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882 Exp Physiol 97.7 (2012) pp 882–894

Research PaperResearch Paper

Food restriction and refeeding induces changes in lipidpathways and fat deposition in the adipose and hepatictissues in rats with diet-induced obesity

Fernanda Oliveira Duarte1,2, Marcela Sene-Fiorese1, Nadia Carla Cheik3,Adriana Simone Lopes Santa Maria1, Antonio Eduardo de Aquino Jr4, Jorge Camargo Oishi1,Elizeu Antonio Rossi5, Ana Claudia Garcia de Oliveira Duarte1,2 and Ana Raimunda Damaso6

1Departamento de Educacao Fısica e Motricidade Humana and 2Departamento de Ciencias Fisiologicas, Centro de Ciencias Biologicas e da Saude,Universidade Federal de Sao Carlos, Sao Carlos/SP, Brazil3Faculdade de Educacao Fısica e Fisioterapia, Universidade Federal de Uberlandia, Uberlandia/MG, Brazil4Programa de Pos-Graduacao em Biotecnologia, Universidade Federal de Sao Carlos, Sao Carlos/SP, Brazil5Departamento de Alimentos e Nutricao, Faculdade de Ciencias Farmaceuticas, Universidade Julio Mesquita Filho, Araraquara/SP, Brazil6 Departamento de Biociencias e Programa de Pos-Graduacao Interdisciplinar em Ciencias da Saude, Universidade Federal de Sao Paulo, Escola Paulistade Medicina, Sao Paulo/SP, Brazil

The aim of this study was to determine the effects of successive cycles of a moderately restrictivediet and refeeding with a high-fat diet on the metabolism of the adipose and hepatic tissues ofobese rats. Rats were assigned to the following groups: a chow diet; a high-fat diet; a moderatecaloric restriction; or a moderate caloric restriction plus refeeding. Some animals in each groupwere given [1-14C]triolein intragastrically, while others received an intraperitoneal injection of3 mCi 3H2O. All animals were killed by decapitation. The retroperitoneal, visceral epididymaland omental white adipose tissues, brown adipose tissue, liver and blood were immediatelyremoved. The lipid uptake from the diet, in vivo rate of lipogenesis, percentage of fat, lipidprofile and leptin concentration were analysed. The high-fat diet promoted an increase in fattyliver (P ≤ 0.05), adiposity mass (P ≤ 0.05) and the plasma concentration of leptin (P ≤ 0.05)and a decreased lipid uptake in white adipose tissue depots (P ≤ 0.05) in relation to the chowdiet. The moderate caloric restriction did not reverse the changes promoted by the high-fat dietbut induced a small decrease in adiposity, which was reversed after refeeding, and the animalsmaintained a dyslipidaemic profile and high fat deposition in the liver. We can conclude that thehigh-fat diet and subsequent moderate caloric restriction plus refeeding increased the risks ofdeveloping visceral obesity, dyslipidaemia and non-alcoholic fatty liver disease, which suggeststhat this type of experimental protocol can be used to study mechanisms related to the metabolicsyndrome.

(Received 2 March 2012; accepted after revision 27 March 2012; first published online 30 March 2012)Corresponding author F. O. Duarte: Departamento de Educacao Fısica e Motricidade-DEFMH, Universidade Federalde Sao Carlos-UFSCar Rodovia Washington Luıs, Km 235-SP-310, Sao Carlos-Sao Paulo-Brasil, CEP: 13565-905. Email:duarte reame@yahoo.com.br

Significant changes in the food industry, in addition tothe higher adherence to a fat-rich diet, has contributed tothe increased number of obesity cases worldwide (Velloso,2006). Therefore, obesity begins as a consequence of apositive energy balance, which is modulated by the actionof many cytokines and hormones, such as leptin, that are

secreted by the adipose tissue (Carnier et al. 2010; Oyamaet al. 2010).

Recent studies have shown that because high-fatdiets promote low satiation, they lead to a positiveenergy balance due to the accumulation of fat mass,particularly abdominal obesity, which is characterized

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Exp Physiol 97.7 (2012) pp 882–894 Food restriction with diet-induced obesity changes in lipid pathways 883

by the deposition of visceral fat. This type of obesityis strongly linked to chronic diseases, such as diabetes,hypertension, dyslipidaemia and non-alcoholic fat liverdisease (NAFLD), and it is also linked to the developmentof the metabolic syndrome (Westerterp, 2006; Duarte et al.2008; Damaso et al. 2011; Ezquer et al. 2011; Vernon et al.2011).

Experimental studies suggest that successive cycles ofweight loss, which are followed by periods of refeedingwith a gain of body weight, facilitate the development ofobesity (Sea et al. 2000; Riccardi et al. 2004). Alternatively,many different studies have indicated that a moderatecaloric restriction (18–30% of normal caloric ingestion) iseffective in reducing complications of obesity and thatweight loss has a long-term beneficial effect on lipidmetabolism, especially on the low-density lipoprotein(LDL) and total cholesterol levels (Poobalan et al. 2004).Additionally, the accentuated reduction of visceral fatimproved the action of insulin in rats (Li et al. 2003;Poobalan et al. 2004).

The hormone leptin, together with insulin, has beenconsidered an important lipostatic modulator, whichregulates food ingestion and energy waste in relation tothe storage of body fat, and its concentration is directlyregulated by adiposity (Hermsdorff et al. 2006; Fonseca-Alaniz et al. 2007). Similar to leptin, ghrelin, which is anorexigenic hormone produced in the gastrointestinal tract,also acts to regulate energy supplies through the centralstimulus and acts antagonistically to the action of leptin(Damaso et al. 2006).

