Resistance exercise induces a greater irisin response than endurance exercise Yoshifumi Tsuchiya, Daisuke Ando, Kaoru Takamatsu, Kazushige Goto PII: DOI: Reference:
S0026-0495(15)00142-0 doi: 10.1016/j.metabol.2015.05.010 YMETA 53213
To appear in:
Metabolism
Received date: Revised date: Accepted date:
12 March 2015 12 May 2015 14 May 2015
Please cite this article as: Tsuchiya Yoshifumi, Ando Daisuke, Takamatsu Kaoru, Goto Kazushige, Resistance exercise induces a greater irisin response than endurance exercise, Metabolism (2015), doi: 10.1016/j.metabol.2015.05.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
T
Resistance exercise induces a greater irisin response than endurance exercise
1
SC
RI P
Yoshifumi Tsuchiya 1, Daisuke Ando 2*, Kaoru Takamatsu 3, Kazushige Goto 1
Graduate School of Sport and Health Science, Ritsumeikan University, 1-1-1, Nojihigashi,
2
MA NU
Kusatsu, Shiga, 525-8577, Japan
Faculty of Education and Human Sciences, University of Yamanashi, 4-4-37, Takeda, Kofu, Yamanashi, 400-8510, Japan University of Tsukuba, 1-1-1, Tennodai, Tsukuba, Ibaraki, 305-8574, Japan
PT
ED
3
CE
Running title: Irisin response to resistance exercise
AC
Conflict of interest: Nothing to declare
*Authors contributed equally to this work.
Corresponding author: Kazushige Goto, Ph. D. Graduate School of Sport and Health Science, Ritsumeikan University 1-1-1, Nojihigashi, Kusatsu, Shiga, 525-8577, Japan E-mail: [email protected] Phone: +81-77-599-4127 Fax: +81-77-599-4127 1
ACCEPTED MANUSCRIPT
Abstract
T
Objective: We determined detailed time-course changes in the irisin response to acute exercise
RI P
using different exercise modes.
SC
Methods: In experiment 1, seven healthy males rested for 12 h (8:00–20:00) to determine the diurnal variation in plasma irisin concentration. In experiment 2, 10 healthy males conducted three
MA NU
exercises to clarify time-course changes in plasma irisin concentration over 6 h, using a randomized crossover design. The resistance exercise (R) trial consisted of eight exercises of 12 repetitions with 3-4 sets at 65% of one repetition maximum (1RM). The endurance exercise (E)
ED
． trial consisted of 60 min of pedaling at 65% of maximal oxygen uptake (VO2max). In the combined
PT
mode (R+E) trial, 30 min of endurance exercise was preceded by 30 min of resistance exercise.
CE
Results: In experiment 1, no significant changes in plasma irisin concentration were observed over 12 h. In experiment 2, the R trial showed a marked increase in plasma irisin concentration 1 h
AC
after exercise (P < 0.05), but not in the E or R+E trials. The area under the curve (AUC) for irisin concentrations for 6 h after exercise was significantly higher in the R trial than in the R+E trial (P < 0.05). The AUC for irisin concentrations was significantly correlated with AUC values for blood glucose, lactate, and serum glycerol (r = 0.37, 0.45, 0.45, respectively. P < 0.05). Conclusions: Resistance exercise resulted in significantly greater irisin responses compared with endurance exercise alone, and resistance and endurance exercises combined. Key words: Irisin, PGC-1α, FNDC5, Myokine, Exercise mode, Time-course change
2
ACCEPTED MANUSCRIPT
1．
Introduction
T
Growing attention has been paid to myokines, which are endocrine metabolic regulators that
RI P
circulate from myocytes into working muscles. Several myokines have been reported, and irisin is
SC
a novel exercise-induced myokine whose level rises in response to expression of peroxisome proliferator receptor γ coactivator-1α (PGC-1α) [1]. Irisin has been proposed to act as a
MA NU
muscle-derived energy expenditure signaling hormone [1]. Importantly, irisin leads to expression of uncoupling protein-1 (UCP1), a thermogenic factor involved in browning of white and beige adipocytes [1, 2]. The increase in irisin augments oxygen consumption, improves glucose
ED
tolerance and insulin sensitivity, and facilitates weight loss compared with those in an untreated
PT
group [1]. Controversies exist among recent publications regarding the importance of irisin and/or
CE
fibronectin type III domain containing 5 (FNDC5) in humans [3-9], and it is necessary to further examine the roles of irisin in metabolic regulation, body composition, and anti-obesity effect.
