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1 JCEM ONLINE Hot Topics in Translational Endocrinology Endocrine Research Exercise-Induced Irisin Secretion Is Independent of Age or Fitness Level and Increased Irisin May Directly Modulate Muscle Metabolism Through AMPK Activation Joo Young Huh,* Vassilis Mougios,* Athanasios Kabasakalis, Ioannis Fatouros, Aikaterina Siopi, Ioannis I. Douroudos, Andreas Filippaios, Grigorios Panagiotou, Kyung Hee Park, and Christos S. Mantzoros Division of Endocrinology, Diabetes, and Metabolism (J.Y.H., A.F., G.P., K.H.P., C.S.M.), Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215; School of Physical Education and Sports Science (V.M., A.K., A.S.), Aristotle University of Thessaloniki, Thessaloniki 54124, Greece; Department of Physical Education and Sport Sciences (I.F., I.I.D.), Democritus University of Thrace, Komotini 69100, Greece; Department of Family Medicine (K.H.P.), Hallym University Sacred Heart Hospital, Hallym University, Gyeonggi-do , Korea; Section of Endocrinology (C.S.M.), Boston VA Healthcare System, Harvard Medical School, Boston, Massachusetts Context: Irisin has been proposed to be a myokine mediating the effect of exercise on adipocyte browning. The physiology of irisin in humans is not completely understood. Objective: To study the physiology of irisin in healthy individuals with different age and fitness levels and to explore the direct effects of irisin on muscle metabolism. Design, Setting, and Subjects: Treadmill exercise studies were conducted to measure circulating irisin at baseline and in response to exercise among old and young, physically active and sedentary individuals. Also, high- and moderate-intensity swimming was performed in adolescent men and women to study the effect of exercise intensity and the time course of irisin induction by acute bouts of exercise. Human myotubes were treated with recombinant irisin, and the effect on gene expression, cell signaling, and metabolism was examined. Results: Baseline circulating irisin was lower in old (vs young) and physically active (vs sedentary) subjects. Despite differences in basal levels, the percentage increase of irisin by acute bouts of exercise was not related to age or fitness level. The time course study revealed that circulating irisin increased immediately after high-intensity interval exercise and declined 1 hour thereafter. In vitro experiments showed that irisin facilitates glucose and lipid metabolism in human muscle through AMP kinase phosphorylation. Conclusions: Despite the differences in basal irisin levels, exercise-induced irisin secretion is independent of age or fitness level. Increased irisin can directly modulate muscle metabolism through AMP kinase activation. (J Clin Endocrinol Metab 99: E2154 E2161, 2014) ISSN Print X ISSN Online Printed in U.S.A. Copyright 2014 by the Endocrine Society Received February 13, Accepted August 4, First Published Online August 13, 2014 * J.Y.H. and V.M. have contributed equally to this work. Abbreviations: AMPK, AMP-activated protein kinase; BMI, body mass index; CME, continuous moderate-intensity exercise; CPT1b, carnitine palmitoyl transferase 1b; FNDC5, fibronectin type III domain-containing protein 5; GLUT4, glucose transporter 4; GYS1, glycogen synthase; HIIE, high-intensity interval exercise; HK2, hexokinase 2; HSMCs, human skeletal muscle cells; PDK4, pyruvate dehydrogenase kinase 4; PEPCK, phosphoenolpyruvate carboxykinase; PGC1, PPAR co-activator 1 alpha; PPAR, peroxisome proliferator-activated receptor; PYGM, glycogen phosphorylase; VO 2 max, maximal oxygen uptake. E2154 jcem.endojournals.org J Clin Endocrinol Metab, November 2014, 99(11):E2154 E2161 doi: /jc

2 doi: /jc jcem.endojournals.org E2155 Exercise is considered a cornerstone in the prevention and treatment of chronic metabolic diseases such as obesity, type 2 diabetes mellitus, and age-related muscle wasting (1, 2). The benefits of exercise are mediated not only by metabolic and molecular remodeling of skeletal muscle (3), but also by the release of cytokines from muscle, termed myokines (4). Myokines communicate with other tissues such as fat, liver, and pancreas and act locally on muscle to exert metabolic effects in an autocrine/paracrine/endocrine manner (4). Irisin is a recently discovered myokine produced by proteolytic cleavage of the membrane protein, fibronectin type III domain-containing protein 5 (FNDC5) (5). Regulated mainly by peroxisome proliferator-activated receptor (PPAR) coactivator 1 (PGC1 ), irisin is suggested to mediate some of the beneficial effects of exercise by inducing uncoupling protein 1 and subsequently increasing energy expenditure in white adipocytes, a process called adipocyte browning (5). Brown fat has been shown to exert antiobesity and antidiabetic effects in murine models and correlate with leanness in humans (6 8). Recent studies have suggested that in addition to classic brown adipocytes within brown fat in human adults, inducible brown adipocytes, or beige/brite adipocytes, may also play a role in human metabolism (9, 10). Thus, irisin has been proposed as an attractive therapeutic tool to treat metabolic disorders. After the discovery of irisin (5), studies have explored the irisin/fndc5 response to exercise. Studies including ours have shown a significant increase in the circulating irisin after an acute bout of exercise (11 14), whereas the effect of exercise training on circulating irisin and muscle FNDC5 expression seems controversial (5, 15 19). In general, the response to exercise depends on intrinsic factors, such as intensity, duration, and mode of exercise; and external factors including age, nutritional status, and body composition (3). Whether the irisin response to exercise differs between old and young subjects, as well as physically active and sedentary subjects remains unknown. In this study, we compared irisin levels at baseline, explored the time course of circulating irisin in response to exercise and compared this response according to age and fitness level. Although muscle is considered the primary organ expressing irisin, the potential existence of a feedback loop and its effects on myocyte metabolism have not been fully investigated in humans. We have previously observed a positive correlation between irisin and follistatin, a myokine that regulates muscle growth, at both mrna and serum levels (20), and showed that irisin regulates muscle growth-related genes through the ERK pathway (21). We also reported that intermittent sprinting, which causes a drop in muscle ATP content, evoked a significant rise in serum irisin, whereas repetition of this exercise for 8 weeks did not cause any changes in either muscle ATP or serum irisin (11). Based on these observations, we hypothesized that irisin may be induced during a state of energy need and, in return, exert metabolic effects on muscle. AMPactivated protein kinase (AMPK) is a metabolic sensor and a key enzyme in muscle metabolism. A number of myokines, including IL-6, have been reported to regulate muscle metabolism (4) through AMPK. To test this hypothesis, we utilized primary human skeletal muscle cells (HSMCs) and studied whether increased irisin regulates AMPK and, subsequently, whether irisin alters the glucose and lipid metabolism in HSMCs. Subjects and Methods Exercise study 1: effect of age and fitness level on circulating irisin Seventy-eight healthy men were recruited and divided into two groups by either age or fitness level as shown in Tables 1 and 2. Basal fitness level was determined by maximal oxygen uptake (VO 2 max), and individuals with VO 2 max higher than 35 ml/ kg/min for old participants and 45 ml/kg/min for young participants were considered physically active. The cutoff points used correspond to 70th percentile of the population and are reasonable for dividing the two groups (22, 23). All participants were nonsmokers, free of musculoskeletal problems who received no antihypertensive medications and had no signs of cardiovascular/respiratory complications. They also had normal dietary habits and did not consume aspirin, antioxidant compounds (including vitamins and minerals), certain medications (ie, probucol, nebivolol, and anti-inflammatory agents), and alcoholic beverages, at least 2 weeks before the study. The exercise protocol consisted of running for 45 minutes on a treadmill at 70 75% of VO 2 max, then at 90% of VO 2 max until exhaustion. At the end of the test, exercise time to exhaustion was recorded in minutes. This single bout of exercise to exhaustion was confirmed to induce significant oxidative and metabolic challenges (23). Blood was collected before and immediately after (in min) exercise for plasma preparation. Table 1. Characteristics of Young vs Old Subjects Young Old P n Age, y Weight, kg BMI, kg/m Fat, % Exercise time to exhaustion, min Irisin, ng/ml 67.9 ( ) 53.1 ( ).01 Data are expressed as means SD or median (interquartile range). P values are from Student s t test or Mann-Whitney U test (for irisin level).

