Journal of Functional Foods 55 (2019) 17–27

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Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Differential effects in male adult rats of lifelong coconut oil exposure versus T
during early-life only
Fernanda Torres Quitetea, Egberto Gaspar de Mouraa, Geórgia Correa Atellab,
Patricia Cristina Lisboaa, Elaine de Oliveiraa,
⁎

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

ARTICLE INFO ABSTRACT

Keywords: We investigated the effects of maternal coconut oil supplementation during breastfeeding on the endocrine-
Coconut oil metabolic profiles of offspring and the impact of continued exposure throughout life. Rat mothers were sepa-
Metabolic programming rated into: soybean oil (SO); and coconut oil (CO) groups and received the oils through gavage (0.5 g/kg of
Obesity BW) and had free access to standard chow. After weaning, half of the pups from CO group continued receiving
Dietary oils
coconut oil in chow (CO + C), while SO and the other half of CO group received standard chow. Offspring were
Functional foods
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, hy-
perleptinemia 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 preg-
nancy, 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 con-
sidered 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 preven-
tion 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 con-
tains 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: elainedeoliveir@pq.cnpq.br, elainedeoliveir@yahoo.com.br (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
Available online 16 February 2019
1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
F.T. Quitete, et al. Journal of Functional Foods 55 (2019) 17–27

