Grape-seed procyanidins prevent low-grade inflammation by modulating cytokine expression in rats fed a high-fat diet.
ABSTRACT The main objective of this study was to evaluate the effect of procyanidin intake on the level of inflammatory mediators in rats fed a hyperlipidic diet, which are a model of low-grade inflammation as they show an altered cytokine production.
Male Zucker Fa/fa rats were randomly grouped to receive a low-fat (LF) diet, a high-fat (HF) diet or a high-fat diet supplemented with procyanidins from grape seed (HFPE) (3.45 mg/kg feed) for 19 weeks and were then euthanized. We determined biochemical parameters, C-reactive protein (CRP) and IL-6 levels in plasma. Adipose tissue depots and body weight were also determined. We assessed CRP, IL-6, TNF-alpha and adiponectin gene expression in liver and white adipose tissue (WAT).
As expected, rats fed the HF diet show an enhanced production of CRP. Our results demonstrate that the HFPE diet decreases rat plasma CRP levels but not IL-6 levels. The decrease in plasma CRP in HFPE rats is related to a down-regulation of CRP mRNA expression in the liver and mesenteric WAT. We have also shown a decrease in the expression of the proinflammatory cytokines TNF-alpha and IL-6 in the mesenteric WAT. In contrast, adiponectin mRNA is increased in this tissue due to the procyanidin treatment. As previously reported, CRP plasma levels correlate positively with its expression in the mesenteric WAT, suggesting that procyanidin extract (PE) modulates CRP at the synthesis level. CRP plasma levels also correlate positively with body weight. As expected, body weight is associated with the adiposity index. Also, TNF-alpha expression and IL-6 expression have a strong positive correlation. In contrast, the expression of the anti-inflammatory cytokine adiponectin correlates negatively with the expression of TNF-alpha and IL-6 in the mesenteric WAT.
These results suggest a beneficial effect of PE on low-grade inflammatory diseases, which may be associated with the inhibition of the proinflammatory molecules CRP, IL-6 and TNF-alpha and the enhanced production of the anti-inflammatory cytokine adiponectin. These findings provide a strong impetus to explore the effects of dietary polyphenols in reducing obesity-related adipokine dysregulation to manage cardiovascular and metabolic risk factors.
- SourceAvailable from: Beatriz Martín-Fernández[Show abstract] [Hide abstract]
ABSTRACT: Aldosterone administration in rats results in several cardiac alterations. Previous studies have demonstrated that proanthocyanidins, phenolic bioactive compounds, have cardioprotective effects. We studied the potential beneficial effects of the proanthocyanidin-rich almond skin extract (PASE) on the cardiac alterations induced by aldosterone-salt treatment, their effects in mineralocorticoid receptor activity and we sought to confirm proanthocyanidins as the specific component of the extract involved in the beneficial cardiac effects. Male Wistar rats received aldosterone (1 mg/Kg/day) +1% NaCl for 3 weeks. Half of the animals in each group were simultaneously treated with either PASE (100 mg/Kg/day) or spironolactone (200 mg/Kg/day). The ability of PASE to act as an antagonist of the mineralocorticoid receptor was examined using a transactivation assay. High performance liquid chromatography was used to identify and to isolate proanthocyanidins. Hypertension and diastolic dysfunction induced by aldosterone were abolished by treatment with PASE. Expression of the aldosterone mediator SGK-1, together with fibrotic, inflammatory and oxidative mediators were increased by aldosterone-salt treatment; these were reduced by PASE. Aldosterone-salt induced transcriptional activity of the mineralocorticoid receptor was reduced by PASE. HPLC confirmed proanthocyanidins as the compound responsible for the beneficial effects of PASE. The effects of PASE were comparable to those seen with the mineralocorticoid antagonist, spironolactone. The observed responses in the aldosterone-salt treated rats together with the antagonism of transactivation at the mineralocorticoid receptor by PASE provides evidence that the beneficial effect of this proanthocyanidin-rich almond skin extract is via as a mineralocorticoid receptor antagonist with proanthocyanidins identified as the compounds responsible for the beneficial effects of PASE.PLoS ONE 10/2014; 9(10):e111104. · 3.53 Impact Factor
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ABSTRACT: Background Hyperglycemia-mediated oxidative stress plays a crucial role in the progression of diabetic neuropathy (DN). Oxidative damage is the most common concluding pathway for various pathogenetic mechanisms of neuronal injury in diabetic neuropathy. Hence, the present study was hypothesized to explore the neuroprotective nature of grape seed extract (GSE) on diabetic rats by assessing markers of brain neurotransmitters secretion, oxidative stress, antioxidant competence and inflammatory marker in alloxan-induced diabetic rats. Methods: Four groups of rats were treated daily for ten weeks : (-ve) control, diabetic-control injected intraperitoneally with 150 mg kg− 1 BW of alloxan monohydrate, diabetic-treated rats injected by alloxan and then treated with GSE 250 mg kg− 1 BW and (+ve) control rats treated with the same previous dose of GSE . Results : In diabetic rats a significant increase in serum glucose and butyrylcholinesterase (BChE), while hypoinsulinemia were recorded. In addition a significant increase in brain neurotransmitters [epinephrine, noradrenaline (NA), serotonin (5-HT) and dopamine], MDA, superoxide dismutase (SOD) were recorded. Whereas there were a significant decrease in brain glutathione (GSH), Vitamin C, nitric oxide levels and glutathione peroxidase (GPx) activities were reported. There was non significant change in catalase (CAT) activity. GSE administration was found to be able to ameliorate most of the biochemical altered parameters in diabetic rats. Conclusion: The present results indicated that experimental diabetes produced metabolic disturbances in glucose, insulin that trigger brain enzymatic and non enzymatic oxidative stress that initiate disturbances in brain neurotransmitter, providing the incidence of nervous manifestation in diabetes. Administration GSE is valuable for enhancing the antioxidant defense against oxidative stress, neuroprotective, resulting in the modulation of brain neurotransmittersJournal of American Science. 01/2012; 8(12).
