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Am J Physiol Endocrinol Metab 294: E251-E260, 2008. First published November 20, 2007; doi:10.1152/ajpendo.00401.2007
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Acutely reduced locomotor activity is a major contributor to Western diet-induced obesity in mice

Mikael Bjursell,1 Anna-Karin Gerdin,1 Christopher J. Lelliott,1 Emil Egecioglu,2 Anders Elmgren,1 Jan Törnell,1,2 Jan Oscarsson,1,2 and Mohammad Bohlooly-Y1,2

1AstraZeneca R&D, Mölndal; and 2Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, Sahlgrenska Academy, Göteborg University, Göteborg, Sweden

Submitted 25 June 2007 ; accepted in final form 13 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of the present study was to investigate the short- and long-term effects of a high-fat Western diet (WD) on intake, storage, expenditure, and fecal loss of energy as well as effects on locomotor activity and thermogenesis. WD for only 24 h resulted in a marked physiological shift in energy homeostasis, including increased body weight gain, body fat, and energy expenditure (EE) but an acutely lowered locomotor activity. The acute reduction in locomotor activity was observed after only 3–5 h on WD. The energy intake and energy absorption were increased during the first 24 h, lower after 72 h, and normalized between 7 and 14 days on WD compared with mice given chow diet. Core body temperature and EE was increased between 48 and 72 h but normalized after 21 days on WD. These changes paralleled plasma T3 levels and uncoupling protein-1 expression in brown adipose tissue. After 21 days of WD, energy intake and absorption, EE, and body temperature were normalized. In contrast, the locomotor activity was reduced and body weight gain was increased over the entire 21-day study period on WD. Calculations based on the correlation between locomotor activity and EE in 2-h intervals at days 21–23 indicated that a large portion of the higher body weight gain in the WD group could be attributed to the reduced locomotor activity. In summary, an acute and persisting decrease in locomotor activity is most important for the effect of WD on body weight gain and obesity in mice.

energy balance; high-fat diet; energy expenditure; calorimetry

RECENT RESEARCH HAS CONFIRMED the presence of a physiological system that has the prime function to maintain homeostasis between energy intake and energy expenditure (EE) involving peripheral signals and the central nervous system (CNS) [reviewed by Flier (15)]. The cause of obesity is an imbalance between food intake and EE. However, studies of food intake and EE are complicated in humans. Therefore, rodents fed a high-fat diet (HFD) have served as a model of human obesity [reviewed by Collins (9)].

The mechanisms behind the increased body weight gain and body fat mass in rodents given a HFD is not clear, since HFD-induced obesity cannot be explained simply by increased energy intake (6, 9, 11, 25, 30). Thus HFD-induced body weight gain and body fat gain seem to be consequences of metabolic changes other than increased energy intake.

EE is composed of basal metabolic rate, the thermic effect of food, and locomotor activity or spontaneous physical activity (SPA). In humans, both SPA and non-exercise activity thermogenesis (NEAT) are factors that clearly contribute to EE and development of obesity (6, 27, 28). Since rodents are smaller, locomotor activity must be less important for total EE because of the much larger body surface-to-body mass ratio in mice than in humans. However, a recent study comparing diet-induced obesity (DIO) rats and a diet-resistant rat strain indicated that locomotor activity also has an important impact on development of weight gain in rodents (33). Nonetheless, it has not been shown whether altered locomotor activity is a consequence or a cause of obesity in rodents given a HFD (6, 41).

It is unclear how locomotor activity is regulated in relation to diet. Interestingly, many peripheral and hypothalamic factors regulating appetite also seem to influence locomotor activity [see review by Castaneda (6)]. It is well known that the dopamine system in the CNS is involved in the regulation of locomotor activity (44). DIO also has been shown to alter the dopamine system in striatum (22), which connects HFD to dopaminergic activity and, hence, locomotor activity. Moreover, endocannabinoids (CB) and the CB receptors have been shown to be involved in regulation of food intake and locomotion. The CB1 receptor is involved in the development of DIO (35), and expression of CB1 has been shown to be reduced by a high cholesterol-containing diet (21). The endogenous CB anandamide (arachidonyl ethanolamide, AEA) is known to be increased by HFD (43) and was recently shown to reduce locomotor activity in mice on a C57Bl/6 genetic background (45).

Thermogenesis is also involved in the energy homeostasis. Leptin increases uncoupling protein-1 (UCP1) expression in brown adipose tissue (BAT) (10, 38), and leptin-deficient mice have a lower body temperature (4, 39). Administration of leptin increases the activity of the sympathetic nervous system (8), which is known to alter diet-induced thermogenesis (2) that is linked to UCP activity in BAT [reviewed in (13)]. High leptin levels can cause leptin resistance over time (30, 47), which may reduce the impact of leptin in regulating energy balance. Thus HFD-induced obesity may affect body temperature via leptin effects.

