S S Daskalopoulou, Irisin and exercise workload in 171:3 343–352

Clinical Study A B Cooke and others young subjects

Plasma irisin levels progressively increase

in response to increasing exercise workloads
in young, healthy, active subjects
Stella S Daskalopoulou1,2,*,†, Alexandra B Cooke1,*, Yessica-Haydee Gomez2,
Andrew F Mutter1, Andreas Filippaios4, Ertirea T Mesfum3 and
Christos S Mantzoros3,4
1
Division of Experimental Medicine, Department of Medicine, Faculty of Medicine, McGill University,
Montreal, Quebec, Canada, 2Division of Internal Medicine, Department of Medicine, Faculty of Medicine,
Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada, 3Endocrinology Section, Correspondence
VA Boston Healthcare System, Boston, Massachusetts, USA and 4Division of Endocrinology, Diabetes and should be addressed
Metabolism, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA to S S Daskalopoulou
*
(S S Daskalopoulou and A B Cooke contributed equally to this work) Email
†
S S Daskalopoulou is now at Montreal General Hospital, 1650 Cedar Avenue, C2.101.4, Montreal, Quebec, stella.daskalopoulou@
Canada H3G 1A4 mcgill.ca
European Journal of Endocrinology

Abstract
Background: Irisin, a recently discovered myokine, has been shown to induce browning of white adipose tissue, enhancing
energy expenditure and mediating some of the beneficial effects of exercise. We aimed to estimate the time frame of
changes in irisin levels after acute exercise and the effect of different exercise workloads and intensities on circulating irisin
levels immediately post-exercise.
Methods: In a pilot study, four healthy subjects (22.5G1.7 years) underwent maximal workload exercise (maximal oxygen
consumption, VO2 max) and blood was drawn at prespecified intervals to define the time frame of pre- and post-exercise irisin
changes over a 24-h period. In the main study, 35 healthy, non-smoking (23.0G3.3 years) men and women (nZ20/15)
underwent three exercise protocols R48-h apart, in random order: i) maximal workload (VO2 max); ii) relative workload
(70% of VO2 max/10 min); and iii) absolute workload (75 W/10 min). Blood was drawn immediately pre-exercise and 3 min
post-exercise.
Results: In the pilot study, irisin levels increased by 35% 3 min post-exercise, then dropped and remained relatively constant.
In the main study, irisin levels post-exercise were significantly higher than those of pre-exercise after all workloads
(all, P!0.001). Post-to-pre-exercise differences in irisin levels were significantly different between workloads (PZ0.001),
with the greatest increase by 34% following maximal workload (PZ0.004 vs relative and absolute).
Conclusions: Circulating irisin levels were acutely elevated in response to exercise, with a greater increase after maximal
workload. These findings suggest that irisin release could be a function of muscle energy demand. Future studies need to
determine the underlying mechanisms of irisin release and explore irisin’s therapeutic potential.

European Journal of
Endocrinology
(2014) 171, 343–352

Introduction
The beneficial effects of regular physical activity have been beneficial effects occur are less well understood. Skeletal
well established in preventing obesity, diabetes, and its muscle has recently been shown to play an active role
complications, as well as in improving health outcomes in the regulation of metabolic homeostasis through
(1), but the molecular mechanisms by which these its ability to communicate with adipose tissue in an

www.eje-online.org Ñ 2014 European Society of Endocrinology Published by Bioscientifica Ltd.

DOI: 10.1530/EJE-14-0204 Printed in Great Britain
 Clinical Study S S Daskalopoulou, Irisin and exercise workload in 171:3 344
A B Cooke and others young subjects

