Acute Partial Sleep Deprivation Due to Environmental Noise Increases Weight Gain by Reducing Energy Expenditure in Rodents

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1 Original Article OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY Obesity Acute Partial Sleep Deprivation Due to Environmental Noise Increases Weight Gain by Reducing Energy Expenditure in Rodents Jennifer B. Parrish 1,2 and Jennifer A. Teske 2,3,4,5,6,7 Objective: Chronic partial sleep deprivation (SD) by environmental noise exposure increases weight gain and feeding in rodents, which contrasts weight loss after acute SD by physical methods. This study tested whether acute environmental noise exposure reduced sleep and its effect on weight gain, food intake, physical activity, and energy expenditure (EE). It was hypothesized that acute exposure would (1) increase weight gain and feeding and (2) reduce sleep, physical activity, and EE (total and individual components); and (3) behavioral changes would persist throughout recovery from SD. Methods: Three-month old male Sprague-Dawley rats slept ad libitum, were noise exposed (12-h light cycle), and allowed to recover (36 h). Weight gain, food intake, sleep/wake, physical activity, and EE were measured. Results: Acute environmental noise exposure had no effect on feeding, increased weight gain (P < 0.01), and reduced sleep (P < 0.02), physical activity (P < 0.03), total EE (P < 0.05), and several components (P < 0.05). Reductions in EE and physical activity persisted during recovery. Conclusions: Reductions in EE during sleep, rest, and physical activity reduce total EE and contribute to weight gain during acute SD and recovery from SD. These data emphasize the importance of increasing physical activity after SD to prevent obesity. Obesity (2017) 25, doi: /oby Introduction Obesity is a global health issue that increases mortality and is positively associated with impaired sleep quality and insufficient sleep time (1,2). Poor sleep is prevalent in society and increases risk for weight gain (3). Exposure to environmental noise reduces sleep and worsens health, and prolonged exposure is associated with higher body mass index and waist circumference (4-7). While sleeping less than recommended increases risk for weight gain through increases in energy intake and eating behavior (3,8,9), effects of poor sleep on total energy expenditure (EE) and its components are less clear (8,10). Components of total EE, including basal metabolic rate (BMR) and physical activity, influence weight gain yet remain largely unexplored in rodent models of sleep deprivation (SD) that exhibit weight gain. Prior work has shown that effects of SD on weight gain depend upon the method used to reduce sleep, the duration of prior wakefulness (acute versus chronic), and accumulated sleep deficit. Chronic SD (>24 h) by exposure to environmental noise (11) and sleep fragmentation (12) increase weight gain, which contrasts weight loss after chronic SD by water immersion (13), forced locomotion (14), total SD, REM SD, or non-rem SD (15). Chronic SD by multiple methods stimulates feeding (11,15,16) with parallel increases in EE after chronic SD with water immersion or forced locomotion (17-20). However, the magnitude of change in weight, feeding, and EE varies based on the method used to chronically deprive sleep. For example, non-rem SD by high amplitude EEG was the least invasive compared with total or REM SD by water immersion (15). Moreover, weight loss was not observed after acute SD (<24 h) by water immersion (21), forced locomotion (3, 6, 12 h) (22,23), or gentle handling (4 6 h) (24,25). Likewise, there was no effect of acute SD on feeding in response to water immersion (21,26) or 5 h of gentle handling (25), although 12 h of gentle 1 United States Air Force Institute of Technology, USA 2 Physiological Sciences Graduate Interdisciplinary Program, University of Arizona, Tucson, Arizona, USA. Correspondence: Jennifer A. Teske (teskeja@ .arizona.edu) 3 Department of Nutritional Sciences, University of Arizona, Tucson, Arizona, USA 4 Neuroscience Graduate Interdisciplinary Program, University of Arizona, Tucson, Arizona, USA 5 Minneapolis VA Health Care System, Minneapolis, Minnesota, USA 6 Department of Food Science & Nutrition, University of Minnesota, Saint Paul, Minnesota, USA 7 Minnesota Obesity Center at the University of Minnesota, Saint Paul, Minnesota, USA. Funding agencies: Funding for this research and publication was supported by the Department of Veterans Affairs [Career Development Award-level 2 (F7212W) to JAT], United States Department of Agriculture (ARZT H and ARZT R to JAT), and a scholarship from the United States Air Force Institute of Technology (to JBP). Disclosure: The authors declared no conflict of interest. Author contributions: JAT contributed to conception and design. JBP acquired the data. JAT and JBP analyzed data, interpreted data, drafted/revised the article, and approved the final version. Received: 19 July 2016; Accepted: 22 September 2016; Published online 29 November doi: /oby Obesity VOLUME 25 NUMBER 1 JANUARY

