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Stress
The International Journal on the Biology of Stress
ISSN: 1025-3890 (Print) 1607-8888 (Online) Journal homepage: http://www.tandfonline.com/loi/ists20
Hypercaloric diet modulates effects of chronic
stress: a behavioral and biometric study on rats
Carla de Oliveira, Cleverson Moraes de Oliveira, Isabel Cristina de Macedo,
Alexandre S. Quevedo, Paulo Ricardo Marques Filho, Fernanda Ribeiro da
Silva, Rafael Vercelino, Izabel C. Custodio de Souza, Wolnei Caumo & Iraci L.
S. Torres
To cite this article: Carla de Oliveira, Cleverson Moraes de Oliveira, Isabel Cristina de Macedo,
Alexandre S. Quevedo, Paulo Ricardo Marques Filho, Fernanda Ribeiro da Silva, Rafael
Vercelino, Izabel C. Custodio de Souza, Wolnei Caumo & Iraci L. S. Torres (2015): Hypercaloric
diet modulates effects of chronic stress: a behavioral and biometric study on rats, Stress
To link to this article: http://dx.doi.org/10.3109/10253890.2015.1079616
Published online: 12 Sep 2015.
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ISSN: 1025-3890 (print), 1607-8888 (electronic)
Stress, Early Online: 1–10
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2015 Taylor & Francis. DOI: 10.3109/10253890.2015.1079616
ORIGINAL RESEARCH REPORT
Hypercaloric diet modulates effects of chronic stress: a behavioral and
biometric study on rats
Carla de Oliveira
1,2,3
*, Cleverson Moraes de Oliveira
1,2,3
*, Isabel Cristina de Macedo
1,3,4
, Alexandre S. Quevedo
1,2,3
,
Paulo Ricardo Marques Filho
1,2,3
, Fernanda Ribeiro da Silva
1,2,3
, Rafael Vercelino
1,4
, Izabel C. Custodio de Souza
1,2
,
Wolnei Caumo
1,2
, and Iraci L. S. Torres
1,2,3,4
1
Pharmacology of Pain and Neuromodulation Laboratory: Animal Models, Department of Pharmacology, Institute of Basic Health Sciences (ICBS),
Federal University of Rio Grande do Sul, ICBS, Porto Alegre, RS, Brazil,
2
Medicine School, Federal University of Rio Grande do Sul, Porto Alegre, RS,
Brazil,
3
Animal Experimentation Unit and Graduate Research Group, Hospital de Clinicas de Porto Alegre, Porto Alegre, RS, Brazil, and
4
Institute of
Basic Health Sciences (ICBS), Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil
Abstract
Obesity is a chronic disease that has been associated with chronic stress and hypercaloric diet
(HD) consumption. Increased ingestion of food containing sugar and fat ingredients (comfort
food) is proposed to ‘‘compensate’’ chronic stress effects. However, this eating habit may
increase body fat depositions leading to obesity. This study evaluated behavioral/physiological
parameters seeking to establish whether there is an association between the effects of HD
intake and stress, and to test the hypothesis that the development of anxious behavior and
obesity during chronic stress periods depends on the type of diet. Sixty-day-old male Wistar
rats (n ¼ 100) were divided into four groups: standard chow, hypercaloric diet, chronic stress/
standard chow and chronic stress/hypercaloric diet. Chronic stress was induced by restraint
stress exposure for 1 h/day, for 80 d. At the end of this period, rat behavior was evaluated using
open-field and plus-maze tests. The results showed that HD alone increased weight gain and
adipose deposition in subcutaneous and mesenteric areas. However, stress reduced weight
gain and adipose tissue in these areas. HD also increased naso-anal length and concurrent
stress prevented this. Behavioral data indicated that stress increased anxiety-like behaviors and
comfort food reduced these anxiogenic effects; locomotor activity increased in rats fed with
HD. Furthermore, HD decreased corticosterone levels and stress increased adrenal weight. The
data indicate that when rats are given HD and experience chronic stress this association
reduces the pro-obesogenic effects of HD, and decreases adrenocortical activity.
Keywords
Anxiety-like, chronic stress, elevated
plus-maze test, hypercaloric diet,
obesity, open-field test
History
Received 9 December 2014
Revised 29 July 2015
Accepted 31 July 2015
Published online 8 September 2015
Introduction
Stress and sweet and fatty food consumption are two common
conditions in today’s society and their association may
become a major health problem. Stress can be a normal
response to alterations in the environment or to events that
may disrupt homeostasis (Dallman et al., 2004). These
organic adaptations may involve physiological and biochem-
ical changes that are influenced by several factors such as
duration, type and severity of the stress condition (Marti et al.,
1994). For example, physical and emotional stressors activate
the hypothalamic–pituitary–adrenal (HPA) axis, thus increas-
ing circulating glucocorticoid concentrations (Pasquali,
2012), which may lead to changes in adiposity and to
growth delays (Welberg & Seckl, 2001). As a consequence,
several lines of research have found a positive relationship
between higher levels of cortisol (corticosterone in rodents)
and different parameters of weight gain, such as obesity
(Rosmond & Bjorntorp, 2000) and visceral fat accumulation
(Rebuffe-Scrive et al., 1985). However, the impact of stress
can be modified by environmental factors such as the type of
diet and time of stress exposure (Maniam & Morris, 2012).
Stress-induced physiological alterations involve a complex
combination of clinical and biochemical aspects that may
result in body size variations that range from weight-loss to
weight-gain (Epel et al., 2010; Pucilowski et al., 1993). These
conflicting results might have independent pathways such as
beta-adrenergic activation (fat-burning), alterations in sym-
pathetic signaling (e.g. towards neuropeptide Y predomin-
ance), or increase in sweet and fatty (comfort-food) intake
(Armario et al., 2012). However, there are interactions
between those mechanisms in which chronic stress is able
to modify palatable food intake (Dallman et al., 2005)
and eating behaviors, which might increase cortisol levels
(Epel, 2009). The increment of glucocorticoid secretion
may induce the metabolic syndrome that leads to obesity
(Lacroix et al., 2004). Moreover, the effects of eating patterns
*These authors contributed equally to this work.