3 E2156 Huh et al Irisin Physiology and Muscle Metabolism J Clin Endocrinol Metab, November 2014, 99(11):E2154 E2161 Table 2. Characteristics of Physically Active vs Sedentary Subjects Physically Active Sedentary P n Age, y Weight, kg BMI, kg/m Fat, % VO 2 rest, ml/ kg/min VO 2 max, ml/ kg/min Exercise time to exhaustion, min Irisin, ng/ml 52.9 ( ) 70.3 ( ).01 Data are expressed as means SD or median (interquartile range). P values are from Student s t test or Mann-Whitney U test (for irisin level). Exercise study 2: effect of exercise intensity on circulating irisin Thirty healthy adolescent swimmers (15 males; age, y; body mass index [BMI], kg/m 2 ; body fat, %; and 15 females; age, y; BMI, kg/m 2 ; and body fat, %) performed continuous moderateintensity exercise (CME; 2000 m of continuous freestyle swimming) and high-intensity interval exercise (HIIE; six 50-m maximal freestyle swimming bouts every 5 min) (24). Swimming time for the 2000-m test was 27 minutes 37 seconds 22 seconds, corresponding to a swimming velocity of m/s, whereas the average swimming time at each of the six maximal 50-m swimming bouts was seconds, corresponding to a swimming velocity of m/s. The two exercise tests were performed on separate days, spaced 1 week apart, in a random order. Blood samples were drawn before exercise, immediately (in less than 5 min), 1 hour, and 24 hours after exercise for plasma preparation. For determination of blood lactate, capillary blood was taken after warm-up and immediately after CME or 2, 4, 6, and 8 minutes after completion of the (the highest value recorded as peak postexercise lactate value). Exercise study 3: exercise-induced gene expression in humans Muscle biopsy (50 mg) was performed in vastus lateralis of young, moderately trained, healthy males (age, y; BMI, kg/m 2 ) before and after 8 weeks of sprint training. The training included a total of four to six 80-m sprint runs per session, three sessions a week (25). The samples were utilized for gene expression analysis. In vitro study in HSMCs Thigh muscle (vastus lateralis) was collected from obese but apparently healthy subjects (age, y; BMI, kg/m 2 ) and cultured as previously described (26). Briefly, tissue was minced and incubated in 37 C for 1 hour in the dissociation media containing 0.1% BSA, 0.25% trypsin-edta, and 0.1% collagenase. Isolated cells were grown in Skeletal Muscle Cell Growth Media (PromoCell), and after reaching 70 80% confluence, growth media was switched to Skeletal Muscle Cell Differentiation Media (PromoCell) for the differentiation of myoblasts into myotubes. The morphology and growth of the isolated myocytes were normal, and their characteristics were maintained until passage 7. To examine the effects of irisin treatment on HSMCs, the cells were incubated in serum-deprived medium containing 10 or 50 nm recombinant irisin (Phoenix Pharmaceuticals) and harvested for gene expression analysis. For AMPK inhibition, 10 M Compound C (Millipore) was added for 2 hours before irisin stimulation. All participants and, for adolescents, their guardians were informed about the details and potential risks of the experimental procedure, after which they provided written consent. The study was approved by the Institutional Review Board at Beth Israel Deaconess Medical Center, Aristotle University of Thessaloniki in accordance with the Declaration of Helsinki. In all studies, samples were stored at 80 C until analysis. Biochemical measurements Irisin was measured with enzyme immunoassay kits (catalog no. EK ; Phoenix Pharmaceuticals). The sensitivity of the assay is 6.6 ng/ml, and intra-assay coefficient of variation was 4 6%. A detailed validation on the kit has been published (11, 27, 28). Insulin, IGF-1, and IGF binding protein 3 were measured with Immulite 1000 automated immunometric assays (Siemens Healthcare Diagnostics). Myostatin was measured using an enzyme immunoassay kit (ALPCO). Lactate was measured by the lactate dehydrogenase enzymatic method (Sigma). Glucose and fatty acid uptake were measured using commercially available kits (Cayman Chemical and Molecular Devices, respectively). ATP was measured in cell lysates with a bioluminescence kit (Promega). Glycolysis was assessed by the release of lactate in the media (Cayman Chemical). Gene expression analysis Total RNA was extracted from human muscle and HSMCs using Trizol (Invitrogen). mrna levels were measured by real-time PCR using TaqMan gene expression assays as described (11). Western blot analysis Western blot was performed as previously described (26). Nitrocellulose membranes were incubated overnight with primary antibodies against phospho- and total AMPK (1:1000 dilutions; Cell Signaling). Statistical analysis Data are expressed as means SE unless stated otherwise. Biomarker concentrations that were not normally distributed (according to the Shapiro-Wilk test) were logarithmically transformed. Baseline differences were determined with Student s t test or Mann-Whitney U test. Changes in irisin levels between types of exercise and over time were examined with two-way (exercise type time) repeated-measures ANOVA, followed by Bonferroni post hoc analysis. Paired t test or Wilcoxon signed rank test was used to compare percentage changes in irisin at the same time point, as appropriate. Comparison of percentage change in irisin levels from baseline was performed with general linear model adjusted for covariates. For in vitro data, mean values obtained from multiple experiments were compared by ANOVA with subsequent Fisher s significant difference method.

