J Physiol 590.

21 (2012) pp 5503–5518 5503

Maternal high-fat diet induces obesity and adrenal and

thyroid dysfunction in male rat offspring at weaning
J. G. Franco1 , T. P. Fernandes2 , C. P. D. Rocha2 , C. Calviño2 , C. C. Pazos-Moura2 , P. C. Lisboa1 ,
E. G. Moura1 and I. H. Trevenzoli1,2
1
Laboratory of Endocrine Physiology, Department of Physiological Sciences, Biology Institute, State University of Rio de Janeiro, Brazil
2
Laboratory of Molecular Endocrinology, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Brazil

Key points
• Perinatal maternal high-fat diet changes milk composition, resulting in obesity and hyper-
glycaemia in male offspring at weaning.
• Offspring obesity is associated with hyperleptinaemia and changes in the central leptin
The Journal of Physiology

signalling pathway in the hypothalamic arcuate nucleus.

• Maternal high-fat diet increased adrenal catecholamines in offspring but reduced liver and
adipose tissue adrenoreceptors, thereby contributing to increased adiposity in these animals.
• Early obesity and hyperleptinaemia in offspring may have a stimulatory effect on the
hypothalamus–pituitary–thyroid axis as an adaptive response to the positive energy balance.
• Both catecholamines and thyroid hormones may impact cardiovascular function, thereby
contributing to the development of hypertension.

Abstract Maternal nutritional status affects the future development of offspring. Both under-
nutrition and overnutrition in critical periods of life (gestation or lactation) may cause several
hormonal changes in the pups and programme obesity in the adult offspring. We have
shown that hyperleptinaemia during lactation results in central leptin resistance, higher adrenal
catecholamine secretion, hyperthyroidism, and higher blood pressure and heart rate in the adult
rats. Here, we evaluated the effect of a maternal isocaloric high-fat diet on breast milk composition
and its impact on leptinaemia, energy metabolism, and adrenal and thyroid function of the
offspring at weaning. We hypothesised that the altered source of fat in the maternal diet even
under normal calorie intake would disturb the metabolism of the offspring. Female Wistar
rats were fed a normal (9% fat; C group) or high-fat diet (29% fat as lard; HF group) for
8 weeks before mating and during pregnancy and lactation. HF mothers presented increased
total body fat content after 8 weeks (+27%, P < 0.05) and a similar fat content at the end of
lactation. In consequence, the breast milk from the HF group had higher concentration of protein
(+18%, P < 0.05), cholesterol (+52%, P < 0.05) and triglycerides (+86%, P < 0.05). At weaning,
HF offspring had increased body weight (+53%, P < 0.05) and adiposity (2 fold, P < 0.05),
which was associated with lower β3-adrenoreceptor content in adipose tissue (−40%, P < 0.05).
The offspring also presented hyperglycaemia (+30%, P < 0.05) and hyperleptinaemia (+62%,
P < 0.05). In the leptin signalling pathway in the hypothalamus, we found lower p-STAT3/STAT3
(−40%, P < 0.05) and SOCS3 (−55%, P < 0.05) content in the arcuate nucleus, suggesting leptin
resistance. HF offspring also had higher adrenal catecholamine content (+17%, P < 0.05), liver
glycogen content (+50%, P < 0.05) and hyperactivity of the thyroid axis at weaning. Our results
suggest that a high fat diet increases maternal body fat and this additional energy is transferred to
the offspring during lactation, since at weaning the dams had normal fat and the pups were obese.


C 2012 The Authors. The Journal of Physiology 
C 2012 The Physiological Society DOI: 10.1113/jphysiol.2012.240655
5504 J. G. Franco and others J Physiol 590.21

The higher fat and protein concentrations in the breast milk seemed to induce early overnutrition
in the HF offspring. In addition to storing energy as fat, the HF offspring had a larger reserve
of glycogen and hyperglycaemia that may have resulted from increased gluconeogenesis. Hyper-
leptinaemia may stimulate both adrenal medullary and thyroid function, which may contribute to
the development of cardiovascular diseases. These early changes induced by the maternal high-fat
diet may contribute to development of metabolic syndrome.
(Resubmitted 10 July 2012; accepted 3 August 2012; first published online 6 August 2012)
Corresponding author I. H. Trevenzoli: Laboratório de Endocrinologia Molecular, Instituto de Biofı́sica Carlos Chagas
Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, sl G0-16, Cidade Universitária–Rio de
Janeiro, RJ, 21941-902 Brazil. Email: haraisis@biof.ufrj.br

Abbreviations AR, adrenoreceptor; BW, body weight; CART, Cocaine - and amphetamine - regulated transcript;
DEXA, dual energy X-ray absorptiometry; FFA, free fatty acid; FT4, free T4; JAK2, Janus tyrosine kinase 2; OBRb, long
form of leptin receptor; PNMT, phenylethanolamine-N -methyl transferase; p-STAT3, phosphorylated signal transducer
and activator of transcription 3; PVN, paraventricular hypothalamic nucleus; SOCS3, suppressor of cytokine signalling
3; STAT3, signal transducer and activator of transcription 3; TH, tyrosine hydroxylase; TT3, total T3.

