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Voluntary running decreases nonexercise activity in lean and diet-induced obese mice
Physiology & Behavior 165 (2016) 249–256
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Voluntary running decreases nonexercise activity in lean and diet-induced obese mice Francine Pereira de Carvalho a, Izabelle Dias Benfato a, Thaís Ludmilla Moretto a, Marcela Barthichoto b, Camila Aparecida Machado de Oliveira c,⁎ a b c
Interdisciplinary Graduate Program in Health Sciences, Federal University of Sao Paulo, Santos, SP, Brazil Graduate Program in Food, Nutrition and Health, Federal University of Sao Paulo, Santos, SP, Brazil Department of Biosciences, Institute of Health and Society, Federal University of Sao Paulo, Santos, SP, Brazil
H I G H L I G H T S • • • •
Voluntary exercise failed to decrease body weight in obese and lean mice. Voluntary exercise causes compensatory behaviors, including decreased SPA. SPA is a key component of daily energy expenditure. By decreasing SPA, voluntary exercise induced-weight reduction also decreases.
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Article history: Received 5 May 2016 Received in revised form 2 August 2016 Accepted 3 August 2016 Available online 04 August 2016 Keywords: Spontaneous physical activity Voluntary exercise Compensation Energy homeostasis High-fat diet
a b s t r a c t Purpose: Determine whether voluntary wheel running triggers compensatory changes in nonexercise activity in lean and high-fat diet fed mice. Methods: C57Bl/6 mice received a control (C) or a high-fat diet (H) and half of them had free access to a running wheel 5 days/week (CE and HE, respectively) for 10 weeks. Energy intake, nonexercise activity (global activity, distance covered and average speed of displacement in the home cage) and energy expenditure (EE) were evaluated at weeks 5 and 10 during the 2 days without the wheels. Results: High-fat diet increased weight gain in H (110%) and HE (60%) groups compared to C and CE groups, respectively, with no effect of exercise. Wheel running increased energy intake (26% CE, 11% HE in week 5; 7% CE, 45% HE in week 10) and decreased distance covered (26% for both CE and HE in week 5; 35% CE and 13% HE in week 10) and average speed (35% CE and 13% HE in week 5; 45% CE and 18% HE in week 10) compared to the respective nonexercised groups. In week 10 there was an interaction between diet and exercise for global activity, which was reduced nearly 18% in CE, H, and HE groups compared to C. Access to a running wheel increased EE in week 5 (11% CE and 16% HE) but not in week 10, which is consistent with the period of highest running (number of turns: weeks 1–5 nearly 100% N weeks 6–10 for CE and HE groups). EE was reduced in H (19%) and HE (12%) groups compared to C and CE, in week 10. Conclusion: Voluntary running causes a compensatory decrease in nonexercise activity and an increase in energy intake, both contributing to the lack of effect of exercise on body mass. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Obesity is a complex disease with genetic, epigenetic, environmental and behavioral components. Between 1980 and 2013, the worldwide prevalence of overweight and obesity increased 27.5% for adults and
⁎ Corresponding author at: Universidade Federal de São Paulo, Campus Baixada Santista, Laboratório de Diabetes Experimental e Sinalização Celular, sala 325, Rua Silva Jardim 136, Vila Mathias, CEP: 11015-020 Santos, SP, Brazil. E-mail address: [email protected] (C.A.M. de Oliveira).
http://dx.doi.org/10.1016/j.physbeh.2016.08.003 0031-9384/© 2016 Elsevier Inc. All rights reserved.
47.1% for children. In the same time span, the number of overweight or obese people rose from 857 million to 2.1 billion [30]. Exercise is an important tool in the fight against obesity as it can create a negative energy balance. However, studies in humans [28,33,34] and rodents [18,21,39] have failed to meet the expected body weight reduction after increasing daily energy expenditure through exercise. Additionally, significant variation in body weight in response to an exercise training protocol has been seen even in a setting of controlled energy intake [6]. Nonexercise activities, or spontaneous physical activity (SPA), are activities of day-to-day living other than exercise per se, including fidgeting, maintenance of posture and other body movements of daily
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life [24,42]. For rodents, SPA refers to all forms of activities, including ambulatory and non-ambulatory behavior [15]. SPA has been shown to protect against weight gain in response to overeating and it seems to have a fundamental role in energy homeostasis [24,41]. High levels of SPA have been shown to protect rats from fat mass gain [41] whereas a decline in SPA contributes to diet-induced obesity in mice [4,41]. In humans, changing SPA toward more sedentary behavior, e.g. sitting or other very low-energy demanding activities, may account for the failure of some exercise programs in reaching the predicted weight loss [28,34]. Morio et al. (1998) found that 14 weeks of progressive endurance training in elderly people did not change their total daily energy expenditure during the training period due to a compensatory decrease in free-living activities. Among the categories of activities they investigated, they found a reduction in time spent walking [28]. The longer the periods in sedentary behaviors, the shorter the time spent in higher intensity physical activities, contributing to an overall reduction in energy expenditure. Sedentary behavior is associated with overweight and obesity, and has other deleterious health effects such as metabolic alterations and premature death [22,32]. High-fat diet fed mice offer an adequate model to study obesity and type 2 diabetes mellitus [2,43], and wheel running has being used to analyze the effects of exercise on diet-induced obesity (DIO) [7,21]. However, wheel running is not always accompanied by changes in body weight or body composition [18,21,39]. The existence of an undefined distance threshold below which voluntary running does not protect from DIO [7], that there is a compensatory increase in energy intake [18,39], or the idea that wheel running just substitutes other forms of equivalent energy-consuming behaviors [18] have been proposed to explain the discrepancy in literature. Only recently a compensatory change in SPA in mice submitted to voluntary exercise has begun to be investigated [1,7,36], but to date, there have been no investigations in DIO mice. Therefore, our objective was to determine the effect of free access to running wheels on nonexercise activity and energy homeostasis in diet-induced obese mice.
five days a week, and rested for 2 consecutive days every week. The wheel (diameter 34.5 cm; width 9 cm) was mounted outside the home cage to preserve animal life space. The total number of wheel rotations was registered daily on an external LE907 individual counter, and the mean value for each week was calculated. Wheel running, commonly used as a model of exercise, is not equivalent to spontaneous physical activity as it engages different neural and physiological mechanisms [31,37].
2. Methods
2.5. Nonexercise or spontaneous physical activity (SPA)
2.1. Animals
SPA was measured by infrared beam sensors [40] using an IR Actimeter system composed of a 2-dimensional (X and Y axes) square frame (25 × 25 cm), each frame containing 16 × 16 infrared beams, 1.3 cm apart (Panlab-Harvard Apparatus, Barcelona, Spain). SPA was recorded individually in the end of the fifth and tenth weeks for 48 consecutive hours and it was determined using the ActiTrack v2.7 software (Panlab-Harvard Apparatus, Barcelona, Spain). The software allowed the determination of global activity, being the sum of stereotypes (the number of samples where the position of the subject is different from its position during the previous sample and equal to its position from the second sample, back in time) and locomotion (the number of samples where the position of the subject is different from its position during the previous sample and different to the position of the second sample, back in time), and also the determination of the distance travelled and average speed of displacement. As for the indirect calorimetry, SPA was analyzed on the days the mice in the CE and HE groups had no access to a running wheel and the areas under the curves of activity in the full (24 h), light (uninterrupted light cycle, from 7:00 to 19:00) and dark cycles (mean of the two dark cycles) were calculated from values of each mouse using the trapezoidal method [26].
The experiments were approved by the Institutional Ethics Committee on Animal Use (CEUA 5042110514). Eight-week-old male C57Bl/6 mice were obtained from the Center for Development of Animal Models for Medicine and Biology (CEDEME, Federal University of São Paulo). They were kept at the laboratory animal facility of the Department of Bioscience in a temperature-controlled room (22 °C) with a 12:12-h lightdark cycle (7:00–19:00 h) and had free access to a control diet with a caloric composition of 16.5% fat, 65.7% carbohydrate, and 17.7% protein, and a caloric density of 3.82 kcal/g. After a four-week adaptation period mice (12 weeks-old) were randomly assigned into one of two diet groups for 10 weeks: they were either kept on the same control diet (C group) or fed a high-fat diet (H group) with a caloric composition of 60.2% fat, 26% carbohydrate, and 13.8% protein, with a caloric density of 5.23 kcal/g. In both diets, the source of carbohydrate was maize starch, dextrinized maize starch and sucrose; protein was from casein and fat was from soybean oil. In the high-fat diet, there was the addition of lard. Half of the mice of both C and H groups stayed in individual cages with free access to a running wheel five days a week (CE and HE groups, respectively). Both diets were purchased from Rhoster (Rhoster Indústria e Comércio LTDA, São Paulo, Brazil). The study was performed in two independent sets of experiments with n = 4–5 mice/group in each (total n = 9, 8, 10 and 8 for C, CE, H and HE groups, respectively). 2.2. Voluntary exercise Mice were housed individually in home cages equipped with a running wheel (Panlab-Harvard Apparatus, Barcelona, Spain) for 10 weeks,
2.3. Body weight and caloric intake Body weight was recorded once a week during the entire experiment. Caloric intake was measured in weeks 5 and 10 while spontaneous physical activity was being monitored (on days without access to a running wheel). Average daily consumption was determined by subtracting the weight of the remaining food removed after 48 h from the weight of food given, with care taken to account for spillage. 2.4. Indirect calorimetry Indirect calorimetry was analyzed for 48 h in the beginning of the fifth (transition from 4th to 5th week) and tenth (transition from 9th to 10th week) weeks at room temperature (22 °C) using an indirect calorimetry system. In short, mice were placed individually in specifically designed calorimeter chambers (Oxylet system, Panlab-Harvard Apparatus, Barcelona, Spain) with free access to diet and water. Oxygen consumption and carbon dioxide production, as well as energy expenditure, were calculated using METABOLISM software (Panlab-Harvard Apparatus, Barcelona, Spain). During indirect calorimetry measurement, mice of the CE and HE groups had no access to a running wheel. Data for energy expenditure were analyzed as areas under the curves of energy expenditure in the full (24 h), light (uninterrupted light cycle, from 7:00 to 19:00) and dark cycles (mean of the two dark cycles) calculated from values of each mouse using the trapezoidal method [26].