AC
Several studies have reported that a single bout of endurance exercise promotes irisin secretion, but these studies are not conclusive. Data from human studies has demonstrated that irisin concentrations increase significantly during [10] and immediately [11-14] after endurance exercise. In contrast, Pekkala et al. [4] reported that irisin concentrations did not change following acute endurance exercise in middle-aged men. The irisin response to resistance training has also been studied [6, 7], however, information about the effect of a single bout of resistance exercise on the irisin response is rather limited. Pekkala et al. [4] showed that resistance exercise increased FNDC5 mRNA expression approximately 1.4-fold in muscle compared with the baseline value, 3
ACCEPTED MANUSCRIPT
while the plasma irisin concentration did not change during the 30-min post-exercise period. In
T
contrast, Huh et al. [14] recently found that resistance exercise (lasting 45 min) induced a greater
RI P
irisin response (measured immediately after exercise) than that of endurance exercise (lasting 36
SC
min). Pekkala et al. [4] also determined the long-term effects of combined training (combination of resistance and endurance training) on the irisin baseline concentration. Moreover, two studies
MA NU
recently reported effect of exercise and dietary intervention on irisin in obese children [15, 16]. However, the irisin response was not examined in response to a single bout of exercise in these studies. As such, we are still unaware of which types of exercise, with equivalent durations,
ED
produce the largest effect on irisin secretion. Moreover, previous studies that have determined the
PT
exercise-induced irisin response took blood samples before and relatively early during the
CE
post-exercise period (< 2 h post-exercise). However, considering that PGC-1α (a primary irisin secretion factor) is upregulated 2–3 h after exercise [11, 17-19], irisin concentration may be
AC
maximized at least 2 h following exercise. The purpose of this study was to compare the magnitude of the irisin response following different types of a single bout of exercise under equivalent exercise durations. We hypothesized that irisin concentrations would be higher in resistance exercise than in endurance exercise when measured 2-3 h after each exercise. To test this hypothesis, we monitored detailed time-course changes in the irisin response during a 6-h post-exercise period.
4
ACCEPTED MANUSCRIPT
2.
Material and methods Subjects
T
2. 1.
RI P
Two experiments were conducted. In experiment 1 (resting experiment), seven physically
SC
active males [mean ± standard error (SE): age; 25 ± 1 yr, height; 171 ± 6 cm, weight; 69 ± 8 kg] were evaluated for diurnal variations in irisin concentration. In experiment 2 (exercise experiment),
MA NU
10 physically active males (mean ± SE: age; 23 ± 1 yr, height; 172 ± 2 cm, weight; 70 ± 2 kg) were evaluated to clarify the effects of a single bout of exercise on time-course changes in irisin concentration. Subjects in both experiments had several years of experience undergoing strenuous
ED
exercise training and had maintained these exercise habits (at least once a week) upon study
PT
initiation. Exclusion criteria for both experiments were coronary heart disease, obesity, metabolic
CE
syndrome, and addictions to prescribed drug or tobacco use. Each subject was informed of the purpose of the study, experimental procedures, and the possible risks involved, and written
AC
informed consent was obtained. All experimental procedures were approved by the Ethical Committee for Human Experiments at Ritsumeikan University in accordance with the Declaration of Helsinki.
2. 2.
Experiment 1 (resting experiment)
2. 2. 1.
Experimental design and measurement procedures
Experiment 1 was designed to determine the diurnal variation in plasma irisin concentration over 12 h. Seven subjects visited the laboratory following an overnight fast. They rested in the 5
ACCEPTED MANUSCRIPT
laboratory for 12 h (8:00–20:00). Control meals (breakfast: 683 kcal, carbohydrate; 62%, protein;
T
17%, fat; 21%, lunch: 729 kcal, carbohydrate; 65%, protein; 16%, fat; 19%) were provided at the
RI P
same time of day to avoid the influence of diet among subjects. The subjects were instructed to
SC
refrain from consuming caffeine and alcohol for 12 h and to perform no physical activity for 24 h before the experiment.