The metabolic activity of the adipose tissue is variableand is dependent on its regional distribution (Hausmanet al. 2001). Additionally, the accumulation of visceraladipose tissues leads to NAFLD in both animals andhumans, including adolescents (Hausman et al. 2001; Daset al. 2004; Cinti, 2005; Damaso et al. 2011).

However, studies to measure lipogenesis and lipidaccumulation due to the diet in both the visceraladipose tissue and liver are scarce, considering thatcaloric restriction and refeeding are nutritional strategiescommonly used by people to promote health. Thus, thegoal of this study was to verify the effects of a moderatelyrestrictive diet and refeeding with a high-fat diet on themetabolism of the adipose and hepatic tissues and theenergy balance of rats with exogenous obesity.

Methods

Animals

The experimental procedures in this study conformed tothe Guide for the Care and Use of Laboratory Animalspublished by the US National Institutes of Health (NIHpublication no. 85-23, revised 1996), and protocols wereapproved by the ethics committee of Universidade Federal

de Sao Carlos (UFSCar; 017/2007). Ninety-six adultmale Wistar rats (90 days old) were supplied by a localbreeding colony (UFSCar). They were individually housedin opaque polypropylene cages (30 cm × 20 cm × 13 cm)with stainless-steel wire lids and fed with food and tapwater ad libitum. The rats were kept at 23 ± 1◦C with a12 h–12 h light–dark photoperiod for 8 weeks.

Protocol and experimental design

The animals (n = 96) were assigned to one of the sixgroups (n ≤ 16 per group) in accordance with the diet andintervention, as follows: a chow diet (C); a hyperlipidicdiet (H); a moderate caloric restriction (M); or refed adlibitum (r). The food for the moderate caloric restrictiongroups was restricted to 25% of the amount consumed onthe previous day by the respective control animals (C andH groups). Except for the C group, all of the other groupswere fed ad libitum with the hyperlipidic diet (3 weeks)before moderate caloric restriction for the development ofexogenous obesity and dyslipidaemia (Duarte et al. 2008).The experimental design is illustrated in Fig. 1.

Body weight and food intake

The body weights and food intake were measured daily.The amount of food offered to the animals in the groupssubjected to a moderate food restriction was calculateddaily from the food consumption of the correspondingcontrol group adjusted per 100 g of body weight.

Diets

The experimental groups received one of the followingdiets: the MP-77 standard rat chow diet (in pellet form)provided by Primor (Sao Paulo, Brazil) that contained(100 g per diet) 23% protein, 49% carbohydrates, 4%total fat and 5% fibre; or a palatable hyperlipidic dietthat consisted of commercial rat chow plus peanuts, milkchocolate and sweet biscuit in the proportion of 3:2:2:1.All components were powdered and mixed to form pellets(Estadella et al. 2004). This diet achieved 20% protein, 20%fat, 48% carbohydrate and 4% fibre. The caloric densityof the diets was determined with an adiabatic calorimeter(IKA-C400, EMBRAPA, Sao Carlos, Brazil). The caloricdensities were 5.12 kcal g−1 for the palatable hyperlipidicdiet and 4.07 kcal g−1 for the chow diet.

Experimental procedure

At the end of 8 week experimental period, in the fedstate, some of the animals in each group (n ≤ 8 pergroup) were administered [1-14C]triolein (∼0.5 g; 0.3 mCiper rat) intragastrically 4 h before decapitation for the

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884 F. O. Duarte and others Exp Physiol 97.7 (2012) pp 882–894

lipidic uptake analysis. The remaining animals (n ≤ 8per group) were fasted for 12 h before receiving anintraperitoneal injection of 3 mCi of 3H2O (0.3 ml)50 min before decapitation. All animals were killed bydecapitation without anaesthetic. Then, whole blood wasdrawn into a heparinized tube for all of the remaininganalyses.

The retroperitoneal (RET), visceral epididymal (EPI)and visceral omental (VIS) white adipose tissues, brownadipose tissue (BAT), liver and intestine were immediatelyremoved by procedures that were adapted for rats (Duarteet al. 2008; Sene-FIiorese et al. 2008), using the anatomicallocalization of the organs that was described by Cinti(2005). The collected tissues were then weighed.

Serum analyses

The total cholesterol, high-density lipoprotein (HDL-cholesterol) and triacylglycerol levels in the plasmawere determined enzymatically by commercial kits(CELM, Barueri, Brazil) using an ultraviolet-visiblespectrophotometer (model UV-160 1PC; Shimadzu,Kyoto, Japan). The plasma leptin level was determinedby a radioimmunoassay using the leptin RIA kit for rats(Linco Research, St Charles, MO, USA).

Intestinal absorption and accumulation in tissues of14C-labelled lipid

The whole intestinal tract and 1 g samples of liver, RET,EPI, VIS and BAT were removed and homogenized.The lipids were saponified, and the free fatty acids

were extracted using the method described by Stansbie(1976). The extract was dissolved in 5 ml of scintillationliquid to determine the amount of 14C-labelled lipidthat accumulated in the tissue and the amount of 14C-labelled triolein that remained in the intestinal tract. Theabsorption of 14C-triolein was determined by subtractingthe remaining radioactivity in the intestinal tract fromthe total amount that was administered (14C-labelled lipidaccumulated per gram of tissue per hour).

