Chapter 125
Microarray Analysis of Hepatic Genes
Altered in Response to Olive Oil Fractions
María Victoria Martínez-Gracia1,2 and Jesús Osada1,2
1
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Veterinaria, Universidad de Zaragoza
2
CIBER de Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, Spain
125.1  Introduction
The Mediterranean diet is associated with a reduced risk
of cardiovascular mortality despite the high intake of fat,
mainly derived from olive oil (Keys, 1995). Olive oil, as a
fruit juice, is a complex mixture where triglycerides are com-
bined with other biologically active substances such as toco-
pherols, phenolic compounds, phytosterols and triterpenoids,
some of which have antioxidant and anti-inflammatory
activities (Visioli et al., 2000; de la Puerta et al., 2001;
Visioli et al., 2003; de la Puerta-Vazquez et al., 2004; Perona
et al., 2006). The current view proposes that these compo-
nents might be responsible for the benefits of virgin olive oil
in animal models ( Calleja et al., 1999; Herrera et al., 2001;
Rodriguez-Rodriguez et al., 2004; Acin et al., 2007) and in
human studies (Ruiz-Gutierrez et al., 1996; Kris-Etherton
et al., 1999; Abia et al., 2001; Perona et al., 2003, 2006). But
still a lot of research needs to be done to understand their
biological action.
125.2  The liver, an organ sensitive
to the diet composition
The liver secretes phospholipids, cholesterol and triglycer-
ides into plasma as lipoprotein complexes (VLDL and HDL)
which allow the transport of those lipids into the aque-
ous medium of blood. Apolipoproteins such as ApoB-100,
ApoA-I, ApoA-II and ApoE are the main protein constitu-
ents of lipoproteins. Furthermore, this organ also secretes the
enzymes (hepatic lipase, lecithin cholesterol acyl transferase
and phospholipid transfer protein) involved in the plasma
transformation of lipoproteins (den Boer et al., 2004).
ApoE-deficient mice lack apolipoprotein E and as a
consequence the elimination of lipoproteins from blood is
impaired. Due to this fact, lipoproteins accumulate in walls
Olives and Olive Oil in Health and Disease Prevention.
ISBN: 978-0-12-374420-3
of vessels, contributing to the development of spontaneous
atherosclerosis (Osada et al., 2000). When fed high-fat diets
these mice responded, inducing changes in plasma apolipo-
proteins (Arbonés-Mainar et al., 2006), hepatic gene expres-
sion being responsible for these variations (Arbones-Mainar
et al., 2006). The hepatic expression of apolipoprotein
A-I was also modified in response to the type and amount
of olive oil provided (Calleja et al., 1999; Acín et al., 2005).
Thus, the liver may undergo important metabolic changes
under the influence of different olive oils. Therefore, the
liver is a central organ in the management of dietary lipids
and an ideal model to verify subtle changes in diet composi-
tion due to its rapid adaptation response. To test the hypoth-
esis that the unsaponifiable fraction of olive oil significantly
influences hepatic gene expression, apoE-deficient mice of
different genetic backgrounds (one from pure C57BL/6J
mice and the other hybrid C57BL/6J x Ola129) were fed
diets supplemented with either 10% (w/w) olive oil or 10%
unsaponifiable fraction-enriched olive oil. Gene expression
was then determined by microarray analysis that allows the
global analysis of all mRNA present in a tissue and later
confirmed by another procedure (quantitative RT-PCR) to
reinforce the validity of results.
125.3  Methodological
consideration
Two-month-old, male, homozygous apoE-deficient mice
with different genetic backgrounds were used, and two
study groups of equal plasma cholesterol were established:
(a) one received a chow diet supplemented with 10% (w/w)
olive oil (diet OO) (n  9), and (b) the other received the
same chow diet but supplemented with 10% (w/w) unsa-
ponifiable-fraction enriched olive oil (diet UEOO; n  8).
This unsaponifiable-fraction enriched olive oil had greater
Copyright © 2010 Elsevier Inc.
1143 All rights of reproduction in any form reserved.
1144 Section  |  II  Major Organ Systems Including Liver and Metabolism
Olive UEOO
(9 mice) (8 mice)
• RNA isolation from liver
• Removal of contaminant DNA
1 pool RNA 1 pool RNA
(9 mice/pool) (8 mice/pool)
Affymetrix array
Selection of genes:
• Detection call, absorbance > 3 times noise