2 Obesity Acute Partial Sleep Deprivation Reduces Energy Expenditure Parrish and Teske handling increased feeding (27). EE has not yet been measured in response to acute SD with a method of SD that increases weight gain. Nonetheless, Caron and Stephenson demonstrated that oxygen consumption did not increase until after 20 h of total SD on a motor-driven treadmill and sleep restricted rats had no change in oxygen consumption (19). This demonstrates that within methods of SD that cause weight loss, the effect of SD on EE is dependent on sleep debit accumulation. Weight loss during SD with water or tactile methods among rodents contrasts societal weight gain due to sleep curtailment (3). Thus, we sought to establish a rodent model for acute SD that causes weight gain. First we tested whether acute environmental noise exposure reduced sleep and increased weight gain. Since chronic SD by this method stimulates hyperphagia (11), we next tested whether acute noise exposure modified energy intake, physical activity, and/or EE (total and individual components including EE during sleep, rest, physical activity). Finally, because a compensatory change in energy metabolism during recovery from SD may also contribute to weight gain associated with SD, we tested whether changes in weight, energy intake, and EE during acute SD continued after SD (i.e., recovery from SD). We hypothesized that acute environmental noise exposure would (1) increase time awake, weight gain, and feeding and (2) reduce physical activity and EE (total and components); and (3) behavioral changes during SD would persist throughout recovery from SD. Methods Animals Three-month-old male Sprague-Dawley rats (Charles River, Kingston, NY) were housed individually in solid-bottom cages with a perforated floor with a 12-h light 12-h dark photoperiod (lights on at 06:00 h) in a temperature controlled room (21 228C). Rodent chow (Harlan Teklad 8604) and water were allowed ad libitum. Rodent chow was chosen instead of a palatable diet since the latter causes hyperphagia itself and preference for a palatable diet may influence feeding results independent of treatment. Studies were approved by the Institutional Animal Care and Use Committee at the University of Arizona. Surgical implantation of EEG/EMG leads Rats were anesthetized with ketamine (50 mg/kg) and xylazine (7 mg/kg). To measure sleep/wake behavioral states, electroencephalogram (EEG) and electromyogram (EMG) leads connected to a radiotelemetry transmitter [F40-EET, Data Sciences International (DSI), St. Paul, MN] were implanted as described (28) followed by >7 days recovery before experimental trials began. Concurrent indirect calorimetry, physical activity, EEG, and EMG recordings Components of total EE, physical activity, sleep/wake, and body temperature were determined simultaneously (29). EE was measured with Promethion-Continuous (Sable Systems, Las Vegas, NV), physical activity (defined as distance traveled) was measured by infrared activity sensors (Sable Systems, Las Vegas, NV) around the calorimetery chamber, and free-moving EEG and EMG were recorded by radiotelemetry (DSI, St. Paul, MN) (28). The calorimeter was calibrated with primary gas standards and chamber airflow was maintained at 2,500 ml/min. Distance traveled, O 2,CO 2, and water vapor were measured every second and reference measurements were determined every 15 min. EE was calculated with Expedata (v1.7.30, Sable Systems, Las Vegas, NV). Body temperature and subjective physical activity (i.e., activity counts) were determined by radiotelemetry with EMG electrodes in neck musculature and the transmitter in the rodent s flank. EEG and EMG signals were visualized with Neuroscore (version 2.1, DSI, St. Paul, MN), and sleep/wake behaviors were scored manually as non-rapid eye movement sleep (NREMS), REMS, active wake (AW), or quiet wake (QW) (28). Based on the manual scoring, the following were calculated by Neuroscore: (a) percent time spent asleep (NREMS 1 REMS), wake (AW 1 QW), NREMS, REMS, AW, and QW; (b) total number of and (c) mean duration of sleep/ wake episodes, and (d) total transitions between sleep/wake states indicative of sleep quality. Quantification of total EE and its components Total EE, individual components of total EE, and BMR [i.e., defined as the minimum or mean metabolic rate in the early light cycle (7 8:30 a.m.) when rats were neither sleeping nor moving] were determined by comparing continuous time-stamped measurementsofeeg/emg(scoredasaw,qw,nrems,rems),radiotelemetric activity counts, distance traveled, and EE measurements (29). EE during REMS and NREMS was calculated as the mean metabolic rate when rats were in REMS and NREMS, respectively. Resting EE was calculated as the mean metabolic rate when rats were neither sleeping nor moving. EE during AW was calculated as the mean metabolic rate when rats were moving based on