Effects of comfort food on food intake, anxiety-like behavior and the stress response in rats

Physiology & Behavior 103 (2011) 487–492

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Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b

Effects of comfort food on food intake, anxiety-like behavior and the stress response in rats☆ D. Ortolani a,b, L.M. Oyama c, E.M. Ferrari b, L.L. Melo a, R.C. Spadari-Bratfisch a,⁎ a b c

Department of Biosciences, Federal University of São Paulo, Santos, SP, Brazil Department of Anatomy, Cell Biology, Physiology and Biophysics, Institute of Biology, State University of Campinas, Campinas, SP, Brazil Department of Physiology, Federal University of São Paulo, São Paulo, SP, Brazil

a r t i c l e

i n f o

Article history: Received 29 January 2011 Received in revised form 23 March 2011 Accepted 27 March 2011 Keywords: Foot-shock stress Comfort food Anxiety Corticosterone Leptin Insulin Behavior

a b s t r a c t It has been suggested that access to high caloric food attenuates stress response. The present paper investigates whether access to commercial chow enriched with glucose and fat, here referred to as comfort food alters behavioral, metabolic, and hormonal parameters of rats submitted to three daily sessions of footshock stress. Food intake, anxiety-like behaviors, and serum levels of insulin, leptin, corticosterone, glucose and triglycerides were determined. The rats submitted to stress decreased the intake of commercial chow, but kept unaltered the intake of comfort food. During the elevated plus maze (EPM) test, stressed rats increased the number of head dipping, entries into the open arms, as well as the time spent there, and decreased the number of stretched-attend posture and risk assessment. These effects of stress were independent of the type of food consumed. Non-stressed rats ingesting comfort food decreased risk assessment as well. Stress and comfort food increased time spent in the center of the open field and delayed the first crossing to a new quadrant. Stress increased the plasma level of glucose and insulin, and reduced triglycerides, although consumption of comfort food increases glucose, triglyceride and leptin levels; no effect on leptin level was associated to stress. The stress induced increase in serum corticosterone was attenuated when rats had access to comfort food. It was concluded that foot-shock stress has an anorexigenic effect that is independent of leptin and prevented upon access to comfort food. Foot-shock stress also has an anxiolytic effect that is potentiated by the ingestion of comfort food and that is evidenced by both EPM and open field tests. © 2011 Elsevier Inc. All rights reserved.

1. Introduction The stress reaction is known to include metabolic changes in an effort of the organism to maintain homeostasis and increase chances of survival. The glucocorticoids and catecholamines orchestrate the stress reaction, altering the activity of most of the organic systems as well as the overall metabolism [1]. Metabolic responses to stress include an increase in plasma glucose, due to the stimulation of gluconeogenesis and glucogenolysis in the liver and the reduction of glucose uptake in insulin dependent tissues; mobilization of amino acids from extra-hepatic tissues; stimulation of lipolysis; and an increase in the metabolic rate [2]. All of these processes enhance the available energy which makes it possible to cope with stress and survive. However, both inadequate control of the stress response and repeated exposure to stress may represent a severe threat to health and well being.

☆ This paper presents part of the work towards a Master's degree of Daniela Ortolani. ⁎ Corresponding author at: Departamento de Biociências, UNIFESP, Avenida D. Ana Costa, 95, CEP: 11060–001, Santos, SP, Brazil. Tel.: + 55 13 3878 3741. E-mail address: [email protected] (R.C. Spadari-Bratfisch). 0031-9384/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2011.03.028

In addition to metabolic alterations, anhedonia, depression [3], and anxiety [4] are also associated with stress. Comorbidity between anxiety and depression is common in humans [5], and both are related to the increased effects of corticotrophin-releasing hormone (CRH) levels and hypothalamic–pituitary–adrenal axis (HPA) activity [4,6]. However, in animals contradictory effects are observed depending on the stress model and the experimental protocol. While some authors have reported enhanced anxiety-related behavior evaluated 24 h or 1 week after one session of foot-shock stress [7], others found no such effects [8]. On the other hand, a decrease in anxiety-related behavior was reported after chronic mild stress [9–12]. One of the behaviors affected by stress is feeding. Studies evaluating animal models or humans have suggested that food intake may either increase, decrease, or even do not change during stressful periods [13,14]. Various factors related to the stress response may be involved in these effects, including the activation of the autonomic nervous system [15]; the stimulation of the HPA axis, which releases CRH, adrenocorticotropic hormone (ACTH) and glucocorticoids [16]; and the release of leptin and insulin [17]. Moreover, during stress, the areas in the nervous system that are involved in the cognitive and emotional aspects of eating behavior related to the effects of reward and motivation are activated [18]. Therefore, it has been proposed

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that the effect of stress on feeding behavior depends on the quality of the food the animals have access to and the combination of plasma levels of glucocorticoids and insulin [14,19]. When rats exposed to restraint stress are allowed to have access to food rich in sugar and lard, the intake of these foods attenuates the endocrine stress response [19–21] although the rats behaviors evaluated in the elevated plus maze and the open field were not altered [22]. We have demonstrated that rats exposed to foot-shock stress reduce food intake, present high serum levels of glucose and insulin, and are insulin resistant [23]. The serum corticosterone level of these animals enhanced immediately after each foot-shock session, and returns to basal levels within 24 h [24]. The mechanism through which food intake has been reduced after foot-shock stress has not been clarified. In the present study we investigate whether the access to comfort food alters the food intake and some other aspects of the behavior of rats submitted to foot-shock stress, as well as the consequences of the intake of comfort food on metabolic markers.

both diets (n = 6 each) were maintained in standard cages for the determination of basal serum corticosterone levels.

