Environmental enrichment and cafeteria diet attenuate the response to chronic variable stress in rats

Physiology & Behavior 139 (2015) 41–49

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Environmental enrichment and cafeteria diet attenuate the response to chronic variable stress in rats N. Zeeni a,⁎, M. Bassil a, G. Fromentin b,c, C. Chaumontet b,c, N. Darcel b,c, D. Tome b,c, C.F. Daher a a b c

Lebanese American University, Department of Natural Sciences, School of Arts and Sciences, PO Box 36, Byblos, Lebanon AgroParisTech, CNRH-IdF, UMR914 Nutrition Physiology and Ingestive Behavior, F-75005 Paris, France INRA, CNRH-IdF, UMR914 Nutrition Physiology and Ingestive Behavior, F-75005 Paris, France

H I G H L I G H T S • • • • •

Rats were housed in standard cages or cages with environmental enrichment (EE). Rats were fed a high-carbohydrate (HC) or a palatable “cafeteria” (CAF) diet. We compared the effect of diet and EE on the response to chronic variables stress. Stress-induced increase in ACTH was attenuated with EE and the CAF diet. Stress-induced rise in corticosterone was reduced by EE and CAF diet combination.

a r t i c l e

i n f o

Article history: Received 22 May 2014 Received in revised form 2 November 2014 Accepted 3 November 2014 Available online 11 November 2014 Keywords: Palatable diet Corticosterone ACTH Dietary intake Body adiposity Blood lipids

a b s t r a c t Exposure to an enriched environment (EE) or the intake of a highly palatable diet may reduce the response to chronic stress in rodents. To further explore the relationships between EE, dietary intake and stress, male Sprague–Dawley rats were fed one of two diets for 5 weeks: high carbohydrate (HC) or “cafeteria” (CAF) (Standard HC plus a choice of highly palatable cafeteria foods: chocolate, biscuits, and peanut butter). In addition, they were either housed in empty cages or cages with EE. After the first two weeks, half of the animals from each group were stressed daily using a chronic variable stress (CVS) paradigm, while the other half were kept undisturbed. Rats were sacrificed at the end of the 5-week period. The effects of stress, enrichment and dietary intake on animal adiposity, serum lipids, and stress hormones were analyzed. Results showed an increase in intra-abdominal fat associated with the CAF diet and an increase in body weight gain associated with both the CAF diet and EE. Furthermore, the increase in ACTH associated with CVS was attenuated in the presence of EE and the CAF diet independently while the stress-induced increase in corticosterone was reduced by the combination of EE and CAF feeding. The present study provides evidence that the availability of a positive environment combined to a highly palatable diet increases resilience to the effects of CVS in rats. These results highlight the important place of palatable food and supportive environments in reducing central stress responses. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Dietary factors are capable of influencing the response to chronic stress and palatable food, specifically, was found to dampen stress responses [1], but more so in the presence of a variety of food items such as in the “cafeteria” diet model [20]. The latter diet refers to the offering of a choice of highly palatable cafeteria-type foods (high fat, energy, and sugar contents) to rodents [32,34,45]. These highly palatable non-monotonous diets were shown to have stress dampening effects possibly by activating the reward circuitry in the brain [39] and ⁎ Corresponding author. Tel.: +961 9547262x2317; fax: +961 9547256. E-mail address: [email protected] (N. Zeeni).

http://dx.doi.org/10.1016/j.physbeh.2014.11.003 0031-9384/© 2014 Elsevier Inc. All rights reserved.

were shown to relieve anxiety-like behavior in rats [23]. Also, it was suggested that the increase in glucocorticoid levels associated with stress, leads to a drive towards pleasurable activities such as ingesting “comfort food” and causing an indirect increase in abdominal fat depots [6]. Harsh environmental conditions can also affect the behavioral, physiological, and biochemical responses of animals exposed to stressors in a strain-dependent manner [17]. On the other hand, environmental enrichment (EE) looks for improving the welfare of animals by reducing stress [4,17,29]. Rodents under enriched conditions developed brain plasticity and better performance on learning tests [41,46]. Animals maintained under EE have also been shown to have reduced aggression, anxiety, fear, stress and excitability, as well as better learning abilities

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N. Zeeni et al. / Physiology & Behavior 139 (2015) 41–49