MA NU
Blood samples were collected from an antecubital vein five times (8:00, 11:00, 14:00, 17:00, and 20:00) during the 12 h of rest. The blood samples were transferred to a tube containing EDTA-Na, and the plasma was obtained after a 10 min centrifugation (3,000 rpm, 4°C) and stored
ED
at −80°C until analyses. Plasma irisin concentrations were determined in duplicate using a
PT
commercially available enzyme-linked immunosorbent assay (ELISA) kit (EK-067-52, Phoenix
CE
Pharmaceuticals, Inc., Darmstadt, Germany), and all blood samples were analyzed within the same plate. Absorbance was measured by wavelength at 450nm [20]. The intra-assay coefficient
AC
of variation (CV) was 1.7%.
2. 3.
Experiment 2 (exercise experiment)
2. 3. 1.
Experimental design
The subjects visited the laboratory five times throughout the experimental period. One ． repetition maximum (1RM) and maximal oxygen uptake (VO2max) were determined [21] on the first and second visits. Different types of acute exercise were conducted on the third to fifth visits. The 1RM values for eight exercises (chest press, lat-pull down, leg press, knee extension, seated 6
ACCEPTED MANUSCRIPT
rowing, shoulder press, arm curl, and triceps press down) were measured using a weight-stack
T
machine (Life Fitness Co., Tokyo, Japan). The subjects stretched for several min before the 1RM
RI P
measurements. Load was increased progressively until the subject was unable to complete a successful lift to determine the 1RM. An incremental pedaling test was performed on the second
SC
． visit to determine VO2max (828E, Monark, Uppsala, Sweden). After a 5 min warm-up at 30 W, the
MA NU
test began at 60 W, and the load was increased progressively by 30 W every 2 min until exhaustion. The test was terminated when the subject failed to maintain a pedaling frequency of ． 60 rpm or reached a VO2 plateau. Respiratory gases were collected and analyzed using an
ED
． automatic gas analyzer (AE300S, Minato Medical Science Co., Tokyo, Japan) to determine VO2,
PT
． ． carbon dioxide output (VCO2), minute ventilation (VE), and the respiratory exchange ratio (RER).
CE
These data were averaged every 30 s. All subjects conducted three different exercise trials with a randomized crossover design on
AC
the third to fifth visits. The trials consisted of resistance exercise (R), endurance exercise (E), and a combined mode of resistance and endurance exercise (R+E). Each trial was conducted during the morning following an overnight fast, and separated by at least 1 week.
2. 3. 2.
Exercise trial protocols
Subjects in the R trial conducted 60 min of resistance exercise (eight exercises: chest press, lat-pull down, leg press, knee extension, seated rowing, shoulder press, arm curl, and triceps press down). Each exercise set consisted of 12 repetitions, with four sets for chest press and lat-pull 7
ACCEPTED MANUSCRIPT
down, and three sets for the remaining six exercises at 65% of 1RM. The subjects rested for 2 min
T
． between sets and exercises. The E trial consisted of 60 min of pedaling at 65% of VO2max.
RI P
Pedaling frequency was set at 60 rpm. Subjects in the R+E trial initially conducted 30 min of four resistance exercises (chest press, lat-pull down, leg press, and shoulder press). Then, 30 min of
SC
． endurance exercise at 65% of VO2max was subsequently performed after a 20 min rest. Therefore,
2. 3. 3.
MA NU
the total exercise duration (60 min) was the same among the three trials.
Blood sampling and analysis
ED
Blood samples were collected before exercise (Pre), immediately after exercise (0 h), and at
PT
0.5, 1, 2, 3, 4, and 6 h after exercise (Figure 1). After drawing blood, serum and plasma samples
CE
were obtained after 10 min of centrifugation (3,000 rpm, 4°C) and stored at −80°C until analyses. Serum insulin, triglycerides (TG), free fatty acids (FFA), glycerol, myoglobin, creatine kinase
AC
(CK), total ketone bodies, and plasma irisin concentrations were measured. Serum insulin concentrations were measured by a chemiluminescent enzyme immunoassay at a clinical laboratory (SRL, Inc., Tokyo, Japan). Serum FFA concentrations were measured using a commercially available enzymatic colorimetric kit (NEFA-HRII, Wako Pure Chemical Industries, Osaka, Japan). Serum glycerol concentrations were determined in duplicate with a commercially available kit (Cayman Chemical Company, Ann Arbor, MI, USA). Serum TG, myoglobin, CK, and total ketone body concentrations were measured at a clinical laboratory (SRL). Plasma irisin concentrations were determined in duplicate using a commercially available ELISA kit 8
ACCEPTED MANUSCRIPT
(EK-067-52, Phoenix Pharmaceuticals, Inc.). All blood samples for each subject were analyzed
T
within the same plate. The intra-assay CV of each measurement was as follows: 4.0%, 3.0%, 1.4%,
Respiratory measurements
MA NU
2. 3. 4.
