Differential effects in male adult rats of lifelong coconut oil exposure versus during early-life only

Journal of Functional Foods 55 (2019) 17–27

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Differential effects in male adult rats of lifelong coconut oil exposure versus during early-life only

T

Fernanda Torres Quitetea, Egberto Gaspar de Mouraa, Geórgia Correa Atellab, ⁎ Patricia Cristina Lisboaa, Elaine de Oliveiraa, a b

Department of Physiological Sciences, Roberto Alcantara Gomes Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, RJ 20551-030, Brazil Medical Biochemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil

ARTICLE INFO

ABSTRACT

Keywords: Coconut oil Metabolic programming Obesity Dietary oils Functional foods

We investigated the effects of maternal coconut oil supplementation during breastfeeding on the endocrinemetabolic profiles of offspring and the impact of continued exposure throughout life. Rat mothers were separated into: soybean oil (SO); and coconut oil (CO) groups and received the oils through gavage (0.5 g/kg of BW) and had free access to standard chow. After weaning, half of the pups from CO group continued receiving coconut oil in chow (CO + C), while SO and the other half of CO group received standard chow. Offspring were killed at postnatal day 180. CO and CO + C offspring had higher body masses, but only CO had higher visceral fat and lower lean mass. CO group exhibited hyperphagia and hyperleptinemia while CO + C group exhibited hypophagia. CO group had higher T3 and TSH. Coconut oil led to long-term overweight, hyperphagia, hyperleptinemia and thyroid dysfunction, whereas the continuous exposure throughout life prevented most of these dysfunctions.

1. Introduction Functional foods are often included in the diets of pregnant and lactating women in an attempt to improve the quality of feeding, be healthier, and reduce the undesirable effects common during pregnancy, such as constipation, nausea and excessive weight gain (Champ & Hoebler, 2009; Choudhary & Grover, 2012). In this context, the supplementation or replacement of some types of oils that are considered to be healthier has been a very common practice during these periods of life. Evidence has emerged that sources of medium-chain saturated fatty acids (MCSFAs), such as coconut oil, may be beneficial in the prevention and treatment of obesity and metabolic syndrome (Cardoso, Moreira, de Oliveira, Raggio Luiz, & Rosa, 2015; Mumme & Stonehouse, 2015; Nagao & Yanagita, 2010). These beneficial effects seem to be involved with their rapid oxidation since MCSFAs are absorbed and transported through the portal vein, reaching the liver, where they are metabolized, providing immediate energy, which reduces their uptake by adipose tissue (Babayan, 1987; Marten, Pfeuffer, & Schrezenmeir, 2006). This process contributes to the improvement of lipid profiles

(Babayan, 1987; Marten et al., 2006). In addition, MCSFAs are also associated with increased satiety and thermogenesis (Baba, Bracco, & Hashim, 1987; Friedman, Harris, Ji, Ramirez, & Tordoff, 1999; St-onge & Jones, 2002) which can contribute to body weight loss. Coconut oil is an important source of MCSFAs, highlighted by the presence of lauric acid (12:0), which contributes between 45 and 50% of coconut oil composition (Assunção, Ferreira, dos Santos, Cabral, & Florêncio, 2009). Virgin coconut oil, produced by unrefined processes, also contains high amounts of antioxidant phenolic compounds, such as caffeic acid, ferulic acid, syringic acid, catechins and epigallocatechins that can contribute to the effects attributed to this oil (Marina, Man, Nazimah, & Amin, 2009). As previously mentioned, some authors suggest that MCSFA intake results in increased satiety and reduces food intake, although these data are still controversial (Dias et al., 2018; Stubbs & Harbron, 1996). The mechanism suggested for these MCSFA effects is associated with an increase in lipid oxidation. A study in rodents showed that a reduction in hepatic fatty acid oxidation was able to stimulate food intake by reducing energy production by the liver (Friedman et al., 1999). It should be considered that nutritional changes exclusively during

Abbreviations: CO, coconut oil group; LAT, L-type amino acid transporters; MCSFA, medium chain saturated fatty acids; MCT, monocarboxylates transporters; OATP, organic anions transporters; CO+C, coconut oil + standard chow supplemented with coconut oil group; SO, soybean oil group ⁎ Corresponding author at: Department of Physiological Sciences – 5° floor, Biology Institute – State University of Rio de Janeiro, Av. 28 de setembro, 87 – Vila Isabel, Rio de Janeiro, RJ 20551-031, Brazil. E-mail addresses: [email protected], [email protected] (E. de Oliveira). https://doi.org/10.1016/j.jff.2019.02.020 Received 2 November 2018; Received in revised form 4 February 2019; Accepted 7 February 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.

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lactation may have a long-term impact, especially when these changes occur only during this period, promoting the metabolic programming phenomenon (Moura & Passos, 2005). Metabolic programming, or ontogenetic plasticity, occurs due to changes in the physiological/environmental pattern (imprinting factor) early in life. This process could have consequences in adult life, causing adaptive changes in physiological processes, since the animals persist with the environmental conditions of early life. However, if the conditions change, then there is a higher risk of disease (Barker, 1995; de Moura, Lisboa, & Passos, 2008; Moura & Passos, 2005). To our knowledge, there are no reports in which the altered conditions during breastfeeding persist in one of the experimental groups until adulthood, since all previous papers changed the imprinting factor after weaning. According to the mismatch theory proposed by Gluckman and Hanson (2008), if the initial conditions are maintained, then the animal could be better adapted to the environment during development. In the literature, the long-term effects of different types of oils, such as fish oil, soybean oil, flaxseed oil, canola oil and palm oil used in critical periods of life (pregnancy and lactation) have been studied (Costa et al., 2011; Fernandes et al., 2012; Guarda et al., 2016; Magri et al., 2015; Misan et al., 2015; Oosting et al., 2010; Quitete, Lisboa, Moura, & de Oliveira, 2018). Nevertheless, the use of oils during critical stages of development can provide a better understanding of the effects of the different oils that are commonly used today because they could alter the endocrine-metabolic parameters of dams and breast milk composition, as well as cause dysfunction in the offspring in the short and long term (Costa et al., 2011; Fernandes et al., 2012; Guarda et al., 2016; Magri et al., 2015; Misan et al., 2015; Oosting et al., 2010; Oosting, Verkade, Kegler, van de Heijning, & van der Beek, 2015; Quitete et al., 2018). Since coconut oil is being heavily prescribed by health professionals and widely used by the general population, even in critical periods of life with no scientific evidence of its effects during these important phases, it is very important to understand the effects of coconut oil supplementation during the critical lactation period. In this context, the present study aims to assess the nutritional status of the dams that received coconut oil supplementation and their pups during lactation, as well as breast milk composition at the end of lactation and endocrinemetabolic parameters of the adult offspring. In addition, this study aims to evaluate whether continuous exposure to coconut oil from weaning until 180 days of life promotes different patterns compared to the offspring that received coconut oil supplementation only during breastfeeding. Based on the metabolic programming theory, we hypothesize that the programming effects of coconut oil exposure only during lactation has a more marked effect than continuous exposure on obesity development.