Correspondence: Iraci Lucena da Silva Torres, Departamento de
Farmacologia, ICBS, UFRGS, Rua Sarmento Leite, 500 sala 202,
90050-170 Porto Alegre, RS, Brazil. Tel: +55 51 3316 3183. Fax: +55
51 3316 3121. E-mail: iracitorres@gmail.com
Downloaded by [179.219.170.170] at 12:53 14 September 2015
(Gibson, 2006; Foster et al., 2009) and the type of diet
(Armario et al., 2012) on body weight may alter in chronically
stressed individuals.
Chronic stress induces behavioral changes that may
include alterations in cognitive abilities (McEwen, 1999),
coping skills (Armario et al., 2012) and emotional responses
(Lister, 1987; McEwen, 2008). Altered emotional states (i.e.
anxiety) have been related to changes in eating behavior (i.e.
food choices) that may lead to obesity (Dallman et al., 2005;
Singh, 2014). For example, negative emotions increase the
preference for palatable food, such as sweet and high-fat diets
(Macht, 2008), which may reduce stress responses (Pecoraro
et al., 2004). This study evaluated the effect of hypercaloric
diet exposure on stressed and nonstressed rats by analyzing
behavioral, physiological and biochemical parameters in order
to test the hypothesis that the development of anxious
behavior and obesity during periods of chronic stress depends
on the type of diet. The behavioral parameters herein
evaluated were locomotor and exploratory activity and
anxiety-like behavior. Moreover, eating behavior was eval-
uated by food consumption (standard chow, cafeteria diet,
water, soft drink). Obesity and growth were assessed using
body weight, weight of adipose tissues (subcutaneous and
mesenteric regions) and naso-anal length.
Methods
Animals
The experiments were carried out on 60-day-old male Wistar
rats (weighing 200–250 g at the beginning of the treatment;
supplied by the Centre for Reproduction and Animal
Experimentation (CREAL) of the Institute of Basic Health
Sciences (ICBS), Universidade Federal do Rio Grande do Sul
(UFRGS)), randomized by body weight and housed in groups
of 3–5 rats per polypropylene cage (49 34 16 cm). All rats
were kept in a standard 12 h:12 h light/dark cycle (lights on at
07:00 h and lights out at 19:00 h), in a temperature-controlled
environment (22 ± 2
C), and had access to water and chow ad
libitum (hypercaloric diet and/or standard rat chow). All
experiments and procedures were approved by the
Institutional Animal Care and Use Committee (Hospital de
Clı
´
nicas de Porto Alegre-HCPA/The Postgraduate Research
Group-GPPG, protocol No. 100382) in compliance with
Brazilian guidelines involving the use of animals for research
(Law No. 11,794). Vigorous attempts were made to minimize
animal suffering and to decrease external sources of pain and
discomfort, as well as to use only the number of rats that was
essential to produce reliable scientific data.
Experimental design
Rats were habituated to the maintenance room for 1 week
before the beginning of the experiment and they were divided
into 4 groups: standard chow (n ¼ 25, with 4–5 rats/cage);
chronic stress/standard chow (n ¼ 25, with 4–5 rats/cage);
hypercaloric diet (n ¼ 24, with 4 rats per cage) and chronic
stress/hypercaloric diet (n ¼ 26, with 3–4 rats/cage). The rats
were weighed weekly, and food intake was recorded daily.
After 80 d of experiment, the rats were exposed to the open-
field apparatus to evaluate locomotor and exploratory activity,
and anxiety-like behavior was evaluated on a plus-maze.
The rats were killed 24 h after the last restraint stress session
(see below), and the adrenal glands and specific subcutaneous
and mesenteric adipose tissues were removed and weighed,
and blood was collected for measurement of serum
corticosterone.
Stress procedure
Chronic stress was induced by exposing the rats to repeated
restraint stress during 80 d (Ely et al., 1997). Each rat was
restrained in a plastic tube (25 7 cm) adjusted to avoid
discomfort, although it limited movements of the rat; the front
part of the tube had holes to allow breathing (Torres et al.,
2001). The rats were stressed in the morning (between 09:00 h
and 12:00 h) for 1 h/d, 5 d/week for 80 d (Ely et al., 1997).
After the stress procedure, the rats were returned to their
home cages. Control rats were kept in their home cages when
experimental rats were subjected to stress.
Experimental diets and food intake
The pelletized standard diet Nuvilab CR-1 (NUVITAL
Õ
)is
composed of 55.0% carbohydrates, 22.0% protein, 4.5% lipids
and other constituents (fiber and vitamins); it contains
2.93 kcal/g (information provided by manufacturer). The
high-calorie diet (cafeteria diet) used was aimed to simulate
modern patterns of human food consumption. This diet has
successfully induced obesity in other experimental studies.
The experimental diet used was adapted from that previously
described (Estadella et al., 2004) and consisted of crackers,
wafers, sausages, chips, condensed milk and a soda drink.
These are highly palatable and highly flavored, which are
features of comfort food (Kumar et al., 2011). The nutritional
composition of this diet was approximately 60.0% carbohy-
drates, 20.0% lipids, 15.0% protein and 5.0% other constitu-
ents (sodium, calcium, vitamins, preservatives and minerals)
providing 4.186 kcal/g (solids) and 0.42 kcal/mL (soda drink;
calculations based on information provided by the manufac-
turer on the package label). The standard diet and the
experimental diet were replaced daily by fresh food. The rats
fed with hypercaloric diet also had access to standard food
and water. The amount of food consumed in each cage was
evaluated by weighing each day the food remaining in the
feeders on a digital scale, and by calculating the food
consumption per cage. To evaluate the weekly caloric
ingestion the amount of cafeteria diet (g) and of soda drink
(mL) were converted into kcal and summed. The intake of
kcal per cage of rats was calculated, and this value was
divided by the number of rats in the cage to estimate the
intake in kcal per rat per cage.
