Nutrition
71
Melatonin is an Ergogenic Aid for Exhaustive Aerobic
Exercise only during the Wakefulness Period
Authors
W. R. Beck1, 2, P. P. M. Scariot2, C. A. Gobatto1, 2
Affiliations
1
Key words
▶melatonin
●
▶ exercise performance
●
▶ ergogenic aid
●
▶ time of day
●
▶ aerobic exercise
●
Abstract
Physical Education, University of Campinas, Campinas, Brazil
Laboratory of Applied Sport Physiology, School of Applied Sciences, University of Campinas, Limeira, Brazil
▼
This study tested the ergogenic effects of acute
administration of melatonin on exhaustive exercise (tlim) at the anaerobic threshold intensity
(iAnT) during periods of lower (L) and higher (H)
spontaneous physical activity in swimming rats.
Additionally, we evaluated the time of day effect
on aerobic exercise tolerance. The periods of L
and H were determined gravimetrically. All animals were subjected to an incremental test to
determine the iAnT. Melatonin was administered
(10 mg.kg − 1, intraperitoneal) and after 30 min,
Introduction
▼
accepted after revision June 30, 2015
Bibliography
DOI http://dx.doi.org/
10.1055/s-0035-1559698
Published online:
October 28, 2015
Int J Sports Med 2016; 37:
71–76 © Georg Thieme
Verlag KG Stuttgart · New York
ISSN 0172-4622
Correspondence
Claudio Alexandre Gobatto
Laboratory of Applied Sport
Physiology
School of Applied Sciences
University of Campinas
Pedro Zaccaria Street, 1.300,
Jardim Santa Luíza
13484 350 – Limeira
São Paulo
Tel.: + 55/19/3701 6669
Fax: + 55/19/3701 6680
[email protected]
Secretion of melatonin, an indoleamine pineal
hormone, is inhibited by environmental light
exposure [22, 33]. As a result, blood melatonin
concentrations are higher at night than during
daylight in entrained mammals [17, 43]. Because
of its daily cycles, melatonin is classically associated with the synchronization of the circadian
rhythms and synchronization of many physiological functions [4, 16, 29]. Similar to the cycles of
melatonin [17], the spontaneous physical activity (SA) of nocturnal rats is high at night (wakefulness) and low during the day (resting/sleep)
[25, 44]. The core body temperature follows a
similar cycle [25, 40].
Anti-inflammatory [13] and antioxidant [35]
properties are also attributed to melatonin. The
intracellular activities of melatonin include
inhibiting lipid peroxidation [12], increasing the
levels of mitochondrial electron transport chain
and Krebs cycle enzymes and maintaining liver
and brain microsomal membrane stability [1]. As
a result, melatonin is thought to have therapeutic
potential for diseases such as insulin resistance
[42] and obesity [16]. Melatonin also has proapoptotic activity in cancer cells [14].
the rats were subjected to tlim during the L (LM)
or H (HM) period. Control groups were called LC
and HC. The criterion of significance was 5 %.
Melatonin enhanced tlim by 169 % during H
(HC = 72 min; HM = 194 min; P < 0.01; ES = 1.23)
and by 90 % during L (LC = 31 min vs. LM = 59 min;
P = 0.39; ES = 1.18), demonstrating a significant
effect on tlim (F = 10.35; P < 0.01) and a strong
effect size (ES). Additionally, tlim was higher during H (F = 14.24; P < 0.01). Melatonin is a reasonable ergogenic aid, particularly during the
wakefulness period, and the exercise tolerance is
dependent on the time of day for swimming rats.