Fig. 1. Experimental protocol. Sixty-four adult male Sprague–Dawley rats were assigned to one of two dietary groups (standard high carbohydrate (HC) versus cafeteria diet (CAF)) and one of two enrichment conditions (enriched versus standard). After the first 2 weeks (baseline period), rats from each dietary and enrichment groups were again divided in two groups: control and stressed. Controls (no CVS) were kept undisturbed in their home cages during the following 3-weeks of treatment (CVS exposure period), and the others were stressed daily for the remaining period of the study using a chronic variable stress (CVS) model.

than those maintained under standard conditions [2,14,15,25,31]. In humans, it was suggested that with a chronic stressful lifestyle, cortisol could potentially change the function of specific neuronal circuits in the brain and that these effects could be modulated and attenuated in enriched environmental conditions [26,27]. The aim of the current study was to evaluate the separate and combined effects of a palatable cafeteria-type diet and environmental enrichment on the response to chronic variable stress (CVS) in rats. CVS is a model in which variable acute stress paradigms are applied unpredictably over several weeks in order to prevent the habituation induced in rodents by chronic application of the same stressor [7,24,36], leading to a diminution of behavioral and physiological responses to the stressor, including attenuated stress-induced hypophagia [18]. Traditionally, CVS has served as a model of depression in rodent studies [16,43] and is considered as a relevant stress model designed to be resistant to

habituation, since it induces chronic hyperactivity of the hypothalamic–pituitary–adrenal (HPA) axis, as indicated by persistent elevated corticosterone levels [24,30]. 2. Methods A summary of the experimental protocol can be found in Fig. 1. 2.1. Animals and diet Sixty-four adult male Sprague–Dawley rats (Lebanese American University Stock) aged initially 8–10 weeks old were housed in groups of four in a temperature and humidity-controlled room under a 12:12 light/dark cycle (lights on at 08:00 h) during the whole length of the experiment. The animals were weight-matched (313 ± 1 g) and assigned

Table 1 Nutrient composition of the standard HC diet and the cafeteria diet food items. Food composition data are provided by the manufacturers' information or food tables.

Protein (wt.%) Carbohydrates (wt.%) Sugars Fat (wt.%) Fat breakdown Saturated fat Mono-unsaturated fat Poly-unsaturated fat Fiber (wt.%) Metabolizable energy (kJ/g) Energy (protein) Energy (carbohydrate) Energy (fat) a b c d e

Standard high-carbohydratea

Chocolateb,e

Biscuitsc,e

Peanut butterd,e

19 65.3 9.2 9.6

7.6 59.1 43.7 26.2

6.3 67.7 13.6 21.8

23.6 12.5 6.3 46.8

18% 29% 47% 4.3 17.7 18% 62% 20%

13% 46% 41% 3 21.1 6% 47% 47%

48% 0% 52% 3.5 20.7 5% 55% 40%

68% 0% 32% 6.2 24.5 17% 9% 75%

Hawa chicken rodent starter diet no 1, Lebanon. Kit Kat, Nestle S.A., Switzerland. Digestive, McVitie's, United Biscuits, UK. Monarch. Items shown to cause hyperphagia in rats fed cafeteria-type diets [34].

N. Zeeni et al. / Physiology & Behavior 139 (2015) 41–49 Table 2 Schedule of stressor agents used during the chronic stress treatment. Day of treatment

Stressor used

Duration

Time of administration

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Flashing light Neighbor cage Cage tilt Space reduction No stressor Flashing light Forced swimming-warm Restraint Cage tilt Flashing light Neighbor cage No stressor Cage tilt Restraint Flashing light Forced swimming—cold Restraint Space reduction No stressor Restraint Forced swimming—warm

4h 14 h 3h 4h x 4h 10 min 1h 4h 3h 14 h x 3h 3h 4h 10 min 2h 5h x 2h 10 min

12:00–16:00 17:00–07:00 10:00–13:00 08:00–12:00 14:00–18:00 12:00–12:10 09:00–10:00 08:00–12:00 12:00–15:00 17:30–07:30 11:00–14:00 10:00–13:00 14:00–18:00 15:00–15:10 12:00–14:00 08:00–13:00 09:00–11:00 16:00–16:10

