Olive Oil and the Hallmarks of Aging

30 Pages·2016·0.31 MB·English

molecules
Review
Olive Oil and the Hallmarks of Aging
LucíaFernándezdelRío1,†,ElenaGutiérrez-Casado1,†,AlfonsoVarela-López2,† and
JoséM.Villalba1,*
1 DepartmentofCellBiology,PhysiologyandImmunology,AgrifoodCampusofInternationalExcellenceceiA3,
UniversityofCórdoba,CampusRabanales,SeveroOchoaBuilding,14014Córdoba,Spain;
[email protected](L.F.R.);[email protected](E.G.-C.)
2 DepartmentofPhysiology,InstituteofNutritionandFoodTechnology“JoséMataix”,
BiomedicalResearchCenter(CIBM),UniversityofGranada,Avda.delConocimientos.n.,Armilla,
18100Granada,Spain;[email protected]
* Correspondence:[email protected];Tel.:[phone]
† Theseauthorscontributedequallytothisworkandmustbeconsideredasfirstauthors.
AcademicEditors:MaurizioBattino,EtsuoNikiandJoséL.Quiles
Received:15December2015;Accepted:22January2016;Published:29January2016
Abstract: Agingisamultifactorialandtissue-specificprocessinvolvingdiversealterationsregarded
as the “hallmarks of aging”, which include genomic instability, telomere attrition, epigenetic
alterations,lossofproteostasis,deregulatednutrientsensing,mitochondrialdysfunction,cellular
senescence,stemcellexhaustionandalteredintracellularcommunication.Virtuallyallthesehallmarks
aretargetedbydietaryoliveoil,particularlybyvirginoliveoil,sincemanyofitsbeneficialeffectscan
beaccountednotonlyforthemonounsaturatednatureofitspredominantfattyacid(oleicacid),but
alsoforthebioactivityofitsminorcompounds,whichcanactoncellsthoughbothdirectandindirect
mechanismsduetotheirabilitytomodulategeneexpression.Amongtheminorconstituentsofvirgin
oliveoil,secoiridoidsstandoutfortheircapacitytomodulatemanypathwaysthatarerelevantfor
theagingprocess. Attenuationofaging-relatedalterationsbyoliveoiloritsminorcompoundshas
beenobservedincellular,animalandhumanmodels. Howoliveoiltargetsthehallmarksofaging
couldexplaintheimprovementofhealth,reducedriskofaging-associateddiseases,andincreased
longevitywhichhavebeenassociatedwithconsumptionofatypicalMediterraneandietcontaining
thisedibleoilasthepredominantfatsource.
Keywords: DNAdamage;fattyacids;hallmarksofaging;Mediterraneandiet;oliveoil;oxidative
stress;polyphenols;senescence
1. Introduction
Agingisatime-dependentprogressivedeclineinphysiologicalfunctionoftheorganismthattakes
placewithdecreasedfertilityandincreasedsusceptibilitytoendogenousandexternalthreats,leading
toawidevarietyofrelateddiseasessuchasdegenerativeandneoplasticdisorders. Nowadays,ithas
beenrecognizedthataging-relatedalterationsaremultifactorialandtissue-specificattheorganismal
level,andinvolvediverseprocessesregardedasthe“hallmarksofaging”. Thesehallmarksinclude
genomicinstability,telomereattrition,epigeneticalterations,lossofproteostasis,deregulatednutrient
sensing,mitochondrialdysfunction,cellularsenescence,stemcellexhaustionandalteredintracellular
communication[1]. Scientistshavedemonstratedastronginterestnotonlyinunravellingthecauses
ofaging,butalsoindiscoveringhowwecanmanipulatepotentialcausesofagingtodecrease,stop,
orevenrevertitsrateofprogression[2,3]. Ofnote,anumberofdietaryapproacheshaverevealed
successfultodecreasetheincidenceofaging-relateddiseasesortoevenincreaselifespan.
