Low protein diet during gestation and lactation increases food reward seeking but does not modify sucrose taste reactivity in adult female rats

Int. J. Devl Neuroscience 49 (2016) 50–59

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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu

Low protein diet during gestation and lactation increases food reward seeking but does not modify sucrose taste reactivity in adult female rats Amanda Alves Marcelino da Silva a,d , Mayara Matias Oliveira b , Taisy Cinthia Ferro Cavalcante c,e , Larissa Cavalcanti do Amaral Almeida d , Julliet Araújo de Souza e , Matilde Cesiana da Silva f , Sandra Lopes de Souza d,g,∗ a

Nursing College—Universidade de Pernambuco—Campus Petrolina-UPE, Recife, PE, Brazil Postgraduate Genetics, Universidade Federal Pernambuco—UFPE, Recife, PE, Brazil c Postgraduate Nutrition, Universidade Federal Pernambuco—UFPE, Recife, PE, Brazil d Postgraduate Neuropsychiatry and Behavioral Sciences, Universidade Federal Pernambuco—UFPE, Recife, PE, Brazil e Nutrition College—Universidade de Pernambuco—Campus Petrolina-UPE, Recife, PE, Brazil f Nutrition College—Universidade Federal de Pernambuco, Centro Acadêmico de Vitória—UFPE-CAV, Vitória de Santo Antão, PE, Brazil g Department of Anatomy, Universidade Federal de Pernambuco—UFPE, Recife, PE, Brazil b

a r t i c l e

i n f o

Article history: Received 22 May 2015 Received in revised form 25 December 2015 Accepted 12 January 2016 Available online 22 January 2016 Keywords: Low protein diet Motivation Taste c-Fos Female Rats

a b s t r a c t Introduction: Nutritional deficiencies during neural development may lead to irreversible changes, even after nutritional rehabilitation, promoting morphological and functional adaptations of structures involved with various behaviours including feeding behaviour. However, the ability of the exposure low protein diet during gestation and lactation to affect the hedonic component of food intake is still poorly understood, especially in females. Methods: Wistar rats were divided into two groups according to the diet offered to the dams during pregnancy and lactation: control female (CF; diet with 17% protein, n = 7) and low protein female (LPF; diet with 8% protein, n = 7). The following parameters were evaluated: (a) body weight during weaning, 30, 45, 60, 75, 90 days of life; (b) standard diet intake from 110 to 132 days of life; (c) fat diet and consumption of simple carbohydrates (HFHS) for 1 h at 145 days of life; (d) incentive runway task 60 days after 82 days of life; (e) taste reactivity at 90 days of life; and (f) neuronal activation in the caudate putamen, amygdala, paraventricular nucleus of the hypothalamus under stimulus HFHS at 145 days of life. Results: The exposure, a low protein diet during gestation and lactation, decreased the body weight throughout the study period from weaning to 90 days of life. However, there was no significant change in the body weight of low protein females from 110 to 132 days of life compared with the control females. There was an increase in the rate of the search for reward and reduced the latency of the perception of bitter taste. The exposure, a low protein diet during gestation and lactation, also promoted hypophagy in adult females compared with control animals. The low protein female had increased HFHS diet consumption compared with the control. Undernutrition increased neuronal activation in response to HFHS diet consumption compared with female controls in the amygdala and in the caudate putamen. Conclusion: Females subjected to the exposure, a low protein diet during gestation and lactation, exhibit hypophagy on a standard diet but a higher consumption of a diet rich in lipids and simple carbohydrates. And also were more motivated by the pursuit of reward and reduced latency of the bitter taste reactivity, and increased the number of immunoreactive cells c-fos protein activated in the caudate putamen, amygdala and paraventricular nucleus. © 2016 Published by Elsevier Ltd. on behalf of ISDN.

∗ Corresponding author at: Av. Prof. Moraes Rego, 1235, Cidade Universitária, Recife 50670901, PE, Brazil. E-mail address: [email protected] (S.L. de Souza). http://dx.doi.org/10.1016/j.ijdevneu.2016.01.004 0736-5748/© 2016 Published by Elsevier Ltd. on behalf of ISDN.