Table 1 Macronutrients and micronutrients composition of standard chow for rats and coconut oil supplemented chow. Standard chowa

Coconut oil chow

Carbohydrate (g/kg) Corn starch (g/kg) % kJ

660.0 – 68.0

570.0 363.3 63.6

Protein (g/kg) Textured soy (g/kg) % kJ

220.0 – 22.7

224.0 303.0 25.0

Fat (g/kg) Coconut oil (ml/kg) % kJ

40.0 – 9.2

45.0 30.3 11.4

Vitamins and Minerals Mineral mixb (g/kg) Vitamin mixb (g/kg) Ca (g/kg) P (mg/kg) Na (mg/kg) Fe (mg/kg) Mn (mg/kg) Zn (mg/kg) Cu (mg/kg) I (mg/kg) Se (mg/kg) Co (mg/kg) F (mg/kg) Vit A (UI/kg) Vit D3 (UI/kg) Vit E (UI/kg) Vit K3 (mg/kg) Vit B1 (mg/kg) Vit B2 (mg/kg) Vit B3 (mg/kg) Vit B5 (mg/kg) Vit B6 (mg/kg) Vit B7 mg/kg) Vit B9 (mg/kg) Vit B12 (mcg/kg) Choline (mg/kg)

– – 10.0–14.0 8000.0 2700.0 50.0 60.0 60.0 10.0 2.0 0.05 1.5 80.0 13000.0 2000.0 34.0 3.0 5.0 6.0 60.0 20.0 7.0 0.05 1.0 22.0 1900.0

9.6 1.6 – – – – – – – – – – – – – – – – – – – – – – – –

Aminoacids Lysine (mg/kg) L-cystine (mg/kg) Methionine (mg/kg)

12000.0 – 4000.0

50.0 875.0 150.0

BHT (mg/kg)

100.0

300.0

a Standard chow to rats (Nuvilab-NUVITAL Nutrientes LTDA, Paraná, Brazil). Composition of diet: Whole corn, soybean bran, wheat bran, calcium carbonate, dicalcium phosphate, sodium chloride, Vitamin A, Vitamin D3, Vitamin E, Vitamin K3, Vitamin B1, Vitamin B2, Vitamin B6, Vitamin B12, niacin, calcium pantothenate, folic acid, biotin, choline chloride, iron sulfate, manganese sulfate, zinc sulfate, copper sulfate, calcium iodate, sodium selenite, cobalt sulfate, lysine, methionine, BHT - butylated hydroxytoluene. b Vitamins and minerals mixture formulated as recommended by the American Institute of Nutrition AIN93G of rodents diet; contains the recommended amount of iodide (Reeves et al., 1993).

2. Materials and methods The experimental design was approved by the Animal Care and Use Committee of the Biology Institute of the State University of Rio de Janeiro (CEUA/001/2014). Wistar rats were kept in a temperaturecontrolled room (25 ± 1 °C) with an artificial light-dark cycle (lights on at 0700 h, lights off at 1900 h). Virgin female rats (n = 30) that were 3 months old were caged with male rats, in a proportion of 2 females to 1 male. After mating, each female rat was placed in an individual cage with free access to water and food until delivery. At birth, litters were adjusted to 6 male pups per dam to maximize the lactation performance (Passos, Ramos, & Moura, 2000).

215898, Lot #G3014) (0.5 g of oil/kg of body weight) via intragastric gavage throughout the lactation period (21 days); CO (coconut oil group, n = 10), dams that received extra virgin coconut oil (Santa Cruz Biotechnology, Inc., TX, USA; sc-214752A, Lot #K0614) (0.5 g of oil/kg of body weight) via intragastric gavage throughout the lactation period; and CO + C (coconut oil + standard chow supplemented with coconut oil group, n = 10), dams that received extra virgin coconut oil (0.5 g of oil/kg of body weight) via intragastric gavage throughout the lactation period, and after the weaning, their offspring received chow supplemented with extra virgin coconut oil for their whole life (180 days). The dams of the three groups during lactation and the offspring of the SO and CO groups for the duration of their lives had free access to standard chow for rodents (Table 1).

2.1. Experimental model At birth, dams and their offspring were randomly divided into the following three groups: SO (soybean oil group, n = 10), dams that received soybean oil (Santa Cruz Biotechnology, Inc., TX, USA; sc18

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Concerning the posterior analysis, only one randomly selected animal per litter was used, except for the body weight and food intake data, for which we used all animals of the litter.

2.6. Analysis of fatty acid profile of breast milk, standard and experimental chow The analysis of the fatty acid fractions by GC-MS was carried as described by Christie (1989). This analysis was performed in quadruplicate and before initiate the lipids extraction, 50 µg of Internal Pd C19 (Sigma-Aldrich) was added. The lipid sample was dissolved in a toluene (1 mL) and sulfuric acid 1% in methanol (2 mL) solution. The mixture was left overnight in a stoppered tube at 50 °C and then 1 mL of sodium chloride 5% was added. The required esters were extracted twice with 2 × 2 mL hexane using Pasteur pipettes to separate the layers. The solvent was removed in a nitrogen stream. Dried FAME was resuspended in 100 µL heptane. GC/MS analysis was carried out on a Shimadzu GCMS-QP2010 Plus system, using an HP Ultra 2 (5% Phenyl - methylpolysiloxane), Agilent (25 m × 0.20 mm × 0.33 µm). Injector was set at 250 °C. Column temperature was programmed from 40 to 160 °C at 30 °C/min, 160–233 °C at 1 °C/min, 233–300 °C at 30 °C/min and held at 300 °C for 10 min. Helium was used as carrier gas with linear velocity of 36.0 cm s−1. A volume of 1 µL of sample was injected into the chromatograph. Electro ionization (EI-70 eV) and a quadruple mass analyzer, operated in scans from 40 to 440 amu. Interface was set at 240 °C and the ion source at 240 °C. The components were identified by comparing their mass spectra with those of the library NIST05 contained in the computer's mass spectrometer. Retention indices were also used to confirm the identity of the peaks in the chromatogram by Supelco 37 Component FAME Mix (Sigma-Aldrich). The fatty acid profiles of breast milk of SO and CO groups are demonstrated in Table 1 of Supporting Information and the fatty acid profiles of standard chow and coconut oil supplemented chow are demonstrated in Table 2 of Supporting Information.

2.2. Coconut oil supplemented chow The standard chow was crushed, added textured soy protein, corn starch and coconut oil according to Table 1 to balance the macronutrients without changing the sources of protein and carbohydrates, thereby maintaining the chow with a normocaloric, normoproteic, normoglicidic and normolipidic profile. Water was added to homogenize and prepare new pellets that were dried in a ventilated oven for 24 h and subsequently stored in a refrigerator. This supplementation was offered to the CO + C group from PN21 to PN180. After weaning, the SO and CO groups had free access to a standard rat chow, and the three groups had free access to water until 180 days of life. The standard chow and the coconut oil supplemented chow were isocaloric and had similar amounts of macronutrients (Table 1). 2.3. Nutritional status evaluation - Body mass: During the 21 days of lactation, the body masses of dams and pups were assessed daily and after weaning were monitored every 4 days until 180 days of age. - Food intake: The food intake of dams during lactation and the offspring after weaning to 180 days old was assessed every 4 days. The amount of food consumed was estimated from the difference between the weight of the food left in the cage and the total quantity put in the cage 4 days before. The cumulative intake was calculated by totaling the amount of chow consumed by dams during lactation and the offspring from weaning until 180 days. - Body composition: At 180 days old, rats were anesthetized with a non-lethal dose of 2,2,2 tribromoethanol (Avertin®) and carried to the Lunar DXA 200368 GE equipment (Lunar, Wisconsin, EUA) with specific software (Encore 2008. Version 12,20 GE Healthcare, Wisconsin, EUA). Total body mass (g), total lean mass (g), total body fat (%) and central fat mass (%) were analyzed. - Central adiposity: Retroperitoneal, epididymal and mesenteric fat were excised and weighed on the sacrifice day. The sum of these fat compartments was represented as visceral fat. A sample of each fat compartment was stored at −80 °C for posterior analysis.

