The Scientific World Journal
Volume 2012, Article ID 780890, 7 pages
Renatade Azevedo Melo Luvizotto,1Andr´ eFerreiradoNascimento,1
MariaTeresa de S´ ıbio,1Regiane Marques CastroOl´ ımpio,1SandroJos´ e Conde,1
andC´ eliaRegina Nogueira1
1Department of Internal Medicine, Botucatu School of Medicine, University of S˜ ao Paulo State, 18618-000 Botucatu, SP, Brazil
2Department of Sports, Center of Physical Education and Sports, Federal University of Espirito Santo (UFES),
29075-910 Vit´ oria, ES, Brazil
Correspondence should be addressed to Renata de Azevedo Melo Luvizotto, email@example.com
Received 29 October 2011; Accepted 27 December 2011
Academic Editors: S. Bernasconi, R. E. Felberbaum, and A. Ferlin
Copyright © 2012 Renata de Azevedo Melo Luvizotto et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
Aims. To analyze the influence of hyperthyroidism on the gene expression and serum concentration of leptin, resistin, and
adiponectin in obese animals. Main Methods. Male Wistar rats were randomly divided into two groups: control (C)—fed with
commercial chow ad libitum—and obese (OB)—fed with a hypercaloric diet. After group characterization, the OB rats continued
receiving a hypercaloric diet and were randomized into two groups: obese animals (OB) and obese with 25μg triiodothyronine
(T3)/100BW (OT). The T3dose was administered every day for the last 2 weeks of the study. After 30 weeks the animals were
euthanized. Samples of blood and adipose tissue were collected for biochemical and hormonal analyses as well as gene expression
of leptin, resistin, and adiponectin. Results. T3treatment was effective, increasing fT3levels and decreasing fT4and TSH serum
concentration.AdministrationofT3promotesweightloss,decreases allfatdeposits,anddiminishesserumlevels ofleptin,resistin,
and adiponectin by reducing their gene expression. Conclusions. Our results suggest that T3modulate serum and gene expression
levels of leptin, resistin, and adiponectin in experimental model of obesity, providing new insights regarding the relationship
between T3and adipokines in obesity.
The thyroid hormones influence energetic metabolism 
and perform a central role in the regulation of adipose tissue
metabolism . Disturbances of these hormones are associ-
ated with alterations of body weight and energy expenditure
. While it is well known that hyperthyroidism leads to
weight loss and hypothyroidism is associated with weight
gain, the changes of thyroid function are discussed contro-
versially in obesity .
Obesity, a public health problem associated with innu-
merable incapacitating and chronic diseases , is defined
as an excessive or abnormal accumulation of adipose tissue
viously considered to be the largest, although inert, energy
store in the body, actively produces a variety of biological
substances. These substances are denominated adipokines
and can influence the function and structural integrity of
other tissues [7, 8]. In obesity, the release of adipokines such
as leptin, resistin, and adiponectin can be altered . Leptin
acts as a signal of satiety in the hypothalamus and thus con-
trols the body weight not only by diminishing the ingestion
of foods but also by increasing energetic expenditure .
meals; this increase is due to direct stimulation of expression
of the ob gene and/or secretion of leptin from adipose tissue
and adipocytes, and present proinflammatory properties
such as TNF-α and IL-6 . Despite being expressed and
secreted in thin individuals, elevated levels are associated
2 The Scientific World Journal
with obesity both in humans and in experimental animal
models . However, the detailed functions of resistin are
still not understood; it has been appreciated that resistin
can cause hepatic insulin resistance and that it may, along
with its closely related homologs, interact with immune
cells as well . Adiponectin is expressed exclusively in
differentiated adipocytes , regulates lipid and glucose
metabolism, suppresses gluconeogenesis, increases insulin
sensitivity, stimulates fatty acid oxidation, protects against
chronic inflammation, and regulates food intake and body
weight . Its reduction is associated with a decrease in
lipid oxidation, increased triglycerides, and a suppression of
insulin-dependent signaling in the liver and muscle, all of
which can contribute to insulin resistance and obesity .
