Olive oil and walnut breakfasts reduce the postprandial inflammatory response in mononuclear cells compared with a butter breakfast in healthy men

The Lipids and Arteriosclerosis Unit, Reina Sofía University Hospital, University of Córdoba, CIBER de Fisiopatologia de la Obesidad y Nutricion (CIBEROBN), Avenida Menéndez Pidal s/n, 14004 Córdoba, Spain.
Atherosclerosis (Impact Factor: 3.99). 09/2008; 204(2):e70-6. DOI: 10.1016/j.atherosclerosis.2008.09.011
Source: PubMed
ABSTRACT
Inflammation is crucial in all stages of atherosclerosis, and few studies have investigated the effect of dietary fat on markers of inflammation related to this disease during the postprandial period.
To evaluate the chronic effects of dietary fat on the postprandial expression of proinflammatory genes in peripheral blood mononuclear cells (PBMCs) in healthy subjects.
20 healthy men followed three different diets for 4 weeks each, according to a randomized crossover design: Western diet: 15% protein, 47% carbohydrates (CHO), 38% fat (22% saturated fatty acid (SFA)); Mediterranean diet: 15% protein, 47% CHO, 38% fat (24% monounsaturated fatty acid (MUFA)); CHO-rich and n-3 diet: 15% protein, 55% CHO, <30% fat (8% polyunsaturated fatty acid (PUFA)). After 12-h fast, volunteers were given a breakfast with a fat composition similar to that consumed in each of the diets-butter breakfast: 35% SFA; olive oil breakfast: 36% MUFA; walnut breakfast: 16% PUFA, 4% alpha-linolenic acid (LNA).
The butter breakfast induced a higher increase in tumor necrosis factor (TNF)-alpha messenger RNA (mRNA) expression than the olive oil or walnut breakfasts (P=0.014) in PBMCs. Moreover, we found a higher postprandial response in the mRNA of interleukin (IL)-6 with the intake of butter and olive oil breakfasts than with the walnut breakfast (P=0.025) in these cells. However, the effects of the three fatty breakfasts on the plasma concentrations of these proinflammatory parameters showed no significant differences (P=N.S.).
Consumption of a butter-enriched meal elicits greater postprandial expression of proinflammatory cytokine mRNA in PBMCs, compared to the olive oil and walnut breakfasts.

Full-text

Available from: Juan A Paniagua
Atherosclerosis 204 (2009) e70–e76
Contents lists available at ScienceDirect
Atherosclerosis
journal homepage: www.elsevier.com/locate/atherosclerosis
Olive oil and walnut breakfasts reduce the postprandial inflammatory response
in mononuclear cells compared with a butter breakfast in healthy men
Yolanda Jiménez-Gómez
a
, José López-Miranda
a,
, Luis M. Blanco-Colio
b
,
Carmen Marín
a
, Pablo Pérez-Martínez
a
, Juan Ruano
a
, Juan A. Paniagua
a
,
Fernando Rodríguez
c
, Jesús Egido
b
, Francisco Pérez-Jiménez
a
a
The Lipids and Arteriosclerosis Unit, Reina Sofía University Hospital, University of Córdoba, CIBER de Fisiopatologia de la Obesidad y Nutricion (CIBEROBN),
Avenida Menéndez Pidal s/n, 14004 Córdoba, Spain
b
The Vascular Research Laboratory, Jiménez Díaz Foundation, Autonomous University of Madrid, Spain
c
The Clinical Analyses Service, Reina Sofía University Hospital, Spain
article info
Article history:
Received 17 August 2007
Received in revised form 4 August 2008
Accepted 9 September 2008
Available online 17 September 2008
Keywords:
Breakfast
Mononuclear cells
Proinflammatory cytokines
Fatty acids
Postprandial lipemia
abstract
Background: Inflammation is crucial in all stages of atherosclerosis, and few studies have investigated the
effect of dietary fat on markers of inflammation related to this disease during the postprandial period.
Objective: To evaluate the chronic effects of dietary fat on the postprandial expression of proinflammatory
genes in peripheral blood mononuclear cells (PBMCs) in healthy subjects.
