Serum irisin levels are regulated by acute strenuous exercise.

ORIGINAL

ARTICLE

Serum irisin levels are regulated by acute strenuous exercise Dennis Löffler1,2, Ulrike Müller3, Kathrin Scheuermann1, Daniela Friebe1, Julia Gesing1, Julia Bielitz1, Sandra Erbs3, Kathrin Landgraf1,2, Isabel Viola Wagner1, Wieland Kiess, and Antje Körner1,2 1

Center for Paediatric Research Leipzig, University Hospital for Children & Adolescents, 2Integrated Research and Treatment Center (IFB) AdiposityDiseases; 3Leipzig Heart Centre, Department of Cardiology, University of Leipzig;

Rationale: The newly discovered myokine irisin has been proposed to affect obesity and metabolism by promoting browning of white adipose tissue. However, clinical and functional studies on the association of irisin with obesity, muscle mass and metabolic status remain controversial. Here we assessed the effect of four distinct exercise regimes on serum irisin levels in children and young adults and systematically evaluated the influence of diurnal rhythm, anthropometric, and metabolic parameters and exercise on irisin. Results: Serum irisin levels did not show diurnal variations, nor were they affected by meal intake or defined glucose load during oral glucose tolerance testing (oGTT). Irisin levels decreased with age. In adults, irisin levels were higher in men than in women and obese subjects had significantly higher levels than lean controls. Irisin levels were closely correlated with muscle-associated bioimpedance parameters like fat-free–mass and body-cell-mass. Of four exercise regimens that differed in duration and intensity, we identified a clear and immediate increase in serum irisin levels after acute strenuous exercise (cycling ergometry) and a 30 minute bout of intensive exercise in children and young adults, whereas longer (six weeks) or chronic (one year) increase in physical activity did not affect irisin levels. Summary: We show that irisin levels are affected by age, gender, obesity, and particularly muscle mass, whereas diurnal rhythm and meals do not contribute to the variation in irisin levels. Short bouts of intensive exercise, but not long-term elevation in physical activity, acutely and transiently increase serum irisin levels in children and adults.

he new myokine irisin was initially described as a muscle-secreted factor promoting browning of adipose tissue in mice (1). It is a cleavage product of the fibronectin-type-III-domain-containing protein 5 (FNDC5), a transmembrane protein mainly present in skeletal muscle (2). The amino-terminal part of the protein is proteolytically released as irisin into the circulation via a so far unknown protease (1). The predominantly muscular origin is reflected by a correlation between serum irisin levels and muscle mass in clinical studies (3, 4), although it is also expressed in the heart (3), salivary glands (5), brain (6),

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and adipose tissue (7). FNDC5 is induced by peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha in response to exercise (1, 8). Subsequent studies were, however, inconsistent describing an increase after acute exercise (8 –10), after one-year lifestyle intervention (11) or a 10-week aerobic exercise program (12), while others failed to verify these results (13, 14). The potential thermogenic action of irisin (1) triggered subsequent studies to evaluate its relationship with obesity and metabolic parameters. Irisin levels were higher in obese individuals (15) and decreased after weight loss

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2015 by the Endocrine Society Received July 15, 2014. Accepted January 22, 2015.

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doi: 10.1210/jc.2014-2932

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Regulation of irisin levels by exercise


(16). Furthermore, irisin levels were correlated with fat mass, body mass index (BMI), and insulin levels (3, 15, 17), although others reported the opposite relationship with BMI (18) or lower levels of irisin in patients with diabetes (19). Overall, although associations between irisin and obesity, muscle mass, and exercise are described, there is high inconsistency between reports, which may derive from different study designs and/or unconsidered factors that contribute to the variation of irisin levels. Particularly, little is known about irisin physiology in children. We aimed to systematically evaluate whether circulating irisin levels are associated with i) muscle mass and obesity and whether they were affected by ii) diurnal regulation, meals, and glucose. iii) Considering the expression in muscle and discrepant results on the effect of exercise on irisin, we systematically evaluated the effect of different exercise schemes on irisin levels in children and young adults. Study designs & Methods Written informed consent was obtained from all subjects aged 12 years and older and/or parents. Blood parameters were assessed by a certified laboratory (Institute of Laboratory Medicine, University Hospital Leipzig). BMI data from children were standardized to age- and sex-specific German reference data (20) and are given as z-score (SDS). A cut off of 1.88 SDS (97th centile) defines obesity in children. Bio-impedance analyses (BIA) were performed using the Nutriguard-MS (Data Input, Germany) and parameter, like fat-free-mass (FFM) or bodycell-mass (BCM) were calculated using NutriPlus Software.