Thyroid hormones are involved in the regulation of adi-
such as resistin, adiponectin, and leptin are involved in reg-
ulation of the energetic balance ; however, the relation-
ship between these hormones in obesity is controversial and
scarcely addressed. In this context, our objective in this study
was to analyze the influence of supraphysiological dose of T3
on the gene expression and serum concentration of leptin,
resistin, and adiponectin in obese animals.
2.1. Animals and Experimental Protocol. This study utilized
male Wistar rats, weighing approximately 150g, supplied
by the Animal Center of the Experimental Laboratory for
Clinical Medicine at the “J´ ulio de Mesquita Filho” Paulista
State University in Botucatu, Sao Paulo, Brazil. The animals
were initially divided into two groups: control (C)—fed with
commercial chow ad libitum—and obese (OB)—fed with a
hypercaloric diet, as previously described , in order to
induce obesity. After the obesity induction, the OB animals
were randomized into two groups: obese animals (OB, n =
10), and obese animals administered a supraphysiological
dose of T3(OT, n = 10) at a concentration of 25μg/100g
body weight (BW) . The T3 was administered by
subcutaneous injections, once a day, during the final 2 weeks
[21, 22]. Appropriate volumes of saline were administered,
by subcutaneous injections, to the OB and C groups. The
animals were housed in individual cages under controlled
ambient temperature (22–26◦C) and lighting (12h light-
dark cycle). Dietary consumption was controlled daily, and
weight was assessed weekly. The experimental protocol was
approved by the Commission for Ethics in Animal Exper-
imentation at the Botucatu—UNESP School of Medicine,
and followed the “Guidelines for the Care and Use of
2.2. Total Body Fat. The total body fat was measured as the
sum of epididymal, retroperitoneal, and visceral fat deposits
. This data point was utilized to confirm obesity in the
animals. In addition, the adiposity index (total body fat
Boustany et al. ) was calculated.
2.3. Biochemical Analysis of Serum. The animals were fasted
for 12 to 15 hours, anesthetized with sodium pentobarbital,
50mg/kg/ip, and sacrificed by decapitation. The blood
was collected in dry tubes and centrifuged at 3000rpm
for 10 minutes. The serum was stored at −80◦C. Serum
concentrations of glucose and triacylglycerol (TG) were
assayed using specific kits (CELM, S˜ ao Paulo, Brazil). Free
fatty acids (FFAs) were determined using a commercial
kit (WAKO, WAKO Pure Chemical Industries Ltd., Osaka,
Japan). Dosing was analyzed by the automated colorimetric
enzyme method (Technicon, RA-XT System, Global Medical
Instrumentation, Minessota, USA).
2.4. Hormonal Measurements. Serum concentrations of
insulin, leptin, resistin, adiponectin, free T3, free T4, and
TSH were measured in all animals. The measurements were
performed by immunoassay, measured with a microplate
reader (Spectra Max 190—Molecular Devices, Sunnyvale,
CA, USA). Commercial kits were utilized for the measure-
ment of leptin, insulin, adiponectin (ELISA kit-Millipore,
St. Charles, MO, USA), resistin (ELISA kit-B-Bridge Inter-
national Inc., Mountain View, CA, USA), and thyroid
pany, Wuhan, China).
2.5. Gene Expression. Whole RNA was extracted from
retroperitoneal adipose tissue using the reagent Trizol (Invit-
rogen, Sao Paulo, Brazil), according to the manufacturer’s
instructions. The SuperScript II First-Strand Synthesis System
the synthesis of 20μL of complementary DNA (cDNA) from
1000ng of whole RNA. The mRNA levels of leptin (assay
Rn 00565158 mL—Applied Biosystems), resistin (assay Rn
Rn 00595250 mL—Applied Biosystems) were determined by
real-time PCR. Quantitative measurements were made with
the commercial kit TaqMan qPCR (Applied Biosystems),
system Applied Biosystems StepOne Plus. Cycling conditions
were as follows: enzyme activation at 50◦C for 2min,
denaturation at 95◦C for 10min, the cDNA products were
amplified for 40 cycles of denaturation at 95◦C for 15s, and
annealing/extension at 60◦C for 1min. Gene expression was
quantified in relation to the values of the C group after
normalization by an internal control (cyclophilin-assay Rn
00690933 mL—Applied Biosystems) by the method 2−ΔΔCT,
as previously described .
