Chapter 95
The Effects of Olive Oils on Hepatic Lipid
Metabolism and Antioxidant Defense
Mechanisms: Insights from Proteomics
Studies
Baukje de Roos, Jose Miguel Arbones-Mainar and Guillermo Rodriguez Gutiérrez
University of Aberdeen, Rowett Insitute of Nutrition and Health, Aberdeen, UK
95.1  Introduction
Olive oil is a natural juice of the olive fruit, and it is a major
fat source in the Mediterranean diet. Although this diet is
associated with a lower rate of coronary heart disease (CHD)
(Keys et al., 1986; de Lorgeril et al., 1999; Kontogianni
et al., 2007), as well as a reduction in all-cause mortality
(Trichopoulou et al., 2003; Knoops et al., 2004), data concern-
ing olive oil consumption and primary end points for cardio-
vascular disease are scarce (Covas, 2007). The Mediterranean
diet might protect against coronary heart disease through
improvement of the lipoprotein profile (Perez-Jimenez,
2005), improvement of the postprandial pro-thrombotic pro-
file (Ruano et al., 2007), lowering of blood pressure, and an
improvement in insulin sensitivity both in healthy and in type
2 diabetic patients (Ros, 2003; Schroder, 2007). These poten-
tial beneficial effects of olive oil suggest that its consumption
affects hepatic lipid and glucose metabolism. Effects of olive
oil on health may be due to both its high content of mono-
unsaturated fatty acids or alternatively, due to its polar, minor
components which are rich in antioxidant phenolics. As natu-
ral dietary antioxidants, these phenolic compounds may pro-
tect the organism against damage caused by oxidant agents
like active oxygen species and free radicals.
95.2  Use of proteomics in nutrition
research
The identification of the bioactive components from olive
oil and defining their mode of action is challenging. This is
mainly due to the complex composition of olive oils, consist-
ing of a mix of bioactive components (Brown and Vander
Olives and Olive Oil in Health and Disease Prevention.
ISBN: 978-0-12-374420-3
Ouderaa, 2007). The physiological effects of individual olive
oils could be mediated through multiple biochemical and
molecular mechanisms, stressing the need to extend the avail-
ability of relevant biomarkers to properly assess their effects.
It is, therefore, difficult to deduce their interactions with
physiological systems using traditional experimental meth-
ods. The global nature of a nutrigenomics technology is a real
advantage for elucidating the complex physiological effects
of food components, nutrients, or a specific diet, which are
often mediated through multiple biochemical and molecular
mechanisms (de Roos and McArdle, 2008). Indeed, the value
of proteomics for nutritional sciences has been recognized
and is now being exploited (Kim et al., 2004; Fuchs et al.,
2005; Kussmann and Affolter, 2006; Kussmann et al., 2006;
Milner, 2007) spreading its use in such research (Figure
95.1). The most widely used technical approach in proteom-
ics to identify changes in individual proteins of tissues, cells
and biofluids upon nutritional intervention is two-dimensional
(2D) gel electrophoresis, coupled with protein spot analysis
by mass spectrometry after an evaporation of peptides and
proteins by electrospray ionization or matrix-assisted laser
desorption ionization (Kim et al., 2004; Fuchs et al., 2005).
Two-dimensional gel electrophoresis yields a physical sepa-
ration of intact polypeptides from each other, providing
information about molecular weight and iso-electric point,
parameters that can be used to narrow down the identification
of the protein (Barnes and Kim, 2004). A disadvantage of this
technique is difficulty in visualization and detection of differ-
ential regulation of low-abundance, very hydrophobic, acidic
or basic proteins (Gygi et al., 2000). Therefore, 2D gel elec-
trophoresis may not always represent the most sensitive tool
to reveal effects of nutritional interventions on inflammatory
Copyright © 2010 Elsevier Inc.
887 All rights of reproduction in any form reserved.
888 Section  |  II  Lipid Aspects