endocrine manner (2). This cross talk has been reported to demographic variables play a role in prediction of irisin’s
mediate many exercise-associated metabolic changes, response to exercise remains to be elucidated. Thus, in our
whereby the contraction of the skeletal muscle causes an pilot study, we aimed to identify the time frame of irisin
enhanced release of several myokines capable of interact- release over a period of 24 h. After determining the time
ing with adipose tissue, such as IL6, IL15, and, most point of maximum increase, we aimed in our main study
recently, irisin (2, 3, 4). to estimate the effect of acute exercise of different
Irisin is a recently discovered hormone identified both workloads and intensities on circulating irisin levels and
in mice and humans (4), the levels of which have been explore effect modification by sex, BMI, exercise capacity,
reported to increase in response to exercise (4). Irisin has and/or baseline physical activity levels.
also been proposed to mediate some of the beneficial
effects of exercise on metabolism (4). Reportedly regulated
Subjects and methods
by peroxisome proliferator-activated receptor (PPAR)-g
co-activator 1a (PGC1a), irisin is proteolytically cleaved The study was approved by the ethics and scientific
from the fibronectin type III domain-containing 5 reviews boards of the McGill University Health Centre
(FNDC5) gene product and secreted into the circulation (MUHC). Written informed consent was obtained from all
from skeletal muscle in response to exercise (4). This newly participants.
identified myokine is proposed to induce the browning of
subcutaneous white adipose tissue and activate thermo-
Participants
genesis by increasing uncoupling protein 1 (UCP1) levels,
both in vitro and in vivo (4). In high-fat diet-fed mice, viral We recruited consecutive young (aged 18–30 years),
European Journal of Endocrinology

injection of full-length Fndc5 resulted in a 15-fold increase healthy, active men and women via notices on Montreal
in liver Fndc5 mRNA levels and a three- to fourfold increase area websites. The International Physical Activity Ques-
in plasma irisin levels (4), which subsequently led to tionnaire (IPAQ) was used to assess baseline physical
reduced fasting insulin, improved glucose tolerance, and activity levels (13); moderate and highly active individuals
decreased body weight (4). Together, these results suggest were recruited. Exclusion criteria were previously diag-
that increased irisin levels can induce the browning of nosed cardiovascular disease, congenital heart diseases,
white adipose tissue, increase energy expenditure, and traditional cardiovascular risk factors (diabetes mellitus,
exert both anti-obesity and anti-diabetic effects (5, 6). hypertension, and dyslipidemia), metabolic syndrome,
More recently, to confirm the above findings, Zhang et al. renal disease, respiratory diseases, inflammatory/autoim-
(7) have cultured murine adipocytes with recombinant mune diseases, obesity (BMIR30 kg/m2), pregnancy, and
irisin and observed a rapid and significant upregulation of history of ever-smoking. Furthermore, subjects who were
brown cell markers, including Ucp1, and Pgc1a. Further- acutely ill or on cardioprotective medications (including
more, they treated mice fed a high-fat diet with daily aspirin) were also excluded.
injections of recombinant irisin for 14 days and observed Upon their first visit, participants completed a
similar induction of browning genes, reduction in body questionnaire, including information about past medical
weight, and improvements in glucose tolerance (7), as was history, current medication use, and lifestyle habits. Their
previously shown using Fndc5 viral vectors (4). These height and weight were measured, as well as waist and hip
findings highlight irisin’s potential to become an attrac- circumferences.
tive therapeutic target to treat metabolic disorders such
as obesity and diabetes (4, 7).
Exercise protocols
To date, few human studies have directly looked at the
effect of acute exercise on circulating irisin levels (8, 9, 10, The assessments were performed in a temperature (22G
11, 12). We previously examined the effects of an acute 1 8C) and humidity (60G5%) controlled environment
bout of exercise (short sprints) in young healthy men (Vascular Health Unit, MUHC). For the pilot study,
(nZ15) and demonstrated that circulating irisin levels participants underwent a maximal workload protocol to
were increased 30 min after completion of acute exercise volitional exhaustion (maximal oxygen consumption
(8). However, whether irisin increases earlier and/or (VO2 max)) using a treadmill and metabolic cart. The
increases in a dose-dependent manner in response to subjects were instructed to perform their regular daily
different workloads and intensities of acute exercise is not activities but avoid vigorous exercise 24 h before the exercise
known. Furthermore, whether important physiological or session and for the duration of the study post-exercise.

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 Clinical Study S S Daskalopoulou, Irisin and exercise workload in 171:3 345
A B Cooke and others young subjects

