J C E M
O N L I N E
Hot Topics in Translational Endocrinology—Endocrine Research
Elevated Endothelin-1 (ET-1) Levels May Contribute to
Hypoadiponectinemia in Childhood Obesity
Carmela Nacci, Valentina Leo, Leonarda De Benedictis, Maria Rosaria Carratù,
Nicola Bartolomeo, Maria Altomare, Paola Giordano, Maria Felicia Faienza, and
Monica Montagnani
Department of Biomedical Sciences and Human Oncology, University of Bari, Medical School, 70124
Bari, Italy
Context: Pediatric obesity is associated with endothelial dysfunction and hypoadiponectinemia,
but the relationship between these two conditions remains to be fully clarified. Whether enhanced
release of endothelin-1 (ET-1) may directly impair adiponectin (Ad) production in obese children
is not known.
Objective: The aim of the study was to explore whether and how high circulating levels of ET-1 may
contribute to impair Ad production, release, and vascular activity.
Design and Participants: Sixty children were included into obese (Ob; n ⫽ 30), overweight (OW; n ⫽
11), and lean (n ⫽ 19) groups. Total and high-molecular-weight Ad, ET-1, vascular cell adhesion
molecule-1, and von Willebrand factor levels were measured in serum samples. Adipocytes were
stimulated with exogenous ET-1 or with sera from lean, OW, and Ob, and Ad production and
release measured in the absence or in the presence of ETA (BQ-123) and ETB (BQ-788) receptor
blockers, p42/44 MAPK inhibitor PD-98059, or c-Jun NH2-terminal protein kinase inhibitor SP600125. Vasodilation to Ad was evaluated in rat isolated arteries in the absence or in the presence
of BQ-123/788.
Results: Total and high-molecular-weight Ad was significantly decreased and ET-1 levels significantly increased in OW (P ⬍ .01) and Ob (P ⬍ .001) children. A statistically significant linear regression (P ⬍ .01) was found between Ad and ET-1. Exposure of adipocytes to exogenous ET-1 or
serum from OW and Ob significantly decreased Ad mRNA and protein levels (P ⬍ 0.001). The
inhibitory effect of ET-1 on Ad was reverted by BQ-123/788 or PD-98059 but not SP-600125. Admediated vasodilation was further increased in arteries pretreated with BQ-123/788.
Conclusions: ET-1-mediated inhibition of Ad synthesis via p42/44 MAPK signaling may provide a
possible explanation for hypoadiponectinemia in pediatric obesity and contribute to the development of cardiovascular complications. (J Clin Endocrinol Metab 98: E683–E693, 2013)
C
hildhood overweight or obesity is associated with adverse long-term outcomes (1, 2) and predicts the development of type 2 diabetes mellitus and cardiovascular
disease (3, 4). Obesity in children has been repeatedly and
independently correlated to markers of endothelial activation, although the relationship between these two con-
ditions remains to be fully clarified (5). Because the cross
talk between the vasculature and the adipose tissue is
known to play a key role in maintaining vascular and
metabolic asset (6), it is likely that dysregulation of the
vascular-adipocyte axis may importantly contribute to
both endothelial dysfunction and metabolic disturbances.
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2013 by The Endocrine Society
Received December 6, 2012. Accepted February 11, 2013.
First Published Online March 1, 2013
Abbreviations: Ach, acetylcholine; Ad, adiponectin; BMI, body mass index; BP, blood pressure; CRP, C-reactive protein; ET-1, endothelin-1; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; HMW, high molecular weight; JNK, c-Jun NH2-terminal protein kinase;
LDL, low-density lipoprotein; LMW, low molecular weight; L-NAME, N-omega-nitro-Larginine methyl ester; MMW, middle molecular weight; MVB, mesenteric vascular bed; NO,
nitric oxide; QUICKI, quantitative insulin-sensitivity check index; SDS, SD score; VCAM,
vascular cell adhesion molecule; vWF, von Willebrand factor.
doi: 10.1210/jc.2012-4119
J Clin Endocrinol Metab, April 2013, 98(4):E683–E693
jcem.endojournals.org
E683
E684
Nacci et al
ET-1 Impairs Adiponectin via MAPK Signaling
For example, adipocyte hypertrophy and hyperplasia
leads to elevated production of proinflammatory adipokines that result in endothelial activation with detrimental
effects in the vasculature (7). On the other hand, impaired
or unbalanced production of endothelial mediators with
effects on metabolic tissues may further exacerbate abnormal adipocyte function (8 –10).
Among endothelial mediators, the potent vasoconstrictor endothelin-1 (ET-1) has been implicated in the development of hypertension, atherosclerosis, and cardiovascular disease (11). Interestingly, circulating levels of ET-1
are increased not only in patients with cardiovascular disturbances but also in obese and diabetic subjects (12, 13).
ET-1 infusion results in hyperinsulinemia and insulin resistance in vivo (14), and chronically elevated ET-1 levels
may contribute to the desensitization of metabolic signaling pathways on adipocytes (15). In addition, ET-1 inhibits adipocyte differentiation (16), reduces lipoprotein
lipase activity (17), inhibits insulin-stimulated glucose uptake (18), and stimulates lipolysis (19). Previous studies
suggest that ET-1 may regulate adiponectin (Ad) production and secretion from adipocytes (20, 21).
Unlike adipokines such as TNF␣ and IL-1, Ad exerts
beneficial vasodilatant and antiinflammatory activities in
the vascular compartment. We and others have demonstrated that Ad directly modulates cardiovascular function through nitric oxide (NO)-dependent (22, 23) and
cyclooxygenase-2-dependent (24, 25) regulatory mechanisms. Disruption of Ad results in impaired endotheliumdependent vasodilation (26) and neointimal formation
(27). In addition, Ad has a regulatory suppressive role on
ET-1-induced cardiomyocyte hypertrophy (28) and significantly limits the hemodynamic and metabolic activity
of ET-1 (29). Consistent with these findings, reduced Ad
levels are associated with accelerated atherosclerosis, hypertension, and coronary artery disease (30 –32).
Dysregulated Ad production in obese adults, adolescents, and children (33, 34) has been linked to increased
levels of TNF␣ (35) and IL-1 (36), but several other factors, including vascular mediators, may contribute to reduce Ad expression and secretion. Our hypothesis is that
the enhanced release of endothelial ET-1 may directly participate in the impaired synthesis of Ad in adipose tissue
under obesity. Therefore, our primary aim was to study
the correlation between circulating levels of ET-1 and Ad
and consequently explore the mechanisms underlying the
inhibitory role of ET-1 on Ad production in prediabetic
obese children.
J Clin Endocrinol Metab, April 2013, 98(4):E683–E693
the University Pediatric Clinic between March 2011 and April
2012. Participants were included into obese (Ob; 30 subjects; 17
males), overweight (OW; 11 subjects; 7 males), and lean (19
subjects, 10 males) groups. Physical activity was not evaluated.