radiotelemetric activity counts. EE due to distance traveled was calculated as the sum of EE during ambulation based on infrared beams (29). Experimental design Rodents were acclimated to chambers identical to the testing chambers (7 10 days) and to the actual testing chambers plus airflow (3 days). Then rats completed the following 72-h protocol: 24 h of baseline sleep/wake ad libitum, 12 h of acute SD by prerecorded environmental noise during the light cycle (11), and 36-h recovery period (e.g., ad libitum sleep/wake). Food intake including uneaten food particles (i.e., spillage) and body weight were measured once daily. EE, physical activity, EEG, and EMG were measured concurrently and continuously. Twenty-four hour EE was not different across the 3 days of acclimation or the 24-h baseline test day as indicated by repeated measures ANOVA (F (3,21) 5 1.2, P ). Change in EE for the 24-h test represents the difference during the 24-h baseline test and the day prior. Advantages of this experimental design include: (1) noise exposure has been shown to stimulate weight gain, (2) procedure lacks tactile stimulation associated with gentle handling, lacks forced locomotion, and lack of thermoregulatory stress associated with water immersion. Statistical analyses Data were analyzed by repeated-measures ANOVA followed by Tukey s multiple comparisons tests (Prism 6.0c, GraphPad Software, 142 Obesity VOLUME 25 NUMBER 1 JANUARY

3 Original Article OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY Obesity Figure 1 Percent time spent awake and asleep during the (A,B) light cycle and (C,D) dark cycle when rats were allowed to sleep ad libitum (Ad lib), exposed to environmental noise for 12 h (partial sleep deprivation, SD), or allowed to recover [(R), i.e., sleep ad libitum] from partial SD. Mean duration of (E) sleep (NREM 1 REM) episodes and (F) NREM sleep episodes across treatments during the dark cycle and the 24-h recording period. Brackets above indicate bars that are significantly different from each other. Data expressed as means 6 SEM; N 5 7. *P < Note different scaling on y-axes. Inc., San Diego, CA). Separate analyses were completed for each time point and end point. EEG/EMG and distance traveled data from one rat were excluded due to technical error based on poor signal quality. An a level of 0.05 was used for all statistical tests. Data are expressed as means 6 SEM. Results Acute noise exposure during the light cycle reduces sleep We first verified that noise exposure increased time spent awake. Time spent awake was significantly different across treatments in the light cycle only (F (2,12) 5 5.7, P dark cycle: F (2,12) , P , Figure 1). Relative to baseline and recovery, time spent awake was significantly greater and time spent asleep was significantly less during noise exposure (P < 0.02 for all comparisons, Figure 1A, B). There was a treatment effect on the mean duration of sleep episodes in the dark cycle and 24-h period only (F (2,12) 5 7.3, P and F (2,12) 5 6.1, P , respectively, Figure 1E, F). Relative to baseline, duration of sleep episodes was significantly longer during SD and recovery in the dark cycle and 24-h period (P < 0.03 for all comparisons, Figure 1E). Duration of REMS episodes was unaffected by treatment (data not shown). However, mean duration of NREMS episodes was significantly different across treatments (dark cycle: F (2,12) , P and 24 h: F (2,12) , P , Figure 1F). Relative to baseline, duration of NREMS episodes in the dark cycle was significantly longer during SD (P ) and remained longer during recovery (P ). Duration of NREMS episodes during recovery was significantly longer than during baseline (P ) and SD (P ) during the 24-h period. Total transitions between sleep/ wake states were unaffected by treatment during all time periods (data not shown). Acute partial SD increases weight gain with no effect on food intake Acute noise exposure significantly affected weight gain (F (2,14) 5 4.4, P ) but not 24-h food intake (F (2,14) 5 0.3, P , Figure 2A, B). Rats gained significantly more weight during acute SD relative to baseline (P ). Rats continued to gain weight during recovery but the differences failed to reach statistical significance (baseline vs. recovery: P SD vs. recovery: P ). Acute partial SD by noise reduces physical activity and total EE Distance traveled was significantly different across treatments during the 24-h period (F (2,12) 5 6.2, P , Figure 2C) and the light cycle (F (2,12) 5 4.4, P ) but failed to reach Figure 2 Acute partial sleep deprivation (SD) due to noise exposure (12 h) increased (A) weight gain but had no effect on (B) food intake corrected for uneaten food. Acute partial SD reduced (C) 24-h distance traveled and (D F) total energy expenditure (EE). Brackets above indicate bars that are significantly different from each other. Data expressed as means 6 SEM; N *P < R, recovery from partial SD. Note different scaling on y-axes. Obesity VOLUME 25 NUMBER 1 JANUARY