In vivo lipogenesis rate in tissues

The in vivo lipogenesis rate (fatty acid synthesis) wasdetermined by the incorporation of 3H2O (tritiatedwater) into saponified lipids according to the method ofRobinson & Williamson (1978). The rate of lipogenesiswas calculated as micromoles of 3H2O incorporated intolipids per gram per hour.

Percentage of fat in tissues

The percentage of lipid content in the tissues wasdetermined by the gravimetric method (Oller doNascimento & Williamson, 1986).

Statistical analysis

Variables with normal distribution are expressed asmeans ± SD. The data analysis was performed usingInstat 3.0 for Windows 95 (GraphPad, San Diego, CA,USA, 1998). Statistical comparisons for unequal samplesizes were performed using a one-way ANOVA. Statistical

Figure 1. Schematic diagram of the experimental protocol

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Exp Physiol 97.7 (2012) pp 882–894 Food restriction with diet-induced obesity changes in lipid pathways 885

significance was set at P < 0.05. Significant differenceswere subjected to a post hoc analysis (Tukey–Kramermultiple comparisons).

Results

Effects of the high-fat diet

The animals in the H group ingested less food (in grams)compared with animals in the C group (Fig. 2Aa), butthese animals did not show a difference in body weight

from the rats fed a standard diet at the end of theexperiment (Fig. 2Ab).

A comparison between the C and H groups(Table 1) showed that the high-fat diet increased therelative weights of the epididymal (C, 0.98 ± 0.04versus H, 1.68 ± 0.08 g (100 g body wt)−1) andomental visceral adipose tissues (C, 1.21 ± 0.03versus H, 1.99 ± 0.10 g (100 g body wt)−1), the centralretroperitoneal adipose tissue (C, 1.23 ± 0.05 versus H,2.29 ± 0.09 g (100 g body wt)−1) and the brown adipose

Figure 2. Food intake and Body weightA, effects of high-fat diet on food intake (a) and body weight (b). B, effects of moderate caloric restrictionon food intake (a) and body weight (b). C, effects of moderate caloric restriction plus refeeding onfood intake (a) and body weight (b). Results are expressed as means ± SD (n ≤ 8 per group). Differentsuperscripts (a versus C; b versus MC; c versus MC-r; and d versus H) are significantly different from eachother at P < 0.05 (Tukey–Kramer multiple comparisons). Abbreviations: bw, body weight; C, chow diet;H, high-fat diet; M, moderate caloric restriction; r, refed.

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886 F. O. Duarte and others Exp Physiol 97.7 (2012) pp 882–894

Tab

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CM

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HM

HM

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I0.

98±

0.04

0.80

±0.

161.

26±

0.29

b1.

68±

0.08

a1.

57±

0.35

b1.

84±

0.49

c82

.43

±2.

9477

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3360

.37

±5.

6ab75

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6.16

78.7

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77.8

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3.48

c

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23±

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0.60

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1.28

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272.

29±

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b2.

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c80

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0776

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63.7

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5.92

ab75

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9580

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2481

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VIS

1.21

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73±

0.11

a1.

32±

0.18

b1.

99±

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a1.

44±

0.32

bd1.

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0.28

ce57

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7630

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52.1

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46.5

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6.66

54.5

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8854

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Live

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72±

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3.20

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24a

3.63

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27b

3.73

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97±

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3.43

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tissue (C, 0.07 ± 0.004 versus H, 0.12 ± 0.005 g (100 gbody wt)−1), but the liver weight did not differsignificantly (C, 3.72 ± 0.05 versus H, 3.73 ± 0.08 g (100 gbody weight)−1; Table 1). However, Table 1 shows thatthere was an increase in the percentage of fat in the liversfrom only the H group compared with the C group (C,3.57 ± 0.53 versus H, 5.00 ± 1.0%).

The high-fat diet promoted a significant decreasein the lipid uptake in the EPI (C, 3.21 ± 1.37 versusH, 0.6 ± 0.11 14C-lipid accumulated (g tissue)−1 h−1),RET (C, 2.37 ± 0.31 versus H, 0.68 ± 0.12 14C-lipidaccumulated (g tissue)−1 h−1) and VIS fat depots (C,1.27 ± 0.27 versus H, 0.65 ± 0.12 14C-lipid accumulated (gtissue)−1 h−1; Fig. 3A), but did not alter the lipid synthesisin these tissues (Fig. 3B). The brown adipose tissue andthe liver (Table 2) did not show a significant difference inthe lipid uptake or lipogenesis rate.

With respect to the lipid profile (Table 3), the high-fatdiet promoted an increase in the total amount of plasmacholesterol (C, 43.4 ± 7.38 versus H, 72.2 ± 7.4 mg dl−1)relative to the chow diet. However, difference in the plasmaHDL-cholesterol and triacylglycerol concentrations wasnot statistically significantly, despite a 32.15% increase inthe triacylglycerol concentration (Table 3).

By analysing the plasma concentration of leptin(Fig. 4), the high-fat diet was found to promote alarge increase in the concentration of this hormonecompared with the standard diet (C, 5.16 ± 0.54 versusH, 12.02 ± 1.39 ng ml−1).

Effects of moderate caloric restriction

The Fig. 2Ba shows that both groups subjected to caloricrestriction (MC and MH) had a lower intake of food (ingrams) relative to the C and H groups, respectively. TheMH group consumed less food than the MC group duringthe last 3 weeks of treatment (Fig. 2Bb). A significantdecrease in the body mass of the animals in the MCand MH groups (Fig. 2Bb) was found when they werecompared with their respective controls, but this decreasewas lower in the MH group than in the MC group(Fig. 2Bb).