This study aimed to assess the acute and chronic effects of WD containing cholesterol and both saturated and unsaturated fats in obesity-prone C57Bl/6J mice on energy homeostasis by studying daily changes in body weight, body fat mass, body temperature, locomotor activity, energy intake, EE, and fecal energy loss. Energy absorption is defined as energy intake minus fecal energy loss. In addition, the acute and chronic effects of WD on the levels of plasma and tissue markers linked to the in vivo parameters were analyzed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and diets. Male C57Bl/6J mice (Harlan, Horst, The Netherlands) were allocated in groups (n = 8 per group) balanced by body weight and housed individually in a temperature-controlled room (22°C) with a 12:12-h light-dark cycle. Mice had access to normal chow (R36; Lactamin, Stockholm, Sweden) and water ad libitum. The R36 diet contains 3.5% cellulose (weight percent) and 22.9% protein, 67.1% carbohydrate, and 9.6% fat (energy percent). The R36 diet is vegetable, and the main sources of proteins are from soy, grain, and potatoes. The carbohydrate source is mainly from grains, and the fat source is from soy and contains linoleic acid. The R36 diet has an energy density of 12.6 kJ/g (according to the supplier). The WD used was RD Western diet (no. D03051901; Research Diets, New Brunswick, NJ) containing 5% cellulose (weight percent) and 16.9% protein, 42.8% carbohydrate, and 40.3% fat (energy percent). The main source of proteins in the WD is casein, and the main source of carbohydrates is corn starch, sucrose, and maltodextrin. The fat consists of (per kg diet) 100 g of milk fat (900 kcal) and 110 g of corn oil (990 kcal). The WD was also supplemented with 0.15% cholesterol. The WD has an energy density of 19.6 kJ/g (according to the supplier). The food quotient (FQ) for the diets was calculated as 0.7 x fat percent, 0.85 x protein percent, and 1 x carbohydrate percent. The FQ for the chow diet was 0.94, and that for WD was 0.85. The experiments were performed according to the approved ethical application of the local ethical committee for animal experimentation.

Study design. Eight-week-old mice were given chow diet (R36) or the WD for 24, 48, or 72 h, and measurements of body weight, energy intake, fecal energy loss, body temperature, and body composition were performed. Two additional groups of mice were given chow diet or WD for 21 days. Body weight, energy intake, fecal energy loss, body temperature, and body composition were measured after 7, 14, and 21 days. After the measurement periods, the mice were killed and the hypothalamus, BAT, and plasma were collected for further analysis. For studies of locomotor activity and indirect calorimetry, 8-wk-old mice were given WD or chow diet and analyzed in connection to the introduction of the diets (occurring at 1600) and then again after 3 and 7 wk. An additional study was performed in which 8-wk-old mice were given chow diet or WD for 5 h (from 1600 to 2100 over dark period) or 24 h (from 0800 to 0800, over light and dark periods). After 24 h, the mice were assessed for body composition by dual-energy X-ray absorptiometry (DEXA). The mice were then killed, and plasma was collected. The gastrointestinal (GI) tract was removed, associated adipose tissue was dissected and returned to the mouse, and the GI-less mouse and the GI tract were independently analyzed by DEXA. Finally, the epididymal fat depots were blotted and weighed.

Energy intake and energy content analysis. Mass of food intake was measured over 24 h in preweighed cages as previously described in nonfasted mice (4) with one small change: no initial incubation (80°C for 1 h) of the cages was done because of the high fat content of the WD. Total feces produced over the measurement period were collected, and energy content of the diets and feces was determined with a bomb calorimeter (C 5000; IKA Werke, Staufen, Germany).

Indirect calorimetry, locomotor activity, body temperature, and composition analysis. Indirect calorimetry was analyzed at room temperature (22°C) as previously described (18) using an indirect calorimetry system (Columbus Instruments, Columbus, OH). In short, the mice were placed in the specifically designed Oxymax calorimeter chambers with ad libitum access to the diets and water for 72 h. The spontaneous locomotor activity was recorded at the same time as the indirect calorimetry, continuously over the measurement period by infrared beam sensors, separated by 0.54 cm from each other. To record activity, we used an OPTO-M3 sensor (Columbus Instruments) to assess x- and z-axis activity. This system provides both total counts (every time a beam is broken) and ambulatory counts (every time a new beam is broken). The recording of ambulatory counts does not include the same beam being broken repeatedly and so measures the actual locomotion but does not include behaviors such as scratching and grooming. Thus the ambulatory readings were used to measure locomotor activity. Rectal body temperatures were recorded in conscious, nonanesthetized mice at midday (1200–1300) as previously described (18). Body composition was analyzed by DEXA in mice anesthetized with isoflurane inhalation as previously described (18). A correlation analysis between locomotor activity and EE was performed as follows. Individual mouse data were collected, and average values in 2-h intervals were calculated for all mice. Average values for mice given chow diet and average values for mice given WD for 21 days were then calculated. Each 2-h point of locomotor activity and EE was used to do the correlation analysis; thus each point represents an average for measurements over 2 h for all mice in the separate groups.

