Am J Physiol Heart Circ Physiol 295: H1514–H1521, 2008.
First published August 8, 2008; doi:10.1152/ajpheart.00479.2008.
Leptin-induced endothelial dysfunction in obesity
Mykhaylo Korda, Ruslan Kubant, Stephen Patton, and Tadeusz Malinski
Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio
Submitted 7 May 2008; accepted in final form 31 July 2008
nitric oxide; endothelium; leptin; nitroxidative stress
that leptin receptors (Ob-R) are expressed in endothelial cells
(4). In a vascular ring model, leptin induced vasorelaxation in
a dose-dependent manner, and this effect was abolished by
eNOS inhibitors (18, 19). The in vitro stimulatory effect of
leptin on NO production in cultured endothelial cells was also
tested by measuring nitrite and nitrate concentrations (30). The
intravenous injection of leptin increased nitrite and nitrate
concentrations in blood serum of normotensive Wistar rats but
not in obese Zucker rats, which have a mutation in their leptin
receptor gene (12). Intraperitoneal leptin administration also
increased plasma concentration and urinary excretion of NO
metabolites, as well as NO second messenger, guanosine 3⬘,5⬘cyclic monophosphate (cGMP) (3). All this data indirectly
indicate that leptin may increase NO production in endothelial
cells and cause these hypotensive effects. It has also been
shown that stimulation of endothelial cells with leptin leads to
an increase in the production of reactive oxygen species, in
particular superoxide (O⫺
2 ), which is known to react rapidly
with NO to form peroxynitrite (ONOO⫺) (5, 35). The leptin
level increases in obesity (7), but the long-term effect of
elevated leptin on the bioavailability of endothelial NO and
cytotoxic ONOO⫺ is not known.
This work was designed to clarify the potential role of NO,
⫺
O⫺
2 , and ONOO in leptin-associated vascular disorders associated with obesity. A system of nanosensors was used for
direct simultaneous measurements of the leptin-induced bio⫺
synthesis of NO, O⫺
in human umbilical vein
2 , and ONOO
endothelial cell (HUVEC) or the endothelium of obese mice.
METHODS
OBESITY IS A SERIOUS RISK factor for vascular disorders such as
hypertension and coronary artery disease (14, 32). Endothelial
dysfunction has been associated with several vascular diseases
(32). Bioavailable nitric oxide (NO) production is diminished
in dysfunctional endothelium, which hinders the L-arginine/NO
pathway and increases vasoconstriction (13).
There have been several reports suggesting the potential
mechanisms for diminished NO production in obesity. It has
been proposed that the elevated free fatty acids levels observed
in obesity may inhibit endothelial NO synthase (eNOS) (8).
Also, the increased levels of cytokines IL-6 and TNF-␣ observed in obesity may affect the phosphorylation of tyrosine
kinases and eNOS expression (16). It also has been suggested
that obesity is associated with an increased production of
reactive oxygen species in the cardiovascular system (31).
Recent evidence indicates that adipose tissue-derived hormone leptin can be involved in vascular tone control (1, 7) and
All materials were purchased from Sigma-Aldrich (St. Louis, MO),
unless otherwise noted.
Cell culture. HUVECs (ATCC No. CRL-1730, Manassas, VA)
were seeded in collagen-coated flasks and incubated in MCDB-131
complete medium at 37°C under an atmosphere of 5% CO2-95% air.
After the formation of a confluent monolayer, the cells were
trypsinized and then resuspended in MCDB-131 and seeded in 12well cell culture clusters. After confluence, MCDB-131 complete
medium was replaced with Eagle’s minimum essential medium without blood serum, and the confluent cells were incubated for 2 or 12 h
with different concentrations of leptin or coincubated with 0.1 ␮mol/l
of leptin and elevated concentrations of L-arginine (3 mmol/l) or
L-arginine (3 mmol/l) and sepiapterin (0.1 mmol/l).
When we cross-check the response of our sensors using enzyme
inhibitors, the cells were preincubated for 2 h before and during
measurements with eNOS inhibitor, 0.3 mmol/l NG-nitro-L-arginine
methyl ester (L-NAME), 2 mmol/l of membrane permeable superoxide dismutase conjugated with polyethylene glycol (PEG-SOD), or 0.01
mmol/l of peroxynitrite decomposition catalyst/scavenger, Mn(III) tetakis,
(1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP), or two oxidase
inhibitors 2.0 mmol/l apocynin or (0.07 mmol/l) 6,8 diallyl 5,7-dihydroxy
Address for reprint requests and other correspondence: T. Malinski, Dept. of
Chemistry and Biochemistry, Ohio Univ., 350 W. State St., Athens, OH
45701-2979 (e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
H1514
0363-6135/08 $8.00 Copyright © 2008 the American Physiological Society
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Korda M, Kubant R, Patton S, Malinski T. Leptin-induced
endothelial dysfunction in obesity. Am J Physiol Heart Circ
Physiol 295: H1514 –H1521, 2008. First published August 8, 2008;
doi:10.1152/ajpheart.00479.2008.—Hyperleptinemia accompanying
obesity affects endothelial nitric oxide (NO) and is a serious factor for
⫺
vascular disorders. NO, superoxide (O⫺
2 ), and peroxynitrite (ONOO )
nanosensors were placed near the surface (5 ⫾ 2 ␮m) of a single
human umbilical vein endothelial cell (HUVEC) exposed to leptin or
aortic endothelium of obese C57BL/6J mice, and concentrations of
⫺
calcium ionophore (CaI)-stimulated NO, O⫺
2 , ONOO were recorded.
