original contributions
nature publishing group
Effect of Red Wine on Adipocytokine Expression
and Vascular Alterations in Fructose-Fed Rats
Marcela A. Vazquez-Prieto1,2, Nicolás F. Renna1,2, Emiliano R. Diez1,2, Valeria Cacciamani1, Carina Lembo1,2
and Roberto M. Miatello1,2
Background
Imbalance in adipocytokines secretion is related to the development
of metabolic syndrome (MS). In addition, moderate consumption of
red wine (RW) decreases the risk of cardiovascular disease. The aim
of this study was to evaluate the effects of moderate consumption
of RW or ethanol (E) on adiponectin and resistin expression, and
vascular alterations in fructose-fed rats (FFRs) as an experimental
model of MS.
NAD(P)H subunits Nox4 expression in mesenteric tissue, plasma
thiobarbituric acid reactive substances (TBARS), and recovered
plasma total antioxidant activity (TAA) compared to F and F+E groups
(P < 0.05). Adiponectin expression decreased, whereas resistin,
vascular cell adhesion molecules-1 (VCAM-1), and nuclear factor-κB
(NF-κB) expression and vascular remodeling in mesenteric arteries
were higher in F than in C group (P < 0.05). Only RW was able to
partially reverse the aforementioned alterations.
Methods
Thirty-day-old male Wistar rats were assigned to control (C), F
(10% fructose in drinking water), F+E (4.5 ml/kg), and F+RW (35 ml/kg
of Malbec RW containing 4.5 ml/kg E). E and RW were administered
during the last 4 weeks of a 10-week period.
Conclusion
In this study, Malbec RW, but not alcohol alone, improved the
balance of adipocytokines and attenuated the oxidative stress and
vascular inflammation in a model of MS, suggesting that nonalcohol
components of RW are responsible for the beneficial effects.
Results
RW administration to F rats was able to significantly decrease insulin
resistance, mesenteric adipose tissue weight, and systolic blood
pressure (SBP) compared to F group. F+E only reduced the SBP
(P < 0.05 vs. F). F+RW also reduced aortic NAD(P)H-oxidase activity,
Keywords: adipocytokines; blood pressure; hypertension; metabolic
syndrome; resistin; adiponectin; red wine
Cardiovascular disease is the leading cause of mortality in
western countries where metabolic syndrome (MS), characterized by hyperglycemia, hyperlipidemia, and hypertension,
is a significant risk factor for cardiovascular disease.1 Fructose
intake has increased dramatically in the past 20 years contributing to the growing worldwide prevalence of MS, ­obesity,
and diabetes.2,3 Fructose-fed rats (FFRs) provide a model of
­dietary-induced insulin resistance, which has been used to
assess the pathophysiological mechanisms involved in the
metabolic and cardiovascular changes associated with MS.4,5
Oxidative stress has been implicated in the pathogenesis
of many cardiovascular diseases. The major sources of reactive oxygen species (ROS) in vascular tissue are membrane­associated NAD(P)H-oxidases, also known as Nox enzymes.
Nox4 subunit expression is strongly correlated with an increase
of NAD(P)H-oxidase activity.6 Our group previously reported
1Department of Pathology, School of Medicine, National University of Cuyo,
Mendoza, Argentina; 2Laboratory of Cardiovascular Pathophysiology, Institute
for Experimental Medical and Biological Research (IMBECU), National Council of
Research (CONICET), Mendoza, Argentina. Correspondence: Roberto M. Miatello
([email protected])
Received 11 June 2010; first decision 1 July 2010; accepted 30 August 2010.
© 2011 American Journal of Hypertension, Ltd.
234
American Journal of Hypertension, advance online publication 30 September 2010;
doi:10.1038/ajh.2010.214
an enhanced NAD(P)H-oxidase aorta activity5 and higher
plasma lipid peroxidation7 in FFR implying that this model of
MS is characterized by oxidative stress.
