It has been well documented that light–dark (LD) cycles can synchronize the expression of circadian clock genes in the suprachiasmatic nucleus of the hypothalamus (SCN) through direct innervation from the retina via the retinohypothalamic tract (15,16). Rodents in which the SCN is ablated lose the ability to synchronize to the LD cycle, and rhythms in locomotor behavior and physiology such as plasma glucagon levels are lost (5,26). These daily endogenous oscillations are thought to be driven by molecular clockwork within the organism (25). The circadian clock in mammals consists of several core components that work together through transcriptional and translational feedback loops and form the molecular basis for circadian rhythms in physiology (25). The core components of the molecular clockwork consist of positive regulators of transcription, i.e., Bmal1 and Clock, and negative components Cry1/2 and Per1/2, whose protein products associate with the BMAL1:CLOCK heterodimer in the nucleus and repress the activity of the transcription factor complex (4,28). These circadian clock genes are expressed within most mammalian cells, are cell autonomous, and continue to oscillate outside of the organism (36). Like any other timepiece, the molecular clockwork can be “set” to a specific time through external manipulation, e.g., environmental time cues.
The most well-characterized time cue or zeitgeber is light. Through manipulation of the LD cycle, molecular and behavioral rhythms can be set experimentally or naturally through seasonal rotation around the sun (25). In addition to light, restricting a rodent’s food availability to a particular time of day has been shown to alter locomotor behavior and shift gene expression of molecular clockwork components in a tissue-specific manner (10,14). Restricted feeding (RF) alters circadian rhythms in both SCN-intact and SCN-ablated mammals suggesting this nonphotic time cue acts independently of the SCN (9). The synchronizing effects of food create robust locomotor responses in the hours preceding food presentation (31). This heightened activity is termed “food-anticipatory activity” (FAA) and is characterized as an intense increase in locomotor behavior approximately 2–4 h before the food is presented (10). RF also affects the expression pattern of molecular clockwork components (8). The effect of RF on circadian clocks seems to be tissue specific, e.g., the molecular clockwork in the liver exhibits altered gene expression in response to RF, whereas there is no change in the SCN (10,31). The ability of restricting feeding to alter animal behavior and molecular and physiological rhythms has lead researchers to conclude that there may exist a network of pacemakers with sensitivity to different environmental stimuli (30).
Scheduled exercise on a treadmill or running wheel has been shown to shift behavioral rhythms in mammals kept under constant darkness as well as entrain the free running rhythm (endogenous rhythm) of an animal placed in constant darkness (11,21). Studies have also demonstrated that scheduled exercise in a novel running wheel can affect how quickly an animal entrains or synchronizes to a new LD cycle. In 2008, Yamanaka et al. (33) demonstrated that scheduled bouts of wheel running could “accelerate reentrainment” of locomotor activity and molecular rhythms in peripheral tissues to 8-h shifts in the LD cycle. The LD cycle was phase advanced by 8 h (one time), and a novel wheel was given at the beginning of the shift. After 4 d in the new lighting schedule, the mice with a running wheel shifted more quickly than the controls. Using Per1-luciferase mice, Yamanaka et al. (33) measured bioluminescence from tissues cultured from the exercised and control mice. Skeletal muscle and lung explants from exercised mice had significantly phase shifted Per1-luciferase bioluminescence rhythms, which is a measure of circadian clock function. This suggests that the molecular clockwork in the skeletal muscle and lung may be sensitive to scheduled exercise as a time cue (33).
In the current study, we wanted to test if scheduled exercise alone, independent of changing the LD cycle, could phase shift locomotor and molecular rhythms to an earlier time of day. Period2::Luciferase (Per2::Luc) mice (36) housed in a 12-h-of-light:12-h-of-dark (12L:12D) light schedule were exposed to scheduled bouts of either voluntary or involuntary exercise for 2 h·d−1 between zeitgeber time (ZT) 4 and 6 for 4 wk. We selected this zeitgeber time (ZT 4–6 h after lights on) on the basis of a previous work with rodents where novel wheel running produced the most significant phase shifts in locomotor activity during the middle of the day (lights on) (24). In a second experiment, we subjected mice to 4 h of RF for 2 wk. The consequences of RF on behavioral and molecular rhythms have been previously demonstrated (10,31); therefore, we decided to compare the effects of exercise with another well-established nonphotic time cue. The primary finding in this study was that there was a significant shift (phase advance) in clock gene expression (PER2::LUC bioluminescence) in three different skeletal muscles and the lung from exercised mice, whereas the PER2::LUC rhythm in the SCN remained unshifted. This study demonstrates that scheduled exercise can alter the molecular clockwork in peripheral tissues of an SCN-intact animal and provides more direct evidence that exercise can act as a nonphotic time cue.