Endothelial NO synthase (eNOS) expression and L-arginine concentrations in HUVEC and aortic endothelium were measured. Leptin did
⫺
release from HUVEC.
not directly stimulate NO, O⫺
2 , or ONOO
However, a 12-h exposure of HUVEC to leptin increased eNOS
expression and CaI-stimulated NO (625 ⫾ 30 vs. 500 ⫾ 24 nmol/l
⫺
control) and dramatically increased cytotoxic O⫺
2 and ONOO levels.
The [NO]-to-[ONOO⫺] ratio ([NO]/[ONOO⫺]) decreased from 2.0 ⫾
0.1 in normal to 1.30 ⫾ 0.1 in leptin-induced dysfunctional endothelium. In obese mice, a 2.5-fold increase in leptin concentration
coincided with 100% increase in eNOS and about 30% decrease in
intracellular L-arginine. The increased eNOS expression and a reduced
L-arginine content led to eNOS uncoupling, a reduction in bioavailable NO (250 ⫾ 10 vs. 420 ⫾ 12 nmol/l control), and an elevated
⫺
(70%). L-Arginine and
concentration of O⫺
2 (240%) and ONOO
sepiapterin supplementation reversed eNOS uncoupling and partially
restored [NO]/[ONOO⫺] balance in obese mice. In obesity, leptin
increases eNOS expression and decreases intracellular L-arginine,
resulting in eNOS an uncoupling and depletion of endothelial NO and
an increase of cytotoxic ONOO⫺. Hyperleptinemia triggers an endothelial NO/ONOO⫺ imbalance characteristic of dysfunctional endothelium observed in other vascular disorders, i.e., atherosclerosis and
diabetes.
LEPTIN-INDUCED ENDOTHELIAL DYSFUNCTION IN OBESITY
eNOS, leptin (10 ␮g/ml) was injected to reach a final concentration of 0.1
␮mol/l in the medium.
⫺
The changes in NO, O⫺
concentration from the
2 , and ONOO
background level with time were measured with a computer-based
Gamry VFP600 multichannel potentiostat (detection limit 1 nmol/l
and resolution time ⬍50 ms for each sensor). Changes in NO, O⫺
2 , and
ONOO⫺ concentrations were calculated by means of calibration
curves constructed for each sensor before and after measurements
with standard solutions (22, 25, 34). The electrochemical sensors
⫺
measure the net concentrations of diffusible NO, O⫺
2 , and ONOO ,
which are not consumed by fast chemical reactions.
eNOS expression measurement. Samples of homogenated cells or
tissue, equalized for protein content, were separated by SDS-PAGE (10%
gels) and transferred to polyvinylidene difluoride membranes. eNOS was
detected with mouse anti-eNOS monoclonal antibody. To compare eNOS
expression with the expression of the reference protein, we analyzed the
expression of ␤-actin by Western blot analysis using a monoclonal
anti-␤-actin antibody (Santa Cruz Biotechnology). Bands were detected
with the horseradish peroxidase-conjugated secondary antibodies and
visualized by chemiluminescence (21).
L-Arginine and cGMP determination. L-Arginine determination
was made using HPLC with fluorescence detection. HUVEC, blood
serum, and aortic endothelial tissue were homogenized in sulfosalicylic acid. After centrifugation and dilution of the supernatant portions, an internal standard (S-carboxymethyl-L-cysteine) was added
and a 1-min derivatization with fluorescent reagent o-phthalaldehyde
was performed. The 10-␮l samples were then injected into an HP1090
liquid chromatograph using a Zorbax Eclipse AAA column, and the
separation of the amino acids was obtained with a two-buffer-system
gradient elution. L-Arginine was detected using a Shimadzu RF 551
Fluorescence HPLC monitor, and the calculations were made using a
standard calibration curve. The concentrations of cGMP were measured in thoracic aortas using an ELISA kit. The protein content of
samples was measured by the bicinchoninic acid protein assay kit
(Pierce, Rockford, IL).
Statistical analysis. The data are presented as the means 1 ␮mol/l
⫾SE from 4 to 6 experiments. Multiple comparisons were evaluated
using ANOVA followed by the Student’s t-test. A P ⬍ 0.05 was
considered as statistically significant.