DIETARY INTERVENTION

2.0

1.5

1.0

0.5
+
0.0

physiological outcomes 2D gel electrophoresis

from biofluids - blood cells - tissues

Database searching

BIOMARKERS MECHANISMS OF ACTION

Figure 95.1  Integration of physiological outcome variables with proteomes from tissues (like liver), plasma, or blood cells, upon dietary intervention
in humans or animal models, to elucidate novel biomarkers for health or disease that are sensitive to such interventions.

pathways. On the other hand, 2D gel electrophoresis and
mass spectrometry has established itself as a valuable tool
in elucidating changes in pathways that relate to glucose and
fatty acid metabolism as well as pathways relating to oxida-
tive stress, antioxidant defense mechanisms and redox status
(de Roos et al., 2005, 2008; Arbones-Mainar et al., 2007b).
Proteomics has the advantage over cDNA micro-arrays
in that it measures the functional product (protein) of gene
expression, and allows the identification of modifications
that may relate to the activation or inactivation of proteins
by dietary interventions. Such proteins may not only play
a major physiological role in target organs, but could also
reflect changes in mechanisms initiated by dietary interven-
tion when circulating in the blood (Figure 95.1).
95.3  Effects of olive oil
consumption on hepatic lipid and
glucose metabolism
We have applied a systems biology approach to gain under-
standing of the mechanisms by which olive oil fatty acids,
Chapter  |  95  The Effects of Olive Oils on Hepatic Lipid Metabolism and Antioxidant Defense Mechanisms 889

or minor constituents of the oil, may affect hepatic meta-
bolic pathways, oxidative stress and eventually atherogen-
esis in Apoe/ mice (Arbones-Mainar et al., 2007b). This
mouse model spontaneously develops atherosclerosis with
features similar to those occurring in humans (Sarria et al.,
2006). Apoe/ mice were given a basal diet supplemented
with 0.15% (w/w) cholesterol and either 20% (w/w) Picual
extra virgin olive oil (EVOO) (90 mg polyphenols kg1),
or 20% (w/w) Arbequina EVOO (25 mg of polyphenols
kg1), or 20% (w/w) palm oil for 10 weeks. Both olive oils
were from olive tree cultivars grown on the same field at
the same time. The percentage of energy provided by mono­
unsaturated fatty acids was 35% for the Picual olive oil
diet, 33% for the Arbequina olive oil diet, and 18% of the
palm oil diet (Arbones-Mainar et al., 2007b).
Both Picual and Arbequina EVOO diets caused a sig-
nificant decrease in aortic root lesion size and a decreased
degree of macrophage infiltration in to the intima (Arbones-
Mainar et al., 2007a). After 10 weeks of intervention, fast-
ing plasma triglycerides were 22% lower (p  0.05) upon
intervention with Picual EVOO (Arbones-Mainar et al.,
2007b), consistent with other studies (Calleja et al., 1999).
The decrease in triglycerides in our study was observed
despite a significant increase of 30% in plasma non-esteri-
fied fatty acids (NEFA) upon intervention with Picual
EVOO, compared with palm oil (Table 95.1) (Arbones-
Mainar et al., 2007b). Plasma glucose concentrations were
26% higher after intervention with Arbequina EVOO com-
pared with palm oil. Intervention with both EVOOs did not
change plasma insulin concentrations compared with palm
oil intervention, nor did it affect the homeostasis model
assessment (HOMA) index of insulin resistance. However,
intervention with Picual EVOO did decrease the revised
quantitative insulin sensitivity check index of insulin sensi-
tivity (revised QUICKI) compared with the palm oil group
(Table 95.1) (Arbones-Mainar et al., 2007b).
The simultaneous decrease in plasma triglyceride lev-
els, increase in plasma NEFA levels, and decrease in insu-
lin sensitivity upon consumption of Picual EVOO suggests
an increase in the accumulation of fat in the liver. Indeed,
average liver weight (expressed as percentage of total final
body weight) was significantly higher upon consumption of
Picual EVOO compared with palm oil, and hepatic fat was
significantly increased by Picual EVOO, but also Arbequina
EVOO, as compared with palm oil (Arbones-Mainar et al.,
2007b). Diets rich in olive oil generally cause higher con-
centrations of liver total cholesterol compared with diets
containing either saturated or polyunsaturated fatty acids.
This may, however, occur only when large amounts of cho-
lesterol are fed in the diets (Beynen, 1988, 1989). Also the
amount of dietary fat administered will be crucial since an
amount of 10% (w/w) dietary olive oil prevented the devel-
opment of fatty livers in another study (Acin et al., 2007).
Proteomics analysis of hepatic tissue revealed significant
five- and two-fold increases in hepatic adipophilin protein
levels upon consumption of Picual and Arbequina EVOO
(Arbones-Mainar et al., 2007b), which may help us under-
stand as to why in this study extra virgin olive oils increased
hepatic fat accumulation. Adipophilin (or adipose differen-
tiation-related protein) is bound to the surface of lipid bod-
ies and lipid bi-layers (Murphy, 2001). The proposed function
of adipophilin is more complex than merely the packaging
of neutral lipids in the cytosol (Figure 95.2). Adipophilin
over-expression in primary liver cells increases the size of

