Download Regulation of adiponectin gene expression in adipose tissue by

Regulation of adiponectin gene expression
in adipose tissue by thyroid hormones
Samira Seifi, Mohammad Reza
Tabandeh, Saed Nazifi, Mehdi Saeb,
Sadegh Shirian & Parisa Sarkoohi
Journal of Physiology and
Biochemistry
Official Journal of the University of
Navarra, Spain
ISSN 1138-7548
Volume 68
Number 2
J Physiol Biochem (2012) 68:193-203
DOI 10.1007/s13105-011-0131-1
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Author's personal copy
J Physiol Biochem (2012) 68:193–203
DOI 10.1007/s13105-011-0131-1
Regulation of adiponectin gene expression in adipose
tissue by thyroid hormones
Samira Seifi & Mohammad Reza Tabandeh &
Saed Nazifi & Mehdi Saeb & Sadegh Shirian &
Parisa Sarkoohi
Received: 5 August 2011 / Accepted: 15 November 2011 / Published online: 30 November 2011
# University of Navarra 2011
Abstract Available experimental data suggest that
adiponectin and thyroid hormones have biological
interaction in vivo. However, the effects of thyroid
hormones on adipose adiponectin gene expression in
thyroid dysfunction are unclear. We induced hyper(HYPER) and hypothyroidism (HYPO) by daily
S. Seifi
Department of Biochemistry, Research and Science Branch,
Islamic Azad University,
Tehran, Iran
M. R. Tabandeh (*) : P. Sarkoohi
Department of Biochemistry and Molecular Biology,
Faculty of Veterinary Medicine,
Shahid Chamran University of Ahvaz,
Ahvaz, Iran
e-mail: [email protected]
S. Nazifi
Department of Clinical Studies, School of Veterinary
Medicine, Shiraz University,
Shiraz, Iran
M. Saeb
Department of Biochemistry,
School of Veterinary Medicine, Shiraz University,
Shiraz, Iran
S. Shirian
Department of Phatobiology,
School of Veterinary Medicine, Shiraz University,
Shiraz, Iran
administration of a 12 mg/l of levothyroxine and 250
mg/l of methimazole in drinking water of rats, respectively, for 42 days. The white adipose tissues and serum
sample were taken on days 15, 28, 42 and also 2 weeks
after treatment cessation. Analysis of adiponectin gene
expression was performed by real-time PCR and 2−ΔΔct
method. The levels of adipose tissue adiponectin mRNA
in the HYPO rats were decreased during the 6-week
treatment when compared to control rats (<0.05) and
were increased significantly 2 weeks after HYPO
cessation (P<0.05). This decline in adiponectin gene
expression occurred in parallel with a decrease in T3,
T4, fT3 and fT4 concentrations (P<0.05). In opposite
to HYPO rats, adipose adiponectin gene expression
was increased in HYPER rats during the 6-week
treatment in parallel with an increase the thyroid
hormones concentrations (P<0.05), and its expression was decreased 2 weeks after HYPER cessation
(P<0.05). Adiponectin gene expression levels showed
significant negative correlations with concentrations of LDL (HYPO; r=−0.806, P=0.001 and
HYPER; r=−0.749, P=0.002), triglyceride (HYPO;
r=−0.825, P=0.001 and HYPER; r=−0.824, P=
0.001) and significant positive correlations with
concentrations of glucose (HYPO; r=0.674, P=
0.004 and HYPER; r=0.866, P=0.001) and HDL
(HYPO; r=0.755, P=0.001 and HYPER; r=0.839,
P=0.001). The current study provides evidence that
adiponectin gene expression in adipose tissue is
regulated by thyroid hormones at the translation level
Author's personal copy
194
and that lipid and carbohydrate disturbances in a
patient with thyroid dysfunction may be, in part, due
to adiponectin gene expression changes.
Keywords Adiponectin . Gene expression . Adipose
tissue . Hypothyroidism . Hyperthyroidism . Rat
Introduction
Adipose tissue was once known primarily as a storage
organ for excess energy in the form of triglycerides
[14]. However, during the past decade, this tissue is
known to secrete various hormones that are recognized as adipocytokines. Adiponectin is a recently
described adipocytokine with multiple functions [4].
This hormone plays an important role in the regulation of whole body energy homeostasis, insulin
sensitivity, lipid/carbohydrate metabolism [19] and
reproduction functions [31, 34, 35]. The thyroid
hormones thyroxin (T4) and triiodothyronine (T3)
also exert several important metabolic actions similar
to adiponectin [7, 10, 30]. Available experimental
data suggest that adiponectin and thyroid hormones
share some biological effects as insulin-sensitizing,
antiatherogenic and antiinflammatory properties [7, 19].
Furthermore, the levels of these hormones change in
different diseases such as cardiovascular diseases,
type 2 diabetes and obesity [14].
Adiponectin might participate in the regulation of
thyroid hormone production. The C-terminal globular
structure of adiponectin can use the gC1q receptor, a
molecule with broad tissue distribution [30]. Some
authors have suggested that adiponectin, via this
receptor found in the mitochondria of the thyroid
cells, may be a regulator of thyroid hormone
production [25]. In agreement with this hypothesis,
human studies have shown that healthy subjects with
high adiponectin levels had higher serum-free T4
levels [12].
Recent reports on the relationship between adiponectin and hypothyroidism have given conflicting
results. Most of the authors have reported that
adiponectin levels remain unmodified in patients with
thyroid hypofunction in comparison with euthyroid
subjects [5, 18, 27]. However, a few numbers of
studies have found low adiponectin levels in hypothyroid subjects [37]. Also, human studies evaluating
the circulating adiponectin in thyroid hyperfunction
S. Seifi et al.
have shown variable results. High adiponectin levels
have been reported accompanying the elevation of
thyroid hormone concentrations in hyperthyroid
patients by some investigators [26, 29], whereas other
authors have found no significant differences in serum
adiponectin between euthyroid subjects and hyperthyroid patients [18, 25, 27].