Abnormalities in adipose tissue have been observed in FFR,
without changes in body weight.8 Adipose tissue as well as
nearby inflammatory cells secrete a variety of bioactive molecules named adipocytokines as tumor necrosis factor-α,
plasminogen activator inhibitor-1, interleukin-6, leptin, adiponectin, and resistin.9 The effects of adipocytokines on vascular function, immune regulation, and adipocyte metabolism
make them key factors in the pathogenesis of MS.10 Previous
studies have shown that lowered levels of plasma adiponectin
induce insulin resistance and atherosclerosis in an experimental model of obesity.11 Studies in human aorta endothelial cells
show that resistin induces the expression of adhesion molecules
such as intercellular cell adhesion molecules and vascular cell
adhesion molecules-1 (VCAM-1) via transcription nuclear
factor-κB (NF-κB) signals, while adiponectin inhibits the resistin effect over endothelial cells.12 The exact mechanism by
which fat accumulation leads to such adipocytokines imbalance
has not been elucidated, but oxidative stress may be implied.
Epidemiological studies have shown a protective effect of
moderate red wine (RW) consumption on ­cardiovascular
February 2011 | VOLUME 24 NUMBER 2 | 234-240 | AMERICAN JOURNAL OF HYPERTENSION
Red Wine and Adipocytokines in Fructose-Fed Rats
disease,13 mainly attributed to antioxidant properties of RW
polyphenols.14,15 Although there is wide evidence showing healthy effects of moderate RW intake, to date there are
no studies that evaluate the effect of RW on adipocytokines
expression in a model of MS.
The aim of this study was to evaluate in an experimental model of MS the effect of moderate consumption of RW
or ethanol (E) on adipocytokines expression in mesenteric
adipose tissue. Additionally, we assessed vascular alteration
through VCAM-1 expression and lumen/media ratio in vascular mesenteric tissue and oxidative stress.
Methods
Reagents. Unless otherwise noted, all reagents were purchased
from Sigma Chemical (St Louis, MO). All other chemicals
were of molecular biology or reagent grade.
Animals, diet, and experimental designs. All procedures were
performed according to institutional guidelines for ­animal
experimentation and were approved by the Technical and
Science Secretary at the National University of Cuyo School of
Medicine. Thirty-day-old male Wistar rats, weighing 90–130 g
were housed during the experimental period of 70 days in a
room under conditions of controlled temperature (21 ± 2 °C)
and humidity with a 12 h light/dark cycle.
At the beginning of the study, 40 rats were randomly distributed into two groups: one control group (C; n = 10) and one
experimental group (F; n = 30) that received 10% fructose
(Saporiti Labs, Buenos Aires, Argentina) solution in drinking
water for 10 weeks. After 6 weeks of fructose administration,
the experimental group was further divided into three groups
(n = 10 each), and for 4 additional weeks received the following
treatments, respectively: F; F+E: F received E 4.5 ml/kg administered daily in drinking water diluted in a 10% fructose solution; and F+RW: F received RW 35 ml/kg (containing 4.5 ml E)
administered daily in drinking water diluted in a 10% fructose
solution. All groups were fed the same standard rat diet (GepsaFeeds, Buenos Aires, Argentina) and tap water ad libitum.
The RW (Malbec grape variety 13% ethanol) was provided
by the National University of Cuyo School of Agricultural
Sciences (Mendoza, Argentina). The phenolic characterization of RW Malbec was evaluated as previously described.16
Total phenol content expressed as gallic acid was 2.9 g/l of RW.
The main phenolic content was (expressed as mg/l): nonflavonoids: 18.2 gallic acid; 2 caffeic acid; 4.2 cis-caftaric acid, transresveratrol: 1.1, flavonoids: 24.1 catechin; 14.2 epicatechin;
procyanidin (11.3 B1; 3.1 B3), flavonols: 4.9 quercetin, and
anthocyanins (344 malvidin-3-glucoside; 16.2 peonidin-3­glucoside; 60.3 delphinidin-3-glucoside). RW doses were calculated on the basis of the recommended daily consumption
of 350 ml for a 70 kg human (5 ml/kg).17 Using an interspecies dose-conversion factor based on equal body surface (7:1
for conversion from rat to human)18 and taking into account
that liquid consumption of FFRs was 35 ml/day, the dose of
RW used was 35 ml/kg per day. RW was administered in the
drinking water diluted to 10% fructose and was held in dark
AMERICAN JOURNAL OF HYPERTENSION | VOLUME 24 NUMBER 2 | February 2011
original contributions
bottles changed out daily in order to prevent oxidation of the
polyphenols.