In addition to the effects of melatonin on circadian rhythms, including reported improvements
in exercise tolerance (i. e., core body temperature
and spontaneous activity), some of the functions
of melatonin may have direct and positive effects
on performance. Exercise-induced inflammation
[13] and oxidative stress [35] are inhibited when
melatonin is administered in both humans [30]
and rats [2]. Melatonin also preserves glycogen
content, stimulating fatty acid oxidation during
exercise [32] and protecting muscle from exercise-induced damage [23]. Nevertheless, the
direct role of melatonin as an ergogenic aid for
exercise performances is not clear in humans
[4, 21, 45] and this topic has not been directly
investigated in rats, despite the fact that this
rodent model allows the researchers to control
all environmental parameters, nutrition, drug
administration and exercise conditions. Furthermore, it is not clear whether melatonin improves
the swimming performance of nocturnal rats
across the circadian periods. This is a critical
issue because previous studies have demonstrated higher swimming performance during
the natural wakefulness period for swimming
nocturnal rats [7]. Therefore, the use of exogenous melatonin as an ergogenic aid to physical
Beck WR et al. Melatonin is an Ergogenic … Int J Sports Med 2016; 37: 71–76
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2
exercise remains unclear, particularly when it is administered at
different times during the day and based on objective exercise
parameters. The aim of this study was to investigate the ergogenic effects of the acute administration of melatonin on exhaustive exercise (tlim) at the anaerobic threshold intensity (iAnT)
during periods of lower and higher spontaneous physical activity in swimming rats. Additionally, we aimed to confirm reports
in the literature suggesting that the time of day has a significant
effect on aerobic exercise tolerance.
Methods
▼
Animals
60 male Wistar rats were obtained from an institutional facility
and maintained under a 12 h light/dark cycle, with the lights
switched on at 06:00 h (zeitgeber time 0). We used a 100 W lamp
during the light period (Phillips® soft white light; 2 700 K; 565–
590 nm; 60 lux). The rats were housed in polyethylene cages
with free access to water and rodent chow. The relative humidity
was kept at 45–55 %, temperature at 22 ± 2 °C and noise levels
below 85 decibels. The study was approved by the institutional
Ethics Committee on the Use of Animals under process 2502–1
and followed the ethical standards of the International Journal of
Sports Medicine as described in Harriss and Atkinson [24]. All
procedures performed were in accordance with the ethical
standards of the institution or practice at which the studies were
conducted.
Experimental design
At 45 days of age, rats were evaluated for spontaneous physical
activity (SA) using gravimetry [11]. The means of the hourly
activity during a 24-h period were used to identify the periods
of lowest and highest SA and to randomly divided the 60 rats
into 2 groups: rats to be assessed at the higher (H, n = 30); and
rats to be assessed at the lower (L, n = 30) SA. All of the subsequent procedures were performed at the time of day corresponding to the specific group. Special environmental
Experimental groups
45 days of age
The signal acquisition system consisted of a primary sensor element (load cell – PLA30Kg, Líder Balanças®, Araçatuba. São
Paulo, Brazil) involving in 2 hard iron platforms located under
the animals’ cage. Due to the sensitive nature of the apparatus,
minimal movements were noted. After amplification (MKTC5–
10®, MK control and instrumentation™, São Paulo, São Paulo,
Brazil) and processing (USB-6008® signal-conditioning module,
National Instruments, Austin, Texas, USA), the collected data
were transmitted to a software program (LabView Signal
Express® 2009, National Instruments™, Austin, Texas, USA) and
Spontaneous physical
activity measurement
L (n = 30)
76 days of age
Water environment and
swimming adaptation
89 days of age
90 days of age
Gravimetric apparatus
Procedure
60 rats
H (n= 30)
illumination was employed for animals assessed during the dark
period ( > 600 nm; < 15 lux), as described elsewhere [8].
From 76 to 89 days of age, all the rats were subjected to a water
environment and swimming adaptation to avoid stress due to
novelty during the exercise tests. The individual swimming
ergometer consisted of a cylindrical PVC tank with a diameter of
30 cm filled to a depth of 100 cm with clean water at 31 ± 1 °C. At
90 days of age, all the animals were subjected to an incremental
test (IT) to determine the anaerobic threshold intensity (iAnT),
according to the time of day of each group. At 92 days of age,
animals that received melatonin 30 min prior to exhaustive
exercise at iAnT (tlim) during L were designated as the LM
(n = 15) group; animals that received melatonin 30 min prior to
exhaustive exercise at iAnT (tlim) during H were designated as
the HM (n = 15) group. Control groups for the effect of melatonin
were included for both times of day (LC, n = 15; HC, n = 15).