SC
ketone bodies, and plasma irisin concentrations, respectively.
RI P
3.9%, 8.2%, 4.0%, 2.6% and 3.5% for serum insulin, TG, FFA, glycerol, myoglobin, CK, total
Five min of resting respiratory gases were collected using an automatic gas analyzer ． ． ． (AE300S, MINATO Medical Science, Osaka, Japan) to determine VO2, VCO2, and VE before
ED
exercise, and at 0.5, 1, 2, 3, 4, 6, and 24 h after exercise (Figure 1). The RER was calculated from
PT
． ． ． ． VO2 and VCO2. Energy expenditure after each exercise was determined from VO2 and VCO2
Statistical analysis
AC
2. 4.
CE
[22].
All experimental data are shown as mean ± standard error. The time-course changes in plasma irisin concentrations in experiment 1 were compared using one-way repeated-measures analysis of variance (ANOVA). Comparisons of the time-course changes in blood parameters and respiratory variables among the three different trials in experiment 2 were made with a two-way repeated-measures ANOVA to identify the interaction (trial × time) and main effect for trial and time. When ANOVA revealed a significant interaction or main effect, a post-hoc analysis (Tukey–Kramer method) was performed to assess differences among trials or times. The area 9
ACCEPTED MANUSCRIPT
under the curve (AUC) values were compared using one-way repeated-measures ANOVA. The
T
correlation between the magnitude of the exercise-induced plasma irisin response and those in
AC
CE
PT
ED
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SC
regression analysis. A p < 0.05 was considered significant.
RI P
other blood parameters was determined by Pearson’s correlation coefficient after multiple linear
10
ACCEPTED MANUSCRIPT
3.
Results Experiment 1 (resting experiment)
T
3. 1.
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Figure 2 shows the time-course changes in plasma irisin concentrations over 12 h. The
SC
plasma concentrations remained stable, and no significant differences were observed among the
3. 2.
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measurement points for 12 h.
Experiment 2 (exercise experiment)
3. 2. 1.
Blood parameters
ED
Figure 3 shows the time-course changes in plasma irisin concentrations before and after
PT
exercise during each trial. No significant difference in plasma irisin concentration at baseline (Pre)
CE
was observed among trials. The E and R+E trials did not show significant changes in plasma irisin concentrations during the 6 h after the exercises. In contrast, the R trial showed a significant
AC
increase in plasma irisin concentration 1 h after exercise (P < 0.05). Furthermore, the plasma irisin concentration 1 h after exercise was significantly higher in the R trial than that in the E and R+E trials (P < 0.05). The AUC value was significantly higher in the R trial (151559 ± 22487 ng/mL･6 h) than that in the R+E trial (123990 ± 18700 ng/mL･6 h, P < 0.05, Figure 4) when exercise-induced irisin responses during the 6-h period after exercise were compared. Table 1 shows the time-course changes in blood variables for each trial. The E trial resulted in significantly lower blood glucose concentration at baseline than those in the R+E trial. Blood glucose concentrations were significantly higher in the R trial than in the E and R+E trials 11
ACCEPTED MANUSCRIPT
immediately after exercise (P < 0.05). Blood lactate concentrations at baseline were significantly
T
higher in the E trial than in the R and R+E trials. Although blood lactate concentrations increased
RI P
significantly after exercise in all trials, the R trial resulted in a significantly greater response than
SC
that observed in the E and R+E trials (P < 0.05). Consequently, the AUC values 6 h after exercise were significantly higher in the R trial than in the E and R+E trials (P < 0.05). Serum FFA
MA NU
concentrations at baseline were significantly higher in the E trial than in the R and R+E trials (P < 0.05). The E trial showed significantly higher serum FFA concentrations compared with those in the R and R+E trials immediately after and at 0.5 h after exercise (P < 0.05). The AUC values
ED
during the 6 h after exercise were significantly lower in the R trial than in the E and R+E trials (P
PT
< 0.05). Serum myoglobin concentrations were significantly elevated after exercise in all trials (P
CE