Behavioral tests
At the end of the experiment, the open field and elevated plus-
maze (EPM) behavioral tests were conducted in a lit and
silent room at 09:00 h and 12:00 h.
Open-field test
The open-field test was used to evaluate the locomotor
activity and anxious behavior state of the rats. The open field
apparatus was a varnished wood cage (60 40 50 cm) with
2 C. de Oliveira et al. Stress, Early Online: 1–10
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a glass front wall (Supplementary Diagram S1). The floor was
covered with linoleum and divided by dark lines into 12
squares of 13 13 cm each. At the start of the test, the rats
were gently placed in the left rear corner of the cage and their
behavior was monitored for 5 min (Bianchin et al., 1993;
Carlini et al., 2002). A rat was recorded as entering a new area
when all four paws crossed the boundary into another marked-
out square. Five measures were taken during the 5-min test
sessions: latency to leave the first square (time in seconds);
number of line crossings (i.e. horizontal activity), outer
crossings (crossing with four paws in the outer squares) and
inner crossings (crossing with four paws in the inner squares);
grooming (time in seconds); number of occurrences of rearing
behavior (i.e. vertical activity) and number of fecal boluses.
After the end of each trial, the box was cleaned with 70%
alcohol, and then water to remove any animal scent.
The following definitions were used for the open-field test:
the number of line crossings (measure of locomotor activity)
(Roesler et al., 1999); the latency to leave the first square
(measure of anxiety) (Lister, 1987); number of rearings (i.e.
standing on rear limbs, considered as exploratory activity)
(Silveira et al., 2005); time of grooming (indicative of
anxiety-like behavior) (Nin et al., 2012). Rearing was defined
from the moment the rat rose up on its hind legs, ending when
one or both front paws touched the floor again (Wells et al.,
2009). Grooming was defined as licking/washing the head and
body (Spruijt et al., 1992).
Elevated plus-maze test
The elevated plus-maze (EPM) test was used to evaluate
anxiety-like behavioral state. The apparatus was made of
black PVC and elevated to a height of 50 cm above the floor
level. The maze had two open arms and two closed arms
(50 40 10 cm) extending from a common central platform
(10 10 cm; Supplementary Diagram S2). At the beginning
of the test, the rat was placed in the central area of the EPM,
facing one of the open arms. The behaviors evaluated in the 5-
min test session in the EPM were: number of open arm entries
(EOA); time spent on open arms (TOA); number of closed
arm entries (ECA); time spent in closed arms (TCA); number
of protected head-dips (PHD: dipping the head over the sides
of the maze from within the central platform or a closed arm);
number of nonprotected head-dips (NPHD: dipping head over
the sides of the maze while in an open arm; Supplementary
Diagram S2); and as for the open-field time grooming;
number of rearings and number of fecal boluses. After each
test, the apparatus was cleaned with 70% alcohol, and then
water to remove any animal scent.
EOA, TOA, ECA and TCA are considered to reflect fear of
entering the open areas, associated with rat anxiety (Krolow
et al., 2010; Ortolani et al., 2011); PHD and NPHD indicate
exploratory activity (Rodgers & Cole, 1993). During the EPM
test, entering a new area was recorded when all four paws
crossed into a new arm or into the central area (Lynn &
Brown, 2009).
Caloric intake (kcal/rat/cage/week)
According to Macedo et al. (2012), food was weighed daily to
assess the amount of food consumed per cage. Using a digital
scale, the daily food consumption (in grams) was calculated
for each cage (amount of food placed in the feeders minus the
remaining food). The food consumption analyses were
calculated according to the type of diet offered to the rats,
the standard chow (standard rat diet 2.93 kcal/g) or hyperca-
loric diet (cafeteria diet 4.18 kcal/g and included the 0.42 kcal/
mL of soda drink) and the kcal intake per rat per cage per
week was calculated (kcal/rat/cage/week).
Body weight
The rats were weighed (g) weekly using a semi-analytical
scale. At the end of the experiment, naso-anal length (cm) was
measured, always by the same experimenter. The specific
subcutaneous and mesenteric adipose tissues were dissected
manually and weighed using a semi-analytical balance. Data
are expressed as grams of tissue per 100 g body weight.
Stress indicators
Serum corticosterone assay
Blood was collected in the morning (07:00 h) for serum
corticosterone assay as follows. The rats were individually
and quickly transferred to a separate room and decapitated
within 1 min without anesthesia; trunk blood was collected
and centrifuged in plastic tubes for 5 min at 5000g at room
temperature. Serum was frozen at 80
C until the assays
were performed. The corticosterone concentrations in the
blood serum samples were determined using a commercially
available ELISA kit (IBL-America #IB79112), and values
were expressed as pg/mL (N ¼ 5 for all groups). This
commercial kit was used as described in the manufacturer’s
protocol. The range of the assay is between 1.63 and
240 nmol/L. The analytical sensitivity of the IBL-
AMERICA ELISA was calculated by subtracting 2 standard
deviations from the mean of 20 replicate analyses of Standard
0 and was 51.63 nmol/L. The intra- and interassay variability
was 3–6%.
Relative weight of the adrenal glands
Adrenal glands were carefully dissected and weighed (N ¼ 8
for all groups) on a scale with 0.0001 g precision (Nyuyki
et al., 2012). Adrenal weights were expressed as grams per
100 g of body weight to correct the weight of the gland in
relation to the weight of the animal.