Most of the above studies measured only plasma
adiponectin levels and thus, could not precisely
identify the molecular mechanism of regulation of
adiponectin gene expression by thyroid hormones. At
the present time, little is known about the effects of
thyroid hormones on adiponectin gene expression in
adipose tissue and present data are conflicting. For
example, Fujimoto found that in cultures of brown
adipose tissue, thyroid hormone presented a small
stimulatory effect on adiponectin mRNA expression
and on hormone secretion [16]. However, T3 treatment did not have any effect on adiponectin gene
expression in 3T3-L1 adipocytes [11].
Up to now, the level of adiponectin gene expression in experimental animal models of hyper- or
hypothyroidism in adipose tissue has not been
studied. A more complete understanding of the
synthesis and regulation of adiponectin secretion by
thyroid hormones will likely lead to better approaches
for the management of obesity, type 2 diabetes,
atherosclerosis and cardiovascular diseases. The studies described herein were intended to examine the
mechanism of regulation of adiponectin gene expression in rat adipose tissue in both hypo- and
hyperthyroidism. We further evaluated the possible
relationship between adiponectin gene expression in
adipose tissue and some metabolic factors in different
thyroid dysfunctions by measurement of plasma
lipoproteins, glucose and NEFA.
Materials and methods
Animal and experimental design
Sixty male adult Sprague–Dawley rats (initial
body weight 200± 50 g) were obtained from the
breeding colony of the animal house of Shiraz
Medical University. Care and use of the laboratory
animals were in accordance with NIH guidelines.
They had free access to commercial chow and tap
water, in a temperature-controlled room (23± 1°C)
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Regulation of adiponectin expression by thyroid hormones
with a 12 h light:12 h darkness cycle (lights on at
07:00 h). The rats were allowed to acclimatize for
1 week before the beginning of the experiment.
They were randomly assigned to one of the three
groups: control or euthyroid (n=20), methimazoltreated group (hypothyroid group) (n = 20) and
levothyroxine-treated group (hyperthyroid group)
(n=20). Based on the duration of hypo- and hyperthyroidism, animals in each treated group were
divided into four subgroups, each composed of five
rats. These subgroups were named as Hyper 15
(hyperthyroidism for 15 days), Hyper 28 (hyperthyroidism for 28 days), Hyper 42 (hyperthyroidism for
42 days), Hypercut (2 weeks after levothyroxine
cessation), Hypo 15 (hypothyroidism for 15 days),
Hypo 28 (hypothyroidism for 28 days), Hypo 42
(hypothyroidism for 42 days) and Hypocut (2 weeks
after methimazol cessation).
Hyperthyroidism was induced by a daily administration of a 12-mg/l solution of levothyroxine in
drinking water during the experiment. To stimulate
the hypothyroid state, 250 mg/l of methimazole was
administered orally every day in drinking water over
the period of experiment. Body weight was measured
at the beginning and 42 days after treatment.
Tissue and serum isolation
Hypothyroid, hyperthyroid and euthyroid rats were
anesthetized (80 mg/kg ketamine hydrochloride and
10 mg/kg xylazine) and killed by decapitation. Five
milliliters of blood was collected by cardiac puncture
and immediately placed into tubes containing EDTA.
The plasma samples were separated by centrifuging
blood samples at 3,000 rpm for 30 min at 4°C and
were stored at −80°C for the subsequent assays. The
retroperitoneal (RET) white adipose tissues were
removed completely and weighed, immediately frozen in liquid nitrogen, stored at −80°C and finally
used for adiponectin mRNA quantification.
Hormone assay
Levels of total T3 and T4 and free T3 (fT3) and free
T4 (fT4) in serum of experimental animals were
determined with the radioimmunoassay kits (Immunotech., Radiová, Czech Republic) according to the
manufacturer’s recommendations. Hormone concentrations were expressed as nanomole per liter for total
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T3 and T4 and as picomole per liter for fT3 and fT4. The
limit of detection was 0.5 pmol/l for both fT3 and fT4.
The limit of detection was 0.5 pmol/l for both fT3 and
fT4. The intraassay coefficients of variation of fT3 and
fT4 were 6.4% and 8.3%, respectively. The interassay
coefficients of variation of fT3 and fT4 were 5.5% and
7.5%, respectively. The limits of detection of total T3
and T4 were 0.1 nmol/l and 13 nmol/l, respectively. The
intraassay and interassay coefficients of variation were
less than 6.2% and 9%, respectively.
Measurement of serum lipids and glucose
Blood glucose was determined by glucose oxidase
method (Ziest Chem, Tehran, Iran). Plasma NEFA
concentration was determined by colorimetric enzymatic assay (RANDOX Laboratories Ltd., Ardmore,
United Kingdom). Plasma triglycerides were measured by GPO-Trinder method (Ziest Chem, Tehran,
Iran) and total cholesterol was measured by CHOD–
PAP method (Eram Teb, Tehran, Iran). Following
precipitation of apoB containing lipoproteins, the
concentration of HDL-cholesterol (HDL-C) was measured by enzymatic colorimetric assay (Ziest Chem,
Tehran, Iran). After dissociation of HDL and VLDL
from LDL by precipitation, the concentration of LDLcholesterol was measured by subtraction of total
cholesterol from HDL and VLDL-cholesterol as
recommended by manufacturer’s procedure (Eram
Teb, Tehran, Iran). Inter- and intracoefficient of
variation (CV) for all parameters (except HDL) were
<3%. For HDL, inter and intraCV were <5%.
RNA isolation
Total RNA was extracted from collected rat adipose
tissue using RNX-Plus reagent according to the
manufacturer’s procedure (Cinnagen Inc., Tehran,
Iran). RNA was treated with DNase I (Fermentas
Inc., Vilnius, Lithuania) to remove any possible DNA
contamination, quantitated by spectrophotometry at 260
nm using the Biophotometer (Eppendorf, Hamburg,
Germany) and finally frozen at −70°C.