Pro-survivalpropertiesofantioxidantshavebeenshowninseveraldiseasemodels[4],although
only a few interventions with antioxidants have successfully increased lifespan so far [5]. Calorie
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restrictionwithoutmalnutritionisclearlythebestcharacterizednon-geneticinterventionthatincreases
maximumlifespanandimproveshealthspan,preventingordelayingtheonsetofpathophysiological
changesinmultiplespecies[6]. Inaddition,prospectivestudieshaveshownthatadherencetoatypical
Mediterraneandietwitholiveoil(OO)asthepredominantfatsourceisassociatedwithimproved
health,lowermortalityandincreasedlongevity,reducedriskofcardiovasculardiseases,cancer,and
the incidence of age-related cognitive decline as Parkinson’s and Alzheimer’s disease [7]. These
benefitshavebeenattributednotonlytoitshighmonounsaturatedfattyacid(MUFA)contentbut
alsotothehealthypropertiesofminorthoughhighlybioactivecomponents,particularlyinthecase
ofvirgin(VOO)andextravirginoliveoil(EVOO).Theseminorcomponentscanbeclassifiedinto
theunsaponifiable(non-polar)andthesoluble(polar)fraction,whichincludesavarietyofphenolic
compoundssuchashydroxytyrosol(HT),tyrosol,caffeicacid,oleuropeinaglycone,andoleocanthal,
amongothers[7,8]. Besidestheseprospectivestudiescarriedoutwithhumanpopulations,animal
studieshavealsoindicatedthatOOphenolshavepreventiveactionsonage-relatedcognitiveandmotor
dysfunction. Furthermore,mechanisticinvitrostudiesalsoindicatethatOOphenoliccompoundsmay
inhibitinflammatorypathways,inducesignalingpathwaysrelatedtocellprotectionandsurvival,and
modulatepathwaysrelatedtoenergymetabolismsimilartoanti-agingsubstances,whichhasbeen
summarizedinanexcellentreview[9]. Thepresentreviewnotonlyconstitutesanupdateincluding
themostrecentadvancesontheresearchlikingOOandthepreventionofaging-relatedalterations,but
alsoitisfocusedonthesystematicevaluationofhowOOanditsminorconstituentsactontherecently
recognized hallmarks of aging. Elucidating how OO targets these hallmarks can help us to better
understandthemolecularandcellularbasisofitsbeneficialactiononagingandaging-relateddiseases.
Moreover,forourreviewwewillconsidernotonlyOO(initsvarieties)andthecompoundscontained
in this edible oil at a pharmacologically relevant concentration, but also olive-related compounds
thatcanbeusedaspurifiedmoleculesorasextractsobtainedfromsourcesasolivesandoliveleafs,
amongothers. Thesesubstanceshavebeenusedforasignificantpartoftheresearchaimedongiving
amechanisticexplanationofhowOOexertsitspositiveeffectsoncellularpathwaysthatarealtered
withaging,andtheycansetupthebasisfordesigningfutureolive-derivedfunctionalfoodswhich
positivelyimpacthumanaging.
2. OliveOilandGenomicInstability
The genetic material tends to accumulate damage during aging because it is continuously
challenged by exogenous and endogenous threats [1]. In an aging context, oxidative damage to
mitochondrialDNA(mtDNA)ismoreimportantthanthedamageexertedtolipidsandproteinsdue
totheabilityofmtDNAtobedisseminatedinthedivisionofmitochondriaandcells,whichamplifies
the physiological consequences of the damage [10]. Traditionally, mtDNA has been considered
highlysusceptibletooxidativeattackbecause: (i)themitochondrialrespiratorychainisasourceofa
continuingfluxofoxygenradicals;(ii)itisnotprotectedbyhistones;and(iii)themitochondriamaybe
lessefficientinrepairingDNAdamageandreplicationerrorsthanthenucleus[11]. Inaddition,aging
isassociatedwithdeletionsofmtDNAinatissue-dependentmanner[12],affectingmainlypostmitotic
tissues like the brain, skeletal muscle and heart [13]. Some studies indicate that multiple mtDNA
deletionsmaybepromotedbydoublestrandbreaks[14].