43

carbohydrate and a choice of highly palatable cafeteria-style foods with known caloric intake: chocolate, biscuits and peanut butter (Table 1). The diets were given ad libitum for 5 weeks. All experimental protocols were approved by the Animal Ethical Committee of the Lebanese American University, which complies with the Guide for the Care and Use of Laboratory Animals [28]. Food intake was monitored 3 times a week and body weight gain was monitored twice a week throughout the study. Food intake, corrected for spillage, including individual CAF diet items, was recorded at 8 am by measuring the difference in food cup weight before and after presentation to the rats. 2.2. Cage enrichment Rats belonging to the enriched groups were housed in cages provided with differently shaped plastic toys, and a running wheel (Ani'Mall Pet Store, Beirut, Lebanon). Toys included colored transparent plastic tunnels and hollow balls that the animals could move in and out of or over, chew toys (nontoxic dog bones and other plastic toys), as well as nesting material. As rats were allowed the freedom to move and exercise voluntarily, with accessibility to complex stimuli, they had more physical and intellectual stimulation than the rats housed in standard laboratory cages. 2.3. Chronic mild stress procedure

to one of two enrichment conditions (non-enriched versus enriched) and one of two dietary groups (standard high carbohydrate (HC) versus cafeteria diet (CAF)). The cafeteria diet consists of the standard high-

After the first 2 weeks of diet (baseline period), rats from each dietary and enrichment groups were again divided in two groups after

Fig. 2. Energy intake (kJ/rat/day) of rats fed a standard high-carbohydrate (HC) or cafeteria (CAF) diet, housed in enriched or non-enriched cages, and either submitted to a chronic variable stress paradigm or not during the CVS exposure period (n = 8). Data are expressed as the mean ± SEM. aStatistical difference with regard to the high-carbohydrate dietary group in the same stress and enrichment conditions. bStatistical difference with regard to the non-stressed group in the same diet and enrichment conditions. cStatistical difference with regard to the non-enriched group in the same diet and stress conditions. *Statistical difference with regard to the same group but during the baseline period.

n.s.s. n.s.s. n.s.s. n.s.s. n.s.s. n.s.s. n.s.s. n.s.s n.s.s. n.s.s. n.s.s. p b 0.001 ± ± ± ± 392.2 1.84 11.3 43.2 9.6 0.22a 0.7 0.6 ± ± ± ± 392 1.92 10.1 40.7 ± ± ± ± b

a

Statistical difference with regard to the high-carbohydrate dietary group in the same stress and enrichment conditions. Statistical difference with regard to the non-stressed group in the same diet and enrichment conditions.

371.8 1.51 9.2 42.1 6.8a 0.21a 0.7a 0.8 ± ± ± ± 402.3 2.02 9.9 38.7 11.5 0.08 0.7 0.7b ± ± ± ± 360 0.71 8.6 45.7 13.5 0.15 0.7 0.9 ± ± ± ± 381.1 0.79 8.9 39.4 Final body weight (g) Abdominal fat (% body weight) Liver weight (g) Adrenal gland weight (mg)

340.5 0.63 8 37.1

± ± ± ±

4.1 0.19 0.7 0.9

331.8 0.79 9.4 41.1

± ± ± ±

4.7 0.15 0.7 0.8b

Stressed

Non-stressed

Non-enriched cages

Stressed

4.9a 0.22a 0.7 0.9b

Non-stressed

Enriched cages

Stressed

13.2a 0.22a 0.7a 1.1

p = 0.003 p b 0.001 p = 0.012 n.s.s.

Effect of enrichment Effect of stress Effect of diet

Statistical significance

Non-stressed

Body weight gain and dietary intake data were analyzed separately using a four-way ANOVA with treatment (No Stress versus Stress), enrichment (non-enriched cages versus enriched cages) and diet (HC versus CAF) as the between subject factors, and time as the withinsubject factor. Body composition, plasma hormones, and blood lipids were analyzed using a three-way ANOVA with treatment (No Stress versus Stress), enrichment (non-enriched cages versus enriched cages) and diet (HC versus CAF) as factors. Descriptive data are presented as means ± standard error of the mean (SEM). Significant main effect differences were tested using Tukey–Kramer's post-hoc test for multiple comparisons. All data were analyzed using the SPSS 19 statistical package, with statistical significance defined as p b 0.05.