Treatment with a moderate caloric restriction usingthe chow diet (Table 1) promoted a significant decreasein the relative weight of the RET (C, 1.23 ± 0.05 versusMC, 0.60 ± 0.22 g (100 g body wt)−1), VIS (C, 1.21 ± 0.03versus MC, 0.73 ± 0.11 g (100 g body weight)−1) andliver (C, 3.72 ± 0.05 versus MC, 3.20 ± 0.24 g (100 g bodyweight)−1) compared with the control group. The MHgroup had decreased VIS (H, 1.99 ± 0.1 versus MH,1.44 ± 0.32 g (100 g body wt)−1) and liver weights (H,3.73 ± 0.082 versus MH, 2.97 ± 0.17 g (100 g body wt)−1)relative to the H group (Table 1). In contrast, the relativeweights of the EPI, RET and VIS were greater in the MHgroup (1.57 ± 0.35, 1.89 ± 0.44 and 1.44 ± 0.32 g (100 g

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body wt)−1, respectively) compared with the MC group(0.8 ± 0.16, 0.6 ± 0.22 and 0.73 ± 0.11 g (100 g bodywt)−1, respectively; Table 1). In the brown adipose tissue,there was no significant difference in weight between thesetwo groups (Table 1). However, the percentage of fat in theVIS (Table 1) of the MC group was significantly reducedcompared with the C group (C, 57.67 ± 7.76 versus MC,39.4 ± 6.69%).

Figure 3A shows that the lipid uptake by the EPIin the MC group was decreased (C, 3.21 ± 1.37 versusMC, 1.12 ± 0.47 14C-lipid accumulated (g tissue)−1 h−1)

when compared with the C group, while in the VIS, thelipid uptake was increased (C, 1.27 ± 0.27 versus MC:1.79 ± 0.32 14C-lipid accumulated (g tissue)−1 h−1). TheEPI and RET adipose tissues did not show any significantdifferences in the lipid uptake for the MH group inrelation to the H group (Fig. 3A); however, the lipid uptakeincreased significantly in the VIS (H, 0.65 ± 0.12 versusMH, 1.38 ± 0.29 14C-lipid accumulated (g tissue)−1 h−1).The lipid uptake was significantly higher in the BAT (Table2) in only the MH group compared with the H group (H,1.53 ± 0.26 versus MH, 2.31 ± 1.0 14C-lipid accumulated

Figure 3. Lipid uptake and Lipogenesis rateA, in vivo lipogenesis rate. B, lipid uptake. Values are expressed as means ± SD (n ≤ 8 per group).Different superscripts (a versus C; b versus MC; c versus MC-r; d versus H; and e versus MH) are significantlydifferent from each other at P < 0.05 (Tukey–Kramer multiple comparisons). Abbreviations: C, chow diet;14C, 14C-labelled triolein; EPI, epididymal; H, hyperlipidic diet; M, moderate caloric restriction; r, refed;RET, retroperitoneal; and VIS, visceral adipose tissues.

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Table 2. Lipid uptake into tissues and in vivo lipogenesis rate of tissues in rats subjected to a moderate food restriction and refeedingwith respect to the standard chow diet or the high-fat diet

Tissues C MC MC-r H MH MH-r

Lipid uptake (14C-lipid accumulated (g tissue)−1 h−1)BAT 3.14 ± 0.99 2.36 ± 1.23 1.28 ± 0.50 1.53 ± 0.26 2.31 ± 1.00d 3.03 ± 1.89Liver 1.32 ± 0.29 1.05 ± 0.16 0.97 ± 0.16 1.26 ± 0.33 1.13 ± 0.32 0.95 ± 0.10

In vivo lipogenesis rate (μmol 3H2O incorporated (g tissue)−1 h−1)BAT 2.20 ± 0.58 55.53 ± 12.70a 13.89 ± 3.17b 1.08 ± 0.92 5.56 ± 2.76b 12.62 ± 5.08d

Liver 4.10 ± 1.18 4.39 ± 1.59 3.23 ± 1.27 3.12 ± 0.57 2.26 ± 0.85b 6.03 ± 0.81cde

Values are expressed as means ± SD (n ≤ 8 per group). Different superscripts (a versus C; b versus MC; c versus MC-r; d versus H; ande versus MH) are significantly different from each other at P < 0.05 (Tukey–Kramer multiple comparisons). Abbreviations: BAT, brownadipose tissue; C, chow diet; 14C, 14C-labelled triolein; H, hyperlipidic diet; M, moderate caloric restriction; and r, refed.

Table 3. Plasma concentrations of total cholesterol, triacylglycerol and high-density lipoprotein cholesterol (in milligrams perdecilitre) in rats subjected to moderate caloric restriction and refeeding

Parameter C MC MC-r H MH MH-r

Cholesterol 43.4 ± 7.38 61.37 ± 8.24a 53.17 ± 8.70 72.20 ± 7.40a 67.86 ± 9.56 63.71 ± 6.07Triacylglycerol 141.2 ± 6.42 85.75 ± 20.05 106.17 ± 18.67 186.6 ± 24.18 184.4 ± 15.06b 166.5 ± 31.27HDL 26.2 ± 3.49 25.25 ± 1.83 25.5 ± 3.89 31.6 ± 2.61 25.67 ± 1.97d 9.0 ± 1.63cde

Values are expressed as means ± SD (n ≤ 8 per group). Different superscripts (a versus C; b versus MC; c versus MC-r; d versus H; ande versus MH) are significantly different from each other at P ≥ 0.05 (Tukey–Kramer multiple comparisons). Abbreviations: C, chowdiet; H, high-fat diet; HDL, high-density lipoprotein; M, moderate caloric restriction; and r, refed.