2.7. Hormone determination in breast milk

2.4. Milk collection

T3 and leptin levels were assessed by radioimmunoassay (RIA) and immunoenzymatic (ELISA) assay, respectively. Total T3 level was measured by a specific RIA kit (MP Biomedicals Diagnostics Division, Orangeburg, NY, USA) with an assay sensitivity of 6.7 ng/dL and an intra-assay coefficient of variation of 4.4%. Leptin was measured in breast milk by commercial ELISA kit (EMD Millipore Corporation, Billerica, MA, USA). The intra-assay coefficient of variation was 2.13% and the assay sensitivity was 0.08 ng/mL.

Milk samples were collected on day 20 of lactation. Dams were separated from their litters for a period of 2 h before milking (Bonomo et al., 2005). After a subcutaneous injection of oxytocin (5 IU) with a non-lethal dose of a ketamine/xylazine mixture, milk was manually collected from all teats. We obtained 0.5–1.0 mL from each lactating rat, and the samples were frozen at −20 °C for further analysis.

Table 2 Macronutrients composition, energy, T3 and leptin levels in breast milk, plasma cholesterol and triglycerides content of dams and plasma leptin of dams and offspring of soybean oil (SO) and coconut oil (CO) groups at the end of lactation.

2.5. Analysis of milk biochemical composition Total milk protein was measured according to the Peterson method (Peterson, 1977) using bovine serum albumin as the standard. Protein concentration was determined based on the Stauffer formula (Stauffer, 1975), and the results were expressed in mg/ml. The cholesterol and triglyceride contents were measured in milk samples by colorimetric assay, using a Bioclin commercial kit. The milk samples were diluted in distilled water (1:25) for triglyceride analysis. The results were expressed in mg/dl. Milk lactose was measured by a colorimetric method using picric acid (Khramov, Kolomeitseva, & Papichev, 2008), using commercial lactose as the standard (Sigma-Aldrich Co, St. Louis, MO, USA). The results were expressed in mg/ml. The total calories of the milk were calculated using the sum of calories from each isolated macronutrient.

SO

CO

Breast Milk Cholesterol (mg/ml) Triglycerides (mg/ml) Lactose (mg/ml) Total protein (mg/ml) Total calories (kJ/ml) T3 (ng/ml) Leptin (ng/ml)

3.67 ± 0.36 36.4 ± 4.06 36.84 ± 3.88 64.52 ± 5.84 2.95 ± 0.29 1.42 ± 0.16 2.10 ± 0.12

5.59 ± 0.54* 70.79 ± 7.80* 33.81 ± 2.78 71.11 ± 2.31 5.27 ± 0.75* 1.84 ± 0.21 1.17 ± 0.10*

Plasma - Dams Cholesterol (mmol/L) Triglycerides (mmol/L) Leptin (ng/ml)

1.61 ± 0.06 0.99 ± 0.07 0.32 ± 0.07

2.17 ± 0.13* 1.43 ± 0.17 0.56 ± 0.08*

Plasma – Pups 21 days old Leptin (ng/ml)

1.21 ± 0.15

1.30 ± 0.20

* p < 0.05. Results expressed as mean ± SEM; n = 10. 19

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2.8. Blood glucose analysis

The homogenates were centrifuged three times at 13.000 rpm for 5 min at 4 °C, and the infranatant was collected and stored at −20 °C until the assay was performed. Protein concentration of the homogenate was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, San Jose, CA, USA) and the result of the ELISA was corrected using the protein content of the respective sample.

Blood glucose was measured from the tail vein with a glucometer after 12 h fasting (ACCU-CHEK Advantage; Roche Diagnostics, Mannheim, Germany) on the sacrifice day. All rats were anesthetized with a non-lethal dose of a ketamine/ xylazine mixture and sacrificed by cardiac puncture after 12 h (dams and adult offspring) or 2 h (pups at 21 days of age) of fasting. The blood was collected in heparinized tubes, centrifuged (4 °C, 20 min, 1.260g) to obtain plasma and stored at −20 °C until analysis. The tissues of interest were excised and stored at −80 °C and were subsequently processed according to the methods described below.

2.14. Western blotting TRβ1 content in liver samples was analyzed by western blotting. For protein extraction, the liver samples were homogenized with a RIPA lysis buffer (50 mM TRIS, 150 mM NaCl, 0,1% SDS, 50 mM NaF, 1 mM sodium orthovanadate, 30 mM sodium pyrophosphate, 5 mM-EDTA, and Triton X-100 1%) that included a protease inhibitor cocktail (Complete EDTA-free - Roche Applied Science, Mannheim, Germany). To homogenize the liver samples, 2 mL of buffer was used, and the homogenates were centrifuged at 13.200 rpm for 25 min at 4 °C. The protein concentration of the homogenate was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, San Jose, CA, USA). Samples were denatured in a sample buffer (50 mM Tris·HCl, pH 6.8, 1% SDS, 5% 2-mercaptoethanol, 10% glycerol, and 0.001% bromophenol blue) and heated at 95 °C for 5 min. Homogenates were analyzed by the SDS-PAGE method. To detect proteins, a 12% polyacrylamide gel was used, and 10 µg of total proteins was added in each slot of gel and electroblotted in a nitrocellulose membrane (Hybond® ECL membrane, Amersham Biosciences, London, UK). Membranes were incubated with Tween-TBS (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 0.1% Tween-20) containing 5% bovine albumin (Sigma-Aldrich, Saint Louis MO, USA) for 45 min to block nonspecific binding sites. Then, membranes were washed with Tween-TBS and incubated overnight with the primary antibodies anti-TRβ-1 (Abcam Inc. Cambridge, MA, USA, rabbit, 1:500) and anti-actin (Sigma-Aldrich, Saint Louis MO, USA, mouse, 1:500). Then, membranes were washed and incubated with the appropriate secondary antibodies conjugated to biotin (SigmaAldrich, Saint Louis MO, USA, 1:10.000 and 1:5000, respectively) for 1 h at room temperature. Subsequently, membranes were washed and incubated for 1 h at room temperature with streptavidin horseradish peroxidase (HRP) conjugate (Millipore, Temecula, CA) in the same dilution of the secondary antibody. The targeted proteins were detected by chemiluminescence (ECL-plus; Amersham Pharmacia Biotech, Piscataway, NJ, USA) with the aid of the Image Quant (LAS 500 GE) apparatus. Finally, the density of the protein bands was quantified by ImageJ 1.34s software (Wayne Rasband NIH, Boston, MA, USA).

2.9. Plasma cholesterol and triglyceride determination The total cholesterol and triglyceride plasma levels were measured by a colorimetric method using a commercial kit (Bioclin, Belo Horizonte, Brazil). 2.10. Hepatic cholesterol and triglyceride determination Liver samples (50 mg) were homogenized in 1 mL of isopropanol (Vetec, Rio de Janeiro, Brazil) and centrifuged (5.900 rpm/10 min/ 4°C). The total cholesterol and triglyceride levels were measured in the supernatant by a colorimetric method using a commercial kit (Bioclin, Belo Horizonte, Brazil). 2.11. Plasma hormone levels determination Plasma levels of each hormone were analyzed using a single test, providing the inter-assay coefficient of variation. Plasma total T3 and free T4 were determined by RIA, using commercial kits (MP Biomedicals Diagnostics Division, Orangeburg, NY, USA). The intra-assay coefficient of variation for T3 was 4.4%, with 6.7 ng/dL as the lower limit of detection, and for T4 was 3.9%, with 0.045 ng/dL as the lower limit of detection. Insulin and corticosterone plasma levels were analyzed by a commercial RIA kit (MP Biomedicals Diagnostics Division, Orangeburg, NY, USA). The intra-assay coefficient of variation for insulin was 7.1%, with 4.6 µIU/mL as the lower limit of detection, and for corticosterone, it was 7.1%, with 7.7 ng/dL as the lower limit of detection. Leptin and thyroid stimulating hormone (TSH) plasma levels were determined by immunoenzymatic assay (ELISA) (EMD Millipore Corporation, Billerica, MA, USA; ALPCO Diagnostics, Salem, NH, USA). The intra-assay coefficient of variation for leptin was 2.13% and for TSH was 3.7%, and the assay sensitivity was 0.08 ng/mL for leptin and 0.1 ng/mL for TSH.