MATERIALS AND METHODS
Twenty-three male and 10 female Per2::Luc mice on C57BL/6 background (36) approximately 6 months old were housed individually in plastic cages measuring 12 × 6 × 5 inches without running wheels. All mice were housed in a light-controlled box with constant air exchange and ad libitum access to food and water. The mice had no prior exposure to running wheels or a treadmill and were housed under the same lighting schedule with 12L:12D (lights on at 6 a.m., eastern standard time). At the conclusion of the exercise study, mice were anesthetized with isoflurane followed by decapitation starting 8 h before lights off (ZT 4–6).
Voluntary locomotor activity in the cage was continuously monitored throughout each experiment using passive infrared motion detectors (Aurora; Ademco®, Syosset, NY) and the ClockLab analysis software (Actimetrics, Wilmette, IL). Activity was recorded in 1-min bins.
All mice had 2 wk of acclimation to the 12L:12D cycle before 4 wk of exercise sessions began. ZT 12 signifies the time of lights off and the onset of locomotor activity for a nocturnal animal. From ZT 4 to 6, mice were removed from the home cages and placed individually in new cages containing voluntary running wheels (n = 10) or involuntary treadmill running (n = 5) or exposed to a locked wheel (n = 6) or nonmoving treadmill (n = 4). Control mice (n = 8) remained in the cage. Mice on the treadmill ran for 2 h, with one 10-min break after the first hour, at a speed of 16 m·min−1 at 0% grade for a total of approximately 1.76 km (Exer 3/6 Treadmill; Columbus Instruments, Columbus, OH). Treadmill speed was chosen on the basis of a previous work (21) with the goal of trying to match running intensities with that reported for C57BL6/J mice in voluntary wheels. Others have demonstrated 60% of V˙O2max for mice running at a similar rate (18). Wheel-run mice had free access to a running wheel for 2 h and averaged approximately 1.06 ± 0.17 km per exercise session, which was similar to the intensity and distance for the treadmill mice and to previously reported values for this strain of mice (13,19).
For the RF experiment, 10 Per2::Luc mice (six males and four females) approximately 6 months old were housed individually with the same lighting schedule as described above. The mice were split into two groups with either free access to food (FF) (n = 5) or RF (n = 5). Mice were housed in a 12L:12D cycle with ad libitum access to food and water for 2 wk. On the first day of RF, food was removed from the RF group at ZT 8 (or 4 h before lights off). On the next day, food was restricted to 4 h between ZT 4 and 8. It is important to note that this design is for restricting the time of feeding but not for calorically restricting the mice. RF mice consumed approximately 3.4 ± 0.2 g of food per day, and a previous study reports that daily food intake for C57BL/6 mice is ∼4 g of chow (3). At the conclusion of the study, body mass was not different from controls (FF = 28.7 ± 2.3 g vs RF = 26.3 ± 1.2 g).
Explants were taken from the Per2::Luc reporter mice on the day after the final exercise session or after the final day of RF and analyzed for 6 d to measure circadian characteristics of the bioluminescence rhythms. Tissue culture dissection media and culturing media were used as reported previously (34–36). Briefly, the brain was removed and placed in chilled Hanks balanced salt solution supplemented with 25 U·mL−1 of penicillin, 25 μg·mL−1 of streptomycin (Invitrogen, Carlsbad, CA), 10 mM of HEPES (Sigma-Aldrich, St. Louis, MO), and 4 mM of NaHCO3 (Fisher Scientific, Hampton, NJ). The brain was sectioned using a vibrating microtome (Vibratome series 1000; EM Corp., Chestnut Hill, MA). The SCN was dissected and placed on a Millicell insert (Millipore) in a 35-mm tissue culture dish (Sigma) containing 1 mL of Dulbecco’s modified eagle medium without phenol red (Invitrogen) supplemented with 4 mM of NaHCO3, 10 mM of HEPES, 25 U·mL−1 of penicillin, 25 μg·mL−1 of streptomycin, 3.5 g of D-glucose (Sigma), 2% B27 (Gibco, Carlsbad, CA), and 0.1 mM of D-luciferin firefly, potassium salt (Biosynth, Lewisville, TX). Peripheral tissues included the lung and three different skeletal muscles: the flexor digitorum brevis (FDB), the extensor digitorum longus (EDL), and the soleus. These tissues were dissected from the mouse; the lung was hand sliced in dissection media, and the muscles were removed from tendon to tendon and cultured in the same culture media containing 5% fetal bovine serum (Invitrogen). Tissue bioluminescence was measured using a LumiCycle (Actimetrics, Wilmette, IL) housed in a light-tight water-jacketed incubator at 36.5°C. The LumiCycle software was used to collect raw bioluminescence data in 1.2-min bins every 10 min that were stored in an attached computer. Similar to what others have published (36), the raw data were smoothed by 0.5-h adjacent averaging using the LumiCycle analysis software. Baseline-subtracted data were then used to calculate the phase of the PER2::LUC bioluminescence rhythms of the cultured tissues, using ClockLab (Actimetrics). The phase was measured as the time of the first peak of the PER2::LUC bioluminescence rhythm after 24 h in culture.
