Extra Virgin Olive Oils Increase Hepatic Fat Accumulation and Hepatic Antioxidant Protein Levels in...

Extra Virgin Olive Oils Increase Hepatic Fat Accumulation and

Hepatic Antioxidant Protein Levels in APOE-/- Mice

Jose Miguel Arbones-Mainar,† Karen Ross,‡ Garry J. Rucklidge,‡ Martin Reid,‡ Gary Duncan,‡

John R. Arthur,‡ Graham W. Horgan,§ Maria A. Navarro,† Ricardo Carnicer,† Carmen Arnal,†

Jesus Osada,† and Baukje de Roos*,‡

Department of Biochemistry, Veterinary School, University of Zaragoza, Spain, Division of Vascular Health,Rowett Research Institute, Aberdeen, United Kingdom, and Biomathematics and Statistics Scotland at the

Rowett Research Institute, Aberdeen, United Kingdom

Received May 28, 2007

We assessed the effects of Picual and Arbequina olive oil, rich and poor in polyphenols, respectively,on plasma lipid and glucose metabolism, hepatic fat content, and the hepatic proteome in femaleApoe-/- mice. Both olive oils increased hepatic fat content and adipophilin levels (p < 0.05), thoughPicual olive oil significantly decreased plasma triglycerides (p < 0.05). Proteomics identified a range ofhepatic antioxidant enzymes that were differentially regulated by both olive oils as compared withpalm oil. We found a clear association between olive oil consumption and differential regulation ofadipophilin and betaine homocysteine methyl transferase as modulators of hepatic triglyceridemetabolism. Therefore, our “systems biology” approach revealed hitherto unrecognized insights intothe triglyceride-lowering and anti-atherogenic mechanisms of extra virgin olive oils, wherein the up-regulation of a large array of anti-oxidant enzymes may offer sufficient protection against lesiondevelopment and diminish oxidative stress levels instigated by hepatic steatosis.

Keywords: proteomics • antioxidant enzymes • insulin resistance • adipophilin • betaine homocysteine methyl-transferase

Introduction

The “Mediterranean Diet” is associated with a lower rate ofcoronary heart disease (CHD),1,2 as well as a reduction in all-cause mortality.3,4 Olive oil is the main source of fat in this typeof diet. Low percentages (5-10%) of extra virgin olive oil(EVOO)-enriched diets on a low cholesterol background haltedthe progression of induced atherosclerosis in rabbits5 and infemale apolipoprotein E knockout (Apoe-/-) mice,6 although ahigher percentage of dietary EVOO (20%) on a high cholesterolbackground diet failed to show a difference in atherosclerosislesion development when compared with a carbohydrate-richdiet.7

The Mediterranean diet might protect against coronary heartdisease by improving the lipoprotein profile.8 The Mediter-ranean diet may also provide additional benefits by acting onother cardiovascular risk factors, including a lowering of bloodpressure, and an improvement in insulin sensitivity both inhealthy and in type 2 diabetic patients.9 The beneficial effectsof olive oil on both lipoprotein metabolism and potentiallyinsulin resistance suggest that consumption of olive oil affects

hepatic lipid and glucose metabolism. To gain understandingin the mechanisms by which olive oil fatty acids, or its minorantioxidant constituents, may affect hepatic metabolic path-ways, oxidative stress and eventually atherogenesis, we haveapplied a systems biology approach. The physiological effectsof the individual olive oils could be mediated through multiplebiochemical and molecular mechanisms, stressing the need toextend the availability of relevant biomarkers to properly assesstheir effects. The current study was carried out in Apoe-/- mice,a well-characterized and widely used model that spontaneouslydevelops atherosclerosis with features similar to those occurringin humans.10

Materials and Methods

The study protocols were approved by the Ethics Committeefor Animal Research of the University of Zaragoza, Spain, andwere conducted in conformity with the Public Health ServicePolicy on Humane Care and Use of Laboratory Animals. Allanimals received humane care according to the criteria outlinedin the “Guide for the Care and Use of Laboratory Animals”published by the NIH in 1985.