• Signal log2 ratio > 3 or < −3
Primer design for qRT-PCR of selected genes
Olive UEOO
individual mRNA expression individual mRNA expression
by qRT-PCR by qRT-PCR
Figure 125.1  Schematic workflow of the protocol. Diagram display-
ing the analytical steps used to prepare the RNA, its use to hybridize
microarrays, critical criteria used to select meaningful genes and a final
confirmation of individual samples by (qRT-PCR) quantitative reverse
transcriptase-polymerase chain reaction. OO, olive oil; UEOO, unsaponi-
fiable fraction-enriched olive oil.
quantities of phytosterols, waxes, triterpenes (erythrodiol,
uvaol and maslinic) and tocopherols (Acin et al., 2007).
For 11 weeks, the animals were fed the experimental diets
which were well-tolerated. After this time period, animals
were sacrificed and the liver removed, frozen in liquid
nitrogen and used to extract its total RNA.
The changes in hepatic gene expression induced by the
unsaponifiable fraction of olive oil were tested by the expres-
sion of 22 690 transcripts represented on the Affymetrix
GeneChip Murine Genome MOE430A array and quantified
in pooled liver samples of nine animals that received the OO
diet and another eight that received the UEOO diet. A sum-
mary of the approach is reflected in Figure 125.1.
The huge amounts of information provided by microar-
rays requires further action be undertaken if meaningful and
manageable data are to be obtained, such as selecting only
the genes with the highest expression changes (Dutta et al.,
2003; Vergnes et al., 2003; Artieda et al., 2005; Calpe-Berdiel
et al., 2005) or those involved in a certain metabolic pathway
(Horton et al., 2003; Kreeft et al., 2005). In the present work,
the first approach has been adopted and only those genes
whose expression was strongly (signal log2 ratio higher or
lower than 3 or 3) modified were considered to be potential
markers of the intake of the unsaponifiable fraction.
125.4  Global changes in gene
expression in livers fed the
different diets using microarrays
The livers of OO animals expressed 10 455 transcripts,
while those of the UEOO animals expressed 10 675 (iden-
tified as ‘present’ by Affymetrix software). Using the
Mann–Whitney ranking feature of the Affymetrix software
to determine significant differences in gene expression
(p  0.01), the increased expression of 660 genes plus the
reduced expression of 324 genes was identified in samples
from the animals on the UEOO diet compared to those on
the OO diet. The complete datasets were deposited in the
GEO database (accession number GSE2261).
Differentially regulated genes with a signal log2 ratio
higher than 3 (for those genes up-regulated) or lower than –
3 (for those repressed) were taken into account. Tables 125.1
and 125.2 list the genes whose mRNAs reflected these
expressions. Six genes fulfilled the criterion of show-
ing increased expression as a response to the unsaponifi-
able fraction of olive oil (Table 125.1). Two of these genes
coded for acute phase proteins (Orm2 and Saa2), one
was involved in signal transduction (Lepr), one coded for
ion-binding proteins (Mt2), one for metabolite transport
proteins (Fabp5), and finally one was an enzyme involved
in nicotinamide metabolism (Nnmt). Six genes met the
criterion of showing a reduced expression as a response
to the presence of the unsaponifiable fraction of olive oil
(Table 125.2). Of these, three were involved in proteolysis
(Chym, Ela2 and Try4), one in lipid metabolism (Pnlip),
one coded for an enzyme involved in carbohydrate metabo-
lism (Gck) and one cell surface protein (Sycn).
To validate the results obtained with the microarray, the
expressions of these 11 genes – Chym, Ela2, Fabp5, Gck,
Lepr, Mt2, Nnmt, Orm2, Pnlip, Saa2, Try4 – were indi-
vidually studied by specific qRT-PCR assays previously
described (Acín et al., 2007) and the results are shown in
Table 125.3. The six up-regulated genes included in the val-
idation analysis – Fabp5, Lepr, Mt2, Nnmt, Orm2, Saa2 –
and the five down-regulated genes selected – Chym, Ela2,
Gck, Pnlip, Try4 – were confirmed by these approaches.
Good agreement was observed between the Affymetrix chip
and qRT-PCR data (r  0.9382, p  0.0001). The present
Chapter  |  125  Microarray Analysis of Hepatic Genes Altered in Response to Olive Oil Fractions 1145