After 6 d in culture, soleus muscles were removed from the LumiCycle and placed in a vial with 4% paraformaldehyde and stored at 4°C for 24 h, then placed in 70% ethanol until embedded in paraffin. Control soleus muscles were prepared immediately after euthanasia. Five-micrometer cross sections were taken starting at the tendon. Sections were stained with hematoxylin and eosin (H&E) (Sigma). Images of the muscle cross sections were taken with a Nikon DS-Ri1 using a Nikon Eclipse (E600; Nikon, Sinjuku, Tokyo, Japan) microscope at 20×.
Animal care and use.
All procedures, which complied with the guidelines of the American Association for Accreditation of Animal Care, were approved by the University of Kentucky Institutional Animal Care and Use Committee. All procedures adhere to the American College of Sports Medicine standards for animal care.
To analyze the locomotor behavior for all experiments, an ANOVA was used with a post hoc Dunnett test. Note that 33 mice began the exercise study, but only 30 were used for locomotor behavioral analysis because of technical problems with the motion detectors. The phase of peak PER2::LUC bioluminescence between exercise groups within each tissue was compared using an ANOVA with a post hoc Tukey test. The phase of peak PER2::LUC bioluminescence was compared using a Student’s t-test.
RF alters the pattern of locomotor activity during the day without decreasing overall cage activity.
Total cage activity was not different between FF and RF mice during RF. During lights on, activity was increased in the RF group, and during lights off, locomotor cage activity was decreased. The double-plotted actograms in Figure 1A represent total locomotor cage activity during the day for the entire experiment. Days are listed on the y axis, and time is listed on the x axis. Zeitgeber time refers to the time of day relative to the LD cycle with ZT 12 indicating the time of lights off. Figure 1B contains cage activity profiles averaged from the last 2 wk of the experiment. In the FF group, activity was relatively low during lights on and higher during lights off. In contrast, the RF mice had significantly increased cage activity during lights on especially during the 2–3 h before food was presented. This anticipatory activity occurred in the hours immediately before food was presented (ZT 1–4; Fig. 1C). Locomotor cage activity, before the start of RF, was averaged for both groups and is presented in Figure 1C (black bars).
RF phase shifts PER2::LUC bioluminescence in peripheral tissues.
Figure 2 includes bioluminescence data from the Per2::Luc mice exposed to RF. Figure 2A is a graph of raw bioluminescence data from two representative soleus muscle explants. The explants remained rhythmic for several days in culture. Figure 2B is a phase plot of quantified bioluminescence data from lung and soleus cultures. In the RF group, PER2::LUC bioluminescence rhythms from both tissues displayed a phase shift to an earlier time of day suggesting that the molecular clockwork in these tissues had been reset to a different time of day after RF. The phase shift was greater in the lung (∼13 h, antiphase to FF) than in the soleus (∼3 h). Figure 2C provides histological data from soleus muscles fixed and stained with H&E either immediately after dissection (control) or after 5 d in culture (LumiCycle). The fibers within the soleus muscles remain structurally intact after the static culture with no indication of necrosis, suggesting that the integrity and organization of the muscle is maintained. These morphological data are consistent with the bioluminescent results and provide confirmation that the muscle explants are transcriptionally and translationally active during the 5 d in culture.