Design: 20 healthy men followed three different diets for 4 weeks each, according to a randomized
crossover design: Western diet: 15% protein, 47% carbohydrates (CHO), 38% fat (22% saturated fatty acid
(SFA)); Mediterranean diet: 15% protein, 47% CHO, 38% fat (24% monounsaturated fatty acid (MUFA));
CHO-rich and n-3 diet: 15% protein, 55% CHO, <30% fat (8% polyunsaturated fatty acid (PUFA)). After 12-
h fast, volunteers were given a breakfast with a fat composition similar to that consumed in each of the
diets—butter breakfast: 35% SFA; olive oil breakfast: 36% MUFA; walnut breakfast: 16% PUFA, 4% -linolenic
acid (LNA).
Results: The butter breakfast induced a higher increase in tumor necrosis factor (TNF)- messenger RNA
(mRNA) expression than the olive oil or walnut breakfasts (P = 0.014 ) in PBMCs. Moreover, we found a
higher postprandial response in the mRNA of interleukin (IL)-6 with the intake of butter and olive oil
breakfasts than with the walnut breakfast (P = 0.025) in these cells. However, the effects of the three fatty
breakfasts on the plasma concentrations of these proinflammatory parameters showed no significant
differences (P = N.S.).
Conclusion: Consumption of a butter-enriched meal elicits greater postprandial expression of proinflam-
matory cytokine mRNA in PBMCs, compared to the olive oil and walnut breakfasts.
© 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Atherosclerosis is the major cause of death in western soci-
eties [1] and there is evidence that inflammation plays a central
role in all phases of the atherosclerotic process [2].Afirststep
in this condition is the adhesion of circulating monocytes to the
endothelium and its migration to the intima layer [3]. A crucial
chemokine responsible for the recruitment of monocytes to inflam-
matory lesions in the vasculature is monocyte chemoattractant
protein-1 (MCP-1) [4,5]. This chemokine is highly expressed in
the macrophage-rich area of the atherosclerotic lesions in human
Corresponding author. Tel.: +34 957 218250; fax: +34 957 218250.
E-mail address: md1lomij@uco.es (J. López-Miranda).
[6] and its expression and secretion from vascular cells have been
proved to account for the increased monocyte chemotatic activ-
ity [7]. Once monocytes have reached the subendothelial space,
the modified LDL and various molecules produced by the T lym-
phocytes, endothelial cells and smooth muscle cells stimulate the
transformation of the monocytes into macrophages, which are
important mediators of inflammation. In the final stage, the plaque
is ruptured in the shoulder [8], area more vulnerable, which is
enriched in T lymphocytes and macrophages.
The diet, and particularly its fat content, can modulate the
cardiovascular risk factors and the mechanisms related to the for-
mation and development of the atheroma plaques [9,10]. However,
the influence of the diet on atherosclerosis goes beyond its known
effects on the classic cardiovascular risk factors [9]. Fatty acids and
other components of the diet modulate the expression of several
0021-9150/$ see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.atherosclerosis.2008.09.011
Page 1
Y. Jiménez-Gómez et al. / Atherosclerosis 204 (2009) e70–e76 e71
genes involved in the inflammatory and immune response, such as
proinflammatory cytokines, adhesion molecules, chemokines and
inflammatory enzymes [11–13].
Changes in postprandial metabolism take place every time we
eat a meal and alterations in this state may play an important role in
the development of cardiovascular and associated diseases [14–16].
During postprandial lipemia, there is an increase in circulating
triacylglycerol-rich lipoproteins (TRL), which may be deposited into
the arterial wall and accumulated in atheromatous plaques [16],
formation of highly oxidisable small, dense LDL and a reduction
in the concentration of HDL [17]. Furthermore, it has been found
that during this phase, when triacylglycerols (TG) and glucose
rise, the neutrophil count increases with the subsequent produc-
tion of proinflammatory cytokines and oxidative stress, with these
changes possibly contributing to endothelial dysfunction [18,19].
Moreover, van Oostrom et al. [20] provided evidence that postpran-
dial triglyceridemia is related to the proinflammatory state due to
the high expression of the activation markers in neutrophils and
monocytes. Our group has also shown that butter and walnuts, but
not olive oil, elicit postprandial activation of nuclear factor-B (NF-
B) in PBMCs in healthy men [21]. Since human beings spend much
of the day in the postprandial state it is important to understand the
inflammatory changes that take place during this period in terms
of the type of fat consumed. Our aim was therefore to evaluate
the chronic effect of the type of fat on the postprandial expression
of proinflammatory genes in PBMCs from healthy men. Because
apolipoprotein (apo)E is an important mediator of the clearance of
circulating TRL by receptor [22] and the apoE E2/E3/E4 polymor-
phism is implicated in a variable lipid postprandial response [23],
we realized the study in subjects with the apoE3/E3 genotype, the
most common allele in the population.