2.6. Carcass lipid and protein content Mice were euthanized by decapitation after CO2 inhalation. Then, retroperitoneal and epididymal adipose tissues were excised and weighed. The carcasses were eviscerated and the remnants, including the retroperitoneal and epididymal fat depots, were weighed and stored at −80 °C. The lipid content was measured as described by Stansbie et al. (1976) and standardized using the method described by Oller Do
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Nascimento and Williamson (1986). The carcass was autoclaved at 120 °C for 90 min and homogenized with water at a volume double the carcass mass. Triplicate aliquots of approximately 3 g were digested in 3 mL of 30% KOH and 3 mL of ethanol for ≥ 2 h at 70 °C in capped tubes. After cooling, 2 mL of 12 N H2SO4 was added and the samples were washed three times with petroleum ether to extract the lipids. The results are expressed as grams of lipid per 100 g of carcass. To measure the protein content, aliquots of 1 g of the same homogenate were heated to 37 °C for 1 h in 0.6 N KOH with constant shaking. After clarification by centrifugation, protein content was measured using the Bradford assay (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as a reference. 2.7. Statistical analyses Results are shown as mean ± standard error of the mean (SE). Twoway ANOVA was employed followed by the Newman-Keuls post hoc test, if necessary, using Statistica 12 software (StatSoft, USA). Comparison of energy intake was carried out by analysis of covariance (ANCOVA) with mass as a covariate. Significance was set at p b 0.05. 3. Results Body weight, total body weight gain and weight gain in the first (basal to week 5) and second half (week 6 to week 10) of the experiment were significantly higher in both groups fed a high-fat diet (H and HE), with no effect of exercise. In the first half of the study, weight gain was nearly 80% higher in high-fat (H and HE groups) compared to standard diet fed groups (C and CE), whereas in the second half, the H group gained 129% more weight than C, and the HE group gained 32% more weight than CE. High-fat diet increased total body weight gain 110% in non-exercised mice and 60% in exercised mice compared to their respective controls (Fig. 1A–B). The number of revolutions on the wheel decreased with time (Fig. 1C). It was approximately twice
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as high in the first half compared with the second half of the experiment, with no difference between the groups (Fig. 1D). Energy intake was evaluated in the fifth and tenth weeks, with body weight as covariate (Fig. 2). No effect of body weight on energy intake was observed. High-fat diet increased energy intake by 57% in mice without access to wheels (H compared to C) and by 38% in mice with access to wheels (HE compared to CE) in week 5, with no effect of diet in the tenth week. Exercise had an effect on energy intake in the fifth and tenth weeks. Voluntary wheel running increased energy intake by 26% and 7% in mice fed a standard diet and by 11% and 45% in mice fed a high-fat diet in weeks 5 and 10, respectively. (Fig. 2). In line with the period of higher running, in the fifth week energy expenditure increased as an effect of exercise in the full cycle (11% higher in CE group compared to C, and 16% higher in HE group compared to H) and dark cycle (25% higher in CE and 23% higher in HE compared with C and H groups, respectively). There was no effect of exercise on energy expenditure in the tenth week (Fig. 3A–D). Mice of the exercised groups (CE and HE) had no access to the wheels during these analyses. High-fat diet reduced energy expenditure in the light cycle (12% and 8% in H and HE groups compared to C and CE groups, respectively) in the 5th week and in the light, dark and full cycles (nearly 19% and 12% reduction in each cycle in H and HE compared to C and CE groups) in the tenth week (Fig. 3B and D). We next analyzed nonexercise activity: global activity, distance travelled and average speed of displacement. Mice of the exercised groups (CE and HE) had no access to the wheels during these measurements, which were performed in the resting days. In week five, global activity was not affected by diet or exercise (Fig. 4A–B). For mice given standard diet, access to wheels decreased distance travelled during full (24 h) cycle on the resting days by approximately 27% and reduced nonexercise activity by 20% during the dark cycle, which is the period of greater activity. Among mice fed a high-fat diet, access to a running wheel decreased the distance covered by 26% during the full and dark cycles. Full cycle average speed decreased 26% similarly in both
Fig. 1. (A) Body weight, (B) total and partial (from basal to week 5 and from week 6 to week 10) body weight gain, (C) average number of wheel revolutions per day throughout the experiment, and (D) average number of revolutions of the running wheel during the first and second halves of the study. Data are shown as mean + SE. Two-way ANOVA; # effect of diet, $ effect of time. p b 0.05. C: control diet fed group (n = 9); CE: control diet fed group with free access to running wheel (n = 8); H: high-fat diet fed group (n = 10); HE: highfat diet fed group with free access to running wheel (n = 8).
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Fig. 2. Food consumption in the fifth and tenth weeks. Data are shown as mean + SE. ANCOVA, with body weight as covariate; # effect of diet, & effect of exercise. p b 0.05. C: control diet fed group (n = 9); CE: control diet fed group with free access to running wheel (n = 8); H: high-fat diet fed group (n = 10); HE: high-fat diet fed group with free access to running wheel (n = 8).
exercised groups (CE and HE) compared with their respective controls (C and H) (Fig. 4C–D). In the 10th week, there was an interaction between the effects of diet and exercise for full and dark cycle global activity. All treatments (high-fat diet and voluntary running) decreased global activity by about 18% in full cycle and by about 15% in dark cycle compared to C mice, but the post-hoc test did not identify significant differences among the groups (Fig. 5A–B). Distance travelled and average speed were both negatively affected by wheel running in the full and dark cycles (Fig. 5C–D). For mice receiving a standard diet, distance covered and average speed of displacement decreased 35% and 45%, respectively. For the group fed a high-fat diet, distance travelled reduced 13% and average speed decreased 18% (Fig. 5C–D). Compared to control diet fed mice (C and CE groups), high-fat diet increased the relative carcass lipid content (30% and 63% increase in H and HE groups compared to C and CE, respectively) and more than doubled the retroperitoneal and epididymal fat pad weight. High-fat diet also negatively affected the relative carcass protein content. This effect, however, was more evident in sedentary than in exercised mice (27% reduction in H compared to C, and 3% reduction in HE compared to CE), which is consistent with the trend for interaction (p = 0.089) we found. Despite the absence of effect of exercise on body weight, it reduced the weight of the epididymal (37% decrease in CE compared to C, and 23% decrease in HE compared to H) and retroperitoneal (24% reduction in both CE and HE compared to C and H, respectively) fat depots. Exercise also decreased the lipid to protein carcass ratio (10% reduction in CE compared to C, and 27% reduction in HE compared to H) (Fig. 6). 4. Discussion The economic and health burdens imposed by obesity, together with unfavorable global projections, reveal the urgency in finding effective ways of combatting excessive weight. Along with caloric restriction, exercise is the most frequently used tool to create a negative energy balance [20]. However, as with other current treatments, exercise commonly fails to achieve the expected results [9,17,34]. Understanding the reasons behind this disappointing outcome may help optimize the strategies targeting weight management. Literature disagrees regarding the protective effect of voluntary running in diet induced obesity [3,7,19,21]. In agreement with other research [18,21,39], we found that free access to a running wheel did not influence body weight. Conversely, Bell et al. (1997) and Brown et
al. (2012) observed a protective effect of voluntary running against DIO. For Brown et al. (2012), mice must cover a minimal (but undefined) distance so that energy expenditure would increase enough to prevent obesity. They intentionally studied female C57Bl/6N mice due to their higher level of activity. However, as our main objective was to investigate the effect of wheel running on SPA, we chose male mice to avoid estrous cycle variation in activity [16]. In our study, as well as in that of Jung and Luthin (2010), wheel running was not homogeneous throughout the investigation. Running decreased during the experiment, being higher in the first than in the last 5 weeks. Careau et al. (2013) have observed a seasonal variation in wheel running [8]. However, we have used two independent sets of mice, and the pattern of running was similar between them. In a review by Ferguson et al. (2013) the authors found little evidence for a seasonal effect on locomotor activity of rats and mice [14]. When body weight gain was analyzed separately in the first and second halves of the 10-week protocol, no difference was found between the exercised groups and their respective controls. We observed two compensatory mechanisms that explain the lack of effects of exercise on body weight, irrespective of the diet used. First, as described by previous studies [18,39], caloric intake was increased by exercise. Secondly, on the days without access to the wheel, distance travelled and average speed of displacement in the home cage were decreased in the exercised groups. Even though we did not measure SPA during the days wheels were available, we believe nonexercise activity was also reduced during the exercising days, as observed recently [10]. Copes et al. (2015) found a substantial decrease in SPA (mainly in SPA intensity) in female mice with free access to a wheel. In spite of this, the total amount of time spent in activity (SPA plus wheel running) was about 30% higher in the exercised groups than in the groups housed without wheels, indicating that the decrease in SPA did not fully compensate for the increase in voluntary exercise in terms of duration of activity. They also observed that access to a wheel increased energy consumption and had no effect on body mass [10]. Of note, our study was the first to describe that voluntary exercise can induce a compensatory decrease in SPA (besides increasing energy intake) on the resting days (days without wheel) in both lean and obese mice. The two compensatory mechanisms occurred in the periods of both higher and lower running (first and second halves of the protocol, respectively). Age, gender and characteristics of exercise might affect compensatory behaviors differently. We used sexually mature 12 week-old male mice which were exercised for 10 weeks. Even though 8–12 weeks has been a common duration for exercise training studies [7,10–12, 21], longer investigations are needed to establish whether the compensation remain or change with time. Also, we measured activity and energy expenditure only at two time points during the 10 weeks mice had access to the wheels. Increasing the frequency of analysis could provide additional information about compensation. Furthermore, varying volumes and intensity of exercise might affect SPA in different ways. Rosenkilde et al. (2012) proposed that a moderate dose of exercise may actually lead to an increase in nonexercise activity thermogenesis without increasing energy intake, as they found an accumulated negative balance 80% greater than expected in moderately overweight individuals. Alternatively, the higher-dose exercise, which was 20% less effective than expected, could have led to an increase in energy intake [34]. Interestingly, Scariot et al. (2016) found recently that aerobic swimming exercise at individualized intensity avoided the decline in SPA observed in non-trained rats after 12 weeks. Thus, knowing how different factors influence compensatory behaviors, including SPA, will allow more effective exercise prescription for body weight reduction. Greater understanding of the role of nonexercise activities on energy homeostasis helps to explain the discrepancy commonly found between the observed and predicted weight loss induced by exercise, even when individuals are kept on a constant daily energy and nutrient intake [6]. As found by Goran and Poehlman (1992) and Morio et al.