lactation may have a long-term impact, especially when these changes Table 1
occur only during this period, promoting the metabolic programming Macronutrients and micronutrients composition of standard chow for rats and
phenomenon (Moura & Passos, 2005). Metabolic programming, or on- coconut oil supplemented chow.
togenetic plasticity, occurs due to changes in the physiological/en- Standard chowa Coconut oil chow
vironmental pattern (imprinting factor) early in life. This process could
have consequences in adult life, causing adaptive changes in physio- Carbohydrate (g/kg) 660.0 570.0
Corn starch (g/kg) – 363.3
logical processes, since the animals persist with the environmental
% kJ 68.0 63.6
conditions of early life. However, if the conditions change, then there is
Protein (g/kg) 220.0 224.0
a higher risk of disease (Barker, 1995; de Moura, Lisboa, & Passos,
Textured soy (g/kg) – 303.0
2008; Moura & Passos, 2005). To our knowledge, there are no reports in % kJ 22.7 25.0
which the altered conditions during breastfeeding persist in one of the
Fat (g/kg) 40.0 45.0
experimental groups until adulthood, since all previous papers changed Coconut oil (ml/kg) – 30.3
the imprinting factor after weaning. According to the mismatch theory % kJ 9.2 11.4
proposed by Gluckman and Hanson (2008), if the initial conditions are Vitamins and Minerals
maintained, then the animal could be better adapted to the environ- Mineral mixb (g/kg) – 9.6
ment during development. Vitamin mixb (g/kg) – 1.6
In the literature, the long-term effects of different types of oils, such Ca (g/kg) 10.0–14.0 –
P (mg/kg) 8000.0 –
as fish oil, soybean oil, flaxseed oil, canola oil and palm oil used in
Na (mg/kg) 2700.0 –
critical periods of life (pregnancy and lactation) have been studied Fe (mg/kg) 50.0 –
(Costa et al., 2011; Fernandes et al., 2012; Guarda et al., 2016; Magri Mn (mg/kg) 60.0 –
et al., 2015; Misan et al., 2015; Oosting et al., 2010; Quitete, Lisboa, Zn (mg/kg) 60.0 –
Moura, & de Oliveira, 2018). Nevertheless, the use of oils during critical Cu (mg/kg) 10.0 –
I (mg/kg) 2.0 –
stages of development can provide a better understanding of the effects Se (mg/kg) 0.05 –
of the different oils that are commonly used today because they could Co (mg/kg) 1.5 –
alter the endocrine-metabolic parameters of dams and breast milk F (mg/kg) 80.0 –
composition, as well as cause dysfunction in the offspring in the short Vit A (UI/kg) 13000.0 –
Vit D3 (UI/kg) 2000.0 –
and long term (Costa et al., 2011; Fernandes et al., 2012; Guarda et al.,
Vit E (UI/kg) 34.0 –
2016; Magri et al., 2015; Misan et al., 2015; Oosting et al., 2010; Vit K3 (mg/kg) 3.0 –
Oosting, Verkade, Kegler, van de Heijning, & van der Beek, 2015; Vit B1 (mg/kg) 5.0 –
Quitete et al., 2018). Vit B2 (mg/kg) 6.0 –
Since coconut oil is being heavily prescribed by health professionals Vit B3 (mg/kg) 60.0 –
Vit B5 (mg/kg) 20.0 –
and widely used by the general population, even in critical periods of
Vit B6 (mg/kg) 7.0 –
life with no scientific evidence of its effects during these important Vit B7 mg/kg) 0.05 –
phases, it is very important to understand the effects of coconut oil Vit B9 (mg/kg) 1.0 –
supplementation during the critical lactation period. In this context, the Vit B12 (mcg/kg) 22.0 –
Choline (mg/kg) 1900.0 –
present study aims to assess the nutritional status of the dams that re-
ceived coconut oil supplementation and their pups during lactation, as Aminoacids
well as breast milk composition at the end of lactation and endocrine- Lysine (mg/kg) 12000.0 50.0
L-cystine (mg/kg) – 875.0
metabolic parameters of the adult offspring. In addition, this study aims Methionine (mg/kg) 4000.0 150.0
to evaluate whether continuous exposure to coconut oil from weaning
BHT (mg/kg) 100.0 300.0
until 180 days of life promotes different patterns compared to the off-
spring that received coconut oil supplementation only during breast- a
Standard chow to rats (Nuvilab-NUVITAL Nutrientes LTDA, Paraná, Brazil).
feeding. Based on the metabolic programming theory, we hypothesize Composition of diet: Whole corn, soybean bran, wheat bran, calcium carbonate,
that the programming effects of coconut oil exposure only during lac- dicalcium phosphate, sodium chloride, Vitamin A, Vitamin D3, Vitamin E,
tation has a more marked effect than continuous exposure on obesity Vitamin K3, Vitamin B1, Vitamin B2, Vitamin B6, Vitamin B12, niacin, calcium
development. pantothenate, folic acid, biotin, choline chloride, iron sulfate, manganese sul-
fate, zinc sulfate, copper sulfate, calcium iodate, sodium selenite, cobalt sulfate,
2. Materials and methods 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 re-
The experimental design was approved by the Animal Care and Use
commended amount of iodide (Reeves et al., 1993).
Committee of the Biology Institute of the State University of Rio de
Janeiro (CEUA/001/2014). Wistar rats were kept in a temperature-
controlled room (25 ± 1 °C) with an artificial light-dark cycle (lights 215898, Lot #G3014) (0.5 g of oil/kg of body weight) via intragastric
on at 0700 h, lights off at 1900 h). Virgin female rats (n = 30) that were gavage throughout the lactation period (21 days); CO (coconut oil
3 months old were caged with male rats, in a proportion of 2 females to group, n = 10), dams that received extra virgin coconut oil (Santa Cruz
1 male. After mating, each female rat was placed in an individual cage Biotechnology, Inc., TX, USA; sc-214752A, Lot #K0614) (0.5 g of oil/kg
with free access to water and food until delivery. At birth, litters were of body weight) via intragastric gavage throughout the lactation period;
adjusted to 6 male pups per dam to maximize the lactation performance and CO + C (coconut oil + standard chow supplemented with coconut
(Passos, Ramos, & Moura, 2000). 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
2.1. Experimental model period, and after the weaning, their offspring received chow supple-
mented with extra virgin coconut oil for their whole life (180 days). The
At birth, dams and their offspring were randomly divided into the dams of the three groups during lactation and the offspring of the SO
following three groups: SO (soybean oil group, n = 10), dams that re- and CO groups for the duration of their lives had free access to standard
ceived soybean oil (Santa Cruz Biotechnology, Inc., TX, USA; sc- chow for rodents (Table 1).

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F.T. Quitete, et al. Journal of Functional Foods 55 (2019) 17–27