2. Materials and methods

2.3. Foot-shock stress protocol

2.1. Animals

The rats were placed in a Plexiglas chamber (26 × 21 × 26 cm) provided with a grid floor made of a stainless-steel rods (0.3 cm in diameter, spaced 1.0 cm apart). During the 30 min sessions, which occurred between 7:30 am and 11:00 am, the foot-shocks were delivered by a constant current source controlled by a microprocessorbased instrument constructed at the Biomedical Engineering Center, State University of Campinas. The current intensity was 1.0 mA, duration 1.0 s with pulses delivered at random intervals of 5–25 s (mean interval of 15 s). The rats were returned to the metabolic cages at the end of the first and the second foot-shock sessions. After the third session, the rats were evaluated in the behavioral tests or sacrificed for blood collection. Those rats designed to blood collection for determination of plasma glucose and insulin were fasted overnight before sacrifice. The rats in the control groups were also placed in the foot-shock cage for the same period of time, but received no foot-shock.

Eighty-six adult male Wistar rats weighing 250 to 300 g at the beginning of the experiments were used. The animals were housed in a temperature-controlled room (22 ± 2 °C), with a 12 h light/dark cycle (lights on at 7:00 am). The rats were placed in individual acrylic metabolic cages (20 cm in diameter × 14 cm in high) with the floor made of stainless-steel rods. The cages were adapted to contain two feeders. During the experiments, the animals were cared for in accordance with the principles for the use of animals in research and education, as laid down in the Statement of Principles adopted by the board of the Federation of American Societies for Experimental Biology (FASEB). The experimental protocols were approved by the Institutional Committee for Ethics in Animal Experimentation of the University of Campinas (certificate number 1409–1). The rats were distributed in two groups according to diet, with half being offered only commercial chow (Labina, Purina®, Evialis Group, Brazil) and the other half given access to comfort food as well; these diets were made available three days before the experiment began and were maintained throughout. Both groups were further subdivided according to stress procedure, with half of each group being maintained under non-stressed conditions, while the other half of each were submitted to inescapable foot-shock stress, as described below. The four resulting experimental groups were designated commercial chow-control; commercial chow-stress; comfort foodcontrol; and comfort food-stress (Fig. 1). Two additional groups of unstressed rats fed with commercial chow only or having access to

Fig. 1. Experimental protocol design.

2.2. Diet composition Commercial chow for rodents (Labina, Purina®, Evialis Group, São Paulo, Brazil) was offered to all rats along with tap water ad libitum. Two groups were also offered the option of comfort food, which was prepared with powdered commercial chow, peanuts (Hikari®, São Paulo, Brazil), milk chocolate (Chocolates Garoto®, Espírito Santo, Brazil) and sweet cookies (Tostines®, Nestlé, São Paulo, Brazil) in a proportion of 3:2:2:1. This diet was pelletized and provided 20% protein, 20% fat, 48% carbohydrate, and 4% fiber. The caloric densities of this diet and of the commercial diet were determined with an adiabatic calorimeter (C400, IKA® Works, São Paulo, Brazil) and were 21.40 kJ/g (35% of calories as fat) and 17.03 kJ/g, respectively [25]. The amount of food consumed by each animal was evaluated by weighing the food remaining in the feeders on a digital scale, every 24 h.

2.4. Behavioral analysis 2.4.1. Elevated plus-maze test (EPM) After the third period in the foot-shock cage, the animals were evaluated in the EPM, under a 60 lx light, as previously described [26,27]. The animals were placed individually in the center of the maze, facing one of the closed arms at the junction between open and closed arms. A video camera was installed to register the animal's behavior for 5 min, and the videos were then analyzed by more than one researcher. The rat was considered to have entered an arm of the maze if all four paws were within that arm. Conventional parameters of anxiety-like behavior were monitored, i.e., the number of entries into the closed arms, entries into the open arms, and the total time spent in each arm. The ratio between time spent in the open (or closed) arms and time spent in the open plus the closed arms was calculated and multiplied by 100, to yield the percentage of time spent in open (or closed) arms. Moreover, the number of each of the following ethological categories was measured: head dipping (dipping of the head below the level of the maze floor), risk-assessment (exiting an enclosed arm with the forepaws and head only, to investigate the surroundings, often, but not necessarily, accompanied by body stretching), stretched-attend postures (when the animal stretches to its full length with the forepaws, keeping the hind paws in the same place and turns back to the anterior position), rearing (rising on the hind limbs), grooming (licking, scratching, and washing of the head and body), and production of fecal bolus. These categories were defined according to previous studies [28–30]. After the removal of each animal, the maze was cleaned with a 10% ethanol solution.