In the main study II, to induce different levels of Cleveland, OH, USA); 15 ml was collected into 15 ml
physical exercise, each participant performed three different conical tubes (Thermo Fisher Scientific, Hampton, NH,
supervised exercise protocols in a random order on three USA). After centrifugation, plasma and urine were pro-
separate study days at least R48 h apart from each other. cessed and stored at K80 8C for the analysis of irisin
Before each session, participants were asked to abstain from (plasma and urine) and lactate (plasma only).
any type of vigorous exercise for 24 h. Participants In the main study, the time of the post-exercise blood
performed their assessments at the same time of day to collection (3 min) was selected as the most appropriate
reduce any effects of possible circadian variability. based on the results of the pilot study demonstrating the
The assessments included: i) maximal workload: the largest increase in irisin levels 3 min post-exercise. There-
same protocol as the one used in the pilot study, which is fore, in the main study, blood was collected from all
a supervised incremental treadmill exercise protocol to participants immediately pre-exercise (10 ml) and 3 min
volitional exhaustion (modified Bruce protocol, validated post-exercise (10 ml) through a peripheral venous
in young healthy individuals) (14). A treadmill (Track- catheter. The blood samples were collected, processed,
master, TMX-055) and an automated metabolic cart and stored in the same manner as indicated earlier.
(Medisoft’s Ergocard) to assess VO2 max were used. Time Plasma irisin levels were quantified using irisin
to exercise cessation was recorded. ii) Relative workload: a ELISA (Aviscera Bioscience (Santa Clara, CA, USA), now
10-min bout of treadmill exercise at 70% of each subject’s provided by Phoenix Pharmaceuticals (Burlingame, CA,
VO2 max, as assessed by the maximal workload session; and USA) under the same cat# EK-067-52) (8). Before using this
iii) absolute workload: a 10-min bout of exercise at 75 W ELISA kit with clinical samples, we performed a number of
resistance on a cycle ergometer (Ergoline, Ergoselect 100). validation studies. The detectable range of the kit was
European Journal of Endocrinology

The order of the maximal and absolute workload 0.066–1024 ng/ml, with intra-assay variation and inter-
assessments was randomized using a web-based list assay variation being 0.6–4.8 and 8.0–10.0% respectively.
randomizer (15). When the maximal workload was Our results showed that serum and plasma concentrations
randomized to the first assessment, the relative and of irisin were relatively similar, and that multiple freeze-
absolute workloads were further randomized. Brachial thaw cycles (0, 2, 4, 8) did not cause any significant
blood pressure was measured according to the guide- changes in irisin levels. The assessment of linearity in
lines (16) using cuff sphygmomanometry (HEM-705CP, serum and plasma samples revealed a 124% recovery
Omron Corp. St. Charles, IL, USA) pre-exercise (baseline) for the 1/2 dilution, and higher recovery (up to 213%)
and 2 min post-exercise. in the 1/4 and 1/8 dilutions. To the best of our knowledge,
no commercially available assay to date can differentiate
between irisin and soluble FNDC5 as there is 100% cross-
Determination of peak metabolic equivalents
reactivity between the two proteins. However, the EK-067-
VO2 max values were calculated as ml/(kg/m2) from the 52 kit by Phoenix Pharmaceuticals is the most widely
breath-by-breath analysis provided by the automated meta- used and validated kit for the assessment of plasma irisin
bolic cart. Peak metabolic equivalents (METs) were then in the current literature.
determined by dividing the calculated VO2 peak by the resting Plasma lactate was measured using automated Roche
metabolic rate (one MET) of each individual participant, as cobas c311 analyzer (Roche Diagnostics) via enzymatic
calculated by the Harris–Benedict equation (17). colorimetric method.
All analyses were performed in a blinded manner
under code by study personnel unaware of the hypotheses
Blood and urine collection
of the study.
In the pilot study, participants had their blood drawn (4 ml)
by a research nurse using a peripheral venous catheter
Statistical analyses
(Becton, Dickinson, and Company, Franklin Lakes, NJ,
USA) immediately pre-exercise, 3 min post-exercise, as well Descriptive statistics were used to present baseline
as 10 min, 30 min 1, 2, 3, 6, 12, and 24 h post-exercise. The characteristics. The distribution of the data was tested
catheter set was introduced and well secured on the forearm and parametric or non-parametric tests were used accor-
30 min pre-exercise and removed at the end of the session. dingly. Differences between pre- and post-exercise irisin
In all four subjects, urine samples were also obtained pre- levels were compared for each session using Wilcoxon
exercise in sterilized urine cups (Starplex Scientific, signed-rank test. The post-to-pre-exercise change in

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 Clinical Study S S Daskalopoulou, Irisin and exercise workload in 171:3 346
A B Cooke and others young subjects

250 187.3G101.0 ng/ml) 3 min post-exercise, compared with

the baseline value. At time points later than 3 min, levels
dropped toward baseline and remained relatively constant
throughout the following time frame of 24 h (Fig. 1).
Irisin levels (ng/ml)