Several studies have tried to test the effect of OO avoiding the age-associated damage in the
DNA,bothinvivoandinvitro. Quilesetal. havetestedtheeffectsoffeedingmaleWistarratswith
diets containing different fat sources as VOO and sunflower oil (SO) [15]. Lower levels of DNA
double-strandbreaksinperipheralbloodlymphocyteswerefoundinyounganimalsfedonVOO,
whichreachedaroundonehalfofthedamagethatwasfoundintheSOgroup.Thesamemeasurements
werecarriedoutinagedratsshowingthat,althoughtheaging-relatedincreaseofDNAdouble-strand
breakslevelsthattookplaceinbothdietarygroups,thedamagewassignificantlylowerinratsfeda
dietcontainingVOO.However,thetwogroupsdidnotdifferineithermeanormaximumlifespan.
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Another study from the same research group was set to analyze the presence of a particular
deletion in liver of rats fed diets containing VOO or SO [16]. Two regions of the mitochondrial
genomewerestudied: ND1andND4genes. Theformerisrarelyaffectedbydeletionsinhumans
andratswhereasthelatterisincludedintheso-calledcommondeletionbothinhumansandrats.
Whileanincreaseofmorethan6-foldinthedeletionwasfoundinthecaseofoldanimalsfedthe
VOO-containing diet, the increase was 60% higher (more than 10-fold) in old animals fed the diet
containingSO.Theseobservationssupportthatdietaryfattypecanmodulatethefrequencyofthe
studieddeletioninratliverandthattheage-relatedincreaseinmtDNAdeletionscouldbeattenuated.
ThelowerincreaseinmtDNAdeletionfrequencyduringagingwasattributedtotheloweramountof
freeradicalsproducedbyVOO.
Fabianietal. havemeasuredtheeffectofOOphenolicsonH O -inducedDNAdamageinhuman
2 2
peripheralbloodmononuclearcells(PBMC)andpromyelocyticleukemiacells(HL-60)[17]. Their
resultsshowedthatHT,acomplexoliveoilphenolextract(OOPE),andolivemillwastewaterphenol
extract(WW-PE)displayedahighlyprotectiveactivityagainstDNAdamageinbothPBMCandHL-60
cells. Furthermore, other purified compounds like one isomer of oleuropein aglycon, oleuropein,
tyrosol,caffeicacidandverbascosidealsodisplayedaprotectiveeffectoncellsalthoughinalower
range. These results suggest that phenols used at low concentrations (1–10 mmol/L), as purified
compoundsorinacomplexcrudeextractindependentlyofthesource(OOorWW-PE),mayprevent
DNAdamageinducedbyH O . Theseconcentrationscouldbeeasilyreachedinthetissueswithan
2 2
ordinaryintakeof50g/dayofoliveoil,becausethesephenoliccompoundsareeffectivelyabsorbedin
humans[18].
AdditionalstudieshavedemonstratedthatpretreatmentofHelacellswithOOPEalsoprevents
nDNAdamageinducedbyH O [19]. ThisprotectionmightbeassociatedwithOOPE’sfreeradical
2 2
scavengingproperties,metalionchelatingpropertiesand/ortheendogenousantioxidantdefenses
andDNArepairsystems[17]. Inaddition,VOOphenolicscouldprotectAPEX1,aDNArepairgene,
byinhibitionofoxidativenDNAdamage[19].
3. OliveOilEffectonTelomereAttrition
Telomeres are nucleoprotein structures that protect the ends of eukaryote chromosomes [20].
Importantly,telomereshorteningisalsoobservedduringnormalagingbothinhumanandinmice[21].
Telomere length is considered to be a biomarker of aging; shorter telomeres are associated with a
decreased life expectancy and increased rates of developing age related chronic diseases [22–24].
