Accepted Manuscript Title: Intermittent food restriction in female rats induces SREBP highexpression in hypothalamus and immediately post-fasting hyperphagia Author: Mariana Rosas Fernández, Carlos Concha Vilca, Leandro Oliveira Batista, Viviane Wagner Ramos, Leonardo Paes Cinelli, Kelse Tibau de Albuquerque PII: DOI: Reference:
S0899-9007(17)30278-2 https://doi.org/10.1016/j.nut.2017.11.026 NUT 10102
To appear in:
Nutrition
Received date: Revised date: Accepted date:
15-5-2017 16-10-2017 11-11-2017
Please cite this article as: Mariana Rosas Fernández, Carlos Concha Vilca, Leandro Oliveira Batista, Viviane Wagner Ramos, Leonardo Paes Cinelli, Kelse Tibau de Albuquerque, Intermittent food restriction in female rats induces SREBP high-expression in hypothalamus and immediately post-fasting hyperphagia, Nutrition (2017), https://doi.org/10.1016/j.nut.2017.11.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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INTERMITTENT FOOD RESTRICTION IN FEMALE RATS INDUCES SREBP HIGH-
2
EXPRESSION
3
HYPERPHAGIA
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Running head: Refeeding cycles and obesity effect
IN
HYPOTHALAMUS
AND
IMMEDIATELY
POST-FASTING
5 6
Authors:
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Mariana Rosas Fernández M.Sc.a, Carlos Concha Vilca M.Sc.b, Leandro Oliveira Batista Ph.D.c,
8
Viviane Wagner Ramos M.Sc.b, Leonardo Paes Cinelli Ph.D.a, Kelse Tibau de Albuquerque
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Ph.D.a,b,c..
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Author affiliation:
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a
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de Janeiro – Macaé, Rio de Janeiro, Brasil.
14
b
Programa de Pós-Graduação em Nutrição, Universidade Federal do Rio de Janeiro, Brasil.
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c
Laboratório de Nutrição Experimental, Universidade Federal do Rio de Janeiro - Macaé, Rio de
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Janeiro, Brasil.
Programa de Pós-Graduação em Produtos Bioativos e Biociências, Universidade Federal do Rio
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Role of each author in the work
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Conception and design of the study: KTA; supervision of post-graduate activities: KTA, LPC; PCR
20
analysis: LOB; generation, collection, assembly of data: MRF and CCV; statistical analysis,
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interpretation data and manuscript: MRF, LOB, VWR and KTA. All authors have read and
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approved the final manuscript.
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Corresponding author: Kelse Tibau de Albuquerque, Laboratório de Nutrição Experimental,
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Universidade Federal do Rio de Janeiro. Rua Aloísio da Silva Gomes, nº 50 - Granja dos
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Cavaleiros, Macaé, Rio de Janeiro, Brasil. CEP: 27930-560, Tel. +55 22 2141-4019. E-mail:
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[email protected]
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Word count: 4435 (including abstract, key words, legend figures and references)
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Number of figures: 4
31 32
Highlights
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The frequency of IFR influences metabolic changes and mass gain
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IFR induces up-regulation of SREBP-1c and 2 in the hypothalamus
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IFR promotes compensatory adipose deposition
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IFR impairs the hypothalamic lipogenic pathway
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Abstract
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Objective: We investigated the effect of intermittent food restriction (IFR) cycles on hypothalamic
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expression of lipogenic proteins and induction of overeating. Methods: Female Wistar rats were
41
distributed in three groups: free access to feed (Control, C), two days feed restriction at 50% of C
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intake followed by three (restricted 3, R3) or five (restricted 5, R5) ad libitum feeding. After 6
43
weeks, the rats were submitted to euthanasia and collected the hypothalamus and blood. The
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deposits of retroperitoneal, mesenteric and gonadal fat were weighed. The expression of the mRNA
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for sterol regulatory element binding protein (SREBP) 1c and 2 and acetyl-CoA carboxylase (ACC)
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in the hypothalamus were determined by real-time PCR and glucose and triacylglycerol were
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evaluated by a commercial kit. Body mass and food intake were measured daily. Results: IFR
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promoted increased expression of SREBP 2 in both treated groups, and in R5 increased expression
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of SREBP 1c. The serum triacylglycerol, the mesenteric deposit and total fat content were higher in
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R3. Neither of the treatment intervals altered the expression of the mRNA of ACC enzyme, but
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induced hyperglycemia and higher food intake immediately after food restriction. Conclusion: IFR
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affected the expression of SREBP-1c in R5 and SREBP-2 in the hypothalamus and caused
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overeating immediately after fasting in both groups. We suggest that hypothalamic and peripheral
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alterations, coupled with compulsive eating behavior in the ad libitum period, indicate risks for
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diabetes mellitus and recovery of body mass after interruption of IFR.