200

Based on these findings, which showed that the greatest

increase in irisin was reported 3 min post-exercise, this
150 time point was selected for blood collection in the main
study. Moreover, we report for the first time the presence
of irisin in the urine at a mean concentration of 14.47G
100
4.46 ng/ml pre-exercise.
3 min 10 min 30 min 1 hr 2h 3h 6h 12 h 24 h
se
ci
er

Post-exercise
ex
e-

Main study
Pr

We recruited 35 subjects (mean ageGS.D.: 23G3.3 years;

Figure 1 20 men and 15 women); baseline characteristics are
Irisin levels pre-exercise and post-exercise, and 24-h. Irisin levels
presented in Table 1. On the maximal workload session,
were measured pre-exercise and post-exercise at 3-, 10-, 30-min
all subjects achieved a respiratory exchange rate (ratio
and at 1-, 2-, 3-, 6-, 12-, and 24-h (nZ4). At each time point irisin
of the volume of carbon dioxide produced and the volume
values are represented as meanGS.E.M.
of oxygen used, i.e. VCO2/VO2) of O1.1 (meanGS.D.: 1.2
European Journal of Endocrinology

G0.1); this indicates adequate exercise effort (secondary

endpoint criterion of reaching VO2 max) (18). The average
circulating irisin levels was calculated and expressed as the exercise time on the maximal workload session was 18.0
difference between post- and pre-exercise irisin levels. G3.1 min.
Irisin levels (pre-exercise levels, post-exercise levels, and The mean pre-exercise irisin level and post-exercise
post–pre difference) were compared among the different irisin level for the three exercise workloads are presented in
exercise intensities using Friedman’s test. Kruskal–Wallis Table 1. There were no significant differences in pre-exercise
test was conducted for comparisons between irisin levels irisin levels on the three exercise workload sessions
across VO2 max tertiles. We further performed Pearson’s or (PZ0.581). The irisin post–pre difference was significant
Spearman’s rank correlations between irisin after maximal in response to all three workloads (P!0.001) (Fig. 2).
workload (pre-exercise levels, post-exercise levels, and
post–pre difference, separately) and BMI, IPAQ, lactate
levels, VO2 max, and other exercise parameters. Analysis of
covariance (ANCOVA) was used to assess between-group Table 1 Subject baseline characteristics. The independent
differences for sex, BMI, IPAQ, and VO2 max in irisin levels samples Student’s t-test was used to compare baseline
on all three workloads. We used repeated measures characteristics between men and women.
ANCOVA and multivariate ANCOVA (MANCOVA) to
Whole P
test interactions between certain variables (sex, BMI, cohort Men Women value
and, IPAQ or VO2 max) and irisin levels on the three
Sex (men/ 20/15 20 15 –
workloads (adjustment included age, sex, BMI, and VO2 women)
max accordingly). IBM SPSS Statistics version 20.0 Age (years) 23.0G3.3 24.2G3.9 21.4G1.4 0.007
(Armonk, NY, USA) was used. Height (cm) 173.8G9.5 180.3G6.3 165.2G4.9 0.000
Weight (kg) 68.01G11.8 75.6G9.1 57.9G5.8 0.000
BMI (kg/m2) 22.4G2.5 23.2G2.5 21.2G1.9 0.015
Waist (cm) 78.1G10.3 83.5G8.0 71.1G8.6 0.000
Hip (cm) 89.1G9.2 91.2G8.6 86.6G9.7 0.164
Results Waist:hip 0.9G0.1 0.9G0.1 0.8G0.1 0.008
ratio
Pilot study IPAQa 2919 (1831– 2750 (1428– 2919 (2239– 0.894
4548) 6152) 3945)
We recruited four young (mean ageGS.D.: 22.5G1.7),
healthy, non-smoking individuals (two men and two
IPAQ, International Physical Activity Questionnaire.
a
women). Irisin levels increased by 35% (137.9G45.7 to Presented as median (interquartile range), all other values are meanGS.D.