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1. Introduction During pregnancy and lactation, the offspring’s developing nervous system undergoes diverse formation and maturation of its physiological and morphological patterns. These periods are considered “critical” because they comprise a sequence of well-defined ontogenetic events that are subject to external agents. This period may occur “set points” (adaptations) that influence later events (Morgane et al., 2002). These adjustments occur in response to environmental demands; in other words, the individual modifies its morphological and functional patterns in response to a stimulus or insult to maintain its survival in its environment (Hales and Barker, 2001). Early exposure to a low protein diet can induce such adaptations because exposure to poor nutrition can result in metabolic changes that encourage the economic and efficient use of scarce energy substrates (Bellinger and Langley-Evans, 2005). Overall, these adaptations are beneficial to organisms that remain in the same metabolic environment they were exposed to in the perinatal period (Bellinger et al., 2006), but long-term damage may occur if there is an excess of nutrients (Bouret and Simerly, 2006). Epidemiological studies and animal model studies have shown that maternal health and nutritional status during pregnancy and lactation affect the offspring’s development of the central and peripheral systems that regulate energy balance and body weight (Desai et al., 2005; Hales and Barker, 1992, 2001). Effects of undernutrition have been implicated in feeding behaviour. The expression of peptides like neuropeptide Y and agouti-related peptide (NPY/AgRP) that stimulate food intake is increased, and levels of peptides like pro-opium-melanocortin and cocaine- and amphetamine-regulated transcript (POMC/CART) that inhibit food intake are decreased, especially in the hypothalamus, which integrates hunger and satiation stimuli (Bouret and Simerly, 2006). In addition, the anorexic actions of leptin and insulin (Fernandez-Twinn et al., 2005) are reduced in animals exposed to malnutrition in early development, resulting in increased food consumption (Vickers et al., 2000). Palatable foods can stimulate food consumption, resulting in a positive energy balance (ErlansonAlbertsson, 2005). Diets high in simple sugars and fat are very palatable and can promote metabolic disorders by stimulating fat synthesis, mainly triglycerides, which are stored in adipose tissue (Estadella et al., 2004). Increased adipose tissue may stimulate the release of adiposity signals such as leptin and insulin (Ashino et al., 2012; Fam et al., 2007). Greater release of these peptides may result in resistance to their cellular actions; thus, their effects are reduced (Kirk et al., 2009; Srinivasan et al., 2006). Another consequence is the increased food intake, as these two factors work together to stimulate satiety (Belgardt and Bruning, 2010). Palatable foods also have organoleptic properties, capable of stimulating food reward pathways and generating a feeling of pleasure (Lowe and Butryn, 2007). The hedonic control components of food intake are translated and integrated through the cortico-mesolimbic system formed by the nucleus accumbens, ventral striatum, ventral tegmental area, prefrontal cortex, hippocampus, and amygdala (Berridge et al., 2009). The perception of palatable information is more complex because it depends on the nutritional value (high in carbohydrates or fat) and sensory properties (sight, smell, taste, texture) (Rolls, 2006). Food information that stimulates sensory receptors is processed in different brain regions depending on the sense (Rolls, 2006). Therefore, sensory receptor stimulation can occur in the temporal cortex (vision), olfactory bulb (smell), thalamus (texture), and nucleus of the solitary tract (taste) (Rolls, 2006). This information will be shaped by the nucleus accumbens and ventral tegmental area (Rolls, 2006). The amygdala transforms the information received from other reward system regions into emotional

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stimuli (pleasure), while the hippocampus records the “pleasurable experience” in memory (Kelley and Berridge, 2002). Increased palatability might involve the stimulation of endogenous opioid activity (Tsujii et al., 1986); in addition, dopaminergic signalling within the reward areas is also influenced by energy-dense food intake (Vucetic et al., 2012). There are few studies of motivation in animals fed a low protein diet. Recently, it was observed that perinatal malnutrition increases motivation for the search of food reward in adulthood (da Silva et al., 2013). This motivation was related to increased neuronal activation in reward system brain regions (da Silva et al., 2013). Therefore, our objective was to study the effect of low protein diet exposure during gestation and lactation periods on food motivation and responsiveness in adult rats. We hypothesized that the low protein diet condition would promote increased food motivation and that taste reactivity changes would be detectable in reward regions of the adult rat brain.

2. Materials and methods 2.1. Animals All experiments were approved by the Ethics Committee on Animal Experiments of UFPE (no process 23076.037409/2011-64) following the rules of CONCEA (National Council for Animal Experiments Control). Wistar albino rats (200–250 g body weight) were reared in the Nutrition Department of the Federal University of Pernambuco. The rats were mated at a ratio of two females to one male. Pregnancy was diagnosed based on the presence of sperm in the vaginal smear and confirmed by body weight gain. After the diagnosis of the pregnancy, the rats were transferred to individual cages, and during pregnancy and lactation, the rats received isocaloric diets (Table 1) with two different protein concentrations: control diet with 17% protein, (n = 7) or low protein diet with 8% protein, n = 7). Sexing was performed to form litters with eight pups of both sexes per dam (four males, four females). Throughout the experiment, the animals were kept in standard vivarium conditions (temperature of 22 ± 1 ◦ C under light/dark inverted 12 h, the light on 18:00, with ad libitum access to food and water. After weaning at 22 postnatal days, pups from both groups were fed the vivarium’s standard diet (Labina Presence® , Paulinia, São Paulo, Brazil). The experimental groups were formed by randomly collecting two female pups from each dam. After the manipulation diet, two perinatal experimental groups were obtained. Control female (CF, n = 14) from dams who received a normal protein diet during the perinatal period and low protein female (LPF, n = 14) from dams who received a low protein diet during the perinatal period.

Table 1 Composition of the experimental diets offered during the period of pregnancy and lactation. Components

Low protein (8%)

Normoprotein (17%)

G% Protein Carbohydrate Lipidis Fiber Vitamin Minerals Metionin Coline % Kcal

100.00 8.20 74.75 7.00 5.00 1.00 3.50 0.30 0.25 394.8

100.0 17.05 65.90 7.00 5.00 1.00 3.50 0.30 0.25 394.8

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Table 2 Composition of the experimental diets used after weaning and adulthood. Nutrients

Proteins Lipids Carbohydratesa Fiber (Celulose) Mineral mix Vitamin mix dl-Metionina Choline bitartrate BHT Total energy (kCal/g)

body weight and food intake. Consumption was calculated as the weight of the feed offered by subtracting the weight of the feed left on the next day (Ferro Cavalcante et al., 2013).