D. Ortolani et al. / Physiology & Behavior 103 (2011) 487–492 Table 1 Daily food (g), caloric intake (kJ) and gain body weight (g) of control and foot-shock stressed rats fed with commercial chow; control and foot-shock stressed rats given an option of commercial chow and comfort food.

Commercial (g) Comfort (g) Total calories (kJ) Gain body weight (g)

Commercial chow

Comfort food

Control (20) Stress (20)

Control (15)

24.05 ± 0.77 – 409.6 ± 13.2 10.80 ± 1.14

Stress (15)

21.90 ± 0.61a 1.43 ± 0.30 0.55 ± 0.18a – 21.77 ± 0.59b 21.87 ± 1.39b 372.9 ± 10.4c 490.2 ± 10.9c 478.7 ± 29.5c 8.95 ± 1.70 8.43 ± 0.83 6.53 ± 1.20

The numbers between parentheses are the number of animals in each group. a Significantly different from control rats fed with commercial chow only (p b 0.05, Student's t test). b Significantly different from the intake of commercial chow for the same group. c Significantly different from control rats fed with commercial chow (p b 0.05, twoway ANOVA and Newman–Keuls post-test).

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Serum corticosterone concentrations were determined by enzyme immunoassay (ELISA) using a commercial kit (Assay Designs, Inc., Ann Arbor, USA). Insulin and leptin were measured using ELISA kits (Linco Research, Inc., Missouri, USA). Serum glucose and triglycerides were evaluated with commercial kits from Labtest Diagnostic S.A. (Minas Gerais, Brazil). 2.6. Statistical analysis Results are expressed as means ± standard error of means. The data were compared using a Student's t test and a two-way Analysis of Variance (ANOVA), followed by a Newman–Keuls's test. The differences were considered significant when p ≤ 0.05. 3. Results 3.1. Food intake

2.4.2. Open field test Immediately after the EPM test, each animal was evaluated in the open field, under a 60 lx light. The apparatus consists of an acrylic cylinder, 72 cm in diameter, and 60 cm high; the base is divided into 12 equal areas (rectangles measuring 13.3 by 15.0 cm). A video camera was installed to register the animal's behavior. Rats were placed individually in the open field for 5 min, and the following parameters were analyzed: latency to first crossing, time spent on the periphery and in the center of the apparatus, number of crossings (times that the animal enters another square with the all paws), number of rearings, number of groomings, and number of fecal bolus [31,32]. After each trial, the apparatus was cleaned with a 10% ethanol solution. 2.5. Biochemical analysis Within 30 s of the end of the last session, rats were euthanized by decapitation. Their blood was collected in plastic vials and centrifuged for 20 min at 4000 rpm, after which aliquots of serum were removed and stored at − 80 °C until assayed for metabolic markers.

Food intake was strongly influenced by stress and the composition of the diet (F[3,69] = 240; p = 0.0001). The rats submitted to footshock stress consumed less commercial chow than did the control rats (p = 0.035, Student's t test). Those rats that could choose between the two diets showed a clear preference for the comfort food. This preference was not altered by stress. However, rats submitted to stress reduced the intake of commercial chow, but did not reduce the intake of the comfort food (Table 1). Despite the differences in food and caloric intake, there was no significant difference in body weight between the groups (F[3,69] = 1.2; p = 0.30, Table 1). 3.2. Behavioral analysis The percentage of entries (F[1,60] = 5.97; p = 0.017) and the time spent in the open arms of the EPM (F[1,60] = 4.59; p = 0.036) were greater in rats submitted to foot-shock stress, although the percentage of entries and the time spent in the closed arms were not altered by stress nor by diet composition (Fig. 2). After foot-shock stress, head dipping was more frequent (F[1,60] = 5.55; p = 0.021), whereas the number of stretched-attend posture was less frequent (F[1,60] = 8.08;

Fig. 2. Percentage of entries in the open and closed arms of the elevated plus maze of control (n = 20) and foot-shock stressed rats (n = 18) fed with commercial chow; control (n = 12) and foot-shock stressed rats (n = 11) given an option of commercial chow and comfort food (⁎significantly different from control groups, p b 0.05, two-way ANOVA and Newman–Keuls post-test).