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ABSTRACT: Micronutrients in rapeseed such as polyphenols, tocopherols, phytosterols and phospholipids in rapeseed exert potential benefit to atherosclerosis. Some part of these healthy components substantially lost during the conventional refining processing. Thus some new processing technologies have been developed to produce various endogenous micronutrient-enriched optimized rapeseed oils. The aim of this study is to assess whether optimized rapeseed oils have positive effects on the atherosclerosis risk factors in rats fed a high-fat diet.Lipids in Health and Disease 10/2014; 13(1):166. · 2.31 Impact Factor
Grape-seed procyanidins prevent low-grade inflammation by modulating
cytokine expression in rats fed a high-fat diet
Ximena Terra, Gemma Montagut, Mario Bustos, Niurka Llopiz, Anna Ardèvol, Cinta Bladé,
Juan Fernández-Larrea, Gerard Pujadas, Josepa Salvadó, Lluís Arola, Mayte Blay⁎
Department of Biochemistry and Biotechnology, Unitat d’Enologia del Centre de Referència en Tecnologia dels Aliments de la Generalitat de Catalunya,
Universitat Rovira i Virgili, 43007 Tarragona, Spain
Received 15 October 2007; received in revised form 4 February 2008; accepted 8 February 2008
Objective: The main objective of this study was to evaluate the effect of procyanidin intake on the level of inflammatory mediators in rats fed
a hyperlipidic diet, which are a model of low-grade inflammation as they show an altered cytokine production.
Design: Male Zucker Fa/fa rats were randomly grouped to receive a low-fat (LF) diet, a high-fat (HF) diet or a high-fat diet supplemented
with procyanidins from grape seed (HFPE) (3.45 mg/kg feed) for 19 weeks and were then euthanized. We determined biochemical
parameters, C-reactive protein (CRP) and IL-6 levels in plasma. Adipose tissue depots and body weight were also determined. We assessed
CRP, IL-6, TNF-α and adiponectin gene expression in liver and white adipose tissue (WAT).
Results: As expected, rats fed the HF diet show an enhanced production of CRP. Our results demonstrate that the HFPE diet decreases rat
plasma CRP levels but not IL-6 levels. The decrease in plasma CRP in HFPE rats is related to a down-regulation of CRP mRNA expression
in the liver and mesenteric WAT. We have also shown a decrease in the expression of the proinflammatory cytokines TNF-α and IL-6 in the
mesenteric WAT. In contrast, adiponectin mRNA is increased in this tissue due to the procyanidin treatment.
As previously reported, CRP plasma levels correlate positively with its expression in the mesenteric WAT, suggesting that procyanidin
extract (PE) modulates CRP at the synthesis level. CRP plasma levels also correlate positively with body weight. As expected, body
weight is associated with the adiposity index. Also, TNF-α expression and IL-6 expression have a strong positive correlation. In contrast,
the expression of the anti-inflammatory cytokine adiponectin correlates negatively with the expression of TNF-α and IL-6 in the
Conclusion: These results suggest a beneficial effect of PE on low-grade inflammatory diseases, which may be associated with the inhibition
of the proinflammatory molecules CRP, IL-6 and TNF-α and the enhanced production of the anti-inflammatory cytokine adiponectin. These
findings provide a strong impetus to explore the effects of dietary polyphenols in reducing obesity-related adipokine dysregulation to manage
cardiovascular and metabolic risk factors.
© 2009 Elsevier Inc. All rights reserved.
Keywords: Procyanidins; IL-6; CRP; TNF-α; Adiponectin; Low-grade inflammation
Procyanidins are phenolic compounds from the flavo-
noids group that are widely found in cereals, vegetables and
fruits like grapes, berries, cocoa and apples. They have a
broad range of biological activities . They function as
powerful antioxidants and exert anti-inflammatory activities
in vitro. Recent studies have shown potent anti-inflammatory
properties of procyanidins on experimental inflammation in
rats and mice [2,3]. Its mechanisms of anti-inflammatory
action remain poorly understood and are relevant to oxygen
free radical scavenging, antilipid peroxidation, inhibition of
the formation of inflammatory cytokines, alterations in cell
membranes receptors, intracellular signaling pathway pro-
teins and modulation of gene expression .