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Fig. 3. Energy expenditure in the fifth (A) and tenth (B) weeks of the study and area under the energy expenditure curves in the full, dark and light cycle in the fifth (C) and tenth (D) weeks. The dark areas in Figs. 3A and B represent the dark cycles (lights off). Data are shown as mean + SE. Statistical analysis was performed only for data of the areas under the curves. Two-way ANOVA; # effect of diet, & effect of exercise. p b 0.05. C: control diet fed group (n = 9); CE: control diet fed group with free access to running wheel (n = 8); H: high-fat diet fed group (n = 10); HE: high-fat diet fed group with free access to running wheel (n = 8).
(1998), an increase in exercise-induced thermogenesis in elderly subjects may be compensated by a subsequent decrease in SPA, so that no change in total daily energy expenditure occurs [17,28]. Discrete variation in movement may account for significant changes in energy expenditure. In humans, when compared with the metabolic rate in the supine position, energy expenditure increased by 4% while sitting motionless and by 54% while sitting fidgeting. Similarly, fidgeting while standing increased energy expenditure by 94% compared to 13% if standing motionless, and, in the same way, increased walking speed is associated with increased energy expenditure [25]. In our study we found wheel running decreased distance travelled and average speed of displacement in the home cage. Thus, as every physical activity has an associated energy cost, reducing SPA implies less energy being expended and, together with the increased energy intake, the decrease in SPA can contribute to the lack of effect of exercise on body weight. Despite the reductions in distance and average speed of locomotion mentioned above, total daily energy expenditure was increased by exercise in the 5th week, and returned to values similar to the respective nonexercised groups in the 10th week. Accordingly, the amount of exercise in the first 5 weeks was twice the amount of the last 5 weeks. Different protocols of endurance exercise have been found to increase
muscle mass [11,23], and non-fat mass is a major factor in determining basal metabolic rate [15,33]. A limitation of our study is that body composition or muscle mass was not determined at week 5, but we hypothesize that in this more active period, lean body mass could have been increased by running, explaining the higher energy expenditure. On the other hand, the much lower amount of exercise in the last half of the study could have led to detraining, when an insufficient stimulus leads to a partial loss of exercise-induced adaptations [29], such as the reversal of the increased resting metabolism [27]. Consequently, no effect of wheel running on both protein content of the carcass and energy expenditure was observed in week 10. However, other adaptations to wheel running must have accounted for the increased energy expenditure. Wheel running has been shown to increase plasma irisin, a thermogenic myokine which drives the browning of white fat [5]. The way in which the amount of exercise modulates irisin production and its consequent effect on thermogenesis warrants further investigation. The relationship between running intensity (speed) and metabolic rate in mice is linear [38]. Brown et al. (2012) found energy expenditure doubled during voluntary running but we only measured energy expenditure in the days mice had no access to the wheels. However, as there was no effect of exercise on body weight, this means the energy intake
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Fig. 4. (A) Global activity, (B) area under the curve of activity, (C) distance travelled, and (D) average speed of displacement of mice in the full, dark and light cycles. The dark areas in Fig. 4A represent the dark cycles (lights off). Data are shown as mean + SE. For global activity, statistical analysis was performed only for data of the areas under the curves. Two-way ANOVA; & effect of exercise. p b 0.05. C: control diet fed group (n = 9); CE: control diet fed group with free access to running wheel (n = 8); H: high-fat diet fed group (n = 10); HE: high-fat diet fed group with free access to running wheel (n = 8).
of mice matched their total daily energy expenditure. In other words, they were in energy balance. Thus, we believe if no compensatory decrease in locomotion (distance travelled and average speed) had
occurred in running mice, energy expenditure would have being greater, favoring a negative energy balance. It must be mentioned, however, that even in the absence of changes in body weight, exercise decreased
Fig. 5. (A) Global activity, (B) area under the curve of activity, (C) distance travelled and (D) average speed of displacement of mice in the full, dark and light cycles. The dark areas in Fig. 5A represent the dark cycles (lights off). Data are shown as mean + SE. For global activity statistical analysis was performed only for data of the areas under the curves. Two-way ANOVA; & effect of exercise, $ interaction between diet and exercise. p b 0.05. C: control diet fed group (n = 9); CE: control diet fed group with free access to running wheel (n = 8); H: high-fat diet fed group (n = 10); HE: high-fat diet fed group with free access to running wheel (n = 8).
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Fig. 6. (A) Carcass fat, (B) weight of the epididymal and (C) retroperitoneal fat pads, (D) carcass protein, and (E) carcass fat/protein ratio at the end of the experiment. Two-way ANOVA; # effect of diet, & effect of exercise. p b 0.05. C: control diet fed group (n = 9); CE: control diet fed group with free access to running wheel (n = 8); H: high-fat diet fed group (n = 10); HE: high-fat diet fed group with free access to running wheel (n = 8).
both visceral adiposity and lipid to protein ratio, a desired outcome to decrease the risk of cardiovascular disease [13,35] and minimized the negative impact of the high-fat diet on body protein content. In this sense, when thinking in exercise and energy homeostasis, the compensatory mechanisms must be taken into account rather than only its energy cost. The observation of a compensatory decrease in SPA in voluntarily running mice may offer an opportunity to better understand SPA reduction mechanistically and to develop strategies to avoid its decline, or even increase nonexercise activity to obtain a greater negative energy balance. Conflict of interest The authors declare no conflict of interest. Results of the present study do not constitute endorsement by the American College of Sports and Medicine. Acknowledgements This work was supported by grants from Fundação de Amparo à Pesquisa no Estado de São Paulo (FAPESP), São Paulo, SP, Brazil (process 2011/05932-3). F.P.C., I.D.B. and M.B. each received a master scholarship from Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasília, DF, Brazil and T.L.M. received a master scholarship from FAPESP (process 2013/01624-8). References [1] W. Acosta, T.H. Meek, H. Schutz, E.M. Dlugosz, K.T. Vu, T. Garland, Effects of earlyonset voluntary exercise on adult physical activity and associated phenotypes in mice, Physiol. Behav. 149 (2015) 279–286. [2] B. Ahrén, G. Pacini, Insufficient islet compensation to insulin resistance vs. reduced glucose effectiveness in glucose-intolerant mice, Am. J. Physiol. Endocrinol. Metab. 283 (4) (2002) E738–E744.