Reverse transcription–polymerase chain reaction
Reverse transcription was done in 20-μl volume using
RevertAid M-MuLV reverse transcriptase (Fermentas
Inc., Vilnius, Lithuania) as recommended by the
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196
S. Seifi et al.
manufacturer. PCRs were performed in a 25-μl
reaction using Taq DNA polymerase and an MJ Mini
thermal cycler (Bio-Rad Laboratories, Hercules, CA,
USA). Specific sets of primers (Macrogen, Seoul,
South Korea) that were used for amplification of rat
adiponectin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as calibrators are detailed in
Table 1. Thermal conditions for amplification of
adiponectin and GAPDH were 35 cycles consisting of
denaturing at 94°C for 30 s, specific annealing for 35 s,
and extension at 72°C for 30 s with an initial denaturing
step at 94°C for 5 min and a final extension step at 72°C
for 5 min. The annealing temperatures were 61.5 and
60°C for adiponectin and GAPDH, respectively. PCR
reactions for each gene were performed at the same time
and with the same batch of Taq DNA polymerase to
reduce the variations in PCR efficiency. A reaction
without cDNA was used as negative control. PCR
product was visualized in an agarose gel (2.5%) and
purified using QIAquick gel extraction kit (Qiagen,
Hilden, Germany), sequenced in both directions
(Macrogen), and its characteristics were determined
using Basic Local Alignment Search Tool (nBLAST) at
http://www.ncbi.nlm.nih.gov.
Quantitative real-time PCR with SYBR Green I®
To evaluate the level of adiponectin gene exprsession
in adipose tissue of different treated animals, quantitative real-time PCR (qRT-PCR) was performed using
the Mini Opticon real-time PCR detection system
(Bio-Rad Laboratories, USA) and qPCR™ Green
Master Kit for SYBR Green I® (Jena Biosciense,
Germany). Relative expression level of adiponectin
transcript was normalized to RNA loading for each
sample using GAPDH mRNA. The characteristics of
primers for qRT-PCR were described in Table 1. PCR
reactions were performed in a final volume of 20 μl in
a 96-well plate containing 2 μl cDNA, 1× buffer
Table 1 Sequences of
adiponectin and GAPDH
primers used in this study
Gene
Adiponectin
GAPDH
F forward primer, R reverse
primer
(10×), 1.5 mM MgCl2, 200 μM dNTPs, sense and
antisense primers (300 nM), 0.025 U/μl Taq DNA
polymerase and 1:66,000 SYBR Green I®. The
reactions were performed with the following settings:
10 min of preincubation at 95°C followed by 40
cycles for 15 s at 95°C and 1 min at 60°C. Reactions
were performed in triplicate. A reaction without
cDNA was performed in parallel as negative control.
Data analysis using the 2−ΔΔCt method
In this work, relative quantification was performed
according to the comparative 2−ΔΔCt method. This
method allows estimating gene copy numbers in
unknown samples and requires a housekeeping gene
of constant copy number in all samples, which
permits normalization of the quantitative data. In this
work, GAPDH served as the housekeeping gene in all
experiments. The ΔΔCt calculation for the relative
quantification of adiponecin gene in different treated
groups was used as follows ΔΔCt=(Ct, target gene−
CtX, GAPDH)x − (Ctc, target gene − Ct, GAPDH),
where x is the unknown sample in treated group and
c is the control sample. After validation of the
method, results for each sample were expressed in
N-fold changes in adiponectin gene expression of
group×copies, normalized to GAPDH. The result for
the gene expression was given by a unitless value
through the formula 2−ΔΔCt [24].
Validation of assay
It was necessary to check that the primers for the
housekeeping gene (GAPDH) and adiponectin had
similar amplification efficiencies. Therefore, each
primer set was prepared with serially diluted cDNA
(1, 1:2, 1:4, 1:6, and 1:8) followed by real-time PCR
in separate tubes. The difference of Ct values (ΔCt)
between the genes of interest and GAPDH for each
Sequence
Accession
number
Product
size (bp)
NM_144744
139
61.5
NM_017008
101
60
F
AATCCTGCCCAGTCATGAAG
R
CATCTCCTGGGTCACCCTTA
F
AGTTCAACGGCACAGTCAAG
R
TACTCAGCACCAGCATCACC
Tm (°C)
61.5
60
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Regulation of adiponectin expression by thyroid hormones
dilution was calculated and the resultant ΔCt values
were plotted against log dilutions. If the slope of the
plot of ΔCt values versus log cDNA dilutions for
each primer set was similar and approximately zero,
the amplification reactions of all genes had the same
efficiency and thus, the ΔΔCt calculation could be
applied (Dorak, 2006).
197
means followed by posthoc Tukey test. Correlation
between adiponectin expression and metabolic factors
were analyzed using Spearman rank correlation coefficients. All experimental data were presented as the
mean±SD. The level of significance for all tests was set
at P<0.05.
Softwares
Results
For DNA sequence analyses and for PCR primer and
probe designing, nBLAST (http://www.ncbi.nlm.nih.
gov) and Beacon designer 7.01 (PREMIER Biosoft
International, Palo Alto,CA, USA) were used, respectively. To analyze qRT-PCR results based on ΔΔCt
method, Opticon Monitor 3 software was used.
Statistical analyses
Data analyses were done using the SPSS 14.0 software
package (SPSS Inc., Chicago, IL, USA). One-way
ANOVA was used to test differences between various
Fig. 1 Adipose tissue
adiponectin gene expression
in hypothyroid (a) and
hyperthyroid (b) rats at days
15 (n=5), 28 (n=5), 42
(n=5) and 2 weeks after
treatment cessation (n=5) in
comparison to control
(untreated) groups. Different
letters above each bar
represent significant
difference at P<.05
Quantitation of adiponectin mRNA levels in adipose
tissue of hyper- and hypothyroid rats by quantitative
real-time PCR revealed that thyroid hormones significantly change adiponectin gene expression. As
shown in Fig. 1a, the level of adipose tissue
adiponectin mRNA in the hypothyroidism rats was
decreased during the 6-week treatment (P<0.05). The
levels of adiponectin mRNA in treated rats were
decreased by about 115% after 15 days, 162% after
28 days and 196% after 42 days, of those found in
control rats, respectively (P<0.05).
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198
S. Seifi et al.
To determine whether the changes in adiponectin
gene expression by hypothyroidism were reversible,
treatment cessation was done 15 days after the last
oral administration of methimazole. Adiponectin
mRNA levels in these rats increased significantly by
114% and 145% in comparison to methimazol-treated
rats for 28 and 42 days, respectively (P<0.05)
(Fig. 1).