Table 125.1  Hepatic genes up-regulated at the level of signal log2 ratio  3 by the unsaponifiable fraction of olive oil.
Biological process GenBank Affymetrix ID Name Gene Olive UE olive Signal log2
Symbol ratio

Signal transduction U42467 1425644_at Leptin receptor Lepr 3 166 5.8

Acute phase response NM_011016 1420438_at Orosomucoid 2 Orm2 71 1497 3.9

Methyl transferase AK006371 1432517_a_at Nicotinamide N- Nnmt 107 1158 3.4

methyltransferase

Acute phase response NM_011314 1449326_x_at Serum amyloid A 2 Saa2 858 4292 3.3

Metal binding protein AA796766 1428942_at Metallothionein 2 Mt2 421 4657 3.1

Transport (fatty acids) BC002008 1416022_at Fatty acid binding Fabp5 33 238 3
protein 5

Data represent intensity of signal for each condition with the Affymetrix chip. ID, identification; UE, unsaponifiable fraction-enriched. Adapted from
Acín et al., Br J Nutr 97: 628–638, Copyright (2007), with permission from the authors.

Table 125.2  Hepatic genes down-regulated at the level of signal log2 ratio   3 by the unsaponifiable fraction of olive oil.
Biological process GenBank Affymetrix ID Name Gene Olive UE olive Signal log2
symbol ratio

Lipid metabolism AI326372 1433431_at Pancreatic lipase Pnlip 858 79 3.3

Proteolysis AK003088 1428062_at Carboxypeptidase A1 Cpa1 800 71 3.3

Proteolysis NM_025583 1448220_at Chymotrypsinogen Chym 778 59 3.3

Proteolysis NM_007919 1448281_a_at Elastase 2 Ela2 666 80 3.3

Carbohydrate metabolism BC011139 1419146_a_at Glucokinase Gck 64 8 3.2

Surface protein BC019567 1451228_a_at Syncollin Sycn 243 14 3

Data represent intensity of signal for each condition with the Affymetrix chip. ID, identification; UE, unsaponifiable fraction-enriched. Adapted from Acín
et al., Br J Nutr 97: 628–638, copyright (2007), with permission from the authors.

data clearly show that pooling RNA from different ani-
mals and using this pool in microarray analysis is a reli-
able screening method for the search of biological effects
in terms of saving samples, time and economic resources,
as other authors have found (Napoli et al., 2002; Dutta
et al., 2003; Artieda et al., 2005; Calpe-Berdiel et al., 2005;
Kreeft et al., 2005). However, the main drawback of this
approach is the lack of information on biological variabil-
ity of individual samples. This limitation may be particu-
larly important in the nutrition field in order to distinguish
dietary responders and non-responders. Therefore, the
experimenter should be aware of this caveat before decid-
ing to pool samples.
125.5  Plasma presence of
unsaponifiable-activated gene
products
Since two of the overexpressed genes (Orm2 and Saa2)
coded for circulating proteins that may influence general
homeostasis, their plasma levels were determined to test
the relevance of these mRNA changes at the protein level.
Figure 125.2 shows the results of Western analysis of serum
amyloid protein concentration. The observed increase of
Saa mRNA expression (Table 125.1) was not reflected in
plasma in either the OO or UEOO mice, suggesting the
absence of an acute phase reaction. However, the increased
1146 Section  |  II  Major Organ Systems Including Liver and Metabolism

Table 125.3  Hepatic genes regulated by the unsaponifiable fraction of olive oil in mice with C57BL/6J x Ola129
genetic background.
Olive (n  9) UE olive (n  8) Fold change Signal log2 ratio