Exclusion criteria were the presence of renal, liver, and/or cardiovascular diseases; hypertension; metabolic and/or endocrine
disorders; genetic syndromes; and the histories of chronic allergies, acute infectious or inflammatory diseases during the last 3
months preceding the study. None of the subjects were taking
any form of medication. Informed assent and consent were obtained from the parents and children. All procedures were in
accordance with the Helsinki Declaration on Human Experimentation and were approved by the local ethic committee.
Anthropometric measurements
Standing height (centimeters) was measured by a wallmounted Harpenden stadiometer and weight (kilograms) measured by an electronic scale with digital readings accurate to 0.1
kg. Body mass index (BMI) was calculated according to standard
methods. International standards for sex- and age-specific BMI
centiles for subjects aged 2–18 years were used (37). The 95th
centile of the BMI reference was the cutoff point for childhood
obesity. OW and Ob children were defined as those with a BMI
higher than the centile curves. The BMI SD score (SDS) was
derived from the available Centers for Disease Control and Prevention standards (38). Blood pressure was measured by a standard Riva-Rocci sphygmomanometer with appropriate size cuff,
and the two last measurements were averaged (39).
Laboratory procedures
After an overnight fast, blood samples were taken from Ob,
OW, and lean subjects. Plasma or serum aliquots were frozen at
⫺80°C until determination. All analyses were performed within
5 months from blood collection in a blinded fashion, and the
intra- and interassay coefficient of variation was less than 8% for
all assays.
Metabolic and endothelial markers
Total, high-density lipoprotein and low-density lipoprotein
(LDL) cholesterol, triglycerides, and glucose concentrations
were measured by an automated analyzer (Roche Diagnostics,
Mannheim, Germany). Serum insulin was measured by a chemiluminescent assay (Diagnostic Products Corp, Los Angeles,
California). Total adiponectin and multimeric high-molecularweight (HMW), middle-molecular-weight (MMW), and lowmolecular-weight (LMW) subfractions were measured by a
commercial ELISA (ELISA 47-ADPH-9755; ALPCO Diagnostics, Salem, Vermont). Endothelin-1 levels were measured
by ELISA (R&D System Europe, Lille, France). The von Willebrand factor (vWF) was measured as vWF antigen by ELISA
(Asserachrom Diagnostica, Stago, France). Insulin sensitivity
was assessed using the quantitative insulin-sensitivity check
index (QUICKI) (40).
Cell culture
Materials and Methods
Patients
Sixty Caucasian children (aged 9.62 ⫾ 2.12 years) were consecutively recruited among those attending the outpatient unit of
Preadipocytes (3T3-L1; American Type Culture Collection,
Manassas, Virginia) were grown in DMEM (Sigma-Aldrich, St
Louis, Missouri) supplemented with fetal bovine serum 10%,
L-glutamine 1%, and penicillin/streptomycin 1% (Euroclone,
Milan, Italy) and differentiated in DMEM containing fetal bo-
doi: 10.1210/jc.2012-4119
vine serum 10%, dexamethasone 1 ␮M, 3-Isobutyl-1-methylxanthine 0.5 mM (Sigma-Aldrich), and insulin 1 ␮g/mL (Novo
Nordisk A/S, Copenhagen, Denmark). Mature adipocytes were
serum starved for 6 hours in DMEM containing 1% glucose and
0.1% fatty acid-free BSA and then left untreated or stimulated
with ET-1 (Sigma-Aldrich) or with sera from lean, OW, or Ob
children (20% of total incubation medium) in the absence or
presence of ETA (BQ-123) and ETB (BQ-788) (Sigma-Aldrich)
receptor blockers (10 ␮M). In a parallel set of experiments,
adipocytes were preincubated with or without the p42/44
MAPK inhibitor PD-98059 (20 ␮M) (Alexis Biochemicals,
San Diego, California), or c-Jun NH2-terminal protein kinase
(JNK) inhibitor SP-600125 (20 ␮M) (Sigma-Aldrich) for 1
hour. Time-course and dose-response curves for exogenous
ET-1 were preliminarily determined (Supplemental Figure 1,
published on The Endocrine Society’s Journals Online web
site at http://jcem.endojournals.org).
jcem.endojournals.org
E685
21 ⫾ 1°C under a 12-hour light, 12-hour dark cycle. Mesenteric
vascular beds (MVBs) were isolated and removed as described
(41). Briefly, MVBs mounted in a temperature-controlled moist
chamber (type 834/1; Hugo Sachs Elektronik, March-Hugstetten, Germany) were perfused with modified Krebs-Henseleit
solution continuously gassed with a mixture of 95% O2 and 5%
CO2 (pH 7.4). A constant flow rate of 5 mL/min through the
MVB was maintained using a peristaltic pump (ISM 833; Hugo
Sachs Elektronik). Drug solutions were infused into the perfusate
proximal to the arterial cannula using another peristaltic pump.
After an equilibration period (30 – 40 minutes), changes in perfusion pressure were measured with a pressure transducer system
(SP 844; Capto, Capto, Norway) and recorded continuously using data acquisition and analysis equipment (PowerLab System;
ADInstruments, Castle Hill, Australia).
Vasodilator Responses in MVB
Western blot analysis
Adiponectin and vascular cell adhesion molecule (VCAM)-1
protein expression was measured in serum samples from lean,
OW, and Ob patients and in conditioned media and cell lysates
from mature adipocytes. Serum samples were pretreated with 50
mM Tris-HCl buffer (pH 6.8) containing 2% sodium dodecyl
sulfate to reduce multimers to dimeric isoforms of adiponectin.
Cell lysates were prepared according to standard methods. Equal
amounts of protein (30 ␮g) were loaded on 10% SDS-PAGE gel
and immunoblotted with the following primary antibodies (dilution 1:1000): c-Jun, ph-c-Jun (Ser73), p42/44 MAPK, ph42/44 MAPK (Thr 202/Tyr 204) (Cell Signaling Technology,
Beverly, Massachusetts); VCAM-1 (Santa Cruz Biotechnology
Inc, Santa Cruz, California); adiponectin (Enzo Life Science, Inc,
Farmingdale, New York). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was from Sigma-Aldrich. Incubation with horseradish peroxidase-linked antimouse and
antirabbit secondary antibodies (Santa Cruz Biotechnology)
(1:3000) was performed for 1 hour at room temperature. Immunoblotting results were visualized by Molecular Imager
ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, California). Images were captured with QuantityOne Software
(Bio-Rad Laboratories) and blots quantified by scanning densitometry (ImageJ, National Institutes of Health, Bethesda,
Maryland).