During the 6-week treatment period, the hypothyroidism group treated with methimazole presented a
significantly (P<0.05) decreased T3, T4, fT3 and fT4
concentrations compared to the control group with the
progression of time (Table 2). Fifteen days after
treatment cessation, there was a remarkable elevation
in T3, T4, fT3 and fT4 concentrations (P<0.05)
(Table 2).
As shown in Fig. 1b, the levels of adipose tissue
adiponectin mRNA in the hyperthyroidism rats was
increased during the 6-week treatment with the
progression of time (P<0.05). The levels of adiponectin mRNA in treated rats were increased by 126%
after 15 days, 145% after 28 days and 175% after
42 days, of those found in control rats, respectively
(P<0.05). To determine whether hyperthyroidism
plays a role in the elevation of adiponectin mRNA
in adipose tissue of levothyroxine-treated rats, treatment cessation was done 15 days after the last oral
administration of levothyroxine. As shown in Fig. 1b,
adiponectin mRNA levels (P<0.05) decreased significantly by 122%, 143% and 163% after treatment
cessation in comparison to days 15, 28 and 42 of
levothyroxine treatment, respectively.
Hyperthyroid rats significantly (P<0.05) increased
their T3, T4, fT3 and fT4 concentrations after 15, 28
and 42 days of treatment (Table 3). Fifteen days after
treatment cessation, there was a remarkable reduction
Table 2 The mean±SD of
thyroid hormone concentrations
in hypothyroid rats at days 15
(n=5), 28 (n=5), 42 (n=5) and
2 weeks after treatment
cessation (n=5) in comparison
to control (untreated) groups
Day 15
Control
Day 28
Within a column, means
with different superscripts are
significantly different (P<0.05)
Hypo
Control
Day 42
a,b,c,d,e
Hypo
Day 55
in T3, T4, fT3 and fT4 concentrations (P<0.05)
(Table 3). These changes in thyroid hormone concentrations after hyperthyroidism induction and cessation
were in accordance with changes in adiponectin
mRNA levels in adipose tissue of treated rats.
The concentrations of plasma cholesterol, LDL and
triglycerides were increased (P<0.05) by treatment
with methimazole, while treatment with levothyroxine
resulted in reduction (P<0.05) of plasma concentrations of these components after 28 and 42 days of
treatment. The concentrations of these components
decreased (P<0.05) in hypothyroid rats and increased
(P<0.05) in hyperthyroid rats 15 days after treatment
cessation.
The concentrations of plasma HDL and NEFA were
decreased (P<0.05) in hypothyroid rats (Table 4),
while in hyperthyroid animals, the plasma concentrations of these components increased (P<0.05) after
28 and 42 days of treatment (Table 5). Treatment
cessation for 15 days caused an elevation (P<0.05) of
plasma concentrations of HDL and NEFA in
methimazole-treated rats (Table 4). The opposite
changes of these plasma components were shown in
levothyroxine treated groups 15 days after treatment
cessation (Table 5).
In hypothyroid rats, plasma glucose concentration
significantly decreased during the treatment (Table 4),
while in hyperthyroid rats, this plasma component
significantly (P<0.05) increased after 28 and 42 days
of treatment (Table 5). Treatment cessation for 15 days
caused an increase (P < 0.05) in plasma glucose
concentration in the hypothyroid group (Table 4)
and a decrease (P < 0.05) in this component in
hyperthyroid rats (Table 5).
Adiponectin gene expression levels in adipose
tissue of hypothyroid rats correlated positively with
T3 (nmol/l)
T4 (nmol/l)
fT3 (pmol/l)
fT4 (pmol/l)
1.02±0.21a
70.66±13.05a
2.29±0.92a
25.51±7.72a
b
0.97±0.27
c
0.55±0.20
b
0.93±0.27
3.92±.87
b
58.11±16.30
a
66.13±26.85
22.21±6.71a
c
16.12±4.68b
b
22.73±8.70a
d
1.50±0.49
3.69±0.94
0.31±0.10
28.60±12.85
1.17±0.69
10.01±4.60c
Control
1.03±0.39b
68.50±10.60a
3.99±1.49b
21.55±6.15a
e
b
a
22.52±6.52a
b
20.78±3.54a
Control
0.83±0.29
b
0.96±0.28
c
b
Hypo
Hypocessation
d
68.33±.95
a
60.07±21.08
66.27±13.11
a
2.25±0.78
3.48±0.74
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Regulation of adiponectin expression by thyroid hormones
Table 3 The mean±SD of
thyroid hormones concentrations
in hyperthyroid rats at days 15
(n=5), 28 (n=5), 42 (n=5) and
2 weeks after treatment
cessation (n=5) in comparison
to control (untreated) groups
199
T3 (nmol/l)
Day 15
Hyper
Control
Day 28
Day 42
Within a column, means
with different superscripts are
significantly different (P<0.05)
Day 55
fT3 (pmol/l)
fT4 (pmol/l)
1.42±0.27a
106±11.31a
2.65±0.82a
23±9.05a
b
b
b
22.2±6.71b
c
4.75±1.96
36.75±8.69c
22.73±8.70b
0.97±0.27
68.33±17.95
c
a
1.88±0.41
Control
0.93±0.27b
64±26.85b
3.69±0.94b
d
c
d
45.70±10.64d
b
21.55±6.15b
b
21.43±4.93b
b
20.78±3.54b
Hyper
117.2±24.75
3.92±0.87
Hyper
2.63±0.49
159.6±32.88
b
Control
a,b,c,d
T4 (nmol/l)
b
1.03±0.39
Hypercessation
68.50±10.60
a
d
1.16±0.21
87.66±21.54
b
Control
b
0.96 ±0.28
T3 (r=0.525, P=0.039), T4 (r=0.564, P=0.023), fT3
(r = 0.497, P = 0.05), fT4 (r = 0.535, P = 0.033)
(Table 6). In hyperthyroid rats, there were significant
correlations between adiponectin gene expression
levels in adipose tissue and T3 (r=0.808, P=0.01),
T4 (r=0.708, P=0.005), fT3 (r=0.716, P=0.002), fT4
(r=0.760, P=0.002) (Table 6).