2.15. Real-time reverse transcription polymerase chain reaction (RT-qPCR) For mRNA studies, total RNA was extracted from the brown adipose tissue (BAT) and liver under RNase-free conditions using a RNeasy Lipid Tissue Mini Kit (QIAGEN GmbH, Hilden, Germany) for BAT and TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) for liver. Total RNA was then quantified via NanoVue™ Plus Spectrophotometer (GE Healthcare, Buckinghamshire). The cDNA was prepared from the total RNA using the Moloney murine leukemia virus reverse transcriptase (M-MLV RT) for RT-PCR and oligo(dT)15 primer (Promega, Madison, WI, USA). The mRNA levels of uncoupling protein 1 (UCP1) (Assay ID: Rn00562126_m1) and deiodinase 2 (DIO2) (Assay ID: Rn00581867_m1) were measured in BAT, and mRNA levels of deiodinase 1 (DIO1) (Assay ID:Rn00572183_m1) were measured in the liver using TaqMan® Fast Universal PCR Master Mix (2X), no AmpErase® UNG (Catalog #: 4324018) (Applied Biosystems®, Foster City, CA, USA) according to the recommendations of the manufacturer. RT-qPCR was carried out in triplicate for each sample using an Applied Biosystems 7500 Real-Time PCR System (Applied BioSystems, Foster City, CA, USA). The oligonucleotide primers and probes were prepared by Applied Biosystems® (Foster City, CA, USA). The co-amplification of mouse β-actin mRNA (Assay ID: Rn00667869_m1), a variant internal

2.12. Insulin resistance index (IRI) The IRI was calculated by the following formula:

IRI = fasting insulin (µIU/ml) × fasting glycemia (mmol/L) 2.13. Leptin level determination in adipose tissue The leptin concentration was analyzed in retroperitoneal adipose tissue by immunoenzymatic assay (ELISA) (EMD Millipore Corporation, Billerica, MA, USA) in a single test, providing the inter-assay coefficient of variation. The intra-assay coefficient of variation was 2.13%, and the assay sensitivity was 0.08 ng/mL. Before the assay, the adipose tissue samples were homogenized with a RIPA lysis buffer (50 mM TRIS, 150 mM NaCl, 0.1% SDS, 50 mM NaF, 1 mM sodium orthovanadate, 30 mM sodium pyrophosphate, 5 mMEDTA, and Triton X-100 1%) that included a protease inhibitor cocktail (cOmplete EDTA-free - Roche Applied Science, Mannheim, Germany). 20

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control, was performed in all of the samples. The results were normalized to the β-actin mRNA levels using the 2ΔΔCT method. This method may be used to calculate relative changes in gene expression determined from real-time quantitative PCR experiments (Livak & Schmittgen, 2001).

ANOVA followed by a Newman-Keuls post hoc test. Other experimental data were analyzed by one-way ANOVA, using Newman Keuls as a posttest. Differences were considered significant when p < 0.05.

2.16. Statistical analysis

3.1. Lactation

Results are reported as the mean ± standard error of mean (SEM). GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analyses and graphics. For the temporal evolution data, each variable was first analyzed by two-way ANOVA with treatment (SO, CO and CO + C) and time (days of lactation for dams and days of life for offspring at 21 and 180 days) as the between-subject factors. If the initial analysis indicated treatment effects or interactions between treatment and time, then data were re-examined by one-way

During lactation, body mass (treatment: F1,107 = 2.53, treatment X time: F5,107 = 0.24; Fig. 1a), weight gain (Fig. 1b), cumulative intake (Fig. 1c) and visceral fat (Fig. 1d) were assessed in dams, and no differences were observed. Body mass (treatment: F1,780 = 2.79, treatment × time: F5,780 = 1.36; Fig. 1e) and weight gain (Fig. 1f) were also evaluated in pups, and no differences were observed between groups. Table 2 shows the biochemistry and hormone composition of breast

3. Results

Fig. 1. Nutritional status of dams and pups at the end of breastfeeding of soybean oil group (SO) and coconut oil group (CO). Body mass (a), weight gain (b) and cumulative food intake of dams (c) and body mass (d) and weight gain (e) of pups during lactation period. Data expressed as mean ± SEM; n = 10. 21

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Fig. 2. Nutritional status and body composition of offspring at 180 days old of soybean oil group (SO), coconut oil group (CO) and coconut oil + chow supplemented with coconut oil group. Body mass (a), weight gain (a-in box), food intake (b) and cumulative food intake (b-in box) during 180 days of life, visceral fat (c) and lean mass (d) content at 180 days of life. Data expressed as mean ± SEM; n = 10; £CO Vs CO + C; ¢CO Vs SO; °CO + C Vs SO; *Vs SO; #Vs CO; p < 0.05. Fig. 3. Evaluation of leptin content in plasma and white adipose tissue (WAT) of soybean oil group (SO), coconut oil group (CO) and coconut oil + chow supplemented with coconut oil group (CO + C) at 180 days of life. Plasma leptin concentration (a) and leptin content in WAT (b). Data expressed as mean ± SEM; n = 10; *Vs SO; #Vs CO; p < 0.05.

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Table 3 Biochemistry and hormonal profiles of soybean oil (SO), coconut oil (CO) and coconut oil + chow supplemented with coconut oil (CO + C) groups at 180 days of life.

180 days-old Plasma cholesterol (mmol/L) Plasma triglycerides (mmol/L) Hepatic cholesterol ((mmol/L)/mg) Hepatic triglycerides ((mmol/L)/mg) Blood glucose (mmol/L) Insulin (µIU/ml) Insulin resistance index (IRI) Corticosterone (ng/ml)

SO

CO

CO + C

1.45 ± 0.15 1.04 ± 0.16 0.05 ± 0.005 0.02 ± 0.001 4.99 ± 0.10 19.74 ± 2.78 98.66 ± 14.62 982.1 ± 148.0

1.44 ± 0.15 1.20 ± 0.16 0.05 ± 0.006 0.02 ± 0.002 5.15 ± 0.06 21.73 ± 2.49 116.30 ± 15.56 1094.0 ± 174.1

1.40 ± 0.13 0.83 ± 0.15 0.04 ± 0.003 0.02 ± 0.001 5.14 ± 0.09 17.11 ± 1.68 89.48 ± 9.90 810.5 ± 66.95

Results expressed as mean ± SEM; n = 40 per group to blood glucose analysis and n = 10 per group to the others analysis.

milk, plasma cholesterol and triglyceride levels of dams and plasma leptin in dams and pups at the end of lactation. No differences were observed in lactose and protein contents in breast milk. However, concerning the lipid profile, the CO group presented increased levels of cholesterol (+52%, p < 0.05; Table 2) and triglycerides (+94%, p < 0.05; Table 2) compared with the SO group. The milk of the CO group was more caloric than the milk of the SO group (+78%, p < 0.05; Table 2). No differences were observed in T3 levels between groups. Leptin levels were lower in the milk of the CO group when compared with the SO group (−45%, p < 0.05; Table 2). In relation to lipids in the plasma of the dams, it was observed that the cholesterol levels in the CO group were higher than in the SO group (+35%, p < 0.05; Table 2). Although the triglycerides levels tended to be higher in the CO group, the trend did not reach significance (+44%,

p = 0.06; Table 2). Plasma leptin was higher in the dams of the CO group (+76%, p < 0.05; Table 2), and no differences were observed in this parameter in pups. 3.2. Offspring at 180 days of age In relation to body mass, there was no interaction between treatment and time after weaning until 180 days of life (F26,1358 = 1.44; Fig. 2a). However, we observed a treatment effect with the CO and CO + C groups presenting higher body mass (+10% for both vs SO, p < 0.05, F2,1358 = 67.88; Fig. 2a) when compared to SO at 180 days of life. The total weight gain was calculated and reproduced the same profile (+11% CO vs SO and +13% CO vs SO, p < 0.05; Fig. 2ainbox).