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 Clinical Study S S Daskalopoulou, Irisin and exercise workload in 171:3 347
A B Cooke and others young subjects

Pre-exercise Post-exercise Correlation between incremental changes of

irisin and lactate levels
P<0.001 P<0.001 P<0.001
The lactate post–pre incremental change was correlated
180 with the corresponding change in irisin levels post–pre on
160 each workload session (Table 3), with the strongest
correlation observed during the maximal workload
140
(rsZ0.523, PZ0.001).
Irisin levels (ng/ml)

120 Significant positive correlations were also observed

100 between lactate post–pre-exercise after maximal workload
and VO2 max (rsZ0.471, PZ0.004), exercise time (rsZ0.528,
80
PZ0.001), and peak METs (rsZ0.477, PZ0.004).
60

40 Interactions between workloads and other parameters

20 in predicting changes in irisin levels
0 No significant interactions were found between the
Maximal Relative Absolute
workload workload workload different exercise workloads and sex, BMI, IPAQ, and
VO2 max on changes in irisin levels (pre-exercise, post-
exercise, and post–pre difference) (PO0.05).
European Journal of Endocrinology

Figure 2
Irisin levels pre-exercise and post-exercise on the three work-
load sessions. Post-exercise irisin levels are significantly higher VO2 max and other exercise parameters
than pre-exercise irisin levels on all three workload sessions
After maximal workload, VO2 max was positively correlated
(nZ35). Data were shown as meanCS.D. P values were obtained
with irisin levels post-exercise (rpZ0.393, PZ0.020) as well
using Wilcoxon signed-rank test to compare post- and
as irisin post–pre difference (rsZ0.414, PZ0.013). Further-
pre-exercise irisin levels.
more, when dividing the study sample into tertiles based
on their VO2 max, we found a trend toward a progressive
Different exercise workloads in relation to increase in irisin post–pre difference across the VO2 max
groups (PZ0.059) (Table 4) on the maximal workload
post–pre-exercise difference in irisin levels
session. Similarly, VO2 max was also positively correlated
A dose–response increase in irisin levels was noted with lactate post–pre difference (rsZ0.471, PZ0.004) after
according to the exercise workload (PZ0.001), with the maximal workload. Lactate levels displayed a progressive
greatest and lowest increase observed after the maximal increase across the VO2 max groups (PZ0.017) (Table 4).
and absolute workload respectively (Table 2). The post–pre
differences in irisin levels between maximum and relative
Table 2 Irisin and lactate levels on all three workload sessions.
workloads, and between maximum and absolute work- Irisin levels (ng/ml), lactate levels (mmol/l). All values are
loads, were all significant (both, PZ0.004). The post–pre meanGS.D. The Friedman’s test was used to compare irisin and
difference in irisin levels, however, was not significantly lactate values between all three workload sessions.
different between the relative and absolute workloads.
Maximal Relative Absolute
workload workload workload P value
Different exercise workloads in relation to
Irisin
post–pre-exercise difference in lactate levels Pre-exercise 84.8G36.1 85.1G35.0 83.4G29.6 0.581
Post-exercise 113.3G52.7 102G44.2 99.1G39.3 0.008
Baseline lactate pre-exercise levels were not significantly Post–pre 28.5G39.3 17.4G14.3 15.7G17.8 0.001
different among the three exercise workload sessions. difference
Lactate
However, the post–pre increment in lactate levels Pre-exercise 2.1G2.4 1.6G0.6 1.5G0.4 0.155
was significantly different across workloads (P!0.001), Post-exercise 12.3G4.5 3.6G2.1 2.5G1.8 !0.001
with the greatest increase after the maximal workload Post–pre 10.2G6.3 2.0G2.1 1.0G1.9 !0.001
difference
protocol (Table 2).

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 Clinical Study S S Daskalopoulou, Irisin and exercise workload in 171:3 348
A B Cooke and others young subjects

Table 3 Correlations between lactate and irisin levels on all expenditure between any two workloads (maximal vs
three workload sessions. Irisin levels (ng/ml), lactate levels relative, maximal vs absolute, and relative vs absolute) was
(mmol/l). A Spearman’s correlations coefficient was used. significant (all P!0.001). On the maximal workload
Significant correlations are in bold. session, there is a trend for a positive correlation
between energy expenditure and irisin post-exercise
Lactate post–
levels (rsZ0.321, PZ0.064) and irisin post–pre difference
Lactate post-exercise pre difference
(rsZ0.307, PZ0.078). Furthermore, energy expenditure
Maximal workload was significantly correlated with lactate post–pre
Irisin post-exercise rZ0.487
PZ0.003 difference (rsZ0.594, P!0.001).
Irisin post–pre rZ0.838 rZ0.523
difference PZ0.000 PZ0.001
Relative workload
Irisin post-exercise rZ0.415 Discussion
PZ0.007
Irisin post–pre rZ0.604 rZ0.372 We report for the first time the time frame of irisin changes
difference PZ0.000 PZ0.014 in plasma and urine, as well as a significant and
Absolute workload progressive increase in the levels of plasma irisin with
Irisin post-exercise rZK0.263
PZ0.064 increasing exercise workload immediately post-exercise.
Irisin post–pre rZ0.508 rZK0.229 More specifically, we showed that irisin is significantly
difference PZ0.000 PZ0.093 elevated early, with the highest irisin levels increased
by 35%, occurring 3 min post-exercise in our pilot study,
European Journal of Endocrinology