Food intake and liquid consumption were recorded throughout the experiment.
At the end of the experimental period, and after overnight fast, the rats were weighed, anesthetized with ketamine
(50 mg/­kg) and acepromazine (1 mg/kg). Blood was collected
from the abdominal aorta into heparinized tubes. Plasma
obtained after centrifugation at 1,000g for 15 min at 4 °C was
frozen at −70 °C until assayed. Arteries and organs from six rats
of each group were excised aseptically for the measurement of
various parameters described below. The remaining four rats
of each group were assigned to histological observations.
Body and mesenteric fat weight. Body weight was measured
weekly. At the end of the experimental protocol, mesenteric
adipose tissues from six rats of each group were dissected and
weighed relative to the total body weight, expressing the ratio
as mesenteric fat tissue (mg)/body weight (g).
Biochemical determinations. Plasma glucose, total cholesterol,
high-density lipoprotein, and triglyceride concentrations were
determined by enzymatic colorimetric methods using commercial kits (GTLab, Buenos Aires, Argentina). Insulin was
measured by radioimmunoassay (Coat-A-Count, Siemens,
Los Angeles, CA) and insulin resistance was assessed using
the homeostasis model assessment (HOMA-IR) originally
described by Matthews et al.19 HOMA-IR was calculated using
the following formula: HOMA-IR (mmol/l × μU/ml) = fasting
glucose (mmol/l) × fasting insulin (μU/ml)/22.5. The systolic
blood pressure (SBP) was monitored indirectly once a week in
conscious, prewarmed, slightly restrained rats by the tail-cuff
method and recorded on a Grass Model 7 polygraph (Grass
Instruments, Quincy, MA).
Measurement of markers of oxidative stress. The measurement
of vascular NAD(P)H-oxidase activity in the aorta was made
by the lucigenin-derived chemiluminescence assay as previously described,20 and the luminescence was measured on a
luminometer (Fluoroskan Ascent FL; Thermo Labsystems,
Waltham, MA). The results were expressed in arbitrary units
(au)/min/mg of aortic tissue.
Thiobarbituric acid reactive substances (TBARS) were
determined as previously described.7 This method is based
on the reaction between plasma malondialdehyde, a product
of lipid peroxidation, and thiobarbituric acid. Total antioxidant activity (TAA) was measured as previously reported.21
The assay relies on the ability of antioxidants in the plasma in
reducing the preformed radical monocation 2,2′-azinobis-(3ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) generated by
oxidation of 7 mmol/l ABTS with 2.45 mmol/l potassium persulfate. The influences of both the concentration of antioxidant
and duration of reaction on the inhibition of the radical cation
absorption were taken into account when determining the
antioxidant activity and the results were expressed as percent
of inhibition of ABTS•+.
235
original contributions
Red Wine and Adipocytokines in Fructose-Fed Rats
Western blot analysis. Adipose and vascular mesenteric tissues
were dissected, weighed, and immediately stored at −70 °C.