< 0.05), but the magnitude of the exercise-induced response did not differ among the three trials. Serum total ketone body concentrations increased significantly after exercise in all trials (P
S0026-0495(15)00142-0 doi: 10.1016/j.metabol.2015.05.010 YMETA 53213
To appear in:
Metabolism
Received date: Revised date: Accepted date:
12 March 2015 12 May 2015 14 May 2015
Please cite this article as: Tsuchiya Yoshifumi, Ando Daisuke, Takamatsu Kaoru, Goto Kazushige, Resistance exercise induces a greater irisin response than endurance exercise, Metabolism (2015), doi: 10.1016/j.metabol.2015.05.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
T
Resistance exercise induces a greater irisin response than endurance exercise
1
SC
RI P
Yoshifumi Tsuchiya 1, Daisuke Ando 2*, Kaoru Takamatsu 3, Kazushige Goto 1
Graduate School of Sport and Health Science, Ritsumeikan University, 1-1-1, Nojihigashi,
2
MA NU
Kusatsu, Shiga, 525-8577, Japan
Faculty of Education and Human Sciences, University of Yamanashi, 4-4-37, Takeda, Kofu, Yamanashi, 400-8510, Japan University of Tsukuba, 1-1-1, Tennodai, Tsukuba, Ibaraki, 305-8574, Japan
PT
ED
3
CE
Running title: Irisin response to resistance exercise
AC
Conflict of interest: Nothing to declare
*Authors contributed equally to this work.
Corresponding author: Kazushige Goto, Ph. D. Graduate School of Sport and Health Science, Ritsumeikan University 1-1-1, Nojihigashi, Kusatsu, Shiga, 525-8577, Japan E-mail: [email protected] Phone: +81-77-599-4127 Fax: +81-77-599-4127 1
ACCEPTED MANUSCRIPT
Abstract
T
Objective: We determined detailed time-course changes in the irisin response to acute exercise
RI P
using different exercise modes.
SC
Methods: In experiment 1, seven healthy males rested for 12 h (8:00–20:00) to determine the diurnal variation in plasma irisin concentration. In experiment 2, 10 healthy males conducted three
MA NU
exercises to clarify time-course changes in plasma irisin concentration over 6 h, using a randomized crossover design. The resistance exercise (R) trial consisted of eight exercises of 12 repetitions with 3-4 sets at 65% of one repetition maximum (1RM). The endurance exercise (E)
ED
． trial consisted of 60 min of pedaling at 65% of maximal oxygen uptake (VO2max). In the combined
PT
mode (R+E) trial, 30 min of endurance exercise was preceded by 30 min of resistance exercise.
CE
Results: In experiment 1, no significant changes in plasma irisin concentration were observed over 12 h. In experiment 2, the R trial showed a marked increase in plasma irisin concentration 1 h
AC
after exercise (P < 0.05), but not in the E or R+E trials. The area under the curve (AUC) for irisin concentrations for 6 h after exercise was significantly higher in the R trial than in the R+E trial (P < 0.05). The AUC for irisin concentrations was significantly correlated with AUC values for blood glucose, lactate, and serum glycerol (r = 0.37, 0.45, 0.45, respectively. P < 0.05). Conclusions: Resistance exercise resulted in significantly greater irisin responses compared with endurance exercise alone, and resistance and endurance exercises combined. Key words: Irisin, PGC-1α, FNDC5, Myokine, Exercise mode, Time-course change
2
ACCEPTED MANUSCRIPT
1．
Introduction
T
Growing attention has been paid to myokines, which are endocrine metabolic regulators that
RI P
circulate from myocytes into working muscles. Several myokines have been reported, and irisin is
SC
a novel exercise-induced myokine whose level rises in response to expression of peroxisome proliferator receptor γ coactivator-1α (PGC-1α) [1]. Irisin has been proposed to act as a
MA NU
muscle-derived energy expenditure signaling hormone [1]. Importantly, irisin leads to expression of uncoupling protein-1 (UCP1), a thermogenic factor involved in browning of white and beige adipocytes [1, 2]. The increase in irisin augments oxygen consumption, improves glucose
ED
tolerance and insulin sensitivity, and facilitates weight loss compared with those in an untreated
PT
group [1]. Controversies exist among recent publications regarding the importance of irisin and/or
CE
fibronectin type III domain containing 5 (FNDC5) in humans [3-9], and it is necessary to further examine the roles of irisin in metabolic regulation, body composition, and anti-obesity effect.