(g tissue)−1 h−1). The liver did not present any changesin uptake patterns upon any of the interventions(Table 2).

The food restriction with chow diet (MC) enhancedlipid synthesis (Fig. 3B) in the EPI, RET and VIS comparedwith the C group. However, in the MH group, this increasein the lipogenesis rate did not occur in the VIS whenwe compared it with the H group. Lipogenesis decreasedin the MH group in the RET, VIS (Fig. 3B) and liver(Table 2) compared with the MC group.

Table 3 demonstrates that the plasma cholesterolconcentration was greater in the MC compared with theC group (C, 43.4 ± 7.38 versus MC, 61.37 ± 8.24 mg dl−1).The triacylglycerol concentration increased significantlyin the MH group compared with the MC group(MC, 85.75 ± 20.05 versus MH, 184.4 ± 15.06 mg dl−1).

Conversely, the HDL-cholesterol concentration was lowerin the MH group than in the H group (H, 31.6 ± 2.61versus MH, 25.67 ± 1.97 mg dl−1).

Caloric restriction with the chow diet decreased theplasma leptin concentration (Fig. 4) compared with theC group (C, 5.16 ± 0.54 versus MC, 2.33 ± 0.91 ng ml−1).The caloric restriction associated with a high-fatdiet (MH) did not alter the leptin concentration inrelation to the H group (H, 12.02 ± 1.39 versus MH,12.38 ± 3.16 ng ml−1). However, leptinaemia was greaterin the MH group relative to the MC group (MC,2.33 ± 0.91 versus MH, 12.38 ± 3.16 ng ml−1).

Effects of moderate caloric restriction plus refeeding

During the refeeding period, the MC-r group ingested agreater amount of food (in grams) relative to the C group

Figure 4. Plasma concentration of leptinValues are expressed as means ± SD (n ≤ 8 per group). Different superscripts (a versus C; b versus MC;c versus MC-r; d versus H; and e versus MH) are significantly different from each other at P < 0.05(Tukey–Kramer multiple comparisons). Abbreviations: C, chow diet; H, high-fat diet; M, moderate caloricrestriction; and r, refed.

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(Fig. 2Ca). Although the MH-r group did not eat morethan the H group, the amount of food intake was lowerthan that of the MC-r group (Fig. 2Ca). Interestingly, thebody weights of the MC-r group were equal to those ofthe C group at the eighth week (Fig. 2Cb). For the MH-r group, the body weights were lower than those for theH group (Fig. 2Cb).

Notably, when comparing the C group with the MC-rgroup (Table 1), the refeeding treatment re-establishedthe relative weights of the EPI (C, 0.98 ± 0.04 versusMC-r, 1.26 ± 0.29 g (100 g body weight)−1), RET (C,1.23 ± versus MC-r, 1.28 ± 0.27 g (100 g body weight)−1),VIS (C, 1.21 ± 0.03 versus MC-r, 1.32 ± 0.18 g (100 gbody weight)−1) and liver tissues (C, 3.72 ± versus MC-r,3.63 ± 0.27 g (100 g body weight)−1). In the MH-r grouprelative to the H group (Table 1), the EPI (H, 1.68 ± 0.08versus MH-r, 1.84 ± 0.49 g (100 g body weight)−1), RET(H, 2.29 ± 0.09 versus MH-r, 2.14 ± 0.6 g (100 g bodyweight)−1) and VIS tissues (H, 1.99 ± 0.10 versus MH-r: 1.75 ± 0.28 g (100 g body weight)−1) also re-establishedtheir relative weights. However, the relative weight of theliver was lower than that of the H group (H, 3.73 ± 0.08versus MH-r, 3.17 ± 0.2 g (100 g body weight)−1; Table 1).

For the MC-r, the percentage of fat (Table 1) was lower inthe EPI (C, 82.43 ± 2.94 versus MC-r, 60.37 ± 5.6%), RET(C, 80.86 ± 6.07 versus MC-r, 63.75 ± 5.92%), VIS (C,57.67 ± 7.6 versus MC-r, 40.57 ± 8.46%) and liver tissues(C, 3.57 ± 0.53 versus MC-r, 3.14 ± 0.38%) comparedwith the C group. Upon refeeding with high-fat diet(MH-r), the percentage of fat in the EPI, RET, VIS andliver tissues (Table 1) did not differ compared with theH group. Nevertheless, the percentages of fat in the EPI(MC-r, 60.37 ± 5.6 versus MH-r, 77.87 ± 3.48%), RET(MC-r, 63.75 ± 5.92 versus MH-r, 81.25 ± 3.06%), VIS(MC-r, 40.57 ± 8.46 versus MH-r, 62.71 ± 1.6%) and livertissues (MC-r, 3.14 ± 0.38 versus MH-r, 6.17 ± 0.98%)were larger in the MH-r group relative to the MC-r group(Table 1).

In the MC-r group, the lipid uptake (Fig. 3A) decreasedsignificantly relative to the C group in the EPI, RET andVIS tissues. For the MH-r group, the lipid uptake did notshow significant changes in the EPI, RET, VIS, BAT andliver tissues relative to the H group or between the MC-rand MH-r groups (Fig. 3A).