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Table 2 Behavior evaluated in open arms of elevated plus maze for control and foot-shock stressed rats fed with commercial chow or given an option of commercial chow and comfort food. The numbers between parentheses are the numbers of animals in each group. Commercial chow

Head dipping Rearing Fecal bolus Stretched-attend posture Risk assessment Grooming

Comfort food

Control (20)

Stress (18)

Control (12)

Stress (11)

13.85 ± 1.61 0 0.35 ± 0.18 13.05 ± 1.26

19.16 ± 2.85 ⁎ 0 0.61 ± 0.31 8.11 ± 1.19 ⁎

12.58 ± 2.19 0 0.00 12.25 ± 1.41

18.45 ± 2.07 ⁎ 0 0.09 ± 0.09 9.54 ± 1.25 ⁎

17.70 ± 0.92 0

13.44 ± 1.23 ⁎ 0

11.50 ± 0.73 ⁎ 0

9.63 ± 0.94 ⁎ 0

⁎ Significantly different from control rats fed commercial chow (p b 0.05, two-way ANOVA and Newman–Keuls post-test).

p = 0.006), and both behaviors were not affected by the diet. Nonstressed rats with access to the comfort food also displayed fewer risk assessments than control rats fed with commercial chow (F[1,60] = 21.25; p = 0.001) and this effect was potentiated by the comfort food (F[1,60] = 7.94; p = 0.006) (Table 2). None of the rats exhibited rearing or grooming behaviors in the open arms of the EPM (Table 2). In rats fed with commercial chow, the parameters evaluated in the open field were not altered by foot-shock stress (Table 3). However, an interaction between stress and comfort food was observed in the latency prior to the first crossing (F[1,60] = 4.79; p = 0.032) and in the time spent in the center of the open field (F[1,60] = 7.76; p = 0.007), that increased, with a corresponding decrease in the time spent on the periphery (F[1,60] = 6.17; p = 0.015). Neither stress nor diet affected the number of crossings, rearings, groomings and production of fecal bolus (Table 3). 3.3. Hormonal and metabolic parameters Foot-shock stress induced an increase in the serum corticosterone concentration of rats fed with the commercial chow (F[1,22] = 14.0; p = 0.0001). Access to the comfort food did not alter the serum corticosterone concentration in control rats. However, the stressinduced increase in serum corticosterone concentration was attenuated in those rats with access to the comfort food (Fig. 3). In order to evaluate if the metabolic cage in itself might represent an additional stressor for the rats, serum corticosterone was also determined in control rats kept in standard cages. No significant differences in the serum corticosterone concentration were found in non-stressed rats kept in standard cages and fed only with commercial chow (65.5 ± 23.2 ng/ml, n = 6) or with access to commercial chow plus comfort food (170.7 ± 35.6 ng/ml, Table 3 Open field behavior of control and foot-shock stressed rats fed a commercial chow or given an option of commercial chow and comfort food. The numbers between parentheses are the numbers of animals.

Latency to first crossing (s) Time spent in center (s) Time spent in periphery (s) Crossing Rearing Grooming Fecal bolus

Commercial chow

Comfort food

Control (20)

Control (12)

Stress (18)

9.05 ± 2.44

8.27 ± 0.99

5.16 ± 0.95

48.95 ± 4.20

46.83 ± 6.33

31.33 ± 4.69

253.75 ± 5.52

253.16 ± 6.33

265.75 ± 4.96

67.25 ± 4.51 39.45 ± 2.73 1.70 ± 0.23 2.00 ± 0.46

74.11 ± 3.88 38.66 ± 2.45 2.16 ± 0.31 0.55 ± 0.32

70.91 ± 9.35 36.75 ± 3.02 1.58 ± 0.19 1.66 ± 0.68

Stress (11)

Fig. 3. Serum corticosterone concentration (ng/ml) of control and foot-shock stressed rats fed with commercial chow; control and foot-shock stressed rats given an option of commercial chow and comfort food (n = 6 per group); (⁎significantly different from control rats fed commercial chow; #significantly different from foot-shock stressed rats fed commercial chow; p b 0.05, two-way ANOVA and Newman–Keuls post-test).

n = 6) and those seen in rats living in metabolic cages (118.3 ± 21.8; 100.0 ± 90.1 ng/ml; respectively). Table 4 shows that serum insulin concentrations were higher in stressed than in control rats (F[1,23] = 14.27; p = 0.001). On the other hand, serum leptin concentration was not affected by stress, although the consumption of the comfort food increased it (F[1,23] = 68.93; p = 0.000). Serum glucose concentrations were higher in rats submitted to foot-shock stress than in control rats fed with commercial chow (F[1,23] = 25.31; p = 0.001) or with comfort food (F[1,23] = 15.47, p = 0.005). The intake of comfort food increased serum glucose levels in non-stressed rats (F[1,23] = 8.23, p = 0.007). On the other hand, the serum triglyceride concentration decreased in rats submitted to stress (F[1,23] = 14.97; p = 0.000), with access to the comfort food increasing it (F[1,23 ] = 9.65; p = 0.005; Table 4). 4. Discussion Data presented here have shown that rats fed with commercial chow and submitted to foot-shock stress decreased their food intake, as has previously been reported for this experimental model [23,24], as well as for other stress protocols [33,34]. The present data also have shown that access to comfort food eliminates this effect of stress on feeding behavior. Accordingly, it has been shown that availability of simple sugars also may influence energy regulation after stress. Rats given access to a 1–2% sucrose solution after exposure to tailshock gain as much weight as do nonstressed controls [35]. Several factors related to the stress response may be involved in the effects of foot-shock stress on feeding behavior. They include the activation of the autonomic nervous system [15], the release of hormones such as CRH, ACTH, glucocorticoids [16], and leptin [17]; as well as the activation of neural systems involved in the cognitive, reward, and emotional aspects of ingestive behavior [18]. However, the anorexigenic effect of foot-shock stress demonstrated here was