4 doi: /jc jcem.endojournals.org E2157 Figure 1. Time course of circulating irisin in response to different exercise intensity in vivo and the effect of irisin on muscle glucose/lipid uptake in vitro. A, Circulating irisin in response to high-intensity interval exercise (HIIE) (6 50-m swimming) and continuous moderate-intensity exercise (CME) (2000-m swimming) in 30 young healthy subjects. Both tests were performed on the same individuals, 1 week apart. *, P.05 vs pre-exercise;, P.001 vs CME immediately after exercise. Values are expressed as means SE. B and C, Differentiated primary human skeletal muscle cells were incubated with 0, 10, or 50 nm irisin for 1 hour, and glucose uptake (B) and fatty acid uptake (C) were measured. Insulin was used as a positive control. Values are expressed as means SE of three or four experiments. *, P.05 vs control. Analyses were performed with the SPSS software, and P values below.05 were considered significant. Results Baseline irisin levels are lower in old (vs young) and physically active (vs sedentary) subjects The characteristics of the subjects are shown in Tables 1 and 2. Weight was comparable, whereas BMI was marginally but significantly different among groups. As expected, body fat percentage was higher in old (vs young) and sedentary (vs physically active) individuals. Baseline irisin levels were lower in the old compared to the young participants, whereas in age-matched groups, active individuals had lower circulating irisin in comparison to sedentary subjects. Moreover, baseline irisin levels were negatively associated with VO 2 max levels (r 0.188; P.019). These differences remained significant after adjustment for BMI, percentage body fat, and age. Circulating irisin levels were increased after an acute bout of treadmill exercise until exhaustion in the entire sample (pre, ; vs post, ng/ml;, P.03). However, there was no significant interaction between time and age (P.69) or fitness level (P.41). The increment of the irisin level, assessed by either change or percentage change from baseline, showed no differences between groups indicating that, despite different baselines, the irisin response to exercise was similar. Irisin is increased immediately after exercise and correlates with exercise intensity To examine the time course of irisin secretion after exercise, we measured plasma irisin in 30 adolescents (15 male and 15 female) for up to 24 hours after CME (2000-m of continuous freestyle swimming) and HIIE (six 50-m maximal freestyle swimming bouts). Postexercise blood lactate levels were for CME and mmol/l for HIIE, distinguishing the two as moderateand high-intensity exercise, respectively. Baseline irisin levels were comparable between the two exercise regimens (CME, ;, ng/ml; P.51). Plasma irisin tended to increase but was not significantly altered by CME at any of the time points observed (Figure 1A). In contrast, irisin was significantly up-regulated by HIIE, increasing by 30% in less than 5 minutes after exercise, remaining high after 1 hour, and then returning to baseline. Comparison between genders showed that male and female adolescents had similar baseline irisin (males, ; females, ng/ml; P.59), but the increment by HIIE was larger in males (Supplemental Figure 1). The change in irisin positively correlated with the change in blood lactate (r 0.39; P.04). In terms of other myokines, plasma myostatin levels were significantly increased, whereas IGF binding protein 3 levels were decreased 24 hours after exercise, with no differences between exercise regimens (Supplemental Figure 2). Irisin directly induces glucose and fatty acid uptake in human muscle The results from exercise studies imply a transient increase in irisin after an acute bout of exercise. The physiological significance of this increase is unknown and is difficult to test in vivo. Therefore, HSMCs were treated with recombinant irisin to directly test whether increased irisin could exert beneficial effect in muscle. As shown in Figure 1, B and C, irisin treatment for 1 hour resulted in increased glucose and fatty acid uptake in HSMCs, comparable to insulin-induced response. Irisin regulates gene expression of metabolic enzymes in human muscle To see whether irisin regulates the transcription of metabolic genes, we treated HSMCs with irisin (control, 10,