Diets Presence® g%

Palatable food g%

23.0 4.0 55.0 5.0 3.7 1.9 0.4 0.2 – 3.48

16.1 17.7 53.3 1.2 1.5 0.5 0.2 0.2 0.0014 4.33

2.4. Behavioural tests 2.4.1. Runway task incentive At 65 days old, the animals were subjected to the runway task incentive to assess “Learning” and “Wanting” (da Silva et al., 2013; Pecina et al., 2003; Silveira et al., 2010) after food deprivation for 4 h. This behavioural paradigm generates learning curves and measures the speed and path travelled to investigate the animal’s motivation for the stimulation reward. The test was performed between 12:00 and 14:00.

Composition according to the total energy

Protein Lipids Carbohydrate

Presence®

HFHS

24 10 66

14.9 36.7 49.2

Source: The calculations of chemical composition were based on nutritional information sent by the supplier of the products and the Brazilian Table of Food Composition (TACO). The standard vivarium diet (Presence® ) from weaning to 144 days of life and HFHS for 1 h at 145 days. a The HFHS experimental diet contains about 50.41% of simple carbohydrates.

2.2. Palatable food The diet formulation (palatable food) was based on the Household Budget Survey (POF) and the AIN-93G, with some adjustments to the chemical composition. Amongst these adjustments, the lipid content and amount of simple carbohydrates were increased (50.41%) to enhance sensory factors, creating a pleasant odour and texture and resembling the Western diet. In addition, the protein content was adequate to maintain the rats’ health (16.1% protein). Studies have revealed that a high-carbohydrate and fat diet provides approximately 20–40% of calories from fat and 10–30% from sucrose (Panchal and Brown, 2011). Table 2 compares the macronutrient compositions of the diets administered during the study. Except for the standard diet (Presence® ), these diets were devised by the Experimental Nutrition Laboratory and Dietetics (LNED) Department of Nutrition, UFPE. The dry ingredients were mixed and sieved for proper homogenization. Then, liquid or semi-solid components were added before water was added. The next step was to dry the diet in an oven with air circulation to 60–70 ◦ C for 24–36 h. The diets were stored at 4 ◦ C until use. 2.2.1. Palatable food intake At 145 days of life, palatable food intake (Table 2) was recorded for 1 h between 11:00 and 12:00. A semi-analytical digital electronic scale was used (Marte XL 500, class II, maximum capacity 500 g, lower division 0.001 g; Marte Científica® , Santa Rita do Sapucaí, Minas Gerais, Brazil) to measure how much food was offered and rejected.

2.4.2. Structure test apparatus The structure of the runway consisted of a runner (140 × 14 × 30 cm) with two boxes (19 × 14 × 30 cm) located at the beginning and end of the runway. The distance varied according to the session day. The boxes were made of acrylic, while the centre was polypropylene. 2.4.3. Runway task training Training sessions (5 min each) were conducted for 11 sessions on alternate days (22 days). In the first three sessions (adaptation), the rats were placed directly on the target box (with the doors closed) for 5 min with access to reward (Chocookies; Nabisco® , East Hanover, NJ, USA). In the fourth training session, the initial box was located 15 cm from the target box. The animal was in the initial box for 30 s with the door closed; when it was raised, the animal could continue to the centre. If the animal did not come out of the initial box within 3 min, it was gently moved to the target box. In session 5, the initial box was 30 cm away from the target box, and this distance was increased to 60 cm in the sixth session, 75 cm in the seventh, 90 cm in the eighth, 120 in the ninth, and 140 cm in the tenth and eleventh. The sessions were defined as previously described (Pecina et al., 2003), with sessions 1–3 considered the adjustment phase (the animal is exposed to a new environment and reward so that the natural neophobic behaviour disappears), sessions 4–6 as the pre-exposure phase (the animal is exposed to the target box and execution centre), sessions 7–9 as the learningincentive or reinforcement phase (encouraged by the stimulus of reward), and sessions 10 and 11 as the fully trained phase (during which the animal confirms their learning by showing total familiarity with the tests and directly accessing the reward). The speed of task completion for each session was calculated by dividing the latency time taken to reach the target box by the length of the track. The speed in retrieving the reward indicates the animal’s motivation. The rat was considered to have left the starting box when all four limbs were out of the box, and it was considered to have entered the target box when all four limbs were within the target box. Once the rat entered the target box and started eating, it was allowed to consume the palatable food for 30 s before being removed.