Statistical analysis
Data normality was tested using the Kolmogorov–Smirnov
test. Parametric and nonparametric tests were used to analyze
data with or without normal distribution, respectively. Body
measurement data (weight, length, tissue weight) and serum
corticosterone concentration were analyzed with two-way
analysis of variance (ANOVA) followed by Fisher’s LSD to
estimate the effect of each factor (chronic stress and type of
diet) and interactions. Among group analyses were performed
using one-way analysis of variance (ANOVA) followed by
Fisher’s LSD to further analyze those parameters. Repeated
measures ANOVA was used for weekly weight and caloric
intake data, to determine differences between groups and
fluctuations within each group over time (11-week period).
DOI: 10.3109/10253890.2015.1079616 Stress, diet, weight gain and anxiety 3
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Afterwards, one-way analysis of variance (ANOVA) followed
by Fisher’s LSD was used to determine differences between
groups at each time point (i.e. each week). Data from open-
field and EPM tests did not show normal distribution and
were analyzed using the Kruskal–Wallis test followed by
Dunn’s post-hoc analysis for group analysis. Nonparametric
Mann–Whitney test was used to compare data from different
groups (i.e. stress + SHD groups vs control + HD groups to
compare stressed vs nonstressed rats) to assess the main
effects of chronic stress and the type of diet.
Data are expressed as mean ± standard error of the mean
(S.E.M.) (parametric data) or median (nonparametric data).
All analyses were performed using SPSS 20.0 packages
(Chicago, IL) for windows. The probability level of p50.05
was set as statistically significant.
Results
Effect of chronic stress and/or hypercaloric diet on
open-field activity
Open-field test data did not show normal distribution
regarding several behaviors. Therefore, nonparametric tests
were used to analyze the results. The Kruskal–Wallis test
was used in an analysis among all groups (Table 1) in order
to further evaluate the effect of chronic stress and
hypercaloric diet on rat behavior. There was a group effect
on anxiety-like behaviors [latency (
2
¼ 9.627; df ¼ 3;
p50.05)] and locomotor activity [outer crossing
(
2
¼ 21.636; df ¼ 3; p50.001) and total crossings
(
2
¼ 21.002; df ¼ 3; p50.001)]. There were no significant
differences for other behaviors (p40.05 in all comparisons).
The analysis of latency by Dunn’s post-hoc test indicated
that stress induced anxiety-like behaviors (increased latency)
(control vs stress groups; p50.01) and the hypercaloric diet
reversed this effect (control vs stress HD groups, (p ¼ 0.2).
Likewise, inner crossing (anxiety-like behavior) showed no
significant difference (
2
¼ 6.866; df ¼ 3; p ¼ 0.07). The
Mann–Whitney test showed that the type of diet altered the
total number of crossings and that hypercaloric diet
increased crossings (U ¼ 633.0; p50.001), but there was
no effect on the other parameters (p40.05 in all compari-
sons). Chronic stress only increased the latency to leave the
first square (U ¼ 879.5; p50.01) and it showed no effects on
the other parameters (p40.05).
Hypercaloric diet alone increased the total number of
crossings (locomotor activity) (control vs HD groups,
p50.01). An increased number of outer crossings was
found in rats fed with hypercaloric diet (HD vs control
groups, p50.001) thus confirming that comfort food
increased locomotion. Similarly to the total crossing data,
chronic stress did not significantly reverse the diet effect on
outer crossings (HD vs stress HD groups, (p ¼ 0.055).
Effect of chronic stress and/or hypercaloric diet on
elevated plus-maze activity
Data from the EPM did not show normal distribution for any
behavior (p40.05). Consequently, nonparametric tests were
used to analyze the effect of chronic stress and hypercaloric
diet (Mann–Whitney test) and for among group analysis
(Kruskal–Wallis test) (Table 2). Only in combination with
HD, chronic stress increased the number of rearings
(U ¼ 869.5; p50.05), indicating increase in exploratory
behaviors under stress conditions (see below). No stress
effect was found for the other parameters (p40.05 in all
comparisons).
Type of diet had no statistically significant effect on
rearing (U ¼ 929; p ¼ 0.07) or other parameters (p40.05 in
all comparisons).
Among group analysis showed a significant difference for
rearing (
2
¼ 9.970; df ¼ 3; p50.05), and no differences for
other behaviors (p40.05 for all). Post-hoc analysis showed
more rearings in the stress HD group than in the control
(p50.01), HD (p50.01) and stress groups (p50.05).
However, this exploratory behavior increased only when
stressed rats were exposed to hypercaloric diet because there
was no difference between control and stress groups (p ¼ 0.6).
There was no statistically significant difference in EOA
(
2
¼ 7.649; df ¼ 3; p ¼ 0.054) or TCA (
2
¼ 6.646; df ¼ 3;
p ¼ 0.08) considered as a group effect.
Caloric intake (kcal/rat/cage/week)
The caloric intake per cage was evaluated weekly for 11
weeks, calculated from kcal/g (food) and kcal/mL (soda
drink), and showed normal distribution at all-time points
(p40.05). Repeated measures ANOVA followed by Fisher’s
LSD was used to test the null hypothesis. Main effects
of time, stress and hypercaloric diet, as well as interactions
Table 1. Open-field test (n ¼ 24–26 rats/group): data are reported as median ± interquartile range in each
behavior.