5 E2158 Huh et al Irisin Physiology and Muscle Metabolism J Clin Endocrinol Metab, November 2014, 99(11):E2154 E2161 whether AMPK signaling was associated with irisin-induced gene regulation, HSMCs were pretreated with AMPK inhibitor compound C. Indeed, compound C effectively prevented irisin-induced AMPK phosphorylation and gene expression (Figure 3 C and D). Figure 2. Effect of irisin on metabolic gene expression in primary human skeletal muscle cells. A C, Differentiated primary human skeletal muscle cells were incubated with 0, 10, or 50 nm irisin for 2 hours (A), 6 hours (B), or 24 hours (C). mrna levels were measured by real-time PCR. Values are expressed as means SE of three or four experiments. *, P.05 vs control. and 50 nm) in a time-dependent (2, 6, and 24 h) manner. As shown in Figure 2, 2 hours of irisin treatment caused only minor changes in metabolic gene expression. However, after 6 hours of treatment, the expression of genes related to glucose metabolism was modulated, including up-regulation of glucose transporter 4 (GLUT4) and hexokinase 2 (HK2), as well as down-regulation of pyruvate dehydrogenase kinase 4 (PDK4), phosphoenolpyruvate carboxykinase (PEPCK), and glycogen phosphorylase (PYGM). This was followed by up-regulation of glycogen synthase (GYS1) and carnitine palmitoyl transferase 1b (CPT1b) at 24 hours after treatment, suggesting a novel function of irisin to modulate metabolic gene expression in HSMCs. Gene modulation was followed by enhanced lactate production (Supplemental Figure 3). Irisin induces AMPK phosphorylation in human muscle To elucidate the mechanism(s) underlying the irisininduced changes in muscle metabolism, we measured intracellular ATP in HSMCs and found a significant decrease after a 1-hour irisin treatment (Figure 3A). Moreover, this decrease coincided with enhanced phosphorylation of AMPK (Figure 3B), indicating that a decrease in ATP may be responsible for triggering AMPK phosphorylation and its downstream effects. To confirm Muscle FNDC5 expression is induced by exercise and regulates metabolic enzymes in vitro and in vivo To examine the effect of exercise on muscle FNDC5 expression in vitro, HSKCs were treated with exercise mimetics. Both FNDC5 and PGC1 mrnas were increased by ionomycin (Ca 2 ionophore) and forskolin (activator of adenylate cyclase) treatment (Figure 4, A and B). FNDC5 was also induced by IL-6, implying that IL-6 could lie upstream of FNDC5 regulation (Figure 4C). In line with the in vitro results, muscle samples obtained from young, moderately trained, healthy males showed that 8 weeks of sprint training significantly induced FNDC5 and PGC1 mrna level (Figure 4D), along with increased metabolic gene expression (Figure 4E). Discussion Irisin has gained attention as exercise hormone on the basis that PGC1 overexpression and endurance exercise training induces irisin in mice (5, 29). However, the physiology of irisin in human remains largely unknown. The present report explored irisin at baseline in subjects with different age and fitness levels, in response to different exercise intensities in adolescent males and females, and its effects on muscle metabolism in humans. The main findings are that: 1) baseline circulating irisin is lower in old (vs young) and active (vs sedentary) subjects; 2) circulating irisin levels peak immediately after exercise, and the increment of irisin depends on exercise intensity; 3) exercise induces FNDC5 gene expression in vitro and in vivo; and 4) increased irisin affects muscle metabolism via AMPK activation. We have previously observed a negative correlation between age and circulating irisin (11) and confirm here that the baseline irisin level is lower in older compared to

6 doi: /jc jcem.endojournals.org E2159 Figure 3. Effect of irisin on metabolic function and AMPK phosphorylation in primary human skeletal muscle cells. A, Intracellular ATP was measured at different time points after irisin treatment. B, Phosphorylated AMPK relative to total AMPK. Representative Western blots are shown at the top. C, Cells were incubated with 10 M compound C (an AMPK inhibitor) for 2 hours before 50 nm irisin treatment for 3 hours. Phosphorylated AMPK and total AMPK were measured. D, Cells were incubated with 10 M compound C for 2 hours before 50 nm irisin treatment for 6 hours. mrna levels were measured by real-time PCR. Values are expressed as means SE of three