2.3. Body weight measurement We assessed weight gain during the weaning period, and at 30, 45, 60, 75, and 90 days of life. Measurements were obtained with on a Marte XL 500, class II scale. 2.3.1. Standard diet intake and body weight from 110 to 132 days of life At 110–132 days of life, the rats received a standard diet (Table 2), and body weights were measured for 22 days (every 3 days) at the same time of day, between 07:00 and 08:00. A semianalytical digital scale (Marte XL 500, class II) was used to measure

2.4.4. Taste reactivity testing The test of reactivity to taste by Grill and Norgren (1978) was performed to study “liking” (Berridge, 2000). This test was performed when the rats were 90 days old. The animal was placed in the arena (25 cm long × 25 cm deep × 20 cm front) with a transparent floor and walls with 1 mL sucrose solution at 30% or quinine solution (C20 H2 4N2 O2 ·HCl·2H2 O; Sigma–Aldrich, St. Louis, MO, USA) to 0.3 M in the left corner of the arena and directly on the floor (Shin et al., 2010). A video camera (Sony Handycam DCR-DVD650 equipped with nightshot function; Sony, Tokyo, Japan) was placed under the transparent floor to record spontaneous facial and body

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reactions that occurred during and after voluntary sucrose or quinine intake. The animals were habituated to the testing arena for 2.5 min before the test. In each session, solutions were placed inside the arena, and the animals were allowed to drink the solution for 2.5 min while their behaviour was filmed for later analysis. Animal reactions that were considered positive or hedonic included rhythmic protrusions of the tongue, lateral protrusion of the tongue, and licking the paws. Negative or aversive reactions were yawning, shaking the head, cleaning the face, “shaking” with the front feet, and rubbing the chin. The tests of the two solutions were performed at least 48 h apart. 2.5. Transcardial perfusion The animals were deeply anesthetized with a combination of ketamine (1 ml/kg) and xylazine (0.1 ml/kg) before the chest cavity was opened to access the left ventricle and introduce the infusion cannula. This cannula remained coupled to a peristaltic pump speed compatible with maintaining blood vessel integrity. Initially, 150 ml saline was infused (NaCl 0.9%) at room temperature to exsanguinate the vessels. This procedure prevents clot formation and provides the correct amount of fixative to the tissues. This was followed by infusion of 400 ml 4% paraformaldehyde (pH 7.4 at 4 ◦ C). Then, the brains were removed from the skull and postfixed in the same fixative solution with added sucrose (20%) for 4 h. After this period, the brains were stored in cryoprotectant solution (sodium phosphate buffer PBS—over 30% sucrose) prior to sectioning. 2.6. c-Fos immunohistochemistry The brains obtained after perfusion were cut into coronal 40-␮m-thick sections using a cryostat (CM1850, Leica, Wetzlar, Germany, belonging to the laboratory of Pathology-Lyka [UFPE] and Core Technology Platform 2 Research Center Aggeu Magalhães—FioCruz). Five series of sections were collected for each animal in an acrylic plate (25 wells) with antifreeze solution and stored at −20 ◦ C. One group of sections was used for c-Fos immunohistochemistry. In this procedure, the coronal sections were removed from the anti-freeze solution and subjected to three series of washes. The first washes were with PBS (3 × 10 min), the second with 0.6% hydrogen peroxide (5 × 1 min) to block endogenous peroxidase activity and nonspecific attachment sites, and the third again in PBS (3 × 10 min) to remove the post anchor residues. The samples were then incubated in solution with primary anti-Fos antibody (made in rabbit) for 48 h. This solution is composed of PBS, Triton-X 100 (0.3%) normal goat serum (NGS 5%) and Fos protein antibody (1:10.000) (Calbiochem, Bad Soden, Germany). During incubation, the slices remained in 1.5-ml vials in a refrigerated incubator shaker (MA83, Marconi, Piracicaba—São Paulo, Brasil) protected from light. After incubation, the sections were subjected to washes in PBS (3 × 10 min) and then incubated in secondary antibody solution for 90 min. This solution consisted of Triton (0.3%), PBS, and biotinylated secondary antibody generated in goats against rabbit (1:200) (catalogue no. B8895; Sigma–Aldrich). Then, the sections were subjected to washing with PBS (3 × 10 min) and further incubation (90 min) with avidin–biotin–peroxidase 1% (Vectastain, Camon, Wiesbaden, Germany). After this incubation period, the sections were washed in PBS (2 × 10 min) and sodium acetate (2 × 10 min) and then held for 5 min. The immunoperoxidase reaction was revealed with 3,3 -diaminobenzidine tetrahydrochloride (DAB, Sigma–Aldrich) diluted in distilled water solution, nickel ammonium sulphate (NAS), sodium acetate (0.2 M), ammonium chloride, and ␤-dglucose. After this reaction, glucose oxidase was added to obtain