Plasma analysis. Blood was taken by cardiac puncture in K-EDTA-coated tubes from nonfasted mice in the afternoon (~1400). The mice were sedated by isoflurane inhalation before blood sampling. Plasma was separated by centrifugation (2,500 rpm, 10 min, 4°C) and stored at –80°C until analyzed. Plasma leptin levels were measured by ELISA (Christal Chemical, Downers Grove, IL). Insulin-like growth factor I (IGF-I) was analyzed by RIA (DSL-2900; Diagnostic Systems, Webster, TX). Total plasma thyroxine (T4) and triiodothyronine (T3) were determined using RIA (Coat-A-Count; Diagnostic Products, Los Angeles, CA). Ghrelin levels were measured using a RIA kit (GHR-89HK; Linco Research, St. Charles, MO). Plasma adiponectin was measured by RIA (Linco Research). Plasma anandamide levels (AEA) were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS tandem mass spectrometry) after extraction with methyl tert-butyl ether/isohexane. An acetonitrile-based gradient was used on a HyPurity C18 column (Thermo Fisher Scientific, Waltham, MA). The Quattro Premier XE mass spectrometer (Waters, Milford, MA) was operated in electrospray positive mode, and levels of AEA and the internal standard AEA-D8 were determined as the multiple reaction monitoring transitions of m/z of 348–362 and 356–363, respectively, using a cone voltage of 27 V and a collision energy of 16 eV.

Determination of dopamine. Brain tissues (striatum, nucleus accumbens, ventral tegmental area, and prefrontal cortex) were dissected 48 h after the switch to WD, weighed, snap frozen in liquid nitrogen, and stored at –80°C until analyzed. The tissue was homogenized with a Sonifier B30 (Branson Sonic Power) in 0.1 M HClO4 containing Na2-EDTA (2.5 mM). After centrifugation (10,000 g, 4°C, 10 min), the supernatant was taken for analysis of dopamine (DA) with a split-fraction HPLC-ED system. DA was detected in one fraction at an oxidizing potential of 0.55 V (Waters 460 detector; Millipore-Waters, Milford, MA). The chromatography software package Chromeleon (Dionex, Sunnyvale, CA) was used for the acquisition and integration of the resulting currents.

Expression level analysis. RNA extraction, cDNA synthesis, and quantification by TaqMan real-time PCR were performed as previously described (3). Gene-specific primer and probe sequences are given in Table 1.


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  		Table 1. Sequences for RT-PCR primers and probes

 
Statistical analysis. All values are means ± SE. Comparison between two groups was done using the Wilcoxon Mann-Whitney U-test. When several groups were studied, a Kruskal-Wallis ANOVA was performed, followed by pairwise comparison using Wilcoxon Mann-Whitney U analysis. Parameters over time were analyzed in a two-way ANOVA model. Values of P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Acute and chronic effects of WD on energy intake, expenditure, and storage. Four groups of 8-wk-old mice were given chow or WD for 24, 48, or 72 h. Two additional groups of mice were analyzed after 7, 14, and 21 days on chow or WD.

WD for 24 h resulted in a significant body weight gain compared with mice given chow. The daily body weight gain was highest during the first 24 h (chow, 2.7% body weight increase; WD, 7.8% body weight increase). Thereafter, the rate of daily body weight gain declined in the WD group, but the body weight gain remained significantly higher in the WD group than in the chow-fed group of mice throughout the study period (Fig. 1A). Body fat mass was already increased after 24 h on WD compared with mice given chow diet. The marked increase in body fat mass after the first 24 h on WD was followed by a smaller increase during the following time period (Fig. 1B). Lean body mass was not significantly different between the groups given WD and the chow diet (Fig. 1C). To exclude the possibility that the dietary content in the GI tract influenced the results of body composition measurements, we performed a control experiment. Mice given WD for 24 h also had increased body fat mass after removal of the GI tract (chow, 2.98 ± 0.15 g; WD, 3.55 ± 0.13 g; P < 0.05) as assessed by DEXA. Also, the weight of the epididymal fat depots was higher in the WD group after 24 h of WD (chow, 0.23 ± 0.02 g; WD, 0.34 ± 0.02 g; P < 0.01). Neither the GI tract alone nor the WD pellets had higher fat mass according to the DEXA scan (data not shown).