RESULTS
⫺
NO, O⫺
2 , and ONOO release in HUVEC. Typical amperometric curves (current proportional to concentration vs. time)
⫺
obtained for NO, O⫺
2 , and ONOO release from HUVEC are
shown in Fig. 1. Leptin did not directly stimulate NO, O⫺
2 , and
ONOO⫺ release from HUVEC. After CaI administration, a
⫺
rapid release of NO, O⫺
2 , and ONOO was observed from both
control and leptin-treated cells. The production of all three of
⫺
Fig. 1. Typical amperometric curves showing nitric oxide (NO; A), superoxide (O⫺
2 ; B), and peroxynitrite (ONOO ; C) release from a single human umbilical
⫺
vein endothelial cell (HUVEC). Leptin (0.1 ␮g/ml; a) or calcium ionophore (CaI; 1 ␮mol/l) was used to stimulate NO, O⫺
generation from a
2 , and ONOO
HUVEC incubated for 2 h without leptin (b) or with leptin (0.1 ␮g/ml; c).
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2-(2-allyl 3-hydroxy 4-methoxyphenyl)1-H benzo(b)pryan-4-one (S17834)
(Institute de Recherches Services, Suresnes, France).
Animals and diets. Four-week-old male C57BL/6J mice were
obtained from Jackson Laboratories (Bar Harbor, ME). Animals were
housed two per cage, kept in a temperature-controlled room (23°C)
with a 12-h:12-h light-dark cycle, and were provided food and water
ad libitum. All mice were randomly assigned to one of two diets for
105 days: low-calorie (D12450B, 10 kcal fats) and high-calorie
(D12492, 60 kcal fats) diet (Research Diets, New Brunswick, NJ).
Mice fed with high-calorie diets were randomly divided into three
groups: group 1, received regular water; group 2, received L-arginine
(100 mg 䡠 kg⫺1 䡠 day⫺1) in their water; and group 3, received L-arginine
(100 mg 䡠 kg⫺1 䡠 day⫺1) in their water and sepiapterin (10 mg䡠kg⫺1 䡠day⫺1)
in their powder chow. The 30-day treatment with L-arginine and/or
sepiapterin started on the 75th day of the experimental period. In
accordance with the protocol approved by the Ohio University Animal
Care and Use Committee, the mice were anesthetized and then
euthanized with an overdose of Penthotal. Aortas were then dissected
to carry out the studies described below.
Serum leptin concentration measurement. Blood (200 ␮l) was
taken from a tail vein of mice on days 15, 45, 75, and 105 of the
experimental period. Serum was isolated and leptin concentrations
were measured using an radioimmunoassay kit for mouse leptin
(Linco, St. Louis, MO).
Thoracic aorta preparation. Following euthanasia, the thoracic
aorta was excised from each animal and placed in modified Hanks’
balanced salt solution (HBSS) at 4°C and pH 7.4. Under the dissection
microscope (Wild M3G; Leica), the aorta was carefully separated
from adhering fat and connective tissue. Then small segments from
the thoracic aorta were cut into rings 3 to 4 mm in length. Rings were
opened longitudinally and pinned on the bottom of a thermostated
organ chamber filled with fresh HBSS buffer.
NO, O2⫺, and ONOO⫺ measurement. Concurrent measurement of
⫺
NO, O⫺
2 , and ONOO were performed with electrochemical nanosensors combined into 1 working unit with a total diameter of 2 to 3 ␮m.
Their design was based on previously developed and well-characterized chemically modified carbon-fiber technology (22, 25, 34).
A HUVEC culture cluster was placed in a well on the stage of an
inverted research microscope (Olympus IX71). The platinum wire auxiliary electrode counter and Ag兩AgCl reference electrodes were then
positioned in the well adjacent to the culture cluster. A module of NO,
⫺
O⫺
2 , and ONOO nanosensors was lowered near the surface (5 ⫾ 2 ␮m)
of a single cell membrane with the aid of a computer-controlled micromanipulator. In a similar manner, during the experiments with aorta, the
auxiliary electrodes of the three-electrode system were positioned in an
oxygenated organ chamber near an endothelial cell in an aortic strip. To
⫺
stimulate the release of NO, O⫺
from HUVEC or aortic
2 , and ONOO
endothelium, eNOS agonist calcium ionophore (CaI) A-23187 (Cal) was
injected with a microinjector to reach a final concentration of 1 ␮mol
CaI/l in the medium. To estimate the ability of leptin to directly stimulate
H1515
H1516
LEPTIN-INDUCED ENDOTHELIAL DYSFUNCTION IN OBESITY
control cells (2.0 ⫾ 0.1) (Fig. 4B). In contrast, HUVEC treated
with the same leptin concentration for 12 h showed a decrease
of the R value to 1.3 ⫾ 0.1. This indicated that there was a
gradual increase in eNOS uncoupling on longer exposure to
leptin. The eNOS uncoupling was partially reversed in the
presence of elevated L-arginine concentration (3 mmol/l) and
sepiapterin (0.1 mmol/l), a precursor of tetrahydrobiopterin (a
cofactor of eNOS) (Fig. 4B). In the presence of L-arginine and
sepiapterin, the R value increased to 2.3 ⫾ 0.1.