Fig. 4. Evaluation of plasma levels of hormones involved with thyroid function of soybean oil group (SO), coconut oil group (CO) and coconut oil + chow supplemented with coconut oil group (CO + C) at 180 days of life. Plasma Free T4 (a), Total T3 (b) and TSH concentrations (c). Data expressed as mean ± SEM; n = 10; * Vs SO; #Vs CO; p < 0.05. 23

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Regarding food intake, we observed an interaction between treatment and time (treatment × time: F26,1358 = 5.61; Fig. 2b) and a treatment effect after weaning, with the CO group presenting hyperphagia (+8% approximately, after weaning, p < 0.05, F2,1358 = 812.20; Fig. 2b), while the CO + C group demonstrated hypophagia (−15% approximately, after weaning, p < 0.05, F2,1358 = 812.20; Fig. 2b) when compared to SO. Cumulative intake was assessed at 180 days and reproduced a similar profile (+7% CO vs SO; −16% CO + C vs SO; −21% CO + C vs CO, p < 0.05; Fig. 2binbox). At PN180, the CO group presented a higher visceral fat content in relation to the SO (+31%, p < 0.05) and CO + C (+40%, p < 0.05) groups (Fig. 2c) and a lower lean mass content in relation to the SO group (−13%, p < 0.05, Fig. 2d). At 180 days of life, the CO group had higher plasma leptin levels than the SO and CO + C groups (2.17 and 1.25-fold-increase, respectively, p < 0.05; Fig. 3a). The same profile was observed in leptin levels in retroperitoneal adipose tissue with the CO group presenting a higher content than the SO and CO + C groups (1.8 and 2.8-fold-increase, respectively, p < 0.05; Fig. 3b). At 180 days of life, no differences were found in blood glucose, plasma insulin, corticosterone, cholesterol, triglycerides, hepatic cholesterol or triglycerides among groups (Table 3). Regarding thyroid function, we observed lower plasma free T4 in

the CO + C group than the CO group (−26%, p < 0.05, Fig. 4a), higher plasma total T3 in the CO group than the SO group (+59%, p < 0.05, Fig. 4b) and higher plasma TSH levels in the CO group than the SO and CO + C groups (+10% and +15%, respectively; Fig. 4c). The TRβ1 content and mRNA expression of DIO1 in the liver (Fig. 5a and b, respectively) and DIO2 and UCP1 gene expression in BAT (Fig. 5c and d, respectively) were also analyzed, but no differences were observed among groups. 4. Discussion Here we demonstrated that maternal exposure to coconut oil during lactation alters the breast milk composition, including its fatty acid profile with high lauric acid content, and programs the adult offspring to develop important disorders, such as obesity, hyperphagia, hyperleptinemia and changes in plasma levels of thyroid hormones. Interestingly, our study shows that if coconut oil exposure persists throughout life, then the animals become better adapted and do not develop the metabolic changes found when this exposure occurs only during lactation, reinforcing the theory that metabolic programming is, in fact, an adaptive phenomenon, which depends on the changes in the initial conditions imprinted during the critical period (de Moura et al., 2008). At weaning, no differences were observed in the body mass of dams

Fig. 5. Evaluation of markers of thyroid function of soybean oil group (SO), coconut oil group (CO) and coconut oil + chow supplemented with coconut oil group (CO + C) at 180 days of life. Deiodinase 1 (DIO1) mRNA expression (a), thyroid hormone receptor beta (TRβ) content (b) in liver; deiodinase 2 (DIO2) (c) and uncoupling protein −1 (UCP-1) (d) mRNA expression in brown adipose tissue. Data expressed as mean ± SEM; n = 9 to mRNA expression analysis and n = 8 to protein content analysis; *Vs SO; p < 0.05. 24

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or pups or in the cumulative food intake of dams, despite the fact that the breast milk of the CO dams was more caloric due to the higher cholesterol and triglyceride contents. We suggest that the higher lipid content in the breast milk of the CO group promoted satiety in the pups of this group, leading to lower breast milk intake. Unfortunately, we did not measure breast milk intake. However, the weight gain in the CO group was similar to that in the SO group, even though the breast milk of CO group was more caloric. We hypothesized that the presence of MCSFAs, transferred to the pups through the breast milk, led to less consumption of milk in this group. In fact, MCSFAs were already associated with an increase in satiety in adult men, reinforcing this hypothesis (Stubbs & Harbron, 1996). This phenomenon was not observed in the dams that received coconut oil. These animals did not present an alteration in food intake. We suggest that this occurred because these dams were in a special phase, lactation, undergoing changes in metabolism, consequent to changes in the hypothalamic circuitry that regulate food intake and energy expenditure (Brogan, Mitchell, Trayhurn, & Smith, 1999; Chen, Williams, Grove, & Smith, 2004). Previous studies have demonstrated that a diet rich in MCSFAs has a cholesterol and triacylglycerol-increasing effect due to an increase in de novo synthesis of long chain fatty acids using the acetyl-CoA of the MCSFAs, after which these fatty acids enter the hepatic fatty acid pool and behave such as dietary long chain fatty acids (Cater, Heller, & Denke, 1997; Tholstrup et al., 2004). This mechanism can explain the increased cholesterol and triglycerides in the breast milk of the CO group. In fact, maternal plasma cholesterol and triglycerides were higher in the CO group than in the SO group and may have contributed to the increase in these fractions in breast milk by passing them from the blood circulation to the milk. Concerning the breast milk, it was observed that the dams of CO group presented lower leptin content in milk compared to the SO group, despite exhibiting a higher leptin plasma level. Since the leptin content in milk is derived from its production in mammary glands (Hassiotou, Savigni, Hartmann, & Geddes, 2014; Weyermann, Beermann, Brenner, & Rothenbacher, 2006) and from the passage of this hormone from the bloodstream to their breast milk, through diffusion or transport mediated by receptors in the mammary tissue (Casabiell et al., 1997; Laud, Gourdou, Bélair, Keisler, & Djiane, 1999), we suggest that the mammary production of leptin, its transport through receptors or both were reduced. However, we did not find data that associated coconut oil or MCSFA intake with these changes. The lower leptin content in the milk did not change leptin plasma concentration in pups at weaning. However, it was suggested that leptin may have direct effects on the gastrointestinal tract, modulating hormones and inflammation (Yarandi, Hebbar, Sauer, Cole, & Ziegler, 2011), and the lower leptin content found in maternal milk of CO group, can represent a possible imprinting factor. Previous studies demonstrated that oral leptin intake during lactation period prevents body weight gain and metabolic features of the metabolic syndrome, such as insulin resistance and glucose intolerance, at adulthood, besides a lower preference to high-fat diet (Picó et al., 2007; Priego, Sánchez, Palou, & Picó, 2010; Sánchez et al., 2008). On the other hand, a subcutaneous infusion of leptin in suckling pups lead to a higher body weight and food intake at adulthood (de Oliveira Cravo et al., 2002), as well as the use of a leptin antagonist (Attig et al., 2008; Beltrand, Sloboda, Connor, Truong, & Vickers, 2012) or leptin antibody (Trotta et al., 2011). Thus, these data show that leptin may play an important role during the earlier stages of life, such as lactation, and the lower leptin content in breast milk of CO dams may programmed the adult offspring to metabolic alterations observed in this study. In adulthood, it was observed that both the CO and CO + C groups presented a higher body mass than the SO group during almost their whole adult life. These findings corroborate a previous study by Magri et al. (2015) that showed that the adult mice offspring of dams fed diets containing interesterified fat or palm oil, rich in MCSFAs, during pregnancy and lactation presented a higher body mass and adiposity