AC
Several studies have reported that a single bout of endurance exercise promotes irisin secretion, but these studies are not conclusive. Data from human studies has demonstrated that irisin concentrations increase significantly during [10] and immediately [11-14] after endurance exercise. In contrast, Pekkala et al. [4] reported that irisin concentrations did not change following acute endurance exercise in middle-aged men. The irisin response to resistance training has also been studied [6, 7], however, information about the effect of a single bout of resistance exercise on the irisin response is rather limited. Pekkala et al. [4] showed that resistance exercise increased FNDC5 mRNA expression approximately 1.4-fold in muscle compared with the baseline value, 3
ACCEPTED MANUSCRIPT
while the plasma irisin concentration did not change during the 30-min post-exercise period. In
T
contrast, Huh et al. [14] recently found that resistance exercise (lasting 45 min) induced a greater
RI P
irisin response (measured immediately after exercise) than that of endurance exercise (lasting 36
SC
min). Pekkala et al. [4] also determined the long-term effects of combined training (combination of resistance and endurance training) on the irisin baseline concentration. Moreover, two studies
MA NU
recently reported effect of exercise and dietary intervention on irisin in obese children [15, 16]. However, the irisin response was not examined in response to a single bout of exercise in these studies. As such, we are still unaware of which types of exercise, with equivalent durations,
ED
produce the largest effect on irisin secretion. Moreover, previous studies that have determined the
PT
exercise-induced irisin response took blood samples before and relatively early during the
CE
post-exercise period (< 2 h post-exercise). However, considering that PGC-1α (a primary irisin secretion factor) is upregulated 2–3 h after exercise [11, 17-19], irisin concentration may be
AC
maximized at least 2 h following exercise. The purpose of this study was to compare the magnitude of the irisin response following different types of a single bout of exercise under equivalent exercise durations. We hypothesized that irisin concentrations would be higher in resistance exercise than in endurance exercise when measured 2-3 h after each exercise. To test this hypothesis, we monitored detailed time-course changes in the irisin response during a 6-h post-exercise period.
4
ACCEPTED MANUSCRIPT
2.
Material and methods Subjects
T
2. 1.
RI P
Two experiments were conducted. In experiment 1 (resting experiment), seven physically
SC
active males [mean ± standard error (SE): age; 25 ± 1 yr, height; 171 ± 6 cm, weight; 69 ± 8 kg] were evaluated for diurnal variations in irisin concentration. In experiment 2 (exercise experiment),
MA NU
10 physically active males (mean ± SE: age; 23 ± 1 yr, height; 172 ± 2 cm, weight; 70 ± 2 kg) were evaluated to clarify the effects of a single bout of exercise on time-course changes in irisin concentration. Subjects in both experiments had several years of experience undergoing strenuous
ED
exercise training and had maintained these exercise habits (at least once a week) upon study
PT
initiation. Exclusion criteria for both experiments were coronary heart disease, obesity, metabolic
CE
syndrome, and addictions to prescribed drug or tobacco use. Each subject was informed of the purpose of the study, experimental procedures, and the possible risks involved, and written
AC
informed consent was obtained. All experimental procedures were approved by the Ethical Committee for Human Experiments at Ritsumeikan University in accordance with the Declaration of Helsinki.
2. 2.
Experiment 1 (resting experiment)
2. 2. 1.
Experimental design and measurement procedures
Experiment 1 was designed to determine the diurnal variation in plasma irisin concentration over 12 h. Seven subjects visited the laboratory following an overnight fast. They rested in the 5
ACCEPTED MANUSCRIPT
laboratory for 12 h (8:00–20:00). Control meals (breakfast: 683 kcal, carbohydrate; 62%, protein;
T
17%, fat; 21%, lunch: 729 kcal, carbohydrate; 65%, protein; 16%, fat; 19%) were provided at the
RI P
same time of day to avoid the influence of diet among subjects. The subjects were instructed to
SC
refrain from consuming caffeine and alcohol for 12 h and to perform no physical activity for 24 h before the experiment.