In addition, we confirmed that the release of NO and ONOO⫺
was related to eNOS activation because eNOS inhibition of the
enzyme by NG-nitro-L-arginine methyl ester (0.3 mmol/l) significantly blocked (about 75%) CaI-stimulated release of both NO
and ONOO⫺ (Fig. 4C). We tested the potential contribution of the
NAD(P)H oxidase-generated O⫺
2 to the overall formation of
ONOO⫺ and the diminished bioavailable NO. First, we measured
NO and ONOO⫺ in the presence of apocynin, a nonspecific
NAD(P)H oxidase inhibitor. We then measured NO and ONOO⫺
in the presence of S-17834, a more selective NAD(P)H oxidase
inhibitor. Treatment of endothelial cells with apocynin or S-17834
increased NO release by about 20 –30% and decreased ONOO⫺
level by 20 –30%. This indicates that about a 20 –30% concentration of O⫺
2 is generated by membrane-bound NAD(P)H oxidase
and about 70 – 80% of O⫺
2 is generated by membrane-bound
eNOS for the production of ONOO⫺ (after CaI stimulation).
⫺
Another potentially major source of NO, O⫺
in
2 , and ONOO
HUVEC is mitochondria. Nevertheless, under experimental conditions used in this study (sensors located 5 ⫾ 2 ␮m from the
outer surface of the cell membrane), it is rather unlikely that
⫺
mitochondrial O⫺
2 or ONOO can diffuse to, and be measured by,
the sensors.
eNOS expression and L-arginine concentration in HUVEC.
As shown in Fig. 5A, eNOS expression increased after cell
incubation with leptin in a dose-dependent manner. HUVEC
treated with leptin for 2 h showed a higher increase of eNOS
expression compared with the cells incubated for 12 h.
Figure 5B shows that during incubation, leptin-treated
HUVEC had significantly lower intracellular L-arginine concentration levels than untreated cells.
Body weight, leptin concentration, and eNOS expression.
The D-12492 mice had statistically significant increased mean
body weight versus D-12450B group as early as 1 mo after diet
initiation (Fig. 6A). Later the high-calorie-fed mice continued
to gain body weight much faster than mice maintained on the
low-calorie diet. After 105 days, the mean body weight of
⫺
Fig. 2. The effect of leptin on CaI (1 ␮mol/l)-stimulated peak concentration of NO (A), O⫺
2 (B), and ONOO (C) released from HUVEC. Cells were incubated
with various concentrations of leptin for 2 h (■) and 12 h (F). *P ⬍ 0.05 vs. control (without leptin); n ⫽ 5 experiments.
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these species was significantly higher in HUVEC incubated
with leptin when compared with control cells.
To investigate the relationship between the eNOS functional
state and leptin concentration, as well as the leptin exposure
⫺
time, we measured the CaI-stimulated NO, O⫺
2 , and ONOO
release from HUVEC treated with different leptin concentrations during different times. The incubation of cells for both 2
and 12 h with increasing leptin concentrations resulted in a
⫺
dose-dependent increase of the peak NO, O⫺
2 , and ONOO
generation (Fig. 2). The curves reflecting the production of
these species reached a semiplateau at about 0.1 ␮g/ml leptin.
It is interesting to report that the incubation of HUVEC with
leptin for 2 h stimulated the NO production to a higher degree
than treatment with leptin for 12 h. A 2-h exposure of cells to
leptin (0.1–10 ␮g/ml) led to a 1.6-fold increase of NO production compared with basal conditions, whereas the same concentrations of leptin exposure for 12 h caused only a 1.3-fold
increase of peak NO release. In contrast to NO, the CaI
⫺
stimulated peak production of O⫺
in the
2 and ONOO
HUVECs incubated with leptin (0.1–10 ␮g/ml) for 12 h was
about 2.0 and 1.7 times higher, respectively, than the control
HUVEC. Whereas there was only a 1.3- and 1.2-fold increase,
⫺
respectively, of the O⫺
2 and ONOO peak productions in the
HUVECs incubated with 0.1–10 ␮g/ml leptin for 2 h.
Analysis of the time-dependent effect of constant leptin
concentration on stimulated peak NO release showed the rapid
rise of NO production for 1 h after the beginning of cell
exposure to the hormone (Fig. 3A). After reaching the maximum (820 ⫾ 35 nmol/l at 2 h), a significant linear decrease in
NO release was observed. In contrast to NO, the O⫺
2 and
ONOO⫺ productions sharply increased at 4 h and steadily
increased thereafter. As expected, in the presence of membrane-permeable PEG-SOD (O⫺
2 scavenger), the concentration
⫺
of O⫺
sharply de2 as well as the concentration of ONOO
creased, whereas NO concentration increased in leptin-treated
cells (Fig. 4A). However, in the presence of MnTMPyP
(ONOO⫺ scavenger), only a significant decrease in ONOO⫺
was observed.
We used the ratio of NO to ONOO⫺ concentration (R ⫽
[NO]/[ONOO⫺]) to quantify the relation between bioavailable
NO and cytotoxic ONOO⫺ in the endothelium. A high R value
indicates strong eNOS coupling and high bioavailable NO
and/or low nitroxidative stress (low level of ONOO⫺).