4 Obesity Acute Partial Sleep Deprivation Reduces Energy Expenditure Parrish and Teske Figure 3 Components of total energy expenditure (EE) when rats were allowed to sleep ad libitum (ad lib), exposed to environmental noise for 12 h (sleep deprivation, SD), or allowed to recover [(R), i.e., sleep ad libitum] from partial SD. (A) EE due to distance traveled (EE-distance traveled), (B) EE during non-rapid eye movement sleep (NREMS), (C) EE during REM sleep (REMS), (D) EE during rest, and (E) EE during active wake. Brackets above bars indicate bars that are significantly different from each other. Data expressed as means 6 SEM; N 5 7. *P < Note different scaling on y- axes. statistical significance during the dark cycle (F (2,12) 5 3.2, P ). Distance traveled was significantly lower during SD and recovery compared with baseline (24 h: P and P light cycle: P and P ). Total EE was significantly affected by treatment (light cycle: F (2,14) 5 6.0, P dark cycle: F (2,14) 5 8.2, P and 24 h: F (2,14) , P ). EE was significantly lower during recovery compared with baseline during the light cycle (P , Figure 2D). During the dark cycle and 24-h period, total EE was significantly lower during SD and recovery relative to baseline (dark cycle: P and P h: P and P , Figure 2E, F). There was no treatment effect on 24-h body temperature (F (2,14) , P ) or BMR regardless of whether BMR was defined as the minimum or mean EE in the early light cycle (F (2,12) 5 2.1, P and F (2,12) 5 2.0, P , respectively). Acute partial SD by noise augments several components of total EE There was a significant effect of treatment on several components of total EE (P < 0.05, Figure 3). EE during distance traveled was significantly different across treatments (24 h: F (2,12) 5 6.5, P light cycle: F (2,12) 5 4.8, P dark cycle: F (2,12) 5 4.5, P , Figure 3A). EE during distance traveled was significantly lower during recovery compared with baseline during the light cycle (P ). During the dark cycle and 24-h period, EE during distance traveled was significantly lower during SD and recovery relative to baseline (dark cycle: P and P h: P and P ). EE during NREMS during the dark cycle was significantly lower during SD compared with baseline (P , Figure 3B). EE during NREMS was significantly lower during recovery relative to baseline during the 24-h period, light cycle, and dark cycle (P , P , and P ). EE during REMS was significantly lower during SD and recovery relative to baseline during the dark cycle (P and P , Figure 3C). Resting EE during SD and recovery was significantly lower relative to baseline in the 24-h period (P and P , Figure 3D). EE during AW was significantly lower during SD and recovery relative to baseline (24 h: P and P dark cycle: P and P , Figure 3E). During the light cycle, EE during AW was significantly lower during recovery compared with baseline and SD (P and P ). 144 Obesity VOLUME 25 NUMBER 1 JANUARY

5 Original Article OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY Discussion We tested whether acute SD due to prerecorded environmental noise exposure, a method of SD previously reported to cause weight gain and hyperphagia, influenced short-term sleep, weight gain, feeding, and EE. Acute noise exposure reduced sleep and increased weight gain by reducing physical activity, total EE, and several components without affecting food intake. Reductions in physical activity and EE continued throughout the recovery period. These data are the first to show acute SD can increase weight gain and demonstrate this method of SD models effects of noise exposure on sleep and factors influencing obesity in humans. Thus, this model of SD may be advantageous relative to other SD methods that cause weight loss for metabolic disease studies. Similar to chronic noise exposure (11), an acute exposure reduced sleep time and quality, which validates this methodology acutely deprives sleep. The mean duration of sleep and NREMS episodes was the only indicator of sleep quality affected by noise. This contrasts another study that showed acute SD with a rotating device reduced sleep quality indicated by increased sleep fragmentation (30). It is plausible that the method used to acutely deprive sleep has independent effects on sleep quality. Sleep deprived rats exhibited prolonged sleeping episodes during the dark cycle following SD, yet total sleep time during recovery was unaltered. Rats did not sleep more in the dark cycle immediately after SD, which suggests that rats failed to compensate with increased total sleep time in response to SD. Thus, prolonging NREMS episode duration may be an indirect mechanism to compensate for acute sleep loss by this method. It was unclear whether acute SD by noise would increase weight gain, and if so, whether it would also modify energy intake and/or EE. We show acute SD by noise increases weight gain with no effect on food intake. These results are consistent with the effect of chronic SD by noise on weight gain (11) yet contrast others that have