2. Methods
2.1. Study subjects
Twenty male medical students all gave informed consent and
underwent a comprehensive medical history, physical examina-
tion and clinical chemistry analysis before enrolment. None of the
subjects showed signs of any chronic disease or obesity, and none
practiced unusually high levels of physical activity. The volunteers
had normal biochemical parameters. They were selected on the
basis of having the apoE3/E3 genotype, in order to avoid the allele
effects of this gene locus on postprandial lipemia [23]. None was
taking medications or vitamins known to affect plasma lipids. The
study protocol was approved by the Human Investigation Review
Committee of the Reina Sofía University Hospital, according to Insti-
tutional and Good Clinical Practice guidelines.
2.2. Study design
Each volunteer in the trial was subjected to three diet inter-
vention periods of 4 weeks of duration, in a randomized crossover
design. The composition of three diets is shown in Table 1. The
PUFA enrichment of the high-CHO diet was achieved via the use of
natural food components rich in -LNA of vegetable origin (based
on walnuts (Juglands regia L.)). The cholesterol content of the diets
was <300 mg/day, and it was kept at a constant level throughout
the three dietary intervention periods.
The composition of the experimental diets was calculated using
the United States Department of Agriculture [24] food tables and
Spanish food composition tables for local foodstuffs [25]. All meals
were prepared in the hospital kitchen and were supervised by a
dietitian. Lunch and dinner were eaten in the hospital dining room,
whereas breakfast and an afternoon snack were eaten in the medi-
cal school cafeteria. Fourteen menus were prepared with regular
solid foods and rotated during the experimental period. Dupli-
cate samples from each menu were collected, homogenized and
stored at 70
C. The study took place during January and March
to minimize seasonal effects and academic stress. Subjects were
encouraged to maintain their regular physical activity and lifestyle
and were asked to record in a diary any event that could affect the
outcome of the study, such as stress, changes in smoking habits
and alcohol consumption or intake of foods not included in the
experimental design.
At the end of the dietary intervention period and after a 12-h
fast, at time 0, the subjects were given a fatty breakfast with a fat
composition similar to that consumed in each of the diets, consist-
ing of 50–66% of the subject’s daily normal intake of calories and
composed of 1 g fat, 7 mg cholesterol and 40 equiv. retinal/kg body
weight, with the following caloric distribution: 60% fat, 15% pro-
tein, and 25% CHO. The composition of three breakfasts is shown in
Table 1. The butter breakfast was based on the consumption of but-
ter, wholemeal bread, hard-boiled egg and whole milk. The olive oil
breakfast was administered in the form of a typical Mediterranean
food with extra virgin olive oil, bread and tomato, accompanied by
skimmed milk and hard-boiled egg. The walnut breakfast consisted
of walnuts (Junglans regia L.), wholemeal bread, jam and skimmed
milk.
The amount of each ingredient was calculated as a function of
individual body weight so that all subjects consumed the same type
of food at different amounts.
2.3. DNA amplification and genotyping of apoE
DNA was extracted from 10 mL blood containing EDTA. A
region of 266-bp of the apoE gene was amplified by PCR
with 250 ng of genomic DNA and 0.2 mmol of each oligonu-
cleotide primer (E1, 5
-GAACAACTGACCCCGGTGGCGGAG-3
, and
E2, 5
-TCGCGGGCCCCGGCCTGGTACACTGCCA-3
) and 10% dimethyl
sulfoxide in 50 L. DNA was denatured at 95
C for 5 min followed
by 30 cycles of denaturation at 95
C for 1 min, annealing at 63
C
for 1.5 min, and extension at 72
C for 2 min. The 20 L of the PCR
product were digested with 10 units of restriction enzyme CfoI (BRL,
MD, U.S.A.) in a total volume of 35 L. Digested DNA was separated
Table 1
Diet and breakfast composition (% of energy intake)
Western diet
richinSFA
Mediterranean diet
enriched in virgin olive oil
High-CHO enriched in
vegetal n-3 fatty acids
Butter breakfast Olive oil breakfast Walnut
breakfast
Protein 15 15 15 15 15 15
CHO 47 47 55 25 25 25
Fat 38 38 <30 60 60 60
SFA 22 <10 <10 35 20 20
MUFA 12 24 12 22 36 24
PUFA 4 4 8 4 4 16
-LNA 0.4 0.4 2 0.7 0.7 4
CHO: carbohydrates; SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid; -LNA: -linolenic acid.