Genes up-regulated

  Fabp5 1.0  0.8 5.5  0.4a   5.3   2.4

  Lepr 0.4  0.5 4.1  0.2a 10.9   3.4

  Mt2 0.5  0.6 13.6  1.5a 29.1   4.8

  Nnmt 0.1  0.3 2.4  0.4a 16.8   4.1

  Orm2 2.4  0.3 75.2  2a 31.8   5.0

  Saa2 3.4  0.3 108.5  1a 32.2   5.0

Genes down-regulated

  Chym 35.1  0.1 5.7  0.3a   0.16 2.6

  Ela2 16.2  0.1 4.2  0.3a   0.26 1.9

  Gck 3.7  0.8 0.8  0.6a   0.21 2.2

  Pnlip 12.2  3 2.0  0.2a   0.16 2.6

  Try4 15.2  0.1 4.6  0.6a   0.30 1.7

Data represent arbitrary units normalized to the -actin expression for each condition with the quantitative reverse transcriptase–polymerase chain
reaction procedure and are mean and standard deviation.
a
p  0.001 according to Mann–Whitney U-test. UE, unsaponifiable fraction-enriched. Adapted from Acín et al., Br J Nutr 97: 628–638, copyright (2007),
with permission from the authors.

1 2 3 4 elicited by these compounds via the induction of Orm2

SAA 14 kDa
Light Ig 22 kDa
Figure 125.2  Plasma SAA levels evidenced by Western blot analysis.
Lane 1 shows a positive control corresponding to rat plasma from an
animal treated with turpentine to induce plasma expression. Lanes 2–4,
plasma from mice consuming the different diets: chow, OO diet and the
UEOO diet respectively. Light chain immunoglobulin detection was
used as a loading control. OO-olive oil; UEOO-unsaponifiable fraction-
enriched olive oil. Adapted from Acín et al, Br J Nutr 97: 628–638, copy-
right (2007), with permission from the authors.
expression. Recent studies have found that subjects with
increased plasma concentrations of this protein have higher
levels of vitamin A (Thurnham et al., 2003). In addition,
in transgenic mice overexpressing Srebp1 and Srebp2, tran-
scriptional factors involved in lipid metabolism, increased
expression of this gene has also been described (Horton
et al., 2003) although at a lower intensity than in mice con-
suming the UEOO diet. Together, these data suggest an
unknown role for orosomucoid in the handling of unsapon-
ifiable compounds.