[3] R.R. Bell, M.J. Spencer, J.L. Sherriff, Voluntary exercise and monounsaturated canola oil reduce fat gain in mice fed diets high in fat, J. Nutr. 127 (10) (1997) 2006–2010. [4] M. Bjursell, A.K. Gerdin, C.J. Lelliott, et al., Acutely reduced locomotor activity is a major contributor to western diet-induced obesity in mice, Am. J. Physiol. Endocrinol. Metab. 294 (2) (2008) E251–E260. [5] P. Boström, J. Wu, M.P. Jedrychowski, et al., A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis, Nature 481 (7382) (2012) 463–468. [6] C. Bouchard, A. Tremblay, J.P. Després, et al., The response to exercise with constant energy intake in identical twins, Obes. Res. 2 (5) (1994) 400–410. [7] J.D. Brown, S.P. Naples, F.W. Booth, Effects of voluntary running on oxygen consumption, RQ, and energy expenditure during primary prevention of diet-induced obesity in C57BL/6N mice, J. Appl. Physiol. (1985) 113 (3) (2012) 473–478. [8] V. Careau, M.E. Wolak, P.A. Carter, T. Garland, Limits to behavioral evolution: the quantitative genetics of a complex trait under directional selection, Evolution 67 (11) (2013) 3102–3119. [9] T.S. Church, C.K. Martin, A.M. Thompson, C.P. Earnest, C.R. Mikus, S.N. Blair, Changes in weight, waist circumference and compensatory responses with different doses of exercise among sedentary, overweight postmenopausal women, PLoS One 4 (2) (2009), e4515. [10] L.E. Copes, H. Schutz, E.M. Dlugosz, W. Acosta, M.A. Chappell, T. Garland, Effects of voluntary exercise on spontaneous physical activity and food consumption in mice: results from an artificial selection experiment, Physiol. Behav. 149 (2015) 86–94. [11] J.M. Costa Júnior, M.R. Rosa, A.O. Protzek, et al., Leucine supplementation does not affect protein turnover and impairs the beneficial effects of endurance training on glucose homeostasis in healthy mice, Amino Acids 47 (4) (2015) 745–755. [12] C.A. de Oliveira, E. Luciano, M.A. de Mello, The role of exercise on long-term effects of alloxan administered in neonatal rats, Exp. Physiol. 90 (1) (2005) 79–86. [13] J.P. Després, Obesity and cardiovascular disease: weight loss is not the only target, Can. J. Cardiol. 31 (2) (2015) 216–222. [14] S.A. Ferguson, K.L. Maier, A review of seasonal/circannual effects of laboratory rodent behavior, Physiol. Behav. 119 (2013) 130–136. [15] T. Garland, H. Schutz, M.A. Chappell, et al., The biological control of voluntary exercise, spontaneous physical activity and daily energy expenditure in relation to obesity: human and rodent perspectives, J. Exp. Biol. 214 (Pt 2) (2011) 206–229. [16] E.D. Giles, M.R. Jackman, G.C. Johnson, P.J. Schedin, J.L. Houser, P.S. MacLean, Effect of the estrous cycle and surgical ovariectomy on energy balance, fuel utilization, and physical activity in lean and obese female rats, Am. J. Phys. Regul. Integr. Comp. Phys. 299 (6) (2010) R1634–R1642. [17] M.I. Goran, E.T. Poehlman, Endurance training does not enhance total energy expenditure in healthy elderly persons, Am. J. Phys. 263 (5 Pt 1) (1992) E950–E957. [18] M. Harri, J. Lindblom, H. Malinen, et al., Effect of access to a running wheel on behavior of C57BL/6 J mice, Lab. Anim. Sci. 49 (4) (1999) 401–405.
256
F.P. de Carvalho et al. / Physiology & Behavior 165 (2016) 249–256
[19] P. Huang, S. Li, M. Shao, et al., Calorie restriction and endurance exercise share potent anti-inflammatory function in adipose tissues in ameliorating diet-induced obesity and insulin resistance in mice, Nutr. Metab. (Lond.) 7 (2010) 59. [20] J.M. Jakicic, K. Clark, E. Coleman, et al., American College of Sports Medicine position stand. Appropriate intervention strategies for weight loss and prevention of weight regain for adults, Med. Sci. Sports Exerc. 33 (12) (2001) 2145–2156. [21] A.P. Jung, D.R. Luthin, Wheel access does not attenuate weight gain in mice fed highfat or high-CHO diets, Med. Sci. Sports Exerc. 42 (2) (2010) 355–360. [22] P.T. Katzmarzyk, Physical activity, sedentary behavior, and health: paradigm paralysis or paradigm shift? Diabetes 59 (11) (2010) 2717–2725. [23] O.J. Kemi, J.P. Loennechen, U. Wisløff, Ø. Ellingsen, Intensity-controlled treadmill running in mice: cardiac and skeletal muscle hypertrophy, J. Appl. Physiol. (1985) 93 (4) (2002) 1301–1309. [24] J.A. Levine, N.L. Eberhardt, M.D. Jensen, Role of nonexercise activity thermogenesis in resistance to fat gain in humans, Science 283 (5399) (1999) 212–214. [25] J.A. Levine, S.J. Schleusner, M.D. Jensen, Energy expenditure of nonexercise activity, Am. J. Clin. Nutr. 72 (6) (2000) 1451–1454. [26] J.N. Matthews, D.G. Altman, M.J. Campbell, P. Royston, Analysis of serial measurements in medical research, BMJ 300 (6719) (1990) 230–235. [27] F. Mazzucatto, T.S. Higa, M.H. Fonseca-Alaniz, F.S. Evangelista, Reversal of metabolic adaptations induced by physical training after two weeks of physical detraining, Int. J. Clin. Exp. Med. 7 (8) (2014) 2000–2008. [28] B. Morio, C. Montaurier, G. Pickering, et al., Effects of 14 weeks of progressive endurance training on energy expenditure in elderly people, Br J. Nutr. 80 (6) (1998) 511–519. [29] I. Mujika, S. Padilla, Detraining: loss of training-induced physiological and performance adaptations. Part II: long term insufficient training stimulus, Sports Med. 30 (3) (2000) 145–154. [30] M. Ng, T. Fleming, M. Robinson, et al., Global, regional, and national prevalence of overweight and obesity in children and adults during 1980–2013: a systematic analysis for the Global Burden of Disease Study 2013, Lancet 384 (9945) (2014) 766–781. [31] C.M. Novak, J.A. Levine, Central neural and endocrine mechanisms of non-exercise activity thermogenesis and their potential impact on obesity, J. Neuroendocrinol. 19 (12) (2007) 923–940.
[32] N. Owen, G.N. Healy, C.E. Matthews, D.W. Dunstan, Too much sitting: the population health science of sedentary behavior, Exerc. Sport Sci. Rev. 38 (3) (2010) 105–113. [33] E.T. Poehlman, A.W. Gardner, M.I. Goran, Influence of endurance training on energy intake, norepinephrine kinetics, and metabolic rate in older individuals, Metabolism 41 (9) (1992) 941–948. [34] M. Rosenkilde, P. Auerbach, M.H. Reichkendler, T. Ploug, B.M. Stallknecht, A. Sjödin, Body fat loss and compensatory mechanisms in response to different doses of aerobic exercise—a randomized controlled trial in overweight sedentary males, Am. J. Phys. Regul. Integr. Comp. Phys. 303 (6) (2012) R571–R579. [35] R. Ross, P.M. Janiszewski, Is weight loss the optimal target for obesity-related cardiovascular disease risk reduction? Can J Cardiol. 24 (Suppl D) (2008) 25D–31D. [36] P.P. Scariot, F.B. Manchado-Gobatto, A.S. Torsoni, I.G. Dos Reis, W.R. Beck, C.A. Gobatto, Continuous aerobic training in individualized intensity avoids spontaneous physical activity decline and improves MCT1 expression in oxidative muscle of swimming rats, Front. Physiol. 7 (2016) 132. [37] C.M. Sherwin, Voluntary wheel running: a review and novel interpretation, Anim. Behav. 56 (1) (1998) 11–27. [38] J.R. Speakman, C. Selman, Physical activity and resting metabolic rate, Proc. Nutr. Soc. 62 (3) (2003) 621–634. [39] J.G. Swallow, P. Koteja, P.A. Carter, T. Garland, Food consumption and body composition in mice selected for high wheel-running activity, J. Comp. Physiol. B. 171 (8) (2001) 651–659. [40] J.A. Teske, C.J. Billington, C.M. Kotz, Neuropeptidergic mediators of spontaneous physical activity and non-exercise activity thermogenesis, Neuroendocrinology 87 (2) (2008) 71–90. [41] J.A. Teske, C.J. Billington, M.A. Kuskowski, C.M. Kotz, Spontaneous physical activity protects against fat mass gain, Int. J. Obes. 36 (4) (2012) 603–613. [42] P.A. Villablanca, J.R. Alegria, F. Mookadam, D.R. Holmes, R.S. Wright, J.A. Levine, Nonexercise activity thermogenesis in obesity management, Mayo Clin. Proc. 90 (4) (2015) 509–519. [43] M.S. Winzell, B. Ahrén, The high-fat diet-fed mouse: a model for studying mechanisms and treatment of impaired glucose tolerance and type 2 diabetes, Diabetes 53 (Suppl. 3) (2004) S215–S219.