Table 4 Serum levels of glucose (mg/dl), triglycerides (mg/dl), insulin (ng/ml) and leptin (ng/ml) in control and foot-shock stressed rats fed commercial chow, or given an option of commercial chow and comfort food. Data are means± sem of 6 animals per group.

15.18 ± 4.53a 85.09 ± 23.37a,b 213.54 ± 23.52a,b 59.90 ± 8.13 30.80 ± 3.79 1.54 ± 0.28 1.36 ± 0.79

a Interaction between stress and comfort food (p b 0.05, two-way ANOVA and Newman–Keuls post-test). b Significantly different from control group.

Insulin Leptin Glucose Triglycerides a

Commercial chow

Comfort food

Control

Control

Stress

0.55 ± 0.07 1.10 ± 0.21a 0.28 ± 0.04 3.50 ± 0.12 3.50 ± 0.28 8.40 ± 1.00b 73.8 ± 3.5 128.3 ± 22.9a 95.27 ± 1.99c 169.80 ± 5.67 157.67 ± 4.84a 209.93 ± 13.05c

Stress 0.92 ± 0.18a 8.60 ± 0.55b 117.95 ± 3.69a 169.23 ± 11.28a,d

Significantly different from control groups. Significantly different from control and stress fed commercial chow. Significantly different from all the other groups. d Significantly different from stressed rats fed a commercial chow and from stressed rats given an option of commercial chow and comfort food (p b 0.05, two-way ANOVA and Newman–Keuls post-test). b c

D. Ortolani et al. / Physiology & Behavior 103 (2011) 487–492

independent of leptin and was not due to anxiety, since the behavior of these foot-shock stressed rats indicated lower levels of anxiety than those found in the control rats. Moreover, the anorexigenic effect of foot-shock stress was restricted to the intake of commercial chow but did not affect the intake of comfort food. It has previously been reported that rats submitted to restraint stress show greater preference for sweet food than do non-stressed rats [36,37] and that the intake of comfort food reduces the stress response [38]. Indeed, rats submitted to 1 h of restraint stress daily for 5 days increased their intake of lard and sucrose [38–41]. However, rats exposed to chronic restraint stress for 40 days showed a consumption pattern for comfort food and commercial chow similar to that of non-stressed control rats, with the intake of comfort food being greater for both groups [22,42]. In the present study, rats in both stressed and non-stressed groups showed a clear preference for comfort food, and this preference was not altered by foot-shock stress. Moreover, foot-shock stressed rats did not decrease their intake of comfort food, which indicates that they did not develop the anhedonia typically observed in rats exposed to chronic unpredictable mild stress [11,43] and in patients with chronic stress and depression [44]. These differences are in line with the hypothesis that the type of stressor and its duration may affect the food intake in various ways [36]. The results presented here also reinforce the hypothesis that the intake of comfort food reduces the stress response [39] because the increase in serum corticosterone levels induced by stress is attenuated in stressed rats with access to comfort food. Both elevated plus maze and open field tests are widely used to assess the neurobehavioral profiles of experimental animals under the influence of anxiogenic/anxiolytic agents [45–47]. The number of entries into the closed arms and the time spent in the open arms of the EPM are considered to reflect the fear of entering open areas and are associated with the anxiety level of the animal. Therefore, in the present study, the greater number of entries and the increased time spent in the EPM open arms suggest that the foot-shock stressed rats have lower levels of anxiety than do the control rats. These stressed rats also performed more head dipping behavior, and exhibited stretched-attend posture and assessment of risk less frequently. These behaviors are associated with exploratory activity [48], with the latter two considered to be strategies for the evaluation of the environment in potentially dangerous situations [49]. The stretched-attend posture has been interpreted as “cautious exploration”, which is a common adaptive behavioral strategy [50]. Hence, the lower number of stretched-attend and risk assessment postures in the stressed rats reinforces the hypothesis that these rats present a lower level of anxiety than do non-stressed rats and are less cautious in exploring the new environment. The limited number of behaviors related to anxiety observed in foot-shock stressed rats was not modified by access to the comfort food. The anxiolytic-like profile observed in the EPM test is similar to what has previously been reported for rats exposed to chronic unpredictable mild stress [9,10,12]. On the other hand, this behavioral profile is in contrast to previous reports showing enhanced anxiety-related behavior [7] or no effects of foot-shock stress on rats' behavior [8]. It has been suggested that these conflicting results might be related to different stress protocols or plasmatic levels of corticosterone induced by different stressors, since the administration of glucocorticoids induced biphasic effects on anxiety related behaviors: anxiolytic effects for low doses and anxiogenic effects for high doses [51]. However, the foot-shock stressed rats in the present study had high levels of corticosterone and low levels of anxiety. Moreover, when these stressed rats have access to comfort food the hormonal response to stress is attenuated, although the profile of anxiety-like behaviors in the EPM remains unaltered. The parameters evaluated in the open field revealed no differences between foot-shock stressed rats and control rats, independent of the diet of the latter, which suggests that locomotor activity has not been impaired by the foot-shocks. On the other hand, there is an interaction