Enriched cages

2.6. Statistical analysis

Stressed

After euthanasia, the intra-abdominal fat (epididymal, mesenteric and retroperitoneal), liver and adrenal glands of the rats were removed and weighed.

Non-stressed

2.5. Body composition

Non-enriched cages

The rats were fasted overnight at the end of the CVS procedure. The following morning they were deeply anesthetized with pentobarbital (100 mg/kg), blood was drawn from the inferior vena cava 5 to 6 min after the induction of anesthesia, and the rats then euthanized by exsanguination. The blood samples were centrifuged at 2000 g for 15 min. The collected serum was then stored at − 80 °C for subsequent analysis. Blood lipid analysis (total cholesterol, HDL cholesterol and LDL cholesterol) was performed using Spinreact kits (Spinreact, Spain). RIA or ELISA kits were used to measure serum corticosterone (CORT) (Double Antibody 125I Radioimmunoassay Kitor rats and mice, MP Biomedicals, LLC, Solon, OH, USA), insulin (DRG diagnostics, Germany) and adrenocorticotropic hormone (ACTH) levels (Calbiotech, Spring Valley, CA, USA). All samples were run in duplicate and analyzed within the same assay.

Cafeteria diet

2.4. Plasma hormones & blood lipid analysis

High-carbohydrate diet

being weight-matched (n = 8): control and stressed. Controls were kept undisturbed in their home cages during the following 3-weeks of treatment, and the others were stressed daily for the remaining period of the study using a chronic variable stress model. This previously used stress model [45] consisted of a 21-day variable stressor paradigm during which different individual stressors were applied each day for different lengths of time (one stressor per day). The following stressors were used: (a) Cage tilt for 3 or 4 h (home cages were tilted to 30° from the horizontal); (b) space reduction for 4 or 5 h (8 rats were placed in a collective cage usually designed for 4 to 5 rats, dimensions 50 × 40 × 21 cm); (c) restraint for 1 to 3 h (rats were placed into a perforated plastic tube of 6 × 20 cm in which they could not turn over); (d) forced swimming for 10 min in cold water (5 rats were placed together in a plastic tank, dimensions 100 cm height × 50 cm diameter containing 75 cm of water at 18 °C) or in (e) warm water (same procedure at 26 °C); (f) flashing light for 3 or 4 h (flashes at 40 W with frequency of 5 flashes/s); and (g) neighbor cages (move to an unclean unoccupied neighbor cage for 14 h). Animals remained on their diet and enrichment conditions throughout stress exposure, unless the stressor was applied outside of the cage. Stress application started at different times every day, in order to minimize its predictability. Control and stressed rats were housed in two separate sections of a large animal room to minimize their excitement from smells and noise exchange. Table 2 reports the detailed stressor schedule.

Interaction effects

N. Zeeni et al. / Physiology & Behavior 139 (2015) 41–49 Table 3 Final body weight, abdominal fat, liver and adrenal gland weights of rats fed a high carbohydrate or cafeteria diet, housed in enriched or non-enriched cages, and either submitted to a chronic variable stress paradigm or not (n = 8). Data expressed as mean ± SEM.

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N. Zeeni et al. / Physiology & Behavior 139 (2015) 41–49

3. Results

45

were housed in non-enriched cages had significantly lower body weight gain (1.7 ± 0.1 g/day) compared to rats that were housed in enriched cages (2.1 ± 0.1 g/day) (Fig. 3).