Adiponectin and resistin were evaluated in adipose tissue, while
Nox4, VCAM-1, and NF-кB protein expression were evaluated
in mesenteric vascular homogenates. Samples were homo­
genized in lysis buffer (containing 2% sodium dodecyl ­sulfate,
10% glycerol, 5% mercaptoethanol, and 6.25 mmol/l Tris
buffer: pH 6.8) for adipose tissue and (10.9% sucrose, 0.037%
EDTANa2, and 0.06% HEPES: pH 7.4) for vascular mesenteric
tissue, both with proteinase inhibitors added (100 μg/ml of
phenylmethanesulfonyl fluoride and 1 μg/ml soybean trypsin
inhibitor). Protein concentrations were quantified in duplicate
using the Bradford method with bovine serum protein as reference protein. Twenty micrograms of protein was loaded per
lane in sodium dodecyl sulfate–polyacrylamide gel (8–12.5%),
electroblotted onto nitrocellulose membranes (Amersham
Biosciences, Buckinghamshire, UK), and incubated with an
appropriate polyclonal antibody: adiponectin and resistin
(1/1,000; Chemicon, Ramona, CA); NF-κB (p65 subunit RelA)
(1/500; Chemicon); VCAM-1 and Nox4 (1/500 and 1/1,000,
respectively, from Santa Cruz Biotechnology, Santa Cruz, CA).
Monoclonal anti-β-actin antibody (1/4,000) was used as control for equal loading. Horseradish peroxidase–conjugated
anti-rabbit and anti-mouse secondary antibodies (1/2,500 and
¼,000, respectively; Dako, Glostrup, Denmark) were used, and
immunoreaction signals were viewed with enhanced chemiluminescence (Hyperfilm ECL; Amersham Biosciences). X-ray
films with protein bands were scanned and densitometric
analysis was performed using the US National Institute of
Health Image 1.66 software (Rasband Wayner et al. Division
of Computer Research and Technology NIH, Bethesda, MD).
Data were expressed in arbitrary units as primary antibody/βactin ratio.
Vascular histological analysis. Mesenteric tissue samples from
four rats of each group were used for the histological study as
previously described.22 Briefly, slices of 5 μm thickness were
cut transversely on a microtome (Microm HM325; Thermo
Scientific, Walldorf, Germany), stained with Masson’s trichrome, and examined under a light microscope (Optiphot-2;
Nikon, Kanagawa, Japan). Images were digitalized with a ­digital
camera (GP-KR222 color CCD; Panasonic, Osaka, Japan) and
processed with an analysis system Scion Image 4.01 (Scion,
Bethesda, MD). The lumen-to-wall media ratio (L/M ratio)
was then calculated.
Statistical analysis. Data were expressed as mean ± s.e.m. The
statistical significance was assessed by one-way analysis of
variance followed by Bonferroni’s Multiple Comparison posttest. GraphPad Prism version 5.00 for Windows (GraphPad
Software, San Diego, CA) was used for all statistical analysis.
Differences were considered significant at P < 0.05.
Results
Fructose solution intake by F, F+E, and F+RW was higher than
water consumption by C group (P < 0.01). All experimental
groups (F, F+E, F+RW) ate less food than C group (P < 0.05).
The addition of E or RW during the last 4 weeks did not modify the liquid or food intake. At week 6, energy intake (kJ) was
higher in F, F+E, and F+RW than C group (P < 0.05). When
the E and RW was added to FFRs, the energy intake increased
in both F+E and F+RW compared to F (P < 0.01) and C groups
(P < 0.001). Although the body weight did not vary among
groups, the mesenteric adipose tissue weight was higher in F
than C group (P < 0.001). Only RW administration reduced
significantly the mesenteric adipose tissue weight (P < 0.01 vs.
F; Table 1).
Chronic administration of fructose displays several alterations included in the cluster of risk factors called MS. At the
end of the study, FFRs had greater SBP than C group (P <
0.001). Administration of both RW and E to FFR during the
last 4 weeks was able to reduced the SPB (P < 0.01), but did not
reach the C group values. F and F+E groups had greater fasting
levels of glycemia, triglyceridemia, insulinemia, and HOMAIR
index than C (P < 0.001). RW administration to FFRs had the
capacity to decrease the insulin levels, HOMAIR index, and
triglycerides compared to F and F+E (P < 0.05). Additionally,
F+RW had greater high-density lipoprotein levels than F (P <
0.01). The L/M ratio in vascular mesenteric tissue, decreased
in F and F+E compared to C group (P < 0.001). F+RW showed
an increase of L/M ratio compared to F and F+E groups (P <
0.01) similar to the C group (Table 2).