Page 2
e72 Y. Jiménez-Gómez et al. / Atherosclerosis 204 (2009) e70–e76
by electrophoresis on an 8% non-denaturing polyacrylamide gel at
150 V for 2 h. Bands were visualized by silver staining.
2.4. Lipid analysis
Venous blood samples were collected in tubes containing
1 mg/mL EDTA in fasting, at time 0, and every 3 h until the 9th
hour after the ingestion of the breakfasts. Plasma was obtained
by low-speed centrifugation (1500 × g)for15minat4
C within
1 h of venipuncture. In order to reduce interassay variation,
plasma samples were stored at 80
C and analyzed at the end
of the study. Lipid parameters were assessed with a DDPPII
Hitachi modular analyzer (Roche, Basel, Switzerland), using spe-
cific reagents (Boehringer-Mannheim, Mannheim, Germany). Total
plasma cholesterol (C) and TG concentrations and lipoprotein frac-
tions were measured by colorimetric enzymatic techniques [26,27].
HDL–C levels were measured using colorimetric assay after precip-
itating the lipoproteins containing apoB with polyethylene glycol
[28]. LDL–C concentrations were calculated by using the Friedewald
formula based on the C, TG, and HDL–C values [29].
2.5. Plasma fatty acid composition
Plasma lipids were first methylated and an aliquot of fatty
acid methyl esters was analyzed by gas chromatograph (Hewlett-
Packard 5890; series II) equipped with a flame ionization detector
and a SP-2380 (Sulpeco, Bellefonte, PA, U.S.A.) fused silica capillary
column (60 m in length and with an internal diameter of 0.25 mm)
coated with cyanopropylpolysiloxane (0.20 m film thickness). The
oven temperature program was isothermal at 160
C for 8 min
before rising to 220
Catarateof2
C/min. The temperature was
kept at 220
C for 12 min. Hydrogen was used as carrier gas at a
column-head pressure of 20 psi. The injector and detector temper-
atures were 210 and 250
C, respectively. Sample injections were
performed in the split mode.
2.6. Adhesion molecules immunoassay
Plasma concentration of MCP-1, IL-6 and TNF- were deter-
mined in duplicate with commercially available enzyme-linked
immunosorbent assay kits (R&D Systems, Inc.) according to the
manufactures guidelines.
2.7. Isolation of PBMCs
Blood samples were diluted 1:1 in PBS, and cells were sep-
arated in 5 mL Ficoll gradient (lymphocyte isolation solution,
Rafer, Zaragoza, Spain) by centrifugation at 2000 × g for 30 min
at 4
C. PBMCs were collected, washed twice with cold PBS,
and resuspended in TRIZOL (Tri
®
Reagent, Sigma, St. Louis, MO).
Approximately 95% of the cells were mononuclear cells (flow
cytometer, data not shown).
2.8. Total RNA isolation and real-time RT-PCR
Total cellular RNA from PBMCs was extracted using the trizol
method according to the recommendations of the manufacturer
(Tri
®
Reagent, Sigma, St. Louis, MO). Next, since PCR can detect
even a single molecule of DNA, RNA samples were digested with
DNase I (AMP-D1, Sigma) before RT-PCR. The expression levels of
the TNF-, IL-6 and MCP-1 genes, and of 18S ribosomal RNA (rRNA)
as a housekeeping gene, were measured by real-time RT-PCR using
a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA,
U.S.A). RT-PCR was performed in two steps as follows: 2 goftotal
RNA underwent random primed reverse transcription for 10 min
at 25
C and 2 h at 37
C using the High-Capacity cDNA kit (Applied
Biosystems) and RNase inhibitor (rRNasin 40 U/L, Promega, Madi-
son, U.S.A.) to synthesize the cDNA. Real-time PCR was realized
with 2 L of cDNA and 18 L of reaction mixture (10 Lof2× Taq-
Man Universal PCR Master Mix (Applied Biosystems), 1 Lof20×
Assays-on-Demand
TM
Gene Expression Assay Mix (Applied Biosys-
tems) and 7 L RNase-free water). After an initial hold of 2 min
at 50
Cand10minat95
C, the samples were cycled 40 times at
95
C for 15 s and 60
C for 60 s. For all quantitative cDNA analysis,
the C
t
technique was utilised [30]. The expression of each target
gene was normalized to housekeeping gene transcript. All measure-
ments were performed in duplicate. Controls consisting of double
distilled H
2
O were negative in all runs.