2.6. Statistical Analysis. Changes in body weight were evalu-
ated by a confidence interval of 95%. Gene expression, bio-
chemistry, and hormone data were analyzed using analysis
of variance (ANOVA) complemented by Bonferroni’s test.
The data are expressed as mean ± standard deviation. A 5%
significance level was adopted.
3.1. Evolution of Body Weight. All the animals had similar
The Scientific World Journal3
0246 8 10 12 14 16 18 20 22 24 26 28 30
Figure 1: Weekly evolution of body weight in the control group
(C, n = 10), obese group (OB, n = 10), and obese with 25μg
T3/100g BW (OT, n = 10). Data are expressed as means with a 95%
Table 1: Composition of body fat: retroperitoneal, epididymal,
visceral, and total fat deposits, and adiposity index.
13.3 ± 2.6b
22.1 ± 6.0b
12.9 ± 4.0b
47.7 ± 10.8b
7.6 ± 1.0b
Epid. fat (g)
Retro fat (g)
Visc. fat (g)
Total fat (g)
8.65 ± 1.8a
9.68 ± 3.2a
6.37 ± 1.5a
24.7 ± 5.6a
5.06 ± 1.1a
7.0 ± 1.5a
9.1 ± 3.9a
5.9 ± 1.8a
22.0 ± 6.8a
4.2 ± 1.4a
Epid. Fat: epididymal fat; Retro. Fat: retroperitoneal fat; Visc. Fat: visceral
fat; totalFat: totalbody fat; Adipos.I: adiposityindex;C: control; OB: obese;
OT: obese with 25μg T3/100gBW. Data expressed as mean ± standard
deviation. ANOVA was utilized, complemented by Bonferroni’s test. Use of
same letters represent P > 0.05; different letters represent P < 0.05.
Table 2: Biochemical analysis: glucose, triglycerides, and free fatty
93.2 ± 6.4a
82.2 ± 15.9a
0.54 ± 0.1a
95.0 ± 5.2a
82.5 ± 17.8a
0.51 ± 0.1a
103.1 ± 9.7b
73.2 ± 14.8a
0.72 ± 0.1b
TG: triglycerides; FFA: free fatty acids; C: control; OB: obese; OT: obese
with 25μg T3/100g BW. Data expressed as mean ± standard deviation.
represent P > 0.05; different letters represent P < 0.05.
were heavier than the C group. After 30 weeks of experiment,
than that of the C group (488g ± 11g). The BW of the OB
+ T3group (489g ± 16g) was significantly lower than the
OB group, but not significantly different from the C group
3.2. Total Body Fat. The hypercaloric diet increased fat de-
posits and the adiposity index. Administration of T3 de-
Similarly, T3 administration decreased total body fat and
adiposity index (Table 1).
3.3. Biochemical Analysis. Table 2 presents the values for
glucose, TG, and FFA. The hypercaloric diet did not alter
the biochemical parameters when compared to the C group.
There was an increase in glucose and FFA levels, but there
was no statistical difference in TG levels in the OT group in
comparison to the OB group.
3.4. Hormonal Measurements. The hypercaloric diet did not
change the thyroid profile; however, an increase in leptin
and resistin levels and a decrease in adiponectin serum
concentration was observed. In the OT group, T3levels were
elevated, yet the serum concentrations of free T4and TSH
were diminished (Table 3). Administration of T3reduced the
serum levels of adipokines, evaluated leptin (Figure 2(a)),
resistin (Figure 2(b)), and adiponectin (Figure 2(c)).
3.5. Gene Expression. Using real-time PCR, gene expression
was analyzed using 6 animals per group. The samples were
normalized by an internal control (cyclophilin), and the C
group was normalized by 1. The gene expression of leptin
(Figure 3(a)) and resistin (Figure 3(b)) was increased in the
OB group, but diminished in the OT group. Compared to
the C group, adiponectin gene expression (Figure 3(c)) was
decreased by hypercaloric diet. Compared to the OB group,
T3administration decreased adiponectin expression levels.