Figure 1
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  		Fig. 1. Body weight gain and body composition. Daily body weight gain (A), body fat mass (B), and body lean mass (C) were measured in C57Bl/6J mice (n = 8 in each group) given chow diet or Western diet (WD) for different time periods. *P < 0.05; **P < 0.01, chow diet vs. WD (Kruskal-Wallis ANOVA followed by pairwise comparison using Wilcoxon Mann-Whitney U analysis).

 
Energy intake was measured over 24-h periods after "acute" (24, 48, and 72 h) and "chronic" (7, 14, and 21 days) WD regimen. The energy intake during the first 24 h was higher in mice given WD, whereas from 24 to 48 h the energy intake was similar in the chow and WD groups. From 48 to 72 h on WD, the energy intake was lower in the WD group compared with mice given chow diet. The energy intake in the WD group remained lower after 7 days but was normalized and similar to the energy intake of the chow-fed mice after 14 and 21 days (Fig. 2A).


Figure 2
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  		Fig. 2. Energy balance and body temperature. Energy intake (A), energy lost in feces (B), energy absorbed (C), food efficiency of absorbed energy (D), and rectal midday body temperature (E) were measured in C57Bl/6J mice (n = 8 in each group) given chow diet or WD for different time periods. *P < 0.05; **P < 0.01, chow diet vs. WD (Kruskal-Wallis ANOVA followed by pairwise comparison using Wilcoxon Mann-Whitney U analysis).

 
Since different diets enriched in fat may affect gastric transit time (42), measurements of fecal production and energy content were performed. Energy content of the feces was lower in the WD group after 24 h, and this continued throughout the study period (Fig. 2B). The amount of dry feces produced by mice on WD was already significantly lower after 24 h compared with that of chow-fed mice (chow, 0.71 ± 0.06 g; WD, 0.52 ± 0.02 g; P < 0.01), and this was also observed during the remaining study period of 21 days (data not shown). However, the lower amount of dry feces correlated to a lower mass of food eaten during the entire study period in the WD group, suggesting that gastric transit time was largely unaffected by the diet switch.

The energy absorbed was calculated as energy intake minus energy lost in feces (Fig. 2C). The mice on WD had an energy absorption of 92% after 24 h and 93–95% on days 7, 14, and 21 on WD. Mice on normal chow had an energy absorption of ~87%. As for the energy intake, absorbed energy was higher after 24 h, lower after 72 h, and normalized after 14 and 21 days on WD compared with mice given chow diet. Therefore, the different energy absorption of WD and chow diet had no significant impact on the differences in energy metabolism between mice on WD and chow diet.

Food efficiency (body weight gain/energy intake) was significantly higher for mice given WD at all time points compared with the mice given chow diet. To exclude differences in energy assimilation, we calculated the food efficiency of absorbed energy (body weight gain/energy absorbed). Mice given WD had a higher body weight gain per amount of energy absorbed at all time points measured over the 21-day study period (Fig. 2D).

Rectal core temperature was increased after 72 h on WD and remained increased after 7 and 14 days of WD. After 21 days on WD, the body temperature was normalized (Fig. 2E).

In another study, a 72-h indirect calorimetry analysis was performed. In the beginning of the first dark period (1700) the diet was switched from chow to WD for eight mice, whereas another eight mice remained on chow diet. After the analysis, the mice remained on their diet (WD or chow) and were reanalyzed after 21 days and 7 wk. As expected, WD feeding lowered the respiratory exchange ratio (RER), especially during nighttime, being significant after 24 h on WD (Fig. 3A). RER was also lower after 21 days and 7 wk on WD (data not shown). The average RER values after 24, 48, and 72 h and 21 days on WD or chow diet were compared with the FQ values of the diets. After 21 days on WD, the RER of the mice given WD almost coincided with the FQ of the WD (Fig. 3B), indicating a slow adaptation to the WD. The total EE was significantly increased after 24 h of WD (Fig. 4A). However, no significant difference in total EE was detected between the groups of mice after 21 days, but EE was significantly lower in the WD group after 7 wk compared with mice given chow diet (Fig. 4, B and C).


Figure 3
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  		Fig. 3. Respiratory exchange ratio (RER) and food quotient (FQ). A: RER was significantly lower in mice given WD than in mice given chow diet, measured over 48 h following food switch. B: comparison of average RER to FQ of the chow diet and WD. Experiments were performed in C57Bl/6J mice (n = 8 in each group) given chow diet or WD. Shaded horizontal bars indicate the dark phase of the 24-h period. Statistics from indirect calorimetry were assessed using a 2-way ANOVA model.