Table 95.1  Levels of fasting plasma triglycerides, non-esterified fatty acids (NEFA), glucose and
insulin concentrations, as well as the insulin resistance marker HOMA and the insulin sensitivity
marker revised QUICKI, in female Apoe/ mice fed a high-fat high-cholesterol diet supplemented
with 20% (w/w) extra virgin Picual olive oil, 20% (w/w) extra virgin Arbequina olive oil, and 20%
(w/w) palm oil (atherogenic control group) for 10 weeks (Arbones-Mainar et al., 2007b).

Picual oil (n  8) Arbequina oil (n  8) Palm oil (n  8)

Total triglycerides (mmol L1) 1.71  0.64* 2.49  0.79 2.21  0.31

NEFA (mg dl1) 5.00  0.96* 4.41  1.29 3.89  0.60

Glucose (mmol L1) 17.41  3.58 18.25  4.34* 14.52  1.85

Insulin (pmol L1) 101.57  32.02 98.29  22.04 96.61  22.93

HOMA 11.26  3.72 11.91  3.84 8.91  2.26

Revised QUICKI 0.22  0.01** 0.22  0.01 0.23  0.01

Values represent the mean  SD. HOMA: homeostasis model for insulin resistance; revised QUICKI: quantitative insulin
sensitivity check index. Significantly different from the palm oil group: *p  0.05; **p  0.01.
890
Adipophilin overexpression
VLDL apoB
NEFA TG
X triglycerides
β-oxidation of fatty acids
Adipophilin knockout
VLDL apoB
NEFA TG
triglycerides
X
β-oxidation of fatty acids
Figure 95.2  Role of adipophilin in hepatic fat metabolism.
cytosolic lipid droplets and decreases the secretion of very-
low-density lipoprotein (VLDL), without influencing the rate
of -oxidation, thereby selectively lessening VLDL assem-
bly (Magnusson, 2006). On the other hand, a knockdown of
adipophilin decreased the pool of cytosolic lipid droplets,
increased the secretion rate of apolipoprotein B-48 VLDL1
and increased -oxidation (Magnusson et al., 2006). In addi-
tion, adipophilin knockout in mice had lower levels of hepatic
triglycerides without a change in plasma lipid profile, and
these animals were protected against the development of a
fatty liver (Chang et al., 2006). Therefore, down-regulation
of adipophilin protein causes a channeling of fatty acids pri-
marily into -oxidation (Figure 95.2). Interestingly plasma
triglycerides were lowest and accumulation of hepatic lipid
droplets was highest upon consumption of Picual EVOO,
which also yielded the highest levels of hepatic adipophi-
lin protein, without a noticeable effect on hepatic proteins
involved in -oxidation of fatty acids. Indeed, hepatic adi-
pophilin protein levels and plasma triglycerides were inversely
correlated (Arbones-Mainar et al., 2007b). Therefore, regula-
tion of adipophilin protein by dietary fatty acids or other com-
pounds may represent an important regulatory pathway in
hepatic lipid metabolism, albeit that regulation of adipophilin
upon dietary intervention may be species-dependent.
Proteomic analysis of hepatic tissue also revealed sig-
nificant increases in several hepatic enzymes involved in the
methionine cycle, especially in betaine homocysteine methyl
transferase (BHMT), by Picual and Arbequina EVOOs,
compared with palm oil (Arbones-Mainar et al., 2007b).
BHMT, which converts homocysteine into methionine
using betaine as a co-factor (Figure 95.3), is up-regulated
during insulin resistance (Wijekoon et al., 2005; Ratnam