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between stress and intake of comfort food, with an increase in the latency prior to the first crossing, as well as in the time spent in the center of the open field. This behavioral profile is indicative of a lower level of anxiety [45]. Therefore, although the data for the EPM test did not discriminate between levels of anxiety for the stressed rats fed with commercial chow and those fed comfort food, the open field revealed the lower level of anxiety of the stressed rats with access to comfort food. In accordance with these data Dess et al., [52] state that reluctance to venture forth into unfamiliar places reduces the likelihood of encountering danger, but endogenous fuel provisioning permits the animal to play it safe for a time. Not only were behavioral parameters altered by foot-shock stress but this led to a widespread metabolic response, with high levels of glucocorticoids, insulin and glucose detected after foot-shock stress. A similar reaction was reported by Verago et al. [53] who evaluated the metabolic profile of rats after each one of the three foot-shock sessions. Moreover, exposure to foot-shock stress led to the development of insulin resistance, as demonstrated in vivo by the oral glucose tolerance test and the reduced response of adipocytes to insulin [54], although the release of insulin by pancreatic islets was not altered [54]. The levels of triglycerides, on the other hand, were lower in the serum of rats submitted to foot-shock stress. Verago et al. [53] reported that triglycerides increased only after the first two sessions of foot-shock stress and was lower than those seen in control rats after the third session. Taken together, these findings indicate that repeated footshock stress leads to a widespread metabolic response aimed at maintaining high plasma levels of glucose by making triglycerides available, probably as an energy source for muscle metabolism. The intake of comfort food represents an additional metabolic challenge, because it increases the level of glucose, triglycerides and leptin. Nevertheless, the present results show that the metabolic response to foot-shock stress is not modified in rats that have access to comfort food, except that the stress-induced increase in the serum levels of corticosterone is attenuated in these rats. Leptin, however, is strongly affected by the intake of comfort food. Secreted by adipocytes, this hormone has many effects on the organism, most of them related to energy homeostasis, such as the signaling of energy stores and the inhibition of food intake [55]. Although the serum leptin concentration was higher in rats with access to comfort food, the intake of this more palatable food was not reduced in control rats nor in foot-shock stressed rats. The anorexigenic effect of stress is thus limited to the intake of commercial chow. Therefore, in foot-shock stressed rats the intake of the high caloric diet is combined with high plasma levels of glucose, triglycerides, insulin, leptin and corticosterone. Concluding, these data suggest that the anorexigenic effect of footshock stress is independent of leptin, and that it is restricted to the ingestion of regular commercial chow, whereas the intake of comfort food is not affected. Moreover, the intake of such more palatable food attenuates the endocrine component of the stress response, as represented by the serum corticosterone levels. An anxiolytic effect of stress that is potentiated by the ingestion of comfort food was also observed. Taken together, these data indicate that the combination of stress and free access to comfort food (both present in the modern society daily life) may represent one of the links between stress and metabolic diseases such as diabetes and atherosclerosis. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Acknowledgements The authors acknowledge the financial support from the “Coordenação de Apoio ao Pessoal de Ensino Superior” (CAPES) and “Fundação para o