Animals and Diets. The design of this study has beendescribed before.11 Briefly, Apoe-/- animals were bred and keptat the Unidad Mixta de Investigacion, Zaragoza, Spain. Animalswere housed in sterile filter-top cages under 12 h light/dark

* To whom correspondence should be addressed. Dr. Baukje de Roos,Rowett Research Institute, Division of Vascular Health, Greenburn Road,Bucksburn, Aberdeen AB21 9SB, United Kingdom. Tel. +44 (0)1224 716636;Fax: +44 (0)1224 716629; E-mail, [email protected].

† University of Zaragoza.‡ Division of Vascular Health, Rowett Research Institute.§ Biomathematics and Statistics Scotland at the Rowett Research Institute.

10.1021/pr070321a CCC: $37.00 2007 American Chemical Society Journal of Proteome Research 2007, 6, 4041-4054 4041Published on Web 09/11/2007

cycles with ad libitum access to food and water. Twenty-fourfemale Apoe-/- mice were randomly distributed into threeexperimental groups matched for baseline plasma cholesterolvalues. Mice were fed a base diet of standard mouse chow (B& K Universal Ltd., Humberside, UK) supplemented with 0.15%(w/w) cholesterol and either 20% (w/w) Picual EVOO (90 mgpolyphenols/kg), 20% (w/w) Arbequina EVOO (25 mg ofpolyphenols/kg), or 20% (w/w) palm oil. The EVOOs were bothfrom Spanish olive tree cultivars grown on the same field atthe same time. The percentage of energy provided by monoun-saturated fatty acids was 35% for the Picual olive oil diet, 33%for the Arbequina olive oil diet, and 18% of the palm oil diet.The diets were prepared weekly and stored under N2 at -20°C until use. The composition of the diet has been describedpreviously.11 At the end of the 10-week intervention period, theanimals were killed by suffocation with CO2 after an overnightfast and blood was obtained thereafter by cardiac puncture.The livers were removed, weighed, frozen in liquid nitrogen,and stored at -80 °C until analysis.

Plasma Analysis. Blood was centrifuged at 3000 rpm for 10min and plasma was collected for the measurement of triglyc-erides,11 non-esterified fatty acids (NEFA) (Waku, Madrid,Spain), insulin, and glucose (rat/mouse insulin ELISA kit, LincoResearch, Missouri ; Glucose RTU, BioMerieux, Lyon, France)validated with standard controls (Calimat, BioMerieux, Lyon,France), according to the manufacturers’ instructions. Fastingplasma insulin and glucose concentrations were used tocalculate the biomarker insulin resistance from the homeostasismodel assessment for insulin resistance (HOMA) [(glucose0 *insulin0)/22.5].12 The biomarker insulin sensitivity was calcu-lated with use of the revised quantitative insulin sensitivitycheck index (QUICKI) [1/(log insulin0 + log glucose0 + logNEFA0)].13

Measurement of Hepatic Fat Content and AdipophilinProtein Levels. Paraffin-embedded liver sections (4 µm) werestained with hematoxylin and eosin and observed using a Nikonmicroscope without prior knowledge of the diet group. Hepaticfat content was evaluated by quantifying the extent of fatdroplets in each liver section with Adobe Photoshop 7.0 andexpressed as percentage of total liver section.

Hepatic adipophilin in cytosolic protein homogenates wasmeasured by an in-house ELISA using a specific polyclonalantibody (Research Diagnostics) as previously described.14

Measurement of Hepatic Glutathione Peroxidase 1 (GPx1),Glutathione S-transferase (GST) and Thioredoxin Reductase(TR) Activity. Hepatic GPx1 and GST activities were measuredby the methods described by Arthur et al.,15 using 1,2 dichloro-4-nitrobenzene as a substrate for the GST activity assay. HepaticTR activity was measured as described by Rigobello et al.16 andadapted for measurement in a 96-well plate.