hepatic Orm2 mRNA levels observed in the chip analysis

and confirmed by qRT-PCR (Table 125.3) were reflected
in the plasma concentration of animals consuming the 125.6  Effects of mouse genetic
UEOO diet (Figure 125.3A). The induction of orosomu- background on the response to
coids has to date been attributed to acute-phase reactions unsaponifiable fraction-enriched
(Hochepied et al., 2003). In this regard, the absence of any olive oil
hepatic steatosis or inflammation described for this model
(Acín et al., 2007), plus a lack of change in SAA after the To investigate which of the selected genes were influenced
administration of the unsaponifiable fraction, suggest that by the genetic background, another study was carried out.
increased orosomucoid plasma levels are a unique response In the latter, 12 homozygous apoE-deficient mice with a
Chapter  |  125  Microarray Analysis of Hepatic Genes Altered in Response to Olive Oil Fractions
* 8
800
600
µg mL–1
400
200
0 2
A Olive UE Olive
10
AU
5
0 on the pattern of gene expression in response to the unsaponifiable frac-
B Olive UE Olive
Figure 125.3  Influence of genetic background on orosomucoid expres-
sion in apoE-deficient mice consuming the different diets. (A), plasma
orosomucoid 2 levels in apoE-deficient mice with C57BL/6J x Ola129
genetic background. (B), orosomucoid 2 mRNA expression in livers of
apoE-deficient mice with C57BL/6J genetic background (determined by
qRT-PCR). Data represent mean and SEM for each group. Means were
compared by the Mann–Whitney U-test. *p  0.001 compared to results
for OO diet. OO, olive oil; UEOO, unsaponifiable fraction-enriched olive
oil. Adapted from Acín et al., Br J Nutr 97: 628–638, copyright (2007),
with permission from the authors.
C57BL6J genetic background received the same diets used
in the previous experiment and their livers were studied for
the expression of these genes by qRT-PCR. Although the
UEOO diets led to a reduction in hepatic Orm2 mRNA lev-
els (Figure 125.3B) surprisingly this was not reflected at the
plasma level (data not shown). The increase in hepatic Orm2
mRNA expression noted in the first strain of mice is proba-
bly a specific response elicited by the unsaponifiable fraction
of olive oil, and is not related to an acute-phase response but
conditioned by the genetic background of the mice.
The results of the other genes, expressed as signal
log2 ratios in both genetic backgrounds, are presented in
Figure 125.4. Interestingly, lesser variability of response
was observed in C57BL/6J background and no Pnlip
expression was detected in the livers of these mice (data not
shown). For the genes Lepr and Saa2, a significant opposite
response was seen in mice of different genetic background.
The expression levels of Lepr, Orm2 and Saa2 as markers
of the presence of the unsaponifiable fraction of olive oil
showed an interaction with genetic background.
In contrast, seven of these genes – Chym, Ela2, Fabp5,
Gck, Mt2, Nnmt and Try4 – were representative markers of
the presence of the unsaponifiable fraction of olive oil in
the diet, although the magnitude of response significantly
differed among them (more pronounced for the Fabp5 gene
in the C57BL/6J background, and less so in genes Gck,
Nnmt and Try4 in the C57BL/6J x Ola129 background) and
for other genes the influence of the UEOO diet was similar
in both types of mice (Mt2, Chym and Ela2 expressions),
so the genetic background did not influence the response.
1147
Hybrid (C57BL/6J × Ola 129)
* * * *
Pure (C57BL/6J)
6
4
0
−2
−4 * *
Nnmt Saa2 Mt2 Lepr Fabp5 Gck Chym Ela2 Try4
Figure 125.4  Influence of genetic background of apoE-deficient mice
tion-enriched olive oil. Data as mean and SD for each group are expressed
as signal log2 ratios of hepatic mRNA expression for each gene in apoE-
deficient mice with C57BL/6J x Ola129 and C57BL/6J genetic back-
grounds consuming either the OO or UEOO diet. Animals receiving the
OO diet were used as the reference against which to compare the effects
of the UEOO diet. *p  0.001 between genetic backgrounds according