2.9. Statistical analysis
Statistical analysis used SPSS statistical software, version 11.0
(SPSS Inc., Chicago, IL). ANOVA for repeated measures was used
to analyze the differences in plasma lipid and lipoprotein concen-
trations and plasma fatty acid composition. ANCOVA for repeated
measures, using the basal values of each mRNA and plasma con-
centration as covariate, was utilised to study the differences in the
expression and production of proinflammatory cytokines under
study. In these analysis, we studied the statistical effects of the
time alone or the change in the variable after ingesting fatty food
over the entire lipemic period (represented as P1) and the effect of
the breakfast (represented as P2), independently of the time in the
postprandial study. We also studied the effect of the interaction
Table 2
Plasma fatty acid composition (percentage relative to the total fatty acids) according to the type of fat consumed during the postprandial
a
Time and breakfast Fatty acid (% relative to the total fatty acids)
14:0 16:0 18:1 18:2 18:3
0h
Butter 0.53 ± 0.18a 21.95 ± 1.40c 18.24 ± 2.13b 23.30 ± 2.15b 0.34 ± 0.07
Olive oil 0.40 ± 0.12b 21.16 ± 1.54 20.12 ± 2.70a 22.00 ± 2.90b 0.35 ± 0.08
Walnuts 0.38 ± 0.16b 20.9 ± 1.04b 17.8 ± 2.42b 25.9 ± 1.64a 0.41 ± 0.15
3h
Butter 1.40 ± 0.43a 23.10 ± 0.71a 18.35 ± 2.30b 23.10 ± 1.10b 0.38 ± 0.12b
Olive oil 0.30 ± 0.15b 20.05 ± 0.84b 27.43 ± 3.98a 21.90 ± 2.28b 0.38 ± 0.04b
Walnuts 0.40 ± 0.15b 20.10 ± 1.38b 17.50 ± 1.21b 30.15 ± 1.60a 1.88 ± 0.63a
Global analysis P values
Time effect 0.001 0.076 0.022 0.129 0.001
Breakfast effect 0.001 0.009 0.001 0.001 0.001
Breakfast × Time effect 0.001 0.001 0.001 0.002 0.001
a
n = 20. Means in a column with different letters are significantly different, P < 0.05 (ANOVA for repeated measures). Values are means ± S.D. (all such values).
Page 3
Y. Jiménez-Gómez et al. / Atherosclerosis 204 (2009) e70–e76 e73
Table 3
Plasma lipid and lipoprotein concentrations according to the type of fat consumed during the postprandial phase
a
Time and breakfast Lipids and lipoproteins (mmol/L)
Total C TG HDL–C LDL–C
0h
Butter 3.85 ± 0.12a 0.78 ± 0.07 1.17 ± 0.05 2.17 ± 0.08a
Olive oil 3.63 ± 0.12b 0.78 ± 0.07 1.18 ± 0.04c 1.91 ± 0.07b
Walnuts 3.57 ± 0.14b 0.72 ± 0.06 1.12 ± 0.05b 1.96 ± 0.10b
3h
Butter 3.62 ± 0.12 1.42 ± 0.12 1.05 ± 0.04 1.76 ± 0.09
Olive oil 3.59 ± 0.12 1.59 ± 0.18 1.04 ± 0.04 1.65 ± 0.09
Walnuts 3.49 ± 0.12 1.34 ± 0.12 1.04 ± 0.04 1.57 ± 0.13
6h
Butter 3.58 ± 0.14 0.88 ± 0.09a 1.06 ± 0.04 1.90 ± 0.09
Olive oil 3.53 ± 0.14 0.64 ± 0.04b 1.07 ± 0.0 4 1.99 ± 0.10
Walnuts 3.46 ± 0.13 0.64 ± 0.04b 1.07 ± 0.04 1.92 ± 0.08
9h
Butter 3.72 ± 0.14 0.59 ± 0.05 1.14 ± 0.04 2.15 ± 0.10d
Olive oil 3.58 ± 0.13 0.53 ± 0.03c 1.12 ± 0.04 2.07
± 0.10
Walnuts 3.55 ± 0.15 0.62 ± 0.04b 1.10 ± 0.04 1.95 ± 0.09b
Global analysis P values
Time effect 0.001 0.001 0.001 0.001
Breakfast effect 0.614 0.862 0.940 0.503
Breakfast × Time effect 0.036 0.006 0.025 0.020
a
n = 20. Means in a column with different letters are significantly different, P < 0.05 (ANOVA for repeated measures). Values are means ± S.E.M. (all such values).