Proteomics. Cytosolic protein homogenates were preparedfrom each individual animal liver as described previously.17,18

Proteins were separated by two-dimensional gel electrophore-sis, and the gels were analyzed using PDQuest software(BioRad). Spots with densities that significantly differed be-tween treatments were excised from the SDS-PAGE gels usingthe robotic BioRad spot cutter. These proteins were trypsinizedusing a protocol of the MassPrep Station (Micromass) andanalyzed by MALDI-TOF and electrospray LC mass spectro-metric methods as described.17,18

Statistical Analysis. Data are presented as means ( SD.Analysis of variance was carried out on plasma parameters andprotein spot data, followed by post-hoc unpaired t-tests based

on the pooled variance. Data were log-transformed beforeanalysis when not normally distributed. Principal componentanalysis was performed after centring and unit variance (UV)scaling of the data in SIGMA P+ (Umetrics Ltd, UK). Analysisof correlations was done with Pearson correlation coefficients.The analysis of multiple hypotheses testing for many combina-tions of variables was done with by determining the falsediscovery rate or q-values19 within Genstat (VSN intl Ltd., UK).q-values estimate the probability that a correlation that is calledsignificant, is false positive. For example, a q-value of 0.05would mean, that we should expect that 5 out of 100 associa-tions that were tested significant, are in fact false positive.

Results

Food Intake and Body Weights. Food intake and bodyweight gain did not differ between the three groups during the10-week intervention period (data not shown). Fasting plasmatriglycerides were 22% lower (p < 0.05) upon intervention withPicual EVOO,11 and NEFA was 30% higher (p < 0.05) uponintervention with Picual EVOO, compared with palm oil (Table1). Plasma glucose concentrations were 26% higher afterintervention with Arbequina EVOO compared with palm oil (p< 0.05). Intervention with both EVOOs did not change plasmainsulin concentrations compared with palm oil intervention,nor did it affect the homeostasis model assessment (HOMA)index of insulin resistance. However, intervention with PicualEVOO did decrease the revised quantitative insulin sensitivitycheck index of insulin sensitivity (revised QUICKI) (p < 0.01)compared with the palm oil group (Table 1).

Liver Weight, Hepatic Fat, and Hepatic Adipophilin. Aver-age liver weight (expressed as percentage of total final bodyweight) was significantly higher in the Picual EVOO group(5.3 ( 0.4%, p < 0.001), but not in the Arbequina EVOO group(4.8 ( 0.6), compared with the palm oil group (4.4 ( 0.4%).Hepatic fat was significantly increased by Picual EVOO (p <0.01) and Arbequina EVOO (p < 0.05), as compared with palmoil (Figure 1).

Table 1. Fasting Plasma Triglycerides, Total Cholesterol, HDLCholesterol, Nonesterified Fatty Acids (NEFA), Glucose andInsulin Concentrations, HOMA and Revised QUICKI, in FemaleApolipoprotein E Knockout Mice Fed a High Fat HighCholesterol Diet Supplemented with 20% (w/w) Extra VirginPicual Olive Oil, 20% (w/w) Extra Virgin Arbequina Olive Oil,and 20% (w/w) Palm Oil (Atherogenic Control Group) for 10Weeksa

Picual oil

(n ) 8)

Arbequina oil

(n ) 8)

palm oil

(n ) 8)

total triglycerides(mmol/L)

1.71 ( 0.64b 2.49 ( 0.79 2.21 ( 0.31

total cholesterol(mmol/L)

40.16 ( 1.89c 37.82 ( 3.75c 31.13 ( 4.04

HDL cholesterol(mmol/L)

1.42 ( 0.06b 1.22 ( 0.12 1.26 ( 0.16

NEFA (mg/dL) 5.00 ( 0.96b 4.41 ( 1.29 3.89 ( 0.60glucose (mmol/L) 17.41 ( 3.58 18.25 ( 4.34* 14.52 ( 1.85insulin (pmol/L) 101.57 ( 32.02 98.29 ( 22.04 96.61 ( 22.93HOMA 11.26 ( 3.72 11.91 ( 3.84 8.91 ( 2.26revised QUICKI 0.22 ( 0.01c 0.22 ( 0.01 0.23 ( 0.01

a Values represent the mean ( SD. HOMA: homeostasis model for insulinresistance; (revised) QUICKI: quantitative insulin sensitivity check index.b Significantly different from the palm oil group: p < 0.05. c Significantlydifferent from the palm oil group: p < 0.01.

research articles Arbones-Mainar et al.

4042 Journal of Proteome Research • Vol. 6, No. 10, 2007

Hepatic adipophilin protein levels were significantly higherupon intervention with Picual EVOO (p < 0.001) and withArbequina EVOO (p < 0.05), as compared with palm oil (Fig-ure 1).