3.1. Energy intake During the baseline period of the experiment, there was a significant effect of diet on energy intake (F(1,47) = 14.9, p b 0.001). Animals fed the CAF diet had significantly higher mean energy intake (636.8 ± 17.4 kJ/day) compared to animals fed the HC diet (491.8 ± 13.9 kJ/day). There was no significant effect of EE on energy intake during the baseline period (Fig. 2). During the CVS exposure period, there was a significant effect of diet (F(1,47) = 10.9, p b 0.001) and stress (F(1,47) = 10.9, p = 0.001) on energy intake. Animals fed the CAF diet (the stressed and nonstressed groups included) had significantly higher mean energy intake (602.4 ± 15.4 kJ/day) compared to animals fed the HC diet (410.3 ± 13.1 kJ/day). Also, stressed rats had a significantly lower mean energy intake (468.5 ± 22.4 kJ/day) compared to non-stressed rats (544.6 ± 16.5 kJ/day) during CVS exposure There was no significant effect of EE on energy intake during the CVS exposure period (Fig. 2). Among stressed animals, energy intake was significantly affected by time: stressed animals ate significantly less during the CVS exposure period (467.5 ± 21.8 kJ/1 day) compared to the baseline period (580 ± 20.7 kJ/day) (F(1,46) = 2.42, p = 0.02) (Fig. 2). When comparing the average energy intake from cafeteria items, it appears that the rats were consuming the biscuits the most followed by HC food, chocolate and peanut butter during the baseline and CVS exposure periods (Table 4). There was no significant effect of stress or enrichment on the consumption of any of the CAF items. However, within stressed animals there was a significant effect of time on the consumption of peanut butter (F(1,7) = 43.1, p b 0.001), chocolate (F(1,7) = 5.5, p = 0.05) and biscuits (F1,7) = 6.1, p = 0.05). Stressed animals housed in both enriched and non-enriched cages consumed less peanut butter and biscuits during the CVS exposure period compared to the baseline period. In addition, stressed animals in non-enriched cages decreased their consumption of chocolate during the CVS exposure period (Table 4).

3.3. Body composition There was a significant effect of stress (F(1,47) = 8.1, p b 0.001) on the adrenal gland weight of the rats, with the rats under CVS having significantly higher adrenal gland weight (43.1 ± 0.9 g) compared to the rats from the non-stressed groups (39 ± 0.8 g). However, there was no significant effect of diet (F(1,47) = 2.03, p = 0.2), or enrichment (F(1,47) = 0.5, p = 0.5) on the adrenal gland weight of the rats (Table 3). There was a significant effect of diet on the liver weight of rats (F(1,47) = 6.9, p = 0.012), with rats from the CAF group having higher liver weights (10.13 ± 0.3 g) than those given the HC diet (8.7 ± 0.4 g). Liver weights were not significantly affected by stress (F(1,47) = 0.4, p = 0.5) or enrichment conditions (F(1,47) = 1.3, p = 0.3) (Table 3). The abdominal fat percent of the rats was significantly affected by diet (F(1,45) = 51.6, p b 0.001). More precisely, rats that were fed the HC diet had significantly lower abdominal fat (0.7 ± 0.11% of body weight) compared to rats that were fed the CAF diet (1.82 ± 0.11% of body weight). However, there was no significant effect of stress (F(1,45) = 0.4, p = 0.5) or enrichment (F(1,45) = 0.08, p = 0.8) on the abdominal fat of the rats (Table 3). 3.4. Serum lipids and glucose Total serum cholesterol levels were significantly affected by diet (F(1,45) = 69.3, p b 0.001). More precisely, rats on the CAF diet had significantly higher total cholesterol levels (136.3 ± 4.9 mg/dl) compared to rats on the HC diet (77 ± 5.1 mg/dl). However, there was no significant effect of stress (F(1,45) = 0.9, p = 0.3) or enrichment (F(1,45) = 0.2, p = 0.6) on the total cholesterol levels of the rats (Table 5). There was a significant effect of diet on HDL levels (F(1,45) = 140.6, p b 0.001) with rats fed the CAF diet having significantly higher HDL levels (43.9 ± 1.6 mg/dl) compared to rats fed the HC diet (17.1 ± 1.6 mg/dl). However, there was no significant effect of stress (F(1,45) = 2.6, p = 0.12) or enrichment (F(1,45) = 2.1, p = 0.17) on the total cholesterol levels of the rats (Table 5). Serum glucose levels were significantly affected by diet (F(1,41) = 4.7, p = 0.04) and by stress (F(1,41) = 5.6, p = 0.02). More precisely, animals fed the CAF diet had higher glucose levels (98.5 ± 3 mg/dl) compared to animal fed the HC diet (89.1 ± 3.1 mg/dl). Also, although still within the normal range, non-stressed animals had significantly higher glucose levels (99 ± 3.1 mg/dl) compared to animals exposed to CVS (88.7 ± 3 mg/dl) (Table 5).