MA NU
Blood samples were collected from an antecubital vein five times (8:00, 11:00, 14:00, 17:00, and 20:00) during the 12 h of rest. The blood samples were transferred to a tube containing EDTA-Na, and the plasma was obtained after a 10 min centrifugation (3,000 rpm, 4°C) and stored
ED
at −80°C until analyses. Plasma irisin concentrations were determined in duplicate using a
PT
commercially available enzyme-linked immunosorbent assay (ELISA) kit (EK-067-52, Phoenix
CE
Pharmaceuticals, Inc., Darmstadt, Germany), and all blood samples were analyzed within the same plate. Absorbance was measured by wavelength at 450nm [20]. The intra-assay coefficient
AC
of variation (CV) was 1.7%.
2. 3.
Experiment 2 (exercise experiment)
2. 3. 1.
Experimental design
The subjects visited the laboratory five times throughout the experimental period. One ． repetition maximum (1RM) and maximal oxygen uptake (VO2max) were determined [21] on the first and second visits. Different types of acute exercise were conducted on the third to fifth visits. The 1RM values for eight exercises (chest press, lat-pull down, leg press, knee extension, seated 6
ACCEPTED MANUSCRIPT
rowing, shoulder press, arm curl, and triceps press down) were measured using a weight-stack
T
machine (Life Fitness Co., Tokyo, Japan). The subjects stretched for several min before the 1RM
RI P
measurements. Load was increased progressively until the subject was unable to complete a successful lift to determine the 1RM. An incremental pedaling test was performed on the second
SC
． visit to determine VO2max (828E, Monark, Uppsala, Sweden). After a 5 min warm-up at 30 W, the
MA NU
test began at 60 W, and the load was increased progressively by 30 W every 2 min until exhaustion. The test was terminated when the subject failed to maintain a pedaling frequency of ． 60 rpm or reached a VO2 plateau. Respiratory gases were collected and analyzed using an
ED
． automatic gas analyzer (AE300S, Minato Medical Science Co., Tokyo, Japan) to determine VO2,
PT
． ． carbon dioxide output (VCO2), minute ventilation (VE), and the respiratory exchange ratio (RER).
CE
These data were averaged every 30 s. All subjects conducted three different exercise trials with a randomized crossover design on
AC
the third to fifth visits. The trials consisted of resistance exercise (R), endurance exercise (E), and a combined mode of resistance and endurance exercise (R+E). Each trial was conducted during the morning following an overnight fast, and separated by at least 1 week.
2. 3. 2.
Exercise trial protocols
Subjects in the R trial conducted 60 min of resistance exercise (eight exercises: chest press, lat-pull down, leg press, knee extension, seated rowing, shoulder press, arm curl, and triceps press down). Each exercise set consisted of 12 repetitions, with four sets for chest press and lat-pull 7
ACCEPTED MANUSCRIPT
down, and three sets for the remaining six exercises at 65% of 1RM. The subjects rested for 2 min
T
． between sets and exercises. The E trial consisted of 60 min of pedaling at 65% of VO2max.
RI P
Pedaling frequency was set at 60 rpm. Subjects in the R+E trial initially conducted 30 min of four resistance exercises (chest press, lat-pull down, leg press, and shoulder press). Then, 30 min of
SC
． endurance exercise at 65% of VO2max was subsequently performed after a 20 min rest. Therefore,
2. 3. 3.
MA NU
the total exercise duration (60 min) was the same among the three trials.
Blood sampling and analysis
ED
Blood samples were collected before exercise (Pre), immediately after exercise (0 h), and at
PT
0.5, 1, 2, 3, 4, and 6 h after exercise (Figure 1). After drawing blood, serum and plasma samples
CE
were obtained after 10 min of centrifugation (3,000 rpm, 4°C) and stored at −80°C until analyses. Serum insulin, triglycerides (TG), free fatty acids (FFA), glycerol, myoglobin, creatine kinase
AC
(CK), total ketone bodies, and plasma irisin concentrations were measured. Serum insulin concentrations were measured by a chemiluminescent enzyme immunoassay at a clinical laboratory (SRL, Inc., Tokyo, Japan). Serum FFA concentrations were measured using a commercially available enzymatic colorimetric kit (NEFA-HRII, Wako Pure Chemical Industries, Osaka, Japan). Serum glycerol concentrations were determined in duplicate with a commercially available kit (Cayman Chemical Company, Ann Arbor, MI, USA). Serum TG, myoglobin, CK, and total ketone body concentrations were measured at a clinical laboratory (SRL). Plasma irisin concentrations were determined in duplicate using a commercially available ELISA kit 8
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(EK-067-52, Phoenix Pharmaceuticals, Inc.). All blood samples for each subject were analyzed
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within the same plate. The intra-assay CV of each measurement was as follows: 4.0%, 3.0%, 1.4%,
Respiratory measurements
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2. 3. 4.