Hepatic GPx1, GST, and TR Activity. Hepatic GPx1 activitywas significantly lower upon intervention with Picual EVOO(0.33 ( 0.04 U/mg protein, mean ( SD) and Arbequina EVOO(0.33 ( 0.03 U/mg protein) compared with palm oil interven-tion (0.40 ( 0.02) (both p < 0.01). Hepatic GST activity wasnot significantly affected by Picual EVOO (8.70 ( 3.54 µM/gprotein) or Arbequina EVOO (6.37 ( 0.82 µM/g protein) ascompared with palm oil intervention (6.04 µM/g protein). TRactivity was significantly increased by Picual EVOO (1.11 ( 0.23U/mg protein) (p < 0.05), but not by Arbequina EVOO (0.94 (0.24 U/mg protein) as compared with palm oil intervention(0.84 ( 0.22 U/mg protein).

Proteomics. Two-dimensional-gel electrophoresis of indi-vidual liver cytosolic protein fractions revealed 80 cytosolicproteins of which levels were significantly up- or down-regulated by Picual EVOO and Arbequina EVOO, comparedwith palm oil. The proteins identified were categorized accord-ing to their major biochemical functions to facilitate theelucidation of pattern changes between treatments (Table 2).Significant increases were observed in levels of a range of anti-oxidant enzymes, enzymes involved in carbohydrate metabo-lism, and enzymes involved in the methionine cycle or glu-tathione synthesis, mainly upon intervention with ArbequinaEVOO.

Principal Component Analysis. Principal component analy-sis (PCA) is a mathematical procedure that transforms anumber of correlated variables into a smaller number ofuncorrelated variables, which may enable the detection of astructure in relationships between them. PCA of the proteomicsresults as well as physiological outcome parameters allowedus to reduce all initial variables into two principal components.These new uncorrelated factors that were successively extracted

revealed that 32% of all variance in the dataset was accountedfor by the first principal component (PC1), and an additional12% was accounted for by the second principal component(PC2) (Figure 2, upper panel). The largest treatment effect onthe first principal component (i.e., the largest distance betweenthe spots representing the dietary intervention groups on theX-axis) was between the palm oil control group (red spots) andthe Arbequina EVOO intervention group (blue spots). Whenconsidering the second principal component (which representsone or more other, independent variables), the Picual EVOOintervention group initiated a specific treatment effect com-pared with the palm oil control group. The loadings plotrevealed that parameters with the highest loadings (i.e., thelargest distance from the 0,0 point) which explained most ofthe treatment effect of the Picual olive oil (green letters) andArbequina olive oil (blue letters) (Figure 2, lower panel). ForPicual olive oil, the proteins that provided the largest contribu-tion to the dietary treatment effects in the principle compo-nent analysis were mostly related to hepatic steatosis. ForArbequina olive oil, the proteins that provided the largestcontribution to the dietary treatment effects in the principalcomponent analysis were mostly related to oxidative stress,carbohydrate metabolism, and the methionine cycle/glu-tathione synthesis.

Pairwise Correlation Analysis. In addition to PCA, weperformed a pairwise correlation analysis over the differenttreatments, including the physiological data measured inplasma lipid as well as the data on hepatic protein levels. Suchan analysis shows which parameters vary in similar waysthroughout the two EVOO treatments, but also highlights whichof the parameters are differentially affected by the two EVOOcultivars. Figure 3 shows a network of all pairwise interactionsfor each of the EVOO diets with a Pearson correlation coef-ficient > 0.66, p value < 0.0005 and a q-value of 0.0025, usingthe software tool Cytoscape.20

Figure 1. Representative images from a liver from (A) the Picual olive oil group, (B) the Arbequina olive oil group, (C) the palm oilgroup, stained with hematoxylin and eosin (magnification ×100) as well as the morphometric quantification of the liver fat content ineach group (results are expressed as percentage of liver covered by fat in each section) (D) and the amount of hepatic adipophilinprotein (E).