56 57 58
Key words: food restriction-refeeding, SREBP, acetyl-CoA carboxylase, hypothalamus,
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hyperphagia post-fasting, intermittent fasting.
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Abbreviations
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IFR
62
Introduction
Intermittent food restriction
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Several studies have demonstrated that high food / energy intake stimulates fat synthesis in
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adipose tissue, whereas the restriction of this leads to mobilization of the fat deposit, with release of
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fatty acids and glycerol [1]. Changes in the composition and amount of food ingested daily may
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determine physiological adjustment mechanisms to maintain a balance between food intake
67
regulation and lipogenesis, resulting in body mass regulation.
68
Intermittent food restriction (IFR) exposes the body to frequent cycles of fasting and
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refeeding [2], causing hormonal changes [3] and constant activation / inhibition of proteins related
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to lipogenesis and regulation of intake [4]. These events determine changes in gene expression in
71
order for the organism to maintain lipogenic control [5,6]. Therefore, changes in gene expression
72
may be consequential to the diet and are important for the organization of the response in various
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tissues.
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The hypothalamus is an important regulatory center of energy homeostasis and sensitive to
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hormones such as insulin and leptin, modulated by adiposity, and exert a central anorectic effect [7].
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The nutritional state in which the organism is found determines the activation of orexigenic or
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anorexigenic neurons resulting in increased or decreased appetite and regulation of energy
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homeostasis [8].
79
The sterol regulatory element binding proteins (SREBPs) are the main transcription factors
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that regulate the expression of genes involved in the biosynthesis of cholesterol, fatty acid,
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phospholipids and triacylglycerol [9]. SREBP-1c, whose main function is the synthesis of fatty
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acids, for its ability to increase the expression of acetyl-CoA carboxylase (ACC) and fatty acid
83
synthase (FAS) enzymes, while SREBP-2 is related to cholesterol synthesis [10], but its increased
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expression may result from reduction in isoform 1c expression [11] allowing the functionality of the
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pathway.
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In the hypothalamus, the lipogenic pathway integrates the regulation of energy homeostasis
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through the action of enzymes, such as ACC, and its product, malonyl-CoA,
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which promotes increased anorectic response, initiated by leptin [12]. Although SREBP-1c is
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expressed in the brain, little is known about nutritional factors that modulate its hypothalamic
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action. Increased insulin concentration in the postprandial period promotes the phosphorylation of
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SREBP-1c and its consequent translocation from the cytoplasm to the nucleus, initiating lipogenic
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activity, while its activity decreases during fasting in response to decreased insulin [6].
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Although it has been shown that IFR promotes body mass loss in humans [13,14] and
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animals [15,16], and that there is a strong relationship between diet, gene expression and
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lipogenesis, it has not been demonstrated whether the practice of food restriction followed by
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refeeding in females Wistar rats affects the hypothalamic lipogenic pathway and promotes
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overeating and can lead to compensatory growth of body mass. In this way, we aimed to evaluate if
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the expression of hypothalamic lipogenic factors is regulated by IFR and if this strategy of weight
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loss determines risk of overeating in non-obese rats.