Introduction as a consequence of the altered secretion of several

Epidemiological and experimental evidence has shown
that malnutrition in early life is associated with the
development of chronic diseases at adulthood, such as
type 2 diabetes and hypertension (Barker et al. 2002;
Moura & Passos, 2005; Moura et al. 2008). During the
gestation and lactation periods, offspring are extremely
sensitive to nutritional, hormonal and environmental
changes that may result in altered physiology of several
tissues and systems throughout life (Simmons, 2005). This
phenomenon is named ‘programming’, and its origins are
related to epigenetic modifications – DNA methylation
and histone acetylation – during critical periods of life
(Burdge et al. 2007).
Obesity is the most frequent characteristic programmed
in humans and experimental models (Spencer, 2012). Both
maternal undernutrition and overnutrition during the
perinatal period may programme obesity development
in the offspring in rodent models (Teixeira et al.
2002; Rodrigues et al. 2009). Rats whose mothers were
undernourished during gestation have low birth weight.
However, they display catch-up growth and become
obese at adulthood (Martin-Gronert & Ozanne, 2010).
When dams are protein-restricted only during lactation,
the offspring have the opposite phenotype (Fagundes
et al. 2009). On the other hand, maternal obesity or
high-fat diet consumption during the perinatal period also
programmes obesity at adulthood (Samuelsson et al. 2008;
Sullivan et al. 2011). These phenotypes may be associated
with changes in leptin levels in early life that may disturb
the development of important regulatory sites of energy
metabolism, such as the hypothalamus (Bouret & Simerly,
2006).
Obesity is characterised by an imbalance between
energy intake and expenditure that results in the
enlargement of adipose tissue mass. Adipocyte hyper-
trophy is associated with many metabolic changes, mainly
hormones and factors, such as increased leptin and serum
free fatty acids (FFAs) and decreased adiponectin (Ahima
& Lazar, 2008).
Leptin is secreted mainly by white adipocytes and
binds to the long form of leptin receptor (OBRb) in
the hypothalamus, reducing appetite and stimulating
energy expenditure through the Janus tyrosine kinase 2
(JAK2)–signal transducer and activator of transcription
3 (STAT3) pathway. There are also leptin receptors in
other tissues, such as mammary glands, placenta, pituitary
gland, adrenals, thyroid and components of the cardio-
vascular system (Bjorbaek & Kahn, 2004), suggesting
that this hormone may also regulate metabolism peri-
pherally. Serum leptin level is positively correlated with
adiposity. However, obese humans and animals usually
do not respond to the anorexigenic effect of leptin
because of the development of leptin resistance in the
hypothalamus.
We have shown that hyperleptinaemia during the
first 10 days of lactation promotes obesity, central leptin
resistance and higher levels of catecholamines, thyroid
hormones, and blood pressure at adulthood (Toste
et al. 2006a,b; Trevenzoli et al. 2007). Currently, a
large number of women of reproductive age are over-
weight or obese (Callaway et al. 2006; Kim et al. 2007).
Here, we hypothesised that maternal consumption of a
high-fat diet during the perinatal period and increased
maternal adiposity would affect leptinaemia and energy
metabolism in their offspring, increasing the risk for
developing obesity and cardiovascular failure. Therefore,
we aimed to elucidate the impact of maternal consumption
of an isocaloric high-fat diet during critical periods
of development, such as gestation and lactation, on
breast milk composition and on the adiposity, glucose
homeostasis, leptinaemia and central leptin sensitivity of
the offspring at weaning to determine if any of these


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J Physiol 590.21 Maternal high fat diet programmes adrenal and thyroid function 5505

At birth, all of the litters were weighed and adjusted to

Table 1. Composition of the control and high-fat diets
six males for each dam, a number that maximises lactation
Macronutrients kcal %kcal performance (Fishbeck & Rasmussen, 1987). Litters that
Control diet (amount per kg) did not have at least six male pups were discarded.
Protein 220 g 880 22.6 At weaning, the dams again underwent DEXA, which
Lipids 40 g 360 9.4 was followed by killing using a rodent guillotine. The
Carbohydrates 660 g 2,640 68 glycaemia of the pups was measured, and then they
Total 1000 g 3,880 100 were killed by cardiac puncture under anaesthesia (55 mg
High-fat diet (amount per kg) (kg BW)−1 ketamine and 100 mg (kg BW)−1 xylazine)
Protein 169.6 g 678.4 17.3 for sample collection. Blood samples were centrifuged
Lipids 214.9 g 1124 28.6 (1000 g, 4◦ C, 20 min), and serum was kept individually
Carbohydrates 530.3 g 2121 54.1 at −20◦ C until hormone quantification assays. The left
Total 1092 g 3923 100 adrenal glands were dissected and kept in 10% acetic
acid solution at −20◦ C for catecholamine quantification.
The right adrenal glands were kept at −80◦ C to measure
the catecholamine-synthesising enzymes tyrosine hydro-
xylase (TH) and phenylethanolamine-N -methyl trans-
parameters were associated with altered adrenal or thyroid ferase (PNMT). Liver samples were collected for glycogen
function. quantification and analysis of β2 -adrenoreceptor (β2AR)
content by Western blotting. We dissected and weighed
three representative depots of white adipose tissue
Methods (retroperitoneal, epididymal and inguinal) to analyse
adiposity in the pups. Samples from the inguinal region
Animals and diets
were taken to analyse leptin, β2AR, and β3AR expression.
The use of animals in our experimental design was The brain was collected for dissection of the arcuate
approved by the Animal Care and Use Committee of the nucleus of the hypothalamus, which was used to quantify
Carlos Chagas Filho Biophysics Institute (process IBCCF leptin signalling proteins. The hearts of the offspring were
114). The handling of the experimental animals followed also weighed.
the principles adopted in the UK and Brazil according For each experimental procedure, animals from at least
the Brazilian Law no. 11.794/2008 (Drummond, 2009; six different litters per group were used to avoid litter
Marques et al. 2009). effects.
Female Wistar rats were kept in an environmentally
controlled room (23 ± 2◦ C and a 12 h light–dark cycle
with lights on from 07.00 h to 19.00 h). Nulliparous
Biochemical and hormonal milk analysis
60-day-old female rats were divided into two groups: the
control group (C), which received a standard diet for Lactose, protein, cholesterol, triglycerides and leptin were
rodents (9% of the calories as fat), and a high-fat group measured in milk samples from the C and HF dams in the
(HF), which received a high-fat diet (28% of the calories as middle (day 11) and at the end (day 21) of lactation (Troina
fat). In the high-fat diet, lard was used as fat source (mainly et al. 2010). Lactating rats were separated from their pups
saturated fat) and we also added 1% soy oil to provide for 2 h and then injected subcutaneously with 5 IU ml−1 of
the minimal amount of n3 fatty acid (mainly unsaturated oxytocin (Eurofarm, São Paulo, SP, Brazil). After 20 min,
fat) for adequate development of rats. The macronutrient the dams were lightly anaesthetised (55 mg (kg BW)−1
composition of the control and high-fat diets is shown in ketamine and 100 mg (kg BW)−1 xylazine), and milk was
Table 1. Both diets contained approximately 3.9 kcal g−1 extracted by gently squeezing the thoracic and abdominal
and followed the AIN-93 recommendations for micro- teats. Lactose content was estimated by a colorimetric
nutrients (Reeves et al. 1993). The female rats were method using commercial lactose (Sigma, St Louis, MO,
fed these diets beginning 8 weeks before mating and USA) as a standard. Protein content was measured
throughout gestation and lactation. After the first 8 weeks by the colorimetric Peterson method using bovine
on the diets, the body composition of the dams was serum albumin (Sigma) as a standard. Total cholesterol
evaluated by dual energy X-ray absorptiometry (DEXA) and triglycerides were measured by an enzymatic and
to confirm the effect of the HF diet in increasing body fat colorimetric method using a commercial kit (Bioclin,
content. Next, the dams were mated in a 3 to 1 ratio. After Belo Horizonte, MG, Brazil). The leptin concentration
mating, pregnant rats were housed in individual standard of the milk was evaluated using a rat leptin-specific radio-
rat cages with free access to water and food until the pups’ immunoassay (RIA) kit with a sensitivity of 0.5 ng ml−1
birth. The dams were weighed weekly. (Linco Research, Inc., St Charles, MO, USA). For these