Locomotor cage activity is decreased during the dark phase in wheel, treadmill, and sham treadmill mice.
Figure 3A shows locomotor cage activity (double-plotted actograms) for control, sham treadmill (ShamT), and treadmill mice for the entire study; dark horizontal bars indicate lights off, and gray vertical bars (ZT 4–6) indicate exercise time. Control mice were primarily active in the dark (ZT 12–24/0) throughout the experiment. Treadmill and wheel mice (wheel data not shown) displayed locomotor cage activity that was similar to controls for the first 2 wk (before exercise) in LD but began to change after the exercise regimen started. Sham wheel (ShamW) mice exposed to wheels during ZT 4–6 displayed home cage locomotor activity that was similar to the 2 wk before exercise began (not shown), whereas ShamT mice placed on a nonmoving treadmill exhibited diminished home cage locomotor activity during the weeks of exercise compared with the weeks before exercise began. Figure 3B shows mean activity profiles for the same three groups in Figure 3A calculated by averaging the home cage locomotor activity during the last 2 wk of exercise (gray vertical bars indicate exercise time, and the shaded area denotes time of lights off). Both Figure 3A and Figure 3B demonstrate that the mice continued to display a short burst of locomotor activity at the dark-to-light transition around ZT 24/0. This pattern is maintained in the exercised and sham animals (as well as control) and is common for C57BL/6 mice (4). Before ZT 4–6 (exercise), values were averaged from all groups during the 2 wk before the start of the exercise paradigm (Fig. 3C, black bars). During the weeks of the exercise treatment, there was no significant increase in locomotor cage activity during lights on and no anticipatory activity between ZT 1 and 4. Total locomotor cage activity (22 h in the home cage) decreased in the wheel (∼40%) and treadmill (∼60%) groups across the lights off period. The decrease in activity in the ShamT (∼40%) group was primarily due to a decrease in activity between ZT 20 and 24 at the end of the lights-off period (Fig. 3C).
Scheduled voluntary or involuntary exercise shifts the circadian clock in skeletal muscles and the lung.
Raw bioluminescence data from three different skeletal muscles demonstrated rhythmic expression of PER2::LUC for 5 d in static culture (Fig. 4A). For the soleus and EDL, representative images are from control, ShamT (sham), and treadmill (exercise). For the FDB, representative images are from the control, ShamW (sham), and wheel (exercise) groups. The phase of the PER2::LUC bioluminescence rhythm was significantly shifted in all three different skeletal muscles explanted from mice exposed to voluntary or involuntary exercise but not controls or shams (Fig. 4B). The phase of the PER2::LUC bioluminescence rhythm in the soleus muscle (postural muscle) was advanced by approximately 3 h in the treadmill and 2 h in the wheel-running groups. In the EDL and FDB muscles (recruited intermittently, used to extend/flex the toes), there were approximately 3- and 2-h (3 h in FDB, wheel only) advances in the treadmill and wheel-running groups, respectively. In the lung, the phase of PER2::LUC bioluminescence was advanced by approximately 2 h in both exercise conditions but was only statistically significant when compared with the ShamW. The phase of PER2::LUC bioluminescence in the SCN remained unchanged with exercise.
In the present study, scheduled exercise during lights on produced significant phase shifts in PER2::LUC bioluminescence rhythms in skeletal muscles and the lung. These data indicate that the circadian clock in skeletal muscle and lung tissues is sensitive to scheduled bouts of exercise while in a normal lighting environment. In addition, we found that 4 wk of low-intensity endurance exercise is sufficient to significantly shift the clock. Similar to what has been published previously (8,10,31), restricted access to food for 4 h·d−1 for 2 wk produced a significant phase shift in PER2::LUC bioluminescence rhythms in skeletal muscle and lung explants. Interestingly, the magnitude of the phase shift (∼2–3 h) in the skeletal muscle (soleus) was comparable in both the RF and exercise studies. However, in the lung, the phase advance was much larger with RF (∼13 h) than with scheduled exercise (∼2 h). This demonstrates that 2 wk of RF and 4 wk of exercise have a similar effect on the clock in skeletal muscle, but RF has a greater effect on the lung. Together, these data support the concept that the endogenous rhythms of circadian clocks in peripheral tissues can be altered by nonphotic environmental stimuli such as RF and exercise; however, the pathways may be different.