Extra Virgin Olive Oil Results in APOE-/- Mice research articles

Journal of Proteome Research • Vol. 6, No. 10, 2007 4043

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7IT

VG

VD

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EA

GQ

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+2

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3



Tab

le2

(Co

nti

nu

ed)

MS/

MS

pep

tid

em

ass

fin

gerp

rin

tin

gfo

ld-c

han

geb

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pro

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iden

tifi

cati

on

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n

Mr

Exp

Mr

Th

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tid

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Pic

ual

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Arb

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ina

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R+

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K+

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Gly

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ase

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53.9

560

.56

6710

251.

922

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eP

1415

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36.3

820

53

LGV

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ydro

gen

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GE

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Mal

ate

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152

37.0

336

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135

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om

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om

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ase

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869

827

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bo

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rate

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Alig

ase

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184

44.5

734

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796

170.

723

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oci

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ed

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roge

nas

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366

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hio

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1507

Glu

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tid

ase

O35

409

52.3

584

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122

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9

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Bet

ain

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om

ocy

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ne

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ase

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45.4

445

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02.

0

4407

Ad

eno

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110

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Tab

le2

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nti

nu

ed)

MS/

MS

pep

tid

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fin

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rin

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Pic

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229

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31.2

672

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Glu

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ase

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61.6

461

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132

1125

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49.8

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111

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8

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Ph

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1633

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829

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Ho

mo

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Th

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4093

628

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29.4

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Tab

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MS

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Exp

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3491

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Tab

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(Co

nti

nu

ed)

MS/

MS

pep

tid

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ass

fin

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rin

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Exp

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Pic

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mis

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us

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tein

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ter

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ican

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05).

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ep

rote

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ore

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10*L

og(

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wh

ere

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the

pro

bab

ility

that

the

ob

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edm

atch

isa

ran

do

mev

ent.

bF

old

-ch

ange

inh

epat

icp

rote

inle

vels

(as

asse

ssed

by

pro

teo

mic

s)o

fp

rote

ins

that

wer

esi

gnif

ican

tly

up

-or

do

wn

regu

late

db

yP

icu

alan

d/o

rA

rbeq

uin

ao

live

oil,

asco

mp

ared

wit

hp

alm

oil.



Discussion

In this study, both olive oils increased liver size and hepaticfat content, probably through an increased supply of NEFA tothe liver and a decreased output of triglycerides from the liver,and no apparent changes in levels of hepatic â-oxidationenzymes as assessed by proteomics. Proteomics identified arange of antioxidant enzymes that were differentially regulatedby both olive oils as compared with palm oil.

Mediterranean populations who generally consume a diethigh in olive oils that is rich in monounsaturated fatty acids(MUFA) have a low prevalence of CHD and low plasmacholesterol levels.21 The substitution of a high-MUFA diet foran average American diet lowers total and low-density lipo-protein (LDL) cholesterol in humans,22 thereby contributing toa reduction in CHD risk. In contrast, both EVOO diets signifi-cantly increased plasma cholesterol levels in Apoe-/- mice,despite causing a significant decrease in aortic root lesion sizeand a decreased degree of macrophage infiltration in to the

intima.11 An olive oil diet low in cholesterol, compared with acoconut oil diet, had no significant effects on total cholesterolwhereas lesion size was significantly decreased in femaleApoe-/- animals only.6 This corroborates our previous observa-tion that dietary cholesterol suppresses the ability of EVOO toimprove the lipoprotein profile in an animal model that isextremely sensitive to diet-induced hyperlipidemia because ofthe absence of the apolipoprotein E protein.10 However, HDLcholesterol was increased upon consumption of Picual EVOO.11

Increased HDL concentrations are considered anti-atherogenicbecause of their ability to promote the efflux of cholesterol fromcells, but they may also have antioxidant, anti-inflammatory,and anti-thrombotic properties.23 In this study, both EVOOsdid not significantly affect the HDL related proteins paraoxo-nase activity and apoA-I levels, but both EVOOs did induce acholesterol-poor, apoA-IV enriched lipoparticle that had en-hanced arylesterase and antioxidant activities.11 Furthermore,Picual EVOO decreased plasma triglycerides, a finding that has