C 2012 The Authors. The Journal of Physiology 
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5506 J. G. Franco and others
analyses, we used six mothers per group for each time
point.
Hormones and free fatty acid (FFA) measurements
Total serum adiponectin was measured by ELISA
(Millipore, Billerica, MA, USA) with an assay sensitivity of
0.155 ng ml−1 . Serum leptin, insulin, corticosterone, total
T3 (TT3) and free T4 (FT4) were measured using specific
commercial RIA kits with the following assay sensitivities:
leptin: 0.5 ng ml−1 (Linco Research, Inc.); insulin:
0.1 ng ml−1 (ICN Pharmaceuticals Inc., Orangeburg, NY,
USA); corticosterone: 50 ng ml−1 (Zen-bio Inc., Research
Triangle Park, NC, USA); and TT3 and FT4: 6.7 ng dl−1
and 0.045 ng dl−1 , respectively (Coat-A-Coat, DPC, Los
Angeles, CA, USA). Serum TSH was measured by specific
rat RIA employing reagents supplied by the National
Hormone and Peptide Program (Torrance, CA, USA) as
previously described (Ortiga-Carvalho et al. 2002). The
intra-assay coefficients of variance of the assays for all
hormones were in the range of 4–7%. Serum FFA was
measured using a colorimetric commercial kit (Zen-bio
Inc.). For each parameter that was evaluated, all of the
measurements were performed in a single assay. For
these assays, we used nine C-offspring pups and ten
HF-offspring pups.
Real-time PCR
Leptin gene expression was quantified as previously
described (Nobre et al. 2011). Total RNA was isolated
from inguinal adipose tissue using a commercial kit
(RNeasy lipid tissue mini kit, Qiagen, Hilden Germany).
For quantitative real-time RT-PCR analysis, reverse
transcription was carried out on 1 μg of total RNA for
all tissues using a SuperScript III kit. The products were
amplified in an Applied Biosystems 7500 real-time PCR
System (Life Technologies Co., Frederick, MD, USA) using
SYBR Green PCR Master Mix (Applied BioSystems, Foster
City, CA, USA). Briefly, after initial denaturation at 50◦ C
for 2 min and 95◦ C for 10 min, the reactions were cycled 40
times using the following parameters for the ob gene: 95◦ C
for 15 s, 53◦ C for 30 s and 70◦ C for 45 s. Product purity
was confirmed by agarose gel analysis. Relative mRNA
levels (2C t ) were determined by comparing the PCR
cycle threshold (C t ) between groups, after normalising
to the housekeeping gene 36β4. The data are expressed
as fold induction over the control group, which was
set to 100%. The sequences of the forward and reverse
primers were 5 -CATCTGCTGGCCTTCTCCAA-3 and
5 -ATCCAGGCTCTCTGGCTTCTG-3 for the leptin
gene, and 5 -TGTTTGACAACGGCAGCATTT-3 and
5 -CCGAGGCAACAGTTGGGTA-3 for 36β4. For these
J Physiol 590.21
measurements, we used nine C-offspring pups and 10
HF-offspring pups.
Microdissection of paraventricular (PVN) and arcuate
(Arc) hypothalamic nuclei
PVN and Arc tissues were obtained by the punch technique
from thick coronal brain sections cut in a cryostat using the
bregma as reference (Palkovits, 1973; Paxinos & Watson,
2007; Helena et al. 2009). Briefly, two subsequent sections
of 1000 μM were made: from −0.6 mm to −1.6 mm for
PVN microdissection, and from −1.6 mm to −2.6 mm
for microdissection of the Arc. The PVN was dissected
from the first section in one punch, obtained with a
1.5 mm-diameter round needle with the top centred
in the third ventricle. The Arc was dissected from the
second section using a 2 mm ‘square puncher’ needle,
centred on the third ventricle, approximately 1 mm dorsal
to the base of the brain. Immediately afterwards, the
tissue samples were kept at −80◦ C until Western blotting
assays.
Western blotting
We used the western blotting assay to detect specific
proteins in various tissues: Arc, PVN, adipose tissue, liver
and adrenal. Samples of each tissue were homogenised in
a specific way, following previous protocols with minor
modifications (Trevenzoli et al. 2010a).
To analyse the leptin signalling pathway and TRH in
hypothalamic punch samples, we used two nuclei pools
to obtain each sample and achieve an adequate protein
concentration in the tissue extracts. The samples were
homogenised in 100 μl of ice-cold lysis buffer (50 mM
Hepes, 1 mM MgCl2 , 10 mM EDTA, 1% Triton X-100
pH 6.4) containing protease inhibitors (Complete, Roche
Diagnostics), using an automatic ultrasonic processor
(2 × 10 s pulse on and 20 s pulse off ). The total protein
concentration of the homogenates was determined using
the BCA Protein Assay Kit (Thermo Scientific, Rockford,
IL, USA).
To analyse the catecholamine-synthesising pathway,
each right adrenal was homogenised manually in 500 μl
of lysis buffer. The homogenates were centrifuged (1120 g,
4◦ C, 5 min), and the supernatants were collected for total
protein quantification.
The liver samples (25 mg) were homogenised in 300 μl
of lysis buffer and centrifuged (1000 g, 4◦ C, 10 min).
Then, the homogenates were diluted 1:3 before protein
quantification. For inguinal adipose tissue, we homo-
genised 40 mg of tissue in 300 μl of the pH 7.4 lysis buffer
(20 mM Tris-HCl, 10 mM NaF, 1% NP40, 150 mM NaCl,
5 mM EDTA, 0.1% SDS) containing protease inhibitors.
The homogenates also underwent an ultrasonic procedure