to the Mann–Whitney U-test. OO, olive oil; UEOO, unsaponifiable frac-
tion-enriched olive oil. Reprinted from Acín et al., Br J Nutr 97: 628–638,
Copyright (2007), with permission from the authors.
The biological change produced by these variations in
gene expression is complex. Thus, three of these genes –
Chym, Ela2 and Try4 – are involved in proteolysis and
showed reduced expression in the UEOO animals. Gck, an
enzyme involved in glucose metabolism and also repressed
in animals receiving high-fat diets (Dutta et al., 2003),
showed similar behavior. The opposite (up-regulation)
was observed for the expressions of Fabp5, Mt2 and Nnmt.
Fabp5 (mal1) is considered to be an epidermal protein
although it is also expressed in adipocytes (Maeda et al.,
2003) and the liver (see GenBank accession AK167389 for
a clone isolated from a liver cDNA library, and the present
data). The exact role of this protein is not yet completely
known, although it has been proposed to bind leukotriene
A4 (Zimmer et al., 2004) and to play a role in systemic
insulin sensitivity (Maeda et al., 2003). The change in its
expression induced by the UEOO diet was particularly
dramatic in the C57BL/6J animals. Mt2 is thought to be
associated with obesity since knock-out mice lacking this
gene develop this problem (Miura and Koizumi, 2005). In
both studied substrates, the expression of this gene was up-
regulated (Figure 125.4). Nnmt has been recently associ-
ated with plasma homocysteine levels (Souto et al., 2005).
Its genetic background-dependent response might explain
the variation in homocysteine levels in different strains of
mice. Taken as a whole, these results suggest that the unsa-
ponifiable components of olive oil play an important role
in controlling the expression of genes with participation in
obesity, insulin sensitivity and cardiovascular risk factors,
and that it deserves further attention.
1148 Section  |  II  Major Organ Systems Including Liver and Metabolism
Summary points
l This nutrigenomic approach clearly illustrates the
important effects of the unsaponifiable fraction of olive
oil influencing the expression of hepatic genes, and
provides further support for the idea that not all MUFA-
containing oils behave in the same way (Kritchevsky
et al., 1984; Kris-Etherton et al., 1999).
l The results suggest that it is no longer appropriate to
speak of monounsaturated fatty-acid-enriched oils (avo-
cado, oleic acid-enriched safflower, oleic acid-enriched
sunflower, olive and peanut oils) as though all had the
same effects. Future studies should be aware of this to
avoid confusion – both to researchers and consumers.
l This approach also shows new connections between
nutrition and gene expression. A gene product with
unknown biological function, orosomucoid, was
up-regulated to an extent depending on the genetic
background of the mice. Fabp5 and Mt2 were strongly
up-regulated while the expression of several proteases
was repressed by the UEOO diet.
l The modifications in gene expression could be used as
markers of the intake of the unsaponifiable fraction of
olive oil.
l The present results also show the usefulness of
Affymetrix chip technology for characterizing gene
expression levels in response to nutritional components
in intact animal systems.
Acknowledgments
This research was supported by grant FEDER-CICYT (SAF2007-60173)
and Redes DGA (B-69). We thank Drs. C. Junquera and L. Osaba of
Progenika Biopharma for performing the microarray analyses. Thanks are
also owed to Angel Beltrán, Jesús Cazo, Jesús Navarro, Carmen Navarro
and Clara Tapia of the Unidad Mixta de Investigación for their invaluable
help in maintaining the experimental animals.
References
Abia, R., Pacheco, Y.M., Perona, J.S., Montero, E., Muriana, F.J., Ruiz-
Gutierrez, V., 2001. The metabolic availability of dietary triacylglycerols
from two high oleic oils during the postprandial period does not depend
on the amount of oleic acid ingested by healthy men. J. Nutr. 131, 59–65.
Acín, S., Navarro, M.A., Carnicer, R., Arbonés, J.M., Guzmán, M.A.,
Arnal, C., Beltrán, G., Uceda, M., Maeda, N., Osada, J., 2005. Dietary