than a control group fed a diet containing soybean oil. In addition, only the CO group presented higher visceral adiposity and lower lean mass content, suggesting an obesity phenotype in this group. Although the CO + C group presented a higher body mass in relation to the SO group, it did not present changes in body adiposity or lean mass content, suggesting that these animals experienced better growth than the other groups and had a better metabolic profile of body composition. In a similar way, Van de Heijning, Oosting, Kegler, and Van der Beek (2017) observed that a diet rich in MCSFA during early life and after weaning was able to prevent the fat accumulation in adipose tissue and high plasma leptin levels in mice that were submitted to a western diet. Some studies have demonstrated that a diet supplemented with virgin coconut oil promotes a reduction in lipogenesis and an enhanced rate of fatty acid catabolism and that these effects are mediated in part by peroxisome proliferator-activated receptor-α (PPARα) dependent pathways (Arunima & Rajamohan, 2014; Ippagunta, Angius, Sanda, & Barnes, 2013). This mechanism can partially explain the body fat changes observed in the CO + C group. It was observed that the CO group presented higher plasma levels of leptin than the SO group, and this result was accompanied by a higher leptin content in visceral adipose tissue. These findings were expected since the CO group animals were obese with high visceral adiposity, and adipose tissue is the main factor responsible for leptin synthesis and its plasma levels (Montague, Prins, Sanders, Digby, & O'Rahilly, 1997). In addition, it seems that when coconut oil is offered throughout the lifetime, these changes do not occur, maybe because coconut oil had a direct effect preventing higher adiposity and hyperleptinemia. The CO group animals presented hyperphagia throughout their entire lifetime. Since the CO animals were obese and hyperleptinemic, we suggest that this hyperphagia resulted from a central leptin resistance, with an impairment in the hypothalamic circuit of food intake control, as demonstrated in different experimental models of metabolic programming (Lima et al., 2011; Nobre et al., 2012; Rodrigues et al., 2011; Trevenzoli et al., 2010). Nevertheless, the CO + C group presented a lower food intake during its whole lifetime compared with the SO and CO groups, demonstrating a hypophagic profile. It has already been demonstrated by Stubbs and Harbron (1996) that a diet rich in MCSFAs provides higher satiety; however, this mechanism is still controversial, and the MCSFAs may cause this effect through increased lipid oxidation since a reduction in hepatic oxidation of fatty acids was able to induce food intake due to the reduced energy production by the liver (Friedman et al., 1999). As previously mentioned, the CO + C group had normalized body fat content and leptin in plasma and adipose tissue. These effects may have also contributed to the reduction in food intake due to the normalization of the suggested hypothalamic resistance to leptin. No differences were observed in plasma and hepatic cholesterol and triglycerides, blood glucose, insulin or corticosterone plasma levels. However, the CO group presented higher plasma TSH and T3 with unchanged T4. Maybe the unchanged T4 levels occur due to high peripheral deiodination, consequently higher T3 is due to both higher TSH and peripheral conversion from T4. However, the mRNA expression of liver DIO1 and BAT DIO2 were not changed, suggesting that this increase deiodination is occurring in other tissues, such as the thyroid and kidney. One limitation is that we did not measure deiodinase activities. Despite higher T3 levels in the CO group, the animals did not present thyroid hyperfunction since no differences were observed in the liver thyroid receptor (TRβ1) or in the BAT UCP1, which are two important markers of thyroid hormone action (Lazar & Chin, 1990; Lazar, 1993; Ribeiro et al., 2010). Here we only evaluated the total T3 that corresponds to free T3 and the hormone bound to transport proteins. It is possible that this increase in the plasma total T3 might be due to an increase in some transport protein, such as thyroid binding globulin (TBG), which would lead to a normal action of T3 on target tissues. Another explanation for the higher TSH in CO group is a tissue-specific thyroid hormone resistance. Thyroid hormones need transporters to 25

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pass through plasma membranes and carry out their transcriptional actions on the nuclei such as monocarboxylate transporters (MCT8 e MCT10), organic anion transporters (OATP1) and L-type amino acid transporters (LAT) (Bernal, Guadaño-Ferraz, & Morte, 2015; Hennemann et al., 2001). In this context, it is possible that the CO group had some impairment in this mechanism, at least in the pituitary gland, because circulating T4 did not correspond to increased TSH in these animals. Nevertheless, the CO + C animals did not present the alterations in thyroid metabolism, suggesting that the exposure to coconut oil during the whole lifetime prevented the thyroid dysfunctions caused by the coconut oil intake only during the lactation period. In summary, we observed that coconut oil supplementation only during the lactation period, a critical period of development, programmed the adult animals to be susceptible to some metabolic disorders, such as obesity, hyperphagia, hyperleptinemia and thyroid dysfunctions. However, it seems that the continuous exposure to this oil during the whole lifetime of the animals was able to prevent most of these disorders. These data reinforce the concept that metabolic programming causes changes in physiological processes, and if an individual persists with the environmental conditions of its early life, then the individual might be better adapted. However, if the conditions are changed, then there is a greater risk of metabolic dysfunctions. In addition, more research is necessary to clarify the molecular mechanisms involved in the alterations caused by coconut oil supplementation during lactation as well as the mechanisms involved in the prevention of these changes when coconut oil is offered throughout the whole lifetime of the animals.

Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2019.02.020. References Arunima, S., & Rajamohan, T. (2014). Influence of virgin coconut oil-enriched diet on the transcriptional regulation of fatty acid synthesis and oxidation in rats – A comparative study. The British Journal of Nutrition, 111(10), 1782–1790. Assunção, M. L., Ferreira, H. S., dos Santos, A. F., Cabral, C. R., Jr., & Florêncio, T. M. (2009). Effects of dietary coconut oil on the biochemical and anthropometric profiles of women presenting abdominal obesity. Lipids, 44(7), 593–601. Attig, L., Djiane, J., Gertler, A., Rampin, O., Larcher, T., Boukthir, S., ... Abdennebi-Najar, L. (2008). Study of hypothalamic leptin receptor expression in low-birth-weight piglets and effects of leptin supplementation on neonatal growth and development. American Journal of Physiology, 295(5), E1117–E1125. Baba, N., Bracco, E. F., & Hashim, S. A. (1987). Role of brown adipose tissue in thermogenesis induced by overfeeding a diet containing medium chain triglyceride. Lipids, 22(6), 442–444. Babayan, V. K. (1987). Medium chain triglycerides and structured lipids. Lipids, 22(6), 417–420. Barker, D. J. (1995). The fetal and infant origins of disease. European Journal of Clinical Investigation, 25(7), 457–463. Beltrand, J., Sloboda, D. M., Connor, K. L., Truong, M., & Vickers, M. H. (2012). The effect of neonatal leptin antagonism in male rat offspring is dependent upon the interaction between prior maternal nutritional status and post-weaning diet. Journal of Nutrition and Metabolism, 2012, 296935. https://doi.org/10.1155/2012/296935. Bernal, J., Guadaño-Ferraz, A., & Morte, B. (2015). Thyroid hormone transporters functions and clinical implications. Nature Reviews- Endocrinology, 11(7), 406–417. Bonomo, I. T., Lisboa, P. C., Passos, M. C., Pazos-Moura, C. C., Reis, A. M., & Moura, E. G. (2005). Prolactin inhibition in lactating rats changes leptin transfer through the milk. Hormome and Metabolic Research, 37, 220–225. Brogan, R. S., Mitchell, S. E., Trayhurn, P., & Smith, M. S. (1999). Suppression of leptin during lactation: Contribution of the suckling stimulus versus milk production. Endocrinology, 140(6), 2621–2627. Cardoso, D. A., Moreira, A. S., de Oliveira, G. M., Raggio Luiz, R., & Rosa, G. (2015). A coconut extra virgin oil-rich diet increases HDL cholesterol and decreases waist circumference and body mass in coronary artery disease patients. Nutricion Hospitalaria, 32(5), 2144–2152. Casabiell, X., Piñeiro, V., Tomé, M. A., Peinó, R., Diéguez, C., & Casanueva, F. F. (1997). Presence of leptin in colostrum and/or breast milk from lactating mothers: A potential role in the regulation of neonatal food intake. The Journal of Clinical Endocrinology and Metabolism, 82(12), 4270–4273. Cater, N. B., Heller, H. J., & Denke, M. A. (1997). Comparison of the effects of mediumchain triacylglycerols, palm oil, and high oleic acid sunflower oil on plasma triacylglycerol fatty acids and lipid and lipoprotein concentrations in humans. American Journal of Clinical Nutrition, 65(1), 41–45. Champ, M., & Hoebler, C. (2009). Functional food for pregnant, lactating women and in perinatal nutrition: A role for dietary fibres? Current Opinion in Clinical Nutrition and Metabolic Care, 12(6), 565–574. Chen, P., Williams, S. M., Grove, K. L., & Smith, M. S. (2004). Melanocortin 4 receptormediated hyperphagia and activation of neuropeptide Y expression in the dorsomedial hypothalamus during lactation. The Journal of Neuroscience, 24(22), 5091–5100. Choudhary, M., & Grover, K. (2012). Development of functional food products in relation to obesity. Functional Foods in Health and Disease, 2(6), 188–197. Christie, W. W. (1989). The analyses of fatty acids. In W. W. Christie (Ed.). Gas chromatography and lipids (pp. 36–47). Bridgwater: The Oily Press. Costa, C. A., Carlos, A. S., dos Santos, A., De, S., Monteiro, A. M., Moura, E. G., & Nascimento-Saba, C. C. (2011). Abdominal adiposity, insulin and bone quality in young male rats fed a high-fat diet containing soybean or canola oil. Clinics (Sao Paulo), 66(10), 1811–1816. de Moura, E. G., Lisboa, P. C., & Passos, M. C. (2008). Neonatal programming of neuroimmunomodulation role of adipocytokines and neuropeptides. Neuroimmunomodulation, 15(3), 176–188. de Oliveira Cravo, C., Teixeira, C. V., Passos, M. C., Dutra, S. C., de Moura, E. G., & Ramos, C. (2002). Leptin treatment during the neonatal period is associated with higher food intake and adult body weight in rats. Hormone and Metabolic Research, 34(7), 400–405. Dias, M. M., Siqueira, N. P., da Conceição, L. L., Reis, S. A., Valente, F. X., Dias, M. M. S., ... Peluzio, M. C. G. (2018). Consumption of virgin coconut oil in Wistar rats increases saturated fatty acids in the liver and adipose tissue, as well as adipose tissue inflammation. Journal of Functional Foods, 48, 472–480. Fernandes, F. S., Sardinha, F. L., Badia-Villanueva, M., Carulla, P., Herrera, E., & Tavares do Carmo, M. G. (2012). Dietary lipids during early pregnancy differently influence adipose tissue metabolism and fatty acid composition in pregnant rats with repercussions on pup's development. Prostaglandins Leukot Essent Fatty Acids, 86(4–5), 167–174. Friedman, M. I., Harris, R. B., Ji, H., Ramirez, I., & Tordoff, M. G. (1999). Fatty acid oxidation affects food intake by altering hepatic energy status. The American Journal of Physiology, 276(4), R1046–R1053. Gluckman, P., & Hanson, M. (2008). Mismatch: The lifestyle diseases timebomb (1st ed.). Oxford: Oxford University Press. Guarda, D. S., de Moura, E. G., Carvalho, J. C., Reis, A. M., Soares, P. N., Lisboa, P. C., &

Ethics statements Our protocol was approved by the Animal Care and Use Committee of the Biology Institute of the State University of Rio de Janeiro (CEUA/ 001/2014). Experiments was carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals. Author contributions Conception and design: F.T.Q., E.O., P.C.L., E.G.M. Collection, analysis and interpretation of data: F.T.Q. Fatty acid profile (GC-MS): G.C.A. Drafting and/or revising the article critically for important intellectual content: E.O., P.C.L., E.G.M., F.T.Q. Acknowledgements All the authors are grateful to Miss Monica Moura, Mrs. Fabiana Gallaulckydio, Mr Ulisses Risso Siqueira and Mrs. Mileane Busch for technical assistance. Funding This research was supported by the ‘National Council for Scientific and Technological Development’ (Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq) and the ‘Carlos Chagas Filho Research Foundation of the State of Rio de Janeiro’ (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro-FAPERJ). Conflict of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. 26

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F.T. Quitete, et al.