RNA extraction and RT-PCR
For RT-PCR, 1 ␮g of total RNA (Fast Pure RNA kit, Takara,
Shiga, Japan) was reversely transcribed into cDNA using oligodeoxythymidine primer (Takara) and PrimeScript reverse
transcriptase (Takara). Equal amounts of each reverse-transcribed cDNA were amplified (Takara Taq), with ␤-actin as the
internal control. Primers for adiponectin (forward, 5⬘-CGTGATGGCAGAGATGGCA, reverse, 5⬘-ACACCTGGAGCCAGACTTG) and ␤-actin (5⬘-ACGAGGCCCAGAGCAAGAGA,
reverse, 5⬘-AAGGTAGTTTCGTGGATGCC) were from Eurofins MWG Operon (Ebersberg, Germany).
Animal experiments
All procedures were in accordance with guidelines and authorization for the use of laboratory animals (Italian Ministry of
Health) and approved by the University Ethical Committee for
Animal Experiments. Male Wistar rats (n ⫽ 12) were housed at
A steady-state perfusion pressure of approximately 100 mm
Hg was obtained 30 – 40 minutes after initial administration of
noradrenaline (NA; 10 ␮M). Dose-response curves measuring
vasodilation (decrease in perfusion pressure) in response to Ad
were obtained by adding Ad (10 –30 ␮M per 30 seconds) to the
perfusate. Vasodilation in response to Ad was compared before
and after 20 minutes treatment with endothelial nitric oxide synthase inhibitor N-omega-nitro-L-arginine methyl ester (LNAME; 100 ␮M) or with ET receptor blockers BQ123/BQ788
(20 ␮M).
Drugs
NA, acetylcholine (ACh), and Ad were from Alexis. Stock
solutions of NA (100 mM) and ACh (10 mM) were in distilled
water. Final dilutions of these drugs were prepared in modified
Krebs-Henseleit solution immediately before use.
Statistical analysis
Our primary aim was to study the correlation between circulating levels of ET-1 and Ad. A power analysis to determine the
number of patients needed for this investigation was based on
Pearson correlation coefficient set at 0.4 or less. A total sample
size of 44 patients was sufficient to detect a significant relationship between ET-1 and Ad with a power of 0.80 and a P ⫽ .05.
The total number of subjects recruited was extended to 60 children (30% increase) to ensure comprehensive statistical analysis.
Quantitative data with standing Gaussian distribution were expressed as mean ⫾ SEM of at least 3 independent experiments for
each condition. An ANOVA model for repeated measures was
used to evaluate difference in values obtained from in vitro studies. A 1-way ANOVA model was used to compare means among
independent groups (analysis on antropometric measurements,
metabolic and endothelial markers on human samples between
BMI groups). Post hoc comparison was performed by Tukey test.
Least squares regression analysis was performed to test the relation between variables. An analysis of covariance model was
used to compare the slope among groups. Values of P ⬍ .05 were
considered to indicate statistical significance. All analysis were
performed using SAS version 9.3 software (SAS Institute, Cary,
North Carolina).
E686
Nacci et al
ET-1 Impairs Adiponectin via MAPK Signaling
J Clin Endocrinol Metab, April 2013, 98(4):E683–E693
Table 1. Clinical and Laboratory Values of lean, Overweight, and Obese Children
Subjects, n
Male
Age, y
Height, cm
Weight, kg
BMI, kg/m2
BMI SDS
Systolic BP, mm Hg
Diastolic BP, mm Hg
Fasting glucose, mg/dL
Fasting insulin, ␮U/mL
Total cholesterol, mg/dL
LDL cholesterol, mg/dL
Triglycerides, mg/dL
QUICKI
Lean
19
10
8.57 ⫾ 3.16
128.74 ⫾ 17.56
29.94 ⫾ 12.31
17.36 ⫾ 2.44
⫺0.19 ⫾ 0.75
100.00 ⫾ 9.13
65.00 ⫾ 12
82.7 ⫾ 0.9
12.5 ⫾ 1.5
153.33 ⫾ 2.16
91.1 ⫾ 3.6
60.9 ⫾ 10.6
0.334 ⫾ 0.006
OW
11
7
10.25 ⫾ 2.91
138.61 ⫾ 15.00
42.90 ⫾ 11.20b
21.76 ⫾ 1.96b
1.04 ⫾ 0.31b
107.50 ⫾ 12.81
68.00 ⫾ 5.93
87.38 ⫾ 3.60
13.95 ⫾ 3.3
159.5 ⫾ 3.8
106.7 ⫾ 3.5
58.6 ⫾ 6.9
0.326 ⫾ 0.011a
Ob
30
17
10.07 ⫾ 3.08
144.04 ⫾ 16.82a
62.68 ⫾ 22.85a
29.03 ⫾ 5.18a,c
2.18 ⫾ 0.45a,c
108.75 ⫾ 17.09
69.00 ⫾ 11.00
85.35 ⫾ 8.29
17.66 ⫾ 8.74
161.8 ⫾ 3.5
98.8 ⫾ 3.0
63.67 ⫾ 31.07
0.319 ⫾ 0.024a
Data shown are mean ⫾ SEM. QUICKI ⫽ 1/关log (insulin) ⫹ log (glucose)兴).
a
P ⬍ .005 vs lean.
b
P ⬍ .005 vs lean.
c
P ⬍ .005 vs OW.
Results
Clinical and biochemical parameters
Children enrolled in the lean, OW, and Ob group
were age and sex matched (Table 1). As expected,
weight, BMI, and BMI SDS values were significantly
higher in the OW and Ob groups with respect to lean
subjects (Table 1). No significant difference was observed in both systolic and diastolic blood pressure (BP)
levels between lean, OW, or Ob children. Importantly,
total cholesterol, LDL cholesterol, and triglyceride levels were not significantly different between groups. Similarly, fasting glucose and fasting insulin levels were
comparable among subjects. However, when assessed
by surrogate QUICKI index, insulin sensitivity was reduced in OW and Ob children with respect to lean subjects (Table 1). Thus, insulin resistance was already
present in OW and Ob children, even if clinical hemodynamic and metabolic parameters were not significantly altered with respect to lean subjects.
Markers of endothelial dysfunction and circulating
levels of Ad
We next evaluated markers of endothelial dysfunction
in serum samples from lean, OW, and Ob children. When
compared with values from lean subjects, levels of vWf
were higher in serum from Ob children, with a trend to
increase in the OW group (Figure 1A). Similarly, increased
release of soluble VCAM-1 was observed in the OW and
Ob groups with respect to lean subjects (Figure 1B). Circulating levels of ET-1 were found significantly increased
in OW and Ob children with respect to lean controls (Figure 1C). Thus, the increased levels of proadhesive and
prothrombotic endothelial mediators suggest the existence of a dysfunctional endothelium in OW and Ob
subjects.