Melatonin (SIGMA-ALDRICH, Co., St. Louis, MO, USA; Reference
M5250, ≥ 98 %, Lot # SLBC7539V) was dissolved in ethanol
( < 0.1 %) and diluted in saline solution (NaCl 0.9 %) to be administered via intraperitoneal at 10 mg.kg − 1, corresponding to a
non-physiological dosage. The control animals received an equal
volume of vehicle alone.
Blood samples were collected before and after tlim to determine
blood lactate concentration levels. The time of exhaustion at
iAnT (tlim) was recorded as the performance parameter. The
experimental design is shown in ●
▶ Fig. 1.
H (n= 30)
L (n = 30)
92 days of age
Incremental test (IT)
Melatonin or vehicle
administration
HC (n = 15) HM (n = 15) LC (n =15)
LM (n =15)
30min
Exhaustive exercise (tlim)
Beck WR et al. Melatonin is an Ergogenic … Int J Sports Med 2016; 37: 71–76
Fig. 1 Experimental design of the study.
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72 Nutrition
Nutrition
73
Fig. 2 Illustration of the apparatus and data
acquisition system for ­measurement of spontaneous physical activity.
USB-6008
1
2
3
SPAN
7
5
MKTC5-10
MK
TC-05
ZERO
6
4
8
9
10
Rats cage
Iron platform
load cell
Exposing animals to proportional incremental loads over time
leads to disproportional increases in blood lactate levels at a
given moment [48]. The IT employed in this experiment consisted of 5-min stages with overloads corresponding to 3, 3.5, 4,
4.5, 5, 5.5, 6 and 6.5 % of the body mass ( % bm), and tail vein
blood samples were collected after each stage from the rat’s distal part of the tail to determine the lactate levels. The lead overloads were inserted in the chest of the rats using an elastic strap.
The blood lactate concentration relative to the exercise intensity
was graphically plotted. Using these data, the alteration of the
proportional blood lactate concentration increases was defined
by visual inspection by 2 experienced researchers, as described
by Matsumoto, Araki, Tsuda, Odajima, Nishima, Higaki, Tanaka,
Tanaka and Shindo [31]. Next, 2 linear regressions were constructed following the described break point, defined as the
intersection of the linear regressions interpolated to the X-axis
[48]. The intensity corresponding to this interpolation was
▶ Fig. 3). This procedure was recently tested
defined as the iAnT ( ●
to determine its validity and reliability [6], eliciting individual
assessment of the swimming intensity for rats.
Analytical procedures for biological specimens
Blood samples (25 µL) were collected using micro heparinized
glass capillaries, and immediately transferred to 1.5 mL plastic
tubes containing 400 µL of trichloroacetic acid [4 %]. The samples
were shaken and centrifuged to use the supernatant, which was
added to a reactive solution (Glycine/EDTA; Hydrazine Hydrate;
Beta-nicotinamide dinucleotide; L-Lactic dehydrogenase bovine
heart; adjusted to a pH of 8.85). After an adequate period of
incubation with controlled temperature, the blood lactate concentrations were determined using the enzymatic method [19],
being the processed samples analyzed by spectrophotometry at
340 nm and compared to a calibration curve.
8
y=2.7208x–9.1479
R2 =0.9791
7
6
5
y=0.5696x + 1.7195
R2 =0.8268
iAnT
4
3
2
3.0
3.5
4.0
4.5
5.0
5.5
Percentage of body mass (% bm)
6.0
6.5
Fig. 3 Incremental swimming test for rats. The intercept of the 2 regression lines extrapolated to the X-axis represents the anaerobic threshold
intensity (iAnT). This figure represents Rat 3 in the experiment, where the
iAnT was 5.05 % bm and the lactate level at iAnT was 4.59 mM.
Statistical methods
The results are presented as the mean ± standard deviation (SD).