et al., 2006). Also, an increase in hepatic BHMT is associ-
ated with an increase in hepatic VLDL and apolipoprotein B
Section  |  II  Lipid Aspects
production rate in rats fed a methionine-deficient diet sup-
plemented with betaine (Sparks et al., 2006). Increased
secretion rates of VLDL1-apolipoprotein B and triglycer-
ides, as assessed by kinetic studies in humans, are a common
feature in insulin resistance. The hepatic VLDL overpro-
duction is believed to be driven by the altered free flow of
NEFAs (Parhofer and Barrett, 2006), and BHMT may well
be involved in this process. However, despite an increase in
plasma NEFA upon consumption of Picual EVOO, plasma
triglycerides were actually lower (Table 95.1), suggesting
that VLDL-­apolipoprotein B secretion was also likely to be
lower (Arbones-Mainar et al., 2007b). This suggests that an
overriding mechanism, perhaps involving adipophilin, is the
prevention of hepatic triglycerides being secreted.
95.4  Effects of olive oil
consumption on antioxidant
defense mechanisms
Extra virgin olive oils contain a wide range of important
polar minor components which are rich in polyphenols. The
total phenolic content can vary widely, ranging from around
232 mg kg1 (Owen et al., 2000) to as high as 614 mg kg1
(Salvador et al., 2000). The major phenolic compounds in
extra virgin olive oil are oleuropein, hydroxytyrosol and
tyrosol, but EVOO also contains a wide range of minor
polyphenols like vanillic acid, p-coumaric acid, vanillin,
demethyloleuropein, homoorientin, verbascoside, rutin,
luteolin 7-O-glucoside, apigenin 7-O-rutinoside, apigenin
7-O-glucoside, luteolin, oleuropein aglycone, cyanidin 3-
O-glucoside, and cyanidin 3-O-rutinoside (Romani et al.,
1999). These polyphenols contribute to the stability of the
oil, and may have anti-inflammatory and anti-atherosclerotic
properties (Carluccio et al., 2003; Gonzalez-Santiago et al.,
2006; Kaliora et al., 2006), possibly mediated by their anti-
oxidant properties. Nevertheless it is important to note that
the bioactivity of phenolic compounds of olive oil is not only
due to their antioxidant properties, but also to their interac-
tion with important enzymatic systems (Tripoli et al., 2005).
Indeed, proteomics revealed that hepatic protein levels
of a wide range of antioxidant enzymes – like thioredoxin
reductase, thioredoxin peroxidase 2, peroxiredoxin 3, super-
oxide dismutase and GST – were 1.5- to 4-fold higher upon
consumption of Picual and Arbequina EVOO compared
with palm oil (Figure 95.3) (Arbones-Mainar et al., 2007b).
In addition, hepatic thioredoxin reductase activity was sig-
nificantly higher upon consumption of Picual EVOO, con-
firming the proteomics findings (Arbones-Mainar et al.,
2007b). A similar coordinated induction of the antioxi-
dant enzymes has been observed in Apoe/ mice in the
period preceding lesion formation (‘t Hoen et al., 2003).
Remarkably, most mRNA levels of these antioxidant
enzymes started to decline as lesions started to develop.
This suggests that the arterial wall delays initial lesion
Chapter  |  95  The Effects of Olive Oils on Hepatic Lipid Metabolism and Antioxidant Defense Mechanisms 891