 

Figure 4
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  		Fig. 4. Energy expenditure. A: energy expenditure was significantly higher measured over 48 h in mice whose diet was switched from chow diet to WD. B: energy expenditure was not different after 21 days of WD compared with chow diet. C: energy expenditure was significantly lower after 7 wk of WD compared with chow diet. Experiments were performed in C57Bl/6J mice (n = 8 in each group) given chow diet or WD. Shaded horizontal bars indicate the dark phase of the 24-h period. Statistics were assessed using a 2-way ANOVA model.

 
The spontaneous locomotor activity was acutely lowered during the 24-h time period following the food switch from chow to WD compared with that in mice that remained on a chow diet (Fig. 5A). After only 3–5 h on WD (1900, dark period), a significantly lower locomotor activity was found among the mice on WD (chow, 706 ± 126 counts; WD, 209 ± 40 counts; P < 0.01; Fig. 5B). The lower locomotor activity persisted after 21 days and 7 wk on WD (Fig. 5, C and D).


Figure 5
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  		Fig. 5. Locomotor activity. A: locomotor activity was significantly lower for mice given WD compared with chow diet measured over 72 h following food switch. B: locomotor activity was acutely lower after food switch in mice given WD than in mice given chow diet, measured during the 3–5 h after the switch. Lower locomotor activity was found in mice given WD for 21 days (C) and 7 wk (D) than in mice given chow diet. Experiments were performed in C57Bl/6J mice (n = 8 in each group) given chow diet or WD. Shaded horizontal bars indicate the dark phase of the 24-h period. Statistics over time were assessed using a 2-way ANOVA model. **P < 0.01, chow diet vs. WD (Wilcoxon Mann-Whitney U analysis).

 
Thus, after 21 days, there were no differences other than decreased locomotor activity (Fig. 5C) and increased body weight gain (Fig. 1A) between chow and WD mice. No difference in total EE was detected (Fig. 4B), showing that the energy cost of locomotor activity was not detectable when calculating total EE. In an attempt to estimate the contribution of the lower locomotor activity to the increased body weight gain in WD fed mice (assuming that the increased weight gain was only a consequence of increased fat mass), we performed a correlation analysis between locomotor activity and EE as described in MATERIALS AND METHODS. When data were correlated from EE and locomotor activity, collected in 2-h periods at days 21–23 after the switch to WD, there was a positive correlation between locomotor activity and EE (Fig. 6). Using the function from the trend line and extrapolating to set the locomotion to zero, we found that the major contribution to total EE was, as expected, basal metabolism (chow, 15.32 kcal·h–1·kg–1; WD, 15.54 kcal·h–1·kg–1). Thus only a small fraction arose from locomotor activity. However, EE was more affected by changes in locomotor activity in mice given WD than in mice given chow diet.


Figure 6
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  		Fig. 6. Correlation analysis between energy expenditure and locomotor activity. Correlation between locomotor activity and energy expenditure was based on average values collected over 72 h. Each dot represents data collected in 2-h intervals from the group of mice given chow diet or WD. Inset (right) is a magnification of the range between 15 and 20 kcal·h–1·kg–1 on the x-axis. The trend line equation for the chow mice is y = 128.16x – 1,963, R2 = 0.8234, and that for the WD mice is y = 91.672x – 1,424.7, R2 = 0.8753.

 
We made the assumption that the mice on WD had the same locomotor activity as the mice on chow diet. As shown by the trend line for the WD mice, the WD mice would have used 18.42 kcal·h–1·kg–1 instead of 17.31·h–1·kg–1 if they had increased their locomotor activity to the same level as the chow mice. Thus, if the WD mice would have had the same locomotor activity as the chow fed mice, they would have used an additional 0.90 kcal·day–1·mouse–1 (by converting the values to days and relating to the body weight of the mice). The daily body weight gain at day 21 after the switch to WD was 0.13 g for chow mice and 0.29 g for WD mice; a difference of 0.16 g, which was almost entirely a consequence of increase in fat mass (Fig. 1). This difference corresponds to ~1.45 kcal of fat mass per day [using the conversion factor previously described (37) and 1 kJ = 1/4.1868 kcal = 0.24 kcal]. Thus it is estimated that 62% of the extra body weight gain in mice fed WD for 21 days can be explained by a reduced locomotor activity.

Acute and chronic effects of WD on plasma parameters. All plasma data are presented in Table 2. A significant increase in plasma leptin was observed after only 24 h and remained high during the 21-day study in mice given WD compared with mice given chow diet. Plasma levels of IGF-I were increased after 24, 48, and 72 h but were not changed after 21 days on WD compared with the chow-fed mice. T3 levels in plasma were acutely increased after 24 h and remained high after 48 and 72 h on WD, but the levels were not different after 21 days on WD compared with the chow mice. Plasma levels of T4 and ghrelin were not significantly altered until after 21 days on WD, when they decreased. No significant changes in plasma adiponectin levels were observed. Mice given WD for 48 h had a 38% increase in plasma AEA concentrations compared with the chow-fed mice (chow, 1.3 ± 0.1 nM; WD, 1.8 ± 0.2 nM; P < 0.05). Since increased AEA could have had importance for the observed decrease in locomotor activity (45), an additional experiment was performed to investigate whether increased AEA had coincided with the observed change in activity at 5 h after the food switch. However, plasma AEA levels were already 18% lower 5 h after the switch to WD compared with the chow-fed mice (nighttime) but 46% higher after 24 h on WD (daytime) (Table 3). Interestingly, nighttime AEA levels were significantly increased compared with the daytime AEA levels when compared with levels in mice given chow diet (Table 3).