Behavior/groups C S HD SHD
Latency (s) 6.00 ± 9.00
a
13.0 ± 16.5
b
5.50 ± 8.25
a
9.00 ± 11.5
a
Outer crossing (n) 64.0 ± 22.5
a
64.0 ± 32.0
a
84.5 ± 22.5
b
76.0 ± 25.5
b
Inner crossing (n) 3.00 ± 5.50 1.00 ± 3.50 2.50 ± 2.75 3.00 ± 5.00
Total crossings (n) 68.0 ± 27.0
a
68.0 ± 32.5
a
90.5 ± 24.5
b
78.5 ± 27.5
b
Rearing (n) 29.0 ± 10.5 26.0 ± 18.0 32.5 ± 9.50 28.0 ± 11.0
Grooming (s) 6.09 ± 12.0 5.72 ± 13.7 5.81 ± 11.6 11.2 ± 27.5
Faecal boluses (n) 3.00 ± 7.00 2.00 ± 5.50 1.00 ± 4.00 2.00 ± 3.00
C, control group; S, stress group; HD, hypercaloric diet group; SHD, stress + hypercaloric diet group.
n ¼ number of occurrences; s ¼ seconds.
Group effect was statistically significant for latency (anxiety-like behavior), outer crossing, and total crossing
(locomotion). Group effect was analyzed by Kruskal–Wallis test followed by Dunn’s post-hoc analysis.
a,b
Statistically significant difference of p50.05 among groups.
4 C. de Oliveira et al. Stress, Early Online: 1–10
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were assessed. The results of the two-way ANOVA for
repeated measures demonstrated that there was a time effect
(F
(10,15)
¼ 8.201, p ¼ 0.001), interactions between time and
stress (F
(10,15)
¼ 3.934, p ¼ 0.002) and interactions between
time and hypercaloric diet factors (F
(10,15)
¼ 9.891,
p ¼ 0.001), but there was no interaction between time, stress
and hypercaloric diet (F
(10,15)
¼ 0.662, p40.05) (Figure 1).
Changes in body parameters
Weekly weight
There were no significant between-group differences in
baseline weight (one-way ANOVA, p40.05, (F
(3,96)
¼ 0.70,
data not shown). The results for repeated measures ANOVA
showed effects of time (F
(11,86)
¼ 94.54, p50.05), chronic
stress (F
(1,96)
¼ 9.31, p50.05) and hypercaloric diet
(F
(1,96)
¼ 9.53, p50.05) (Figure 2, Panel A). Additionally,
there was an interaction between time and chronic stress
(F
(11,86)
¼ 2.25, p50.05) and between time and hypercaloric
diet (F
(11,86)
¼ 3.59, p50.05). However, there were no
interactions between chronic stress and hypercaloric diet
(F
(1,96)
¼ 0.03, p40.05) or between time, chronic stress and
hypercaloric diet (F
(11,86)
¼ 1.22, p40.05).
Body weight
Body weight variations showed normal distribution (p50.05).
The two-way ANOVA showed significant effects of chronic
stress (F
(1,50)
¼ 20.214, p ¼ 0.009) and hypercaloric diet
(F
(1,48)
¼ 28.420, p ¼ 0.001) but no interaction was found
between these factors (F
(1,99)
¼ 4.549, p40.05). Furthermore,
one-way ANOVA followed by Fisher’s LSD post-hoc test
showed a significant group effect (F
(3,96)
¼ 16.328,
p50.0001). Weight increase was greater in the HD group
when compared with control (F
(3,96)
¼ 16.328, p50.01), stress
(F
(3,96)
¼ 16.328, p50.0001) and stress HD groups
(F
(3,96)
¼ 16.328, p50.05). The stress group showed weight
loss when compared with the control group (F
(3,96)
¼ 16.328,
p50.05). Moreover, there was no difference between control
and stresss HD groups indicating the reversibility of stress
effects caused by hypercaloric diet, or vice versa
(F
(3,96)
¼ 16.328, p50.05) (Figure 2, Panel B).
Body length
The group analysis (F
(3,96)
¼ 8.053, p50.001) indicated that
the hypercaloric diet increased the length of the rats (control
vs HD groups, (F
(3,96)
¼ 8.053, p50.001), and chronic stress
disrupted this effect (HD vs stress HD group, F
(3,96)
¼ 8.053,
p50.001) (Figure 2, Panel C). However, there was no
significant effect of chronic stress alone (F
(3,96)
¼ 8.053,
p ¼ 0.08).
Adipose tissue deposition
Overall, the main effects were that hypercaloric diet increased
adipose tissue in subcutaneous (F
(1,96)
¼ 46.763; p50.001)
and mesenteric (F
(1,96)
¼ 47.385; p50.001) areas (Figure 2,
Panel D). Compared with controls, chronic stress decreased
adipose tissue in both areas [subcutaneous, (F
(1,96)
¼ 46.763;
p50.001) and mesenteric, (F
(1,96)
¼ 11.582; p50.01)]
(Figure 2, Panel D: I and II). There were no interactions
Table 2. Plus-maze test (n ¼ 24–26 rats/group): data are reported as median ± interquartile range for each behavior.
Behavior/group C S HD SHD
PHD (n) 2.00 ± 5.25 1.00 ± 4.50 3.00 ± 3.00 1.00 ± 2.00
NPHD (n) 0.50 ± 2.25 1.00 ± 3.50 2.50 ± 4.75 0.00 ± 1.50
EOA (n) 0.00 ± 2.25 1.00 ± 1.00 1.00 ± 2.00 0.00 ± 1.00
ECA (n) 3.50 ± 3.75 3.00 ± 3.50 3.00 ± 5.50 3.00 ± 4.00
TOA (s) 9.72 ± 33.3 10.4 ± 34.7 21.7 ± 36.9 8.53 ± 17.9
TCA (s) 290.6 ± 38.4 289.0 ± 32.8 278.7 ± 35.1 291.9 ± 19.4
Grooming (s) 4.50 ± 28.4 6.06 ± 19.3 10.6 ± 27.3 7.81 ± 51.1
Rearing (n) 17.0 ± 10.0
a
17.0 ± 9.0
a
16.5 ± 7.5
a
20.5 ± 17.2
b
Fecal boluses (n) 0.00 ± 3.25 0.00 ± 1.00 0.00 ± 1.00 0.00 ± 1.25
a,b
Statistically significant difference of p50.05 between groups. There was a significant difference among groups in
rearing (exploratory behavior).