Apolipoprotein B

methionine
adenosyl transferase

S-Adenosyl
Methionine Methionine

X (phosphatidyl ethanolamine)
Dimethylglycine
S-adenosyl-L-methionine-
betaine homocysteine dependent methyltransferase
methyltransferase
Betaine Choline X-CH3(phosphatidyl choline)

S-Adenosyl
Homocysteine
Homocysteine
GST

S-adenosyl homocysteine
hydrolase

Cysteine Glutamyl Cysteine GSH GSSG

glutathione glutathione
synthase peroxidase 1

Cysteine-sulfinic hydroxyacyl
acid decarboxylase glutathione
hydroxylase

S-D-Lactoyl-
Taurine Glutamate Glutathione

glutamate
dehydrogenase

Oxoglutarate
Figure 95.3  Schematic overview of pathways involved in methionine and glutathione metabolism, of which some were differentially regulated by
Picual and Arbequina extra virgin olive oil intervention, as compared with palm oil intervention.

formation by stimulating the expression of antioxidant
enzymes, but when this defense capacity collapses a greatly
accelerated development of atherosclerosis occurs (‘t Hoen
et al., 2003). Therefore, polyphenol compounds in EVOO
may well be able to delay the onset of atherosclerotic
lesions by the combat of oxidative stress.
Proteomics also revealed the up-regulation of gluta-
mate dehydrogenase, glutathione synthase and hydroxyacyl
glutathione hydrolase, as well as the down-regulation of
cysteine-sulfinic acid decarboxylase, indicating an increased
production of glutathione by predominantly Arbequina
EVOO (Figure 95.3) (Arbones-Mainar et al., 2007b). In
Apoe/ mice, depletion of glutathione via a decreased
synthesis precedes lipid peroxidation and atherogenesis,
and glutathione is severely depleted in the atheroma-prone
aortic arch of male compared with wild-type mice after
10 weeks (Biswas et al., 2005). Therefore, glutathione defi-
ciency might be central to the failure of the intracellular
antioxidant defenses and is causally implicated in the
pathogenesis of atherosclerosis. The apparent increase
in glutathione production and other antioxidant defense
mechanisms upon consumption of EVOO may thus repre-
sent a mechanism by which olive oil components diminish
oxidative stress.
In contrast, protein and activity levels of hepatic GPx1
were lower upon consumption of Picual and Arbequina
EVOO compared with palm oil (Figure 95.3) (Arbones-
Mainar et al., 2007b). In patients with coronary artery dis-
ease, a low level of red-cell GPx1 activity is independently
associated with an increased risk of cardiovascular events
(Blankenberg et al., 2003). However, it is questionable
whether GPx1 plays a key role in lesion formation at the
site of the aortic root. A specific deficiency in GPx1 was not
accompanied by an increase in markers of oxidative damage
or increased atherosclerosis in mice (de Haan et al., 2006).
Also, GPx1 expression in the aortic arch of Apoe/ mice,
892
as compared with wild-type mice, decreases rapidly in the
period preceding lesion development, resulting in hardly
any detectable GPx1 expression in atherosclerotic tissue
(‘t Hoen et al., 2003). Moreover, GPx1 enzyme activity was
absent in human atherosclerotic lesions (Lapenna et al.,
1998). However, based on previous studies we cannot rule
out a role for GPx1 in atherogenesis at other sites (de Haan
et al., 2006). GPx1 may indeed play different roles in different
organs (Arthur, 2000) and the lower protein and activity
levels of hepatic GPx1 upon olive oil consumption may
simply reflect a lower degree of hepatic oxidative stress
due to the up-regulation of other antioxidant enzymes.
95.5  Mechanisms of protection:
does hormesis play a role?
Hormesis is described as the capacity of an organism to dis-
play a stimulatory response to a compound at low doses,
but to show an inhibitory response to the same compound at
higher doses (Figure 95.4) (Lindsay, 2005). Phytochemicals,
like olive oil polyphenols, act through mechanisms that
operate in response to other xenobiotic substances normally