Table 1 | Body and adipose tissue weight, fluid, food, and energy intake from C, F, F+E, and F+RW rats
Groups
Body weight (g)
Mesenteric adipose tissue (mg)/body weight (g)a
Fluid intake (ml/day)
Food intake (g)
C
F
358 ± 7
369 ± 9
F+E
349 ± 7
3.9 ± 0.2
6.5 ± 0.4**
5.5 ± 0.1*
26.5 ± 1.2
35.5 ± 2.4**
35.1 ± 2.1**
F+RW
353 ± 7
4.5 ± 0.2***,†
35.2 ± 2.2**
18.3 ± 0.6
16.4 ± 0.4*
15.9 ± 0.5*
15.7 ± 0.6*
Energy intake (initial to week 6) (kJ)
246.1 ± 9.2
280.3 ± 9.4*
279.1 ± 9.8*
277.4 ± 9.7*
Energy intake (week 6–10) (kJ)
243.5 ± 8.0
277.6 ± 9.3*
328.8 ± 10.2**,***
327.4 ± 11.7**,***
Values are expressed as mean ± s.e.m., n = 10 in all variables except an = 6.
C, control; E, ethanol; F, fructose; RW, red wine.
*P < 0.05 (vs. C); **P < 0.01 (vs. C); ***P < 0.05 (vs. F); †P < 0.05 (vs. F+E).
236
February 2011 | VOLUME 24 NUMBER 2 | AMERICAN JOURNAL OF HYPERTENSION
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Red Wine and Adipocytokines in Fructose-Fed Rats
Table 2 | Systolic blood pressure, metabolic variables, and lumen:media ratio in mesenteric arteries from C, F, F+E, and F+RW rats
Groups
C
SBP (mm Hg)a
116.2 ± 1.1
F
F+E
132.5 ± 1.2**
F+RW
123.8 ± 1.2*,***
121.0 ± 1.3*,***
7.6 ± 0.2**
5.9 ± 0.4†
Plasma glycemia (mmol/l)
5.0 ± 0.4
6.8 ± 0.5*
Plasma insulin (pmol/l)
69 ± 7
185 ± 18**
HOMA-IR
2.2 ± 0.3
7.6 ± 0.8**
7.4 ± 0.3**
4.2 ± 0.3*,***,†
Plasma triglycerides (mmol/l)
0.68 ± 0.02
1.12 ± 0.07**
1.10 ± 0.07**
0.70 ± 0.07***,†
Plasma high-density lipoprotein (mmol/l)
1.00 ± 0.04
0.92 ± 0.04
1.07 ± 0.02
1.10 ± 0.01***
Plasma total cholesterol (mmol/l)
1.78 ± 0.04
1.79 ± 0.06
1.76 ± 0.08
L/M ratio in mesenteric arteriesb
17.6 ± 0.7
12.5 ± 0.7**
13.8 ± 0.3**
1.81 ± 0.08
■■
16.3 ± 0.5••
111 ± 4*,***,†
154 ± 4**
Values are expressed as mean ± s.e.m.; n = 6 in all variables except an = 10, bn = 4.
C, control; E, ethanol; F, fructose; HOMA-IR, homeostasis model assessment; L/M: lumen to media; RW, red wine; SBP, systolic blood pressure.
*P < 0.05 (vs. C); **P < 0.01 (vs. C); ***P < 0.05 (vs. F); †P < 0.05 (vs. F+E) ••P <0.01 (vs. F); ■■P <0.01 (vs. F+E).
*
**
3.0
**
2.5
2.0
1.5
1.0
0.5
0.0
b
C
F
C
F+E
F
F+E
F+RW
β-Actin
Mesenteric vascular tissue
Nox4/β-actin ratio
20
**
**
10
5
0
C
F
F+E
b
5
**
4
*
3
2
1
0
C
F
F+E
F+RW
100
80
**
**
F
F+E
60
40
20
0
C
F+RW
F+RW
Nox4
15
a
% Inhibition of ABTS . +
NAD(P)H oxidase activity
(au.min−1.mg−1 aortic tissue)
3.5
Plasma TBARS µmol.l−1
a
F+RW
Figure 1 | NAD(P)H-oxidase activity and expression. (a) NAD(P)H-oxidase
aorta activity and (b) Nox4 protein expression in vascular mesenteric tissue
from C, F, F+E, and F+RW rats. Bar graph represents mean ± s.e.m., (n = 6).