of both factors breakfast and time which is indicative of the
magnitude of the postprandial response in each meal (represented
as P3). When statistically significant effects were found, an ANOVA
was used to identify group differences in each time. A study of
the relation among parameters was carried out using Pearson’s
linear correlation coefficient. A probability of less than 0.05 was
considered significant. All data presented in the text and tables are
expressed as mean ± S.E.M.
3. Experimental results
3.1. Plasma fatty acid composition
The data suggest that the type of diet consumed during the
dietary intervention period and the intake of the fatty breakfasts
at the end of each period has a direct influence on plasma fatty
acid composition (Table 2). At the end of the dietary intervention
period, we observed that the Western diet raised the proportion of
palmitic and myristic fatty acids (P < 0.05). On the other hand, the
Mediterranean diet produced an increase in oleic acid (P < 0.05),
while the high-CHO diet enriched in n-3 fatty acids augmented
linoleic (P < 0.05) and linolenic acids. In the postprandial period,
we found an increase (P < 0.05) of palmitic and myristic fatty acids
and of oleic acid with the butter and olive oil breakfasts, respec-
tively. Moreover, we showed that the walnut breakfast increased
concentrations of linolenic and linoleic fatty acids (P < 0.05).
3.2. Diet intake and postprandial lipemia
At the end of the dietary intervention period and after a 12-h
fast (Table 3), we observed an increase in total C and LDL–C with
the Western diet, compared with the Mediterranean diet and the
high-CHO diet rich in -LNA (P < 0.05), as well as, a reduction in
HDL–C concentrations with the high-CHO diet rich in -linolenic
acid respect to the Mediterranean diet (P = 0.015). Throughout the
whole period of postprandial lipemia, we demonstrated an increase
in total TG and a reduction in C, HDL–C and LDL–C concentrations
(P < 0.001). Furthermore, we found a higher increase in total post-
prandial TG with the butter breakfast than with the walnut- or
olive oil-enriched breakfasts at 6 h (P < 0.05). By 9 h, there was a
lower concentration of TG with the olive oil breakfast than with the
walnut breakfast (P < 0.05), as well as an increase in LDL–C concen-
trations with the butter breakfast compared to the walnut breakfast
(P = 0.013) (Table 3).
3.3. Diet intake and proinflammatory cytokines
In order to determine whether the intake of the three fat-load
breakfasts could regulate the expression of different inflammation
parameters in PBMCs during the postprandial phase, we studied
the mRNA levels for TNF-, IL-6 and MCP-1 in these cells (Fig. 1).
We observed that the ingestion of the butter breakfast induced a
higher postprandial expression of mRNA TNF- than the olive oil
or walnut-enriched breakfasts (P = 0.014). At 3 h after the intake
of the olive oil and walnuts breakfasts, we found a lower post-
prandial expression of mRNA TNF- than after the butter-rich
breakfast (P < 0.05). When we studied the expression of mRNA IL-
6, we showed a higher postprandial response in the mRNA of this
cytokine with the intake of the butter and olive oil breakfasts than
with the walnut breakfast (P = 0.025). We observed an increase in
the mRNA IL-6 after the butter- and olive oil-enriched breakfasts
compared to the walnut breakfast af ter 3 h (P < 0.05). Finally, we did
not find any significant differences in mRNA MCP-1 expression fol-
lowing the intake of the three types of fatty breakfasts throughout
the postprandial phase.