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Material and methods
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Animals conditions and groups
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Thirty female Wistar rats, nine weeks old, at the beginning of the investigation, were housed
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in individual cages at 22° C + 1° C under a light: dark (12/12h) cycle with lights on at 8:00 a.m. The
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rats were maintained with water ad libitum and in the following food protocols: free access to feed
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(Control, C, n=10), two days feed restriction at 50% of C intake followed by three (Restricted 3,
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R3, n=10) or five (Restricted 5, R5, n=10) days of ad libitum access to commercial feed (Nuvilab®,
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São Paulo, Brazil). On the day of the experiment the rats were submitted to euthanasia by
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decapitation in guillotine between 8am and 10pm. Hypothalamus samples were collected and stored
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immediately at -80 ° C for analysis, except for glucose and triglycerides, which were analyzed two
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hours after euthanasia. All procedures were approved by the Ethics and Animal Use Commission
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(CEUA) of the Federal University of Rio de Janeiro - Campus Macaé (MAC030).
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Food intake and visceral fat pad measurements
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Food intake and body mass were monitored daily in the experimental period. Each day the
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amount of feed to be offered to the restricted groups (50% of the C consumption) and food behavior
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were identified and evaluated. On the day of euthanasia, deposits of visceral mesenteric,
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retroperitoneal and gonadal fat (perirenal and periovary) were removed and weighed.
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Biochemical assays
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Serum glucose and triacylglycerol concentrations were analyzed by microassay with
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colorimetric kit (Glicose PAP Liquiform / Triglycerides Liquiform, Labtest Diagnostica, MG,
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Brazil). The assays were performed according to the manufacturer's instructions and absorbance
121
readings performed on a spectrophotometer (Asys Expert 96, Biochrom, Cambridge, UK).
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Protein expression analysis
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The total mRNA was extracted from the hypothalamus samples with triazole reagent. The
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cDNA was synthesized from total RNA (2mg) in 20 μl of reaction using the commercial High-
125
Capacity cDNA kit (Applied Biosystems, California, USA), using the parameters provided by the
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manufacturer. Amplification of the mRNA was performed by the PCR technique (Real Time PCR or
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polymerase chain reaction) in real time through the StepOne Plus device (Applied Biosystems,
128
California, USA). Custom initiators SREBP-1c, SREBP-2 and ACC were used, and using the 18S
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as housekeeping gene. The reactions were performed with 10 μL TaqMan Universal mastermix
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TaqMan (Applied Biosystems, California, USA), 1 μL of the initiator and 9 μL of the synthesized
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cDNA, the plate was sealed and centrifuged at 2,500 rpm for 5 minutes to eliminate air bubbles and
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loaded in the apparatus at 50 ° C for 2 minutes for incubation followed by 95 ° C for 10 minutes for
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polymerase and PCR activation (40 cycles) at 95 ° C for 15 seconds to denature and 60 ° C for 1
134
minute for extension. Analyzes were performed in triplicate and the results were analyzed using
135
ΔΔCT, the expression data were compared with the 18S gene as the reference gene.
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Statistical analysis
137
For statistical analysis, we used software (GraphPad, CA, USA) and the results were
138
expressed as mean + standard error of the mean. For analysis of multiple comparisons, we used
139
one-way analysis of variance followed by the post hoc analysis test Bonferroni. Statistical
140
significance was set at p <0.05.
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Results
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Food behavior, body weight gain in the intermittent food restriction
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Feeding IFR for 6 weeks induced lower total food intake (Figure 1A) in both restricted
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groups compared to C, but the restricted ones did not differ from each other. However, the same
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groups presented increased food intake immediately after fasting (Figure 1C and 1D) in all weeks
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vs. C. We observed that the lower frequency of food restriction (group R5) promoted lower body
158
mass gain (Figure 1B) compared to C, while group R3 did not differ between the groups.