Available online at www.sciencedirect.com
Journal of Nutritional Biochemistry 20 (2009) 210–218
⁎Corresponding author. Department of Biochemistry and Biotechnol-
ogy, 43007 Tarragona, Spain. Tel.: +34 977 558497; fax: +34 977558232.
E-mail address: email@example.com (M. Blay).
0955-2863/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
Obesity is associated with a state of chronic inflammation
characterized by macrophage infiltration of muscle and
adipose tissue and abnormal production of proinflammatory
mediators. In addition to adipocytes, adipose tissue contains
fibroblasts, preadipocytes, tissue-resident macrophages and
vascular constituents. Macrophages are known to be crucial
contributors to inflammation, but more recently, it has been
recognized that adipocytes demonstrate significant intrinsic
inflammatory properties as well. Like macrophages, the
adipocyte is exquisitely sensitive to infectious disease agents
and cytokine-mediated inflammatory signals. In turn, these
stimuli induce the expression of inflammatory mediators
such as IL-6, TNF-α and SAA. Although many of these
activities are restricted to autocrine and paracrine effects,
some of these cytokines that are secreted from adipocytes
and adipose-resident macrophages make significant con-
tributions to systemic inflammation .
Adipose tissue is not usually thought of as an immune or
inflammatory organ. However, the discovery of elevated
secretion of these factors from obese adipose tissue provided
the first evidence of a direct connection between obesity and
systemic inflammation .
The altered production of proinflammatory molecules
(so-called “adipokines”) by adipose tissue has been impli-
cated in the metabolic complications of obesity .
Compared with adipose tissue of lean individuals, adipose
tissue of obese individuals expresses increased amounts of
proinflammatory proteins such as TNF-α, IL-6, inducible
nitric oxide synthase, C-reactive protein (CRP), soluble
ICAM and monocyte chemotactic protein-1, as well as
reduced adiponectin expression .
CRP is an acute-phase protein that binds specifically to
phosphorylcholine as a component of microbial capsular
polysaccharide and participates in the innate immune
response against microorganisms. CRP is the most exten-
sively studied marker of systemic inflammation in humans.
A large number of studies have further strengthened the
association of elevated CRP levels with nearly all the
important cardiovascular risk factors, including insulin
resistance, diabetes, metabolic syndrome, hypertension,
smoking and dyslipidemia. The regulation of this protein
in the liver is believed to be driven by IL-6, IL-1 and TNF-α
 from visceral adipose tissue draining directly into the
portal system that causes the obesity-associated rise of CRP
production. Furthermore, in addition to liver-derived CRP,
newer data show that adipose tissue itself may contribute to
obesity-associated increased CRP levels [9,10].
Adiponectin, the most abundantly secreted adipocytokine
from differentiated adipocytes, has potent vasculoprotective,
angiogenic, anti-inflammatory and antiatherogenic proper-
ties. High adiponectin levels are associated with a reduced
risk of myocardial infraction in men, while low serum
adiponectin levels are reported in obese individuals and in
those with hypertension, coronary artery disease and type 2
diabetes . Adiponectin has inflammatory-modulating
activities demonstrated in clinical studies showing inverse
associations between adiponectin levels and serum markers
of inflammation . Although it is not clear how or whether
adiponectin itself has anti-inflammatory properties, it is clear
that adiponectin production by adipose can be inhibited by
systemic inflammation and confers protection against the
metabolic syndrome and diabetes [13,14].
TNF-α, a proinflammatory cytokine originally defined by
its antitumor activity, has a strong link with obesity. Some
authors have reported that adipocytes directly express TNF-
α in rodents and led to the concept of a role for inflammation
in obesity. These observations were paralleled by human
studies showing increased TNF-α expression in the adipose
tissue of individuals who were obese and decreased TNF-α
expression after weight loss. Evidence supporting a key role
for TNF-α in obesity-related insulin resistance came from
studies showing that ob/ob mice (leptin-deficient mice with
evidence of insulin resistance) that were also deficient for
TNF-α or TNF receptors (TNFRs) had improved insulin
sensitivity in diet-induced obesity compared with TNF-α-
and TNFR-sufficient ob/ob mice .
IL-6, a stress-induced inflammatory cytokine, is directly
implicated in atherogenesis. High levels of IL-6 are thought
tobe responsiblefor theincreaseinacute-phase proteins seen
in obese patients, in particular, CRP . Obesity-associated
induction of adipose IL-6 production induces CRP secretion,
and there are data that suggest that IL-6 decreases lipoprotein
lipase activity, which results in increased macrophage uptake
of lipids . In addition, IL-6 was significantly associated
with body mass index, waist circumference and visceral
adiposity in obese subjects. Adipocytes and macrophages
both contribute to white adipose tissue (WAT)-derived IL-6,
although the ultimate stimulus for IL-6 production in the
presence of high adiposity is currently unknown.
Understanding the mechanisms that lead from obesity to
inflammation will have important implications for the design
of the new therapies to reduce the morbidity and mortality of
obesity. The main objective of the present study was to
examine the putative modulatory effects of procyanidin
extract (PE) on cytokine expression and CRP and IL-6
release in rats fed the high-fat (HF) diet to gain insight on the
mechanisms that underlie the anti-inflammatory effects
ascribed to procyanidins.