Intriguingly, the R value was 2.8 ⫾ 0.1 in HUVEC incubated for 2 h with 0.1 ␮g/ml leptin when compared with
LEPTIN-INDUCED ENDOTHELIAL DYSFUNCTION IN OBESITY
H1517
D-12492 mice was about 1.4 times higher than that of the
control group. No influence of treatment with L-arginine on
body weight was detected.
The shape of curves reflecting the increase of leptin concentration in serum of mice fed with high- and low-calorie diets
(Fig. 6B) were very similar to the curves reflecting the gain of
body weight in corresponding animal groups. Whereas the
leptin concentration at the end of the experimental period (105
days) was 6.9 ⫾ 0.8 ng/ml in the control group, the maximal
leptin concentration at the same time was 2.5 times higher
(16.1 ⫾ 1.5 ng/ml) in the mice provided with the high-calorie
diet and untreated water. As in the case with body weight,
L-arginine did not significantly affect the leptin concentration
in obese mice. A slight linear increase (about 8% maximal) in
leptin concentration with the body weight was observed in
mice on a low-calorie diet (Fig. 6C). Nonetheless, the increase
in leptin concentration with the body weight was exponential
for obese mice. It is interesting to note that the most remarkable increase in leptin concentration was observed in obese
mice at a weight exceeding the maximal weight (about 35 g) of
the low-calorie diet D-12450B mice. In addition, the 2–5 fold
increase in leptin concentration correlated with the increase in
eNOS in obese mice expression (about a 2-fold increase
compared with control). The expression of inducible NO synthase in the endothelium did increase significantly in obese
mice (data not shown). The cGMP level (aortic wall) correlated
inversely with eNOS expression (Fig. 7, A and B) and directly
reflected the diminished NO concentration observed in obese
mice (Fig. 2A).
⫺
NO, O⫺
release in aortic endothelium. To
2 , and ONOO
deeper investigate the ability of leptin to affect the functional
state of endothelial cells, the CaI-stimulated NO, O⫺
2 , and
ONOO⫺ production in aortic endothelium of normal (control)
and obese C57BL/6J mice was measured (Fig. 7C). NO concentration released from the aortic endothelium of control mice
was 420 ⫾ 12 nmol/l, about 1.7 times higher than from aortic
endothelium of mice with obesity (250 ⫾ 11 nmol/l). Obese
mice treated with L-arginine showed higher NO production
(297 ⫾ 10 nmol/l, P ⬍ 0.05) compared with untreated obese
mice. A further increase (⬃25%) in NO levels was observed
after L-arginine plus sepiapterin treatment.
⫺
⫺
⫺
Fig. 4. A: maximal NO, O⫺
concentration produced by HUVEC in the presence of O⫺
scavengers. NO, O⫺
releases
2 , and ONOO
2 , and ONOO
2 or ONOO
were stimulated by CaI (1 ␮mol/l) from a HUVEC incubated for 12 h with leptin (0.1 ␮mol/l) or with polyethylene glycol (PEG)-SOD (100 U/ml) (gray bar)
or MnTMPyP (0.02 mmol/l) (black bar); control (white bar), *P ⬍ 0.0001 vs. control; n ⫽ 4 to 5 experiments. B: the ratio (R) of peak NO concentration to the
peak concentration of ONOO⫺ (R ⫽ [NO]/[ONOO⫺]). NO and ONOO⫺ releases were stimulated by CaI (1 ␮mol/l) from HUVEC incubated for 2 h without
leptin (control, white bar) or incubated with leptin (0.1 ␮g/ml) for 2 h (gray bar) or 12 h (black bar) at normal and elevated L-arginine (L-Arg; 3 mmol/l) or
L-arginine (3 mmol/l) and sepiapterin (0.1 mmol/l). *P ⬍ 0.05 vs. control and **P ⬍ 0.01 vs. 12-h incubation without L-arginine, sepiapterin; n ⫽ 4 to 5
experiments. C: CaI-stimulated maximal NO and ONOO⫺ concentration released from HUVEC in the absence (control, white bars) or in the presence (black
bars) of NG-nitro-L-arginine methyl ester (L-NAME; 0.3 mmol/l) and apocynin (3 mmol/l) or S-17834 (0.07 mmol/l). The concentration of NO and ONOO⫺ was
measured after 12 h incubation with leptin (0.1 ␮g/ml). *P ⬍ 0.001 vs. control, n ⫽ 4 to 5 experiments.
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⫺
Fig. 3. CaI (1 ␮mol/l)-stimulated peak concentration of NO (A), O⫺
2 (B), and ONOO (C) as a function of incubation time. HUVECs were incubated with leptin
(0.1 ␮g/ml) (■) or without leptin (F). *P ⬍ 0.05 vs. control (without leptin); n ⫽ 4 to 5 experiments.
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LEPTIN-INDUCED ENDOTHELIAL DYSFUNCTION IN OBESITY
Fig. 5. A: endothelial NO synthase (eNOS)
expression in HUVEC incubated with various
concentrations of leptin for 2 h (white bars) and
12 h (black bars). *P ⬍ 0.05 vs. control (without leptin); n ⫽ 4 to 5 experiments. B: timedependent effect of constant leptin concentration (0.1 ␮g/ml) on L-arginine level in HUVEC.