Table 1.

To address different potential regulators, we applied several study cohorts. Cohort specific information is given in the following description and in table 1 All studies were approved by the ethics committee of the University of Leipzig (registration no. 096 –11– 07 032 011, 029 –2006). 1.) Diurnal variation, effect of meals, and glucose load in young healthy adults The study was performed in 28 young healthy adults aged 18 to 35 years under controlled in-house conditions. Subjects received meals of standardized compositions at defined times (08:30, 12:30, 18:30 and 10:00). Blood sampling was started at 08:00 (with fasting samples) and subsequently continued with hourly samples for 24h. Additional samples were taken 30min after meals. During the day, subjects engaged in sedentary activities (eg, reading, watching video). On the following day, an oGTT was performed with blood samples collected directly before and 10, 20, 30, 60, 90, and 120min after oral glucose intake (1.75g/kg body weight, max. 75g). Samples were immediately processed and stored at – 80°C. A schema of the daily routine and blood sampling is given in Figure 1A. 2.) Leipzig Atherobesity Childhood cohort We recruited 105 lean and obese children (tab.1a) and performed detailed anthropometric, clinical, metabolic, and cardiovascular assessments including oGTT as described previously (21). We measured serum irisin levels in fasting samples. Participants were aged between 8 and 21 years, free of diseases and without any medication. (NCT01605123)

Characteristics of the 4 different intervention cohorts Sex (m/f)

lean owⴙobese

20/22 26/37

Age BMI/BMI SDS a) Leipzig Atherobesity Childhood cohort

Pub PH

14.5 ⴞ -0.32 ⴞ 2.73 (9.65–21.17) 0.86 (-1.69 –1.22) 14.0 ⴞ 2.74 (8.08 –19.01) 2.59 ⴞ 0.57 (1.48 –3.96) b) Diurnal variation in young healthy adults

3.7 ⴞ 1.55 (1– 6) 3.9 ⴞ 1.69 (1– 6)

lean 7/7 24.14 ⫾ 2.97 (19.82–30.28) 21.29 ⫾ 1.39 (18.67–23.57) obese 7/7 25.8 ⫾ 5.27 (17.48 –34.85) 38.23 ⫾ 5.88 (30.95– 49.67) c) 6 week in-house intervention - Leipzig Atherobesity Childhood Intervention cohort obese 23/35 12.7 ⫾ 2.28 (7.61–16.94) d) Long term low-grade intervention in school children control intervention competitive sports

12/17 20/14 16/9

11.79 ⫾ 0.37 (11.17–12.73) 11.8 ⫾ 0.47 (11.02–12.84) 11.65 ⫾ 0.66 (10.43–12.81)

2.42 ⫾ 0.41 (1.65–3.36)

3.36 ⫾ 1.76 (1– 6)

0.02 ⫾ 0.94 (-1.75–2.10) 0.11 ⫾ 1.11 (-2.39 –1.99) ⫺0.2 ⫾ 0.76 (-1.99 –1.35)