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Apoio da Pesquisa do Estado de São Paulo” (FAPESP). The authors also acknowledge Fabio Montesano for the statistical analysis and Linda Gentry El-Dash for the linguistic revision. References [1] Tsigos C, Chrousos GP. Hypothalamic-pituitary-adrenal axis, neuroendocrine factors and stress. J Psychosom Res 2002;53:865–71. [2] Appel JK, Dikeman ME, Minton JE, McMurphy RM, Fedde MR, Leith DE, et al. Effects of restraint and isolation stress and epidural blockade on endocrine and blood metabolite status, muscle glycogen metabolism, and incidence of dark-cutting longissimus muscle of sheep. J Anim Sci 1995;73(8):2295–307. [3] Kendler KS, Karkowski LM, Prescott CA. Causal relationship between stressful life events and the onset of major depression. Am J Psychiatry 1999;156:837–41. [4] Müller MB, Zimmermann S, Sillaber I, Hagemeyer TP, Deussing JM, Timpl P, et al. Limbic corticotropin-releasing hormone receptor 1 mediates anxiety-related behavior and hormonal adaptation to stress. Nat Neurosci 2003;6:1100–7. [5] American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorder (DSM-IV-TR)4th ed; 1994. Washington, DC. [6] De Kloet ER. Hormones, brain and stress. Endocr Regul 2003;37:51–68. [7] Kavushansky A, Ben-Shachar D, Richter-Levin G, Klein E. Physical stress differs from psychosocial stress in the pattern and time-course of behavioral responses, serum corticosterone and expression of plasticity-related genes in the rat. Stress 2009;12(5):412–25. [8] Maclean RR, Datta S. The relationship between anxiety and sleep–wake behavior after stressor exposure in the rat. Brain Res 2007;1164:72–80. [9] D'Aquila PS, Brain P, Willner P. Effects of chronic mild stress on performance in behavioural tests relevant to anxiety and depression. Physiol Behav 1994;56(5): 861–7. [10] Rossler AS, Joubert C, Chapouthier G. Chronic mild stress alleviates anxious behaviour in female mice in two situations. Behav Processes 2000;49:163–5. [11] Willner P. Chronic mild stress (CMS) revisited: consistency and behavioural– neurobiological concordance in the effects of CMS. Neuropsychobiology 2005;52: 90–110. [12] Schweizer MC, Henniger MS, Sillaber I. Chronic mild stress (CMS) in mice: of anhedonia, ‘anomalous anxiolysis’ and activity. PLoS One 2009;4(1):e4326. [13] Cottone P, Sabino V, Steardo L, Zorrilla EP. Consummatory, anxiety-related and metabolic adaptations in female rats with alternating access to preferred food. Psychoneuroendocrinology 2009;34(1):38–49. [14] Dallman MF. Stress-induced obesity and the emotional nervous system. Trends Endocrinol Metab 2010;21(3):159–65. [15] Berthoud HR, Morrison C. The brain, appetite, and obesity. Annu Rev Psychol 2008;59:55–92. [16] Adam TC, Epel SE. Stress, eating and the reward system. Physiol Behav 2007;91: 449–58. [17] Baranowska B, Baranowska-Bik A, Bik W, Martynska L. The role of leptin and orexins in the dysfunction of hypothalamo-pituitary-gonadal regulation and in the mechanism of hyperactivity in patients with anorexia nervosa. Neuro Endocrinol Lett 2008;29(1):37–40. [18] Torres SJ, Nowson CA. Relationship between stress, eating behavior, and obesity. Nutrition 2007;23:887–94. [19] Warne JP. Shaping the stress response: interplay of palatable food choices, glucocorticoids, insulin and abdominal obesity. Mol Cell Endocrinol 2009;300(1–2): 137–46. [20] Dallman MF, Pecoraro N, La Fleur SE. Chronic stress and comfort foods: selfmedication and abdominal obesity. Brain Behav Immun 2005;19:275–80. [21] Maniam J, Morris MJ. Palatable cafeteria diet ameliorates anxiety and depression-like symptoms following an adverse early environment. Psychoneuroendocrinology 2009:1–12. [22] Krolow RCG, Noschang D, Arcego AC, Andreazza W, Peres CA, Gonçalves C, et al. Consumption of a palatable diet by chronically stressed rats prevents effects on anxiety-like behavior but increases oxidative stress in a sex-specific manner. Appetite 2010;55(1):108–16. [23] Farias-Silva E, Grassi-Kassisse DM, Wolf-Nunes V, Spadari-Bratfisch RC. Stressinduced alteration in the lipolytic response to β-adrenoceptor agonists in rat white adipocytes. J Lipid Res 1999;40(9):1719–27. [24] Farias-Silva E, Sampaio-Barros MM, Amaral MEC, Carneiro EM, Boschero AC, Grassi-Kassisse DM, et al. Subsensitivity to insulin in adipocytes from rats submitted to foot shock stress. Can J Physiol Pharmacol 2002;80(8):783–9. [25] Estadella D, Oyama LM, Dâmaso AR, Ribeiro EB, Do Nascimento CO. Effect of palatable hyperlipidic diet on lipid metabolism of sedentary and exercised rats. Nutrition 2004;20:218–24. [26] Melo LL, Brandão ML. Involvement of 5-HT1A and 5-HT2 receptors of the inferior colliculus in aversive states induced by exposure of rats to the elevated plus-maze test. Behav Pharmacol 1995;6(4):413–7.