and by 34% in our main study of 35 subjects. Irisin is a

Exercise time and peak METs were significantly
myokine released in response to exercise in rodents, but
different between VO2 max tertiles, with the highest values few studies to date have looked at the effect of acute
observed in subjects who reached a higher VO2 max. exercise in humans. We demonstrated in a previous acute
Furthermore, irisin post–pre difference after maximal exercise study in humans that circulating irisin levels
exercise was correlated with peak METs (rsZ0.451, increase 30 min after short sprints in young healthy
PZ0.007) and trended toward significance with exercise moderately trained men (8). Norheim et al. (10) also
time (rsZ0.301, PZ0.079), while post-exercise irisin performed an acute exercise test (45 min of ergometer
was significantly correlated with exercise time cycling at 70% VO2 max) in young healthy subjects (nZ13)
(rpZ0.357, PZ0.035). and showed significantly higher levels (1.2-fold) immedi-
Energy expenditure (kcal) was significantly different ately after exercise, which returned to pre-exercise levels at
between the three different workload sessions (maximal: 2 h post-exercise. They did not observe any effect of acute
180.27G80.90 kcal; relative: 111.90G34.32 kcal; absolute: exercise on FNDC5 mRNA; however, PGC1a was 7.4-fold
62.08G18.29 kcal) (P!0.001). Furthermore, energy higher 2 h post-exercise when compared with pre-exercise

Table 4 Exercise parameters, irisin, and lactate levels in the VO2 max groups (low, moderate, and high) on the maximal workload
sessions. All values are meanGS.D. The Kruskal–Wallis test was used to compare exercise parameters, irisin values, and lactate levels
between VO2 max groups.

Low VO2 max (nZ12) Moderate VO2 max (nZ12) High VO2 max (nZ11) P value

VO2 max 38.5G4.1 48.5G3.0 60.6G5.6 !0.001

Exercise time 15.3G1.9 18.0G1.3 21.1G2.6 !0.001
Peak METs 11.3G1.5 14.4G1.3 17.6G1.6 !0.001
Energy expenditure 84.2G17.3 133.8G45.7 227.1G52.1 !0.001
Irisin pre-exercise 74.3G18.7 83.4G16.0 97.8G58.6 0.345
Irisin post-exercise 91.6G27.9 113.2G51.8 137.1G66.8 0.101
Irisin post–pre difference 17.3G20.3 29.8G42.6 39.3G19.7 0.059
Lactate pre-exercise 2.4G3.2 2.5G2.6 1.3G0.2 0.081
Lactate post-exercise 10.1G4.3 12.0G5.4 14.9G2.1 0.018
Lactate post–pre difference 7.7G6.9 9.5G7.4 13.6G2.1 0.017

Energy expenditure (kcal); exercise time (min); irisin levels (ng/ml); lactate levels (mmol/l); peak METs, peak metabolic equivalent; VO2 max, maximal oxygen
consumption (ml/kg per min).