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  		Table 2. Plasma levels of hormones

 

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  		Table 3. Plasma levels of anandamide

 
Gene expression and DA levels in CNS. Expression of genes was determined to correlate our physiological findings with expression of the neuropeptides regulating food intake and metabolism. All expression data from the hypothalamus are collected in Table 4. After 24 h on WD, the gene expression levels of hypothalamic neuropeptide Y (NPY) and Agouti-related protein (AGRP) were significantly decreased (18 and 39%, respectively) compared with the levels in mice given chow diet. The expression levels of these genes remained lower up to 21 days of WD. However, no differences in expression levels of hypothalamic proopiomelanocortin, orexin, or the CB receptors CB1, CB2, and GPR55 were observed between mice fed WD or chow diet at any of the time points (data not shown). Expression level analysis in BAT revealed a significant increase of UCP1 expression after 24 and 72 h on WD (percentage of control: chow 24 h, 1.00 ± 0.17; WD 24 h, 1.40 ± 0.14; WD 48 h, 1.30 ± 0.13; WD 72 h, 1.55 ± 0.10; P < 0.05). A significant increase in UCP1 mRNA was still present in mice given WD for 21 days (percentage of control: chow 21 days, 1.00 ± 0.09; WD 21 days, 1.50 ± 0.02; P < 0.01).


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  		Table 4. mRNA expression levels in hypothalamus

 
Brain levels of DA were determined to investigate a potential explanation to changed locomotor activity. However, levels of DA were not significantly different between mice given WD for 48 h compared with and chow-fed mice in any of the brain regions examined (striatum, nucleus accumbens, ventral tegmental area, and prefrontal cortex; data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The aim of this study was to investigate aspects of the energy balance derangement engendered in mice by a high-fat WD. The time course of the changes in energy intake, EE, locomotor activity, body composition, and energy losses in feces was studied. The WD fed mice had increased body weight, body fat, energy intake, and energy absorption after only 24 h. Interestingly, decreased locomotor activity was observed during the first dark period only a few hours after the animals were presented with WD. The increase in EE observed after the first 24 h of WD was in line with increased body temperature, UCP1 expression in BAT, and plasma T3 levels. Although the body weight gain was increased during the entire 21-day study period, energy intake and absorption, total EE, and body temperature were normalized between 7 and 21 days on WD. However, the locomotor activity was acutely and persistently decreased by WD. An estimation of the contribution of decreased locomotor activity to EE was done by correlating locomotor activity and EE at each 2-h interval on days 21–23. The estimation indicated that more than half of the differences in body weight gain between mice given chow and WD could be attributed to reduced locomotor activity. Therefore, it is likely that the higher body weight gain in the WD group during the entire study period to a large extent is explained by the diet-induced reduction in locomotor activity.

It is well known that mice given HFD increase their body weight as a result of increased body fat accumulation, resulting in obesity-related symptoms (1, 9, 11, 16, 30, 31). However, the cause of the HFD-induced obesity is not simply explained by increased energy intake (9, 11, 25, 30). Interestingly, HFD for 4 mo resulted in higher body weights in the C57Bl/6J mice strain compared with the A/J mice strain despite similar calorie intake and higher activity in the C57Bl/6J mice (5). In contrast to our findings, no significant effect of HFD on physical activity in C57Bl/6J mice after 1 mo was observed compared with baseline. The reason for this discrepancy is unclear but might be due to subtle differences between the C57Bl/6J strains used or the diet composition. However, short-term adaptation to WD and the explanation for increased body weight following a switch to WD is, to the best of our knowledge, not fully investigated.

This study was designed to investigate the mechanisms behind increased body weight gain observed when animals are given a WD. The WD fedmice used in the present study has a higher content of neutral fat. However, it must be pointed out that there are other differences between the diets that could have influenced the results in this study. The WD contains cholesterol that has been shown to influence body weight gain in the context of liver X receptor deficiency (23). The major protein sources of the chow diet are soybean, grain, and potatoes, whereas the main source of protein in the WD is casein. Thus the influence of proteins and the cholesterol content of the WD on the parameters investigated in this study needs to be investigated in a separate study.