With respect to the in vivo lipogenesis rate (Fig. 3B andTable 2), the refeeding with chow diet (MC-r) did not alterthis parameter in any of the tissues analysed relative to theC group. However, the MH-r group had an increased rateof lipogenesis in the EPI, RET, VIS (Fig. 3B), BAT and livertissues (Table 2) relative to the H group. Additionally, therewas an increased rate of lipogenesis in the EPI (Fig. 3B)and liver tissues (Table 2) between the MC-r and MH-rgroups.

In Table 3, the plasma concentrations of cholesterol,triacylglycerol and HDL did not differ between the MC-

r and C groups. There was a significant decrease inthe plasma concentration of HDL in the MH-r groupcompared with the H group (H, 31.6 ± 2.61 versus MH-r, 9.0 ± 1.63 mg dl−1) and with the MC-r group (MC-r,25.5 ± 3.89 versus MH-R, 9.0 ± 1.63 mg dl−1; Table 3).

The plasma leptin concentration (Fig. 4) was lowerin the MC-r group relative to the C group. However,leptinaemia was not significantly different between theMH-r group and the H group. Conversely, the leptinconcentration was higher in the MH-r group comparedwith the MC-r group (Fig. 4).

An outline of the main results from this studyconcerning the high-fat diet is shown in Fig. 5.

Discussion

Effects of the high-fat diet

Many studies have been performed to understand theeffects of high-fat diet consumption on visceral adiposityand development of obesity and dyslipidaemia and todetermine possible treatments using food restriction andexercise as non-pharmacological interventions for obesity(Duarte et al. 2006, 2008; Sene-Fiorese et al. 2008; de Pianoet al. 2010a; de Mello et al. 2011).

In the present study, we noted that the increased energyconsumption (data not available) induced hypophagiain the animals after 8 weeks of eating the high-fatdiet based on the decreased consumption of food thatoccurred. In animals, Kretschmer (2005) and Duarteet al. (2008) observed that a progressive increase in theconsumption of a diet with a high caloric density ledthe animals to consume a progressively smaller amountof food. Additionally, this diet stimulates an increase inthe metabolic efficiency, which results in an adaptationof the lipid pathway to promote body mass regulation(Estadella et al. 2004). In our study, we observed thatafter eating the high-fat diet for 8 weeks, the rats hadincreased adiposity but not body weight relative in relationto the rats fed a chow diet and without changes in therelative weight of the gastrocnemius muscle (data notshown).

It is known that the size of the adipose tissue is regulatedby the energy balance and that chronic ingestion of a high-fat diet contributes to the development of hypertrophyand hyperplasia in this tissue (Guerra et al. 2007).Accordingly, the adipose tissue plays an important rolein the control of metabolism and in the central regulationof food ingestion because it produces and secretes manyadipokines, including leptin, which are highly relatedto the adipose mass (Damaso et al. 2006; Trayhurn &Bing, 2006). Leptin plays a role in the regulation ofenergy balance by acting primarily on the arcuate nucleusof the hypothalamus, which stimulates the expressionof neurotransmitters and hormones that regulate food

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ingestion and the increased energy waste (Hermsdorffet al. 2006). In this sense, we noted that the leptinaemiaincreased twofold with the consumption of the high-fatdiet in this study and the animals presented increased fatmass but did not develop morbid obesity at the end ofthe experimental period confirming the relationship ofleptin with increased fat mass and its modulatory actionmechanisms of food intake.

In fact, Lin et al. (2001) observed that the peripheralresponse to leptin in rats was abolished with ingestionof a high-fat diet and that the leptin resistance wasinduced within a short period of time. Normally, leptindecreases the mRNA of neuropeptide Y (NPY), whichis an orexigenic neuropeptide, in the hypothalamus,but that does not occur in rats fed a high-fat dieteven with an increase in circulating leptin. Leptinaffects some neuropeptide systems in the hypothalamus,releases the α-melanocyte-stimulating hormone andinhibits the hypothalamic neurons that produce NPYand agouti-related peptide, which have an orexigenicaction. Additionally, leptin increases the expression ofpro-opiomelanocortin, which controls food ingestionand energy balance (Stanley et al. 2005; Velloso, 2006).Thus, the absence of effects on NPY mRNA in rats fed

with a high-fat diet, which was observed by Lin et al.(2001), provides evidence for leptin resistance in thehypothalamus. Additionally, in rodents with diet-inducedobesity, the peripheral administration of leptin failedto reduce food ingestion because the leptin transportinto the brain was saturable and the clearance of leptinwas enhanced (El-Haschimi et al. 2000; Lin et al.2001). Moreover, energy storage may also be regulatedthrough a central stimulus by ghrelin (an orexigenichormone produced in the gastrointestinal tract), whichpromotes modulations in the neurons that produce NPY,agouti-related peptide and pro-opiomelanocortin. Thecirculating concentration of this hormone is inverselyrelated to adiposity. Thus, anorexic people show a highconcentration of this hormone, which is then normalizedafter an increase in weight. Conversely, the circulatingconcentration of ghrelin is reduced in obesity (Cummingset al. 2002; Hansen et al. 2002; Murphy & Bloom,2004). Accordingly, previous studies on obese teenagersdemonstrated similar results, and it was observed thatalthough these hormones have antagonistic actions, theycompete for the same sites of action on the neurons(Damaso et al. 2006). Finally, the ingestion of a high-fat diet may induce resistance to the action of leptin even

Figure 5. Outline of the main resultsA, changes in lipid pathways due to the high-fat diet. An increase in the adipose tissue due to the high-fat diet promotes the development of hyperleptinaemia. The signal to the hypothalamus is impaired,which leads to a decrease in energy expenditure and increased fat storage in the liver and adipocytes.B, a moderate food restriction paired with the high-fat diet cannot reverse the deleterious effects thatwere caused by the intake of fat. A small decrease occurs in adipose tissue without a concomitantdecrease in the leptin levels. C, the period of refeeding after the food restriction stimulates overeatingand, consequently, decreases energy expenditure to restore the fat mass. Changes in the lipid pathwayscan re-establish leptin and the lipid levels.