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Physiology & Behavior journal homepage: www.elsevier.com/locate/phb
Voluntary running decreases nonexercise activity in lean and diet-induced obese mice Francine Pereira de Carvalho a, Izabelle Dias Benfato a, Thaís Ludmilla Moretto a, Marcela Barthichoto b, Camila Aparecida Machado de Oliveira c,⁎ a b c
Interdisciplinary Graduate Program in Health Sciences, Federal University of Sao Paulo, Santos, SP, Brazil Graduate Program in Food, Nutrition and Health, Federal University of Sao Paulo, Santos, SP, Brazil Department of Biosciences, Institute of Health and Society, Federal University of Sao Paulo, Santos, SP, Brazil
H I G H L I G H T S • • • •
Voluntary exercise failed to decrease body weight in obese and lean mice. Voluntary exercise causes compensatory behaviors, including decreased SPA. SPA is a key component of daily energy expenditure. By decreasing SPA, voluntary exercise induced-weight reduction also decreases.
a r t i c l e
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Article history: Received 5 May 2016 Received in revised form 2 August 2016 Accepted 3 August 2016 Available online 04 August 2016 Keywords: Spontaneous physical activity Voluntary exercise Compensation Energy homeostasis High-fat diet
a b s t r a c t Purpose: Determine whether voluntary wheel running triggers compensatory changes in nonexercise activity in lean and high-fat diet fed mice. Methods: C57Bl/6 mice received a control (C) or a high-fat diet (H) and half of them had free access to a running wheel 5 days/week (CE and HE, respectively) for 10 weeks. Energy intake, nonexercise activity (global activity, distance covered and average speed of displacement in the home cage) and energy expenditure (EE) were evaluated at weeks 5 and 10 during the 2 days without the wheels. Results: High-fat diet increased weight gain in H (110%) and HE (60%) groups compared to C and CE groups, respectively, with no effect of exercise. Wheel running increased energy intake (26% CE, 11% HE in week 5; 7% CE, 45% HE in week 10) and decreased distance covered (26% for both CE and HE in week 5; 35% CE and 13% HE in week 10) and average speed (35% CE and 13% HE in week 5; 45% CE and 18% HE in week 10) compared to the respective nonexercised groups. In week 10 there was an interaction between diet and exercise for global activity, which was reduced nearly 18% in CE, H, and HE groups compared to C. Access to a running wheel increased EE in week 5 (11% CE and 16% HE) but not in week 10, which is consistent with the period of highest running (number of turns: weeks 1–5 nearly 100% N weeks 6–10 for CE and HE groups). EE was reduced in H (19%) and HE (12%) groups compared to C and CE, in week 10. Conclusion: Voluntary running causes a compensatory decrease in nonexercise activity and an increase in energy intake, both contributing to the lack of effect of exercise on body mass. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Obesity is a complex disease with genetic, epigenetic, environmental and behavioral components. Between 1980 and 2013, the worldwide prevalence of overweight and obesity increased 27.5% for adults and
⁎ Corresponding author at: Universidade Federal de São Paulo, Campus Baixada Santista, Laboratório de Diabetes Experimental e Sinalização Celular, sala 325, Rua Silva Jardim 136, Vila Mathias, CEP: 11015-020 Santos, SP, Brazil. E-mail address: [email protected] (C.A.M. de Oliveira).
http://dx.doi.org/10.1016/j.physbeh.2016.08.003 0031-9384/© 2016 Elsevier Inc. All rights reserved.
47.1% for children. In the same time span, the number of overweight or obese people rose from 857 million to 2.1 billion [30]. Exercise is an important tool in the fight against obesity as it can create a negative energy balance. However, studies in humans [28,33,34] and rodents [18,21,39] have failed to meet the expected body weight reduction after increasing daily energy expenditure through exercise. Additionally, significant variation in body weight in response to an exercise training protocol has been seen even in a setting of controlled energy intake [6]. Nonexercise activities, or spontaneous physical activity (SPA), are activities of day-to-day living other than exercise per se, including fidgeting, maintenance of posture and other body movements of daily
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life [24,42]. For rodents, SPA refers to all forms of activities, including ambulatory and non-ambulatory behavior [15]. SPA has been shown to protect against weight gain in response to overeating and it seems to have a fundamental role in energy homeostasis [24,41]. High levels of SPA have been shown to protect rats from fat mass gain [41] whereas a decline in SPA contributes to diet-induced obesity in mice [4,41]. In humans, changing SPA toward more sedentary behavior, e.g. sitting or other very low-energy demanding activities, may account for the failure of some exercise programs in reaching the predicted weight loss [28,34]. Morio et al. (1998) found that 14 weeks of progressive endurance training in elderly people did not change their total daily energy expenditure during the training period due to a compensatory decrease in free-living activities. Among the categories of activities they investigated, they found a reduction in time spent walking [28]. The longer the periods in sedentary behaviors, the shorter the time spent in higher intensity physical activities, contributing to an overall reduction in energy expenditure. Sedentary behavior is associated with overweight and obesity, and has other deleterious health effects such as metabolic alterations and premature death [22,32]. High-fat diet fed mice offer an adequate model to study obesity and type 2 diabetes mellitus [2,43], and wheel running has being used to analyze the effects of exercise on diet-induced obesity (DIO) [7,21]. However, wheel running is not always accompanied by changes in body weight or body composition [18,21,39]. The existence of an undefined distance threshold below which voluntary running does not protect from DIO [7], that there is a compensatory increase in energy intake [18,39], or the idea that wheel running just substitutes other forms of equivalent energy-consuming behaviors [18] have been proposed to explain the discrepancy in literature. Only recently a compensatory change in SPA in mice submitted to voluntary exercise has begun to be investigated [1,7,36], but to date, there have been no investigations in DIO mice. Therefore, our objective was to determine the effect of free access to running wheels on nonexercise activity and energy homeostasis in diet-induced obese mice.
five days a week, and rested for 2 consecutive days every week. The wheel (diameter 34.5 cm; width 9 cm) was mounted outside the home cage to preserve animal life space. The total number of wheel rotations was registered daily on an external LE907 individual counter, and the mean value for each week was calculated. Wheel running, commonly used as a model of exercise, is not equivalent to spontaneous physical activity as it engages different neural and physiological mechanisms [31,37].
2. Methods
2.5. Nonexercise or spontaneous physical activity (SPA)
2.1. Animals
SPA was measured by infrared beam sensors [40] using an IR Actimeter system composed of a 2-dimensional (X and Y axes) square frame (25 × 25 cm), each frame containing 16 × 16 infrared beams, 1.3 cm apart (Panlab-Harvard Apparatus, Barcelona, Spain). SPA was recorded individually in the end of the fifth and tenth weeks for 48 consecutive hours and it was determined using the ActiTrack v2.7 software (Panlab-Harvard Apparatus, Barcelona, Spain). The software allowed the determination of global activity, being the sum of stereotypes (the number of samples where the position of the subject is different from its position during the previous sample and equal to its position from the second sample, back in time) and locomotion (the number of samples where the position of the subject is different from its position during the previous sample and different to the position of the second sample, back in time), and also the determination of the distance travelled and average speed of displacement. As for the indirect calorimetry, SPA was analyzed on the days the mice in the CE and HE groups had no access to a running wheel and the areas under the curves of activity in the full (24 h), light (uninterrupted light cycle, from 7:00 to 19:00) and dark cycles (mean of the two dark cycles) were calculated from values of each mouse using the trapezoidal method [26].
The experiments were approved by the Institutional Ethics Committee on Animal Use (CEUA 5042110514). Eight-week-old male C57Bl/6 mice were obtained from the Center for Development of Animal Models for Medicine and Biology (CEDEME, Federal University of São Paulo). They were kept at the laboratory animal facility of the Department of Bioscience in a temperature-controlled room (22 °C) with a 12:12-h lightdark cycle (7:00–19:00 h) and had free access to a control diet with a caloric composition of 16.5% fat, 65.7% carbohydrate, and 17.7% protein, and a caloric density of 3.82 kcal/g. After a four-week adaptation period mice (12 weeks-old) were randomly assigned into one of two diet groups for 10 weeks: they were either kept on the same control diet (C group) or fed a high-fat diet (H group) with a caloric composition of 60.2% fat, 26% carbohydrate, and 13.8% protein, with a caloric density of 5.23 kcal/g. In both diets, the source of carbohydrate was maize starch, dextrinized maize starch and sucrose; protein was from casein and fat was from soybean oil. In the high-fat diet, there was the addition of lard. Half of the mice of both C and H groups stayed in individual cages with free access to a running wheel five days a week (CE and HE groups, respectively). Both diets were purchased from Rhoster (Rhoster Indústria e Comércio LTDA, São Paulo, Brazil). The study was performed in two independent sets of experiments with n = 4–5 mice/group in each (total n = 9, 8, 10 and 8 for C, CE, H and HE groups, respectively). 2.2. Voluntary exercise Mice were housed individually in home cages equipped with a running wheel (Panlab-Harvard Apparatus, Barcelona, Spain) for 10 weeks,
2.3. Body weight and caloric intake Body weight was recorded once a week during the entire experiment. Caloric intake was measured in weeks 5 and 10 while spontaneous physical activity was being monitored (on days without access to a running wheel). Average daily consumption was determined by subtracting the weight of the remaining food removed after 48 h from the weight of food given, with care taken to account for spillage. 2.4. Indirect calorimetry Indirect calorimetry was analyzed for 48 h in the beginning of the fifth (transition from 4th to 5th week) and tenth (transition from 9th to 10th week) weeks at room temperature (22 °C) using an indirect calorimetry system. In short, mice were placed individually in specifically designed calorimeter chambers (Oxylet system, Panlab-Harvard Apparatus, Barcelona, Spain) with free access to diet and water. Oxygen consumption and carbon dioxide production, as well as energy expenditure, were calculated using METABOLISM software (Panlab-Harvard Apparatus, Barcelona, Spain). During indirect calorimetry measurement, mice of the CE and HE groups had no access to a running wheel. Data for energy expenditure were analyzed as areas under the curves of energy expenditure in the full (24 h), light (uninterrupted light cycle, from 7:00 to 19:00) and dark cycles (mean of the two dark cycles) calculated from values of each mouse using the trapezoidal method [26].