or four experiments. *, P.05 vs control;, P.05 vs irisin-treated cells. younger subjects. Muscle being the primary organ of FNDC5 expression, it is possible that lower muscle mass in older subjects has resulted in a lower level of irisin. Interestingly, in age-matched groups, irisin levels were lower in active compared to sedentary subjects, which seems paradoxical. We and others have reported that, whereas acute exercise induces circulating irisin levels, Figure 4. Regulation of FNDC5 in skeletal muscle in vitro and in vivo. A C, FNDC5 and PGC1 mrna were measured at 2 hours after ionomycin (1 M), forskolin (1 M), or IL-6 (100 ng/ml) treatment. Values are expressed as means SE of three or four experiments. *, P.05 vs control. D, Muscle FNDC5 and PGC1 mrna before and after 8 weeks of high-intensity interval training. E, Muscle metabolic gene expression after 8 weeks of training. *, P.05 vs before training. Values are expressed as means SE of 10 subjects. TFAM, mitochondrial transcription factor A; CYCS, cytochrome c. chronic exercise training results in either unchanged or even down-regulated basal irisin levels (11, 13). Higher VO 2 max values in active subjects imply that they have improved cardiovascular fitness and thus would require less feedback from muscle (30, 31). Indeed, the negative association between VO 2 max and irisin levels makes it plausible that lower irisin in active subjects is a result of an adaptive response to higher muscle capacity. A similar phenomenon has been reported with other cytokines, including IL-6 (32). Therefore, not only muscle mass but also capacity may be involved in regulation of basal irisin level. Because muscle mass and capacity have not been measured in the current study, the direct cause-and-effect relationship needs to be further examined. Despite the apparent differences in baseline irisin levels, the increased irisin in response to treadmill exercise was unrelated to age or fitness level. Rather, comparison between high- and moderate-intensity swimming revealed that exercise intensity, evidenced by postexercise lactate levels, seems to be closely associated with the irisin response. Similar time response was observed when subjects performed 90-minute treadmill exercise at 60% of VO 2 max (12). In this report, irisin was measured before, during (54 min), and after (90 min) exercise, and irisin was significantly increased only at 54 minutes. Therefore, it is possible that irisin bursts out at the initial stage of the exercise and declines thereafter, meaning that no change could be observed at the final stage in exercise that lasts more than 1 hour. It is interesting to note a larger increase in irisin levels in male compared to female adolescents immediately after acute swimming. The larger increment in males might be due to their higher muscle mass, which would have served as a greater source of irisin secretion. Whether there is a difference in the regulation of irisin secretion between genders needs to be clarified in larger studies. The transient increase in irisin after an acute bout of exercise is not

7 E2160 Huh et al Irisin Physiology and Muscle Metabolism J Clin Endocrinol Metab, November 2014, 99(11):E2154 E2161 likely to be effective in browning, which is more of a chronic effect. Therefore, it is important to find out the physiological significance of irisin secretion and its target in a matter of minutes to hours. The clinical study has limitations in correlating the irisin increase with metabolic outcomes; thus, we employed a primary culture system. In HSMCs, irisin down-regulated ATP levels, triggering AMPK phosphorylation, with maximum phosphorylation noted at 3 hours. This led to dose- and time-dependent effects of irisin on downstream metabolic gene expression and function. Up-regulation of GLUT4 and HK2 and down-regulation of PDK4 and PEPCK all point to facilitation of glucose use as an energy source. Expression of PPAR, suggested to act downstream of FNDC5 to partly induce uncoupling protein 1 in adipocytes, was also upregulated. Decreased glycogen phosphorylase and increased GYS1 suggest a glycogen sparing effect of irisin, possibly as a compensation for glycogen depletion after exercise. Muscle gene expression before and after endurance exercise training also showed that exercise-induced FNDC5 is related to muscle metabolic gene regulation. We