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before modification of body composition (Levin & Dunn-Meynell, 2002; Sahu, 2003).

In addition to its interaction with hypothalamicneurons for the control of food ingestion, leptin alsoparticipates in the regulation of energy balance throughits action on the BAT. Leptin stimulates an increase inthe expression of mitochondrial uncoupling protein 1,which uncouples the phosphorylation function in BATmitochondria and plays an important role in regulatingbody temperature (Westerterp, 2006). Thus, the increasedrelative weight of the BAT that was observed in this studymay be due to the ingestion of the high-fat diet and to thehyperleptinaemia that promotes increased expression ofuncoupling protein 1, which dissipates the proton gradientof oxidative phosphorylation and generates heat (Cinti,2005).

The high-fat diet may also have contributed to theincreased adiposity due to changes in the metabolicactivity of the adipose tissue. In addition, the high-fat diet reduced the activity of lipogenic enzymes andthe rate of lipogenesis in the retroperitoneal fat depots,but it increased the lipoprotein lipase activity in theomental visceral fat (Estadella et al. 2004). It is importantto mention that in the present study, the high-fat dietpromoted a substantial increase in lipid deposition inthe various white adipose tissues that were analysedand caused a statistically insignificant increase in lipidsynthesis in these tissues. Although lipid synthesis inthe liver was not modified, there was a significantincrease in the fat deposition in this tissue (40%). Whenexcess fat accumulates in the hepatic tissues, it resultsin alterations between the amount of triacylglyceroluptake and release by this tissue, which suggests anincrease in lipogenesis and in the intrahepatic deposition,thus inducing a vicious cycle. Moreover, a concomitantdecrease in triacylglycerol clearance through alterationsin β-oxidation and the production of very low-densitylipoprotein may occur. Indeed, in addition to the lipidaccumulation in our study, there were increased levels ofcholesterol and circulating triacylglycerol, which suggestedthe development of hepatic steatosis and dyslipidaemia(Qureshi & Abrams, 2007; de Piano et al. 2010b).

Therefore, it was demonstrated that both the insulin andleptin hepatic resistance is implicated in the accumulationof triacylglycerol in this tissue (Angulo, 2007; Fishmanet al. 2007). Hepatic insulin resistance is associated withthe development of hepatic steatosis in both experimentalanimals and humans, not only because of the increasedfatty acid influx due to release by the visceral adiposetissue, but also because of the increased secretion ofnumerous metabolites from the adipose tissue, includingpeptides and cytokines, such as adiponectin, leptin andresistin, that directly affect the action of insulin on thehepatic tissue (Cohen & Friedman, 2004; Medina et al.2004). Therefore, the results from this study demonstrated

that non-alcoholic hepatic steatosis is a phenomenon thatoccurs quickly when the animals are subjected to a high-fat diet. From a clinical point of view, our results suggestthe importance of reducing the consumption of high-fat diets, even when there has been no weight gain, toavoid the development of non-alcoholic hepatic steatosisand dyslipidaemia, which are both components of themetabolic syndrome (Gauthier et al. 2006; Tock et al.2006).

Effects of moderate caloric restriction

After the animals were given the high-fat diet for 3 weeks,they were subjected to caloric restriction. Moderate andsevere regimes of food restriction lower the amount oftotal energy ingested, which results in a negative energybalance and is characterized by an imbalance betweenthe consumed energy and the oxidized energy (Duarteet al. 2008). Therefore, a decrease in the adipose massdeposition occurs, as previously noted in this study.Evidence demonstrates that leptin is involved in therestoration of the negative energy balance through hungermodulation, and during the food restriction period, theexogenous administration of leptin lowers the rodentappetite (Westerterp-Plantenga et al. 2001). Our resultsshowed that with food restriction, leptinaemia decreased54.84% and the adiposity of the white adipose tissuesdecreased compared with those of animals fed a standarddiet. Conversely, leptinaemia was not altered when foodrestriction was applied with the high-fat diet, whichreinforces the hypothesis of the existence of an interactionbetween the adipose mass and leptinaemia. Flier (2004)stated that decreases in leptinaemia in situations of loweradipose mass act as a signal from the adipose tissue to thebrain that the body is experiencing food restriction.