Cells were incubated with leptin (■) or without
leptin (F) leptin for 12 h. *P ⬍ 0.05, n ⫽ 4 to
5 experiments.
DISCUSSION
It is generally accepted that obesity is closely associated
with an increased risk of hypertension and heart failure, but the
mechanisms involved are still not fully understood (14, 32). It
has been reported that the adipocyte-derived hormone leptin is
involved in the regulation of blood vessel tonus (21). However,
the effect of leptin on NO production in endothelial cells has
been assessed only with the use of indirect methods (3, 12, 18,
19). In contrast, this study used highly sensitive, selective,
millisecond response-time electrochemical nanosensors (22,
25, 34) to directly measure the concentration of CaI-stimulated
⫺
NO, O⫺
2 , and ONOO release from HUVEC treated with leptin
as well as from the aortic endothelium of normal and obese
mice with hyperleptinemia.
Our results clearly demonstrated that leptin does not
⫺
directly stimulate NO, O⫺
release from the
2 , or ONOO
endothelium. However, the exposure of endothelial cells to
leptin resulted in the dose-dependent upregulation of eNOS
expression and an enhanced NO production that lasted for
about 2 h.
Leptin is known to increase cell proliferation, which profoundly influences the abundance of the eNOS transcript. Thus
any factors that influence endothelial cell growth rate may
confound the interpretation of the primary effects on eNOS
gene expression (30). Therefore, the leptin-enhanced expres-
Fig. 6. A: body weight of C57BL/6J mice maintained on low-calorie diet (■) or high-calorie (60 kcal fat) diet with (E) or without (F) supplementation of
L-arginine. B: leptin concentration changes in blood plasma of C57BL/6J mice maintained on low-calorie diet (■) or high-calorie D-12492 (60 kcal fat) diet with
(E) or without supplementation (F) of L-arginine (F). *P ⬍ 0.05 vs. control mice (low-calorie diet); n ⫽ 4 to 5 mice. C: leptin vs. body weight linear plot (r ⫽
0.93, P ⬍ 0.05) for the low-calorie diet (■, solid line) or exponential plot (r2 ⫽ 0.95, P ⬍ 0.0001) for high-calorie diet D-12492 (60 kcal fat) diet (E, dashed
line).
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⫺
The CaI-stimulated peaks of O⫺
2 and ONOO release were
significantly elevated in obese mice. The maximal O⫺
2 production in aortic endothelium of mice maintained on a high-calorie
diet and untreated water was about 3.4 times higher than in
control mice. The peak ONOO⫺ release was 1.7 times higher
in obese mice than in control mice. A high-caloric diet supplementation with L-arginine or L-arginine plus sepiapterin
⫺
resulted in the decrease of O⫺
2 and ONOO and an increased
NO production in the aortas of obese mice by 30% and 17%,
respectively. The high-calorie diet L-arginine supplementation
⫺
resulted in the decrease of O⫺
2 and ONOO . This effect was
even more pronounced after supplementation with L-arginine
plus sepiapterin (Fig. 7C).
The R value decreased about 64% in obese mice compared
with control mice (Fig. 8A). L-Arginine treatment increased the
R value about 30% over the untreated obese mice, and treatments with L-arginine plus sepiapterin doubled the R value
over the untreated obese mice.
L-Arginine concentration in blood and aortic endothelium.
The level of L-arginine in the blood of obese mice did not
change significantly compared with the control mice (Fig. 8B).
In contrast, obesity resulted in about a 30% decrease of
L-arginine level in aortic endothelium of obese mice compared
with control mice. L-Arginine supplementation caused a significant increase of L-arginine level both in blood (70%) and in
aortic endothelium (30%) of the obese mice.
LEPTIN-INDUCED ENDOTHELIAL DYSFUNCTION IN OBESITY
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sion of eNOS in endothelial cells can be explained, at least in
part, by a transcriptional and/or posttranscriptional mechanism.
We acknowledge, however, that further work beyond the limits
of the present study is required to verify the mechanism of
leptin-enhanced eNOS expression in the endothelium.
A short-time exposure of HUVEC to leptin did not increase
⫺
O⫺
levels significantly. This indicates that an
2 and ONOO
acute exposure of endothelial cells to leptin may have beneficial effects on the cardiovascular system by increasing the
potential for the generation of bioavailable NO.
In contrast, long-term (12 h) exposure of endothelial cells to
leptin decreased CaI-stimulated bioavailable NO despite a
twofold increase in eNOS expression in the endothelium. This
apparent paradox can be explained by the enhanced generation
⫺
of O⫺
2 and ONOO after long-term exposure of the HUVEC to
leptin. Indeed, a wide variety of proatherogenic risk factors like
obesity and other cardiovascular disease states can be associ-
ated with increased oxidative stress and diminished NO bioavailability (10).