Obesity is a condition that has reached epidemic levels in
recent years . It is a complex disease, where lifestyle
interacts with genetic susceptibility to produce the obese
phenotype. Nowadays, the substantial rise in obesity indices
appears to be due to the lifestyle of the population, partic-
ularly due to inappropriate diets and the lack of physical
activity . Lifestyle is highly recognized as playing a central
role in the etiology of chronic diseases . Furthermore,
obesity is associated with several chronic diseases including
coronary arterial disease, hypertension, diabetes mellitus
type 2, and some forms of cancer .
The homogenous behavior of the animals is not assured
in experimental studies, even when they are maintained
under laboratory conditions. In this context, rats, given
normocaloric or hypercaloric rations in models of diet-
induced obesity, can present different responses with com-
mon characteristics . Thus, classification errors may
occur, such that animals submitted to a normocaloric diet
can be classified as controls, when in fact they exhibit
responses similar to animals that became obese via a
hypercaloric diet, or vice versa. For this reason, it becomes
necessary to establish a criterion that would enable the
separation of animals into control or obese. A study in
our laboratory showed that the best indicator of obesity is
bodily adiposity, but this index is obtained after the animal
is euthanized . However, BW, evaluated in vivo, presents
a good correlation with the adiposity index . In this
4The Scientific World Journal
Leptin serum concentration
Resistin serum concentration
C OB OT
Figure 2: Influence of different doses of T3on serum concentration of leptin (a), resistin (b), and adiponectin (c). C: control (n = 10);
OB: obese (n = 10); OT: obese with 25μg T3/100g BW (n = 10). Data expressed as mean ± standard deviation. ANOVA was utilized,
complemented by Bonferroni’s test. Use of same letters represent P > 0.05; different letters represent P < 0.05.
context, the control and obese groups were constituted by
applying BW as the classification criterion of the study.
Administration of supraphysiological doses of T3 aug-
mented the serum concentration of T3 . In the OT
animals, free T4and TSH levels were diminished compared
to OB animals, not surprisingly as exogenous T3suppresses
the endogenous secretion of TSH and T4by the thyroid ,
showing the effectiveness of treatment.
Hypercaloric diets induce accentuated weight gain and
adiposity. Consumption of diets rich in fat does not augment
lipid oxidation in the same proportion, which leads to the
elevation of body weight due to the deposition of triacylglyc-
erol in adipose tissue [30, 31]. The supraphysiological doses
of T3diminished the weight of OB animals and reduced the
adiposity index (Figure 1, Table 1). Hormones and cytokines
induce distinct metabolic responses in different fat deposits
, and this study shows a similar mobilization of all fat
deposits in the OT group (Table 1).
Thyroid hormones regulate the metabolism of lipids 
the plasmatic concentration of intermediate lipids and lipid
peroxidation . Concentrations of free fatty acids are used
to indicate the mobilization of fat . In the present study,
T3administration elevates lipolysis in the OT group but did
not alter TG levels (Table 2). These data are in agreement
with other reports of TG levels not being influenced by
thyroid hormone .
Excess of thyroid hormones augments plasma glucose
levels . The administration of T3produced a significant
diminution of plasma insulin levels in the animals treated
thyroxine (T4), and thyroid-stimulating hormone (TSH).
0.12 ± 0.03a
44.8 ± 1.2b
13.3 ± 0.8b
0.13 ± 0.06a
45.8 ± 1.2b
13.9 ± 0.9b
0.22 ± 0.04b
37.8 ± 1.8a
11.0 ± 1.2a
TSH: thyroid stimulating hormone; T3triiodothyronine; T4thyroxine; C:
control; OB: obese; OT: obese with 25μg T3/100g BW. Data expressed
as mean ± standard deviation. ANOVA was utilized, complemented by
Bonferroni’s test. Use of same letters represent P > 0.05; different letters
represent P < 0.05.
hormones on insulin sensitivity and glucose metabolism
remains controversial . However, one important factor
to highlight in this study is that the thyroid hormones induce
weight loss in a manner related to improving of resistance to
insulin (data not shown) .