cholesterol suppresses the ability of olive oil to delay the develop-
ment of atherosclerotic lesions in apolipoprotein E knockout mice.
Atherosclerosis 182, 17–28.
Acin, S., Navarro, M.A., Perona, J.S., Arbones-Mainar, J.M., Surra, J.C.,
Guzman, M.A., Carnicer, R., Arnal, C., Orman, I., Segovia, J.C.,
Osada, J., Ruiz-Gutierrez, V., 2007. Olive oil preparation determines
the atherosclerotic protection in apolipoprotein E knockout mice.
J. Nutr. Biochem. 18, 418–424.
Acín, S., Navarro, M.A., Perona, J.S., Surra, J.C., Guillen, N., Arnal, C.,
Sarría, A.J., Arbonés-Mainar, J.M., Carnicer, R., Ruiz-Gutiérrez, V.,
Osada, J., 2007. Microarray analysis of hepatic genes differentially
expressed in the presence of the unsaponifiable fraction of olive oil in
apolipoprotein E-deficient mice. Br. J. Nutr. 97, 628–638.
Arbonés-Mainar, J.M., Navarro, M.A., Acín, S., Guzmán, M.A., Arnal, C.,
Surra, J.C., Carnicer, R., Roche, H.M., Osada, J., 2006. trans-10, cis-12-
and cis-9, trans-11-conjugated linoleic acid isomers selectively modify
HDL-apolipoprotein composition in apolipoprotein E knockout mice.
J. Nutr. 136, 353–359.
Arbones-Mainar, J.M., Navarro, M.A., Guzman, M.A., Arnal, C., Surra, J.C.,
Acin, S., Carnicer, R., Osada, J., Roche, H.M., 2006. Selective effect of
conjugated linoleic acid isomers on atherosclerotic lesion development
in apolipoprotein E knockout mice. Atherosclerosis 189, 318–327.
Artieda, M., Cenarro, A., Junquera, C., Lasierra, P., Martinez-Lorenzo, M.J.,
Pocovi, M., Civeira, F., 2005. Tendon xanthomas in familial
hypercholesterolemia are associated with a differential inflamma-
tory response of macrophages to oxidized LDL. FEBS Lett. 579,
4503–4512.
Calpe-Berdiel, L., Escola-Gil, J.C., Ribas, V., Navarro-Sastre, A., Garces-
Garces, J., Blanco-Vaca, F., 2005. Changes in intestinal and liver
global gene expression in response to a phytosterol-enriched diet.
Atherosclerosis 181, 75–85.
Calleja, L., Paris, M.A., Paul, A., Vilella, E., Joven, J., Jimenez, A.,
Beltran, G., Uceda, M., Maeda, N., Osada, J., 1999. Low-cholesterol
and high-fat diets reduce atherosclerotic lesion development in ApoE-
knockout mice. Arterioscler. Thromb. Vasc. Biol. 19, 2368–2375.
de la Puerta-Vazquez, R., Martinez-Dominguez, E., Sanchez Perona, J.,
Ruiz-Gutierrez, V., 2004. Effects of different dietary oils on inflam-
matory mediator generation and fatty acid composition in rat neu-
trophils. Metabolism 53, 59–65.
de la Puerta, R., Martinez Dominguez, M.E., Ruiz-Gutierrez, V., Flavill, J.A.,
Hoult, J.R., 2001. Effects of virgin olive oil phenolics on scavenging
of reactive nitrogen species and upon nitrergic neurotransmission.
Life Sci. 69, 1213–1222.
den Boer, M., Voshol, P.J., Kuipers, F., Havekes, L.M., Romijn, J.A., 2004.
Hepatic steatosis: a mediator of the metabolic syndrome. Lessons from
animal models. Arterioscler. Thromb. Vasc. Biol. 24, 644–649.
Dutta, R., Singh, U., Li, T.B., Fornage, M., Teng, B.B., 2003. Hepatic
gene expression profiling reveals perturbed calcium signaling in
a mouse model lacking both LDL receptor and Apobec1 genes.
Atherosclerosis 169, 51–62.
Herrera, M.D., Perez-Guerrero, C., Marhuenda, E., Ruiz-Gutierrez, V.,
2001. Effects of dietary oleic-rich oils (virgin olive and high-oleic-
acid sunflower) on vascular reactivity in Wistar-Kyoto and spontane-
ously hypertensive rats. Br. J. Nutr. 86, 349–357.
Hochepied, T., Berger, F.G., Baumann, H., Libert, C., 2003. Alpha(1)-acid
glycoprotein: an acute phase protein with inflammatory and immu-
nomodulating properties. Cytokine Growth Factor Rev. 14, 25–34.
Horton, J.D., Shah, N.A., Warrington, J.A., Anderson, N.N., Park, S.W.,
Brown, M.S., Goldstein, J.L., 2003. Combined analysis of oligonucle-
otide microarray data from transgenic and knockout mice identifies direct
SREBP target genes. Proc. Natl. Acad. Sci. U S A 100, 12027–12032.
Keys, A., 1995. Mediterranean diet and public health: personal reflections.
Am. J. Clin. Nutr. 61 (Suppl), 1321S–1323S.
Kreeft, A.J., Moen, C.J., Porter, G., Kasanmoentalib, S., Sverdlov, R., van
Gorp, P.J., Havekes, L.M., Frants, R.R., Hofker, M.H., 2005. Genomic