Although plasma cholesterol concentrations had
negative correlation with adiponectin gene expression
levels in hypothyroid (r= −0.321, P=0.226) and
hyperthyroid rats (r=−0.450, P=0.106), these correlations were not significant (Table 6). Adiponectin
gene expression levels in adipose tissue of hypothyroid rats showed significant negative correlations with
concentrations of LDL (r=−0.806, P=0.001), triglyceride (r=−0.825, P=0.001) and significant positive
correlations with concentrations of glucose (r=0.674,
P=0.004) and HDL (r=0.755, P=0.001) (Table 6).
Highly significant positive correlations were found
between adiponectin gene expression levels in adipose tissue of hyperthyroid rats and plasma
63.27±13.11
6.9±2.08
3.99±1.49
3.84±0.55
3.48±0.74
concentrations of glucose (r=0.866, P=0.001) and
HDL (r=0.839, P=0.001) (Table 6). In addition,
plasma concentrations of LDL (r=−0.749, P=0.002)
and triglycerides (r=−0.824, P=0.001) had significant positive correlations with adiponectin gene
expression levels in adipose tissue of hyperthyroid
rats (Table 6). Although hypothyroid and hyperthyroid rats had lower and higher body weights in
comparison with euthyroid rats, respectively, these
differences were not significant.
Discussion
Although at the present time, there are numerous data
from clinical and experimental studies about the
changes of serum adiponectin concentrations in
different thyroid dysfunction, these data are
conflicting and little is known about the molecular
relationship between thyroid hormones actions and
the gene expression pattern of adiponectin in different
Table 4 The mean±SD of serum cholesterol, LDL, HDL, triglyceride, NEFA and glucose concentrations in hypothyroid rats at days
15 (n=5), 28 (n=5), 42 (n=5) and 2 weeks after treatment cessation (n=5) in comparison to control (untreated) groups
Cholesterol (mg/dl) LDL (mg/dl) HDL (mg/dl) Triglyceride (mg/dl) Glucose (mg/dl) NEFA (nmol/l)
Day 15 Hypo
Control
Day 28 Hypo
Control
Day 42 Hypo
Control
Day 55 Hypocessation
Control
a,b,c,d
60.18±1.97a
42.21±0.94a
37.49±1.46a
52.9±3.92a
69.96±1.43a
0.38±0.025a
41.03±1.42b
41.22±0.54a
38.44±0.77a
44.9±2.35b
70.27±1.88a
0.42±0.015b
c
b
27.14±5.48
b
c
b
0.25±0.036c
40.10±5.48
a
a
0.44±0.02b
25.34±2.28
b
a
76.44±2.72
b
40.77±1.31
d
115.9±21.3
b
53.8±1.04
a
42.23±1.01
c
58.02±0.84
a
69.02±4.87
62.11±2.52
b
69.46±0.72
d
c
43.86±0.59
92.85±5.43
b
0.2±0.026d
56.31±2.7
43.36±0.72
a
71.71±3.43
0.43±0.007b
0.24±0.026c
41.19±0.78
39.98±0.28
37.03±0.72
96.16±10.05e
42.14±1.6a
33.33±1.15a
53.74±5.13a
68.39±2.74a
a
b
a
b
39.65±1.74
a
40.26±1.23
37.24±1.65
71.28±2.32
Within a column, means with different superscripts are significantly different (P<0.05)
71.28±2.32
0.43±0.03b
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200
S. Seifi et al.
Table 5 The mean±SD of serum cholesterol, LDL, HDL, triglyceride, NEFA and glucose concentrations in hyperthyroid rats at days
15 (n=5), 28 (n=5), 42 (n=5) and 2 weeks after treatment cessation (n=5) in comparison to control (untreated) groups
Cholesterol (mg/dl) LDL (mg/dl) HDL (mg/dl) Triglyceride (mg/dl) Glucose (mg/dl) NEFA (nmol/l)
39.42±0.63a
Day 15 Hyper
Control
b
41.03±1.42
41.22±0.54
34.2±1.71b
Day 28 Hyper
b
40.77±1.31
42.23±1.01
c
Day 42 Hyper
b
39.98±0.28
b
a
32.18±1.25
38.1±1.2
39.65±1.74a
Control
73.19±2.16a
0.49±0.021a
b
44.9±2.35
71.27±1.88
a
0.42±0.015b
34.64±2.03c
79.29±1.92b
0.57±0.029c
b
69.46±0.72
a
0.44±0.02b
85.37±3.02
c
0.64±0.031d
73.71±3.43
a
0.43±0.007b
39.11±1.47
73.21±2.88
a
0.54±0.032c
42.28±1.65b
71.28±2.32a
38.44±0.77
b
40.10±5.48
d
33.12±2.41
a
41.19±0.78
Day 55 Hypercessation
40.04±0.33a
b
c
28.73±1.45
Control
45.34±1.72a
32.07±1.23c 54.67±1.77c
a
Control
a,b,c,d
36.62±0.7a
a
62.1±1.76
b
37.03±0.72
50.36±2.52
40.26±1.23b 37.24±1.65b
Table 6 Multiple correlations performed between adipose
tissue adiponectin gene expression and T3, T4, fT3, fT4,
cholesterol, LDL, HDL, triglyceride, NEFA and glucose
concentrations in hypo- and hyperthyroid rats. Correlation
coefficient (r) and statistical significance (P) are indicated
Adipose tissue adiponectin mRNA
Variables
Hypothyroidism
Hyperthyroidism
r
r
p
p
T3 (nmol/l)
0.525*
0.039
0.808**
0.01
T4 (nmol/l)
*
0.564
0.023
0.708**
0.005
fT3 (pmol/l)
0.497
0.05
0.716**
0.002
fT4 (pmol/l)
0.535*
0.033
0.760**
0.002
Cholesterol (mg/dl)
−0.321
0.226
−0.450
0.106
LDL (mg/dl)
−0.806**
0.001
−0.749**
0.002
HDL (mg/dl)
0.755**
0.001
0.839**
0.001
−0.825**
0.001
−0.824**
0.001
0.367
0.161
0.757**
0.002
0.004
**
0.001
Triglyceride (mg/dl)
NEFA (nmol/l)
Glucose (mg/dl)
**
0.674
P=0.05 (statistical significance)
**
c
33.69±1.28
b
43.36±0.72
a
0.43±0.03b
Within a column, means with different superscripts are significantly different (P<0.05)
tissues. To our knowledge, this is the first in vivo
study determining whether the expression of adiponectin is altered in adipose tissue by experimental
hypo- and hyperthyroidism.