53

a light brown/purple colour. The sections were then washed with sodium acetate (2 × 10 min) and PBS (3 × 10 min) to neutralize the reaction. The sections were mounted on gelatinized slides, subjected to dehydration and diaphanization, and finally covered with a coverslip and DPX. In the dehydration process, the sections were immersed in a solution in a sequence of increasing alcohol concentrations (50%, 70%, 95% - 3 min each, in 100% - 10 min) followed by diaphanization in xylene, 10 min). Neurons were identified and quantified from photomicrographs acquired with a digital camera (SHC-410NAD, Samsung, Seoul, South Korea) adapted to a microscope (BX50, Olympus, Tokyo, Japan) and a computer. Image acquisition was performed using a 10× objective and with the help of TV Tuner Application software. The Stereotaxic Rat Atlas was used as a reference (Watson, 2005) to identify and delineate the brain regions of interest. The areas chosen for this study were the caudate putamen (CPU), central and basolateral amygdala (AMY), and paraventricular nucleus (PVN) of the hypothalamus. The CPU and AMY were identified based on obvious markings −2.64 from the bregma. The PVN was considered to range from −1.72 to −1.92 from the bregma. Quantitative analysis of c-Fos-immunoreactive cells was performed using ImageJ software (version 1.45, http://rsbweb.nih.gov/ij/; National Institutes of Health, Bethesda, MD, USA) for each region of interest on both sides of the brain, with dark-brown or black staining considered positive. 2.6.1. c-Fos immunoreactivity in response to diet At 145 days, c-Fos protein immunoreactivity in reward system areas was examined in response to food intake of a high-fat, high-sucrose (HFHS) diet. The Fos protein belongs to the group of nuclear proteins that bind to promoters and DNA regulatory sites controlling the transcription of target genes and numerous effectors (Curran, 2002). This protein is encoded by the proto-oncogene immediate activation (GSEs) cfos, a transient and quickly expressed gene (between 20 and 90 min) in many tissues in response to various stimuli such as cell proliferation, neuronal depolarization, apoptosis, nociceptive stimulation, and food (Del-Bel et al., 1993; Hoffman et al., 1993; Morgan and Curran, 1989). The fos-protein labelling technique is widely used to evaluate the effects of environmental stimuli, map cell activity, and identify certain brain areas because it participates in information storage, external changes in perception, and the adaptive process to influences neuronal activity that can result in lasting nervous system changes (Sagar et al., 1988). The animals were divided into two groups: control females (CF, n = 4) and low protein females (LPF, n = 4). Both groups consumed palatable food for 60 min. During the test, animals were exposed to food for 60 min between 11:00 and 12:00 after fasting for 12 h. Afterwards, the animals were anesthetised for transcardial perfusion. 2.7. Data analysis Data for the 1-h food consumption, taste reactivity test, and quantitative analysis of immunoreactive cells are presented as the mean and standard error, and Student’s t-tests were used to compare each group with its control. For analysis of body weight, weight gain pattern, food consumption, and runway analysis task incentive, we performed two-way analysis of variance (ANOVA) followed by Bonferroni tests to compare the effect of the low protein diet over the test days. In all evaluations, the level of significance was p ≤ 0.05. All data were analysed using

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A.A.M. da Silva et al. / Int. J. Devl Neuroscience 49 (2016) 50–59

300

Body weight (g)

250

Control

*

*

200

Low Protein

*** 150

*** 100

***

50

**

0 22

30

45 60 Days of life

75

90

Fig. 1. Evaluation of body weight of females submitted to exposure low protein diet during gestation and lactation. Two way ANOVA was used followed by Bonferroni test, * p < 0.05, *** p < 0.001. Data presented as mean ± SEM. CF, n = 10; LPF, n = 10.

Speed (cm/s)

40

Incentive Learning

Pre-exposure goal

***

30

3.2. Behavioural tests

Trained

*** ***

Control Low Protein

*** 20

***

10 0

***

*** 4

5

6

7 8 Session

9

10

3.2.1. Runway task incentive (seconds) Two-way ANOVA revealed an interaction in females (F1,202 = 94.57, p < 0.001) between maternal diet and motivation sessions during behavioural testing. From session 6 during pre-exposure to the target stage, the speed to complete the test was strongly affected by maternal diet (F1,78 = 219.00 p < 0.001). This influence was maintained over the following stages of training (F1,74 = 32.34, p < 0.001) and trained (F1,50 = 6.31, p < 0.001; Fig. 2).

11

Fig. 2. Test food motivation in females submitted to exposure low protein diet during gestation and lactation. Data presented as mean ± SEM, n = 14. Two way ANOVA was used followed by Bonferroni test, *** p < 0.001.

GraphPad Prism 5 software, version 7 (GraphPad Inc., La Jolla, CA, USA).

3.2.2. Taste reactivity No significant differences were observed with regard to positive reaction time in LPF (40.70 ± 1.56, n = 10) compared with CF (36.89 ± 1.48, n = 9, p = 0, 0812; Fig. 3A). However, less time was spent making negative reactions by LPF rats (41.11 ± 1.40, n = 9) compared with CF rats (56.00 ± 1.20, n = 9, p < 0.0001; Fig. 3B). 3.3. Food intake and body weight

3. Results 3.1. Body weight (grams) As shown in Fig. 1, exposure a low protein diet decreased body weight (F1,108 = 1.43, p < 0.0001) over the study period from weaning (CF = 44.01 ± 0.78, n = 10; LPF = 33, 58 ± 0.64, n = 10, p < 0.01) to 90 days of age (CF = 204.0 ± 4.47, n = 10; LPF = 193.8 ± 1.80, n = 10, p < 0.05).

Standard diet consumption was evaluated for 22 consecutive days when the animals were 110–132 days old. The results showed that exposure to the low protein diet (F1,112 = 1.580, p < 0.0001) promoted hypophagy (day 1: 16.56 ± 0.43 n = 8; day 22: 16.23 ± 0.57, n = 8, p < 0.001) compared with the CF group (day 1: 20.35 ± 0.87, n = 8; day 22: 19.81 ± 0.69, n = 8, p < 0.001; Fig. 5). However, there was no significant change in body weight between the LPF (day 1: 232.47 ± 2.92, n = 10; day 22: 247.35 ± 2.52, n = 10; p > 0.05) and CF groups during the same trial period during standard diet con-

A 50

B Control Female Low protein Female

80 60

Time (s)

Time (s)

40 30 20

20

10 0

***

40

Sucrose

0

Quinine

Fig. 3. Reactivity test the taste in females submitted to exposure low protein diet during gestation and lactation. Student t-test was used, *** p < 0.001. The data were expressed as mean and standard error. Groups: control female (CF), n = 9; low protein female (LPF), n = 10.