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J Physiol 590.21 Maternal high fat diet programmes adrenal and thyroid function 5507

(2 × 10 s pulse on and 20 s pulse off ) followed by two Catecholamine quantification

subsequent centrifugations (9500 g, 4◦ C, 15 min). The
Total catecholamine (adrenaline and noradrenaline)
total protein content of the supernatants was quantified
content was quantified using a trihydroxyindole
using a BCA kit (Thermo Scientific). Both liver and
method with some modifications (Trevenzoli et al.
adipose tissue were used for analysis of AR content.
2007). We used 50 μl of the supernatant from the
All samples were denatured in the sample buffer (50 mM
homogenised glands to quantify the catecholamines.
Tris-HCl, pH 6.8, 1% SDS, 5% 2-mercaptoethanol, 10%
Adrenaline was used as standard. Briefly, 50 μl of the
glycerol, 0.001% bromophenol blue) and heated at 95◦ C
standard/supernatant/medium was mixed with 250 μl
for 5 min.
of 0.5 M phosphate buffer (pH 7.0) and 25 μl of 0.5%
Supernatants were analysed by SDS-PAGE, with
potassium ferricyanate; the mixing was followed by
a 10% or 12% polyacrylamide gel, and electro-
incubation (20 min; ice bath). The reaction was stopped
blotted onto PVDF membranes (Hybond P ECL
with 500 μl of 60 mg ml−1 ascorbic acid/5 N NaOH (1:19
membrane; Amersham Pharmacia Biotech, NJ, USA).
proportion). The parameters used in the fluorometer
The membranes were incubated with Tris-buffered
(Victor2, PerkinElmer, Turku, Finland) were 420 nm for
saline–Tween 20 (T-TBS) containing 5% non-fat dry
excitation and 510 nm for emission. The results were
milk for 90 min to block non-specific binding sites.
obtained by plotting the values as a linear regression of
Then, the membranes were washed with T-TBS and
the standard adrenaline curve. For this assay, we used nine
incubated overnight with specific primary antibodies.
adrenals from the C offspring and 10 adrenals from the
For the arcuate nucleus, anti-OBR (1:1000), anti-JAK2
HF offspring.
(1:500), anti-STAT3 (1:1000), anti-pSTAT3 (1:1000),
anti-suppressor of cytokine signalling 3 (SOCS3; 1:250)
(Santa Cruz Biotechnology, Santa Cruz, CA, USA) and
Liver glycogen content
anti-cyclophilin (1:3000) (Affinity BioReagents, IL, USA)
were used. For the PVN, anti-Pro TRH (1:500) (Life- Liver glycogen was measured using a protocol adapted
Span BioSciences, Seattle, WA, USA) and anti-cyclophilin from the Miller method (Trevenzoli et al. 2010b). Liver
(1:3000) were used. For adrenal gland, anti-TH (1:1000) samples were homogenised with 10% trichloroacetic acid
and anti-PNMT (1:1000) (Sigma-Aldrich, St Louis, MO, (100 mg of tissue/0.3 ml) and centrifuged (1000 g at 4◦ C
USA) were used. For adipose tissue, anti-β2AR (1:1000) for 10 min). To extract the glycogen, 200 μl of super-
(Sigma-Aldrich) and anti-β3AR (1:1000) (Santa Cruz natant was mixed with 667 μl of absolute ethanol. The
Biotechnology) were used. For liver, anti-β2AR (1:1000) mixture was maintained at 4◦ C for 24 h for glycogen
(Sigma-Aldrich) was used. Next, the membranes were precipitation and then centrifuged (1000 g at 4◦ C for
washed and incubated with the appropriate secondary 10 min); the resulting supernatant was then discarded.
antibodies, which were conjugated to biotin (Santa Cruz The glycogen pellets were hydrolysed with 0.1 ml of 1 M
Biotechnology; diluted in 0.5% non-fat dry milk/TBS) or HCl for 30 min at 100◦ C. Then, the mixtures were placed
horseradish peroxidase (HRP; Amersham, UK), for 1–2 h in an ice bath, and 0.1 ml of 1 M NaOH was added
at room temperature. The membranes that were incubated for neutralisation. The glucose produced by glycogen
in biotin-antibodies were washed and incubated for 1 h hydrolysis was measured using an enzymatic colorimetric
at room temperature with streptavidin HRP-conjugates method (Glucox commercial kit, Doles, Goiás, Brazil)
(Zymed, Camarillo CA, USA). Immunoreactive proteins and compared to a standard glucose curve. We used
were visualised using HRP substrate (ECL-plus; GE, Little nine C-offspring pups and 10 HF-offspring pups for this
Chalfont, Buckinghamshire, UK) and then exposed to analysis.
X-ray film. Finally, the area and density of the protein
bands were quantified using ImageJ 1.34s software (Wayne
Rasband, National Institutes of Health, Bethesda, MD, Statistical analysis
USA). All proteins were normalised by the cyclophilin
The data are expressed as the mean ± SEM. We used
content in each sample, except those from adipose
two-way ANOVA to analyse the body mass curve before
tissue, which were normalised by Ponceau staining (total
mating. The other variables were analysed using Student’s
protein).
unpaired t test. Results were considered significant when
For each protein analysed by Western blotting, we
P < 0.05.
used six or seven samples per group, and each sample
was obtained from a different animal. For analysis of
proteins in the hypothalamus, we used two animals
Results
(2 hypothalamic nuclei) to obtain each sample, and
five or six samples per group were used in the Wistar female rats fed with the high-fat diet did not show
experiments. increased body mass after 8 weeks of treatment (before