Interestingly, moderate food restriction led to decreasedvisceral adiposity and increased lipid uptake from the dietand lipid synthesis in this tissue, which suggests that theseare compensatory mechanisms that are attempting to re-establish the visceral fat depot. The other fat pads (RETand EPI) present a different response to the moderatefood restriction regarding lipid uptake, confirming thatthe metabolic activity of the adipose tissue may varydepending on its regional distribution. Additionally, theglycogen stores are depleted and the insulin concentrationdecreases during food restriction, which leads to anincrease in fatty acid oxidation (Mars et al. 2006). As shownby Li et al. (2003), moderate food restriction in obeserats may prevent the accumulation of visceral fat, whichresults in an improvement in insulin action. The decreasedvisceral adiposity is an important factor because the releaseof free fatty acids from omental visceral fat and theirtransport to the liver through the hepatic vein may lead toliver dysfunction and the development of dyslipidaemia

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and non-alcoholic hepatic steatosis (Qureshi & Abrams,2007).

According to our results, moderate food restrictionassociated with a standard diet improved the dyslipidaemicprofile of the animals. Conversely, the application ofmoderate food restriction with the high-fat diet did notnormalize the cholesterol concentration or the circulatingtriacylglycerol level. Therefore, these results and previousstudies reinforce the importance of eating balanced dietsfrom a young age and controlling body mass and visceraladiposity because of its known deleterious effects related tothe increased risk of obesity (Hausman et al. 2001; Einsteinet al. 2005; Caranti et al. 2007; de Piano et al. 2010b).

Effects of moderate caloric restriction plus refeeding

In our study, after the caloric restriction period, theenergy deficit was quickly restored upon refeeding. Marset al. (2006) suggests that the leptin concentration isinversely proportional to appetite; therefore, chroniccaloric restriction leads to overfeeding after the refeedingperiod (Ogawa et al. 2005), which promotes an increasein the adipose mass and leptinaemia (Stanley et al. 2005).The accentuated increase of food ingestion was probablydue to the increased activation of the NPY system with anincrease in NPY mRNA expression by the arcuate nucleus,which is associated with an upregulation of the receptors(Beck, 2006). Additionally, NPY may have an importantrole in the management of body mass and the regulationof food intake by hormonal factors, including the actionsof leptin and ghrelin (Beck, 2006).

Therefore, leptin participates in the negative feedbackin order to restore the body mass in the energy balance.Additionally, there is leptin suppression in response tofood restriction, which suggests an increase of ghrelin(a strong appetite stimulator in animals and humans)and, consequently, a stimulus for increased food ingestion(English et al. 2002; Pardo et al. 2010). According to someauthors (Mars et al. 2006; Duarte et al. 2008), the yo-yo effect (characterized by alternating periods of weightloss and weight gain) may lead to a worsening of theleptin action to promote food homeostasis and may resultin a decrease in the total energy expenditure, therebyfacilitating the development of obesity (Archer & Mercer,2007), as was observed in our study.

When the animals were refed, the lipogenic activity wasgreater and the synthesized endogenous and exogenoustriglycerides obtained from the diet were efficientlystocked in the adipose tissue, which restored the adiposity(especially visceral) of the tissues to control levels.Adaptive mechanisms also occurred for lipid uptake,which varied depending on the adipose depot, whichis possibly due to the variations in lipoprotein lipaseactivity that were observed in the different fat depots

with the different types of intervention and demonstratea correlation with regional differences (Sea et al. 2000).

Some studies have demonstrated that rats with high-fatdiet-induced obesity developed a hypothalamic resistanceto leptin and insulin, which are the main anorexigenichormones, that led to the progressive loss of balancebetween food intake and thermogenesis and resulted inbody mass gain in mice but not in Wistar rats (Lin et al.2001; Munzberg et al. 2004; De Souza et al. 2005).

Additionally, obese individuals have alteredpostprandial ghrelin concentrations because theycannot properly interrupt a meal, which again suggests aresistance to the action of leptin, particularly consideringthe existence of an inverse association between the energybalance and these hormones (English et al. 2002; Pardoet al. 2010). In this study, we observed that leptin levelsdid not change after refeeding with high-fat diet whenthe weight was restored. Moreover, the high-fat diet andrefeeding resulted in increased visceral adiposity, fattylivers and changes in the lipid profile, as reported by otherauthors (Duarte et al. 2008; Sene-Fiorese et al. 2008). It isalso known that a large quantity of visceral adipose tissueis associated with a worsening of the resistance to insulinaction and the metabolic syndrome (Gallagher et al.2005). According to Moraes et al. (2009), the functionalresistance to leptin and insulin in the hypothalamus is aconsequence of a diet-induced activation of inflammatorysignalling, specifically at this site of the brain, and leadsto the molecular impairment of leptin and insulin signaltransduction. Thus, we hypothesized that the refeedingwith a high-fat diet increased the deleterious effects ofchronic ingestion of fat, which led to the development ofobesity, particularly visceral obesity, in these animals.

Conclusion

As discussed, a general analysis of the metabolic andhormonal effects demonstrated that the high-fat dietand a subsequent moderate caloric restriction plusrefeeding increased the risks of developing visceral obesity,dyslipidaemia and non-alcoholic hepatic steatosis. Also,although it is currently established that hyperleptinaemiais indicative of leptin resistance in obese humans, acomplete understanding of the molecular components ofthe regulatory pathways of energy homeostasis is necessaryfor the development of specific therapeutic treatments tocontrol obesity and the metabolic syndrome.

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Acknowledgements

This work was supported by Conselho Nacional deDesenvolvimento Cientfico e Tecnologico CNPq (141486/2003-3) and Fundacao de Amparo a Pesquisa do Estado de Sao PauloFAPESP (99/12981-7), Universidade Federal de Sao Carlos. Wewish to thank Keico Okino Nonaka, PhD (Departamento deCiencias fisiologicas, Universidade Federal de Sao Carlos).

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