2.6. Carcass lipid and protein content Mice were euthanized by decapitation after CO2 inhalation. Then, retroperitoneal and epididymal adipose tissues were excised and weighed. The carcasses were eviscerated and the remnants, including the retroperitoneal and epididymal fat depots, were weighed and stored at −80 °C. The lipid content was measured as described by Stansbie et al. (1976) and standardized using the method described by Oller Do
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Nascimento and Williamson (1986). The carcass was autoclaved at 120 °C for 90 min and homogenized with water at a volume double the carcass mass. Triplicate aliquots of approximately 3 g were digested in 3 mL of 30% KOH and 3 mL of ethanol for ≥ 2 h at 70 °C in capped tubes. After cooling, 2 mL of 12 N H2SO4 was added and the samples were washed three times with petroleum ether to extract the lipids. The results are expressed as grams of lipid per 100 g of carcass. To measure the protein content, aliquots of 1 g of the same homogenate were heated to 37 °C for 1 h in 0.6 N KOH with constant shaking. After clarification by centrifugation, protein content was measured using the Bradford assay (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as a reference. 2.7. Statistical analyses Results are shown as mean ± standard error of the mean (SE). Twoway ANOVA was employed followed by the Newman-Keuls post hoc test, if necessary, using Statistica 12 software (StatSoft, USA). Comparison of energy intake was carried out by analysis of covariance (ANCOVA) with mass as a covariate. Significance was set at p b 0.05. 3. Results Body weight, total body weight gain and weight gain in the first (basal to week 5) and second half (week 6 to week 10) of the experiment were significantly higher in both groups fed a high-fat diet (H and HE), with no effect of exercise. In the first half of the study, weight gain was nearly 80% higher in high-fat (H and HE groups) compared to standard diet fed groups (C and CE), whereas in the second half, the H group gained 129% more weight than C, and the HE group gained 32% more weight than CE. High-fat diet increased total body weight gain 110% in non-exercised mice and 60% in exercised mice compared to their respective controls (Fig. 1A–B). The number of revolutions on the wheel decreased with time (Fig. 1C). It was approximately twice
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as high in the first half compared with the second half of the experiment, with no difference between the groups (Fig. 1D). Energy intake was evaluated in the fifth and tenth weeks, with body weight as covariate (Fig. 2). No effect of body weight on energy intake was observed. High-fat diet increased energy intake by 57% in mice without access to wheels (H compared to C) and by 38% in mice with access to wheels (HE compared to CE) in week 5, with no effect of diet in the tenth week. Exercise had an effect on energy intake in the fifth and tenth weeks. Voluntary wheel running increased energy intake by 26% and 7% in mice fed a standard diet and by 11% and 45% in mice fed a high-fat diet in weeks 5 and 10, respectively. (Fig. 2). In line with the period of higher running, in the fifth week energy expenditure increased as an effect of exercise in the full cycle (11% higher in CE group compared to C, and 16% higher in HE group compared to H) and dark cycle (25% higher in CE and 23% higher in HE compared with C and H groups, respectively). There was no effect of exercise on energy expenditure in the tenth week (Fig. 3A–D). Mice of the exercised groups (CE and HE) had no access to the wheels during these analyses. High-fat diet reduced energy expenditure in the light cycle (12% and 8% in H and HE groups compared to C and CE groups, respectively) in the 5th week and in the light, dark and full cycles (nearly 19% and 12% reduction in each cycle in H and HE compared to C and CE groups) in the tenth week (Fig. 3B and D). We next analyzed nonexercise activity: global activity, distance travelled and average speed of displacement. Mice of the exercised groups (CE and HE) had no access to the wheels during these measurements, which were performed in the resting days. In week five, global activity was not affected by diet or exercise (Fig. 4A–B). For mice given standard diet, access to wheels decreased distance travelled during full (24 h) cycle on the resting days by approximately 27% and reduced nonexercise activity by 20% during the dark cycle, which is the period of greater activity. Among mice fed a high-fat diet, access to a running wheel decreased the distance covered by 26% during the full and dark cycles. Full cycle average speed decreased 26% similarly in both
Fig. 1. (A) Body weight, (B) total and partial (from basal to week 5 and from week 6 to week 10) body weight gain, (C) average number of wheel revolutions per day throughout the experiment, and (D) average number of revolutions of the running wheel during the first and second halves of the study. Data are shown as mean + SE. Two-way ANOVA; # effect of diet, $ effect of time. p b 0.05. C: control diet fed group (n = 9); CE: control diet fed group with free access to running wheel (n = 8); H: high-fat diet fed group (n = 10); HE: highfat diet fed group with free access to running wheel (n = 8).
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Fig. 2. Food consumption in the fifth and tenth weeks. Data are shown as mean + SE. ANCOVA, with body weight as covariate; # effect of diet, & effect of exercise. p b 0.05. C: control diet fed group (n = 9); CE: control diet fed group with free access to running wheel (n = 8); H: high-fat diet fed group (n = 10); HE: high-fat diet fed group with free access to running wheel (n = 8).
exercised groups (CE and HE) compared with their respective controls (C and H) (Fig. 4C–D). In the 10th week, there was an interaction between the effects of diet and exercise for full and dark cycle global activity. All treatments (high-fat diet and voluntary running) decreased global activity by about 18% in full cycle and by about 15% in dark cycle compared to C mice, but the post-hoc test did not identify significant differences among the groups (Fig. 5A–B). Distance travelled and average speed were both negatively affected by wheel running in the full and dark cycles (Fig. 5C–D). For mice receiving a standard diet, distance covered and average speed of displacement decreased 35% and 45%, respectively. For the group fed a high-fat diet, distance travelled reduced 13% and average speed decreased 18% (Fig. 5C–D). Compared to control diet fed mice (C and CE groups), high-fat diet increased the relative carcass lipid content (30% and 63% increase in H and HE groups compared to C and CE, respectively) and more than doubled the retroperitoneal and epididymal fat pad weight. High-fat diet also negatively affected the relative carcass protein content. This effect, however, was more evident in sedentary than in exercised mice (27% reduction in H compared to C, and 3% reduction in HE compared to CE), which is consistent with the trend for interaction (p = 0.089) we found. Despite the absence of effect of exercise on body weight, it reduced the weight of the epididymal (37% decrease in CE compared to C, and 23% decrease in HE compared to H) and retroperitoneal (24% reduction in both CE and HE compared to C and H, respectively) fat depots. Exercise also decreased the lipid to protein carcass ratio (10% reduction in CE compared to C, and 27% reduction in HE compared to H) (Fig. 6). 4. Discussion The economic and health burdens imposed by obesity, together with unfavorable global projections, reveal the urgency in finding effective ways of combatting excessive weight. Along with caloric restriction, exercise is the most frequently used tool to create a negative energy balance [20]. However, as with other current treatments, exercise commonly fails to achieve the expected results [9,17,34]. Understanding the reasons behind this disappointing outcome may help optimize the strategies targeting weight management. Literature disagrees regarding the protective effect of voluntary running in diet induced obesity [3,7,19,21]. In agreement with other research [18,21,39], we found that free access to a running wheel did not influence body weight. Conversely, Bell et al. (1997) and Brown et
al. (2012) observed a protective effect of voluntary running against DIO. For Brown et al. (2012), mice must cover a minimal (but undefined) distance so that energy expenditure would increase enough to prevent obesity. They intentionally studied female C57Bl/6N mice due to their higher level of activity. However, as our main objective was to investigate the effect of wheel running on SPA, we chose male mice to avoid estrous cycle variation in activity [16]. In our study, as well as in that of Jung and Luthin (2010), wheel running was not homogeneous throughout the investigation. Running decreased during the experiment, being higher in the first than in the last 5 weeks. Careau et al. (2013) have observed a seasonal variation in wheel running [8]. However, we have used two independent sets of mice, and the pattern of running was similar between them. In a review by Ferguson et al. (2013) the authors found little evidence for a seasonal effect on locomotor activity of rats and mice [14]. When body weight gain was analyzed separately in the first and second halves of the 10-week protocol, no difference was found between the exercised groups and their respective controls. We observed two compensatory mechanisms that explain the lack of effects of exercise on body weight, irrespective of the diet used. First, as described by previous studies [18,39], caloric intake was increased by exercise. Secondly, on the days without access to the wheel, distance travelled and average speed of displacement in the home cage were decreased in the exercised groups. Even though we did not measure SPA during the days wheels were available, we believe nonexercise activity was also reduced during the exercising days, as observed recently [10]. Copes et al. (2015) found a substantial decrease in SPA (mainly in SPA intensity) in female mice with free access to a wheel. In spite of this, the total amount of time spent in activity (SPA plus wheel running) was about 30% higher in the exercised groups than in the groups housed without wheels, indicating that the decrease in SPA did not fully compensate for the increase in voluntary exercise in terms of duration of activity. They also observed that access to a wheel increased energy consumption and had no effect on body mass [10]. Of note, our study was the first to describe that voluntary exercise can induce a compensatory decrease in SPA (besides increasing energy intake) on the resting days (days without wheel) in both lean and obese mice. The two compensatory mechanisms occurred in the periods of both higher and lower running (first and second halves of the protocol, respectively). Age, gender and characteristics of exercise might affect compensatory behaviors differently. We used sexually mature 12 week-old male mice which were exercised for 10 weeks. Even though 8–12 weeks has been a common duration for exercise training studies [7,10–12, 21], longer investigations are needed to establish whether the compensation remain or change with time. Also, we measured activity and energy expenditure only at two time points during the 10 weeks mice had access to the wheels. Increasing the frequency of analysis could provide additional information about compensation. Furthermore, varying volumes and intensity of exercise might affect SPA in different ways. Rosenkilde et al. (2012) proposed that a moderate dose of exercise may actually lead to an increase in nonexercise activity thermogenesis without increasing energy intake, as they found an accumulated negative balance 80% greater than expected in moderately overweight individuals. Alternatively, the higher-dose exercise, which was 20% less effective than expected, could have led to an increase in energy intake [34]. Interestingly, Scariot et al. (2016) found recently that aerobic swimming exercise at individualized intensity avoided the decline in SPA observed in non-trained rats after 12 weeks. Thus, knowing how different factors influence compensatory behaviors, including SPA, will allow more effective exercise prescription for body weight reduction. Greater understanding of the role of nonexercise activities on energy homeostasis helps to explain the discrepancy commonly found between the observed and predicted weight loss induced by exercise, even when individuals are kept on a constant daily energy and nutrient intake [6]. As found by Goran and Poehlman (1992) and Morio et al.
