as base-2 log of mean fold change±s.e.m. relative to control cells (n¼6–10). ***Po0.001, **Po0.01 and *Po0.05 versus control
cells in a one-way Kruskal–Wallis and Dunn’s post hoc tests.
Effect of exercise mimetic signaling compounds on myokine mRNA level in human myotubes. Cells were treated with forskolin
treated with forskolin (4mM), ionomycin (0.5mM) or vehicle (control) for 24h. Culture media was tested for FGF21 (a) and apelin (b). Data are
presented as mean±s.e.m. (n¼6). **Po0.01 versus control cells in a one-way Kruskal–Wallis and Dunn’s post hoc tests.
Effect of exercise mimetic signaling compounds on myokine concentrations in the culture medium of human myotubes. Cells were
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& 2013 Macmillan Publishers Limited International Journal of Obesity (2013) 1–7
specific skeletal muscle isoform cannot be excluded. It can be
hypothesized that apelin is acutely released by skeletal muscle
during exercise to act locally on its own receptor as an autocrine/
paracrine regulator. In humans, APLN and APJ mRNA levels are
known to be regulated by insulin in adipocytes.27We found here
no direct effect of insulin, PPAR-a and/or PPAR-d agonists on APLN
expression in human myotubes (Supplementary Figure 1). These
data also indicate a differential regulation of APLN expression
between fat and muscle cells. The upregulation of apelin
expression in skeletal muscle is in agreement with apelin
transgenic and knockout mice data demonstrating a positive role
of apelin in skeletal muscle vascular mass and mitochondrial
biogenesis,35and in the maintenance of insulin sensitivity.43
Considering the potential role of apelin in the regulation of lipid
metabolism and insulin sensitivity,27
investigate the functional and metabolic role of apelin in the
human skeletal muscle.
In summary, these data highlight apelin as a novel exercise-
regulated myokine in humans. Apelin is expressed, secreted and
responsive to exercise-activated signaling pathways in cultured
human primary myotubes. Skeletal muscle APLN expression is
upregulated by 8 weeks of endurance exercise training in obese
male subjects and might contribute to exercise training-mediated
improvement of whole-body insulin sensitivity. Collectively, these
data suggest that apelin may be locally produced by skeletal
muscle fibers in response to exercise and acts locally to improve
muscle metabolism and function. Future studies should investi-
gate the influence of acute exercise on skeletal muscle apelin
expression, as well as its metabolic role in the human skeletal
future studies should
CONFLICT OF INTEREST
The authors declare no conflict of interest.
We are very grateful to the staff of Toulouse Clinical Investigation Centre and to the
study participants. This study was supported by grants from the National Research
Agency ANR-12-JSV1-0010-01 (CM) and ANR-09-GENO-0018-01 (DL), European
Federation for the Study of Diabetes/Novo Nordisk and Socie ´te ´ Francophone du
Diabe `te (CM), Inserm DHOS Recherche Translationnelle and AOL Ho ˆpitaux de
Toulouse (DL), Fondation pour la Recherche Me ´dicale (DL), Inserm DHOS Recherche
Translationnelle 2009 (CT, DL), AOL 08 163 02 Ho ˆpitaux de Toulouse (CT, DL) and
Glaxo Smith Kline (DL).
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Supplementary Information accompanies this paper on International Journal of Obesity website (http://www.nature.com/ijo)
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