Abbreviations: BMI, body mass index; ow, overweight, PH, pubic hair


doi: 10.1210/jc.2014-2932


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Blood samples were taken immediately before and after exercise. c) 6 week in-house intervention in obese children - Leipzig Atherobesity Childhood intervention cohort We recruited 58 obese children (23 boys/35 girls) who volunteered to take part in a 4 to 6 week intervention program at a Centre in BadFrankenhausen (tab.1c). The program consisted of nutritional and psychological counseling and daily physical activity. Children were 7 to 17 years old and anthropometric data and medical history were recorded. Blood samples were obtained before and at the end of the intervention. (NCT01605123) d) Long term low-grade intervention in school children with sustained increase in activity We evaluated 88 children (tab.1d), who participated in the previously described school children intervention (22) aimed at promoting Figure 1. Sampling schedule for diurnal variation and validation of irisin physical activity by increasing numdetection (A) Schedule of blood sampling during 24 hours including meals (red arrows) followed by oGTT (blue) and on exercise program (green) at day two. B, Spike control of the used ber of exercise lessons in the interirisin-ELISA. Rec-irisin (from a distinct commercial source) was spiked into a human serum sample vention group (n ⫽ 34) with one ad(white bar) at indicated concentrations (black bars). The white bar represents the endogenous ditional sport unit per day compared irisin serum concentration of the donor the black bars reveal nearly full recovery of the rec-irisin, verifying the specificity of the used antibody. C, Immunoblot of human muscle lysate, paired to the control group (n ⫽ 29) with serum sample, mouse serum sample, and rec irisin revealed a distinct band at around 25 kDa, two regular sport units per week. In representing irisin. Serum samples were albumin depleted. D, Immunoblot of rec irisin before (-) addition, we compared a specialized and after (⫹) treatment (left side) with the deglycosylation enzyme mix from NEB revealed a school for competitive sports (n ⫽ band at around 17 kDa after treatment. Immunoblot of a human serum sample before (-) and after (⫹) treatment (right side) with the same enzyme mix showed a week band at around 17 25) with 12 U per week. kDa after treatment, representing deglycosylated irisin. All children started with the baseline examination at an age around 3.) Exercise/intervention regimes 11–12 years (table 1d), followed by a 1, 2, and 3 year a) Acute maximal exercise (cycling ergometry) follow-up examination. Fasting blood samples were taken 29 volunteers out of the Leipzig Atherobesity Childannually but not immediately before or after exercise. Anhood cohort (lean: 11 boys/8 girls, obese: 2 boys/8 girls) thropometric data and ergometry data were collected anperformed a 15min maximum cycling spiroergometry. Irinually. All pupils in the class had to adhere to the respecsin was assessed in blood samples taken directly before and tive regime. immediately after exercise. b) Acute short term intensive exercise (30min) Young healthy adults’ from the diurnal variation cohort (tab.1b) conducted an intensive 30min physical program (10min jogging, 10min gymnastics, 10min sprint). Physical exhaustion was verified by serum lactate increase.

Quantification of irisin in serum samples Serum irisin levels were quantified using a commercial enzyme-linked immunosorbent assay (ELISA) kit directed against amino acids 31–143 of the FNDC5 protein according to the manufacturer’s protocol (EK-067–52 Phoe-


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nix Pharmaceuticals). Absorbance was measured spectrophotometrically at 450nm (FLUROstar OPTIMA, BMG Labtech, Germany). Immunoblotting Human muscle biopsies were lysed in lysis buffer (50 mM HEPES pH7.5, 150 mM NaCl, 10 mM EDTA. 1% TritonX-100, Roche complete). Serum albumin was depleted using the SweliGel-Kit (PIERCE, USA) according to the manufacturer’s protocol. Human recombinant irisin (32–143aa) was purchased from Adipogen (AG-40B0136; San Diego, USA). For deglycosylation experiments, samples were treated with the Protein Deglycosylation Mix (NEB, Germany) according to manufactures protocol. All samples were separated by SDS-PAGE, transferred to a PVDF-membrane and detected by chemiluminescence. An antibody directed against irisin was taken from the irisin ELISA Kit. Statistical Analysis Statistical analyses were performed using Statistica 7.1 (Stat-Soft, Tulsa). Non-normally distributed data were log-transformed (log) before analysis. For comparison of quantitative traits between two groups, Student’s t test was applied. Paired sample t-tests were used to assess differences before and after exercise. Correlation analyses were performed using Pearson correlation or partial correlation. For multiple regression analyses, the stepwise forward model was employed. Threshold for statistical significance was P ⬍ .05.