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Fig. 3. Energy expenditure in the fifth (A) and tenth (B) weeks of the study and area under the energy expenditure curves in the full, dark and light cycle in the fifth (C) and tenth (D) weeks. The dark areas in Figs. 3A and B represent the dark cycles (lights off). Data are shown as mean + SE. Statistical analysis was performed only for data of the areas under the curves. Two-way ANOVA; # effect of diet, & effect of exercise. p b 0.05. C: control diet fed group (n = 9); CE: control diet fed group with free access to running wheel (n = 8); H: high-fat diet fed group (n = 10); HE: high-fat diet fed group with free access to running wheel (n = 8).
(1998), an increase in exercise-induced thermogenesis in elderly subjects may be compensated by a subsequent decrease in SPA, so that no change in total daily energy expenditure occurs [17,28]. Discrete variation in movement may account for significant changes in energy expenditure. In humans, when compared with the metabolic rate in the supine position, energy expenditure increased by 4% while sitting motionless and by 54% while sitting fidgeting. Similarly, fidgeting while standing increased energy expenditure by 94% compared to 13% if standing motionless, and, in the same way, increased walking speed is associated with increased energy expenditure [25]. In our study we found wheel running decreased distance travelled and average speed of displacement in the home cage. Thus, as every physical activity has an associated energy cost, reducing SPA implies less energy being expended and, together with the increased energy intake, the decrease in SPA can contribute to the lack of effect of exercise on body weight. Despite the reductions in distance and average speed of locomotion mentioned above, total daily energy expenditure was increased by exercise in the 5th week, and returned to values similar to the respective nonexercised groups in the 10th week. Accordingly, the amount of exercise in the first 5 weeks was twice the amount of the last 5 weeks. Different protocols of endurance exercise have been found to increase
muscle mass [11,23], and non-fat mass is a major factor in determining basal metabolic rate [15,33]. A limitation of our study is that body composition or muscle mass was not determined at week 5, but we hypothesize that in this more active period, lean body mass could have been increased by running, explaining the higher energy expenditure. On the other hand, the much lower amount of exercise in the last half of the study could have led to detraining, when an insufficient stimulus leads to a partial loss of exercise-induced adaptations [29], such as the reversal of the increased resting metabolism [27]. Consequently, no effect of wheel running on both protein content of the carcass and energy expenditure was observed in week 10. However, other adaptations to wheel running must have accounted for the increased energy expenditure. Wheel running has been shown to increase plasma irisin, a thermogenic myokine which drives the browning of white fat [5]. The way in which the amount of exercise modulates irisin production and its consequent effect on thermogenesis warrants further investigation. The relationship between running intensity (speed) and metabolic rate in mice is linear [38]. Brown et al. (2012) found energy expenditure doubled during voluntary running but we only measured energy expenditure in the days mice had no access to the wheels. However, as there was no effect of exercise on body weight, this means the energy intake
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Fig. 4. (A) Global activity, (B) area under the curve of activity, (C) distance travelled, and (D) average speed of displacement of mice in the full, dark and light cycles. The dark areas in Fig. 4A represent the dark cycles (lights off). Data are shown as mean + SE. For global activity, statistical analysis was performed only for data of the areas under the curves. Two-way ANOVA; & effect of exercise. p b 0.05. C: control diet fed group (n = 9); CE: control diet fed group with free access to running wheel (n = 8); H: high-fat diet fed group (n = 10); HE: high-fat diet fed group with free access to running wheel (n = 8).
of mice matched their total daily energy expenditure. In other words, they were in energy balance. Thus, we believe if no compensatory decrease in locomotion (distance travelled and average speed) had
occurred in running mice, energy expenditure would have being greater, favoring a negative energy balance. It must be mentioned, however, that even in the absence of changes in body weight, exercise decreased
Fig. 5. (A) Global activity, (B) area under the curve of activity, (C) distance travelled and (D) average speed of displacement of mice in the full, dark and light cycles. The dark areas in Fig. 5A represent the dark cycles (lights off). Data are shown as mean + SE. For global activity statistical analysis was performed only for data of the areas under the curves. Two-way ANOVA; & effect of exercise, $ interaction between diet and exercise. p b 0.05. C: control diet fed group (n = 9); CE: control diet fed group with free access to running wheel (n = 8); H: high-fat diet fed group (n = 10); HE: high-fat diet fed group with free access to running wheel (n = 8).
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Fig. 6. (A) Carcass fat, (B) weight of the epididymal and (C) retroperitoneal fat pads, (D) carcass protein, and (E) carcass fat/protein ratio at the end of the experiment. Two-way ANOVA; # effect of diet, & effect of exercise. p b 0.05. C: control diet fed group (n = 9); CE: control diet fed group with free access to running wheel (n = 8); H: high-fat diet fed group (n = 10); HE: high-fat diet fed group with free access to running wheel (n = 8).
both visceral adiposity and lipid to protein ratio, a desired outcome to decrease the risk of cardiovascular disease [13,35] and minimized the negative impact of the high-fat diet on body protein content. In this sense, when thinking in exercise and energy homeostasis, the compensatory mechanisms must be taken into account rather than only its energy cost. The observation of a compensatory decrease in SPA in voluntarily running mice may offer an opportunity to better understand SPA reduction mechanistically and to develop strategies to avoid its decline, or even increase nonexercise activity to obtain a greater negative energy balance. Conflict of interest The authors declare no conflict of interest. Results of the present study do not constitute endorsement by the American College of Sports and Medicine. Acknowledgements This work was supported by grants from Fundação de Amparo à Pesquisa no Estado de São Paulo (FAPESP), São Paulo, SP, Brazil (process 2011/05932-3). F.P.C., I.D.B. and M.B. each received a master scholarship from Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasília, DF, Brazil and T.L.M. received a master scholarship from FAPESP (process 2013/01624-8). References [1] W. Acosta, T.H. Meek, H. Schutz, E.M. Dlugosz, K.T. Vu, T. Garland, Effects of earlyonset voluntary exercise on adult physical activity and associated phenotypes in mice, Physiol. Behav. 149 (2015) 279–286. [2] B. Ahrén, G. Pacini, Insufficient islet compensation to insulin resistance vs. reduced glucose effectiveness in glucose-intolerant mice, Am. J. Physiol. Endocrinol. Metab. 283 (4) (2002) E738–E744.