Symbols indicate *P < 0.05 and **P < 0.01 (vs. C); filled circle: P < 0.05 and
two filled circles: P < 0.01 (vs. F); filled square: P < 0.05 and two filled squares:
P < 0.05 (vs. F+E). C, control; E, ethanol; F, fructose; RW, red wine.
Aortic NAD(P)H-oxidase activity, Nox4 expression in
v­ ascular mesenteric tissue (Figure 1a,b, respectively) and
plasma TBARS levels were higher, while plasma TAA was
lower in F and F+E than the C group (P < 0.05; Figure 2a,b,
respectively). RW administration to F group significantly lowered aortic NAD(P)H-oxidase activity, vascular Nox4 expression, and plasma TBARS levels, while increased the plasma
TAA in comparison with F and F+E (P < 0.05).
When the adipocytokines expression was examined
in mesenteric adipose tissue by western blotting, lower
AMERICAN JOURNAL OF HYPERTENSION | VOLUME 24 NUMBER 2 | February 2011
Figure 2 | Markers of oxidative stress. (a) Plasma TBARS levels and (b) total
antioxidant activity (TAA) from C, F, F+E, and F+RW rats. Bar graph represents
mean ± s.e.m., (n = 6). Symbols indicate *P < 0.05 and **P < 0.01 (vs. C);
filled circle: P < 0.05 and two filled circles: P < 0.01 (vs. F); filled square:
P < 0.05 and two filled squares: P < 0.01 (vs. F+E). ABTS, 2,2′-azinobis-(3ethylbenzothiazoline-6-sulfonic acid; C, control; E, ethanol; F, fructose; RW, red
wine; TBARS, thiobarbituric acid reactive substances.
a­ diponectin expression and greater resistin expression was
observed in F and F+E groups than in C group (P < 0.01 and
P < 0.05, respectively). The administration of RW to F group
significantly increased adiponectin and decreased resistin
expression (P < 0.05 vs. F and F+E groups; Figure 3a,b).
Protein expression of VCAM-1 was also higher in F and
F+E groups than in C group (P < 0.01). This increase was
reversed in F+RW group (P < 0.05 vs. F+E and P < 0.01 vs.
F; Figure 4a). To explore the main transcriptional regulator of
VCAM-1, NF-кB (NF-кB p65) protein was examined in vascular mesenteric tissue homogenates (Figure 4b). In agreement with the pattern observed for VCAM-1 expression, we
observed an enhancement of NF-кB protein expression in F
and F+E groups (P < 0.01 vs. C group). The increased expression of NF-кB was attenuated when RW was administrated to
FFRs (P < 0.01 vs. F and F+E).
Discussion
In this study, we found that moderate consumption of RW prevents the imbalance of adiponectin and resistin expression in
adipose tissue. RW also attenuates VCAM-1 expression, vascular remodeling, and oxidative status. All these effects could
be associated with actions of RW nonalcoholic compounds on
the mechanisms of oxidative stress enhanced in FFRs.
237
original contributions
a
C
F
F+E
Red Wine and Adipocytokines in Fructose-Fed Rats
F+RW
Adiponectin
a
**
VCAM-1/β-actin ratio
Adiponectin/β-actin ratio
30
5.0
**
2.5
C
F
C
F+E
F
F+E
F+RW
**
**
10
0
C
C
b
F
F+E
F+RW
F
F+E
F+RW
**
**
F
F+E
NF-κB p65
β-Actin
β-Actin
*
*
NF-κB p65/β-actin ratio
7.5
Resistin/β-actin ratio
F+RW
20
F+RW
Resistin
5.0
2.5
0.0
F+E
β-Actin
7.5
b
F
VCAM-1
β-Actin
0.0
C
C
F
F+E
F+RW
30
20
10
0
C
F+RW
Figure 3 | Adipocytokine expression in visceral adipose tissue.