Results 1. Verification of irisin detection by immunoassay Due to recent controversies about circulating irisin including difficulties of its specific detection in human serum (14), we first evaluated the specificity of the applied immunoassay and the expression of irisin. Recombinant irisin (obtained from a distinct commercial vendor) was adequately recovered when spiked at different concentrations into human serum (Figure 1B), indicating that the antibody detects irisin. With Western blot on human muscle protein, a corresponding serum sample, a mouse serum sample, and rec-irisin using the antibody of the immunoassay (Figure 1C), we detected a strong band at around 25 kDa in all samples corresponding with recirisin and a much weaker band around 12 kDa in the human muscle lysate and mouse serum. An additional 37 kDa band was detected in the muscle cell lysate and a 50 kDa band in the serum samples. As irisin may undergo protein modification by glycosylation we performed


deglycosylation experiments using several enzymes (Figure 1D). Following this treatment, the 25 kDa rec-irisin (left side) signal showed a shift to approximately 17–18 kDa. Also, we detected a week band (marked by arrow) of the same size in a deglycosylated human serum sample, indicating that irisin circulates, at least in part, in a glycosylated form. Based on these results we were confident to apply the immunoassay for assessment of irisin in our clinical study samples. 2. Diurnal variation and effect of glucose load and standardized meals on irisin in young adults We confirmed typical patterns of cortisol diurnal variation, with a peak at 8:00 am and a gradual decline over 12–16h, in all subjects (Figure 2A). We did not see a systematic diurnal regulation of irisin in lean or obese men or women (Figure 2B). However, a higher variability in irisin serum concentrations was detected in obese subjects, as indicated by noticeable peaks within the curve and a higher coefficient of variation (Figure 2C) compared to lean subjects. Meal intake or a defined glucose load during an oGTT did not affect irisin (Supplemental Figure 1). Hence, irisin has no diurnal variation and is not affected by food intake. Obese subjects do, however, appear to have a higher variability. 3. Association of irisin with obesity and metabolic profile In lean adults, 24h mean irisin levels were higher in men than in women (Figure 3A). Obese women had higher irisin levels than lean controls. In contrast, in children lean girls had slightly increased serum irisin levels compared to lean boys (Figure 3B), which were more than twice as high as in lean women, suggesting a potential association of irisin with age. Irisin levels were higher in young children (113.3 ⫾ 43.4ng/ml) compared to young adults (82.3 ⫾ 35.9ng/ml; P ⬍ .001). Accordingly, we found a negative association between age and serum irisin levels (Figure 3C) across all cohorts spanning an age range from young childhood to adulthood. Pubertal development did not affect irisin levels (Figure 3D). Irisin serum concentrations showed a positive correlation with BMI (Figure 3E) and waist-to-hip ratio (r ⫽ 0.57; P ⫽ .001) in young adults. We also observed a positive correlation between irisin and bio-impedance markers of muscle mass body-cell-mass (BCM) (Figure 3F) and fatfree-mass (Table 2), which was not present in children (Supplemental tab.1). In multiple regression analyses, BCM was a positive predictor of irisin concentrations, independent of gender


doi: 10.1210/jc.2014-2932

and age in adults, but not in children (Supplemental tab.2). When merging adults and children, age was the strongest independent negative predictor of irisin levels. Hence, irisin increases with muscle mass in adults and declines with age. Finally, we assessed the correlation of irisin with metabolic parameters. In adults, we identified correlations of irisin with 2h blood glucose and triglycerides (tab.2) and


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HDL-cholesterol. However, none of these parameters withstood adjustment to BMI, age, and gender in partial correlation analysis. In children, we did not observe any correlation of serum irisin with metabolic or cardiovascular parameters (Supplemental tab.2.).

Figure 2. Diurnal course of serum irisin levels (A) Cortisol serum levels showed the expected diurnal fluctuations in lean and obese subjects. B, Irisin is not regulated in a diurnal manner neither in men (B) nor in women (C). Data are presented as group-average of all participants (n ⫽ 7 subjects per subgroup) at every measured time point (every full hour ⫹ 30 minutes after meals) ⫾ SEM. D, Coefficient of variation in obese subjects shows a higher variability. Lean (white squares) and obese (black squares) variability is presented as coefficient of variation %.