Under normal conditions, Ad circulates in LMW
trimers, MMW hexamers, and HMW multimers, these
latter considered the most biologically active Ad isoforms (42). Both total and HMW Ad levels were significantly lower in OW and Ob groups than in lean
subjects (Figure 1, B and D). When the entire population
was considered, a statistically significant inverse correlation was observed between ET-1 and total Ad values
(r ⫽ ⫺0.36, P ⫽ .022). Similar results were obtained
when the correlation coefficient was calculated between
ET-1 and HMW Ad (Figure 2A). Moreover, a statistically significant linear regression (F ⫽ 6.77, P ⫽ .0124)
was found in a model considering HMW Ad as the dependent variable and ET-1 as the independent one (␤ ⫽
⫺.35; r2 ⫽ 0.13), thus suggesting that the decreased
HMW Ad depends, at least in part, on increased ET-1
values. Regression analysis shows a significant direct
relationship for BMI with respect to ET-1/total Ad (r ⫽
0.62, P ⬍ .0001) as well as ET-1/HMW Ad (r ⫽ 0.52,
P ⬍ .0001) (Figure 2B). Although the analysis of covariance analysis did not reach a statistically significant
difference among slopes between lean, OW, and Ob
groups (F ⫽ 0.61, P ⫽ .55), post hoc comparison of least
square shows a statistically significant difference of Ob
vs lean (P ⫽ .0085) and a trend in lean vs OW (P ⫽
.0564). No significant difference was measured in Ob vs
OW (P ⫽ .070). These findings suggest the existence of
a reciprocal interference between ET-1 and Ad and
doi: 10.1210/jc.2012-4119
A
jcem.endojournals.org
120
vWFAg (%)
§
B
1
90
VCAM-1
60
Total Ad
30
HMW Ad
Lean
OW
5
4
Ob
Ob
D
#
3
2
1
Total
HMW
MMW
LMW
10
Adiponectin (µ
µ g/ml)
§
4
ET-1 (pg/ml)
3
Lean OW Ob
0
C
2
E687
8
*
§
6
*
4
§
2
0
0
Lean
OW
Ob
Lean
OW
Ob
Figure 1. Circulating Ad levels and markers of endothelial dysfunction in OW and Ob children. A, Levels of vWf measured in plasma samples of
lean, OW, and Ob children and expressed as a percentage of vWF antigen. B, Representative immunoblots of soluble VCAM-1, total Ad, and
HMW protein expression in serum from lean, OW, and Ob groups. C, Levels of ET-1 measured in serum samples of lean, OW, and Ob children. D,
Total Ad and HMW, MMW, and LMW Ad subfraction levels in serum samples from lean, OW, and Ob children. Bar graphs indicate the mean
value ⫾ SEM. #P ⬍ .05, *P ⬍ .01, §P ⬍ .001 vs lean group.
prompted us to further investigate whether ET-1 may
directly contribute to impaired adipocyte-mediated Ad
production.
The inhibitory effect of ET-1 on Ad expression is
abrogated by ET-1 receptor blockade
To evaluate the role of ET-1 on Ad production and
secretion, mRNA and protein levels of Ad were measured
in adipocytes serum starved for 6 hours and then stimulated with increasing concentrations of exogenous ET-1
(1–20 nM) for 6, 12, and 24 hours. ET-1 dose dependently
reduced both Ad mRNA and protein levels, with maximal
effect achieved at 24 hours with ET-1 10 nM (Figure 3A).
Pretreatment with either ETA receptor blocker BQ-123 or
ETB receptor blocker BQ-788 alone slightly but significantly ameliorated Ad secretion in culture medium (Figure
3B, upper panel) and Ad protein levels in cell lysates (Figure 3B, middle panel). In cells pretreated with a combination of BQ-123 and BQ-788, the ET-1-dependent inhibition of Ad production was completely abrogated (Figure
3B). These findings suggest that ET-1 directly and dose
dependently inhibits Ad production in adipocytes via activation of its specific membrane receptors.
Exposure to serum from OW and Ob children
decreases Ad expression in adipocytes
As shown in Figure 1B, levels of ET-1 are significantly higher in OW and Ob subjects. We therefore
evaluated whether stimulation of adipocytes with sera
from lean, OW, or Ob children would affect Ad production and secretion. Exposure of adipocytes to either
exogenous ET-1 or serum from OW and Ob children
reduced Ad production and secretion (Figure 3C). Interestingly, pretreatment with a combination of BQ123/BQ-788 was able to ameliorate impaired Ad levels
in both lysates (Figure 3D) and culture medium (Figure
3E) of adipocytes exposed to serum from OW or Ob
children. Thus, as for the results obtained with exogenous ET-1, the increased levels of endogenous ET-1 in
OW and Ob children may significantly impact on adipocyte-mediated production of Ad.
The ET-1-dependent inhibitory effect on Ad
expression is mediated by p42/44 MAPK
We then explored signaling pathways activated
downstream ET-1 receptors. When compared with
basal conditions, phosphorylation levels of p42/44
MAPK were increased in adypocytes stimulated with
E688
Nacci et al
ET-1 Impairs Adiponectin via MAPK Signaling
A
J Clin Endocrinol Metab, April 2013, 98(4):E683–E693
9
Lean
Overweight
Obese
8
Ad HMW (µg/ml)
7
6
5
y = -0.35x + 4.64
R² = 0.13
4
3
2
1
0
0
1
2
3
4
5
6
7
ET-1 (pg/ml)
B
Figure 2. A, Inverse relationship between circulating levels of ET-1 and HMW adiponectin subfraction (Ad HMW) in pediatric subjects (r ⫽ ⫺0.35;
P ⫽ .0124). B, Direct relationship between ET-1/HMW Ad vs BMI (left; r ⫽ 0.52; P ⫽ .0001) and ET-1/total Ad vs BMI (right; r ⫽ 0.62; P ⫽ .0001)
in pediatric subjects.
exogenous ET-1 (Figure 4A) as well as with serum from
OW or Ob children (Figure 4B). A slight increase in
phosphorylated c-jun, a substrate of JNK kinase, was
also observed (Figure 4C). Pretreatment with BQ-123/
BQ-788 restored Ad production in adypocytes stimulated with either exogenous ET-1 or serum from Ob
children (Figures 3, D and E) and concomitantly reduced phosphorylation of p42/44 MAPK (Figure 4, A
and B). Pretreatment with the MAPK inhibitor PD
98059 abrogated p42/44 MAPK phosphorylation in response to stimulation with either exogenous ET-1 or
serum from Ob children (Figure 4, A and B) and concurrently increased levels of Ad (Figure 4, A and B).
Conversely, although pretreatment with the JNK inhibitor SP600125 reduced levels of phosphorylated c-jun in
adipocytes stimulated with ET-1, no significant change
was observed in Ad levels under these conditions (data
not shown). Altogether, these results suggest that acti-
vation of p42/44 MAPK signaling may represent a potential mechanism for ET-1-mediated inhibition of Ad
production in adipocytes.