In this study, a room temperature of 22°C was used for all measurements. However, this is not the thermoneutral zone for mice, and thus the total EE includes all metabolic processes. Interestingly, it was previously found that locomotor activity was increased by a thermoneutral temperature in a B6 mouse strain placed on a HFD for 7 days (12). However, the oxygen consumption (VO2) was lower at thermoneutrality, reflecting a minor contribution of locomotor activity to changes in VO2 under these conditions. Thus thermogenesis and basal metabolism are indeed parameters that are influenced by both ambient temperature and HFD. Hence, the results in the present study might have been affected if a thermoneutral setup would have been used.

In the present study, the energy intake and energy absorption was increased after 24 h, lower after 72 h, and normalized between 7 and 14 days in mice on WD compared with chow-fed mice. Interestingly, an acute and persistent downregulation of the hypothalamic orexigenic signals NPY and AGRP was observed in mice fed WD. Thus these orexigenic signals from the CNS are lowered to levels at which the mass of WD consumed (grams of diet eaten) corresponds to normalization of energy intake. These hypothalamic peptides are regulated by peripheral signals including ghrelin and leptin, among others (7, 24). Ghrelin increases NPY and AGRP expression (26). Plasma ghrelin levels did not decrease before 21 days of WD. Therefore, ghrelin could not explain the early changes in NPY or AGRP expression. However, plasma leptin levels were elevated after only 24 h of WD, which is in line with lower AGRP, NPY, and mass WD consumption. The increased leptin levels and decreased hypothalamic NPY and AGRP expression could help to explain the decrease in mass WD consumption that occurred after 24–48 h on WD.

It is well known that high leptin levels can cause leptin resistance (47). One week of WD did not affect leptin sensitivity, whereas 8 wk of WD induced leptin resistance (30). Thus it is likely that the normalization process of energy intake and absorption observed in this study may partly be a consequence of reduced leptin sensitivity. Leptin is also known to increase EE and body temperature (14, 19, 36). The higher EE and body temperature and the normalization of these parameters over time may thus be an effect of reduced leptin sensitivity.

T3 level increased after 24 h of WD but normalized after 21 days on the diet. In contrast, T4 level tended to decrease after 24 h of WD, but the effect was not significant until 21 days on WD. The transient increase in T3 level is in line with the transient change in body temperature and EE. The changes in T3 and T4 levels are largely consistent with an increased T4/T3 conversion. However, the normalization of T3 levels and decreased T4 levels after 21 days on WD cannot be explained solely by changed T4/T3 conversion. Further studies are needed to identify changes in deiodinase activity or plasma thyroid-stimulating hormone (TSH) levels after the switch to WD. The UCP1 expression in BAT was significantly increased after 72 h on WD, which is in line with increased body temperature after 72 h. T3 is known to increase UCP1 expression in vitro (17) and to increase BAT UCP1 expression to normal levels in hypothyroid fetus (34). In this study, the increased T3 levels in the WD mice may explain the increased UCP1 expression. Changes in plasma leptin and T3 levels may therefore help to explain the transient increase in body temperature.

In addition to the rapid changes in plasma leptin and T3 levels and hypothalamic gene expression levels, the body weight gain and body fat gain were rapid, occurring only 24 h after the switch to WD. HFD can reduce the gastric transit time (42) and potentially explain acutely increased body weight. However, the reduced mass intake of the WD correlated with a lower amount of feces, showing that increased GI content could not explain the higher body weight gain in the WD mice. A control experiment was performed to exclude the possibility that the GI content, after the switch to WD, influenced the measures of body fat. Surgical removal of the GI tract did not influence the result of the DEXA measurements of body fat mass after 24 h on WD. Also, the weight of the dissected fat depots was increased in mice given WD for 24 h. Thus the increase in body weight gain and body fat gain after only 24 h on WD is explained by increased fat mass. Although no major impact was found on reduced GI transit time by the WD, the energy absorption was higher after the first 24 h on WD. To what extent the components of the WD affect nutrient uptake is not clear, and it is unclear whether a reduction in the GI transit time would have increased the absorption even more during the initial 24 h on WD. Although energy absorption is an estimation of energy intake minus energy lost in feces, the feces energy is only a fraction of the energy balance, and the major part of the consumed energy is absorbed.