The Kolmogorov-Smirnov and Liliefors tests confirmed the normality of all the variables; we therefore used parametrical statistics. The SA data were expressed as the 60-min means over
24 h to determine the moments of higher and lower spontaneous activity. Percentage analysis and t-tests for dependent samples were conducted to analyze the differences in SA between a
period of total daylight and a period of total darkness. The t-test
for independent samples was used to analyze the differences
between the iAnT and blood lactate levels with the iAnT determined during the H and L periods. 2-way analysis of variance
was used for the tlim variables (tlim and blood lactate levels
before and after tlim) to test for a i) the main effect of the drug
[2, melatonin and placebo] and ii) the main effect of SA [2, higher
and lower] and a possible interaction. It was also calculated and
included the effect size (ES) for the studied variables. The Newmann-Keuls post hoc was used when appropriated. The criterion for significance was P < 0.05. All statistical procedures were
performed using MatLab® 7.0 (MathWorks™).
Beck WR et al. Melatonin is an Ergogenic … Int J Sports Med 2016; 37: 71–76
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Incremental swimming test
9
Lactate concentration (mM)
continuously recorded at 30 Hz over 24 h. The apparatus was
previously calibrated using 14 known loads from 17.12 to
6,395.98 g. This procedure provided a linear relationship regression (R2 = 0.99), employing a unit conversion from millivolts
(mv) to kilograms (kg). Next, the digital signals were treated as
described elsewhere [11], using MatLab® 7.0 (MathWorks™).
The SA scores were adjusted by the rat’s body mass (kg.g − 1) and
statistically processed. The apparatus is illustrated in ●
▶ Fig. 2.
74 Nutrition
Discussion
▼
The main finding of the present study was the significant performance-enhancing effect of melatonin on the tested exercise. The
time to exhaustion at the anaerobic threshold intensity was
higher in animals that received melatonin than in control animals, which is a novel result. However, it is important and interesting to note that the melatonin was significantly effective only
when it was administered during the period of higher spontaneous activity (H). Additionally, performances were significantly
Beck WR et al. Melatonin is an Ergogenic … Int J Sports Med 2016; 37: 71–76
Spontaneous physical activity
0.70
0.60
Light period
Dark period
0.50
0.40
0.30
0.20
0.10
0.00
6h
7h
8h
9
10 h
11 h
12 h
13 h
14 h
15 h
16 h
17 h
18 h
19 h
20 h
21 h
22 h
23 h
24 h
h
1h
2h
3h
4h
5h
Fig. 4 Spontaneous physical activity results recorded continuously for 24 h.
Aerobic endurance
300
*
250
200
150
100
50
0
LC
LM
HC
HM
Fig. 5 Descriptive data of the time to exhaustion at the anaerobic
threshold intensity of groups that were assessed during periods corresponding to lower (L) and higher (H) levels of spontaneous physical
activity and that received melatonin (M) or vehicle (C), expressed as the
means and standard deviation. * P < 0.05 in relation to all other groups.
The effect size for HM vs. HC was 1.23 and for HM vs. LM 1.18.
better during H compared with L, which corroborates a previous
study [7].
Spontaneous physical activity
The SA data were used to objectively determine the periods of
higher and lower activity throughout the day, as initially proposed. We confirmed the nocturnal activity of albino Wistar rats
that has been reported in the literature [37], as 75.24 % of the
total daily spontaneous physical activity occurred during the
dark period, with a peak at 20:00 h (zeitgeber time 14). Our data
are consistent with those reported by Ikeda, Sagara and Inoue
[25], who showed that approximately 70 % of daily activity
occurs during the dark period.
The SA was recorded using a device that was undetectable to the
rats and under identical the same environmental conditions,
thus avoiding potential influence from handling during the SA
assessments. These measurements estimate the natural resting
(or sleep period) and wakefulness periods at times when physiological [25, 28, 29, 40] and behavioral [15, 27] modifications are
systematically observed under laboratory conditions in
entrained animals.
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The spontaneous activity during periods of total daylight was
significantly lower when compared to that during periods of
total dark (P < 0.01; ES = 0.87). This result is not surprising, given
that 75.24 % of the rat’s entire daily activity is performed at
night. The lowest SA values were observed at 12:00 h (mean values of activity from zeitgeber time 6–7), and the highest values
were observed at 20:00 h (zeitgeber time 14–15), as illustrated
in ●
▶ Fig. 4. These results were used to determine the time of day
to begin all of the assessments for the L and H groups, respectively.