Figure 2. Scores plot (upper panel) and loadings plot (lower panel) of an unsupervised principal component analyses of hepatic cytosolicproteins of which levels were significantly up-or down regulated upon intervention with Picual olive oil or Arbequina olive oil, ascompared with palm oil. The scores plot reveals the treatment effects between each of EVOO interventions compared with the palmoil intervention (i.e., the largest distance between the spots representing the dietary intervention groups on the X-axis) on the first andsecond principal component. The loadings plot reveals the proteins and physiological outcome parameters that provided the largestpositive and negative contribution to the dietary treatment effects in the principal component analysis. These were for palm oil (inRED): A ) lesion size, B ) GPx1 activity, C ) cysteinic sulfinic acid decarboxylase, D ) succinyl CoA ligase, E ) adenosylhomocysteinase,F ) revised QUICKI, G ) glycyl-tRNA synthetase, H ) HSP110, I ) lactamase beta; for Picual olive oil (in GREEN): A ) adipophilin(Western Blot), B ) adipophilin (ELISA), C ) liver weight, D ) GST activity, E ) 2-hydroxyphytanoyl CoA lyase; for arbequina olive oil(in BLUE): A ) malate dehydrogenase, b ) acyl-protein thioesterase, c ) phosphatidylcholine transfer protein, D ) superoxide dismutase,E ) S-adenosyl-L-methionine dependent methyltransferase, F ) S-adenosyl-L-homocysteine hydrolase, G ) thioether-S-methyltrans-ferase, H ) aldehyde dehydrogenase, I ) glutathione synthase, J ) thioperoxin peroxidase, K ) methionine adenosyltransferase, L )fructose 1,6-bisphosphatase, M ) glutamate carboxypeptidase.



been observed before.6 The decrease in triglycerides in ourstudy was observed despite a significant increase in plasmaNEFA (Table 1) and a significant increase in hepatic fat content(Figure 1).

This is, to our knowledge, the first study that reports anincrease in hepatic fat accumulation upon consumption ofEVOO in this mouse model of atherosclerosis. It has been foundpreviously that diets rich in olive oil generally cause higher

concentrations of liver total cholesterol compared with dietscontaining either saturated or polyunsaturated fatty acids,which may, however, occur only when large amounts choles-terol are fed in the diets.24,25 Also the amount of dietary fatadministered will be crucial since an amount of 10% (w/w)dietary olive oil prevented the development of fatty livers in aprevious study.26 Liver weight was significantly higher uponconsumption of Picual EVOO, and the hepatic fat content was

Figure 3. Correlation plot indicating all pairwise correlations between plasma lipid and liver protein levels that had a Pearson correlationhigher than 0.66, a p value lower than 0.0005, and a q-value lower than 0.0025, using the software tool Cytoscape as described inMaterials and Methods. The color code indicates the percentage increase or decrease in plasma levels or hepatic protein mass in eachdietary olive oil intervention group as compared to the palm oil control group.



significantly increased upon consumption of both Picual andArbequina EVOOs. In addition, there were significant five-and2-fold increases in hepatic adipophilin protein levels uponconsumption of Picual and Arbequina EVOO, respectively(Figure 1). Adipophilin (or adipose differentiation-relatedprotein) is bound to the surface of lipid bodies and lipid bi-layers.27 The proposed function of adipophilin appears morecomplex than merely the packaging of neutral lipids in thecytosol. Adipophilin overexpression in primary liver cellsincreases the size of cytosolic lipid droplets and reduces thesecretion of VLDL, without influencing the rate of â-oxidation,thereby selectively decreasing VLDL assembly.28 On the otherhand, a knockdown of adipophilin decreased the pool ofcytosolic lipid droplets, increased the secretion rate of apoli-poprotein B-48 VLDL1, and increased â-oxidation.28 Also,adipophilin knockout in mice had lower levels of hepatictriglycerides without a change in plasma lipid profile, and theseanimals were protected against the development of a fattyliver.29 Therefore, down-regulation of adipophilin proteincauses a channeling of fatty acids primarily into â-oxidation.Interestingly, we found that plasma triglycerides were lowestand accumulation of hepatic lipid droplets was highest in thePicual EVOO group that had the highest levels of hepaticadipophilin protein, without a noticeable effect on hepaticproteins involved in â-oxidation of fatty acids. Indeed, hepaticadipophilin protein levels and liver weight were two of the mainparameters responsible for the treatment effect of Picual oliveoil in the principal component analysis (Figure 3), and hepaticadipophilin protein levels and plasma triglycerides were in-versely correlated (r ) -0.433, p < 0.05). The two EVOOsproduced differential effects on hepatic adipophilin protein,possibly because of a different dietary oleic acid content or adifference in the amount and types of polyphenols. In one ofour earlier studies, a decrease in hepatic adipophilin proteinwas associated with decreased serum and hepatic triglyceridesand increased â-oxidation of fatty acids upon consumption offish oil.18 Therefore, regulation of adipophilin protein by dietaryfatty acids or other compounds may represent an importantregulatory pathway in hepatic lipid metabolism, albeit thatregulation of adipophilin upon dietary intervention may bespecies dependent.