Concerning the posterior analysis, only one randomly selected an-
imal per litter was used, except for the body weight and food intake
data, for which we used all animals of the litter.
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 macro-
nutrients without changing the sources of protein and carbohydrates,
thereby maintaining the chow with a normocaloric, normoproteic,
normoglicidic and normolipidic profile. Water was added to homo-
genize 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 off-
spring 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 spe-
cific 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.
2.4. Milk collection
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 quad-
ruplicate 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 chro-
matograph. Electro ionization (EI-70 eV) and a quadruple mass ana-
lyzer, 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 con-
tained 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 de-
monstrated in Table 1 of Supporting Information and the fatty acid
profiles of standard chow and coconut oil supplemented chow are de-
monstrated in Table 2 of Supporting Information.
2.7. Hormone determination in breast milk
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 Table 2
et al., 2005). After a subcutaneous injection of oxytocin (5 IU) with a Macronutrients composition, energy, T3 and leptin levels in breast milk, plasma
non-lethal dose of a ketamine/xylazine mixture, milk was manually cholesterol and triglycerides content of dams and plasma leptin of dams and
collected from all teats. We obtained 0.5–1.0 mL from each lactating offspring of soybean oil (SO) and coconut oil (CO) groups at the end of lacta-
rat, and the samples were frozen at −20 °C for further analysis. tion.
SO CO
2.5. Analysis of milk biochemical composition
Breast Milk
Cholesterol (mg/ml) 3.67 ± 0.36 5.59 ± 0.54*
Total milk protein was measured according to the Peterson method
Triglycerides (mg/ml) 36.4 ± 4.06 70.79 ± 7.80*
(Peterson, 1977) using bovine serum albumin as the standard. Protein Lactose (mg/ml) 36.84 ± 3.88 33.81 ± 2.78
concentration was determined based on the Stauffer formula (Stauffer, Total protein (mg/ml) 64.52 ± 5.84 71.11 ± 2.31
1975), and the results were expressed in mg/ml. The cholesterol and Total calories (kJ/ml) 2.95 ± 0.29 5.27 ± 0.75*
triglyceride contents were measured in milk samples by colorimetric T3 (ng/ml) 1.42 ± 0.16 1.84 ± 0.21
Leptin (ng/ml) 2.10 ± 0.12 1.17 ± 0.10*
assay, using a Bioclin commercial kit. The milk samples were diluted in
distilled water (1:25) for triglyceride analysis. The results were ex- Plasma - Dams
Cholesterol (mmol/L) 1.61 ± 0.06 2.17 ± 0.13*
pressed in mg/dl. Milk lactose was measured by a colorimetric method
Triglycerides (mmol/L) 0.99 ± 0.07 1.43 ± 0.17
using picric acid (Khramov, Kolomeitseva, & Papichev, 2008), using Leptin (ng/ml) 0.32 ± 0.07 0.56 ± 0.08*
commercial lactose as the standard (Sigma-Aldrich Co, St. Louis, MO,
Plasma – Pups 21 days old
USA). The results were expressed in mg/ml. The total calories of the Leptin (ng/ml) 1.21 ± 0.15 1.30 ± 0.20
milk were calculated using the sum of calories from each isolated
macronutrient. * p < 0.05. Results expressed as mean ± SEM; n = 10.

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F.T. Quitete, et al.
2.8. Blood glucose analysis
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 in-
terest were excised and stored at −80 °C and were subsequently pro-
cessed according to the methods described below.
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 com-
mercial 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 com-
mercial 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.
2.12. 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 mM-
EDTA, and Triton X-100 1%) that included a protease inhibitor cocktail
(cOmplete EDTA-free - Roche Applied Science, Mannheim, Germany).
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Journal of Functional Foods 55 (2019) 17–27
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.
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% bro-
mophenol blue) and heated at 95 °C for 5 min. Homogenates were
analyzed by the SDS-PAGE method. To detect proteins, a 12% poly-
acrylamide 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 (Sigma-
Aldrich, 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 di-
lution 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).
2.15. 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 deiodi-
nase 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
F.T. Quitete, et al. Journal of Functional Foods 55 (2019) 17–27

control, was performed in all of the samples. The results were nor-
malized 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 post-
test. Differences were considered significant when p < 0.05.
3. Results

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 treat-
ment (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), cumu-
lative 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

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.

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F.T. Quitete, et al. Journal of Functional Foods 55 (2019) 17–27

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.

22
F.T. Quitete, et al. Journal of Functional Foods 55 (2019) 17–27

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.
SO CO CO + C

180 days-old
Plasma cholesterol (mmol/L) 1.45 ± 0.15 1.44 ± 0.15 1.40 ± 0.13
Plasma triglycerides (mmol/L) 1.04 ± 0.16 1.20 ± 0.16 0.83 ± 0.15
Hepatic cholesterol ((mmol/L)/mg) 0.05 ± 0.005 0.05 ± 0.006 0.04 ± 0.003
Hepatic triglycerides ((mmol/L)/mg) 0.02 ± 0.001 0.02 ± 0.002 0.02 ± 0.001
Blood glucose (mmol/L) 4.99 ± 0.10 5.15 ± 0.06 5.14 ± 0.09
Insulin (µIU/ml) 19.74 ± 2.78 21.73 ± 2.49 17.11 ± 1.68
Insulin resistance index (IRI) 98.66 ± 14.62 116.30 ± 15.56 89.48 ± 9.90
Corticosterone (ng/ml) 982.1 ± 148.0 1094.0 ± 174.1 810.5 ± 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 treat-
ment 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. 2a-
inbox).