Leptin can influence the interaction between genes and
environmental factors. Diets rich in fat raise leptin levels
can differentially affect body composition even with similar
diets. However, the rise in leptin levels is better explained
by increase in body fat . Experimental studies suggest
that sensitivity to leptin can be controlled by hormonal and
nutritional factors . The literature shows a clear positive
correlation between adipose tissue and leptin expression.
Diet-induced obesity elevates the gene expression of leptin
The Scientific World Journal5
Leptin gene expression
C OB OT
Resistin gene expression
C OB OT
Adiponectin gene expression
Figure 3: Influence of different T3doses on the gene expression of leptin (a), resistin (b), and adiponectin (c). C: control (n = 6); OB: obese
(n = 6); OT: obese with 25μg T3/100g BW (n = 6). Data expressed as mean ± standard deviation. ANOVA was utilized, complemented by
Bonferroni’s test. Use of same letters represent P > 0.05; different letters represent P < 0.05.
[41, 42]. However, the effects of T3on the gene expression
of leptin present inconsistent results; despite the in vitro
data showing that T3 produces a dose-dependent rise in
leptin expression , our data reveal that, following in
vivo hyperthyroidism, leptin gene expression was reduced.
In concordance, Pinkney et al.  and Zabrocka et al. 
observed a diminution of leptin expression in response to
treatment with T3.
The physiological role of adiponectin has not yet been
completely elucidated. Experimental data suggest that adi-
ponectin augments sensitivity to insulin and can present an-
established that adiponectin levels are inversely proportional
to the degree of adiposity , and weight loss elevates
the endogenous production of adiponectin . Here we
demonstrate that the OB group had decreased adiponectin
serum levels when compared to the C group, and the admin-
istration of T3, interestingly, even diminishing the body fat
mass, presented lower levels of adiponectin (Figures 2(c)
and 3(c)). Confirming this data Cabanelas et al.  show
reduced adiponectin gene expression in inguinal explants
of normal rats; also, we have demonstrate, recently that
adiponectin levels are decreased in calorie-restricted obese
rats . However, in contrast, an experimental study of rats
with hyperthyroidism showed an important rise in serum
adiponectin . Our data show that supraphysiological T3
doses alter adiponectin expression in obesity, suggesting that
T3causes undesirable effects on adipose tissue.
Resistin prejudices glucose homeostasis and insulin ac-
diary role between obesity and insulin resistance in rodents
although this role is still questioned in humans . In
this study, a hypercaloric diet increases the serum levels of
resistin, while T3treatment decreases it. The significant dim-
inution of serum resistin in the OT group corroborates
the first study performed on humans, in which patients
with hyperthyroidism exhibited low serum resistin concen-
trations . However, these initial findings contrast with
subsequent studies that report high resistin levels in hyper-
thyroidism patients [54, 55], showing a divergence in the
data. Nevertheless, resistin gene expression is almost unde-
tectable in rats with hyperthyroidism . Our results show
that administering supraphysiological doses of T3 decrease
resistin expression to the C group levels (Figure 3(b)).
The exogenous treatment with T3is effective in augmenting
serum levels of free T3 and diminishing concentrations of
free T4 and TSH. Administration of T3 promotes weight
loss and decreases adiposity. Following administration of
T3, there was a decrease in serum concentration of leptin,
resistin, and adiponectin, as well as a reduction in the gene
expression. Our data demonstrate that T3 acts, directly or
indirectly, on adipose tissue-derived adipokines which can
6The Scientific World Journal
insights regarding the relationship between T3and adipok-
ines in obesity model.
The authors thank Sandra F´ abio, Jos´ e Georgete, M´ ario
Bruno, and Sueli Clara for their technical assistance. They
are grateful to Dijon HS Campos and Ricardo L Damatto
for their contribution in the animals’ euthanasia. They
also thank FAPESP for the financial support (processes:
06/58177-0 and 07/50041-4). This manuscript has been
proofread and edited by native English speakers with related
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