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5508
mating) compared to their controls (Fig. 1A). However,
the DEXA analysis showed that they had higher total body
fat content (+27%, P < 0.05; Fig. 1B). At weaning, the
control and HF mothers had similar body masses and fat
contents (Fig. 1C and D).
Breast milk from the HF group contained more lactose
(+20%, P < 0.05) and triglycerides (+35%, P < 0.05)
and less cholesterol (−19%, P < 0.05) than the C group,
without any changes in protein content, on the 11th day
of lactation (Fig. 2). At weaning (21st day of life), the
breast milk had higher concentrations of proteins (+18%,
P < 0.05), cholesterol (+52%, P < 0.05) and triglycerides
(+86%, P < 0.05). The leptin concentration in the milk
was similar between the two groups (Fig. 2).
The maternal HF diet did not impact the birth
weight of the offspring (Fig. 3A). In contrast, at
weaning, the HF male offspring were evidently obese,
characterised by higher body mass (+53%, P < 0.05;
Fig. 3B) and increased fat mass in different adipose
tissue depots: retroperitoneal (2.3-fold, P < 0.05; Fig. 4A),
epididymal (3.4-fold, P < 0.05; Fig. 4A) and inguinal
(2-fold, P < 0.05; Fig. 4A). This increased adiposity
was associated with higher leptin expression in the
inguinal adipose tissue (2.5-fold, P < 0.05; Fig. 4B) and
hyperleptinaemia (+62%, P < 0.05; Fig. 4C) in the HF
Figure 1. Maternal body weight and adiposity
Body mass evolution and fat content/DEXA of control (filled bars; n = 6) and high-fat (open bars; n = 9) dams
before gestation (A and B, respectively), and body mass and fat content at weaning (C and D, respectively). Results
are expressed as means ± SEM; ∗ P < 0.05.
J. G. Franco and others J Physiol 590.21
offspring. In addition, the HF offspring presented higher
heart weights (ConOff: 0.190 ± 0.0087 g, and HFOff:
0.256 ± 0.006 g, P < 0.05). When analysed as a relative
proportion of body weight, the fat mass remained higher
in the HF offspring (P < 0.05), but the heart weight was
similar between the groups (data not shown).
To verify whether hyperleptinaemia was associated with
alterations of central leptin signalling, we evaluated key
proteins of the leptin pathway in the hypothalamic arcuate
nucleus. HF offspring had similar amounts of OBRb
and JAK2 compared with the controls (Fig. 5). However,
they had lower phosphorylated STAT3 (p-STAT3)/STAT3
(−40%, P < 0.05; Fig. 5C), which is the main target
of the leptin pathway. This change was associated with
reduced SOCS3 in the HF offspring (−56%, P < 0.05;
Fig. 5D).
The adrenal function evaluation showed that the
HF offspring had higher adrenal catecholamine content
(+20%, P < 0.05; Fig. 6A) without changes in TH or
PNMT (Fig. 6C and D). Corticosteronaemia was similar
between the groups (Fig. 6B).
To evaluate the possible impact of the higher levels
of catecholamines in HF offspring on other tissues, we
evaluated the expression of different adrenoceptors (ARs).
HF offspring had less β3AR (−40%, P < 0.05; Fig. 7B),

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J Physiol 590.21 Maternal high fat diet programmes adrenal and thyroid function 5509

without changes in β2AR (Fig. 7A), in the inguinal adipose
tissue.
Despite the higher adiposity, which suggests insulin
resistance, HF offspring presented hyperglycaemia
(+30%, P < 0.05; Fig. 8A) and no alterations in serum
insulin, adiponectin or FFA (Fig. 8B–D).
HF offspring also presented higher absolute liver weight
(+46%, P < 0.05; Fig. 9A) and glycogen content (+50%,
P < 0.05; Fig. 9B). This alteration was accompanied by
reduced β2AR in the liver (−24%, P < 0.05; Fig. 9C).
When analysed as a relative proportion of body weight,
liver weight trended toward being higher in the HF
offspring (P = 0.08; data not shown).
Figure 10 shows parameters of the hypothalamus–
pituitary–thyroid (HPT) axis. HF offspring had higher
serum TT3 (+15%, P < 0.05) and FT4 (+40%, P < 0.05)
without significant changes in TSH. This profile was
accompanied by increased pro-TRH in the PVN (+28%,
P < 0.05).
Discussion
Normal leptinaemia in the perinatal period is an
important factor for adequate development of the
central nervous system, especially the hypothalamus
(Pinto et al. 2004; Bouret & Simerly, 2006), and also
for the physiology of peripheral tissues, such as adrenal
gland, liver, adipose tissue and thyroid, in rodents (Moura
& Passos, 2005; Vickers et al. 2005). Both hyperleptinaemia