through the maternal diet during lactation. Journal of Nutritional Science, 4(e19), 1–10. Passos, M. C. F., Ramos, C. F., & Moura, E. G. (2000). Short and long term effects of malnutrition in rats during lactation on the body weight of offspring. Nutrition Research, 20(11), 1603–1610. Peterson, G. L. A. (1977). A simplification of the protein assay method of Lowry et al. which is more generally applicable. Analytical Biochemistry, 83(2), 346–356. Picó, C., Oliver, P., Sánchez, J., Miralles, O., Caimari, A., Priego, T., & Palou, A. (2007). The intake of physiological doses of leptin during lactation in rats prevents obesity in later life. International Journal of Obesity (Lond), 31(8), 1199–1209. Priego, T., Sánchez, J., Palou, A., & Picó, C. (2010). Leptin intake during the suckling period improves the metabolic response of adipose tissue to a high-fat diet. International Journal of Obesity (Lond), 34(5), 809–819. Quitete, F. T., Lisboa, P. C., Moura, E. G., & de Oliveira, E. (2018). Different oils used as supplement during lactation causes endocrine-metabolic dysfunctions in male rats. Journal of Functional Foods, 48, 43–53. Reeves, P. G., Nielsen, F. H., & Fahey, G. C., Jr. (1993). AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123(11), 1939–1951. Ribeiro, M. O., Bianco, S. D., Kaneshige, M., Schultz, J. J., Cheng, S. Y., Bianco, A. C., & Brent, G. A. (2010). Expression of uncoupling protein 1 in mouse brown adipose tissue is thyroid hormone receptor-beta isoform specific and required for adaptive thermogenesis. Endocrinology, 151(1), 432–440. Rodrigues, A. L., de Moura, E. G., Passos, M. C., Trevenzoli, I. H., da Conceição, E. P., Bonono, I. T., ... Lisboa, P. C. (2011). Postnatal early overfeeding induces hypothalamic higher SOCS3 expression and lower STAT3 activity in adult rats. Journal of Nutritional Biochemistry, 22(2), 109–117. Sánchez, J., Priego, T., Palou, M., Tobaruela, A., Palou, A., & Picó, C. (2008). Oral supplementation with physiological doses of leptin during lactation in rats improves insulinsensitivity and affects food preferences later in life. Endocrinology, 149(2), 733–740. Stauffer, C. E. (1975). A linear standard curve for the Folin Lowry determination of protein. Analytical Biochemistry, 69(2), 646–648. St-onge, M. P., & Jones, P. J. (2002). Physiological effects of medium-chain triglycerides: Potential agents in the prevention of obesity. Journal of Nutrition, 132(3), 329–332. Stubbs, R. J., & Harbron, C. G. (1996). Covert manipulation of the ratio of medium to long-chain triglycerides in isoenergetically dense diets: Effect on food intake in ad libitum feeding men. International Journal of Obesity and Related Metabolic Disorders, 20(5), 435–444. Tholstrup, T., Ehnholm, C., Jauhiainen, M., Petersen, M., Høy, C. E., Lund, P., & Sandström, B. (2004). Effects of medium-chain fatty acids and oleic acid on blood lipids, lipoproteins, glucose, insulin, and lipid transfer protein activities. American Journal of Clinical Nutrition, 79(4), 564–569. Trevenzoli, I. H., Rodrigues, A. L., Oliveira, E., Thole, A. A., Carvalho, L., Figueiredo, M. S., ... Moura, E. G. (2010). Leptin treatment during lactation programs leptin synthesis, intermediate metabolism, and liver microsteatosis in adult rats. Hormone and Metabolic Research, 42(7), 483–490. Trotta, P. A., Moura, E. G., Franco, J. G., Lima, N. S., de Oliveira, E., Cordeiro, A., ... Passos, M. C. (2011). Blocking leptin action one week after weaning reverts most of the programming caused by neonatal hyperleptinemia in the adult rat. Hormone and Metabolic Research, 43(3), 171–177. Van de Heijning, B. J. M., Oosting, A., Kegler, D., & Van der Beek, E. M. (2017). An increased dietary supply of medium-chain fatty acids during early weaning in rodents prevents excessive fat accumulation in adulthood. Nutrients, 9(631), 1–16. Weyermann, M., Beermann, C., Brenner, H., & Rothenbacher, D. (2006). Adiponectin and leptin in maternal serum, cord blood, and breast milk. Clinical Chemistry, 52(11), 2095–2102. Yarandi, S. S., Hebbar, G., Sauer, C. G., Cole, C. R., & Ziegler, T. R. (2011). Diverse roles of leptin in the gastrointestinal tract: Modulation of motility, absorption, growth, and inflammation. Nutrition, 27(3), 269–275.

Figueiredo, M. S. (2016). Maternal flaxseed oil intake during lactation changes body fat, inflammatory markers and glucose homeostasis in the adultprogeny: Role of gender dimorphism. The Journal of Nutritional Biochemistry, 35, 74–80. Hassiotou, F., Savigni, D., Hartmann, P., & Geddes, D. (2014). Mammary cells synthesize appetite hormones that may contribute to breast milk. The FASEB Journal, 28(1). Hennemann, G., Docter, R., Friesema, E. C., de Jong, M., Krenning, E. P., & Visser, T. J. (2001). Plasma membrane transport of thyroid hormones and its role in thyroid hormone metabolism and bioavailability. Endocrine Reviews, 22(4), 451–476. Ippagunta, S., Angius, Z., Sanda, M., & Barnes, K. M. (2013). Dietary CLA-induced lipolysis is delayed in soy oil-fed mice compared to coconut oil-fed mice. Lipids, 48(11), 1145–1155. Khramov, V. A., Kolomeitseva, A. S., & Papichev, N. V. (2008). Jaffe color test-based microtechnique for determination of milk lactose. Gigiena i Sanitariia, 3, 86–87. Laud, K., Gourdou, I., Bélair, L., Keisler, D. H., & Djiane, J. (1999). Detection and regulation of leptin receptor mRNA in ovine mammary epithelial cells during pregnancy and lactation. FEBS Letters, 463(1–2), 194–198. Lazar, M. A. (1993). Thyroid hormone receptors: Multiple forms, multiple possibilities. Endocrine Reviews, 14(2), 184–193. Lazar, M. A., & Chin, W. W. (1990). Nuclear thyroid hormone receptors. The Journal of Clinical Investigation, 86(6), 1777–1782. Lima, N., Da, S., de Moura, E. G., Passos, M. C., Nogueira Neto, F. J., Reis, A. M., de Oliveira, E., & Lisboa, P. C. (2011). Early weaning causes undernutrition for a short period and programmes some metabolic syndrome components and leptin resistance in adult rat offspring. British Journal of Nutrition, 105(9), 1405–1413. Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25(4), 402–408. Magri, T. P., Fernandes, F. S., Souza, A. S., Langhi, L. G., Barboza, T., Misan, V., ... Tavares do Carmo, Md. (2015). Interesterified fat or palm oil as substitutes for partially hydrogenated fat in maternal diet can predispose obesity in adult male offspring. Clinical Nutrition, 34(5), 904–910. Marina, A. M., Man, Y. B., Nazimah, S. A., & Amin, I. (2009). Antioxidant capacity and phenolic acids of virgin coconut oil. International Journal of Food Sciences and Nutrition, 60(2), 114–123. Marten, B., Pfeuffer, M., & Schrezenmeir, J. (2006). Medium-chain triglycerides. International Dairy Journal, 16(11), 1374–1382. Misan, V., Estato, V., de Velasco, P. C., Spreafico, F. B., Magri, T., dos Santos, R. M., ... Tavares do Carmo, Md. (2015). Interesterified fat or palm oil as substitutes for partially hydrogenated fat during the perinatal period produces changes in the brain fatty acids profile and increases leukocyte-endothelial interactions in the cerebral microcirculation from the male offspring in adult life. Brain Research, 1616, 123–133. Montague, C. T., Prins, J. B., Sanders, L., Digby, J. E., & O'Rahilly, S. (1997). Depot- and sex-specific differences in human leptin mRNA expression: Implications for the control of regional fat distribution. Diabetes, 46(3), 342–347. Moura, E. G., & Passos, M. C. F. (2005). Neonatal programming of body weight regulation and energetic metabolism. Bioscience Reports, 25(3–4), 251–269. Mumme, K., & Stonehouse, W. (2015). Effects of medium-chain triglycerides on weight loss and body composition: A meta-analysis of randomized controlled trials. Journal of the Academy of Nutrition and Dietetics, 115(2), 249–263. Nagao, K., & Yanagita, T. (2010). Medium-chain fatty acids: Functional lipids for the prevention and treatment of the metabolic syndrome. Pharmacological Research, 61(3), 208–212. Nobre, J. L., Lisboa, P. C., Lima, N., Da, S., Franco, J. G., Nogueira Neto, J. F., de Moura, E. G., & de Oliveira, E. (2012). Calcium supplementation prevents obesity, hyperleptinaemia and hyperglycaemia in adult rats programmed by early weaning. British Journal of Nutrition, 107(7), 979–988. Oosting, A., Kegler, D., Boehm, G., Jansen, H. T., van de Heijning, B. J., & van der Beek, E. M. (2010). N-3 long-chain polyunsaturated fatty acids prevent excessive fat deposition in adulthood in a mouse model of postnatal nutritional programming. Pediatric Research, 68(6), 494–499. Oosting, A., Verkade, H. J., Kegler, D., van de Heijning, B. J., & van der Beek, E. M. (2015). Rapid and selective manipulation of milk fatty acid composition in mice

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