3.2. Body weight gain During the baseline period of the experiment, there was a significant effect of diet on the body weight gain of the animals (F(1,42) = 22.2, p b 0.001). Rats that were fed the HC diet had significantly lower body weight gain (1.6 ± 0.2 g/day) compared to rats that were fed the CAF diet (2.6 ± 0.2 g/day). There was no significant effect of stress or EE on body weight gain during the baseline period (Fig. 3). There was a significant effects of stress (F(1,47) = 3.9, p = 0.05) on the body weight gain of rats during the CVS exposure period. Stressed rats had significantly lower body weight gain (1.6 ± 0.13 g/day) compared to non-stressed rats (2.2 ± 0.12 g/day) (both diets and enrichment conditions included). During the CVS exposure period, there was a significant interaction effect of diet and EE on the body weight gain of rats (F(1,47) = 7.1, p = 0.01). Rats that were fed the HC diet had significantly lower body weight gain (1.4 ± 0.1 g/day) compared to rats that were fed the CAF diet (2.4 ± 0.1 g/day). In addition, rats that

3.5. Plasma hormones 3.5.1. ACTH Stressed animals (both diets and enrichment conditions included) showed increased serum ACTH levels (20.3 ± 0.3 pg/ml) compared to

Table 4 Energy intake (kJ/rat/day) from different cafeteria items of rats fed a cafeteria diet, housed in enriched or non-enriched cages and either submitted to a chronic variable stress paradigm or not during weeks 3 to 5. Data are expressed as the mean ± SEM. Baseline period (weeks 1–2)

CVS exposure period (weeks 3–5)

Enriched

Non-stressed rats

Non-enriched

Enriched Standard HC Biscuits Peanut butter Chocolate

122.3 80.7 21.3 44.9

± ± ± ±

4 2.35 2.4 2.7

120.9 76.5 19.25 39.25

± ± ± ±

3.9 2.9 1.25 2.8

115.1 80 24.9 38.4

± ± ± ±

4.8 2.2 2.1 4.3

⁎ Statistical difference with regard to the baseline period in the same enrichment condition (p b 0.05).

Stressed rats Non-enriched

Enriched

Non-enriched

126.4 80.1 17.3 40.2

100.2 ± 4.9⁎ 74.4 ± 3.6 13 ± 1.1⁎ 41.1 ± 3.3

94.3 75.3 14.2 33.3

± ± ± ±

5.5 3.5 2.3 2.4

± ± ± ±

3.7⁎ 3.9 2.8⁎ 2.2⁎

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N. Zeeni et al. / Physiology & Behavior 139 (2015) 41–49

Fig. 3. Weight gain (g/day) of rats fed a standard high-carbohydrate (HC) or cafeteria (CAF) diet, housed in enriched or non-enriched cages, and either submitted to a chronic variable stress paradigm or not during the CVS exposure period (n = 8). Data are expressed as the mean ± SEM. aStatistical difference with regard to the high-carbohydrate dietary group in the same stress and enrichment conditions. bStatistical difference with regard to the non-stressed group in the same diet and enrichment conditions. *Statistical difference with regard to the same group but during the baseline period.

non-stressed animals (18.5 ± 0.3 pg/ml), (F(2,42) = 16.1, p b 0.001). There were no significant effects of diet or EE on serum ACTH levels. However, there were significant interaction effects of diet and stress (F(2,42) = 11.3, p = 0.002) as well as enrichment and stress (F(2,42) = 4.7, p = 0.04) on ACTH level. More precisely, the increase in ACTH associated with CVS was attenuated in the presence of EE as well as in the presence of the CAF diet (Fig. 4). 3.5.2. Corticosterone (CORT) Stressed animals (both diets and enrichment conditions included) showed increased serum CORT levels (129.5 ± 3.2 ng/ml) compared to non-stressed animals (96.8 ± 3.4 ng/ml), (F(2,42) = 49.5, p b 0.001). In addition, there was a significant interaction effect of diet and enrichment (F(2,42) = 4.7, p = 0.036) on serum CORT levels. More precisely, stressed animals fed the CAF diet had significantly lower CORT levels (115.2 ± 4.5 ng/ml) than those on the HC diet (143.7 ± 4.5 ng/ml) and those housed in enriched cages had significantly lower CORT levels (110.9 ± 4.5 ng/ml) than those housed in non-enriched conditions (148 ± 4.5 ng/ml) (Fig. 5). In order to further understand the interaction between diet and enrichment on corticosterone levels, data for the stressed and unstressed groups were tested separately. In non-stressed animals, there was a