Together, several of the acute changes that occurred as a result of WD were normalized during the first 3 wk. The changes that may contribute to the increased body weight gain and body fat gain in mice during the first 24 h of WD include increased energy intake, energy absorption, food efficiency, and acutely lowered locomotor activity. However, WD also resulted in higher EE accompanied by increased body temperature over the first 72 h. Thus, although locomotor activity was decreased, total EE was increased over the first days in mice given WD. Hence, the increase in body temperature (and basal metabolism) obscures the effect of lower locomotor activity on total EE during the first days on WD. After 21 days of WD, EE, body temperature, and energy absorption were normalized, but the body weight gain was still increased, mainly as a consequence of increased fat mass. Interestingly, the locomotor activity continued to be lower after 21 days on WD. To investigate to what extent the reduction in locomotor activity affected EE and whether reduced activity could explain the higher body weight gain, we performed a correlation analysis between locomotor activity and EE. It must be pointed out that several assumptions were made to understand the potential role of decreased locomotor activity for the higher body weight gain on day 20–21 in the WD group. First, it was assumed that the mice given WD had the same locomotor activity as the mice given chow diet. Second, the energy cost of the hypothetically increased locomotor activity among mice given WD was based on the trend line produced by correlating locomotor activity and EE in the WD group. This analysis showed that the same locomotor activity in the WD group as in the chow group would have resulted in a 0.90 kcal·day–1·mouse–1 higher EE. Third, we assumed that the mice given WD would have had a body weight gain in the Oxymax system similar to that observed in ordinary cages (cf., Fig. 1A) and the fourth assumption was that the body weight gain on day 20–21 was solely in the form of increased fat mass (cf., Fig. 1, B and C). Therefore, the finding that ~60% of the higher body weight gain in the WD group on day 20–21 could be explained by reduced locomotor activity must be interpreted with caution. However, the results of these calculations clearly indicate that reduced locomotor activity following a switch to WD could have a major impact on body fat gain in mice.

Lower RER was observed after the switch to WD, indicating increased use of lipids as energy source. As expected, average RER for the chow-fed mice coincided well with the FQ for the chow diet. However, not until after 21 days on WD did RER for mice fed WD reach the FQ value for the WD. This "lag" in matching fat intake to utilization would dispose toward the observed body fat accumulation. Interestingly, it has been shown in humans that physical activity increases fat oxidation and reduces the time to match RER and FQ when a HFD is introduced (20, 40). The lowered locomotor activity in the WD mice may therefore contribute strongly to our observation of a prolonged lag time to match fat intake and utilization.

The DA system in the CNS is influenced by ingestive behaviors, body weight, and HFD (22, 32, 46) and is known to influence locomotor activity (44). However, no significant differences in DA were found after 48 h on WD in prefrontal cortex, ventral tegmental area, nucleus accumbens, or stratum, parts of the CNS that are suggested to be important for the DA effects on reward and locomotor activity (44). Hence, changes in DA levels cannot explain the reduced locomotor activity during WD. Anandamide (AEA), an endogenous CB, is known to be increased by HFD (29, 43). Recently, it was found that AEA decreases locomotor activity (45) and, hence, also might play a role in alterations in locomotor activity after a switch to WD. In the present study, no differences in mRNA expression of the CB receptors CB1, CB2, and GPR55 were observed as a result of WD exposure. The plasma levels of AEA were lower in mice given WD for 5 h but increased after 24 and 48 h on WD. Since a decrease in locomotor activity occurred only 5 h after the start of WD, it is unlikely that changed circulating levels of AEA could explain the acute drop in locomotor activity. However, the diurnal changes in AEA levels in mice given chow, but not in mice given WD, indicate that WD changes diurnal rhythm of AEA. Further careful studies are needed to clarify the influence of the WD to disrupt the diurnal pattern of plasma AEA levels and how this would influence locomotor activity and ingestive behaviors.

In summary, this study investigated the acute and chronic effects of a Western diet on a number of metabolic parameters. Our findings indicate that several parameters are acutely altered by WD feeding, including increased body weight, body fat, thermogenesis, and decreased locomotor activity. In addition, several of the parameters are normalized when the mice eventually adapt to the novel diet, but the body weight gain is still higher. Calculations of the contribution of locomotor activity on EE revealed that a major part of the energy gain in mice given WD could be explained by reduced locomotor activity. We believe that these novel findings have shed light on the question whether changed locomotor activity is a cause or a consequence of obesity in mice given WD and thus may be important for the general understanding of the effects of WD on body weight regulation.


   ACKNOWLEDGMENTS
 
We acknowledge Magnus Kjaer for assistance with the statistical analysis and Lennart Svensson, Lena Amrot Fors, Anna Tuneld, Charlotte Lindgren, Anne-Cristine Carlsson, Martina Johansson, Gisela Häggblad, and Marie-Louise Berglund Zackrisson for kindly helping with the plasma assays. In addition, we thank Marie Jönsson, Maria Lennerås, and Daniel Andersson for assistance with the experiments. Stephan Hjorth and Len Storlien are acknowledged for scientific advice. Finally, Göran Hansson is acknowledged for analysis of AEA levels.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Bjursell, AstraZeneca R&D Mölndal, S-43183 Mölndal, Sweden (e-mail: mikael.bjursell{at}astrazeneca.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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