To better specify the relationship between leptin, eNOS
expression, and obesity in animals, another portion of our
experiments involved an inbred C57BL/6J strain of obese
mice. These mice were not obese on a standard chow diet but
became obese and developed hyperleptinemia when fed a
high-fat diet (28). We clearly documented the exponential
increase in serum leptin concentration with body weight of
obese mice. The increased leptin was especially dramatic when
the weight of obese mice exceeded the maximal weight of
low-calorie diet mice.
The present study provides direct evidence that obesity
causes an increase in leptin level that inversely correlates with
NO bioavailability in the aortic endothelium and with cGMP in
the aortic wall. Simultaneously, with the suppression of NO
production in obese mice, we registered the considerable in-
Fig. 8. A: the ratio of NO concentration to the concentration of ONOO⫺ (R ⫽ [NO]/[ONOO⫺]). NO and ONOO⫺ releases were stimulated by CaI (1 ␮mol/l)
from endothelial cells of the aortas of C57BL/6J mice maintained for 105 days on low-calorie diet (1) or on high-calorie (60 kcal fat) diet (2) with
supplementation of L-arginine (100 mg 䡠 kg⫺1 䡠 day⫺1) (3) or with supplementation with L-arginine (100 mg 䡠 kg⫺1 䡠 day⫺1) and sepiapterin (10 mg/kg/day) (4).
*P ⬍ 0.01 vs. control (low-calorie diet); #P ⬍ 0.05 vs. obese mice without supplementation with L-arginine or L-arginine and sepiapterin; n ⫽ 5 mice.
B: L-arginine concentration in serum and aortic endothelium of C57BL/6J mice maintained for 105 days on low-calorie diet (1) or on high-calorie (60 kcal fat)
without (2) or with supplementation with L-arginine (100 mg 䡠 kg⫺1 䡠 day⫺1) (3). *P ⬍ 0.01 vs. control (low-calorie diet); #P ⬍ 0.05 vs. obese mice without
supplementation with L-arginine; n ⫽ 5 mice.
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Fig. 7. A: eNOS expression in aortic endothelial cells of C57BL/6J mice maintained on a low-calorie diet (control, white bars) and high-calorie diet (60 kcal
fat; black bars) for 105 Days. B: cGMP content in aortic wall of C57BL/6J mice maintained on a low-calorie diet (control, black bars) and a high-calorie diet
⫺
(60 kcal fat; white bars) *P ⬍ 0.005, n ⫽ 4 to 5 mice. C: CaI (1 ␮mol/l)-stimulated NO, O⫺
release from aortic endothelial cells of C57BL/6J
2 , and ONOO
mice maintained for 105 days on low-calorie diet (black bars) or high-calorie (60 kcal fat, white bars) with or without supplementation with L-arginine (100
mg 䡠 kg⫺1 䡠 day⫺1) or L-arginine (100 mg 䡠 kg⫺1 䡠 day⫺1) and sepiapterin (10 mg 䡠 kg⫺1 䡠 day⫺1). *P ⬍ 0.01 vs. control (low-calorie diet); #P ⬍ 0.05 vs. obese mice
without L-arginine supplementation; n ⫽ 4 to 5 mice.
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LEPTIN-INDUCED ENDOTHELIAL DYSFUNCTION IN OBESITY
AJP-Heart Circ Physiol • VOL
Furthermore, in our experiments, the obese mice treated
with L-arginine or L-arginine and sepiapterin (a precursor of
tetrahydrobiopterin) showed significantly higher NO pro⫺
duction and lower O⫺
release from the aortic
2 and ONOO
endothelium compared with the untreated obese mice.
The most potentially damaging property of the eNOS dimer
(called anticooperative bonding) occurs because the two eNOS
monomers can act independently (24). During moderate Larginine or tetrahydrobiopterin deficiencies, after activation, it
is very probable that one monomer of an eNOS dimer could be
coupled and rapidly making NO, while the adjacent monomer
of the same eNOS dimer is uncoupled and rapidly making O⫺
2.
The near diffusion-limited reaction of O⫺
with
NO
to
form
2
ONOO⫺ is even faster than the dismutation of O⫺
2 by superoxide dismutase. Unlike NO and O⫺
2 , which are not strong
oxidants, ONOO⫺ is a potent oxidant. ONOO⫺ is relatively
unstable in its anionic form. However, when protonated, the
peroxynitrous acid (HONOO) at high concentrations may
freely diffuse several cell diameters before it rearranges to
form nitric acid (HNO3) or undergoes homolytic or heterolytic
cleavage to generate highly toxic oxidative species, including
˙NO2, NO⫹
2 , and ˙OH. These products are among the most
reactive and damaging species in biological systems. They may
initiate a cascade of events leading to the oxidation of proteins,
DNA, and lipids and finally resulting in cytotoxicity and
cellular dysfunction (2). The sum of unfavorable events developed in cells due to ONOO⫺ hyperproduction is collectively
called “nitroxidative stress,” which is a major component of
oxidative stress.
There is another contributing mechanism that may partially
help explain our results. It has been suggested that increased
free fatty acid concentration in obesity may be related to
increased levels of asymmetric dimethylarginine (ADMA), an
endogenous inhibitor of NO synthase (2). L-Arginine competes
with ADMA for the eNOS enzyme (29). Therefore, an increase
in NO production after supplementation with L-arginine to
obese mice may be due in part to an increase of the arginineto-ADMA ratio in endothelial cells and the reduction of the
ADMA inhibitory effect.