(a) Adiponectin and (b) resistin protein expression in visceral adipose
tissue assessed by western blotting from C, F, F+E, and F+RW rats. Bar
graph represents mean ± s.e.m., (n = 4). Symbols indicate *P < 0.05 (vs. C),
**P < 0.01 (vs. C), filled circle: P < 0.05 (vs. F) and filled square: P < 0.05 (vs. F+E).
C, control; E, ethanol; F, fructose; RW, red wine.
Figure 4 | Vascular inflammation markers. (a) VCAM-1 and (b) NF-кB protein
expression in vascular mesenteric tissue assessed by western blotting from
C, F, F+E, and F+RW rats. Bar graph represents mean ± s.e.m., (n = 4). Symbols
indicate **P < 0.01 (vs. C), two filled circles: P < 0.01 (vs. F) and filled square:
P < 0.05 (vs. F+E). C, control; E, ethanol; F, fructose; NF-κB, nuclear factor-κB;
RW, red wine; VCAM-1, vascular cell adhesion molecules-1.
According to previous studies,4,7,22 fructose-fed animals
develop insulin resistance, hypertriglyceridemia, increased
SBP, and oxidative stress. The decrease of insulin ­sensitivity
and the hypertriglyceridemic effect of a high-fructose diet
have been widely studied.8,23 In this study, RW administration
to FFRs reversed the development of insulin resistance, hypertriglyceridemia, and also lowered the SBP. It has been shown in
diabetic type 2 patients that moderated RW consumption for
2 weeks decreased the insulin resistance state.24 Conversely, E
administration to FFR only decreased the SBP. Similar results
in SBP without changes in ROS production were found in FFR
treated with E alone.25 The vascular effect of alcohol could be
attributed to the involvement of extracellular and intracellular
Ca2+ mobilization of vascular smooth muscle cells.26
The effect of RW on body weight reduction had been
assessed. Monteiro et al.27 found a reduction of body weight in
normal male rats treated with RW or E, but only RW decreased
the adiposity size associated with an increase of the adipose
tissue aromatase expression. A similar effect of RW on adipose
tissue reduction was observed in male Zucker rats fed a highfat diet.28 We did not observe significant differences in body
weight among groups, although the F group consumed fewer
calories during the last 4 weeks than F+E and F+RW. Moreover,
we observed higher mesenteric adipose tissue weight in F
rats consistent with previous data reports,8,23 and only RW
administration to F group had lowered visceral adiposity. It is
important to note that both E and RW reduced body weight
but only RW reduced adipose tissue suggesting that nonalcohol content of RW is responsible. It has been reported that
polyphenols such as catechin reduces body weight in obese
mice and increases the metabolic rate and fat oxidation levels
in humans.29,30 Resveratrol inhibited preadipocyte proliferation and adipogenic differentiation through sirtuin-1-pathway,
an important regulator on energy homeostasis, and also influences adipose tissue mass and function in human adipocytes.31
Moreover, in a rat model of obesity, RW polyphenols corrected
vascular alterations, especially endothelial dysfunction, by
increasing NO availability resulting from enhanced NO production and reduced ROS release via decreased expression of
NAD(P)H-oxidase membrane subunit, Nox1.32
Consistent with previous studies,7,23 the present results
demonstrate that RW administration reduced aortic NAD(P)
H-oxidase activity and Nox4 expression in mesenteric ­tissue.
RW also decreased plasma TBARS and enhanced the TAA in
FFRs. Furthermore, in this study RW attenuated the expression of VCAM-1 and NF-кB, and decreased L/M ratio in
mesenteric tissue. Conversely, ethanol alone did not modify
the oxidative and vascular inflammation status, although it
did reduce the SBP. This highlights the importance of the role
of inflammation and oxidative stress on vascular damage.