We observed that adiponectin gene expression level
in adipose tissue of hypothyroid rats decreased by
the 6-week methimazol treatment. Two weeks after
treatment cessation, the level of adiponectin gene
expression in adipose tissue of these animals begin to
rise. This response is probably due to either a direct
*
c
43.86±0.59
P=0.01 (statistical significance)
0.866
effect of thyroid hormones on adiponectin regulation or
to weight changes induced by thyroid hormones. The
latter seems unlikely, since in our hypo- and hyperthyroid rats, a significant weight change was not observed.
Our results indicate that hyperthyroidism induction
by the 6-week levothyroxine treatment resulted in a
concomitant increase in adiponectin gene expression
level in rat adipose tissue. These changes in adiponectin mRNA level following hyperthyroidism induction were reversible after 2-week treatment cessation.
Changes in adiponectin mRNA levels in adipose
tissue of hypo- and hyperthyroid rats were in
accordance with changes in thyroid hormone concentrations after hypo- and hyperthyroidism induction
and cessation. The concentrations of serum thyroid
hormones including T3, T4, fT3 and fT4 increased
during the 6-week hypothyroid state progression and
begin to decline 2 weeks after hypothyroidism
cessation. We observed opposite patterns in thyroid
hormone concentrations in hyperthyroid rats. These
changes in adiponectin gene expression in adipose
tissue of hypo- and hyperthyroid rats suggest that
thyroid hormones act at the translation level to
regulate the adiponectin production in vivo.
Few studies have shown that thyroid hormones had
different, and sometimes, opposite effects on adiponectin gene expression in different adipose cell lines.
In agreement with our data, Fujimoto et al. found that
in cultures of brown adipose tissue, thyroid hormone
presented a small stimulatory effect on adiponectin
messenger RNA expression and on hormone secretion
[16]. However Fasshauer reported that addition of T3
to the culture medium of 3T3-L1 adipocytes did not
have any effect on adiponectin gene expression [11].
Author's personal copy
Regulation of adiponectin expression by thyroid hormones
It has been reported that thyroid hormones can induce
the expression of different signal transduction regulatory factors including peroxisome proliferator-activated
receptors (PPARs) [32], mature sterol regulatory
element-binding protein-1 (SREBP-1) [39] and adipocyte determination and differentiation-dependent
factor 1/sterol regulatory element-binding protein 1c
transcription factor (ADD1/SREBP1c) [39]. These
regulatory factors can bind the promoter regions of
several lipogenic genes such as adiponectin and can
controls adiponectin gene expression in differentiated
adipocytes [28, 38]. For example, PPARγ stimulation
increases serum adiponectin by transcriptional induction
in adipose tissue because there is a functional PPARresponsive element in human adiponectin promoter
[38]. With this background, we hypothesize that changes
in adiponectin gene expression that occurs during the
experimental hypo- and hyperthyroidism in adipose
tissue may be due to direct action of thyroid hormones
on PPARs, SREBP-1 and ADD1/SREBP1c expression.
To date, no previous studies have evaluated the effects
of thyroid hormones on adiponectin gene expression;
however, there are numerous conflicting reports about
the effects of hypo- and hyperthyroidism on the serum
adiponectin in human and animal. For example, human
studies have shown that healthy subjects with high
adiponectin levels had higher serum-free T4 levels [12].
In rats with propylthiouracil-induced hypothyroidism,
serum adiponectin levels were significantly increased
as compared to untreated rats [3, 20]. These serum
adiponectin increases in hypothyroid rats may be as
an adaptive response to reduce body weight associated with thyroid hormone changes because adiponectin
concentrations have inverse relationship with body
weight changes [4, 17]. Experimental hypothyroidism
induced by methimazole treatment in rats was not
accompanied by any significant change in serum
adiponectin concentrations [3]. Serum adiponectin
concentrations have been studied in patients with
hypothyroidism, and most of the authors have
reported that adiponectin levels remain unmodified
in patients with thyroid hypofunction in comparison
with euthyroid subjects [5, 18, 27]. However, a few
numbers of studies have found low adiponectin levels
in hypothyroid subjects [25, 27]. Iglesias et al .
reported that in hypothyroid subjects, restoration of
normal thyroid hormone levels after levothyroxine
therapy was not accompanied by significant changes
in circulating adiponectin levels [18].
201
Human studies evaluating to circulating adiponectin in thyroid hyperfunction have shown variable
results. High adiponectin levels have been reported
accompanying the elevation of thyroid hormone
concentrations in hyperthyroid patients by some
investigators [26, 29, 37], whereas other authors have
found no significant differences in serum adiponectin
between euthyroid and hyperthyroid patients [2, 18,
25]. Data from animal investigation showed that
serum adiponectin levels in levothyroxine-treated rats
increased in comparison to euthyroid rats [3], whereas
Kokkinos et al. reported that serum adiponectin levels
were not significantly altered in the plasma of the
hyperthyroid rats [20].
These results demonstrate that adipocytes show
different responses to thyroid hormone in different in
vivo and in vitro conditions. In addition, discrepancies among the studies in humans, as mentioned
above, may be due to different etiology of thyroid
dysfunction in the analyzed population.
Thyroid hormones appear to be important regulators of lipid and carbohydrate metabolism. Hyper- or
hypocholestremia, triglyceride and glucose level
changes are usually associated with thyroid dysfunction in human and animals [33, 36]. However, precise
molecular mechanisms of these disturbances are still
poorly understood. One major finding of our study
was the decrease in the serum concentrations of
cholesterol, LDL, triglyceride and glucose and the
increase of HDL serum concentrations in parallel with
the increase in thyroid hormone concentrations and
adiponectin gene expression levels in hyperthyroid
rat. As far as we know, adiponectin can induce the
stimulation of glucose uptake in muscle, fatty acid
oxidation in muscle and liver and can inhibit hepatic
glucose production, cholesterol and triglyceride synthesis and lipogenesis [8, 13, 36, 38]. Taking these
findings together, we hypothesized that, in part, some
biological effects of thyroid hormones on lipid and
carbohydrate metabolism are modulated by changes
in adiponectin production from adipose tissue. In
agreement with this hypothesis, we found that a
decrease in adiponectin gene expression in adipose
tissue of hypothyroid rats was associated with
increased plasma cholesterol, LDL, triglycerides and
glucose concentrations and with decreased serum
HDL concentration. This view is supported by recent
reports in which low adiponectin levels significantly
predict the risk to develop lipid and carbohydrate
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202
disturbances such as insulin resistance and type 2
diabetes [21–23].