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ketone bodies, and plasma irisin concentrations, respectively.
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3.9%, 8.2%, 4.0%, 2.6% and 3.5% for serum insulin, TG, FFA, glycerol, myoglobin, CK, total
Five min of resting respiratory gases were collected using an automatic gas analyzer ． ． ． (AE300S, MINATO Medical Science, Osaka, Japan) to determine VO2, VCO2, and VE before
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exercise, and at 0.5, 1, 2, 3, 4, 6, and 24 h after exercise (Figure 1). The RER was calculated from
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． ． ． ． VO2 and VCO2. Energy expenditure after each exercise was determined from VO2 and VCO2
Statistical analysis
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2. 4.
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[22].
All experimental data are shown as mean ± standard error. The time-course changes in plasma irisin concentrations in experiment 1 were compared using one-way repeated-measures analysis of variance (ANOVA). Comparisons of the time-course changes in blood parameters and respiratory variables among the three different trials in experiment 2 were made with a two-way repeated-measures ANOVA to identify the interaction (trial × time) and main effect for trial and time. When ANOVA revealed a significant interaction or main effect, a post-hoc analysis (Tukey–Kramer method) was performed to assess differences among trials or times. The area 9
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under the curve (AUC) values were compared using one-way repeated-measures ANOVA. The
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correlation between the magnitude of the exercise-induced plasma irisin response and those in
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regression analysis. A p < 0.05 was considered significant.
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other blood parameters was determined by Pearson’s correlation coefficient after multiple linear
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3.
Results Experiment 1 (resting experiment)
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3. 1.
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Figure 2 shows the time-course changes in plasma irisin concentrations over 12 h. The
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plasma concentrations remained stable, and no significant differences were observed among the
3. 2.
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measurement points for 12 h.
Experiment 2 (exercise experiment)
3. 2. 1.
Blood parameters
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Figure 3 shows the time-course changes in plasma irisin concentrations before and after
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exercise during each trial. No significant difference in plasma irisin concentration at baseline (Pre)
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was observed among trials. The E and R+E trials did not show significant changes in plasma irisin concentrations during the 6 h after the exercises. In contrast, the R trial showed a significant
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increase in plasma irisin concentration 1 h after exercise (P < 0.05). Furthermore, the plasma irisin concentration 1 h after exercise was significantly higher in the R trial than that in the E and R+E trials (P < 0.05). The AUC value was significantly higher in the R trial (151559 ± 22487 ng/mL･6 h) than that in the R+E trial (123990 ± 18700 ng/mL･6 h, P < 0.05, Figure 4) when exercise-induced irisin responses during the 6-h period after exercise were compared. Table 1 shows the time-course changes in blood variables for each trial. The E trial resulted in significantly lower blood glucose concentration at baseline than those in the R+E trial. Blood glucose concentrations were significantly higher in the R trial than in the E and R+E trials 11
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immediately after exercise (P < 0.05). Blood lactate concentrations at baseline were significantly
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higher in the E trial than in the R and R+E trials. Although blood lactate concentrations increased
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significantly after exercise in all trials, the R trial resulted in a significantly greater response than
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that observed in the E and R+E trials (P < 0.05). Consequently, the AUC values 6 h after exercise were significantly higher in the R trial than in the E and R+E trials (P < 0.05). Serum FFA
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concentrations at baseline were significantly higher in the E trial than in the R and R+E trials (P < 0.05). The E trial showed significantly higher serum FFA concentrations compared with those in the R and R+E trials immediately after and at 0.5 h after exercise (P < 0.05). The AUC values
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during the 6 h after exercise were significantly lower in the R trial than in the E and R+E trials (P
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< 0.05). Serum myoglobin concentrations were significantly elevated after exercise in all trials (P
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< 0.05), but the magnitude of the exercise-induced response did not differ among the three trials. Serum total ketone body concentrations increased significantly after exercise in all trials (P