PHD, protected head-dipping movements; NPHD, non-protected head-dipping movements; EOA, entries into open
arms; ECA, entries into closed arms; TOA, time on open arms; TCA, time on closed arms; C, control group; S,
stress group; HD, hypercaloric diet group; SHD, stress + hypercaloric diet group.
n ¼ number of occurrences; s ¼ seconds.
Figure 1. Assessment of caloric intake. Effect of chronic stress and
hypercaloric diet on caloric intake (kcal/rat/cage/week) (n ¼ 6–7 cages/
group). Data are mean ± SEM. Significant effect of time; time chronic
stress interaction, and time hypercaloric diet interaction (repeated
measures ANOVA, p50.05). C, control group (n ¼ 25, with 4–5 rats/
cage); S, stress group (n ¼ 25, with 4–5 rats/cage); HD, hypercaloric diet
group (n ¼ 24, with 4 rats/cage); SHD, stress + hypercaloric diet group
(n ¼ 26, with 3–4 rats/cage).
DOI: 10.3109/10253890.2015.1079616 Stress, diet, weight gain and anxiety 5
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between factors for subcutaneous (F
(1,96)
¼ 0.047; p ¼ 0.8) or
mesenteric (F
(1,96)
¼ 1.188; p ¼ 0.2) areas.
The hypercaloric diet increased adipose tissue in subcuta-
neous (control vs HD groups, F
(3,96)
¼ 9.438; p50.001) and
mesenteric (control vs HD groups, F
(3,96)
¼ 23.304; p 50.05)
areas (Figure 2, Panel D I and II). Furthermore, the group
analysis showed that combined stress and HD reduced the
increase seen with HD alone or the decrease seen with stress
Figure 2. Assessment of weight parameters. Panel A: effect of chronic stress and hypercaloric diet on weekly weight (n ¼ 24–26 animals/group). Data
are mean ± SEM. Significant effect of time, chronic stress and hypercaloric diet (repeated measures ANOVA, p50.05). Time chronic stress and
time hypercaloric diet interactions were observed (repeated measures ANOVA, p50.05). C, control group (n ¼ 25, with 4–5 rats/cage); S, stress
group (n ¼ 25, with 4–5 rats/cage); HD, hypercaloric diet group (n ¼ 24, with 4 rats/cage); SHD, stress + hypercaloric diet group (n ¼ 26, with 3–4 rats/
cage). Panel B: effect of chronic stress and hypercaloric diet on final body weight of rats (n ¼ 24–26 rats/group). Data are mean ± SEM. Different
superscript letters (a–c) indicate a statistically significant difference of p50.05 among groups. Values with the same superscript letter are not
significantly different. Group analysis was performed by one-way ANOVA followed by Fisher’s LSD. C, control group; S, stress group; HD,
hypercaloric diet group; SHD, stress + hypercaloric diet group. Panel C: naso-anal length (cm) measured at the end of the experiment (n ¼ 24–26 rats/
group). Data are mean ± SEM. Different superscript letters (a and b) indicate a statistically significant difference of p50.05 among groups. Group
analysis was performed by one-way ANOVA followed by Fisher’s LSD. C, control group; S, stress group; HD, hypercaloric diet group; SHD,
stress + hypercaloric diet group. Panel D: adipose tissue deposit weights (n ¼ 23–26 animals/group). Data are mean ± SEM. For each figure, different
letter superscripts (a–c) indicate a statistically significant difference of p50.05 among groups analyzed by two-way ANOVA followed by Fisher’s LSD.
C, control group; S, stress group; HD, hypercaloric diet group; SHD, stress + hypercaloric diet group. I Subcutaneous region. II Mesenteric region.
6 C. de Oliveira et al. Stress, Early Online: 1–10
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alone in the subcutaneous (control vs stress groups,
F
(3,96)
¼ 9.438; p50.05) and mesenteric (control vs stress
groups, F
(3,96)
¼ 23.304; p50.05) adipose tissues.
Serum corticosterone concentrations
Two-way ANOVA showed a significant main effect of the
type of diet, indicating that hypercaloric diet decreased
corticosterone concentrations (measured only at the end of the
80-d experiment) (F
(1,16)
¼ 12.835, p50.05). However, there
was no chronic stress effect (F
(1,16)
¼ 0.850, p ¼ 0.37) or
interaction between these factors at this time (F
(1,16)
¼ 1.325,
p ¼ 0.267) (Figure 3, Panel A).
Changes in relative adrenal weight
The study found main effects of chronic stress
(F
(1,28)
¼ 5.288, p50.05) and hypercaloric diet
(F
(1,28)
¼ 6.197, p50.05) on the relative adrenal weight
(two-way ANOVA) as well as interaction between these
factors (F
(1,28)
¼ 4.182, p50.05). Group analysis
(F
(3,28)
¼ 5.283, p50.01) showed that chronic stress increased
the relative adrenal weight (control vs stress groups,
F
(3,28)
¼ 5.283, p5 0.01). However, rats fed with hypercaloric
diet, stressed and nonstressed showed no change in relative
adrenal weight [control vs stress HD groups, (F
(3,28)
¼ 5.283,
p ¼ 0.8) and control vs HD groups; (F
(3,28)
¼ 5.283, p ¼ 0.8)]
(Figure 3, Panel B).