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 Clinical Study S S Daskalopoulou, Irisin and exercise workload in 171:3 349
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values (10). In a study by Kraemer et al. (9) young subjects levels amongst individuals with different levels of physical
(nZ12) completed 90 min of treadmill exercise at 60% of fitness, but this may be due to the inclusion of only
VO2 max and irisin levels were measured during exercise physically active individuals, as defined by IPAQ scores.
(at 0, 54, and 90 min) as well as at 20 min post-exercise. We do, however, report a greater increase in irisin levels in
They observed significantly higher irisin levels at 54 min response to higher workload exercise, with the greatest
into exercise test (20.4% in young males and 22.5% in increase observed in more physically fit individuals who
young women). However, irisin levels were no longer were able to reach a higher VO2 max within the maximal
elevated at 90 min post-exercise, as well as 20 min post- workload session.
exercise. These results contribute new evidence to We have demonstrated that short-term, high-
regulation of irisin levels during steady-state acute intensity exercise leads to the most significant increase
physical activity, suggesting that irisin signaling occurs in plasma irisin levels. These findings lend support to the
during the first hour of exercise (9). To date, the majority positive effects of high-intensity exercise as an effective
of studies have measured irisin levels in plasma or serum; and time-efficient method of improving cardiorespiratory
however, Aydin et al. (12) provide the first demonstration fitness and cardiac mass in sedentary adults (20). However,
of irisin in the saliva, supported by their results showing we have also demonstrated for the first time that even a
strong irisin immunoreactivity in human submandibular total duration of 10 min of sub-maximal exercise (relative
glands. Interestingly, they observed a significant increase and absolute workloads) increase circulating irisin levels.
in irisin levels in the saliva after moderate outdoor exercise This is especially interesting since maximal exercise to
(5.5 km/45 min) in a middle-aged normal weight popu- exhaustion is not a common daily occurrence for most
lation (nZ14) (12). There remain two studies (11, 12) people. In fact, several studies have suggested that shorter
European Journal of Endocrinology

which have shown no significant changes in plasma irisin bouts of exercise accumulated through several episodes
in response to acute exercise. However, the timing of (!10 min) are as beneficial as longer bouts of activity
blood draw post-exercise was either not exactly specified (O30 min) (21). Recent health guidelines have stated that
(12) or 3 h after the exercise test (11). In addition to the the accumulation of at least 150 min of aerobic physical
findings from Kraemer et al. (9), the results from our activity, even if done in short bouts of 10 min, has
pilot study lend support to an acute and transient increase beneficial health outcomes, namely effective weight loss
in plasma irisin, which may explain some of the and decreased risk for diabetes (22). It remains to be
inconsistent results when irisin was measured at later evaluated whether these beneficial effects are at least
time points post-exercise. partly attributed to smaller and more frequent irisin
We showed for the first time that the increase in irisin release after several short bouts of low workload exercise.
levels after maximal workload protocol was significantly We noted significant elevated irisin levels post-
greater in more fit individuals, who exercised longer and exercise on all three workloads. Physiologically, it remains
reached a higher percentage of VO2 max and peak METs unclear whether this rapid increase in circulating irisin
post-exercise providing the dose–response criterion for levels results from a de novo production of FNDC5 in
causality as per Bradford Hill criteria (19). These subjects response to exercise, or whether FNDC5 is pre-translated,
also had a greater increase in lactate in response to stored, and only released into the circulation as muscle
maximal exercise, suggestive of the greater muscle strain demand increases. Alternatively, exercise may rapidly
they underwent. The change in irisin and lactate levels activate a cleavage factor specific to FNDC5, leading to
correlated, and both increased with higher exercise work- the rapid release of irisin into the circulation following
load, suggesting that greater increase in irisin levels in exercise initiation. We can hypothesize from a study
these individuals could perhaps be a function of muscle reported by Norheim et al. (10) that the acute increase in
energy demands. Furthermore, our hypothesis that muscle plasma irisin levels is probably independent of an increase
demand may play a role in the physiological regulation of in the de novo production of FNDC5, as the muscle FNDC5
irisin concurs with the findings reported by Huh et al. (8), mRNA levels were unchanged after the acute exercise
who showed that the increase in irisin after acute exercise bout. Further studies, however, will be needed to confirm
correlated with a decrease in muscle ATP levels. This the mechanisms behind the very rapid release of irisin into
suggests that lower ATP levels and/or higher ADPC the bloodstream.
phosphate group (Pi) may act to signal irisin release to To date, there is no evidence in the literature
help restore ATP homeostasis in the working muscle (8). regarding irisin metabolism, and whether the kidney
We did not observe significant differences in baseline irisin may play a role in its excretion. We have for the first