Abnormalities in lipid storage in hepatic and adipose tissuegives rise to a multitude of undesirable effects includingincreased levels of plasma lipids, including NEFA, and insulinresistance.30 The reason for the increase in plasma NEFA levelsin particularly the Picual olive oil group as compared with thepalm oil group is not clear, but might involve the disturbanceof hormonal regulation of lipolysis in adipose tissue, possiblydue to a decrease in insulin sensitivity.31 Indeed, a significantincrease in plasma NEFA and a decrease in insulin sensitivityoccurred in the Picual EVOO group that had also the highestlevels of hepatic fat and adipophilin protein. In addition, weobserved significant increases in several hepatic enzymesinvolved in the methionine cycle, especially in betaine ho-mocysteine methyl transferase (BHMT), by both EVOOs (Table2). BHMT, which converts homocysteine into methionine usingbetaine as a cofactor (Figure 4), is up-regulated during insulinresistance.32,33 Also, an increase in hepatic BHMT is associatedwith an increase in hepatic very low-density lipoprotein (VLDL)and apolipoprotein B production rate in rats fed a methionine-deficient diet supplemented with betaine34 (Figure 4). Increasedsecretion rates of VLDL1-apolipoprotein B and triglycerides, asassessed by kinetic studies in humans, are a common feature

in insulin resistance. The hepatic VLDL overproduction isbelieved to be driven by the altered free flow of NEFAs,35 butBHMT may well be involved in this process. However, despitean increase in plasma NEFA upon consumption of PicualEVOO, plasma triglycerides were actually lower (Table 1),suggesting that VLDL-apolipoprotein B secretion was also likelyto be lower. This suggests that an overriding mechanism,possibly involving adipophilin, is preventing the hepatic trig-lycerides from being secreted.

EVOOs contains a wide range of important minor antioxidantcompounds, such as polyphenols, that contribute to thestability of the oil and that can have anti-inflammatory andanti-atherosclerotic properties.36,37 Consumption of polyphenolshas been associated with prevention from and/or treatmentof atherosclerosis.38 Our proteomics approach revealed a rangeof antioxidant enzymes, like TR, thioredoxin peroxidase 2,peroxiredoxin 3, superoxide dismutase and GST, whose hepaticprotein levels were 1.5-4-fold higher upon consumption ofboth EVOOs compared with palm oil. In addition, hepatic TRactivity was significantly higher upon consumption of PicualEVOO, confirming the proteomics findings. A similar coordi-nated induction of the antioxidant enzymes has been observedin Apoe-/- mice in the period preceding lesion formation.39

Remarkably, most mRNA levels of these antioxidant enzymesstarted to decline as lesions started to develop. This suggeststhat the arterial wall delays initial lesion formation by stimulat-ing the expression of antioxidant enzymes, but when thisdefense capacity collapses a greatly accelerated developmentof atherosclerosis occurs.39 Therefore, compounds in EVOO(like for example the polyphenols) may well be able to delaythe onset of atherosclerotic lesions by the combat of oxidativestress.