ET-1 blockade increases Ad-mediated vasodilation
in isolated rat resistance arteries
To evaluate whether ET-1 may interfere with Admediated vasodilation, isolated and perfused rat mesenteric arteries were preconstricted with NA (10 ␮M)
and subsequently stimulated with increasing concentrations of Ad (10 –30 ␮g) (Figure 5). Consistent with
Ad ability to stimulate production and release of endothelial NO (22), acute stimulation with Ad dose dependently increased vasodilation. This effect was abrogated when vessels were pretreated with the NO
synthase inhibitor L-NAME (Figure 5A). Interestingly,
Ad-mediated vasodilation was further increased in vessels pretreated with ET-1 receptor blockers BQ-123/
jcem.endojournals.org
E689
Discussion
B
1
Adiponectin/GAPDH
A
Adiponectin/GAPDH
doi: 10.1210/jc.2012-4119
2
(arbitrary units)
(arbitrary units)
(arbitrary units)
Adiponectin/GAPDH
The recognition that metabolic and
endothelial function are fully inte0.5
1
grated and reciprocally controlled is
* *
*
a key concept for understanding the
pathophysiological mechanisms linking
0
0
obesity to its related morbidities
1
2 3 4
5
1 2
3
4
5
(43). To our knowledge, this is the
Ad (medium)
Ad
first study demonstrating that endoβ-actin
thelial dysfunction with increased
Ad (lysate)
circulating levels of ET-1 is associAd (lysate)
ated with hypoadiponectinemia in
GAPDH
GAPDH
Ob and OW children. Our results
ET-1
- + + +
+
strongly suggest that ET-1 signifiET-1 (nM)
- 1 5 10 20
BQ-123
- +
+
cantly inhibits both Ad protein exBQ-788
- +
+
pression in adypocytes and Ad vasodilator effects on resistance arteries.
medium
D
C
1
lysate
1
2
3
4
Moreover, findings obtained here
**
**
suggest that the ET receptor-medi*
Ad (lysate)
ated activation of p42/44 MAPK sig0.5
**
GAPDH
naling pathways is a potential mechLean OW Ob
Ob
anism for ET-1-mediated inhibition
0
+ BQ
of Ad production.
1
2
3
4
5
In addition to representing the
Ad (medium)
E
earliest marker of vascular abnor1
2
3
4
5
malities, endothelial dysfunction is a
Ad (lysate)
Ad (medium)
common feature of metabolic disturbances such as insulin resistance and
GAPDH
Lean ET-1 ET-1 Ob Ob
glucose intolerance. The hypothesis
+ BQ
+ BQ
Basal ET-1 Lean OW Ob
explored here is that, rather than
Figure 3. The inhibitory effect of ET-1 on Ad expression is abrogated by ET-1 receptors blockade. A,
representing a mere consequence of
Differentiated 3T3-L1 adipocytes were serum starved for 6 hours and then stimulated with increasing
concentrations of exogenous ET-1 (1–20 nM) for 24 hours. mRNA and protein Ad levels were
metabolic abnormalities, unbalanced
measured by RT-PCR and Western blotting, respectively, as described in Materials and Methods.
release of vascular mediators may trigRepresentative immunoblots from at least 3 independent experiments are shown. Each bar represents
ger hypoadiponectinemia. We sethe mean ⫾ SEM of densitometric analysis for Ad protein normalized to GAPDH expression. B,
lected metabolically normal OW and
Representative immunoblots for results obtained in mature adipocytes preincubated for 1 hour in the
absence or presence of the ETA receptor antagonist BQ-123 (10 ␮M), the ETB receptor antagonist
Ob children to dissect the contribution
BQ-788 (10 ␮M), or the combination of both ET-receptor blockers and then stimulated with or
of a dysfunctional endothelium to the
without ET-1 10 nM for 24 hours. Each bar represents the mean ⫾ SEM of densitometric analysis for
impaired secretion of Ad before cliniAd protein normalized to GAPDH expression in at least 3 independent experiments. *P ⬍ .05 vs basal
conditions. C, Cell lysates and conditioned media collected from adipocytes treated with exogenous
cally relevant dislipidemia and hyperET-1 (10 nM) or with serum from lean, OW, or Ob children (20% of total incubation medium) were
glycemia develop. The existence of ensubjected to immunoblotting for Ad and GADPH as described in Materials and Methods.
dothelial dysfunction in OW and Ob
Representative immunoblots from at least 3 independent experiments are shown. Bar graphs indicate
the mean ⫾ SEM of densitometric analysis for Ad protein normalized to GAPDH expression. *P ⬍
children was suggested by the in.05, **P ⬍ .01 vs basal conditions. D, Mature adipocytes were treated as described in C and then
creased levels of soluble VCAM-1 propreincubated for 1 hour in the absence or presence of a BQ-123/BQ-788 (10 ␮M) combination. Ad
tein and vWf and by the abnormal proand GADPH expressions were evaluated by immunoblotting in cell lysates. E, Ad expression in the
duction of ET-1. Interestingly, of the
conditioned media of mature adipocytes treated with exogenous ET-1 or Ob serum in the absence or
presence of a BQ-123/BQ-788 (10 ␮M) combination. Representative immunoblots from at least 3
various endothelial mediators anaindependent experiments are shown.
lyzed, ET-1 was the first to be significantly modified not only in Ob subBQ-788 (Figure 5B). These findings suggest that ET-1 jects but also in OW subjects.
physiologically counteracts vasodilation induced by Ad
The possibility of a reciprocal interference between
and imply that elevated ET-1 levels may contribute to ET-1 and circulating Ad (both total and HMW Ad isoform) was supported by a statistically significant inverse
reduce vascular beneficial effects of Ad.
Nacci et al
ET-1 Impairs Adiponectin via MAPK Signaling
A
B
*
2
ph/total MAPK
(arbitrary units)
ph/total MAPK
*
J Clin Endocrinol Metab, April 2013, 98(4):E683–E693
1
0
1
2
3
4
5
(arbitrary units)
E690
*
2
3
4
OW
Ob
*
1
0
1
6
Ad
*
2
5
6
7
Ad
ph-p42/44 MAPK
ph-p42/44 MAPK
p42/44 MAPK
p42/44 MAPK
Basal
ET-1
ET-1
+ BQ
Basal ET-1 ET-1
+ PD
Lean
ph/total c jun
(arbitrary units)
C
2
*
*
Ob Ob
+ BQ
Ob Ob
+ PD
*
1
0
1
2
3
4
5
Ad
ph-c jun
c jun
Basal
ET-1 Lean OW
Ob
Figure 4. The ET-1-dependent inhibitory effect on Ad expression is mediated by p42/44 MAPK. A, Mature adipocytes were treated without or
with exogenous ET-1 (10 nM, 6 hours) in the presence or in the absence of ET-1 receptor blockers (BQ) or p42/44 MAPK inhibitor PD-98059 (20
␮M, 1 hour preincubation). Cell lysates were subjected to immunoblotting with antibodies for Ad and phosphorylated and total isoforms of p42/
44 MAPK. B, Mature adipocytes were stimulated with serum from lean, OW, or Ob children (20% of total incubation medium) under conditions
described in A. C, Mature adipocytes were treated without or with exogenous ET-1 (10 nM) or with serum from lean, OW, or Ob children. Cells
lysates were subjected to immunoblotting with antibodies for Ad and phosphorylated and total isoforms of c-jun. Representative immunoblots
from at least 3 independent experiments for each setting are shown. Bar graphs indicate the mean ⫾ SEM of densitometric analysis for
phosphorylated isoforms normalized to respective total protein expression. *P ⬍ .05 vs respective basal conditions.