2. Materials and methods
Grape seed PE was provided by Les Dérives Résiniques et
PE contained essentially monomeric (21.3%), dimeric
(17.4%), trimeric (16.3%), tetrameric (13.3%) and oligomeric
(5–13 units) (31.7%) procyanidins and phenolic acids (4.7%).
Semipurified diets were obtained from Research Diets
(USA). Briefly, three diets were used (Table 1). The low-fat
211X. Terra et al. / Journal of Nutritional Biochemistry 20 (2009) 210–218
(LF) diet, the hyperlipidic (HF) diet and the hyperlipidic with
PE (HFPE) diet had equal protein percentage. The standard
control diet was the LF diet. The HFPE diet differs from the
HF diet in PE content, which was 0.32 mg of PE per gram of
amount of procyanidins that humans consume daily.
2.3. Experimental design and euthanasia
Male Zucker Fa/fa rats (Charles River Laboratories,
Spain) were used in all studies (n=30). Rats were left 1 week
in quarantine. At ~15 weeks of age, rats were randomly
assigned to receive the LF (n=10), HF (n=10) or HFPE
(n=10) diet ad libitum. Rats were housed in cages by pairs
and subjected to a standard 12-h light:12-h dark cycle. The
experimental period lasted 19 weeks. After rats were
weighed, they were anesthetized by sodium pentobarbital
(100 mg/kg ip) and euthanized by exsanguination after 6 h of
fasting. Blood was obtained from abdominal aorta. The
entire liver and adipose tissues were dissected out, weighed
and snap frozen in liquid N2and stored at −80°C. All the
procedures were performed with the approval of the ethics
committee of our center and followed the laws concerning
animal experimentation of the Government of Catalonia.
2.4. Measurement of adiposity, food intake and body
weekly during the whole experiment. Adipose tissue fat pads
(mesenteric, retroperitoneal and epididymal) were excised
separately and weighed. Adiposity index was calculated as
total adipose tissue weight versus total body weight.
2.5. Measurement of biochemical parameters
After sacrifice, blood was collected and heparinized
plasma was obtained by centrifugation. Total cholesterol
levels and total plasma glucose levels were measured by
enzymatic colorimetric methods (QCA S.L.). Determination
of the GSH/GSSG ratio was assessed by colorimetric assay
from Oxford Biomedical Research according to the manu-
2.6. Measurement of CRP levels
Plasma CRP levels were quantified using a specific
enzyme immunoassay (EIA) according to the manufacturer's
instructions (Helica Biosystems). The assay is a double
polyclonal antibody sandwich EIA.
2.7. Measurement of IL-6 and adiponectin plasma levels
Plasma IL-6 and adiponectin levels were quantified using
specific EIAs according to the manufacturer's instructions
(Biosource International, Inc.). The assays are based on a
2.8. mRNA analysis of CRP, IL-6, TNF-α and adiponectin
genes by real-time RT-PCR
RNA from liver tissue was isolated with High Pure RNA
Isolation Kit from Roche. RNA from adipose tissue was
isolated using Trizol reagent (Invitrogen) following the
manufacturer's instructions. cDNA was synthesized from
1 μg of total RNA using oligo-dTand Superscript II Reverse
Transcriptase (Life Technologies). cDNA (20 ng) was
subjected to quantitative RT-PCR amplification using
SYBR Green Master Mix (Applied Biosystems). The
forward and reverse primers for rat genes are shown in
Table 2. Reactions were run on a quantitative Real-Time
PCR System (Applied Biosystems); the thermal profile
settings were 50°C for 2 min and 95°C for 2 min and then
40 cycles at 95°C for 15 s and 60°C for 2 min. Relative
expression levels of the mRNA of the target genes were
normalized to GAPDH mRNA levels.
2.9. Calculations and statistical analysis
Results are expressed as mean±S.E.M. Effects were
assessed using ANOVA or Student's t test. We used Tukey's
test for honestly significant differences to make pairwise
comparisons. Spearman's rank correlation test between the
Composition of the LF, HF and HFPE test diets
Protein (% energy)
Carbohydrate (% energy)
Fat (% energy)
Rat-specific primer sequences
F: 5′ TTTGTGCTATCTCCAGAACAGATCA 3′
R: 5′ GCCCGCCAGTTCAAAACAT 3′
F: 5′ CCCAACTTCCAATGCTCTCCTAATG 3′
R: 5′ GCACACTGAGTTTGCCGAATAGACC 3′
F: 5′ GGCCGTTCTCTTCACCTACG 3′
R: 5′ GGCTCCATGCTCCTCCATCT 3′
F: 5′ CGTCAGCCGATTTGCCATTTC 3′
R: 5′ TGGGCTCATACCAGGGCTTGAG 3′
F: 5′ CAT GGC CTT CCG TGT TCC T 3′
R: 5′ CCT GCT TCA CCA CCT TCT TGA 3′
212X. Terra et al. / Journal of Nutritional Biochemistry 20 (2009) 210–218
three experimental groups was assessed. All calculations
were performed using SPSS 14.0 software.