In summary, we have provided direct evidence that, despite
leptin-induced increased eNOS expression, bioavailable NO is
⫺
reduced, whereas O⫺
levels are increased in
2 and ONOO
⫺
⫺
obesity. Increased O2 and ONOO production that contributes
to high oxidative/nitroxidative stress in endothelial cells is
most likely due to a discrepancy between enhanced eNOS
expression and diminished intracellular L-arginine concentration in obesity. Thus long-term exposure of endothelium to
leptin may lead to enzymatic uncoupling of the oxidative and
reductive domains of eNOS. L-Arginine and sepiapterin treatment during the long term hyperleptinemia accompanying
obesity increases L-arginine (and most likely tetrahydrobiopterin) concentration in aortic endothelium and significantly
⫺
reduces eNOS uncoupling (decreasing O⫺
pro2 and ONOO
duction and preserving bioavailable NO). Leptin-induced
[NO]/[ONOO⫺] imbalance in the vascular endothelium observed in obesity is similar to the redox state that has been
reported in other endothelium-impaired function disorders,
including hypertension, atherosclerosis, diabetes, and others (9,
32, 33).
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⫺
crease in O⫺
generation. The most probable
2 and ONOO
mechanism for the NO release diminishment in this case, like
in the experiments with cell culture, is the overproduction of
⫺
O⫺
2 , resulting in the fast reaction with NO to form ONOO .
This direct quantitative data are concordant with previous
indirect studies that indicate the role of oxidative stress in
obesity (11, 27). Dobrian et al. (9) recently reported that there
was a ⬃1.8-fold decrease of the plasma and urine nitrate and
nitrite levels and the same increase in O⫺
2 production from the
aortas from obese Sprague-Dawley rats compared with lean
ones. At the same time, the eNOS expression in aorta of obese
animals was increased ⬃8-fold compared with control. This
apparent discrepancy can be clearly explained with the data
presented in this study.
To explain the differences in the effect of leptin exposure on
⫺
NO production versus O⫺
2 and ONOO production, it may help
to realize that the NO sensor used in this study detects only the
net concentration of NO (i.e., NO that is not consumed in fast
chemical reactions and can freely diffuse to a target cell and
trigger cGMP production). This net concentration depends not
only on the activity of eNOS but also on the production of O⫺
2.
The rapid CaI stimulation of eNOS produces a large NO
release accompanied by O⫺
2 production, suggesting that some
of the production of O⫺
is
calcium dependent, similar to the
2
production of NO by eNOS.
Also, it is helpful to realize that both membrane-bound
eNOS and NAD(P)H oxidase may contribute to the overall
generation of ONOO⫺ (23, 33). At low to moderate activity of
eNOS, the main generator of O⫺
2 in the vascular wall is
NAD(P)H (23). In this study, after rapid CaI-stimulation of
⫺
eNOS, the contribution of eNOS generated O⫺
2 to the ONOO
production was about three times higher than the contribution
of NAD(P)H oxidase generated O⫺
2 . Furthermore, endothelial
mitochondria can be a generator of O⫺
2 and subsequently
ONOO⫺. But, the nanotechnological set-up used in this study
⫺
is not suitable for measuring O⫺
directly in
2 or ONOO
mitochondria without disturbing cell membranes and stimulating NO and O⫺
2 release. These studies elucidate mainly NO,
O⫺
2 generation by membrane-bound eNOS.
One of the most probable mechanisms underlying the
leptin-stimulated endothelial dysfunction may be the imbalance between eNOS expression and intracellular L-arginine
and/or tetrahydrobiopterin level. According to our results,
leptin significantly increases the amount of eNOS protein in
HUVEC and endothelium of obese mice. This enhanced
eNOS expression in sustained hyperleptinemia was also
recently observed by other investigators (9, 26, 36). Coincident with the increase in eNOS expression, we observed a
tendency toward diminishment of the L-arginine concentration in leptin-incubated HUVEC and a significantly diminished L-arginine level in obese animals with hyperleptinemia. L-Arginine is required not only as a substrate for NO
synthesis but also as a stabilizer of eNOS, preventing it from
uncoupling. The relative L-arginine and/or eNOS cofactor
tetrahydrobiopterin insufficiencies result in the uncoupling
of the oxidative and reductive domains of eNOS, which
results in O⫺
2 generation (6, 17). The net effect of the
reaction between NO and increased O⫺
2 caused the diminishment of NO bioavailability. The [NO]/[ONOO⫺] ratios
were significantly decreased in both HUVEC treated with
leptin for 12 h and aortic endothelium of obese mice.
LEPTIN-INDUCED ENDOTHELIAL DYSFUNCTION IN OBESITY
GRANTS
This study was supported by National Heart, Lung, and Blood Institute
Grant HL-55397, the Marvin White Endowment, and the Biomedical Nanoscience Nanotechnology Program (Ohio University).
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