significant effect of diet (F(1,46) = 9.2, p = 0.007) but no significant effect of EE (F(1,46) = 0.3, p = 0.6) or the interaction of diet and EE (F(1,46) = 4.1, p = 0.06). On the other hand, in stressed animals, there was a significant effect of diet (F(1,46) = 14.1, p = 0.001) as well as EE (F(1,46) = 24, p b 0.001), but there was no significant interaction effect (F = 1.8, p = 0.2). This result indicates that that the interaction between diet and EE is significant only when we control for stress. In addition, the effect of diet on CORT was tested separately, by running the model on all animals without taking into account enrichment conditions. There was a significant effect of diet (F = 12.6, p = 0.001) as well as stress (F = 27.9, p b 0.001). Also, the separate effect of EE was tested by running the model on all animals without taking into account diet conditions. In this case, there was a significant effect of the interaction of stress with EE (F = 11.08, p = 0.02). When removing this interaction effect from the model, there was a significant effect of EE (F = 12.2, p = 0.02) and stress (F = 30.2, p b 0.001). Therefore, the significance of the interaction between diet and EE that we had initially obtained (F(2,42) = 4.7, p = 0.036) was not as large as the unitary effect of each variable (F = 8.7, p = 0.005 for diet and F = 12.2, p = 0.02 for EE), indicating that the effect on corticosterone was not synergistic but rather additive.

n.s.s. n.s.s. n.s.s. n.s.s. n.s.s. n.s.s. n.s.s. n.s.s. n.s.s. n.s.s. n.s.s. p = 0.024 p b 0.001 p b 0.001 n.s.s. p = 0.04 9.8a 3.2a 5.2 5.6b ± ± ± ± 127 47.3 53 91 9a 3.1a 5.2 5.6a ± ± ± ±

3.5.3. Insulin Serum insulin levels were not affected by dietary treatment (F(2,42) = 0.6, p = 0.80), stress (F(2,42) = 0.09, p = 0.76) or enrichment (F(2,42) = 2, p = 0.16) (Table 5).

9.8a 3.1a 5.6 6.9 ± ± ± ± 144.6 50.4 49.4 97.2 b

Statistical difference with regard to the high-carbohydrate dietary group in the same stress and enrichment conditions. Statistical difference with regard to the non-stressed group in the same diet and enrichment conditions.

4. Discussion

a

9.8a 3.2a 6.3 7.3 ± ± ± ± 150.2 46.5 63.9 106.3 10.8 2.2 5.6 6.2 ± ± ± ± 77.9 16 52.7 85.5 10.8 3.4 5.6 6.2 ± ± ± ± 91.6 18.8 49.9 86.4 9.8 3.1 5.6 6.2b ± ± ± ±

Stressed Stressed

63.2 19 62.9 84.4 9.8 3.1 5.6 6.2 ± ± ± ± 75 14.6 56 100.3 Total cholesterol (mg/dl) HDL (mg/dl) Serum insulin (μg/L) Serum glucose (mg/dl)

Stressed Non-stressed

Non-stressed Non-stressed

Non-enriched cages

47

Fig. 4. Serum ACTH concentration (pg/ml) of rats fed a standard high-carbohydrate (HC) or cafeteria (CAF) diet, housed in enriched or non-enriched cages, and either submitted to a chronic variable stress paradigm or not (n = 8). Data are expressed as the mean ± SEM.*Statistical difference with regard to the non-stressed group having the same diet and enrichment conditions. p b 0.05 is considered significant.

123.2 31.5 52.2 112

Effect of stress Non-stressed

Stressed

Effect of diet

Statistical significance Enriched cages Cafeteria diet

Non-enriched cages Enriched cages

High-carbohydrate diet

Table 5 Serum cholesterol, HDL-cholesterol, insulin and glucose of rats fed a cafeteria diet, housed in enriched or non-enriched cages and either submitted to a chronic variable stress paradigm or not during weeks 3 to 5.

Effect of enrichment

Interaction effects

N. Zeeni et al. / Physiology & Behavior 139 (2015) 41–49

The objective of this study was to investigate the separate and combined effects of a palatable cafeteria diet and environmental enrichment on the metabolic and hormonal responses to CVS in rats. The present findings show a significant interaction between CAF diet and EE resulting in reduced serum corticosterone levels in stressed rats.