analysis of the response of mouse models to high-fat feeding shows a
major role of nuclear receptors in the simultaneous regulation of lipid
and inflammatory genes. Atherosclerosis 182, 249–257.
Kris-Etherton, P.M., Pearson, T.A., Wan, Y., Hargrove, R.L., Moriarty, K.,
Fishell, V., Etherton, T.D., 1999. High-monounsaturated fatty acid
Chapter  |  125  Microarray Analysis of Hepatic Genes Altered in Response to Olive Oil Fractions
diets lower both plasma cholesterol and triacylglycerol concentra-
tions. Am. J. Clin. Nutr. 70, 1009–1015.
Kritchevsky, D., Tepper, S.A., Klurfeld, D.M., Vesselinovitch, D.,
Wissler, R.W., 1984. Experimental atherosclerosis in rabbits fed
cholesterol-free diets. Part 12. Comparison of peanut and olive oils.
Atherosclerosis 50, 253–259.
Maeda, K., Uysal, K.T., Makowski, L., Gorgun, C.Z., Atsumi, G.,
Parker, R.A., Bruning, J., Hertzel, A.V., Bernlohr, D.A.,
Hotamisligil, G.S., 2003. Role of the fatty acid binding protein mal1
in obesity and insulin resistance. Diabetes 52, 300–307.
Miura, N., Koizumi, S., 2005. Gene expression profiles in the liver
and kidney of metallothionein-null mice. Biochem. Biophys. Res.
Commun. 332, 949–955.
Napoli, C., de Nigris, F., Welch, J.S., Calara, F.B., Stuart, R.O., Glass, C.K.,
Palinski, W., 2002. Maternal hypercholesterolemia during pregnancy
promotes early atherogenesis in LDL receptor-deficient mice and alters
aortic gene expression determined by microarray. Circulation 105,
1360–1367.
Osada, J., Joven, J., Maeda, N., 2000. The value of apolipoprotein E
knockout mice for studying the effects of dietary fat and cholesterol
on atherogenesis. Curr. Opin. Lipidol. 11, 25–29.
Perona, J.S., Cabello-Moruno, R., Ruiz-Gutiérrez, V., 2006. The role of
virgin olive oil components in the modulation of endothelial function.
J. Nutr. Biochem. 17, 429–445.
Perona, J.S., Canizares, J., Montero, E., Sanchez-Dominguez, J.M., Ruiz-
Gutierrez, V., 2003. Plasma lipid modifications in elderly people after
administration of two virgin olive oils of the same variety (Olea euro-
paea var. hojiblanca) with different triacylglycerol composition. Br. J.
Nutr. 89, 819–826.
Rodriguez-Rodriguez, R., Herrera, M.D., Perona, J.S., Ruiz-Gutierrez, V.,
2004. Potential vasorelaxant effects of oleanolic acid and erythrodiol,
1149
two triterpenoids contained in ‘orujo’ olive oil, on rat aorta. Br. J. Nutr.
92, 635–642.
Ruiz-Gutierrez, V., Muriana, F.J., Guerrero, A., Cert, A.M., Villar, J.,
1996. Plasma lipids, erythrocyte membrane lipids and blood pressure
of hypertensive women after ingestion of dietary oleic acid from two
different sources. J. Hypertens. 14, 1483–1490.
Souto, J.C., Blanco-Vaca, F., Soria, J.M., Buil, A., Almasy, L., Ordonez-
Llanos, J., Martin-Campos, J.M., Lathrop, M., Stone, W., Blangero,
J., Fontcuberta, J., 2005. A genomewide exploration suggests a new
candidate gene at chromosome 11q23 as the major determinant of
plasma homocysteine levels: results from the GAIT project. Am. J.
Hum. Genet. 76, 925–933.
Thurnham, D.I., McCabe, G.P., Northrop-Clewes, C.A., Nestel, P., 2003.
Effects of subclinical infection on plasma retinol concentrations and
assessment of prevalence of vitamin A deficiency: meta-analysis.
Lancet 362, 2052–2058.
Vergnes, L., Phan, J., Strauss, M., Tafuri, S., Reue, K., 2003. Cholesterol
and cholate components of an atherogenic diet induce distinct
stages of hepatic inflammatory gene expression. J. Biol. Chem. 278,
42774–42784.
Visioli, F., Galli, C., Grande, S., Colonnelli, K., Patelli, C., Galli, G.,
Caruso, D., 2003. Hydroxytyrosol excretion differs between rats and
humans and depends on the vehicle of administration. J. Nutr. 133,
2612–2615.
Visioli, F., Galli, C., Plasmati, E., Viappiani, S., Hernandez, A., Colombo, C.,
Sala, A., 2000. Olive phenol hydroxytyrosol prevents passive smoking-
induced oxidative stress. Circulation 102, 2169–2171.
Zimmer, J.S., Dyckes, D.F., Bernlohr, D.A., Murphy, R.C., 2004. Fatty acid
binding proteins stabilize leukotriene A4: competition with arachidonic
acid but not other lipoxygenase products. J. Lipid Res. 45, 2138–2144.