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280

Body Weight (g)

present investigation demonstrate that exposure to low protein diet during gestation and lactation can affect feeding behaviours in adult female rats. Body weight involvement and reduced standard diet consumption was observed in females subjected to a low protein diet. However, their consumption of a fatty diet increased, and they were faster to perceive bitter tastes in behavioural tests. In addition, the LPF group had more motivation for the reward search although the latency time for sucrose perception was not different compared to the CF group. Our results also show more c-Fos-immunoreactive cells in the AMY, CPU, and PVN and the LPF group. Maternal low protein diet manipulation compromises female body weight gain of female, albeit transiently (Qasem et al., 2012; Zambrano et al., 2006). Others have reported that exposure to low protein diets during pregnancy and/or lactation leads to considerable reductions in body weight without necessarily affecting birth weight (da Silva et al., 2013; Remmers et al., 2008). Protein energy deficiency during lactation can also induce changes in milk quality and quantity, which is reflected in offspring body weight gain (Patel and Srinivasan, 2011). Most experimental research has studied the consequences of early handling in males. However, studies that compared the effects of manipulating a low protein diet in both males and females found that weight loss is common in both sexes (Zambrano et al., 2006). In our study, this difference in body weight was not maintained throughout adulthood. This might be related to the fact that females subject to malnutrition exhibited greater weight gain from weaning to 90 days old. This may have been a mechanism involved along the group of females ages low protein diet were able to recover body weight. Some authors have shown that females with early life protein restriction exhibit growth retardation followed by catch-up growth. It is possible that this catch-up growth causes delayed body weight recovery (Whatson et al., 1978; Williams et al., 1974). The hypothalamus is an important structure in the control of energy balance, and in rats, its nuclei can be malformed following lower consumption of milk during lactation (Plagemann et al., 2000). The circadian hypothalamic expression of genes involved in the control of eating behaviour such as NPY and POMC is changed in males subjected to perinatal undernutrition, with concomitant increases and decreases in the expression of orexigenic and anorexigenic hypothalamic peptides, respectively (OrozcoSolis et al., 2010). This change is reflected by the high dietary intake

Control Low protein

270 260 250 240 230 220 1

4

7

10 13 Days

16

19

55

22

Fig. 4. Evolution of body weight of 110–132 days of life in females submitted to exposure low protein diet during gestation and lactation. Two way ANOVA was used followed by Bonferroni test, * p < 0.05, *** p < 0.001. Data presented as mean ± SE; CF, n = 10; LPF, n = 10.

sumption (day 1: 240.11 ± 4.26, n = 10; day 22 250.42 ± 3.59, n = 10; p > 0.05; Fig. 4). 3.3.1. Palatable food intake LPF exposure was associated with increased palatable food intake in 1 h compared with the control (3.76 ± 0.46, n = 10 vs. 1.93 ± 0.18, n = 8, respectively; p = 0.0038; Fig. 6). 3.3.2. c-Fos immunoreactivity in response to diet In the AMY and CPU, the LPF group showed greater neuronal activation in response to the HFHS diet (AMY: 505.75 ± 48.73, n = 4; CPU: 1000.25 ± 0.25, n = 4) compared with the GF group (AMY: 318. 25 ± 20.84, n = 4; CPU: 650 ± 20.41, n = 4; p > 0.001). There was also a significant difference in the numbers of activated neurons in the PVN of low-protein females (LPF PVN: 134. 75 ± 2.93, n = 4; CF: 101.50 ± 5.04, n = 4, p = 0.0013; Fig. 7). 4. Discussion Numerous studies have attempted to elucidate the impact of the insults occurring in development and early life (Chaudhary et al., 2013; da Silva et al., 2013; Dyer and Rosenfeld, 2011). In this sense, there is thought to be a strong relationship between an inadequate diet, imbalanced neurotransmission, and important neurological disorders (Chaudhary et al., 2013). The results of the

Control Low Protein

25

Food intake (g)

20

*** 15

**

***

***

*

***

*

***

10 5 0

1

4

7

10

13

16

19

22

Days Fig. 5. Effect of the exposure low protein diet during gestation and lactation on food intake pattern vivarium diet (Purina Labina) in females during 110–132 days. Two way ANOVA was used followed by Bonferroni test, * p < 0.05, *** p < 0.001. Data presented as mean ± SEM. CF, n = 8; LPF, n = 8.

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Food intake (g)

5

Control Low Protein

**

4 3 2 1 0

Fig. 6. Effect of the exposure low protein diet during gestation and lactation on the HFHS diet intake in adult females. Was used Student t-test for comparison between groups, * p < 0.05, *** p < 0.001. Data presented as mean ± SEM. CF, n = 8; LPF = 10.