Figure 2. Breast milk composition

Milk content of protein (A), lactose (B), triglycerides (C), cholesterol (D) and leptin (E) of control (filled bars; n = 6)
and high-fat (open bars; n = 6) dams at the 11th day of lactation (middle lactation) and at weaning. Results are
expressed as means ± SEM; ∗ P < 0.05.


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5510 J. G. Franco and others J Physiol 590.21

(Toste et al. 2006b; Trevenzoli et al. 2010a,b) and

Figure 3. Effect of maternal HF diet on the offspring body
mass
Birth weight (A) and body mass at weaning (B) of the control (C;
n = 9) and high-fat (HF; n = 10) offspring. Results are expressed as
means ± SEM; ∗ P < 0.05.
blockage of leptin action (Attig et al. 2008) during
lactation programme a cluster of metabolic dysfunctions
throughout life, including obesity and leptin resistance.
Maternal nutritional status may impact leptinaemia and
the metabolic parameters of the offspring with short- and
long-term consequences. Undernutrition during lactation
results in abolition of the leptin surge in the middle of
lactation and increased leptinaemia at weaning (Teixeira
et al. 2002). These changes are associated with central
resistance to the anorexigenic effect of leptin (Passos et al.
2004). However, the normalisation of leptin level pre-
vents the programming effects of undernutrition (Vickers
et al. 2005). In the present study, we found that a
maternal high-fat diet resulted in hyperleptinaemia in the
offspring at weaning, which was associated with higher
body weight and adiposity. These effects were independent
of maternal obesity at weaning, at which point the HF
mothers showed no change in body fat, suggesting a
specific effect of saturated fat on offspring metabolism.
The HF diet did markedly change the breast milk
composition, and the high concentrations of proteins,
lactose and triglycerides in the breast milk seemed
to induce overnutrition in the offspring throughout
lactation. Cunha et al. (2009) have shown that breast
milk from dams with pups that are raised in small

Figure 4. Effect of maternal HF diet on the offspring adiposity and leptin production
Fat mass of retroperitoneal, epididymal and inguinal compartments (A), leptin RNAm expression in inguinal fat
pad (B) and leptinaemia (C) of the control (C; n = 9) and high-fat (HF; n = 10) offspring at weaning. Results are
expressed as means ± SEM; ∗ P < 0.05.


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J Physiol 590.21 Maternal high fat diet programmes adrenal and thyroid function 5511

litters (a recognised programming model of neonatal
overnutrition) also present high levels of triglycerides. A
maternal HF diet increases the n3:n6 ratio and insulin
level in breast milk from non-human primates without
changing leptin content (Grant et al. 2011). Thus, our
results suggest that the precocious obesity programmed
by a maternal HF diet is a consequence of changes in
the macronutrients in breast milk rather than changes
in the leptin concentration of milk that might disturb
the leptin surge in the middle of lactation. Although
many researchers are investigating the disturbances to
the progeny caused by maternal malnutrition, most of
them have used a drastic diet in terms of fat content (e.g.
50–60% of calories from fat), whereas the diet used in
the present study was isocaloric and moderately high-fat.
Our data show that even consumption of a normocaloric
diet during pregnancy and lactation may cause deleterious
effects in the offspring if the quality of the diet is not
appropriate.
As a consequence of early overnutrition, the HF
offspring presented increased visceral and subcutaneous
fat mass. Serum leptin level is positively correlated with
body weight and the amount of adipose tissue (Friedman
& Halaas, 1998), and the HF offspring presented hyper-
leptinaemia associated with higher expression of leptin
in the inguinal adipose tissue. A similar phenotype was
observed in rats raised in small litters during lactation
(Rodrigues et al. 2009).
The anorexigenic effect of leptin is mediated
by the JAK2–STAT3 pathway in the hypothalamus.

Figure 5. Effect of maternal HF diet on leptin signalling pathway in the arcuate nucleus of the offspring
Content of OBRb (A), JAK2 (B), p-STAT3 (C) and SOCS3 (D) in samples of arcuate hypothalamic nuclei of control
(C; n = 6) and high-fat (HF; n = 6) offspring at weaning. Representative blots of leptin pathway proteins and
cyclophilin (control load) are shown (E). Results are expressed as means ± SEM; ∗ P < 0.05.