The iAnT values were 4.86 ± 0.43 % bm for the LC group,
4.74 ± 0.35 % bm for the LM group, 5.49 ± 0.41 % bm for the HC
group and 5.19 ± 0.43 % bm for the HM group, with significant
differences between the groups. The iAnT of the HC group was
higher than that of the LC group (P < 0.01; ES = 1.48), and the iAnT
of the HM group was higher than that of the LM group (P < 0.01;
ES = 1.16). The blood lactate levels at iAnT were 4.60 ± 1.22 mM
for the LC group, 3.76 ± 1.09 mM for the LM group, 3.57 ± 1.41 mM
for the HC group and 4.34 ± 1.29 mM for the HM group, resulting
in no significant differences among the groups (P > 0.05). Melatonin had no significant effect (F = 3.58; P = 0.07) on iAnT; however, the time of day significantly influenced iAnT (F = 23.77;
P < 0.01), and there was no interaction between effects (F = 0.61;
P = 0.44). Neither the time of day (F = 0.43; P = 0.51) nor melatonin (F = 0.01; P = 0.90) influenced the blood lactate concentration at iAnT.
Descriptive results from exhaustive exercise at anaerobic threshold intensity (tlim) are shown in ●
▶ Fig. 5. In summary, the tlim of
the HM group was 169 % higher than the HC group (P < 0.01), and
the tlim for the LM group was 90 % higher than the LC group
(P = 0.39). For tlim, the effects of melatonin were significant
(F = 10.35; P < 0.01; Melatonin > Vehicle), as were the effects of
the time of day (F = 14.24; P < 0.01; higher spontaneous activity > lower spontaneous activity), resulting in a significant interaction between the effects (F = 4.05; P = 0.04).
The blood lactate concentrations collected at rest just before
tlim, were 0.92 ± 0.06 mM in the LC group, 1.29 ± 0.08 mM in the
LM group, 1.47 ± 0.03 mM in the HC group and 2.02 ± 0.18 mM in
the HM group. The administration of melatonin (F = 17.31;
P < 0.01) and assessment during higher spontaneous activity
(F = 33,28; p < 0.01) were associated with significantly increased
blood lactate concentration, with no interaction between the
effects (F = 0.72; P = 0.39). The blood lactate concentrations after
exercise were 7.01 ± 0.48 mM for the LC group, 6.75 ± 0.31 mM for
the LM group, 6.93 ± 0.46 mM for the HC group and 6.28 ± 0.60 mM
for the HM group. There were no significant effects of melatonin
(F = 0.80; P = 0.37) or time of day (F = 0.29; P = 0.59) and there was
no interaction between the effects (F = 0.14; P = 0.70).
Time to exhaustion (min)
▼
Spontaneous physical activity (Kg .g –1)
Results
Swimming exhaustive bouts at iAnT (tlim): the SA
effect
The effects of circadian rhythm on athletic performance are currently under investigation, and there is considerable controversy
related to the variability of environmental factors and differences among the types of exercise [18, 20, 36, 38, 47]. According
to Drust, Waterhouse, Atkinson, Edwards and Reilly [18], both
field and laboratory studies are necessary to appropriately
understand the effects of biological rhythms on exercise, and the
control of environmental factors is crucial for study validity. Our
experiment showed that the aerobic exercise tolerance was
improved when the rats were evaluated during the period of
high spontaneous physical activity. Other experiments compared exercise during both circadian periods and systematically
found that rats performed better at night [5, 7]. In addition to the
effect of the time of day, it is important to note that the environmental light employed for the exercise testing during the dark
period (higher spontaneous physical activity, wakefulness
period of nocturnal rats) can also influence the performance [8].
Because the focus of this experiment was to investigate the
effects of melatonin on performance, we will briefly refer the
reader to published work discussing the time-of-day effect on
tlim [7].