In addition, we also analyzed plasma levels of TNF-,IL-6and
MCP-1 during the postprandial phase (Fig. 2). Comparisons of the
effect of the time, the fatty breakfast, and the interaction of both fac-
tors (time and breakfast) on the concentrations of these parameters
revealed no significant differences (P = N.S.).
3.4. Correlations between proinflammatory and lipid
parameters/fatty acid composition
We studied the relationship between the mRNA levels for TNF-
and IL-6, and lipid and lipoprotein concentrations, as well as
between these proinflammatory cytokines and the plasma fatty
acid composition during the first 3 h after the intake of a fatty
Page 4
e74 Y. Jiménez-Gómez et al. / Atherosclerosis 204 (2009) e70–e76
Fig. 1. Mean (±S.E.M.) mRNA TNF- (A), IL-6 (B) and MCP-1 (C) expression in PBMCs, n = 20. Results are expressed in arbitrary units. ANCOVA for repeated measures: P1:
time effect; P2: breakfast effect; P3: breakfast by time interaction.
a
P < 0.05 butter vs. olive oil and walnuts breakfasts and
b
P < 0.05 walnut vs. butter and olive oil breakfasts
at that specific point in time.
Fig. 2. Mean (±S.E.M.) plasma TNF- (A), IL-6 (B) and MCP-1 (C) levels, n = 20. Results are expressed in arbitrary units. ANCOVA for repeated measures: P1: time effect; P2:
breakfast effect; P3: breakfast by time interaction.
Page 5
Y. Jiménez-Gómez et al. / Atherosclerosis 204 (2009) e70–e76 e75
breakfast without considering the type of fat consumed. We did
not find correlation among the different studied parameters (data
no shown).
4. Discussion
Ours results show that a butter-enriched breakfast increases
postprandial expression of mRNA TNF- in PBMCs from healthy
men with apoE3/E3 genotype compared with a breakfast rich in
olive oil or walnuts. Moreover, we observed a higher postprandial
response of mRNA IL-6 in these cells with the butter and olive oil
breakfasts compared to the breakfast rich in walnuts.
Few studies have investigated changes in inflammatory mark-
ers related to atherosclerosis during the postprandial state [31,32],
which is the normal metabolic condition of the human beings
throughout the day, and none of them considered the effect of
the type of fat on such response. Our group has already demon-
strated that the consumption of an olive oil-enriched breakfast
does not activate NF-B in PBMCs as do butter- and walnut-
enriched breakfasts [21]. Nevertheless, the chronic effect of the
type of dietary fat on the postprandial inflammatory response is
not known. MCP-1 regulates the transmigration of monocytes and
other mononuclear cells on inflammatory sites [33]. Moreover,
MCP-1 also recruits monocytes into atherosclerotic lesions and into
the infarct zone after myocardial infarction [34]. However, when we
analyzed this chemokine, we did not observe significant differences
in the expression and plasma levels of MCP-1 throughout the post-
prandial phase. This result may be explained, at least in part, by the
characteristics of our sample population, which consisted of com-
pletely healthy young people. On the other hand, TNF- and IL-6
can mediate the systemic effects of inflammation, including fever,
loss of appetite, mobilization of protein and fat, and acute phase
protein synthesis. The production of sufficient amounts of these
cytokines is clearly beneficial in response to infection, but inap-
propriate quantities or overproduction may be harmful. In order
to alleviate inflammation, therefore, it is important to inhibit the
production of proinflammatory cytokines. Nutrition strategies may
be desirable to manipulate the secretion of these cytokines and,
in this regard, our present study showed a higher expression of
proinflammatory cytokines in PBMCs following a butter-enriched
breakfast than after those rich in olive oil or walnuts. However, we
did not observe significant postprandial differences in plasma lev-
els of these proinflammatory cytokines. The fact that we only found
differences in the expression of TNF- and IL-6 at mRNA levels in
PBMCs following the intake of the three breakfasts may be due to
that the synthesis and secretion processes of these proteins do not
happen simultaneously, and to the short half-life of both cytokines
[35,36].