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5512 J. G. Franco and others J Physiol 590.21

When activated, p-STAT3 increases the synthesis of
anorexigenic neuropeptides (α-melanocyte-stimulating
hormone (α-MSH) and cocaine and amphetamine
regulated transcript (CART)) and reduces the production
of orexigenic factors (neuropeptide Y (NPY) and
agouti-related peptide (AgRP)). In rodents, these neural
pathways that regulate food intake and energy expenditure
are not completely developed until the third postnatal
week (Grove et al. 2005); however, in humans, they develop
mainly prenatally (Djiane & Attig, 2008). In the present
study, dams were fed experimental diets until the end of
lactation, when the pups were 3 weeks old, specifically
to include this critical period of brain development in
rodents.
The JAK2–STAT3 pathway also stimulates the
transcription of SOCS3, a negative regulator of leptin
signalling (Bjorbaek et al. 1997, 1998). Hyperleptinaemia
is frequently associated with central leptin resistance
with important consequences, such as hyperphagia
and decreased energy expenditure (Ahima & Lazar,
2008). In our experimental model, the analysis of the
leptin pathway in the hypothalamic arcuate nucleus
of the offspring showed that the maternal HF diet
reduced STAT3 activation and SOCS3 content. These
results indicate the beginning of leptin resistance, which
may contribute to lower energy expenditure and the
development of obesity. Because central feeding circuits
are still undeveloped during lactation (Grove & Smith,
2003), we believe that leptin has a larger effect on
energy metabolism than on regulating food intake in this
period.
Leptin resistance is frequently selective in obese humans
and animals. It is characterised by a lack of anorexigenic
and metabolic leptin responses and overactivation of
other systems influenced by this hormone, such as the
sympathetic nervous system (SNS).

Figure 6. Effect of maternal HF diet on adrenal function of the offspring

Adrenal content of catecholamines (A), serum corticosterone (B), adrenal content of tyrosine hydroxylase (TH;
C) and phenylethanolamine n-methyl transferase (PNMT; D) of control (C; n = 7–9) and high-fat (HF; n = 7–10)
offspring at weaning. Representative blots of catecholamine synthesising enzymes and cyclophilin (control load)
are shown (E). Results are expressed as means ± SEM; ∗ P < 0.05.


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J Physiol 590.21 Maternal high fat diet programmes adrenal and thyroid function
The sympathoadrenal system is an important
component of physiological adaptation to challenging
situations. Changes in maternal diet, leptin levels or
nicotine exposure during lactation are stressful events,
resulting in alterations in both the corticosterone and
catecholamine levels of the offspring throughout life
(Vieau et al. 2007; Fagundes et al. 2009; Trevenzoli et al.
2010a; Laborie et al. 2011; Pinheiro et al. 2011). In
addition, these hormones make relevant contributions to
energy metabolism and cardiovascular function because
they affect glucose production and utilisation and fat
storage and protein metabolism. Thus, the HPA and
sympathoadrenal system seems to be profoundly involved
with the developmental origins of obesity and cardio-
vascular disease.
Leptin increases the sympathetic tone to brown
adipose tissue, kidneys, hind limbs and adrenal glands.
Hypertension associated with obesity may have a great
Figure 7. Effect of maternal HF diet on content of
adrenoreceptors in adipose tissue of the offspring
Content of β2 (A) and β3 (B) adrenoreceptors in the inguinal
adipose tissue of control (C; n = 7) and high-fat (HF; n = 7) offspring
at weaning. Representative blots of ARs and Ponceau staining
(control load) are shown (C). Results are expressed as means ± SEM;
∗ P < 0.05.

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5513
contribution of leptin action (Kalil & Haynes, 2012). We
have shown that leptin treatment during the first 10 days of
lactation increases catecholamine levels in the short and
long term. These alterations are associated with higher
blood pressure and heart rate at adulthood (Trevenzoli
et al. 2007). In this study, we showed that a maternal
high-fat diet also increased adrenal catecholamine content
in the pups at weaning, which may contribute to future
programming of the cardiovascular system. Samuelsson
et al. (2010) also demonstrated that maternal obesity
induces hypertension development that is associated with
SNS overactivity in 30-day-old rats. Thus, we suggest
that adrenal catecholamines may contribute to this
phenotype.
The catecholamine synthesis pathway is regulated by
TH and PNMT activities among other steps (Haycock &
Wakade, 1992). We did not find differences in the content
of these key enzymes between the groups that could have
explained the higher adrenal catecholamine content in the
HF offspring. However, the activities of TH and PNMT are
regulated by serine phosphorylation, and leptin directly
stimulates both TH expression and activity. This effect
is mediated by PKC and MAPK (Takekoshi et al. 2001;
Utsunomiya et al. 2001). The hyperleptinaemia found in
the HF offspring and/or the increased sympathetic tone to
the medulla may stimulate TH activity.
To investigate whether higher catecholamine
production in the HF offspring was associated with
tissue catecholamine effects, we measured ARs in tissues
that are profoundly regulated by these hormones.
We observed a lower content of β3AR in the adipose
tissue from the HF offspring, without an alteration in
β2AR. Lipolysis in white adipose tissue is stimulated by
catecholamines through βARs and the PKA pathway
that activates hormone-sensitive lipase (Langin, 2006).
Thus, our data suggest that the reduced lipolysis in the
HF offspring may have contributed to the development
of early obesity. Mice whose mothers were obese and
diabetic have lower ARβ2 and β3AR mRNA levels
and higher peroxisome proliferator-activated receptor
γ (PPARγ) mRNA levels in the inguinal fat tissue at
adulthood, suggesting decreased lipolysis and increased
lipogenesis (Samuelsson et al. 2010). It is possible
that high levels of catecholamines induce differential
downregulation of ARs. In addition, adipocytes are also
able to produce catecholamines, which might have an
important paracrine role on adiposity regulation and
deserves further investigation (Vargovic et al. 2011;
Kvetnansky et al. 2012).
Increased adiposity is strongly associated with insulin
resistance in humans and rodents. In fact, we observed
hyperglycaemia in our experimental model at weaning,
suggesting glucose intolerance. However, serum insulin,
adiponectin and FFAs were unchanged in these animals.
We first tested the hypothesis that HF offspring would
5514 J. G. Franco and others J Physiol 590.21