159 160 161 162
Effects of intermittent food restriction on visceral fat and biochemical parameters
163
In the R3 group we observed a higher total fat content and mesenteric fat deposit (Figure 2)
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and a 26.8% increase in total visceral fat in the R5 group compared to the C group, suggesting
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limited regulation of the visceral adiposity of the restricted groups. We observed in the restricted
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groups elevated serum glucose and triacylglycerol concentration in the R3 group and higher
167
triacylglycerol in the R5 group, compared to the C group (Figure 3).
168 169 170 171 172 173 174 175 176 177
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Effect of intermittent food restriction on lipogenic hypothalamic proteins
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The expression of SREBP-1c mRNA from group R5 was greater compared to groups C and
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R3, and SREBP 2 was higher in both restricted groups vs C, while we did not observe alteration in
185
the expression of the ACC enzyme in the restricted groups (Figure 4).
186 187 188 189 190 191 192
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194 195
Discussion
196
We demonstrated that intermittent food restriction, as an anti-obesogenic strategy, in female
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Wistar rats reduced body mass, but promoted greater visceral adiposity and damage to the
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hypothalamic lipogenic pathway. In fact, the treatment increased the expression of the lipogenesis
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transcription factors SREBP-1c and SREBP-2. Food restriction cycles and refeeding promote
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repeated changes in hormone secretions and activation or inhibition of neuropeptides that act on
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energy homeostasis and body mass control [17,18].
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Restricted groups had lower accumulated consumption during the treatment period;
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however, only the R5 group showed lower final body mass gain. Lenglos et al. showed that female
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rats submitted to IFR had elevated plasma corticosterone levels, as well as high expression of
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corticotropin-releasing factor and relaxin-3, involved in stress response [19]. Similarly, continuous
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food restriction in humans for three weeks increased the total output of cortisol and perceived stress
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[20]. Chronic stress causes dysregulation on the hypothalamic-pituitary-adrenal axis, resulting in
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weight loss difficulty [21]. In the present study, both groups showed lower food intake and the
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weight difference in the treated groups may be due to different food restriction frequencies. Thus,
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the R3 group, with more frequent restriction and refeeding cycles, was submitted to greater stress,
211
which did not allow evidence the lowest weight gain observed in the R5 group. However, the IFR
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promoted higher food intake immediately after the restriction period, regardless of the refeeding
213
time. Data from our laboratory confirm hyperphagic behavior on the two post-restriction days in
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groups R3 and R5 (data not shown). Restriction and refeeding cycles may lead to disturbances in
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food intake by determining orexigenic response stimulated by neuropeptide Y and agouti-related
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protein [4,22] suggesting compulsive eating of the rats in this study.
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Fasting induces the production of glucose and accumulation of hepatic lipid, products
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consequent to gluconeogenesis and lipolysis of adipose tissue; so that the verified elevated levels of
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glucose and serum triacylglycerols are signs of metabolic adjustment and also appear to represent a
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protective mechanism for hepatic lipotoxicity [23].
221
Shorter restriction and refeeding cycles, as in the case of R3, induced an increase in
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triacylglycerol, and glucose intolerance. Hyperglycemia, common to restricted groups, may be due
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to hepatic gluconeogenesis and peripheral insulin resistance. IFR increases insulin resistance by
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decreasing its signaling and reducing glycogen-phosphorylase expression [24]. The mechanism of
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glucose-lactate detection of the brain regulates the hepatic production of lipid and glucose [25], so
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that frequent reductions in serum glucose caused by fasting can favor early rupture of homeostasis,
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resulting in simultaneous increase in the production of glucose and triacylglycerols.