reported no effect of acute SD by tactile methods on weight gain (21-25). The lack of an effect of acute SD on 24-h food intake here is consistent with the delayed stimulatory effect of chronic noise exposure on cumulative food intake (11). While effects of acute SD on feeding have been inconsistent across methods (21,25-27), it is plausible that SD by noise modifies meal architecture without influencing overall food intake. In contrast to our study, others reported elevated EE during acute SD with forced locomotion (19). Discrepancies for weight gain, feeding, and total EE between studies may be related to study design, compensatory thermogenesis (31), failure to quantify uneaten food (32), availability of sleep opportunities (19), stress, or circadian misalignment. The duration of SD, method used to deprive sleep, timing of measurements, and the method to quantify EE differ between studies. Work is needed to systematically determine how these aforementioned methodological details influence the overall effect of SD on energy metabolism. While lower total EE during and after acute SD in our study agrees with lower EE during recovery from SD in adults (33-35), our results contrast other clinical studies (8). The lack of congruency for EE results between our rodent study and clinical studies may be related to species differences and methodological details previously reviewed (8). Nonetheless, reduced total EE following SD may be one mechanism that would be expected to contribute to the observed weight gain here and after chronic SD by noise in rodents (11) and humans (4-7), but this remains to be tested. This is first study to test whether changes in individual components of total EE contributed to acute SD-induced weight gain in a rodent model. We used high-resolution indirect calorimetry coupled with EEG/EMG and physical activity measurements during SD and recovery to quantify subtle changes in components of EE during bouts of sleep, rest, and movement. The reduction in EE during distance traveled, sleep, rest, and AW in response to acute SD in rodents is novel. To account for the reduction in total EE, one would expect a decrease in one or several of these components. That we show reductions in several components of EE suggests an overall effort to recover from SD by conserving energy during rest, subsequent sleep opportunities, and while performing physical activity. This is supported by our finding of lower resting EE during SD and recovery periods, which agrees with lower resting EE during or after SD in adults (34,36). Finally, these data suggest that more than 1 day of recovery sleep after acute SD by this method would be required to normalize EE. We hypothesized that lower physical activity and resultant EE would contribute to reductions in total EE. Physical activity, EE during distance traveled, and AW were significantly lower during SD and recovery here. The reduction in physical activity during SD and recovery would be expected to have contributed to reduction in total EE. It is plausible that SD also reduced the efficiency of movement. In humans, sleep restriction has been shown to reduce physical activity (37), intensity of physical activity (38), and time spent in vigorous activity (39) and to increase ratings of physical exhaustion during recovery from SD (40). Nonetheless, it remains to be tested whether noise exposure modifies the efficiency of movement and its potential contribution to total EE. There was no effect of treatment on BMR regardless of the operational definition. We acknowledge it is unclear how to best define BMR in rodents. In humans, BMR is defined as the EE during the first arousal from sleep. Unlike most humans with one consolidated 5- to 8-h episode of sleep, rodents have several short sleep episodes. Thus, it is unclear whether defining BMR in rats in a manner similar to humans is physiologically relevant. Last, we acknowledge limitations of our study include: brief intervention length, use of standard rodent diet only, sequential treatment order, infrequent body weight measurements, lack of meal pattern analysis, or the assessment of the impact of stress hormones and circadian rhythms on the results (31). In conclusion, in contrast to studies that report weight loss with acute SD, environmental noise increases weight gain without changing food intake but instead reduces EE during sleep, resting, and physical activity. These results support the restorative function of sleep to conserve energy by demonstrating that with limited available sleep time, the body compensates by conserving energy in other ways.o Acknowledgments This material is based upon work supported in part by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development. The contents of the publication/presentation do not represent the views of the Department of Veterans Affairs or the United States Government. VC 2016 The Obesity Society Obesity Obesity VOLUME 25 NUMBER 1 JANUARY

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