Discussion
Anxiety-like behaviors
This study showed that, in rats exposed for 80 d to stress and a
comfort food diet, chronic stress increases anxiety-like
behaviors and the hypercaloric diet can reverse these
anxiogenic effects (Adam & Epel, 2007; Dallman et al.,
2005). This inference corroborates other studies that indicate
stressful events may induce behavioral alterations and
anxiety-like symptoms (Kormos & Gaszner, 2013; Lister,
1987), and that hypercaloric diet can modify physiological
stress responses (Krolow et al., 2010). Additionally, this study
indicates that chronic stress and hypercaloric diet are
associated with behavioral, physiological and biochemical
changes.
The lack of chronic stress effects in the rats fed with
hypercaloric diet might involve multistep mechanisms to
maintain homeostasis (Dallman et al., 2004). This study
shows a type of diet-dependent effect on serum corticosterone
concentrations, and suggests that this result reflects a rat’s
preference for a hypercaloric diet (i.e. carbohydrates and fat),
and/or augmented neuropeptide Y (NPY) expression, an
orexigenic protein, in the hypothalamus (Dallman et al.,
2004). Moreover, the type of diet influenced also locomotor
activity since group analysis indicated that hypercaloric diet
increased locomotion in an open field. Similar to our data on
rats, another study using mice fed with high-fat diet showed
increase in the locomotor activity rhythm (Kohsaka et al.,
2007).
A number of animal studies indicate that stress exposure
alters the activity of the neuroendocrine and neurotransmitter
systems linked to behaviors such as anxiety-like and fear
behaviors (Bergstrom et al., 2008; Wood et al., 2008).
Chronic stress exposure can lead to alterations in the central
dopaminergic and serotonergic nervous systems and to HPA
axis deregulation, which are associated with psychiatric
disorders (Nikolaus et al., 2009). The central serotonergic
signaling system has been shown to play an important role in
the observed changes in appetite control and food intake
regulation (Mann, 1999). Thus, our findings may provide a
better understanding of the pathogenesis of emotional and
eating disorders that can be related to central dopaminergic
and serotonergic systems, but further investigations on these
neurotransmitters are needed.
The chronically stressed rats fed with hypercaloric diet
showed increase in the number of rearings in the elevated plus
Figure 3. Effects of chronic stress and hypercaloric diet on corticosterone and adrenal weight. Panel A: serum corticosterone concentration. Measured
24 h after the last session of chronic stress. Data are mean ± SEM and n ¼ 5 rats/group). p50.05 was considered to be statistically significant. The main
effect of the type of diet and chronic stress was evaluated by two-way ANOVA. Superscript letter (a) indicates a significant effect of hypercaloric diet
on corticosterone concentration (p50.05). There was no chronic stress effect or interaction (p40.05). C, control group; S, stress group; HD,
hypercaloric diet group; SHD, stress + hypercaloric diet group. Panel B: the relative adrenal gland weight (g/100 g body weight) (n ¼ 8 rats/group).
Data are mean ± SEM. Different superscript letters (a and b) indicate statistically significant difference of p50.05 between the groups. Values with the
same superscript letter are not significantly different. The main effect of the type of diet and chronic stress was evaluated by two-way ANOVA followed
by Fisher’s LSD. Group analysis was performed by one-way ANOVA followed by Fisher’s LSD. C, control group; S, stress group; HD, hypercaloric
diet group; SHD, stress + hypercaloric diet group.
DOI: 10.3109/10253890.2015.1079616 Stress, diet, weight gain and anxiety 7
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maze test (Table 2). This indicates that when both factors
were combined, there was intensification in exploratory
activity in a novel environment. Previous studies have
suggested that rearing is a behavior associated with emotional
factors (Walsh & Cummins, 1976), and reflects exploratory
activity in animal models (Cruz et al., 1994). Moreover, the
results demonstrate that time in the open arms on the EPM
increases in rats fed with hypercaloric diet. Thus, we suggest
that comfort food changes behaviors by reducing the rats’ fear
to enter open areas and increasing willingness to remain in
open environments (Krolow et al., 2010; Ortolani et al.,
2011). These findings corroborate other studies showing that
the ingestion of hypercaloric food for a few months reduces
sympathetic responses to stressors, indicating lower anxiety
levels (Prasad & Prasad, 1996; Singh, 2014).
Caloric intake, body weight and length
The caloric intake among rats fed with hypercaloric diet
supplement, as expected, was greater than in rats fed with
only standard chow. This increased consumption contributed
to a greater increase in body weight and adiposity. These
results confirm a previous study of our group using the same
chronic stress protocol for twelve weeks, which was long
enough to increase adiposity (de Oliveira et al., 2014; Macedo
et al., 2015). However, rats fed with hypercaloric diet and
submitted to chronic stress did not show the same effect on
caloric intake, indicating that the chronic stress exposure
modulates the diet-induced increase in caloric intake. It is
well established that duration, type and severity of stress may
change an animal’s responses to the available diet (Maniam &
Morris, 2012). More severe stressors usually decrease food
intake when animals are exposed to either standard or
hypercaloric diet (Pucilowski et al., 1993). The diverse
effects of chronic stress on food intake by animals could be
due to the different composition between the two types of
diet. For example, chronically stressed rats most often reduce
sucrose consumption (Dalm et al., 2008) and this finding
could explain the lower food intake found in the current study.
Chronic stress through the 80 d of the experiment reduced
weight gain of rats regardless of the diet that was offered to
them (Figure 2, Panel A, B). However, rats on the hypercaloric
diet showed the greatest increase in body weight (control vs
HD groups). Moreover, when stressed rats were fed the
hypercaloric diet, the stress effect in decreasing weight gain
was reversed by the comfort food (control vs stress HD
groups). Access to a hypercaloric diet is considered a major
environmental factor that contributes to the current obesity
epidemic (Berthoud et al., 2011), and it has been shown
experimentally to minimize stress effects, hence acting as
comfort food (Dallman et al., 2005). It has been proposed that
the exposure to chronic stress and hypercaloric diet could reset
homeostasis over time, and that these imbalances are, in turn,
associated with clinically manifested impairments in meta-
bolic and physiological processes (de Oliveira et al., 2014).