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4. Regulation of serum irisin by physical exercise We compared four distinct intervention regimes differing in intensity and duration of physical activity (Figure 4A). a) Acute maximal exercise. After a 15min maximum


effort test by spiroergometry, serum irisin levels coherently increased in all subjects to an average of 123% (Figure 4B). Stratification for gender and obesity did not affect the results (Supplemental Figure 2A) except for lost sta-

Figure 3. Association of irisin with age, gender, and obesity (A) Lean men and obese women have significantly higher irisin levels compared to lean women. B, In children, only lean girls have considerable higher irisin levels compared to lean boys. Data are presented as mean⫾SEM. Serum irisin levels are negatively correlated with age (C) but pubertal development does not significantly affect irisin levels (D). Irisin is positively correlated with BMI (E) and muscle-associated BIA parameters (F) like body cell mass (BCM) in young adults. Black dots are marks for outliner and were analyzed by Tukey’s post test. Whiskers represent the fifth-95th percentile. Pearson correlation coefficient r and p value are shown in each scatterplot.


doi: 10.1210/jc.2014-2932


Table 2. Correlation of serum irisin levels with anthropometric, metabolic and cardiovascular parameters in 28 young adults (aged 17.5 to 34.9 yr, mean age 24.97 ⫾ 4.28y) Parameter

r P rb Anthropometric parameters

Age BMI (kg/m2) Waist-to-hip ratio

0.11 0.41 0.57

Pb

0.575 0.030 0.15

0.487

Skinfold subs. 0.17 (mm) Fat free mass 0.60 (kg) BCM 0.70 Metabolic parameters

0.389

0.001

⫺0.42

0.042

0.002

0.29

0.202

⬍0.001

0.38

0.105

Fasting blood glucose 120-min blood glucose Fasting plasma insulin Peak insulin HOMA-IR Quicki Triglycerides HDL cholesterol LDL cholesterol Cholesterol Cardiovascular

0.34

0.073

0.25

0.246

0.39

0.043

0.26

0.212

0.26

0.173

⫺0.07

0.740

0.23 0.28 ⫺0.28 0.44 ⫺0.46

0.248 0.147 0.143 0.020 0.014

0.02 ⫺0.04 0.02 0.25 ⫺0.18

0.925 0.866 0.918 0.230 0.417

⫺0.05 0.819 ⫺0.05 0.800 parameter

⫺0.15 ⫺0.17

0.486 0.419

Systolic BP 0.39 (mmHg) 0.28 Diastolic BP (mm Hg) Hormonal parameter

0.060

⫺0.03

0.991

0.185

⫺0.06

0.799

⫺0.32 0.28

0.187 0.247

0.22 ⫺045

0.422 0.078

E2 Testosterone

Abbreviations: BMI, body mass index; FFM, fat free mass; BCM, body cell mass; ECM, extra cellular mass b

partial correlation analysis after adjustment for age, sex and BMI

tistical significance in obese boys, apparently due to the small sample size (N ⫽ 2, data not shown). b) Acute short-time exercise in adults. The 30min shortterm exercise unit increased irisin significantly in 70% of the participants (Figure 4C). After stratification, a significant irisin increase was pertained in lean men and obese women (Supplemental Figure 2B) but not in lean women or obese men. The increase was only immediately detectable after exercise and disappeared 30min after exercise. c) In-house intervention. A 4 to 6 week exercise program was successful in reducing body weight (by 6.0 ⫾ 2.4%), cholesterol, HDL, and LDL (Supplemental tab.3). In the entire cohort we detected slightly decreased irisin levels after intervention (Figure 4D). After stratification