The RF mice demonstrated anticipatory activity in the hours preceding the feeding time cue unlike what we saw in the mice given the scheduled exercise time cue. The mechanisms behind anticipatory activity are not well understood, but FAA has been observed in rodents with SCN lesions or lacking core clock genes suggesting that FAA is not dependent on the canonical clock or SCN-driven pathways (8,32). Some evidence suggests that FAA is the result of RF altering circulating metabolic and hormonal factors (e.g., glucagon, insulin, corticosterone, and melatonin), thereby setting peripheral oscillators that then feed back to the brain (12). Scheduled exercise did not produce anticipatory activity demonstrating that nonphotic environmental cues alter locomotor activity differentially and may be driven by separate pathways. Both RF and scheduled exercise are able to “set” behavioral rhythms when rodents are housed in the absence of light, but the results of this study indicate that only RF can phase shift locomotor activity in the presence of a normal LD cycle (8,11).
Although exercise did not induce anticipatory activity, locomotor behavior in the wheel-running group, the treadmill group, and, interestingly, the ShamT group was reduced throughout the dark phase. Although exercise did not induce anticipatory activity, we did see a reduction in cage activity behavior in the wheel-running group, the treadmill group, and the ShamT group but not the ShamW group throughout the dark phase. The observation that the ShamT group also exhibited a lower cage activity indicates that this effect is not solely due to exercise. For this study, the mice in the wheel running cages were moved to a separate light box similar to the one containing the home cages. In contrast, the treadmill exercise and ShamT mice were moved outside the box but in the same room in the animal facility for that 2-h period during the light phase. The reason that ShamT mice were less active in the home cage whereas the ShamW mice (moved to a new cage with a locked wheel) showed no change in locomotor activity levels is not clear. One explanation could be the increased stress of moving the mice out of the light box into the room with the treadmill. One study demonstrated that mice exposed to social stress had decreased activity around the home cage (7). We cannot determine whether the ShamT mice were more stressed; however, the fact that the wheel mice but not the ShamW mice had less locomotor activity during the dark argues that this lowered behavior is not as simple as one factor. It is also important to point out that the phase of the molecular circadian clock was not altered in the peripheral tissues of the ShamT mice, so whatever factor(s) influenced locomotor cage activity did not contribute to the nonphotic cue with exercise.
It has been well demonstrated that altering lighting conditions and restricting an animal’s feeding to a specific time of day can shift locomotor behavior and molecular clockwork gene expression as well as clock-controlled genes with tissue specificity (27,37). The consequences of scheduled exercise on the circadian transcriptome in skeletal muscle are unknown at this time; however, it has been demonstrated that several genes involved in metabolism are expressed in a circadian manner (22,23). In skeletal muscle, genes involved in fatty acid metabolism such as PGC-1β, UCP3, Dbt, Dgat2, and Acat2 have been shown to oscillate in a circadian manner (period = ∼24 h) and are important for metabolic functions such as fatty acid oxidation (17,20) and fatty/cholesterol synthesis (1,6). Because daily activities for most species occur with some circadian rhythmicity (active phase–inactive phase every 24 h) and skeletal muscle is a highly metabolic tissue, it is reasonable that genes involved in energy storage/utilization would also be under circadian control to anticipate the daily demands on the system. The present study has demonstrated that low-intensity scheduled exercise can shift the circadian molecular clock in skeletal muscle, and we suggest that this could lead to altered metabolic processes, in the presence of an LD cycle. With increasing associations between clock disruption and metabolic disease, scheduled exercise may be useful as a lifestyle treatment to synchronize rhythms in humans (2,29).
A substantial amount of work has been done in the field of chronobiology to understand the relationship between time cues and central and peripheral circadian clocks within the organism (12,25). The exercised mice in this study were exposed to two zeitgebers: a constant LD cycle and scheduled exercise. We have demonstrated that the molecular clocks in peripheral tissues in mice with an intact central clock (SCN) were shifted with exposure to voluntary or involuntary exercise for 2 h·d−1 for 4 wk. These data provide further evidence that exercise alone has effects on the molecular clock and strengthens the hypothesis that exercise has the ability to influence the network of oscillators that exist within the mammalian system.
Support for this study was provided by the National Institutes of Health, grants AR055246 and ES018636 to K.A.E., and the T32 Fellowship, grant HL086341-02 to G.W.
The authors thank Cynthia Long and Mellani Lefta for assistance with histological sections.
There are no disclosures.
The results from this study do not constitute endorsement by the American College of Sports Medicine.
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