confirmed that irisin-mediated gene regulation is dependent on AMPK, by the use of an AMPK inhibitor. Therefore, irisin stimulation leads to many of the classical exercise-induced changes in muscle metabolism. A recent study has reported that in mouse muscle cells, irisin induces mitochondrial biogenesis and uncoupling (33), further supporting the hypothesis that irisin may regulate muscle metabolism. There are reports suggesting that white adipose tissue is a source of irisin secretion (16, 34), but the exercise-induced burst in irisin is likely accounted for by skeletal muscle release alone. Furthermore, although the exerciseinduced change in circulating irisin is around 10 30%, the local concentration in skeletal muscle during exercise may well exceed those measured in the circulation. If confirmed, this will increase the relevance of our findings with regard to the effect of irisin on muscle. Significant upregulation of circulating irisin in less than 30 minutes implies that irisin release would have resulted from translational or post-translational regulation, but we also report here that FNDC5 transcription can be induced by exercise, which may prolong the effect of irisin in muscle. In other words, irisin may function as an exercise signal to facilitate muscle metabolism, irrespective of effects on adipocyte browning (13). Also, the rapid regression of the exerciseinduced irisin implies that there may also be a mechanism for irisin uptake or clearance from circulation, which has not been assessed herein. Some studies have questioned whether irisin is an exercise-induced factor, and a large portion of this controversy is accounted for in the use of different assay kits. We and others have validated the kit that we have used in this study, and this kit has been successful in detecting changes in irisin levels in the circulation (11, 26 28). Another reason for the inconsistency could be the differences in study design and lack of prior knowledge on the timeframe over which irisin is increased. In this respect, some studies have failed to detect a rise in irisin levels due to delayed sampling time (16, 18). Several aspects of irisin physiology remain to be elucidated. Although we have found that age and fitness level do not affect irisin levels in response to exercise, it needs to be shown whether subjects with metabolic complications such as diabetes have impaired irisin response. Moreover, the role of irisin in metabolic regulation indicates the existence of an irisin receptor that has yet to be identified, which triggers downstream AMPK activation. In conclusion, baseline irisin levels are associated with age and fitness level but exercise-induced irisin secretion is independent. The possibility of direct regulation of muscle metabolism by irisin opens a new avenue of investigation on local effects of irisin in metabolic organs and also provides the potential for future therapeutics in subjects with obesity or diabetes. Whether irisin mimics the exerciseinduced response in humans and/or may prove to have clinical applications for obese/diabetic patients needs to be studied further in future observational and interventional studies. Acknowledgments Address all correspondence and requests for reprints to: Christos Mantzoros, MD, DSc, Harvard Medical School, Boston, MA cmantzor@bidmc.harvard.edu. This work was supported by an award from the Clinical Science Research and Development Service of the VA Office of Research and Development. Part of this study was supported by a donation received from the Bodosakis Foundation (Greece) for instrument purchase. Disclosure Summary: The authors have nothing to disclose. References 1. Colberg SR, Sigal RJ, Fernhall B, et al. Exercise and type 2 diabetes: the American College of Sports Medicine and the American Diabetes Association: joint position statement. Diabetes Care. 2010;33: e147 e Sigal RJ, Kenny GP, Wasserman DH, Castaneda-Sceppa C, White RD. Physical activity/exercise and type 2 diabetes: a consensus statement from the American Diabetes Association. Diabetes Care. 2006; 29: Egan B, Zierath JR. Exercise metabolism and the molecular regulation of skeletal muscle adaptation. Cell Metab. 2013;17: Pedersen BK, Febbraio MA. Muscles, exercise and obesity: skeletal muscle as a secretory organ. Nat Rev Endocrinol. 2012;8:

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