However,telomerelengthvariesconsiderablyamongindividuals[25]. DNAinthesestructuresis
particularlysusceptibletoage-relateddeterioration[26]. Oneofthereasonsforthisisthatreplicative
DNApolymeraseslackthecapacitytoreplicatecompletelytheterminalendsoflinearDNAmolecules,
afunctionthatisproprietaryofaspecializedDNApolymeraseknownastelomerase. However,most
mammaliansomaticcellsdonotexpresstelomerase,andthisleadstotheprogressiveandcumulative
lossoftelomere-protectivesequencesfromchromosomeends[1]. Moreover,telomeresareboundby
acharacteristicmultiproteincomplexknownasshelterin[27]thatpreventtheaccessofDNArepair
proteinstothetelomeres. Althoughtelomerelengthisstronglyinfluencedbygeneticfactors,telomere
attritionmightbemodulatedbyenvironmentalandlifestylefactors,includingolderage,cigarette
smoking,gender,ethnicity[28],education[29]andotherindicatorsofsocioeconomicstatus[30]and
diet[31].Studieshavesuggestedthattelomereattritionismodifiable,assubstantialvariabilityexistsin
therateoftelomereshorteningthatisindependentofchronologicalage[32–34]. Therefore,variability
oftelomerelengthmaybepartiallyexplainedbylifestylepractices,includingdietarypatterns.
Some reports have indicated a positive relationship of leukocyte telomere length and
Mediterrraneandietadherenceintwodifferenthumanpopulationsubsets[35,36]. Theseincluded
a cohort of elderly subjects from the South Italy [35] and a group of nurses aged 30–55 years [35].
Similarly, a study comparing between Greek and Dutch elderly men has reported that the latter
presentedlowerleukocytetelomerelength[37]. Whenmeasured,telomeraseactivityhasalsoshowna
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positiveassociationthatcorrelateswithleukocytetelomerelength[35]. Oxidativestressmayinfluence
therateoftelomeresshortening[37]. Inthatsense,ithasbeenreportedthattelomeraseactivitywas
negativelymodulatedbyinflammationandoxidativestress[35]. Incontrast,norelationshipbetween
leukocytetelomerelengthandindicatorsofoxidativestressandplasmaantioxidantswasfoundin
thestudycomparingDutchandGreekelderlymen,althoughtheendogenousserumantioxidants
albuminanduricacidwerepositivelyassociatedwithtelomerelength[37].
Studies directly evaluating the effects of OO intake on leukocyte telomere length are absent.
Ofnote,regressionanalysisshowedthattheMediterraneandietadherencewasnotassociatedwith
leukocyte telomere length in the overall study population after adjusting for age, sex, education,
ethnicity,caloricintake,smoking,andphysicalandleisureactivities.However,asignificantassociation
with leukocyte telomere length was recognized among non-Hispanic whites. Furthermore, it was
foundthathigheradherencetoaMediterraneandietwasassociatedwithlongerleukocytetelomere
lengthamongwhites,butnotamongAfricanAmericansandHispanics[38]. Thus,resultsmaynotbe
generalizedtootherpopulations.
Anotherinterventionhasbeenperformedonelderlysubjectsthatconsumedthreediets,each
for four weeks: a saturated fatty acid (SFA) diet, a low-fat and high-carbohydrate diet, and a
MediterrraneandietenrichedinMUFAfollowingarandomizedcrossoverdesign. Humanumbilical
endothelialcellswereincubatedwithserumfromeachpatient. Amongthese,theMUFA-richdiet
inducedlowerpercentageofcellswithtelomereshortening,comparedwiththebaselineandtheother
two diets. Moreover, this effect correlated with lower intracellular production of reactive oxygen
species(ROS)anddiminishedcellularapoptosis[39].
The Prevención con Dieta Mediterránea (PREDIMED) trial consists in a 5-year nutritional
intervention with three groups: Mediterranean diet specifically supplemented with EVOO,
Mediterraneandietsupplementedwithmixednuts,oralow-fatdietascontrolgroup. Inasubset
fromthisstudy, higherbaselinetelomerelengthsignificantlypredictedagreaterdecreaseinbody
weightbodymassindex(BMI),waistcircumferenceandwaisttoheightratio. Inaddition,changes
in telomere length during the 5-year intervention were inversely associated with changes in the
fouranthropometricvariables. Thereductioninadiposityindicesduringtheinterventionthatwas
associatedwithincreasedtelomerelengthwasevenhigheramongsubjectswiththelongesttelomeres
atbaseline. Logisticregressionanalysisshowedthattheriskofremainingobeseafter5yearswas
lowerinthoseparticipantswhoinitiallyhadthelongesttelomeresandincreasedtheirtelomerelength
afterintervention[40].