3.1. Food intake, body weight and adipose tissue weight
Zucker Fa/fa rats fed the HF diet had significantly
higher body weights than control rats fed the LF diet
(Pb.05; Table 3). In spite of fat intake being significantly
increased, the total energy intakes of the three groups of
rats were comparable (PN.05), indicating that higher body
weight gains in the HF group may be related to higher fat
intake but not to higher energy intake.
The epididymal and retroperitoneal fat pad weights of rats
fed the HF diet were higher than the weights of those fed the
LF diet (Pb.05; Table 3), although mesenteric weight
remained unchanged. Adiposity index in the three groups
(LF, HF and HFPE) was unchanged.
A positive correlation was found between adiposity index
and body weight (ρ=.408, Pb.05) (Table 6).
3.2. Diet effect on metabolic variables and GSH/GSSG ratio
The total plasma cholesterol levels of Zucker rats fed the
HFPE, HF or LF diet did not change significantly, neither by
diet nor by procyanidin ingestion. Glucose plasma levels
were not significantly increased by the HF diet compared to
the LF diet, but the HFPE diet reduced glucose levels signi-
ficantly (Table 4).
In plasma analysis, rats fed the HF and HFPE diets
showed a reduced GSH/GSSG ratio compared to those fed
the LF diet, whereas no significant difference was found by
PE treatment (Table 4).
3.3. PE modulates CRP and adiponectin plasma levels in
rats fed the hyperlipidic diet without modifying IL-6 levels
CRP plasma levels were increased in HF rats, thus
indicating a low-grade inflammation similar to that found in
overweight/obese individuals. Moreover, HFPE administra-
tion to rats, that is, a daily ingestion per animal of nearly
0.070 mg of PE during 19 weeks of treatment, resulted in an
important decrease in CRP that is in the range found in rats
fed a standard diet (LF). In contrast to most adipocyte
hormones, the anti-inflammatory cytokine adiponectin is
decreased in obesity and increased in response to weight
reduction. In this work, we found a decrease in adiponectin
plasma levels in HF rats. Furthermore, adiponectin plasma
levels were increased significantly in rats fed the HFPE diet
(Table 4). We then measured IL-6 levels in the plasma of
animals and found no difference in IL-6 levels between the
Body weight gain, food and fat intake and adipose weight of Zucker rats fed LF, HF and HFPE diets for 19 weeks
LF (n=10) HF (n=10)HFPE (n=10)
Body weight gain (%)
Fat intake (kcal)
Total energy intake (kcal)
Tissue weight (g)
Values are expressed as mean±S.E.M. The significance of difference among the three groups was analyzed by ANOVA. Values not showing a superscript letter
among the three diet groups are not significantly different (Pb.05).
Plasma analysis of markers of oxidative stress, inflammation and metabolic variables
Total cholesterol (mg/ml)
133.63±31.81 (n=6)6.67±2.39⁎(n=8) 11.96±1.84⁎(n=8)
Student's t test was used. Values are expressed as mean±S.E.M.
⁎Pb.05 compared to LF.
⁎⁎Pb.05 compared to HF.
213X. Terra et al. / Journal of Nutritional Biochemistry 20 (2009) 210–218
HF group and the HFPE group, but both groups had higher
IL-6 levels than rats receiving the standard LF diet.
3.4. PE acts by down-regulating mRNA CRP levels in the
liver and mesenteric adipose tissue
Real-time PCR analysis of CRP in the liver showed
differences between HF and HFPE rats. As expected, pro-
cyanidins caused a decrease in the synthesis of CRP mRNA
in the liver with respect to rats receiving the same diet
without procyanidins (Fig. 1).
Because CRP from adipose tissue is also an important
source of this pentraxin in obese rats, we determined the
CRP levels in adipose tissue of different origins: mesenteric,
epididymal and retroperitoneal.
In mesenteric adipose tissue, we found that HF rats had
higher CRP mRNA levels than control LF rats and HFPE
rats. Thus, the HF diet increased CRP levels that were
diminished by procyanidin treatment. In retroperitoneal and
epididymal adipose tissue, nonsignificant differences were
found between procyanidin treatment and diet (Table 5).
3.5. PE modulates gene expression in the mesenteric
Quantitative RT-PCR analysis of the mRNAs for CRP, IL-
6, TNF-α and adiponectin genes in the mesenteric adipose
tissue of rats fed the standard LF diet, HF diet or HFPE diet
was performed. As we have previously described, procya-
nidin treatment modified not only CRP levels in mesenteric
adipose tissue (Fig. 2A) but also IL-6, TNF-α and
adiponectin gene expression. Our results show that IL-6
gene expression level was up-regulated by the HF diet and
reduced significantly by procyanidin treatment (Fig. 2B).
TNF-α gene expression level was also reduced significantly
by procyanidins (Fig. 2C). On the contrary, the anti-
inflammatory cytokine adiponectin was increased by PE
treatment (Fig. 2D).
3.6. Correlations of CRP, IL-6, adiponectin and TNF-α
expression in mesenteric adipose tissue and CRP and IL-6
To test possible associations between mRNA levels of
CRP, IL-6, adiponectin and TNF-α in mesenteric adipose
tissue; CRP and IL-6 plasma levels; body weight; and
adiposity index, we performed Spearman's rank correlation
test, analyzing the data from the three experimental groups.