In contrast, protein and activity levels of hepatic GPx1 werelower upon olive oil consumption. In patients with coronaryartery disease, a low level of red-cell GPx1 activity is indepen-dently associated with an increased risk of cardiovascularevents.40 However, it is questionable whether GPx1 plays a keyrole in lesion formation at the site of the aortic root. A specific

Figure 4. Schematic overview of pathways involved in methio-nine and glutathione metabolism, of which some were differen-tially regulated by Picual and Arbequina olive oil intervention,as compared with palm oil intervention.



deficiency in GPx1 was not accompanied by an increase inmarkers of oxidative damage or increased atherosclerosis.41

Also, GPx1 expression in the aortic arch of Apoe-/- mice, ascompared with wild type mice, decreases rapidly in the periodpreceding lesion development, resulting in hardly any detect-able GPx1 expression in atherosclerotic tissue.39 Moreover, GPx1enzyme activity was absent in human atherosclerotic lesions.42

However, based on previous studies we cannot rule out a rolefor GPx1 in atherogenesis at other sites.41 In our study, thepairwise correlation analysis revealed a clustering of hepaticGPx1 activity with lesion size, hepatic adipophilin levels, liverweight, hepatic fat content and body weight, indicating for thefirst time that their regulatory mechanisms may be related toeach other (Figure 3). GPx1 may indeed play different roles indifferent organs,43 and the lower protein and activity levels ofhepatic GPx1 upon olive oil consumption may simply reflect alower degree of hepatic oxidative stress due to the up-regulationof other antioxidant enzymes.

Proteomics also revealed the up-regulation of glutamatedehydrogenase, glutathione synthase, and hydroxyacyl glu-tathione hydrolase, as well as the down-regulation of cysteine-sulfinic acid decarboxylase, indicating an increased productionof glutathione by predominantly Arbequina EVOO (Table 2 andFigure 4). Other studies in Apoe-/- mice showed that depletionof glutathione via a decreased synthesis precedes lipid peroxi-dation and atherogenesis, and glutathione is severely depletedin the atheroma-prone aortic arch of male compared with wildtype mice after 10 weeks.44 Therefore, glutathione deficiencymight be central to the failure of the intracellular antioxidantdefenses and is causally implicated in the pathogenesis ofatherosclerosis. The apparent increase in glutathione produc-tion and other antioxidant defense mechanisms upon con-sumption of EVOO may thus represent a mechanism by whicholive oil components diminish oxidative stress.

In conclusion, olive oil decreased atherosclerosis in Apoe-/-

mice despite an increase in plasma total cholesterol and thedevelopment of hepatic steatosis. These are conflicting findings,and it would be intriguing if olive oil consumption would affectthe interaction between similar mechanisms in humans, ashepatic lipid loading over a longer time period may havedetrimental effects. A systems biology approach, as presentedhere, is crucial in unraveling the complex interactions betweenpathways that are on one hand involved in the beneficialreduction in plaque formation and on the other hand involvedin the potentially less beneficial development of hepaticsteatosis. We found, for example, a significant up-regulationof a large array of antioxidant enzymes upon consumption ofEVOO that may diminish oxidative stress instigated by hepaticsteatosis and in addition, may slow down the development ofatherosclerosis. Indeed, the accumulation of triglycerides maynot pose a major challenge to the liver, and represent arelatively safe way to store triglycerides, as long as the anti-oxidant capacity is adequate to prevent lipotoxicity.45 Inaddition, our proteomics results revealed, for the first time, thattwo different EVOOs instigated distinct effects on hepatic lipidmetabolism by regulation of hepatic adipophilin. This mech-anism may override any regulation by insulin or BHMT toincrease the production of VLDL apolipoprotein B that isnormally observed upon development of hepatic steatosis andearly symptoms of insulin resistance.

Acknowledgment. This research was supported bygrants FEGA-FEOGA (CAO99-014), CICYT (SAF2004-08173-C03-

02), FISS 01/0202, Redes DGA (A-26), and FISS de investigacioncooperativa C03- 01 and G03-140 and by Fundacion Espanoladel Corazon. B.d.R., K.R., G.R., M.R., G.D., and J.A. are fundedby the Scottish Executive Environment and Rural AffairsDepartment. J.M.A.-M., R.C., and M.A.N. were recipients of CAI-EUROPA, DGA, and FEGA-FEOGA fellowships. We thank Dr.Gerald Lobley for his help with the interpretation of theproteomics data and Dr. Uceda for his analysis of diet com-position. We thank Angel Beltran, Jesus Cazo, Jesus Navarro,Carmen Navarro, and Clara Tapia from Unidad Mixta deInvestigacion for their invaluable help in maintaining animals.

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