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 Clinical Study S S Daskalopoulou, Irisin and exercise workload in 171:3 350
A B Cooke and others young subjects

time successfully measured irisin levels at physiological represents higher muscle mass in these young, healthy,
concentrations in the urine, which suggests a role for the active subjects. We did not measure ATP levels; however,
kidney in the metabolism and excretion of circulating we calculated energy expenditure using VO2 and respir-
irisin. This topic will be part of our future research. atory exchange ratio (RER) values to indirectly assess
We reported no significant interactions between muscle energy expenditure and ATP utilization (25).
different exercise workloads and BMI, IPAQ, and VO2 max Furthermore, we used a cycle ergometer to produce
on irisin levels, demonstrating that elevated irisin levels the exercise protocol that corresponded on average to
after exercise is driven by the differences in exercise 41.32G18.8% VO2 max of every subject (obtained by
workload alone. BMI was previously shown to be the calculating each subject’s average VO2/min for each minute
main contributor to circulating irisin levels at rest (8, 23, 24). of exercise divided by their VO2 max on the maximal
In a wide range of BMI (8.9–84.6 kg/m2), significant workload session). We used this design by convention, so as
correlations of irisin with body weight, BMI, fat mass, to study not only a low workload protocol but also evaluate
and muscle mass were noted (23). Huh et al. (8) report a the effect of absolute workload on irisin levels. The cycle
trend toward a positive correlation between irisin and BMI ergometer allowed for greater control in setting the same
in healthy middle-aged women, as well as a decrease in absolute workload for all subjects, as it enables adjustment
irisin levels in obese individuals 6 months postbariatric to each individual’s pedal speed to provide the necessary
surgery. However, a negative correlation was found in a resistance to maintain a 75 W output. Lastly, we did not
study of 29 morbidly obese men (24). We did not observe obtain hematocrit and hemoglobin values and were not
any relationship between BMI and irisin levels at baseline able to adjust for delta plasma volume in our study pre-
or after exercise; however, individuals in our study had exercise and post-exercise. To date, only one other irisin
European Journal of Endocrinology

a narrow and healthy range of BMI (22.4G2.5 kg/m2). study has examined plasma volume shift after acute
Interestingly, men had significantly higher BMI than exercise and found only a K3.0G6.09% shift in plasma
women. Although non-significant, possibly due to small volume after 54 min at treadmill exercise at 60% VO2 max in
sample size, they also had higher baseline irisin levels and young healthy subjects (9). In our study, the mean exercise
a greater increase post-exercise. This could perhaps be time on the maximal workload session was 18:04 min, and
explained by increased muscle mass in men, because Huh the two other sessions were only 10 min, therefore the
et al. (8) showed that circulating irisin levels were minimal shift that might have occurred during our study is
positively correlated with bicep circumference, a surrogate unlikely to increase plasma irisin to the magnitude that
marker of muscle mass. This suggests that increased we have reported.
muscle mass may be the main predictor of irisin levels; Strengths of the study include the blinded assessment
however, larger studies will be necessary to draw more of irisin levels by personnel who were not aware of the
definite conclusions. study hypothesis. Furthermore, our pilot study allowed
Limitations of this study include a relatively small and us to identify the optimal time frame that circulating
arbitrary sample size, because no prior studies were irisin should be measured post-exercise (3 min). Also, as
available on which to base calculations. However, the described in the Methods section in detail, we carried out
sample size is larger than that in most previous studies and our own validation studies on the Aviscera/Phoenix
the several measurement time points increased the power Pharmaceutical kit to ensure it would be able to reliably
of the study. These data could be used for calculating and accurately assess plasma irisin levels in our samples.
sample size in future studies. Based on our results, to have This is currently the most widely used and validated kit for
adequate power to determine sex differences in irisin levels measuring plasma irisin (8, 10, 11, 12, 24, 26, 27, 28, 29).
in response to exercise we would need 62 subjects per These validation studies help to clarify some of the
group; however, a much larger sample size would be controversies in the literature regarding the validity of
necessary to show significant differences for BMI, IPAQ, the various assays used for measurements of plasma irisin.
and VO2 max. As a result of strict inclusion criteria, we were For instance, Erickson (30) and Raschke et al. (31) have
only able to assess a very narrow and healthy range of BMI, challenged the existence of irisin in humans, and have
and overall, a very active group of young individuals. questioned the initial results of Bostrom et al. on the basis
Although it was beyond the scope of this study, assessing of poor assay validity. However, Bostrom et al. (32) have
the above-mentioned associations is an interesting future addressed many of these controversies in a recent editorial
direction. We did not directly measure muscle mass, and with supporting evidence from the literature. Western blot
we can only speculate that higher BMI in our group analysis using the Aviscera/Phoenix antibody detects

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 Clinical Study S S Daskalopoulou, Irisin and exercise workload in 171:3 351
A B Cooke and others young subjects

a 25 kDa band in plasma, which represents the fully

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Received 12 March 2014

Revised version received 22 May 2014
Accepted 11 June 2014

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