Numerous evidence supports that hypothyroidism
is associated with atherosclerosis, ischemic heart
diseases, atherogenic lipid profile, diastolic hypertension and impaired endothelial function [1, 9, 15].
Recent in vivo studies have shown that adiponectin
attenuate formation of atherosclerotic plaques in the
injured vessel wall by suppression of the expression
of VCAM-1 and class A scavenger receptors [36] and
endothelial nitric oxide (NO) generation [6]. Based on
our data and the above-mentioned evidence, we
suggest that decreased adiponectin gene expression
in adipose tissue of hypothyroid subjects is likely to
be important in the pathophysiology of atherosclerosis
in these patients.
The current study provides evidence that adiponectin gene expression in adipose tissue is regulated
by thyroid hormones at the translation level. In
addition, we observed a close relationship between
adipose adiponectin gene expression in thyroid
dysfunctions with serum carbohydrate and lipid
disturbances. This finding supports the notion that
induction or suppression of adiponectin gene expression or secretion in patients with thyroid dysfunction
may be a candidate novel therapeutic protocol in these
patients. Moreover, additional research is needed to
confirm whether adiponectin and its receptor gene
expression are regulated in different tissues such as
muscle and liver by thyroid hormones.
Acknowledgment This study was financially supported by
the Shahid Chamran University of Ahvaz (Grant No. 89/3/02/
44305) and Shiraz Unuversity.
References
1. Agdeppa D, Macaron C, Mallik T, Schnuda ND (1979)
Plasma high density lipoprotein cholesterol in thyroid
disease. J Clin Endocrinol Metnb 49:726–729
2. Altinova AE, Toruner FB, Akturk M, Bukan N, Cakir N,
Ayvaz G, Arslan M (2006) Adiponectin levels and
cardiovascular risk factors in hypothyroidism and hyperthyroidism. Clin Endocrinol 65:530–535
3. Aragao CN, Souza LL, Cabanelas A, Oliveira KJ, PazosMoura CC (2007) Effect of experimental hypo- and
hyperthyroidism on serum adiponectin. Metabolism 56:6–
11
4. Berg AH, Combs TP, Scherer PE (2002) ACRP30/
adiponectin: an adipokine regulating glucose and lipid
metabolism. Trends Endocrinol Metab 13:84–89
S. Seifi et al.
5. Botella-Carretero JI, Alvarez-Blasco F, Sancho J, EscobarMorreale HF (2006) Effects of thyroid hormones on serum
levels of adipokines as studied in patients with differentiated thyroid carcinoma during thyroxine withdrawal.
Thyroid 16:397–402
6. Chen H, Montagnani M, Funahashi T, Shimomura I, Quon
MJ (2003) Adiponectin stimulates production of nitric
oxide in vascular endothelial cells. J Biol Chem
278:45021–45026
7. Dimitriadis G, Baxter B, Marsh H, Mandarino L, Rizza R,
Bergman R, Haymond M, Gerich J (1985) Effect of thyroid
hormone excess on action, secretion, and metabolism of
insulin in human. Am J Physiol 248:593–601
8. Dogru T, Sonmez A, Tasci I, Yilmaz MI, Kilic S, Ozgurtas
T, Eyileten T, Erbil MK, Kocar IH (2006) Plasma
adiponectin and insulin resistance in new onset hypertension. Endocrine 29:405–408
9. Dunajska K, Milewicz A, Jcedrzejuk D, Szymczak J,
Kuliczkowski W, Salomon P, Biały D, Poczcatek K,
Nowicki P (2004) Plasma adiponectin concentration in
relation to severity of coronary atherosclerosis and cardiovascular risk factors in middle-aged men. Endocrine
25:215–221
10. Duntas LH (2002) Thyroid disease and lipids. Thyroid
12:287–293
11. Fasshauer M, Klein J, Neumann S, Eszlinger M, Paschke R
(2002) Hormonal regulation of adiponectin gene expression
in 3T3-L1 adipocytes. Biochem Biophys Res Commun
290:1084–1089
12. Fernández-Real JM, López-Bermejo A, Casamitjana R,
Ricart W (2003) Novel interactions of adiponectin with the
endocrine system and inflammatory parameters. J Clin
Endocrinol Metab 88:2714–2718
13. Fruebis J, Tsao TS, Javorschi S, Ebbets-Reed D, Erickson
MR, Yen FT, Bihain BE, Lodish HF (2001) Proteolitic
cleavage product of 30-kDa adipocyte complement-related
protein increases fatty acid oxidation in muscle and causes
weight loss in mice. Proc Natl Acad Sci USA 98:2005–
2010
14. Fruhbeck G, Gomez-Ambrosi J, Muruzabal FJ, Burrell MA
(2001) The adipocyte: a model for integration of endocrine
and metabolic signaling in energy metabolism regulation.