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During periods of fasting, energy stocks coming from glycogen are mobilized [3,23,26] and
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followed by lipolysis of adipose tissue, with release of triacylglycerols and production of glucose
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from glycerol [27,28]. It was observed that acute food restriction, with two days of refeeding, was
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not able to restore serum triglyceride content after fasting; but four days of refeeding were sufficient
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to double the serum concentration of this substrate [29], which reinforces the high triacylglycerol
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levels observed. Increased lipolysis causes increased levels of diacylglycerol in the cell, which
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raises the content of protein kinase C, which is related to insulin resistance by inhibiting the
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phosphorylation of insulin receptor substrate 1 [30,31].
236
Stress can induce resistance to insulin and leptin, and may favor gain of body mass and fat
237
[32]. The higher frequency of restriction cycles (R3) promotes repeated feelings of hunger stress,
238
which may have been enough to limit body mass loss and also promote an increase in the deposit of
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mesenteric fat. Although little is known about the relationship between IFR and resting energy
240
expenditure, it was observed that IFR in slim and healthy people for two weeks decreased the
241
resting energy expenditure [33], suggesting that IFR may alter one of the main components of
242
energy expenditure and be related to body mass in humans.
243
Animal studies have shown that IFR leads to increased fat deposits [4,15] and in the present
244
study we found a higher mesenteric deposition and total visceral fat content in the R3 group.
245
Different adipocyte-related genes are regulated by the diet, and IFR may promote the deposition of
246
triacylglycerols in visceral adipose tissue, since 3 days of fasting and 3 days of refeeding in male
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Wistar rats resulted in increased regulation of Fsp27 gene expression in the visceral epididymal
248
tissue, which is involved in lipid deposition [16].
249
During the fasting period, the ACC enzyme is phosphorylated by AMP-activated protein
250
kinase (AMPK), inhibiting its activity, with the consequent decline of the malonyl-CoA content in
251
the hypothalamus. On the other hand, in the fed period, the decrease in AMPK activity allows the
252
activation of the ACC enzyme, increasing malonyl-CoA and potentiating the anorectic effect of
253
leptin [34]. It was observed that fasting for 45 hours and refeeding for 3 hours did not promote
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alteration in the expression of this enzyme in male Wistar rat brains [6] and it was observed that
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acute food restriction did not alter the expression of FAS and ACC enzymes in the brain [35].
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Karami et al. (2006) demonstrated a decrease in ACC activity in the brain, both in the presence and
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absence of citrate in male mice [36]. However, the effects of repeated restriction and refeeding
258
cycles on the enzymatic activity of ACC in the central nervous system (CNS) have not yet been
259
clarified. In the present study, we showed that the IFR for 6 weeks did not modify the expression of
260
mRNA for ACC, despite the evident alteration in the SREBP expression.
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The fasting for 48 hours followed by refeeding was able to increase the activity of lipogenic
262
enzymes in the epididymal and retroperitoneal visceral deposits [29]. Similarly, other studies have
263
indicated hepatic steatosis and increased mRNA of lipogenic enzymes in white adipose tissue
264
[16,29,37].
265
Although the lipogenic byproduct, malonyl-CoA, is a regulator of food intake, the
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hypothalamus is not the center of intense lipogenic activity, therefore nutritional changes such as
267
fasting and refeeding intervals can more easily alter the expression of lipogenic enzymes in
268
peripheral tissues than in the hypothalamus, by the lipogenic dynamics of the tissue itself. It is
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suggested that the expression of lipogenic enzymes in the brain is not regulated by the peripheral
270
nutritional condition [6] and that the regulation of these enzymes in the hypothalamus differs
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according to their location in the hypothalamic nuclei [38].