As shown in Table 6, a significant positive correlation
between CRP expression and CRP plasma levels (ρ=.623,
Pb.001) was found as expected. Furthermore, TNF-α
expression and IL-6 expression correlated positively
There were significant negative correlations between IL-6
and adiponectin expression (ρ=−.436, Pb.05) and between
TNF-α and adiponectin expression (ρ=−.457 Pb.05).
Finally, body weight correlated positively with CRP plasma
levels (ρ=.475, Pb.05).
There was no significant correlation of IL-6 plasma levels
with any of the parameters measured. Liver CRP expression
level was also examined, but no significant correlation
between any of the parameters measured was found (data
The primary function of adipose tissue is to store energy
in the form of triglycerides during periods of energy excess
and to release energy during fasting or starvation as free fatty
acids and glycerol. Adipose tissue secretes a variety of
peptides called adipokines including leptin, adiponectin,
TNF-α, IL-6 and resistin, which have endocrine, autocrine
and paracrine effects on the brain, liver and skeletal muscles
. Dysfunction of adipose tissue can result in insulin
resistance and obesity-linked metabolic and vascular dis-
eases. Obesity is associated with a chronic inflammatory
response, which is characterized by abnormal cytokine pro-
duction, increased synthesis of acute-phase reactants, such
as CRP, and the activation of proinflammatory signaling
Fig. 1. Diet effect on CRP expression in liver. Liver mRNA was extracted,
corresponding cDNA was synthesized and CRP gene expression was
measured by quantitative real-time RT-PCR. Student's t test was used to
evaluate significance between groups (Pb.05).
Effect of the HFPE diet on CRP relative expression in adipose tissue
Adipose tissue mRNAwas extracted, corresponding cDNAwas synthesized
and CRP gene expression was measured by quantitative real-time RT-PCR.
Results are expressed as relative expression levels normalized to the
expression of the control group (LF). Values are expressed as mean±S.E.M.
The significance of difference among the three groups was analyzed by
ANOVA. Values not showing a superscript letter among the three groups are
not significantly different (PN.05).
214X. Terra et al. / Journal of Nutritional Biochemistry 20 (2009) 210–218
pathways . It remains highly likely that adipokines
contribute to obesity-associated systemic inflammation and
remain potentially important targets for prevention of
inflammation-induced insulin resistance or vasculopathy.
Procyanidins have been postulated to possess anti-
inflammatory and immunomodulatory activities in vitro
and in vivo . In this work, we show that PE acts as an
anti-inflammatory substance in vivo. To assess the effect of
procyanidins, we compared the ability of PE to modify
inflammatory parameters in Zucker Fa/fa rats after 19 weeks
on a non-hyperlipidic diet, a hyperlipidic diet or a
hyperlipidic diet with PE.
We found that feeding rats with the hyperlipidic diet
resulted in a moderate increase in body weight as expected,
as well as a less pronounced increase in rats receiving
procyanidins, as we demonstrated before . Body weight
was positively related with adiposity index, comparing all
the experimental groups. Moreover, biochemical parameters
measured in plasma indicate that the HF diet produced a
marked increase in oxidative stress, although it was not
attenuated by procyanidin treatment. In contrast, total
cholesterol was unchanged and glucose levels were reduced
in HFPE-fed rats.
Previous studies indicate that a chronic low-grade
inflammation is involved in the pathogenesis of athero-
sclerosis, and an elevated, highly sensitive CRP level is a risk
factor for coronary artery disease. CRP is also a well-known
systemic marker for inflammation in human and rats .
Plasma CRP levels were also strongly associated with
obesity and obesity-related diseases, including insulin
resistance, diabetes mellitus and hyperlipidemia. In this
work, we found that CRP plasma levels were increased
because of the HF diet up to 472 μg/ml, which is at the upper
range in normal laboratory healthy rats . We demonstrate
that ingestion of procyanidins diminishes CRP levels, thus
reducing the diet-induced low-grade inflammation. In
addition, plasma CRP levels were positively associated
with total body fat mass and CRP expression levels in
mesenteric adipose tissue. We examined CRP expression in
liver where procyanidins reduced its mRNA. Some authors
have recently reported the same properties of red wine
phenolics that reduced CRP expression in the human hepatic
cell line Hep3B .
In the mesenteric adipose tissue, CRP was also down-
regulated by the HFPE diet, while in retroperitoneal and
epididymal adipose tissue, it was unchanged by either the HF
Fig. 2. Diet effect on gene expression in mesenteric adipose tissue. mRNAwas extracted, corresponding cDNAwas synthesized and CRP, IL-6, adiponectin and
TNF-α gene expression was measured by quantitative real-time RT-PCR. ANOVA test was used to evaluate significance between groups (Pb.05).
215 X. Terra et al. / Journal of Nutritional Biochemistry 20 (2009) 210–218
diet or procyanidins. These variations found in CRP
expression between the adipose tissues examined might be
due to the different degree of macrophage infiltration in the
WATs and the resultant different pattern of cytokine release
[7,23], although the reason for this difference is unclear.