Fig. 5. Serum corticosterone concentration (ng/ml) of rats fed a standard highcarbohydrate (HC) or cafeteria (CAF) diet, housed in enriched or non-enriched cages, and either submitted to a chronic variable stress paradigm or not (n = 8). Data are expressed as the mean ± SEM.*Statistical difference with regard to the non-stressed group having the same diet and enrichment conditions. p b 0.05 is considered significant. There was a significant interaction effect of diet and EE on CORT levels: the combined effect of EE and the CAF diet had a greater reducing impact on CORT levels than each effect taken alone.

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Among stressed animals, food intake was lower during the CVS exposure period compared to the control period. This result is in accordance with other studies that show a reduction in food intake in response to CVS [10,35,45]. Rats from the CAF groups showed higher overall food consumption owing to the increased palatability as well as the higher saturated and trans-fat content and had similar preference patterns before and during stress. Rats from the CAF groups were consuming biscuits the most, followed by standard HC food, chocolate and finally peanut butter. Similar consumption patterns were found for these items in other studies [34,45]. Body weight gain was affected by CVS but also by the CAF diet and EE conditions especially during the CVS exposure period. Indeed, stress was associated with a decrease in the rate of weight gain during the CVS exposure period (weeks 3–5). Previous studies have also shown a prominent decrease in the amount and rate of weight gain in stressed rats [11,44]. In agreement with previous studies showing that body weight and fat depot, particularly intra-abdominal fat increase after access to a palatable diet [9,33,45], this study shows that animals fed the cafeteria diet had higher body weight, liver weight and intraabdominal fat percentages compared to animals on the HC diet. Interestingly, rats housed in enriched cages gained more weight than those in non-enriched cages without having higher abdominal adiposity. Similar weight gain and adiposity patterns were found with enriched cages in rodents [17,22], possibly due to an increase in physical activity leading to an increase in lean body mass [12]. CAF-fed animals had higher total cholesterol and HDL cholesterol levels as well as higher liver weights compared to the other animals. Similar effects of cafeteria diets on blood lipids have previously been described [21,37,45]. The increase in liver weight may be due to triglyceride accumulation as a consequence of the high fat content of the CAF diet, and suggests impaired liver metabolism and steatosis [21]. In addition to decreased weight gain, CVS was associated with increased ACTH and CORT levels as it was already found in similar rodent chronic stress models [24,30,45]. Moreover, the results show that CVS was associated with an increase in the adrenal gland weight of the animals. This increase may be due to hyperplasia and hypertrophy occurring in adrenal sub-regions because of stress-related ACTH secretions [38]. On the other hand, EE did not have any effect on liver or adrenal gland weight which is consistent with recent findings in rats and mice [17,22]. Rodents housed with EE were previously shown to have lower baseline ACTH and CORT concentrations [2] as well as lower fecal CORT metabolite levels [12] compared to animals housed in standard conditions. In addition, previous findings have suggested that palatable food consumption reduces the overall impact of stress in rats [8, 23,45] and in mice [19]. In the present study, while CVS alone tends to increase ACTH levels, its combination with EE or with a CAF diet leads to an attenuation of the stressed-induced ACTH upsurge. In addition, there was a significant interaction between diet and EE on CORT levels, resulting in reduced serum corticosterone levels in stressed rats. It must be noted that the relatively high baseline hormone values obtained in the present experiment may be due to the overnight fast of the animals before their sacrifice as well as the pentobarbital injection which was previously shown to increase serum CORT and ACTH levels in rats [40] . However, significant effects of the independent variables were still observed. CVS models are known to induce chronic hyperactivity of the hypothalamic–pituitary–adrenal (HPA) axis leading to an increase in adrenal gland weight as well as elevated corticosterone levels as seen in the current study [24,30]. Diet palatability could attenuate the response to CVS through effects on the HPA axis stress response. Indeed, the HPA axis is a conductor for stress response and is also tightly intertwined with the endocrine regulation of appetite. Palatable food can stimulate an endogenous opioid mechanism that decreases the activity of the HPA axis and thus attenuates the response to stress [1,3,38,45]. In addition, EE may also attenuate activation of the HPA axis via the hippocampus [5]. In fact, while chronic stress alters hippocampal structure by changing its

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