have increased glycogenolysis induced by catecholamines,
by assessing the glycogen content and expression of β2-AR
in the liver. HF offspring presented higher amounts
of glycogen with lower content of β2-AR, suggesting
reduced glycogenolysis. Thus, the hyperglycaemia found
in the HF offspring seems not to be a consequence of
increased glycogenolysis. On the other hand, a maternal
high-fat diet has been associated with an increased
gluconeogenic capacity of offspring at birth through
epigenetic modifications, which could lead to excessive
hepatic glucose production and altered insulin sensitivity
throughout life (Strakovsky et al. 2011). In addition, a
maternal high-fat diet or maternal obesity can trigger
liver lipotoxicity in rodents (Oben et al. 2010) and
non-human primates (McCurdy et al. 2009), which
indicates liver insulin resistance and contributes to hepatic
glucose production. This phenotype at adulthood (high
glycogen content and liver steatosis) may be associated
with high leptin levels in the neonatal period, as we
have shown previously (Trevenzoli et al. 2010b). We also
cannot discard the possibility of muscle insulin resistance
decreasing glucose uptake and contributing to the
hyperglycaemia.
Thyroid hormones classically have important roles
in early development. Our group have shown that
changes in thyroid function appear to be important for
early adaptation in different experimental programming
models (Toste et al. 2006a; Oliveira et al. 2009; Rodrigues
et al. 2009). The HPT axis activity may be stimulated by
leptin under physiological conditions (Legradi et al. 1997;
Nowak et al. 2002; Ortiga-Carvalho et al. 2002). Thus,
we suppose that in our experimental model, the higher
levels of thyroid hormones in HF offspring occurred as
a consequence of hyperleptinaemia induced by obesity.
Hyperleptinaemia in obese humans (Pacifico et al. 2011)
and rodents (Perello et al. 2010) is associated with
high circulating thyroid hormones, suggesting that leptin
still activates the HPT axis in these individuals even
though they are resistant to the anorexigenic effect of
leptin.
Despite having high levels of thyroid hormones, the
HF offspring were markedly obese, which seems counter-
intuitive because thyroid hormones increase the metabolic
rate and energy expenditure (Yen, 2001). However, in
obese animals, these effects may be counterbalanced by an
increase in food intake induced by T3 (Kong et al. 2004)
and possibly by an impairment of the metabolic actions of
thyroid hormones, among other characteristics of obesity.
Interestingly, a feature of the HF offspring was the small
but significant central activation of thyroid function, as
suggested by the higher pro-TRH content in the PVN,
which may have caused the higher levels of T3 and T4.

Figure 8. Effect of maternal HF diet on glucose homeostasis of the offspring

Glycaemia (A), insulinaemia (B), adiponectinaemia (C) and serum free fatty acids (D) of control (C; n = 9) and
high-fat (HF; n = 10) offspring at weaning. Results are expressed as means ± SEM; ∗ P < 0.05.


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J Physiol 590.21 Maternal high fat diet programmes adrenal and thyroid function 5515

Figure 9. Effect of maternal HF diet on the liver content of glycogen and adrenoreceptor of the offspring
Liver weight (A), glycogen content (B) and content of β2 adrenoreceptor (C) in the liver of control (C; n = 7–9)
and high-fat (HF; n = 7–10) offspring at weaning. Representative blots of β2AR and cyclophilin (control load) are
shown (C). Results are expressed as means ± SEM; ∗ P < 0.05.

Figure 10. Effect of maternal HF diet on the thyroid function of the offspring
Serum total T3 (A), free T4 (B), TSH (C) and pro-TRH content in the hypothalamic paraventricular nucleus (D)
of control (C; n = 5–9) and high-fat (HF; n = 5–10) offspring at weaning. Representative blots of pro-TRH and
cyclophilin (control load) are shown (D). Results are expressed as means ± SEM; ∗ P < 0.05.


C 2012 The Authors. The Journal of Physiology 
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5516 J. G. Franco and others
These data are consistent with the results demonstrated by
Perello et al. (2010) in DIO rats; they found a dissociation
between leptin sensitivity in the arcuate and PVN hypo-
thalamic nuclei, with a lack of anorexigenic response but
preserved stimulation of TRH induced by leptin. Thus,
our data suggest that the hyperleptinaemia observed in
the HF offspring may have contributed to their higher
thyroid hormone levels. The normal serum TSH level
suggests a lower sensitivity of the pituitary to negative
feedback from thyroid hormone; the HF offspring may
also have had a new set point for regulation of the HPT
axis.
In the present study, we demonstrate for the first time
that a perinatal maternal high-fat diet, through changes
in milk composition, has short-term consequences, such
as increased adrenal medullary and thyroid function,
higher adiposity, hyperleptinaemia, and hyperglycaemia
in the offspring. These changes occur even with a normal
maternal caloric intake and may contribute, as priming
factors, to the development of metabolic and cardio-
vascular diseases.
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Author contributions
Experiments were performed mainly in the Laboratory of
Molecular Endocrinology of the Federal University of Rio de
Janeiro and some procedures made in the Laboratory of End-
ocrine Physiology of the State University of Rio de Janeiro. J.G.F.,
T.P.F., C.P.D.R., C.M.C., and I.H.T. participated in collection,
analysis and interpretation of data. J.G.F., C.C.P.-M., P.C.L.,
E.G.M., and I.H.T. performed drafting and critical revising of
the manuscript. All authors have approved the final version of
the manuscript.
Acknowledgements
Research was supported by Conselho Nacional de
Desenvolvimento Cientı́fico e Tecnológico (CNPq),
Coordenação de Aperfeiçoamento de Pessoal de Nı́vel
Superior (CAPES) and Fundação de Amparo à Pesquisa do
Rio de Janeiro (FAPERJ). T.P.F. was recipient of a FAPERJ
fellowship; C.C. was recipient of a CAPES fellowship and J.G.F.
was recipient of a CNP fellowship.

C 2012 The Authors. The Journal of Physiology 
C 2012 The Physiological Society