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for gender we only found this decrease of irisin in boys, but not in girls (Supplemental Figure 2C). However, changes in irisin were extremely variable, with 14 out of 39 girls (35.9%) and 8 out of 23 boys (34.8%) showing increased irisin levels at the end of the intervention, whereas the others remained unchanged or decreased. Hence, there is no clear and consistent effect in this cohort. d) Sustained increase in physical activity. We compared 88 children subjected to either regular school sport (control), one additional sport unit per day (intervention) or competitive sport school children (sport school) over a period of 3 years. All groups significantly increased bodycell-mass and VO2max (Supplemental tab.4). Even though a significant increase of irisin appeared in the control boys after 12 month and the intervention girls after 36 month (Supplemental Figure 2D), there was no consistent effect across all groups over the entire time span and children from the sport school did not have higher irisin levels (Figure 4E). Hence, we found an acutely increasing effect of short bouts of intensive exercise on irisin levels, whereas longer regimens did not affect irisin consistently.

Discussion We show that in adults irisin levels are affected by age, gender, obesity, and muscle mass while diurnal rhythm and meals do not contribute to the variation in irisin levels. Considering the expression in muscle and discrepant results on the effect of exercise on irisin, we particularly evaluated the effect of different exercise schemes on irisin levels in children and young adults. Our results show that short bouts of intensive exercise, but not long-term elevation in physical activity, acutely and transiently increase serum irisin levels. Due to the controversies regarding the protein properties of circulating irisin and its detection by immunoassays, we assessed the specificity of the used immunoassay. Our data on adequate retrieval of irisin detection from spiking experiments and the detection by Western-blot was reassuring for us to use the assay. In our experiments, irisin protein signals in human and mouse serum and muscle samples revealed a distinct band with a size of 25 kDa which would correspond to glycosylated irisin, but also to the mass of the main FNDC5 isoform (7, 23). The Flag-tag of the rec-irisin is likely to explain the slightly different signal in the immunoblot analysis. The shift of the 25 kDa band to app. 17 kDa after deglycosylation treatment of recombinant irisin and in human serum indicates that irisin circulates at least in part in glycosylated state and strengthens our interpretation that


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the 25 kDa signal corresponds to irisin. Nevertheless, we cannot exclude that FNDC5 exists in human serum, since


we still detect the 25 kDa signal after deglycosylation and the antibody we used has 100% cross-reactivity with sol-

Figure 4. Regulation of serum irisin by physical exercise (A) Schematic illustration of physical exercise duration (x-axis) and intensity (y-axis) in the four investigated cohorts (marked with B-E). (B) Acute maximal exercise (Leipzig Atherobesity Childhood cohort): 15 minutes short term intervention increased serum irisin 1.2 fold. (C) Acute short-time exercise in adults: 30 minutes short term intervention increased serum irisin 1.2 fold. (D) 6 week in-house intervention: A 4 – 6 week obesity intervention in a rehab clinic slightly decreased irisin serum level (0.95 fold). ␦-irisin was extremely heterogeneous. 34.8% of the boys (N ⫽ 23) and 35.9% of the girls (N ⫽ 39) have increased serum levels, others are unchanged or decreased. (E) Long term low-grade intervention in school children: Additional sport units per day (intervention group) or in sum 12 sport units per week (sport school) over a period of 3 years nearly have no effect on irisin serum concentrations. Graphs provide information of the entire subgroup comprising lean and obese men and women before and after intervention, respectively. Basal concentrations are set 1, postintervention concentrations are calculated as fold change. Data are presented as fold change ⫾ SEM of all intervention participants. Statistical significance was assessed by a paired Student’s t test. Stratification for weight status and gender is given in supplemental Figure 2.