Taken together, the increased CRP expression in mesen-
teric adipose tissue may partially account for the elevation of
plasma CRP and the effect of procyanidins on the adipose
tissue may be responsible for the reduction of CRP protein
and expression levels shown with the HFPE diet.
On the contrary, we have not detected changes in IL-6
plasma levels due to procyanidin treatment, although IL-6
levels were increased by the HF diet. There were no
positive correlations between IL-6/CRP levels as could be
It has been recently reported that adiponectin-deficient
mice exhibit severe diet-induced insulin resistance and
enhanced neointimal thickening after vascular injury .
These findings suggest that adiponectin has anti-inflamma-
tory properties and acts as an endogenous modulator of
obesity-related diseases. Furthermore, administration of
adiponectin to obese or diabetic mice causes weight loss
and also enhances insulin sensitivity and reduces the plasma
glucose level by suppressing hepatic glucose production
. Previous studies in our group  demonstrate that PE
acts as an enhancer of the glucose uptake in 3T3-L1
adipocytes and show that an acute gavage of PE (250 mg
PE/kg body weight) significantly reduced blood glucose
levels in streptozotocin-induced diabetic rats. In this work,
adiponectin plasma levels and adiponectin expression in
mesenteric adipose tissue of HFPE rats were highly
increased compared with HF rats. Then, the reduction of
glucose plasma levels may be driven as a consequence of
the enhanced adiponectin expression that PE produces.
These findings suggest that procyanidins act as anti-
inflammatory molecules in vivo by increasing adiponectin
expression. In agreement with our results, the monomeric
procyanidin, catechin, has been recently described as an
inducer of adiponectin expression in the adipocyte cell line
In the current study, procyanidin treatment decreased IL-6
mRNA levels in the mesenteric WAT. In addition, IL-6
expression was negatively correlated with adiponectin
expression, suggesting that the expression of IL-6 was
negatively regulated by adiponectin in adipose tissue, as
shown by other investigators .
Spearman's correlation coefficients (ρ) of CRP, IL-6, adiponectin and TNF-α expression in mesenteric adipose tissue, plasma levels of CRP and IL-6 and mass
mRNA levels in mesenteric adipose tissuePlasma levelsMass parameters
IL-6Adiponectin TNF-αCRPIL-6 Body weightAdiposity
1921 18 2222
216 X. Terra et al. / Journal of Nutritional Biochemistry 20 (2009) 210–218
Our results show that TNF-α expression in adipose tissue
was also reduced by the HFPE diet and that this mRNA
expression had a strong negative association with adiponec-
tin expression. TNF-α suppresses the transcription of
adiponectin in an adipocyte cell line, which might explain
the lower levels of serum adiponectin in individuals who are
The mechanisms regulating CRP synthesis at extrahepatic
sites are unknown. CRP induction in hepatocytes is
principally regulated at the transcriptional level by the
cytokine IL-6. This cytokine controls expression of many
acute-phase protein genes through activation of the tran-
scription factors STAT3, C/EBP family members and Rel
proteins (NF-kB) . In searching for the mechanisms
involved in inflammation-associated diseases, we have
previously demonstrated that PE inhibits NF-kB activation
in vitro .
We propose that the inhibition of the NF-kB pathway
produced by procyanidins down-regulates TNF-α and IL-6
expression, which may explain the increase of adiponectin
expression and the indirect reduction of CRP plasma and
In summary, we have shown for the first time that
procyanidins prevent low-grade inflammation in vivo, by
adjusting adipose tissue cytokine imbalance, enhancing anti-
inflammatory molecules and diminishing proinflammatory
ones. Further studies are needed to elucidate the mechanism
by which procyanidins may act as anti-inflammatory agents
in obese humans.
This study was supported by grant number CO3/O8
from the Fondo de Investigación Sanitaria and AGL2005-
04889/ALI from the Ministerio de Educación y Ciencia of
the Spanish Government. X. Terra is the recipient of a
fellowship from the Rovira i Virgili University in
 Rasmussen SE, Frederiksen H, Krogholm KS, Poulsen L. Dietary
proanthocyanidins: occurrence, dietary intake, bioavailability, and
protection against cardiovascular disease. Mol Nutr Food Res 2005;49:
 Sakaguchi Y,Shirahase H,KunishiroK,IchikawaA,KandaM, Uehara
Y. Effect of combination of nitric oxide synthase and cyclooxygenase
inhibitors on carrageenan-induced pleurisy in rats. Life Sci 2006;79:
 Li WG, Zhang XY, Wu YJ, Tian X. Anti-inflammatory effect and
mechanism of proanthocyanidins from grape seeds. Acta Pharmacol
 Kris-Etherton PM, Lefevre M, Beecher GR, Gross MD, Keen CL,
Etherton TD. Bioactive compounds in nutrition and health-research
methodologies for establishing biological function: the antioxidant and
anti-inflammatory effects of flavonoids on atherosclerosis. Annu Rev
 Berg AH, Scherer PE. Adipose tissue, inflammation, and cardiovas-
cular disease. Circ Res 2005;96:939–49.
 Kontogianni MD, Zampelas A, Tsigos C. Nutrition and inflammatory
load. Ann NYAcad Sci 2006;1083:214–38.