272
Okamoto et al. (2006) reported that animals, both lean and obese, had an acute food
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restriction of 45 hours and a 3-hour refeeding, and observed that there were no changes in SREBP-1
274
in the brain, indicating that, at least acutely, independently of the nutritional status, the food
275
restriction does not alter this transcription factor in the CNS [6]. The present study showed
276
increased expression of SREBP-1c in R5 and SREBP-2 in both restricted groups. However, the fact
277
that the R3 group did not modify SREBP-1c, but only SREBP-2, may be a mechanism to regulate
278
the synthesis of malonyl-CoA and to limit the anorectic response, since this group had a higher
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frequency of restriction, which adds to the higher serum concentration of triacylglycerol probably
280
due to lipolysis and hepatic synthesis.
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The brain is rich in cholesterol which is mostly formed by de novo synthesis, and since the
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blood-brain barrier limits its uptake from the circulation [39,40], we suggest that the expression of
283
SREBP 2 in the groups may arise from the need to increase synthesis rather than serum uptake. As a
284
result of the intermittent restriction cycles, this regulation ensures the maintenance of lipid levels in
285
the structures of the hypothalamic tissue [41,42]. Changes in SREBP-1c expression can be
286
compensated by SREBP-2 [11].
287
It is considered that increase of SREBP-1c is decisive in the transcription and final
288
translation of the lipogenic protein ACC [5] and with this the increase of its malonyl-CoA product,
289
which in the hypothalamus can lead to anorectic response. Studies suggest that chronic mild food
290
restriction leads to increased phosphorylation of AMPK and ACC, followed by reduction of
291
hypothalamic malonyl-CoA levels [43]. This result signals the development of IFR on the
292
mechanisms of regulation of food intake and body mass, since the reduction of malonyl-CoA limits
293
the anorectic response.
294
Transcriptional changes, especially in the hypothalamus, are detrimental to the energy
295
balance, which makes it understandable that such changes can be observed immediately in the
296
performance of food restriction in the peripheral tissues [16,29,37], while in CNS, such changes are
297
less evidenced by the acute practice of food restriction [35], however, this study showed
298
transcriptional changes with chronic intermittent food restriction.
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We conclude that, although IFR was effective in promoting lower body mass gain in female
300
Wistar rats, it affected the expression of lipogenic transcription factors SREBP-1c and 2 in the
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hypothalamus, demonstrating impairment of lipogenic activity.
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Funding
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This work was supported by the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do
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Rio de Janeiro - FAPERJ and the Fundação Educacional de Macaé - FUNEMAC.
306
Conflict interesting: the authors Leonardo Paes Cinelli and Kelse Tibau de Albuquerque were
307
equally important for the accomplishment of this work.
308
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Figure 1. Food consumption in the period (A); body mass gain (B), post-restriction food intake (C
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and D). The results are expressed as mean ± standard error of the mean (n = 10 / group). * versus C
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for p <0.05 (post hoc Bonferroni test for multiple comparisons.). C, control; R3, restricted 3 and
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R5, restricted 5.
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Figure 2. Deposits of visceral fat: mesenteric, retroperitoneal, gonadal and total visceral adiposity. The results are expressed as mean ± standard error of the mean (n = 10 per group). * versus C for p <0.05 (post hoc Bonferroni test for multiple comparisons.). C, control; R3, restricted 3; R5, restricted 5; MES, mesenteric; RET, retroperitoneal; GN, gonadal.
Figure 3. Serum glucose and triacylglycerol concentration. The results are expressed as mean ± standard error of the mean (n = 10). * versus C for p<0.05 (post hoc Bonferroni test for multiple comparisons.). C, control; R3, restricted 3; R5, restricted 5.
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Figure 4. Expression of the mRNA of the transcription factors SREBP 1c (n = 6) and 2 (n = 4) and of the ACC enzyme (n = 8) in the hypothalamus. The results are expressed as mean + standard error of the mean. * versus C and # versus R3 for p<0.05 (post hoc Bonferroni test for multiple comparisons). C, control; R3, restricted 3; R5, restricted 5; SREBP, sterol regulatory element binding protein, isoforms -1c and; ACC, acetyl-CoA carboxylase; AU, arbitrary units.
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