Furthermore, chronic stress situations increase the activity of
corticotropin-releasing hormone (CRH), having anorexic
effects (Pasquali, 2012) and involving reduced orexigenic
peptide (NPY) expression in the hypothalamus (Heinrichs
et al., 1993). These mechanisms might be involved in the
inhibition of body weight gain in the stressed groups in the
present experiment.
Rats fed the hypercaloric diet for 80 d showed the greatest
nose-to-tail length (control vs HD group) (Figure 2, Panel C).
Furthermore, this increase caused by the hypercaloric diet
(Estadella et al., 2004), as well as the increase in body weight,
was inhibited by chronic stress. In addition, the hypercaloric
diet increased the subcutaneous and mesenteric adipose tissue
depots (control vs HD groups) (Figure 2, Panel D I and II).
This followed increased kcal intake from eating the high-fat
and carbohydrate diet, with a major impact on energy
metabolism, consequent increased lipid deposition in adipose
tissue and body weight gain (Kohsaka et al., 2007). These
effects might involve risks of obesity, such as dyslipidemia
and metabolic syndrome.
Conversely, chronic stress offset the effect of availability of
the kcal-rich diet on the subcutaneous and mesenteric adipose
tissues (Figure 2, Panel D I and II). Chronic stress exposure
has been generally reported to reduce food intake, body
weight gain and adiposity (Macedo et al., 2012; Solomon
et al., 2011). Thus, we suggest that chronic stress exposure
(specifically restraint) induces metabolic changes in rats that
reduce obesity parameters when comfort food is eaten.
Adrenal weight and serum corticosterone
concentration
Chronic stress inhibited body weight gain and increased
relative adrenal weight. However, combining chronic stress
with the hypercaloric diet prevented the adrenal hypertrophy
seen with stress alone. Notably, the single serum cortico-
sterone measurement at the end of the chronic stress exposure
(for 80 d) showed no significant increase in serum cortico-
sterone concentration; nonetheless, availability of the hyper-
caloric diet significantly reduced the terminal serum
corticosterone concentration in both the stressed and
unstressed rats (Figure 3, Panel A). This effect could be due
to direct or indirect inhibitory effects of the chronic
hypercaloric diet on the HPA axis. For example, abdominal
fat stores exert a negative feedback action on the HPA axis to
decrease corticosterone levels (Dallman et al., 2003, 2005).
The present data corroborate other studies performed by our
research group (Macedo et al., 2012; Torres et al., 2001),
which showed no main effects of chronic stress on cortico-
sterone levels. This indicates HPA axis habituation. However,
after 11 weeks of chronic-stress exposure, rats present a
greater increase in the daily peak release of corticosterone
(19:00 h), suggesting incomplete HPA axis adaptation to
chronic-stress exposure (de Oliveira et al., 2014). Importantly,
highly palatable fat- and carbohydrate-rich foods have been
shown to decrease the stress response in rats exposed to
chronic stress (Pecoraro et al., 2004). Furthermore, sweet and
fatty foods, which show low-protein levels, may also provide
relief from stress in vulnerable people by improving function
of the serotonergic system (Gibson, 2006). However, humans
(Adam & Epel, 2007) or rats (Dallman et al., 2003) may
increase their food intake due to stress or negative emotions
(Appelhans et al., 2011). In this case, the food eaten tends to
have high-sugar and/or high-fat levels (Tomiyama et al.,
2012). The relationship between food and stress depends on
8 C. de Oliveira et al. Stress, Early Online: 1–10
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the type of stressor, and the effect can be the converse. For
example, chronic stress induces both increased consumption
of palatable and rewarding foods, thus leading to obesity or
decreased appetite, leading to weight loss. Moreover, expos-
ure to a stressor associated with the intake of palatable foods
can reduce the signs of stress and anxiety, as reported here.
Conclusion
This study confirms that exposure to chronic stress triggers
anxiety-like behaviors, and chronic hypercaloric diet reverses
some features of anxious behaviors. Most importantly, chronic
stress alone reduces food intake, body weight gain and
adipose storage, suggesting that the stress response can lead to
suppression of appetite and food intake. Conversely, rats fed
highly palatable foods rich in fat (and carbohydrate) increased
food intake, this being a determining factor for the increase in
adiposity. Interestingly, the combination of chronic stress and
cafeteria diet reduced the signs of stress and anxiety. These
findings may be of considerable clinical importance because
stressful events not only stimulate HPA axis activity, but
when associated with comfort food ingestion can become a
powerful inhibitor of stress-induced HPA axis activation.
Consequently, we can suggest that the HPA axis is not only
the indicator of an appropriate stress response, but is also
interconnected with neuroendocrine parameters that regulate
appetitive behaviors. Last, we can infer that chronic stress
may offset consequences of a comfort food diet on increasing
body weight and adiposity. It is known that the mechanisms
may include changes in corticosterone secretion and action
induced by chronic stress. Curiously, there was no effect of
chronic stress on corticosterone levels if a comfort food diet
was provided. Furthermore, the mechanisms by which these
events occur still need to be addressed.
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of the paper. The
research was supported by the following Brazilian funding
agencies: National Council for Scientific and Technological
Development – CNPq (Dr. I.L.S.T, W.C); Committee for the
Development of Higher Education Personnel – CAPES
(I.C.C.S, C.M.O, C.O. and I.C.M.); The Postgraduate
Research Group – GPPG/Hospital de Clı
´
nicas de Porto
Alegre – HCPA (I.L.S.T. – Grant 100382), Docfix CAPES/
FAPERGS/09/2012 Grant, A.S.Q. and R.V.).
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Supplementary material available online
10 C. de Oliveira et al. Stress, Early Online: 1–10
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