Biologically, TNF- acts as a trigger that activates a cascade of
cytokine production. A numb er of regulatory agents, including glu-
corticoids, acute-phase proteins, eicosanoids and soluble receptors
[37], limit TNF- production. Lipids have been shown to be potent
modulators of inflammation since a large number of the modula-
tory compounds cited previously are derived from the hydrolysis
of membrane phospholipids. Because of this effect of lipids in the
inflammation and, to the fact that apoE genotype is know to have
significant effects on postprandial lipemia [23], we study only indi-
vidual homozygous for the most common allele, apoE3/E3. When
we investigated whether the effect observed in the proinflamma-
tory cytokines was due to the change in the lipid profile [38] or in
the plasma fatty acid composition, we did not observe a correlation
between these parameters and mRNA for TNF- and IL-6. There-
fore, it is possible that other factors take part in the process. We
speculated that, as indicated in our previous study [21], an increase
in activation of NF-B may be one of the main mechanisms of the
inflammatory properties of the butter breakfast, since this nuclear
factor plays a central role in regulating the cytokine network. It
has also been observed that the modulatory effect of fatty acids
on the synthesis of proinflammatory cytokines may be due to a
peroxisome proliferative activated receptor (PPAR)-dependent
mechanism. Desreumaux et al. [39] showed that activation of PPAR-
in the colon inhibits mucosal production of IL-1 and TNF- by
downregulation of the NF-B and mitogen-activated protein kinase
signal pathways. Furthermore, we observed a higher mRNA IL-6
expression in PBMCs following the butter and olive oil breakfasts
than with the walnut breakfast. A mechanism possibly capable of
explaining this result is that n-3 polyunsaturated fatty acids induce
changes in both cycloxygenase and lipoxygenase products, such as
a reduction in the production of prostaglandin E
2
and leukotriene
B
4
[40]
.
Since both metabolites enhance the release of IL-6 in vitro
[41], a reduction in these eicosanoids could explain the reduction
in mRNA IL-6 that we observed in this study with the walnut-rich
breakfast.
To summarize, this study has shown that breakfasts rich in olive
oil or walnuts had an anti-inflammatory effect, which may provide
an additional beneficial mechanism of both types of fat in the pri-
mary and secondary prevention of cardiovascular disease. The final
objective in the prevention and treatment of coronary atheroscle-
rosis is to reduce the risk of new heart attacks and mortality due
to cardiovascular failure. Identifying a suitable diet is thus funda-
mental for avoiding the development of this disease and associated
cardiovascular events.
Acknowledgments
Supported in part by research grants from the MCYT (AGL
2004/07907, AGL2006-01979/ALI to JL-M and SAF2003-05770 to
FP-J), the Spanish Ministry of Health (FIS PI041619 to CM, FIS 01/449
to JL-M, and CB06/03/0047 (CIBER Fisiopatologia de la Obesidad y
Nutricion is an initiative of ISCIII) to FP-J), Consejería de Salud, Junta
de Andalucía (03/75, 04/238 to JL-M, 03/73, 04/191 to FP-J, 05/396
to CM), the Diputación Provincial de Córdoba (to FP-J), the CAM
(08.4/0021.1/2003 to JE) and the Spanish Cardiovascular Network
(to JE). We extend our thanks to Canoliva (Antonio Cano e Hijos
SA, Luque, Córdoba), who generously donated the olive oil for the
dietary experiments, and also to the volunteers.
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    • "There was a higher postprandial response in the mRNA of IL-6 with the butter and olive oil breakfasts, however, the plasma concentrations of these pro-inflammatory markers showed no significant differences. The authors reported that these three diets did not activate NF-B or affect plasma or expression levels of MCP-1 [134]. "
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  • Source
    • "The TNFα promoter is susceptible to regulation by cytosine methylation [37]. TNFα has been demonstrated to be modified in response to the intake of several foods or nutrients, such as fruits and vegetables [38], olive oil and walnuts [39], fructose [40] and polyunsaturated fatty acids [41]. Obese/overweight subjects were recruited at the Center for Obesity and Work. "
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    • "These diets do not have to be low energy, and notably, the Mediterranean diet is proportionately high in energy from cold pressed, high polyphenolcontent oil, in addition to fats in dairy, meat, fish, and nuts. The typical traditional Mediterranean contains approximately 38% of olive oil, equivalent to the fat percentage of a typical western diet [23]. Note that saturated fat has been present in our diets for millions of years [24]. "
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