correlation between these 2 variables and by a significant
direct relationship between ET-1/Ad and BMI. Undoubtedly, this does not exclude that several other cytokines
including TNF-␣, IL-1 and IL-6, plasminogen activator
inhibitor 1, and C-reactive protein (CRP) may contribute
to lower Ad secretion in these children. However, in a
previous study, we reported that pediatric obesity is associated with high levels of CRP and TNF-␣ (34), but we
failed to ascertain a direct correlation between these inflammatory mediators and the reduced Ad levels. Conversely, current findings point to a direct interference of
ET-1 on Ad production.
Although overexpression of ET-1 might help to elucidate the role played on Ad, transgenic ET-1 mice display
vascular, cardiac, and renal abnormalities (44 – 47) that
may complicate, rather than simplify, the interpretation of
ET-1 effects on Ad expression. Recently the lack of ET-1
has been associated with the preservation of circulating Ad
levels in vascular endothelial cell-specific ET-1 knockout
mice (48). These findings indirectly support our idea that
endothelial dysfunction with excessive ET-1 release may
importantly contribute to dysregulation of adipose tissue
signaling.
An acute stimulatory role of ET-1 has been described
for adipocyte secretion of leptin (49) and IL-6 (50). In a
previous study, Clarke et al (20) have reported that ET-1
acutely stimulates Ad production through the ETA receptor. However, the same authors observed that stimulation
with ET-1 time dependently decreases Ad levels (20). Similarly, Bedi et al (21) demonstrated that chronic ET-1 stimulation inhibits Ad secretion via a mechanism involving
jcem.endojournals.org
30
10
20
Adiponectin (µM)
ACh
30
20
Adiponectin (µM)
10
A
ACh
doi: 10.1210/jc.2012-4119
90
60
L-NAME
30
0
120
90
100
110
30
Adiponectin (µM)
20
30
Adiponectin (µM)
80
10
40
ACh
30
20
B
20
10
10
ACh
Perfusion Pressure (mmHg)
120
90
60
BQ123/788
30
0
10
20
30
40
80
90
100
110
Time (min)
Figure 5. ET-1 blockade increases Ad-mediated vasodilation in
isolated rat resistance arteries. Rat mesenteric vascular arteries isolated
and perfused as described in Materials and Methods were
submaximally precontracted with NA (10 ␮M). In each preparation,
endothelial integrity was initially assessed by evaluating vasodilation
obtained with a maximally stimulating dose of ACh (1 ␮M). A, Doseresponse curves for Ad-induced vasorelaxation were obtained by
adding increasing concentrations of Ad (10 –30 ␮M per 30 seconds) to
the perfusate in the absence or presence of the nitric oxide synthase
inhibitor L-NAME (100 ␮M, 30 minutes). B, Dose-response curves for
Ad-induced vasorelaxation as in A were repeated in the absence and
presence of ET-1 receptor blockers BQ123/BQ788 (20 ␮M, 30
minutes). Representative tracings are shown for experiments that were
independently repeated at least 3 times.
vesicular trafficking and depleting plasma membrane
phosphatidylinositol 4,5-bisphosphate.
In line with this, we found that exposure up to 24
hours to exogenous ET-1 or to serum from OW and Ob
children inhibited Ad production and secretion. Interestingly, endogenous ET-1 was more effective than exogenous ET-1 to inhibit Ad expression in our experiments. Because commercially available ET-1 is from
suine and analogous ET-1 concentrations are required
to regulate IL-6 (51), leptin (49), and suppressor of cytokine signaling-3 gene expression (52), it is likely that
homology sequence/receptor binding affinity of porcine
ET-1 may be slightly different from that of human ET-1.
In our experiments, both ETA and ETB receptors seem
equally involved in the ET-1-dependent inhibition of Ad
because complete restoration of Ad expression was observed only in cells pretreated with a combination of
ETA and ETB receptor blockers. Although the specific
role of ETA and ETB in ET-1-mediated Ad modulation
was not explored here, it is possible that chronic exposure to ET-1 may activate feedback regulatory mechanisms and turn ET-1 initial secretory effect into a strong
inhibition of Ad synthesis and release.
E691
Downstream ET receptors, ET-1 stimulation leads to
the activation of multiple signaling pathways in adipocytes, including the phosphatidylinositol 3-kinase/AKT
pathways and the p42/44 MAPK pathways. In a very recent study, JNK signaling has been suggested to mediate
some effects of ET-1 on the regulation of suppressor of
cytokine signaling-3, a large family of genes involved in
modulation of adipokine synthesis (52). When we explored signaling pathways activated by ET-1 in adipocytes, we found that both p42/44 MAPK and c-jun, a
substrate of the JNK kinase, were phosphorylated in response to exogenous ET-1 or serum from OW and Ob
children. It is important to note that the pharmacological
inhibition of ET receptors substantially counteracted the
activation of MAPK signaling by either exogenous ET-1
and serum from OW and Ob children. Conversely, only a
slight inhibitory effect on JNK signaling was observed in
adipocytes stimulated with serum from OW and Ob children. This is not surprising, considering that serum from
OW and Ob children contains several other mediators
(including TNF-␣, IL-1 and IL-6, plasminogen activator
inhibitor 1, and CRP) able to stimulate JNK signaling
pathways independent from ET-1 receptors. The ET-1stimulated Ad inhibition was demonstrated to be dependent on p42/44 MAPK signaling but not on JNK-mediated
pathways. This is evident because pretreatment with PD98059 not only significantly inhibited ET-1-stimulated
MAPK phosphorylation but also restored Ad production
in adipocytes. Conversely, although pretreatment with the
JNK inhibitor SP600125 reduced levels of phosphorylated
c-jun, no significant change in Ad levels was observed in
cells stimulated with ET-1. Thus, increased production of
ET-1 may significantly contribute to impair Ad synthesis
and secretion via activation of p42/44 MAPK signaling.