Swimming exhaustive bouts at iAnT (tlim): the
melatonin effect
According to the data shown in ●
▶ Fig. 5, the animals that received
melatonin and were evaluated during the wakefulness period
(HM) presented the highest exercise tolerance (P < 0.01). The
time to exhaustion of HM was 169 % higher than that of the HC
group (P < 0.01). The tlim of the LM group, although not statistically significant, was 90 % higher than that of the LC group
(P = 0.39). These results demonstrate that the ergogenic effect of
melatonin was dependent on the time of day for swimming rats
subjected to the proposed exercise and testing periods described
in this study. This interesting result suggests that there is an
additive effect of SA and melatonin on performance. Additionally, the importance of melatonin and the time of day effect on
such result was confirmed by the interaction between the effects
(F = 4.05; P = 0.04).
Although some studies have been performed in humans, the literature on the effects of melatonin in exercised rats is scarce,
and no experiments have directly investigated melatonin as an
ergogenic intervention in these animals. Mazepa, Cuevas, Collado and Gonzalez-Gallego [32] elegantly analyzed the metabolic effects of melatonin on energy substrates and postulated
that melatonin preserves glycogen by increasing the use of lipids
as energy substrates during exercise. However, the exercise
parameters were not individualized, and the exercise outcomes
were not shown. Other studies have investigated the effects of
melatonin on energy metabolism in rats during exercise using
different assays [10, 26, 34]. However, no studies have directly
investigated the ergogenic effects of melatonin, and several
studies failed to control the exercise intensity or subjected the
animals to swimming trials individually. According to the American Physiological Society, these are important limitations in
studies measuring exercise swimming in rats [3].
Regarding the drug concentration, other studies have used melatonin dosages of 10 mg.kg − 1 for rats [9, 41, 49] and intraperitoneal administration is appropriate because of the rapid
bioavailability [46]. There are many possible mechanisms that
could explain the ergogenic effects of melatonin [21, 39, 45]. This
hormone acts by reducing exercise-induced inflammation and
oxidative stress [2, 30], and alters energy availability during
exercise [10, 26, 32]. Therefore, it is plausible to suggest that
melatonin will have the most robust ergogenic effects in situations where inflammation, oxidative stress and a lack of available energy are important limitations. Due to the circadian
cycling of physiological, cognitive and behavioral parameters, it
also seems also plausible that exercise tolerance might be
improved during the normal wakefulness period, compared to
the resting or sleep period, for both humans and rats. Nevertheless, from the viewpoint of comparative physiology, the level of
endogenous melatonin is typically increased during wakefulness
for entrained nocturnal rats, but this is not the case for humans.
It is possible that the combination of endogenous and exogenous
melatonin is required for the ergogenic effect observed in our
experiment. These effects may differ for humans, who frequently possess low endogenous melatonin concentrations during the wakefulness period.
It is important to note that despite our methodological attempts
to objectively determine the SA, drug administration and exercise parameters, our study has limitations. Because this is the
first study to show the ergogenic effects of melatonin on exercise
tolerance in rats, new experimental designs are needed to confirm the consistency of this phenomenon and to understand the
role of melatonin in acute exercise tolerance (i. e., anti-inflammatory effects, antioxidant effects, modulation of energy substrates, alertness, core body core temperature and circadian
rhythms). Such studies should use different dosages of melatonin and exercise intensities/durations.
Conclusion
▼
Finally, our results strongly suggested that melatonin is a reasonable ergogenic aid for swimming rats, particularly during the
wakefulness period. This work also supports the assertion that
exercise tolerance is dependent on time of day for swimming
rats assessed under individualized exercise intensity regiments
with strict control of the laboratory’s environmental conditions.
Acknowledgements
▼
This work was supported by the [FAPESP – Fundação de Amparo
à Pesquisa do Estado de São Paulo] under grant [number
2009/08535-5, 2010/13377-7, 2011/13226-1 and 2012/205011]; and [CNPQ – Conselho Nacional de Desenvolvimento Científico e Tecnológico] under grant [number 305650/2009-2 and
484174/2011-8].
Conflict of interest: The authors have no conflict of interest to
declare.
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