Previous studies have shown the key role of NAD(P)H-oxidase
in the vascular oxidative stress, vascular smooth muscle cells
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February 2011 | VOLUME 24 NUMBER 2 | AMERICAN JOURNAL OF HYPERTENSION
Red Wine and Adipocytokines in Fructose-Fed Rats
proliferation, endothelial dysfunction, and development of
hypertension.4,5,20 Moreover, Nyby et al.33 have shown that
increased ROS production by NAD(P)H-oxidase was associated with vascular inflammation assessed through VCAM-1
mRNA expression and vascular dysfunction in FFR. On the
other hand, Sarr et al.34 showed that RW polyphenols prevented endothelial dysfunction, vascular ROS production, and
NAD(P)H-oxidase expression in rats infused with angiotensin
II–induced hypertension.
Activation of the vascular endothelium constitutes the initiating event in vascular inflammation and the development of
vascular disease. Once activated, endothelium expresses cellular adhesion molecules and cytokines that work in concert to
recruit circulating inflammatory cell into vessel wall. Increased
production of ROS promotes endothelial inflammatory activation. Moreover, oxidative stress promotes vascular remodeling
in human and animal experimental studies.35 All this effects
are attributed mainly through activation of NF-кB pathway,
playing a key role in the development of vascular dysfunction.
The corresponding gene products mediate important biological functions such as immune and inflammatory reactions,
smooth muscle cell proliferation, and angiogenesis.36 A limitation of this study could be that we cannot answer whether
the decreased L/M ratio and VCAM-1 expression is possibly
an improvement in vascular function.
Also, some adipocytokines play a key role in vascular
inflammation and vascular function.37 Adiponectin inhibits
the expression of adhesion molecules, such as VCAM-1, the
tumor necrosis factor-α that promotes monocyte adhesion
to endothelial cells, and also inhibits the proliferation and
migration of smooth muscle cells.10 Contrarily, resistin has
the opposite effect of adiponectin. Resistin induces the production of proinflammatory cytokines by endothelial cells
and by ­macrophages, and promotes migratory activity of vascular smooth muscle via activation of the NF-кB pathway,38
suggesting that the balance of adipocytokines concentration
may determine the inflammatory vascular status. In this
study, F rats increased resistin expression and decreased adiponectin expression compared to the control group. Only
RW administration partially reversed the imbalance of adipocytokine expression. Excess of ROS and visceral adiposity
appears to be associated with an adipocytokine imbalance
and vascular inflammation. Recent studies have shown that
polyphenols from RW such as resveratrol have anti-inflammatory effects on adipocytokine expression and secretion
in human adipose tissue in vitro through the sirtuin-1-pathway.39 Resveratrol also modulated adipocytokines expression
and improved insulin sensitivity in adipocytes via suppression of extracellular receptor–activated kinase and NF-кB
activation.40 On the other hand, procyanidins from grape
seed also attenuated the expression of inflammatory molecules and enhanced adipo­nectin expression in white adipose
tissue in rats fed a high-fat diet.41 Our results suggest that
RW attenuated oxidative stress and decreased the adipose tissue, thus reducing imbalance of adipocytokines and markers
of vascular inflammation.
AMERICAN JOURNAL OF HYPERTENSION | VOLUME 24 NUMBER 2 | February 2011
original contributions
In summary, consumption of RW counters oxidative stress,
the imbalance of adipocytokines, VCAM-1 expression, and
vascular remodeling, supporting the hypothesis that RW has
other nonethanol-related beneficial effects that may be linked
with antioxidant properties of its nonalcoholic constituents.
Further research is necessary for understanding the ­molecular
mechanisms of RW or its constituents on adipocytokines
imbalance in this pathological condition, focusing on the
regulation of adipocytokine expression and its mechanisms of
action.
Acknowledgments: This work was supported by a Grant from National
Council of Research (PIP 5192) and Program I+D from the National University
of Cuyo. The authors thank Susana Gonzalez and Cristina Lama for their
technical assistance.
Disclosure: The authors declared no conflict of interest.
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February 2011 | VOLUME 24 NUMBER 2 | AMERICAN JOURNAL OF HYPERTENSION