Ad-mediated endothelial release of NO favors vasodilation, thereby ameliorating tissue perfusion and metabolic delivery of nutrients. Increased ET-1 production may
not only directly contribute to impaired production of Ad
but also significantly reduce Ad-dependent vasodilation
and endothelial protection. We found that acute stimulation with Ad dose dependently increased vasodilation in
isolated resistance arteries by a mechanism that was endothelial nitric oxide synthase dependent. This small but
significant vasodilatant effect was further increased in vessels pretreated with ET-1 receptor blockers, suggesting
that ET-1 counteracts Ad-dependent vasodilation. These
findings are in agreement with recent observations suggesting that Ad pretreatment is able to inhibit the increased
perfusion pressure and associated metabolic stimulation
caused by ET-1 (29) and further support the concept of a
direct cross talk between vascular and metabolic factors.
Thus, on top of direct promitogenic actions, circulating
E692
Nacci et al
ET-1 Impairs Adiponectin via MAPK Signaling
high levels of ET-1 may reduce cardiovascular protection
by inhibiting Ad-mediated NO-dependent vasodilation.
Overall, this study provides evidence that increased circulating levels of ET-1 in overweight and obese children
may contribute, at least in part, to reduce levels of Ad and
suggests that ET-mediated activation of p42/44 MAPK
signaling pathways may be one possible mechanism for
ET-1-dependent inhibition of Ad synthesis. Imbalance in
the relationship between Ad and ET-1 may provide a possible explanation for hypoadiponectinemia in pediatric
obesity and contribute to the development of both metabolic disturbances and cardiovascular complications.
J Clin Endocrinol Metab, April 2013, 98(4):E683–E693
12.
13.
14.
15.
16.
17.
Acknowledgments
Address all correspondence and requests for reprints to: Monica
Montagnani, MD, PhD, Department of Biomedical Sciences and
Human Oncology, Pharmacology Section, Medical School University of Bari “Aldo Moro,” Policlinico, P.zza G. Cesare, 1
70124 Bari, Italy. E-mail: [email protected].
This work was supported in part by the Juvenile Diabetes
Research Foundation Grant CDA 2-2006-32 (to M.M.).
Disclosure Summary: The authors have nothing to disclose.
18.
19.
20.
21.
References
1. Wang Y, Lobstein T. Worldwide trends in childhood overweight
and obesity. Int J Pediatr Obes. 2006;1:11–25.
2. van Vliet M, Heymans MW, von Rosenstiel IA, Brandjes DP, Beijnen JH, Diamant M. Cardiometabolic risk variables in overweight
and obese children: a worldwide comparison. Cardiovasc Diabetol.
2011;10:106.
3. Franks PW, Hanson RL, Knowler WC, Sievers ML, Bennett PH,
Looker HC. Childhood obesity, other cardiovascular risk factors,
and premature death. N Engl J Med. 2010;362:485– 493.
4. Juonala M, Magnussen CG, Berenson GS, et al. Childhood adiposity, adult adiposity, and cardiovascular risk factors. N Engl J Med.
2011;365:1876 –1885.
5. Montero D, Walther G, Perez-Martin A, Roche E, Vinet A. Endothelial dysfunction, inflammation, and oxidative stress in obese children and adolescents: markers and effect of lifestyle intervention.
Obes Rev. 2012;13:441– 455.
6. Eringa EC, Bakker W, Smulders YM, Serne EH, Yudkin JS, Stehouwer CD. Regulation of vascular function and insulin sensitivity by
adipose tissue: focus on perivascular adipose tissue. Microcirculation. 2007;14:389 – 402.
7. Zhou J, Qin G. Adipocyte dysfunction and hypertension. Am J Cardiovasc Dis. 2012;2:143–149.
8. Saint-Marc P, Kozak LP, Ailhaud G, Darimont C, Negrel R. Angiotensin II as a trophic factor of white adipose tissue: stimulation of
adipose cell formation. Endocrinology. 2001;142:487– 492.
9. Mohite A, Chillar A, So SP, Cervantes V, Ruan KH. Novel mechanism of the vascular protector prostacyclin: regulating microRNA
expression. Biochemistry. 2011;50:1691–1699.
10. Sansbury BE, Cummins TD, Tang Y, et al. Overexpression of endothelial nitric oxide synthase prevents diet-induced obesity and
regulates adipocyte phenotype. Circ Res. 2012;111:1176 –1189.
11. Marasciulo FL, Montagnani M, Potenza MA. Endothelin-1: the yin
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
and yang on vascular function. Curr Med Chem. 2006;13:1655–
1665.
Cardillo C, Campia U, Bryant MB, Panza JA. Increased activity of
endogenous endothelin in patients with type II diabetes mellitus.
Circulation. 2002;106:1783–1787.
Barath A, Turi S, Nemeth I, et al. Different pathomechanisms of
essential and obesity-associated hypertension in adolescents. Pediatr Nephrol. 2006;21:1419 –1425.
Wilkes JJ, Hevener A, Olefsky J. Chronic endothelin-1 treatment
leads to insulin resistance in vivo. Diabetes. 2003;52:1904 –1909.
Ishibashi KI, Imamura T, Sharma PM, Huang J, Ugi S, Olefsky JM.
Chronic endothelin-1 treatment leads to heterologous desensitization of insulin signaling in 3T3-L1 adipocytes. J Clin Invest. 2001;
107:1193–1202.
Hauner H, Petruschke T, Gries FA. Endothelin-1 inhibits the adipose differentiation of cultured human adipocyte precursor cells.
Metabolism. 1994;43:227–232.
Ishida F, Saeki K, Saeki T, et al. Suppressive effects of the endothelin
receptor (ETA) antagonist BQ-123 on ET-1-induced reduction of
lipoprotein lipase activity in 3T3-L1 adipocytes. Biochem Pharmacol. 1992;44:1431–1436.
Usui I, Imamura T, Babendure JL, et al. G protein-coupled receptor
kinase 2 mediates endothelin-1-induced insulin resistance via the
inhibition of both G␣q/11 and insulin receptor substrate-1 pathways in 3T3-L1 adipocytes. Mol Endocrinol. 2005;19:2760 –2768.
Juan CC, Chang CL, Lai YH, Ho LT. Endothelin-1 induces lipolysis
in 3T3-L1 adipocytes. Am J Physiol Endocrinol Metab. 2005;288:
E1146 –E1152.
Clarke KJ, Zhong Q, Schwartz DD, Coleman ES, Kemppainen RJ,
Judd RL. Regulation of adiponectin secretion by endothelin-1.
Biochem Biophys Res Commun. 2003;312:945–949.
Bedi D, Clarke KJ, Dennis JC, et al. Endothelin-1 inhibits adiponectin secretion through a phosphatidylinositol 4,5-bisphosphate/actin-dependent mechanism. Biochem Biophys Res Commun. 2006;
345:332–339.
Chen H, Montagnani M, Funahashi T, Shimomura I, Quon MJ.
Adiponectin stimulates production of nitric oxide in vascular endothelial cells. J Biol Chem. 2003;278:45021– 45026.