1500

IR- FOS-positive cells

Control Low Protein

***

1000

***

500

* 0

PVN

Amygdala

CPU

Fig. 7. Quantitative analysis of the number of c-FOS positive cells in the paraventricular nucleus, amygdala and caudate putamen of animals to the exposure low proteindiet during gestation and lactation. Was used Student t-test to compare the values groups represented as mean ± SEM, p < 0.05, CF, n = 4; LPF, n = 4.

of undernourished animals (Orozco-Solis et al., 2010). In contrast, estradiol treatment increases PVN expression of anorexigenic peptides (POMC, CART), which might mediate the inhibitory effect of estradiol on food intake (Silva et al., 2010). Estradiol also alleviates the orexigenic effects of ghrelin and NPY (Butera, 2010). Thus, the hypophagic effects of a low protein diet during gestation and lactation in females that consume a standard diet in adulthood may reflect a heightened role of orexigenic neuropeptides by dysregulation of the hypothalamic axis, and a maintained low weight is a consequence of hypophagy. These data suggest that exposure to a low protein diet during gestation and lactation differentially affect the homeostatic control of food intake by males and females. It is important to note that we did not evaluate the oestrous cycle, which must be investigated in future work. Palatable food can override the homeostatic control regulation, which primarily occurs in the hypothalamus (Erlanson-Albertsson, 2005). A palatable stimulus is also able to extend the meal by activating the food reward system mediated by dopamine and opioids (Erlanson-Albertsson, 2005). Maternal nutrition promotes changes in the system involved in appetite control and palatability perception (Bellinger et al., 2004). In addition, perinatal undernutrition determines the preference for a high-fat diet (Bellinger et al., 2004). We found that malnutrition early in life promoted hypophagia in the presence of a standard diet (homeostatic component) and greater consumption of an HFHS diet (hedonic component). This result demonstrates the vulnerability of pathways that regulate food intake in subjects with a history of malnutrition because satiety is dependant on an appropriate balance between the homeostatic and hedonic components. The evaluation of the search for reward through the runway task incentive demonstrated that exposure to a low protein diet during gestation and lactation stimulates the search for reward during the pre-exposure phase in females. It is known that undernutrition can change hedonic

aspects of eating behaviour (Tonkiss et al., 1990). In a recent study, da Silva et al. (2013) concluded that perinatal protein undernutrition leads to delayed task learning and that, once a task is learnt, the speed to complete it is longer in undernourished male animals (da Silva et al., 2013). The same authors reported increased neuronal activation in reward system areas such as the CPU and AMY (da Silva et al., 2013). Likewise, undernutrition increases the action of substances that act on the opioid system, such as dopamine and (Lindblom et al., 2006; Valdomero et al., 2007). These two systems are the main circuits responsible for pleasure produced by drugs and palatable food (Mahler et al., 2007; Solinas and Goldberg, 2005). Dopamine is involved in the motivational aspect of search, especially in expenditure and energy gain reward (Barbano et al., 2009), while opioids are involved with food palatability requirements, which comprise the target of motivation (Barbano et al., 2009). The analysis of exploratory activity in undernourished females with the elevated plus-maze test showed that undernutrition increased their exploration activity (Almeida et al., 1996). This can be a facilitating factor of motivation to search for reward, as noted in the present study. Some investigators have attempted to identify biological markers of vulnerability to cocaine addiction and craving for palatable food. It was found that overstimulation of the hydroxylase fumarate enzyme in the nucleus accumbens can lead to reduced motivation by drugs (del Castillo et al., 2009). On that basis, the authors concluded that hyperstimulation of fumarate hydratase in the nucleus accumbens results in reduced sensitivity to reward (del Castillo et al., 2009). An analysis of this enzyme in females subjected to undernutrition revealed increased mitochondrial hydroxylase fumarate in the nucleus accumbens region (Lizarraga-Mollinedo et al., 2013). This upregulation observed in females was not sufficient to prevent enhanced motivation in females; however, it demonstrates