 WeisbergSP, McCannD, Desai M,RosenbaumM,LeibelRL, Ferrante
Jr AW. Obesity is associated with macrophage accumulation in adipose
tissue. J Clin Invest 2003;112:1796–808.
 Yuan M, Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M,
et al. Reversal of obesity- and diet-induced insulin resistance with
salicylates or targeted disruption of Ikkbeta. Science 2001;293:
 Fain JN. Release of interleukins and other inflammatory cytokines by
human adipose tissue is enhanced in obesity and primarily due to the
nonfat cells. Vitam Horm 2006;74:443–77.
 Ouchi N, Kihara S, Funahashi T, Nakamura T, Nishida M, Kumada
M, et al. Reciprocal association of C-reactive protein with adipo-
nectin in blood stream and adipose tissue. Circulation 2003;107:
 Xavier Pi-Sunyer F. The relation of adipose tissue to cardiometabolic
risk. Clinical Cornerstone 2006;8:S14–S23.
 Koh KK, Han SH, Quon MJ. Inflammatory markers and the metabolic
syndrome: insights from therapeutic interventions. J Am Coll Cardiol
 Shimada M, Mochizuki K, Sakurai N, Goda T. Dietary supplementa-
tion with epigallocatechin gallate elevates levels of circulating
adiponectin in non-obese type-2 diabetic Goto–Kakizaki rats. Biosci
Biotechnol Biochem 2007;71:2079–82.
 Kasim-Karakas SE, Tsodikov A, Singh U, Jialal I. Responses of
inflammatory markers to a low-fat, high-carbohydrate diet: effects of
energy intake. Am J Clin Nutr 2006;83:774–9.
 Tilg H, Moschen AR. Adipocytokines: mediators linking adipose
tissue, inflammation and immunity. Nat Rev Immunol 2006;6:
 Khuseyinova N, Koenig W. Biomarkers of outcome from cardiovas-
cular disease. Curr Opin Crit Care 2006;12:412–9.
 Garg A. Adipose tissue dysfunction in obesity and lipodystrophy. Clin
 Terra X, Valls J, Vitrac X, Merrillon JM, Arola L, Ardevol A, et al.
Grape-seed procyanidins act as antiinflammatory agents in endotoxin-
stimulated RAW 264.7 macrophages by inhibiting NFkB signaling
pathway. J Agric Food Chem 2007;55:4357–65.
 Vadillo M, Ardèvol A, Fernández-Larrea J, Pujadas G, Bladé C,
Salvadó MJ, et al. Moderate red-wine consumption partially prevents
body weight gain in rats fed a hyperlipidic diet. J Nutr Biochem 2006;
 Cho WC, Yip TY, Chung WS, Leung AW, Cheng CH, Yue KK.
Differential expression of proteins in kidney, eye, aorta, and
serum of diabetic and non-diabetic rats. J Cell Biochem 2006;99:
 Yamaguchi Y, Yamada K, Yoshikawa N, Nakamura K, Haginaka J,
Kunitomo M. Corosolic acid prevents oxidative stress, inflammation
and hypertension in SHR/NDmcr-cp rats, a model of metabolic
syndrome. Life Sci 2006;79:2474–9.
 KaurG,RaoLVM,AgrawalA,PendurthiUR.Effectof winephenolics
on cytokine-induced C-reactive protein expression. J Thromb Haemost
 Yu R, Kim CS, Kwon BS, Kawada T. Mesenteric adipose tissue-
derived monocyte chemoattractant protein-1 plays a crucial role in
adipose tissue macrophage migration and activation in obese mice.
 Malavazos AE, Corsi MM, Ermetici F, Coman C, Sardanelli F, Rossi
A, et al. Proinflammatory cytokines and cardiac abnormalities in
uncomplicated obesity: relationship with abdominal fat deposition.
Nutr Metab Cardiovas 2007;17:294–302.
 Combs TP, Berg AH, Obici S, Scherer PE, Rossetti L. Endogenous
glucose production is inhibited by the adipose-derived protein Acrp30.
J Clin Invest 2001;108:1875–81.
217X. Terra et al. / Journal of Nutritional Biochemistry 20 (2009) 210–218
 Pinent M, Blay M, Blade MC, Salvado MJ, Arola L, Ardevol A.
effect in streptozotocin-induced diabetic rats and insulinomimetic
activity in insulin-sensitive cell line. Endocrinology 2004;145:
 Cho SY, Park PJ, Shin HJ, Kim YK, Shin DW, Shin ES, et al. (−)-
Catechin suppresses expression of Kruppel-like factor 7 and in-creases
expression and secretion of adiponectin protein in 3T3-L1 cells. Am J
Physiol Endocrinol Metab 2007;292:E1166–72.
 Maeda N, Shimomura I, Kishida K, Nishizawa H, Matsuda M,
Nagaretani H, et al. Diet-induced insulin resistance in mice lacking
adiponectin/ACRP30. Nat Med 2002;8:731–7.
 Black S, Kushner I, Samols D. C-reactive protein. J Biol Chem 2004;
218 X. Terra et al. / Journal of Nutritional Biochemistry 20 (2009) 210–218