Hattori Y, Suzuki M, Hattori S, Kasai K. Globular adiponectin upregulates nitric oxide production in vascular endothelial cells. Diabetologia. 2003;46:1543–1549.
Shibata R, Sato K, Pimentel DR, Takemura Y, Kihara S, Ohashi K,
Funahashi T, Ouchi N, Walsh K. Adiponectin protects against myocardial ischemia-reperfusion injury through AMPK- and COX-2dependent mechanisms. Nat Med. 2005;11:1096 –1103.
Addabbo F, Nacci C, De Benedictis L, Leo V, Tarquinio M, Quon
MJ, Montagnani M. Globular adiponectin counteracts VCAM-1mediated monocyte adhesion via AdipoR1/NF-␬B/COX-2 signaling
in human aortic endothelial cells. Am J Physiol Endocrinol Metab.
2011;301:E1143–E1154.
Ouchi N, Ohishi M, Kihara S, et al. Association of hypoadiponectinemia with impaired vasoreactivity. Hypertension. 2003;42:
231–234.
Kubota N, Terauchi Y, Yamauchi T, et al. Disruption of adiponectin
causes insulin resistance and neointimal formation. J Biol Chem.
2002;277:25863–25866.
Fujioka D, Kawabata K, Saito Y, et al. Role of adiponectin receptors
in endothelin-induced cellular hypertrophy in cultured cardiomyocytes and their expression in infarcted heart. Am J Physiol Heart Circ
Physiol. 2006;290:H2409 –H2416.
Bussey CT, Kolka CM, Rattigan S, Richards SM. Adiponectin opposes endothelin-1-mediated vasoconstriction in the perfused rat
hindlimb. Am J Physiol Heart Circ Physiol. 2011;301:H79 –H86.
Kumada M, Kihara S, Sumitsuji S, et al. Association of hypoadiponectinemia with coronary artery disease in men. Arterioscler
Thromb Vasc Biol. 2003;23:85– 89.
Iwashima Y, Katsuya T, Ishikawa K, et al. Hypoadiponectinemia is
doi: 10.1210/jc.2012-4119
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
an independent risk factor for hypertension. Hypertension. 2004;
43:1318 –1323.
Han SH, Sakuma I, Shin EK, Koh KK. Antiatherosclerotic and antiinsulin resistance effects of adiponectin: basic and clinical studies.
Prog Cardiovasc Dis. 2009;52:126 –140.
Riestra P, Garcia-Anguita A, Lasuncion MA, Cano B, de Oya M,
Garces C. Relationship of adiponectin with metabolic syndrome
components in pubertal children. Atherosclerosis. 2011;216:467–
470.
Giordano P, Del Vecchio GC, Cecinati V, et al. Metabolic, inflammatory, endothelial and haemostatic markers in a group of Italian
obese children and adolescents. Eur J Pediatr. 2011;170:845– 850.
Kamon J, Yamauchi T, Muto S, et al. A novel IKK␤ inhibitor stimulates adiponectin levels and ameliorates obesity-linked insulin resistance. Biochem Biophys Res Commun. 2004;323:242–248.
Engeli S, Feldpausch M, Gorzelniak K, et al. Association between
adiponectin and mediators of inflammation in obese women. Diabetes. 2003;52:942–947.
Cole TJ, Bellizzi MC, Flegal KM, Dietz WH. Establishing a standard
definition for child overweight and obesity worldwide: international
survey. BMJ. 2000;320:1240 –1243.
Kuczmarski RJ, Ogden CL, Grummer-Strawn LM, et al. 2000 CDC
growth charts: United States. Adv Data:1–27.
Williams CL, Hayman LL, Daniels SR, et al. Cardiovascular health
in childhood: A statement for health professionals from the Committee on Atherosclerosis, Hypertension, and Obesity in the Young
(AHOY) of the Council on Cardiovascular Disease in the Young,
American Heart Association. Circulation. 2002;106:143–160.
Katz A, Nambi SS, Mather K, et al. Quantitative insulin sensitivity
check index: a simple, accurate method for assessing insulin sensitivity in humans. J Clin Endocrinol Metab. 2000;85:2402–2410.
Potenza MA, Gagliardi S, De Benedictis L, et al. Treatment of spontaneously hypertensive rats with rosiglitazone ameliorates cardiovascular pathophysiology via antioxidant mechanisms in the vasculature. Am J Physiol Endocrinol Metab. 2009;297:E685–E694.
jcem.endojournals.org
E693
42. Wang Y, Lam KS, Yau MH, Xu A. Post-translational modifications
of adiponectin: mechanisms and functional implications. Biochem J.
2008;409:623– 633.
43. Kim JA, Montagnani M, Koh KK, Quon MJ. Reciprocal relationships between insulin resistance and endothelial dysfunction: molecular and pathophysiological mechanisms. Circulation. 2006;113:
1888 –1904.
44. Shindo T, Kurihara H, Maemura K, et al. Renal damage and saltdependent hypertension in aged transgenic mice overexpressing endothelin-1. J Mol Med (Berl). 2002;80:105–116.
45. Amiri F, Virdis A, Neves MF, et al. Endothelium-restricted overexpression of human endothelin-1 causes vascular remodeling and
endothelial dysfunction. Circulation. 2004;110:2233–2240.
46. Amiri F, Paradis P, Reudelhuber TL, Schiffrin EL. Vascular inflammation in absence of blood pressure elevation in transgenic murine
model overexpressing endothelin-1 in endothelial cells. J Hypertens.
2008;26:1102–1109.
47. Leung JW, Wong WT, Koon HW, et al. Transgenic mice over-expressing ET-1 in the endothelial cells develop systemic hypertension
with altered vascular reactivity. PLoS One. 2011;6:e26994.
48. Iwasa N, Emoto N, Widyantoro B, Miyagawa K, Nakayama K,
Hirata K. Knockout of endothelin-1 in vascular endothelial cells
protects against insulin resistance induced by high-salt diet in mice.
Kobe J Med Sci. 2010;56:E85–E91.
49. Xiong Y, Tanaka H, Richardson JA, et al. Endothelin-1 stimulates
leptin production in adipocytes. J Biol Chem. 2001;276:28471–
28477.
50. Chai SP, Juan CC, Kao PH, Wang DH, Fong JC. Synergistic induction of interleukin-6 expression by endothelin-1 and cyclic AMP in
adipocytes. Int J Obes (Lond). 2013;37(2):197–203.
51. Chai SP, Chang YN, Fong JC. Endothelin-1 stimulates interleukin-6
secretion from 3T3-L1 adipocytes. Biochim Biophys Acta. 2009;
1790:213–218.
52. Chang HH, Huang YM, Wu CP, et al. Endothelin-1 stimulates suppressor of cytokine signaling-3 gene expression in adipocytes. Gen
Comp Endocrinol. 2012;178:450 – 458.