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which neural mechanisms are involved in preventing the deleterious effects of undernutrition. Sucrose reactivity was not significantly different in females subjected to undernutrition, but increased sensitivity to aversive substances was observed in the LPF group. Taste is unique within the sensory system because it has innate associations with reward mechanisms and an aversion in response to the qualities of substances (Yamamoto, 2008). This sense serves as the gateway to meal selection and is influenced by caloric intake, sensory satiety specifics, and caloric needs (Berridge, 1991). In undernourished animals, the selection pressure for energy-dense foods is exacerbated, leading to hyperphagia. Consequently, the neural adjustments of food intake control lead to a vicious cycle over two properties: food palatability and energy value. The energy value of perception mechanisms is independant of their properties, namely, continuing to eat until satiation is exclusively dependant on the animal’s state of hunger and satiety. Thus, the organism first selects food to maintain energy. However, the study of “wanting” reveals an important concept to the incentive given to reward (palatable food) and its predictive properties, which help to determine the motivation value (Berridge, 2009). These “tracks” are closely related to simply imagining the smell, sight, and palatability of food (Berridge, 2009). At that moment, what matters is the pleasure that the food will provide the individual (Berridge, 2009). Our findings suggest that the main motivation in bodies subjected to undernutrition is the result of neural adjustments made in gustatory system pathways. The gustatory insular cortex responds to two neuronal populations of taste. The first type is related to flavour perception, showing increasing discharges in response to one or more flavours (Yamamoto et al., 1989). Another neuronal population exhibits excitatory responses to palatable stimuli (e.g., sucrose) and inhibitory responses to aversive stimuli (e.g., quinine) (Yamamoto et al., 1989). One study found that undernutrition significantly reduces the number and dendritic trees in the insula, which may suggest a possible mechanism underlying flavour perception and hedonic responses (Salas et al., 2012). The nucleus of the solitary tract is another region that experiences deleterious effects of undernutrition, leading to reductions in the number and size of dendritic extensions (Rubio et al., 2004). The adaptation of the motivation of behaviour requires discrimination between beneficial and harmful stimuli (McCutcheon et al., 2012). This discrimination leads to the generation of an approach or rejection response, as appropriate, and allows for behavioural organization to maximize reward and minimize punishment (McCutcheon et al., 2012). The perception of bitter protects organisms against eating potentially toxic substances (Chandrashekar et al., 2000). In this study, undernutrition influenced the perception of aversive substances. Specifically, the LPF group exhibited reduced reactivity to the bitter taste of quinine. This result indicates that undernutrition impairs the perception of bitterness in males and improves it in females. The nucleus accumbens is associated with positive reward responses but is also responsible for processing aversive stimuli (Roitman et al., 2010). Aversive stimulus signalling in the nucleus accumbens is largely dependant on dopamine (McCutcheon et al., 2012). The mechanisms by which dopamine mediates increased motivation are not completely elucidated. Some authors have hypothesized that there are different neuronal excitation thresholds for each stimulus (Badrinarayan et al., 2012; McCutcheon et al., 2012; Wheeler et al., 2011). In addition to being present in the oral mucosa, T2R receptors that perceive bitter tastes are also expressed in neurons in the brainstem (Dehkordi et al., 2012). This can be considered a body’s defence mechanism to quickly minimize harmful effects to the body (Chandrashekar et al., 2000). How these mechanisms of “disliking” are blunted remains unclear, but the present work suggests that these events may be affected during

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any phase of gustatory system development, causing alterations in adulthood. Our analysis of c-Fos immunoreactivity neurons demonstrated that perinatal undernutrition might enhance neuronal activation in areas that control food intake in female rats. The CPU and AMY are reward system regions related to motivation and learning, respectively (da Silva et al., 2013). Increased neuronal activation in these areas suggests that malnourished females have a heightened response to palatable food in a manner similar to that observed by da Silva et al. in undernourished males (da Silva et al., 2013). Likewise, greater neuronal activation was observed in the PVN of the hypothalamus; this area receives signals from other hypothalamic nuclei and is involved in signalling satiety (Williams et al., 2001). This increased neuronal activation in response to palatable food may have been a compensation mechanism to inhibit HFHS diet consumption. 5. Conclusion We conclude that exposure low protein diet during gestation and lactation increases the motivation for a food reward, though not by modifying the perception of sucrose in females. However, we did not observe the same response with regard to aversive properties. There was a modification of eating control depending on the type of diet, leading to a hypophagy front in the standard diet but hyperphagia in a fat diet. Neural responses were also modified by further activation of hedonic areas followed by compensatory responses via the hypothalamus. Therefore, it is noteworthy that this is the first work to study the influence of the exposure low protein diet in early life periods on reactivity to taste. Changes here can be a strong predictor of high consumption of energy-dense foods. As described in other studies, high dietary intake has been the main factor related to metabolic diseases, and bodies subject to low protein diet during pregnancy and lactation are more susceptible to such diseases. Conflict of interest The authors have no conflict of interest. Acknowledgements We appreciate the financial support FACEPE (Foundation for Science and Technology of the State of Pernambuco), CAPES and CNPq. We thank the Pathology Laboratories LIKA-UFPE (Immunopathology Keiso Asami) and the Center for Technology Platform 2 Aggeu Magalhães Research Center—FioCruz for technical and structural support. References Almeida, S.S., Tonkiss, J., Galler, J.R., 1996. Prenatal protein malnutrition affects exploratory behavior of female rats in the elevated plus-maze test. Physiol. Behav. 60, 675–680. Ashino, N.G., Saito, K.N., Souza, F.D., Nakutz, F.S., Roman, E.A., Velloso, L.A., Torsoni, A.S., Torsoni, M.A., 2012. Maternal high-fat feeding through pregnancy and lactation predisposes mouse offspring to molecular insulin resistance and fatty liver. J. Nutr. Biochem. 23, 341–348. Badrinarayan, A., Wescott, S.A., Vander Weele, C.M., Saunders, B.T., Couturier, B.E., Maren, S., Aragona, B.J., 2012. Aversive stimuli differentially modulate real-time dopamine transmission dynamics within the nucleus accumbens core and shell. J. Neurosci. 32, 15779–15790. Barbano, M.F., Le Saux, M., Cador, M., 2009. Involvement of dopamine and opioids in the motivation to eat: influence of palatability, homeostatic state, and behavioral paradigms. Psychopharmacology (Berl.) 203, 475–487. Belgardt, B.F., Bruning, J.C., 2010. CNS leptin and insulin action in the control of energy homeostasis. Ann. NY Acad. Sci. 1212, 97–113. Bellinger, L., Langley-Evans, S.C., 2005. Fetal programming of appetite by exposure to a maternal low-protein diet in the rat. Clin. Sci. (Lond.) 109, 413–420.

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