doi: 10.1210/jc.2014-2932

uble FNDC5. However, the precise size of irisin is still controversial and one recent study reported irisin signals at ⬃12 kDa (9) according to the predicted size of the primary structure, which we could also detect at very low levels in the human muscle lysate and mouse serum. Additional bands above 15 kDa and around 37 kDa in the muscle biopsy lysate correspond in size with signals in mouse muscle of the same study (9). We can, however, not exclude cross reacting serum proteins as we detect a band around 50 kDa in the serum samples and irisin measurements with different ELISA kits produce very different results (24). Nevertheless, we concluded that irisin and its precursor FNDC5 is expressed in humans and was detectable by the ELISA we applied. Another potential explanation for discrepant results from studies may be confounding or regulating factors that have not been considered in previous studies. For example, physical activity patterns vary over the day, which may affect circulating levels of myokines as shown for FGF21 (25). Skeletal muscle has been identified as main source of serum irisin (2, 7) and the release was hypothesized to be related to exercise and muscle contraction (1). So far, little is known about the diurnal regulation of myokines. Very recently a diurnal regulation of irisin levels was shown in young lean soldiers (26) with an irisin peak at 9 p.m., even though there were fewer time points than in our study. We did, however, not find any rhythmicity or significant diurnal variation of irisin levels over the day in lean or obese normal healthy young adults under controlled sedentary conditions. The different findings may be explained by the higher fitness level of the military group. Considering the induction of irisin by PGC1-␣ (1), which itself responds to environmental stimuli such as nutritional status (27), we assessed whether meals or nutrients can affect irisin release. We did not find evidence for the regulation by food intake in general or by glucose load in our study. This is somewhat contradicting to reports demonstrating that glucose and palmitate decreased FNDC5 mRNA and irisin release from myotubes in vitro (4). However, this study investigated isolated muscle cells in vitro over a period of several days. Glucose stimulation during an oGTT might be too short. We noticed appreciably higher serum irisin levels in children than in adults and we detected an independent negative association of irisin with age in healthy lean subjects over a wide age span from childhood to (young) adulthood. This corresponds with findings from another adult cohort (28), whereas two other studies in obese subjects did not report an age dependency in obese children (29) or obese adults (30). In our study, the gender difference with higher irisin


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levels in men compared to women was detectable in adults, but not so clear in children, even though lean girls have slightly higher irisin levels than boys. This is in accordance with previous studies in obese children (29), while others found differences (31). Whether irisin is associated with fat and/or muscle mass is also a matter of debate. We found higher irisin levels in obese compared to lean adults. This was further supported by positive correlations between serum irisin BMI and waist-to-hip-ratio (WHR) in this cohort and is in line with other studies (3, 16). Besides muscle, an association between fat mass and irisin has been described (32). However, the positive correlations of irisin with muscle parameters like BCM, a good marker of muscle mass derived from BIA (33), and fat-free–mass, but not with fat-mass in our cohort rather supports a primary association with muscle mass, which is in line with previous findings (16). In multivariate analyses, BCM was the only significant predictor of irisin levels, indicating that indeed muscle mass is the determining factor. Considering that obese subjects also have significantly higher BCM, the association with muscle mass may underlie the association with obesity. Interestingly, we did not detect a positive association of irisin with fat-free-mass nor with BCM in children. However, it has to be considered that the body composition in children is distinct from that in adults and that massive dynamic alterations occur during development and puberty. BCM is not completely matured in children and adolescents (34), since it reflects all active cells in the body including osteoblasts and osteoclasts to a much higher percentage compared to adults. Hence, a more direct marker for muscle mass (eg, from MRI data) would be desirable, but was not available in our study. Several authors have questioned the regulatory role of exercise on circulating irisin in adults (4, 8, 10) and children (29). In the present study, we aimed to achieve a better understanding of potential association by comparing distinct exercise regimens which differed in duration and intensity. We observed a transient exercise-induced increase in irisin immediately after acute strenuous exercise, while there were no significant changes after long term exercise. Similar findings were described in two recent studies. In mice, irisin was increased in serum and skeletal muscle immediately after exercise (9). In a human study irisin was increased ⬃1.2-fold immediately after acute exercise but not after 12 weeks of training (8). The increase in circulating irisin by exercise in our study appeared to be short and transient and was already completely restored after 30min as similarly found by others (10). There are two possible explanations. First, the intervention was too short to induce a sustained irisin ele-