[27] Pellow S, Chopin P, File SE, Briley M. Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods 1985;14(3): 149–67. [28] Rodgers RJ, Cole JC. An ethological analysis of chlordiazepoxide and bretazenil (Ro16-6028) in the murine elevated plus-maze. Behav Pharmacol 1993;4:573–80. [29] Cruz AP, Frei F, Graeff FG. Ethopharmacological analysis of rat behavior on the elevated plus-maze. Pharmacol Biochem Behav 1994;49(1):171–6. [30] Anseloni VCZ, Brandão ML. Ethopharmacological analysis of behaviour of rats using variations of the elevated plus-maze. Behav Pharmacol 1997;8:533–40. [31] Walsh RN, Cummins RA. The open-field test: a critical review. Psychol Bull 1976;83(3):482–504. [32] Capaz FR, Vanconcellos LE, De Moraes S, Neto JP. The open field: a simple method to show ethanol withdrawal symptoms. Arch Int Pharmacodyn Ther 1981;251(2): 228–36. [33] Martí J, Armário A. Effects of chronic stress on food intake in rats: influence of stressor intensity and duration of daily exposure. Physiol Behav 1994;55:747–53. [34] Vallès A, Martí O, García A, Armário A. Single exposure to stressors causes longlasting, stress-dependent reduction of food intake in rats. Am J Physiol Regul Integr Comp Physiol 2000;279:R1138–44. [35] Dess NK. Divergent responses to saccharin vs. sucrose availability after stress in rats. Physiol Behav 1992;52(1):115–25. [36] Kant GJ, Bauman R. Effect of chronic stress and time of day on preference for sucrose. Physiol Behav 1993;54:499–502. [37] Young JB. Effects of neonatal handling on sympathoadrenal activity and body composition in adult male rats. Am J Physiol Regul Integr Comp Physiol 2000;279: R1745–52. [38] Pecoraro N, Reyes F, Gomez F, Bhargava A, Dallman MF. Chronic stress promotes palatable feeding, which reduces signs of stress: feedforward and feedback effects of chronic stress. Endocrinology 2004;145(8):3754–62. [39] Dallman MF, Pecoraro N, Akana SF, La Fleur SE, Gomez F, Houshyar H, et al. Chronic stress and obesity: a new view of “comfort food”. Proc Natl Acad Sci USA 2003;100:11696–701. [40] La Fleur SE, Akana SF, Manalo SL, Dallman MF. Interaction between corticosterone and insulin in obesity: regulation of lard intake and fat stores. Endocrinology 2004;145:2174–85. [41] Liang S, Byers DM, Irwin LN. Sex and diet affect the behavioral response of rats to chronic mild stressors. Physiol Behav 2008;93:27–36. [42] Fachin A, Silva RK, Noschang CG, Pettenuzzo L, Bertinetti L, Billodre MN, et al. Stress effects on rats chronically receiving a highly palatable diet are sex-specific. Appetite 2008;51:592–8. [43] Moreau JL, Bourson A, Jenk F, Martin JR, Mortas P. Curative effects of the atypical antidepressant mianserin in the chronic mild stress-induced anhedonia model of depression. J Psychiatry Neurosci 1994;19(1):51–6. [44] Forbes C, Blanchard JJ, Bennett M, Horan WP, Kring A, Gur R. Initial development and preliminary validation of a new negative symptom measure: the Clinical Assessment Interview for Negative Symptoms (CAINS). Schizophr Res 2010;124(1–3):36–42. [45] Prut L, Belzung C. The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. Eur J Pharmacol 2003;463:3–33. [46] Carobrez AP, Bertoglio LJ. Ethological and temporal analyses of anxiety like behavior: the elevated plus-maze model 20 years on. Neurosci Biobehav Rev 2005;29:1193–205. [47] Teegarden SL, Bale TL. Decreases in dietary preference produce increased emotionality and risk for dietary relapse. Biol Psychiatry 2007;61(9):1021–9. [48] Rodgers RJ, Cao BJ, Dalvi A, Holmes A. Animal models of anxiety: an ethological perspective. Braz J Med Biol Res 1997;30(3):289–304. [49] Blanchard RJ, Yudko EB, Rodgers RJ, Blanchard DC. Defense system psychopharmacology: an ethological approach to the pharmacology of fear and anxiety. Behav Brain Res 1993;58(1–2):155–65. [50] Rodgers RJ, Haller J, Holmes A, Halasz J, Walton TJ, Brain PF. Corticosterone response to the plus-maze: high correlation with risk assessment in rats and mice. Physiol Behav 1999;68:47–53. [51] Vafaei AA, Rashidy-Pour A, Taherian AA. Peripheral injection of dexamethasone modulates anxiety related behaviors in mice: an interaction with opioidergic neurons. Pak J Pharm Sci 2008;21(3):285–9. [52] Dess NK, Choe S, Minor TR. The interaction of diet and stress in rats: high-energy food and sucrose treatment. J Exp Psychol Anim Behav Process 1998;24(1):60–71. [53] Verago JL, Grassi-Kassisse DM, Spadari-Bratfisch RC. Metabolic markers following beta-adrenoceptor agonist infusion in foot shock stressed rats. Braz J Med Biol Res 2001;34(9):1197–207. [54] Farias-Silva E, Santos IN, Amaral MEC, Grassi-Kassisse DM, Spadari-Bratfisch RC. Glucocorticoid receptor and beta-adrenoceptor expression in epididymal adipose tissue from stressed rats. Ann NY Acad Sci 2004;1018:328–32. [55] Anubhuti, Arora S. Leptin and its metabolic interactions — an update. Diabetes Obes Metabo 2008;10:973–93.