[3] R.R. Bell, M.J. Spencer, J.L. Sherriff, Voluntary exercise and monounsaturated canola oil reduce fat gain in mice fed diets high in fat, J. Nutr. 127 (10) (1997) 2006–2010. [4] M. Bjursell, A.K. Gerdin, C.J. Lelliott, et al., Acutely reduced locomotor activity is a major contributor to western diet-induced obesity in mice, Am. J. Physiol. Endocrinol. Metab. 294 (2) (2008) E251–E260. [5] P. Boström, J. Wu, M.P. Jedrychowski, et al., A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis, Nature 481 (7382) (2012) 463–468. [6] C. Bouchard, A. Tremblay, J.P. Després, et al., The response to exercise with constant energy intake in identical twins, Obes. Res. 2 (5) (1994) 400–410. [7] J.D. Brown, S.P. Naples, F.W. Booth, Effects of voluntary running on oxygen consumption, RQ, and energy expenditure during primary prevention of diet-induced obesity in C57BL/6N mice, J. Appl. Physiol. (1985) 113 (3) (2012) 473–478. [8] V. Careau, M.E. Wolak, P.A. Carter, T. Garland, Limits to behavioral evolution: the quantitative genetics of a complex trait under directional selection, Evolution 67 (11) (2013) 3102–3119. [9] T.S. Church, C.K. Martin, A.M. Thompson, C.P. Earnest, C.R. Mikus, S.N. Blair, Changes in weight, waist circumference and compensatory responses with different doses of exercise among sedentary, overweight postmenopausal women, PLoS One 4 (2) (2009), e4515. [10] L.E. Copes, H. Schutz, E.M. Dlugosz, W. Acosta, M.A. Chappell, T. Garland, Effects of voluntary exercise on spontaneous physical activity and food consumption in mice: results from an artificial selection experiment, Physiol. Behav. 149 (2015) 86–94. [11] J.M. Costa Júnior, M.R. Rosa, A.O. Protzek, et al., Leucine supplementation does not affect protein turnover and impairs the beneficial effects of endurance training on glucose homeostasis in healthy mice, Amino Acids 47 (4) (2015) 745–755. [12] C.A. de Oliveira, E. Luciano, M.A. de Mello, The role of exercise on long-term effects of alloxan administered in neonatal rats, Exp. Physiol. 90 (1) (2005) 79–86. [13] J.P. Després, Obesity and cardiovascular disease: weight loss is not the only target, Can. J. Cardiol. 31 (2) (2015) 216–222. [14] S.A. Ferguson, K.L. Maier, A review of seasonal/circannual effects of laboratory rodent behavior, Physiol. Behav. 119 (2013) 130–136. [15] T. Garland, H. Schutz, M.A. Chappell, et al., The biological control of voluntary exercise, spontaneous physical activity and daily energy expenditure in relation to obesity: human and rodent perspectives, J. Exp. Biol. 214 (Pt 2) (2011) 206–229. [16] E.D. Giles, M.R. Jackman, G.C. Johnson, P.J. Schedin, J.L. Houser, P.S. MacLean, Effect of the estrous cycle and surgical ovariectomy on energy balance, fuel utilization, and physical activity in lean and obese female rats, Am. J. Phys. Regul. Integr. Comp. Phys. 299 (6) (2010) R1634–R1642. [17] M.I. Goran, E.T. Poehlman, Endurance training does not enhance total energy expenditure in healthy elderly persons, Am. J. Phys. 263 (5 Pt 1) (1992) E950–E957. [18] M. Harri, J. Lindblom, H. Malinen, et al., Effect of access to a running wheel on behavior of C57BL/6 J mice, Lab. Anim. Sci. 49 (4) (1999) 401–405.
256
F.P. de Carvalho et al. / Physiology & Behavior 165 (2016) 249–256
[19] P. Huang, S. Li, M. Shao, et al., Calorie restriction and endurance exercise share potent anti-inflammatory function in adipose tissues in ameliorating diet-induced obesity and insulin resistance in mice, Nutr. Metab. (Lond.) 7 (2010) 59. [20] J.M. Jakicic, K. Clark, E. Coleman, et al., American College of Sports Medicine position stand. Appropriate intervention strategies for weight loss and prevention of weight regain for adults, Med. Sci. Sports Exerc. 33 (12) (2001) 2145–2156. [21] A.P. Jung, D.R. Luthin, Wheel access does not attenuate weight gain in mice fed highfat or high-CHO diets, Med. Sci. Sports Exerc. 42 (2) (2010) 355–360. [22] P.T. Katzmarzyk, Physical activity, sedentary behavior, and health: paradigm paralysis or paradigm shift? Diabetes 59 (11) (2010) 2717–2725. [23] O.J. Kemi, J.P. Loennechen, U. Wisløff, Ø. Ellingsen, Intensity-controlled treadmill running in mice: cardiac and skeletal muscle hypertrophy, J. Appl. Physiol. (1985) 93 (4) (2002) 1301–1309. [24] J.A. Levine, N.L. Eberhardt, M.D. Jensen, Role of nonexercise activity thermogenesis in resistance to fat gain in humans, Science 283 (5399) (1999) 212–214. [25] J.A. Levine, S.J. Schleusner, M.D. Jensen, Energy expenditure of nonexercise activity, Am. J. Clin. Nutr. 72 (6) (2000) 1451–1454. [26] J.N. Matthews, D.G. Altman, M.J. Campbell, P. Royston, Analysis of serial measurements in medical research, BMJ 300 (6719) (1990) 230–235. [27] F. Mazzucatto, T.S. Higa, M.H. Fonseca-Alaniz, F.S. Evangelista, Reversal of metabolic adaptations induced by physical training after two weeks of physical detraining, Int. J. Clin. Exp. Med. 7 (8) (2014) 2000–2008. [28] B. Morio, C. Montaurier, G. Pickering, et al., Effects of 14 weeks of progressive endurance training on energy expenditure in elderly people, Br J. Nutr. 80 (6) (1998) 511–519. [29] I. Mujika, S. Padilla, Detraining: loss of training-induced physiological and performance adaptations. Part II: long term insufficient training stimulus, Sports Med. 30 (3) (2000) 145–154. [30] M. Ng, T. Fleming, M. Robinson, et al., Global, regional, and national prevalence of overweight and obesity in children and adults during 1980–2013: a systematic analysis for the Global Burden of Disease Study 2013, Lancet 384 (9945) (2014) 766–781. [31] C.M. Novak, J.A. Levine, Central neural and endocrine mechanisms of non-exercise activity thermogenesis and their potential impact on obesity, J. Neuroendocrinol. 19 (12) (2007) 923–940.
[32] N. Owen, G.N. Healy, C.E. Matthews, D.W. Dunstan, Too much sitting: the population health science of sedentary behavior, Exerc. Sport Sci. Rev. 38 (3) (2010) 105–113. [33] E.T. Poehlman, A.W. Gardner, M.I. Goran, Influence of endurance training on energy intake, norepinephrine kinetics, and metabolic rate in older individuals, Metabolism 41 (9) (1992) 941–948. [34] M. Rosenkilde, P. Auerbach, M.H. Reichkendler, T. Ploug, B.M. Stallknecht, A. Sjödin, Body fat loss and compensatory mechanisms in response to different doses of aerobic exercise—a randomized controlled trial in overweight sedentary males, Am. J. Phys. Regul. Integr. Comp. Phys. 303 (6) (2012) R571–R579. [35] R. Ross, P.M. Janiszewski, Is weight loss the optimal target for obesity-related cardiovascular disease risk reduction? Can J Cardiol. 24 (Suppl D) (2008) 25D–31D. [36] P.P. Scariot, F.B. Manchado-Gobatto, A.S. Torsoni, I.G. Dos Reis, W.R. Beck, C.A. Gobatto, Continuous aerobic training in individualized intensity avoids spontaneous physical activity decline and improves MCT1 expression in oxidative muscle of swimming rats, Front. Physiol. 7 (2016) 132. [37] C.M. Sherwin, Voluntary wheel running: a review and novel interpretation, Anim. Behav. 56 (1) (1998) 11–27. [38] J.R. Speakman, C. Selman, Physical activity and resting metabolic rate, Proc. Nutr. Soc. 62 (3) (2003) 621–634. [39] J.G. Swallow, P. Koteja, P.A. Carter, T. Garland, Food consumption and body composition in mice selected for high wheel-running activity, J. Comp. Physiol. B. 171 (8) (2001) 651–659. [40] J.A. Teske, C.J. Billington, C.M. Kotz, Neuropeptidergic mediators of spontaneous physical activity and non-exercise activity thermogenesis, Neuroendocrinology 87 (2) (2008) 71–90. [41] J.A. Teske, C.J. Billington, M.A. Kuskowski, C.M. Kotz, Spontaneous physical activity protects against fat mass gain, Int. J. Obes. 36 (4) (2012) 603–613. [42] P.A. Villablanca, J.R. Alegria, F. Mookadam, D.R. Holmes, R.S. Wright, J.A. Levine, Nonexercise activity thermogenesis in obesity management, Mayo Clin. Proc. 90 (4) (2015) 509–519. [43] M.S. Winzell, B. Ahrén, The high-fat diet-fed mouse: a model for studying mechanisms and treatment of impaired glucose tolerance and type 2 diabetes, Diabetes 53 (Suppl. 3) (2004) S215–S219.