Am J Physiol 280:827–847
15. Fortuño A, Rodríguez A, Gómez-Ambrosi J, Frühbeck G,
Díez J (2003) Adipose tissue as an endocrine organ: role of
leptin and adiponectin in the pathogenesis of cardiovascular
diseases. J Physio Biochem 59:51–60
16. Fujimoto N, Matsuo N, Sumiyoshi H, Yamaguchi K,
Saikawa T, Yoshimatsu H, Yoshioka H (2005) Adiponectin
is expressed in the brown adipose tissue and surrounding
immature tissues in mouse embryos. Biochim Biophys
Acta 1731:1–12
17. Heilbronn LK, Smith SR, Ravussin E (2003) The insulinsensitizing role of the fat derived hormone adiponectin.
Curr Pharm Des 9:1411–1418
18. Iglesias P, Alvarez FP, Codoceo R, Diez JJ (2003) Serum
concentrations of adipocytokines in patients with hyperthyroidism and hypothyroidism before and after control of
thyroid function. Clin Endocrinol 59:621–629
19. Kershaw EE, Flier JS (2004) Adipose tissue as an
endocrine organ. J Clin Endocrinol Metab 89:2548–2556
Author's personal copy
Regulation of adiponectin expression by thyroid hormones
20. Kokkinos A, Mourouzis I, Kyriaki D, Pantos C, Katsilambros
N, Cokkinos DV (2007) Possible implications of leptin,
adiponectin and ghrelin in the regulation of energy homeostasis by thyroid hormone. Endocr 32:30–32
21. Kubota N, Terauchi Y, Yamauchi T, Kubota T, Moroi M,
Matsui J, Eto K, Yamashita T, Kamon J, Satoh H, Yano W,
Nagai R, Kimura S, Kadowaki T, Noda T (2002)
Disruption of adiponectin causes insulin resistance and
neointimal formation. J Biol Chem 277:25863–25866
22. Okamoto Y, Kihara S, Ouchi N, Nishida M, Arita Y,
Kumada M, Ohashi K, Sakai N, Shimomura I, Kobayashi
H, Terasaka N, Inaba T, Funahashi T, Matsuzawa Y (2002)
Adiponectin reduces atherosclerosis in apolipoprotein Edeficient mice. Circulation 106:2767–2770
23. Pérez-Echarri N, Pérez-Matute P, Martínez JA, Marti A,
Moreno-Aliaga MJ (2005) Serum and gene expression levels
of leptin and adiponectin in rats susceptible or resistant to dietinduced obesity. J Physio Biochem 61:333–342
24. Pfaffl MW (2001) A new mathematical model for relative
quantification in real-time RT-PCR. Nucleic Acids Res
29:45
25. Pontikides N, Krassas GE (2007) Basic endocrine products
of adipose tissue in states of thyroid dysfunction. Thyroid
17:421–431
26. Saito T, Kawano T, Saito T, Ikoma A, Namai K, Tamemoto
H, Kawakami M, Ishikawa S (2005) Elevation of serum
adiponectin levels in Basedow disease. Metabolism
54:1461–1466
27. Santini F, Marsili A, Mammoli C, Valeriano R, Scartabelli G,
Pelosini C, Giannetti M, Centoni R, Vitti P, Pinchera A (2004)
Serum concentrations of adiponectin and leptin in patients
with thyroid dysfunctions. J Endocrinol Invest 27:5–7
28. Seo JB, Moon HM, Noh MJ, Lee YS, Jeong HW, Yoo EJ,
Kim WS, Park J, Youn BS, Kim JW, Park SD, Kim JB
(2004) Adipocyte determination- and differentiationdependent factor 1/sterol regulatory element-binding protein 1c regulates mouse adiponectin expression. J Biol
Chem 279:22108–22117
29. Sieminska L, Niedziolka D, Pillich A, Kos-Kudla B, Marek
B, Nowak M, Borgiel-Marek H (2008) Serum concentrations of adiponectin and resistin in hyperthyroid Graves’
disease patients. J Endocrinol Invest 31:745–749
30. Soltys BJ, Kang D, Gupta RS (2000) Localization of P32
protein (gC1q-R) in mitochondria and at specific extramitochondrial locations in normal tissues. Histochem Cell
Biol 114:245–55
203
31. Tabandeh MR, Hosseini A, Saeb M, Kafi M, Saeb S (2010)
Changes in the gene expression of adiponectin and
adiponectin receptors (AdipoR1 and AdipoR2) in ovarian
follicular cells of dairy cow at different stages of
development. Theriogenology 73:659–669
32. Weitzel JM, Hamman S, Jauk M, Lacey M, Filbry A,
Radtke C, Iwen KA, Kutz S, Harneit A, Lizardi PM, Seitz
HJ (2003) Hepatic gene expression patterns in thyroid
hormone-treated hypothyroid rats. J Mol Endocrinol
31:291–303
33. Wilcox HG, Keyes WG, Hale TA, Frank R, Morgan DW,
Heimberg M (1982) Effects of triiodothyronine and
propylthiouracil on plasma lipoproteins in male rats. J
Lipid Res 23:1159–1166
34. Wu X, Motoshima H, Mahadev K, Stalker TJ, Scalia R,
Goldstein BJ (2003) Involvement of AMP-activated protein
kinase in glucose uptake stimulated by the globular domain
of adiponectin in primary rat adipocytes. Diabetes
52:1355–1363
35. Yamauchi T, Kamon J, Minokoshi Y, Ito Y, Waki H,
Uchida S, Yamashita S, Noda M, Kita S, Ueki K, Eto K,
Akanuma Y, Froguel P, Foufelle F, Ferre P, Carling D,
Kimura S, Nagai R, Kahn BB, Kadowaki T (2002)
Adiponectin stimulates glucose utilization and fatty-acid
oxidation by activating AMP-activated protein kinase.
Nature Med 8:1288–1295
36. Yamauchi T, Kamon J, Waki H, Imai Y, Shimozawa N,
Hioki K, Uchida S, Ito Y, Takakuwa K, Matsui J, Takata M,
Eto K, Terauchi Y, Komeda K, Tsunoda M, Murakami K,
Ohnishi Y, Naitoh T, Yamamura K, Ueyama Y, Froguel P,
Kimura S, Nagai R, Kadowaki T (2003) Globular adiponectin protected ob/ob mice from diabetes and apoEdeficient mice from atherosclerosis. J Biol Chem
278:2461–2468
37. Yaturu S, Prado S, Grimes SR (2004) Changes in adipocyte
hormones leptin, resistin, and adiponectin in thyroid
dysfunction. J Cell Biochem 93:491–496
38. Yilmaz MI, Sonmez A, Caglar K, Gok DE, Eyileten T,
Yenicesu M, Acikel C, Bingol N, Kilic S, Oguz Y (2004)
Peroxisome proliferator-activated receptor γ (PPAR-γ)
agonist increases plasma adiponectin levels in type 2
diabetic patients with proteinuria. Endocrine 25:207–214
39. Zhang Y, Yin L, Hillgartner FB (2003) SREBP-1 integrates
the actions of thyroid hormone, insulin, cAMP, and
medium-chain fatty acids on ACCalpha transcription in
hepatocytes. J Lipid Res 44:356–368