classified as toxic. Many of the naturally occurring food
phytochemicals are metabolized and excreted using the
same system of phase I and phase II enzymes that metab-
olize toxic compounds (Lindsay, 2005). The majority of
toxic compounds exhibit hormesis (Calabrese and Baldwin,
2003). There is good evidence that exposure to dietary phy-
tochemicals produces a mild stress response, which might
result in the adaptation to that stress response if the exposure
optimum intake for beneficial effects
benefit
amount of
dietary polyphenols
Figure 95.4  Potential U-shaped hormetic response in polyphenol
intake. The optimal intake of olive oil polyphenols that results in a ‘ben-
eficial’ response may only be those obtained through consumption of
a Mediterranean diet. Any substantial increase in olive oil polyphenol
intake, for example though intake of supplements, may eliminate any
beneficial effects.
Section  |  II  Lipid Aspects
is prolonged. Such situations may cause a permanent altera-
tion in cellular homeostasis, or redox state, after which the
cells are better able to respond to subsequent stress chal-
lenges. Up-regulation of antioxidant responses, as seen
in our study with Picual and Arbequina EVOO (Arbones-
Mainar et al., 2007b), is indeed a mechanism that cells
utilize to adapt to their temporal changes in their environ-
ment as part of the hormesis response which make the cells
more able to resist similar challenges in future (Lindsay,
2005). However, as with any U-shaped hormetic responses,
the optimal intake of olive oil polyphenols that results in a
‘beneficial’ response may only be those obtained through
consumption of a Mediterranean diet. Any substantial
increase in polyphenol intake, for example though intake of
supplements, could eliminate any beneficial effects associ-
ated with consumption of the compounds in olive oil.
Summary points
l Extra virgin olive oil has anti-atherosclerotic properties,
at least in Apoe/ mice, but may also increase plasma
total cholesterol levels and the development of hepatic
steatosis when large amounts of dietary fat and choles-
terol are fed in the diets. These are conflicting findings
and currently it is unknown if olive oil consumption
would affect the interaction between similar processes
in humans.
l The systems biology approach in our study was crucial
to unraveling the complex interactions between path-
ways that are on one hand involved in the beneficial
reduction in plaque formation, and on the other hand
involved in the potentially, less beneficial, development
of hepatic steatosis.
l A significant up-regulation of a large array of antioxi-
dant enzymes upon consumption of EVOO may dimin-
ish oxidative stress instigated by hepatic steatosis and,
in addition, may slow down the development of athero-
sclerosis. Indeed, the accumulation of triglycerides
may not pose a major challenge to the liver, and may
represent a relatively safe way to store triglycerides, as
long as the antioxidant capacity is adequate to prevent
lipotoxicity (Slawik and Vidal-Puig, 2006).
l Our proteomics results revealed, for the first time,
that two different EVOOs instigated distinct effects
on hepatic lipid metabolism by regulation of hepatic
adipophilin. This mechanism may override any regula-
tion by insulin or BHMT to increase the production of
VLDL apolipoprotein B that is normally observed upon
development of hepatic steatosis and early symptoms of
insulin resistance.
l The optimal intake of polyphenols from extra virgin
olive oil that results in a ‘beneficial’ anti-oxidant
response may only be those obtained through consump-
tion of a Mediterranean diet.
Chapter  |  95  The Effects of Olive Oils on Hepatic Lipid Metabolism and Antioxidant Defense Mechanisms
Acknowledgments
The authors’ work is funded by the Scottish Government Rural and
Environment Research and Analysis Directorate (RERAD).
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