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vation. Secondly, only a short bout of exercise is necessary to mobilize all the irisin from its precursor and a further increase would only occur if new FNDC5 was recruited to the membrane. So far little is known about the signaling of irisin (35) and whether a small transient increase upon exercise is of physiological relevance is unknown. However, there are several myokines that are released acutely by muscle exercise and this increase is not a result of muscle damage. For example, IL-6 increases rapidly in response to exercise and declines in the postexercise period. The magnitude is related to exercise duration and intensity. Also, prolonged exercise (eg, one year) does not alter IL-6 (36). A recent study showed, that myokines altered differentially by acute exercise vs. prolonged training revealed only a limited overlap. They identified chemokine-(C-X3-C-motif)ligand1 (CX3CL1) and chemokine-(C-C-motif)-ligand2 (CCL2) as myokines induced by acute exercise and suggested those may be involved in communication between skeletal muscle and other organs (37). Our long-term exercise studies in children showed no clear effect on irisin. This is in contrast to a recent study describing mean increased irisin levels (12%) after a oneyear classical life-style intervention in obese children (29). In that study, obese children significantly lost weight. Hence, body fat mass may have decreased in favor of muscle mass. The authors did, unfortunately not measure body composition. Also, the increase in irisin was not consistent for all children but was present in approximately one third of the children. In summary, we show that in adults irisin levels are affected by age, gender, obesity, and muscle mass while diurnal rhythm and meals do not contribute to the variation in irisin levels. Short bouts of intensive exercise, but not long-term increase in physical activity, acutely increase irisin levels transiently in children and young adults.

Acknowledgments We thank Antje Berthold for the technical assistance and Matthias Blüher for the appropriation of human muscle samples. Address all correspondence and requests for reprints to: Corresponding author and person to whom reprint request should be addressed: Prof. Dr. Antje Körner, MD, Center for Pediatric Research, Hospital for Children & Adolescents, University of Leipzig, Liebigstraße 21, 04 103 Leipzig, Germany, phone: ⫹49 –341–9 726 500, fax: ⫹49 –341–9 726 509, email: [email protected]. Disclosure Statement: The authors have nothing to disclose. This work was supported by grants from the Federal Ministry of Education and Research (BMBF), Germany, FKZ: 01EO1001 (IFB AdiposityDiseases), the German Research Council (DFG) for the Clinical Research Center “Obesity Mechanisms”


CRC1052/1 C05, the Leipzig Research Center for Civilization Diseases (LIFE), and the German diabetes society (DDG).

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Circulating Irisin Levels Are Not Regulated by Nutritional Status, Obesity, or Leptin Levels in Rodents.

Serum irisin levels in patients with psoriasis.

Irisin is elevated in skeletal muscle and serum of mice immediately after acute exercise.

Serum irisin levels and thyroid function--newly discovered association.

Serum irisin levels, precocious myocardial infarction, and healthy exceptional longevity.

Strenuous Exercise Worse Than Sedentarism?

Muscle carnitine after strenuous endurance exercise.

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Irisin Levels are Not Affected by Physical Activity in Patients with Anorexia Nervosa.

Serum irisin levels are lower in patients with breast cancer: association with disease diagnosis and tumor characteristics.

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Resistance exercise induces a greater irisin response than endurance exercise.

Effect of Yoga Practice on Levels of Inflammatory Markers After Moderate and Strenuous Exercise.

Strenuous exercise and the heart: are we not seeing the wood for the trees?

Exercise-Induced Rhabdomyolysis with Acute Renal Failure After Strenuous Push-UPS.

Plasma irisin levels progressively increase in response to increasing exercise workloads in young, healthy, active subjects.

Post-exercise serum hepcidin levels were unaffected by hypoxic exposure during prolonged exercise sessions.

The Impaired Function of Macrophages Induced by Strenuous Exercise Could Not Be Ameliorated by BCAA Supplementation.

Maternal and fetal cardiovascular responses to strenuous bicycle exercise.

Serum irisin and myostatin levels after 2 weeks of high-altitude climbing.

Maternal serum and fetal cord blood irisin levels in gestational diabetes mellitus.

The effects of acute moderate exercise on lymphocyte function and serum immunoglobulin levels.

Impact of acute aerobic exercise and cardiorespiratory fitness on visuospatial attention performance and serum BDNF levels.