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 sup-
plemented 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
F.T. Quitete, et al. Journal of Functional Foods 55 (2019) 17–27

Regarding food intake, we observed an interaction between treat- the CO + C group than the CO group (−26%, p < 0.05, Fig. 4a),
ment and time (treatment × time: F26,1358 = 5.61; Fig. 2b) and a higher plasma total T3 in the CO group than the SO group (+59%,
treatment effect after weaning, with the CO group presenting hyper- p < 0.05, Fig. 4b) and higher plasma TSH levels in the CO group than
phagia (+8% approximately, after weaning, p < 0.05, the SO and CO + C groups (+10% and +15%, respectively; Fig. 4c).
F2,1358 = 812.20; Fig. 2b), while the CO + C group demonstrated hy- The TRβ1 content and mRNA expression of DIO1 in the liver (Fig. 5a
pophagia (−15% approximately, after weaning, p < 0.05, and b, respectively) and DIO2 and UCP1 gene expression in BAT
F2,1358 = 812.20; Fig. 2b) when compared to SO. Cumulative intake (Fig. 5c and d, respectively) were also analyzed, but no differences were
was assessed at 180 days and reproduced a similar profile (+7% CO vs observed among groups.
SO; −16% CO + C vs SO; −21% CO + C vs CO, p < 0.05; Fig. 2b-
inbox).
4. Discussion
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)
Here we demonstrated that maternal exposure to coconut oil during
groups (Fig. 2c) and a lower lean mass content in relation to the SO
lactation alters the breast milk composition, including its fatty acid
group (−13%, p < 0.05, Fig. 2d).
profile with high lauric acid content, and programs the adult offspring
At 180 days of life, the CO group had higher plasma leptin levels
to develop important disorders, such as obesity, hyperphagia, hy-
than the SO and CO + C groups (2.17 and 1.25-fold-increase, respec-
perleptinemia and changes in plasma levels of thyroid hormones.
tively, p < 0.05; Fig. 3a). The same profile was observed in leptin le-
Interestingly, our study shows that if coconut oil exposure persists
vels in retroperitoneal adipose tissue with the CO group presenting a
throughout life, then the animals become better adapted and do not
higher content than the SO and CO + C groups (1.8 and 2.8-fold-in-
develop the metabolic changes found when this exposure occurs only
crease, respectively, p < 0.05; Fig. 3b).
during lactation, reinforcing the theory that metabolic programming is,
At 180 days of life, no differences were found in blood glucose,
in fact, an adaptive phenomenon, which depends on the changes in the
plasma insulin, corticosterone, cholesterol, triglycerides, hepatic cho-
initial conditions imprinted during the critical period (de Moura et al.,
lesterol or triglycerides among groups (Table 3).
2008).
Regarding thyroid function, we observed lower plasma free T4 in
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
F.T. Quitete, et al.
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 as-
sociated with an increase in satiety in adult men, reinforcing this hy-
pothesis (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 meta-
bolism, consequent to changes in the hypothalamic circuitry that reg-
ulate 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 medi-
ated by receptors in the mammary tissue (Casabiell et al., 1997; Laud,
Gourdou, Bélair, Keisler, & Djiane, 1999), we suggest that the mam-
mary 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. How-
ever, it was suggested that leptin may have direct effects on the gas-
trointestinal 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 intol-
erance, 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 pro-
grammed 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
25
Journal of Functional Foods 55 (2019) 17–27
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 re-
sistance, 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 pre-
sented 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 con-
troversial, 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 adi-
pose 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 per-
ipheral 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 in-
crease 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 corre-
sponds 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
F.T. Quitete, et al.
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 co-
conut 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, pro-
grammed the adult animals to be susceptible to some metabolic dis-
orders, 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 pro-
gramming causes changes in physiological processes, and if an in-
dividual 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 ad-
dition, 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.
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 in-
tellectual 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
Journal of Functional Foods 55 (2019) 17–27
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jff.2019.02.020.
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