Telomerase itself could be a potential target to prevent telomere length attrition. Actually,
telomerase deficiency in humans has associated with premature development of diseases, such
as pulmonary fibrosis, dyskeratosis congenita, and aplastic anemia, which involve the loss of the
regenerativecapacityofdifferenttissues[41]. Twophenoliccompounds, oleaceinandoleuropein,
mainlypresentinoliveleaves,seeminterestingatthisrespectaccordingtotheresultsobtainedin
aninvitroassay. Namely,expositionofendothelialprogenitorcellstoanyofthesecompoundsprior
to angiotensin II treatment increased telomerase activity. This effect was also associated with an
increase of proliferation and a decrease in the percentage of senescent cells and intracellular ROS
formation[42],asdescribedinthe“StemCellExhaustion”section. However,ithastobetakeninto
accountthattheamountofoleuropeininEVOOisverylow. Furthermore,whetherOOintakemay
affecttelomeraseactivityisstillpoorlyinvestigatedandnoinvivoeffectshavebeenreported,which
warrantsfutureresearch.
4. EpigeneticChangesInducedbyOliveOil
Epigenetic modifications are heritable changes in gene function that are not caused by
variation in DNA sequence [43]. These changes mainly involve alterations in DNA methylation
patterns,posttranslationalmodificationofhistones,andchromatinremodeling. Therearemultiple
enzymaticsystemsassuringthegenerationandmaintenanceofepigeneticpatterns,includingDNA
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methyltransferases,histoneacetylases,deacetylases,methylases,anddemethylases,aswellasprotein
complexesimplicatedinchromatinremodeling. Aging-associatedtranscriptionalsignaturesalsoaffect
noncodingRNAs,includingmicroRNAs(miRNAs)[1]. Epigeneticprocessesaredynamicandmaybe
affectedbyenvironmentalfactorssuchasexerciseanddiet[44,45]. Actually,dietisthemorestudied
environmentalfactorinepigenetics[46].
OneofthemostcommonepigeneticmarksisDNAmethylation. DNAmethylationmayplayan
importantroleincausingdiseasebysilencinggenesthroughhypermethylationoractivatinggenes
through hypomethylation. Cytosine DNA methylation, the most studied epigenetic mechanism,
is usually considered as a flexible method for repressing gene expression. Different dietary and
lifestylefactorsareabletomodulatethemethylationofspecificCpGsitesingenepromotersevenin
theadultage[45]. ChangeshavebeenobservedinDNAmethylationpatternsduringaging[47,48],
althoughhypermethylationhasbeennotedinsomelociwhilehypomethylationhasbeenalsonotedin
others[49].
In relation to OO, an observational study performed on Greek adolescents has been recently
publishedwithveryinterestingresults. Agenome-widemethylationprofileinbloodrevealedthat
theMUFAtoSFAratiointhedietwasassociatedwith26islandshoresand158site[50]. Thisdietary
featureistypicalofMediterraneandiets,likelyduetothehighintakeofOO[51]. Inthatsense,dietary
interventionswithMediterraneandietshavealsoshownthatDNAmethylationlevelswereassociated
withadherencetothediet[52]. Accordingtopreviousfindings, itispossiblethatOOwasableto
induce epigenetic changes by means of its MUFA content. However, an invitro study carried out
withhumancoloncancercells(Caco-2)hasreportedverysimilareffectsforVOOanditsphenolic
compounds[46].
AlongwithDNAmethylations,EVOOcouldalsoinduceepigeneticchangesbyhistoneacetylation
processes. Insupportofthis,aninvitroassayhasshownthatsomephenoliccompoundscontained
in EVOO, namely secoiridoids (i.e., oleuropein and its conjugated forms), are able to induce
hyperacetylationofhistoneH3incellcultures,amongmanyothereffects[53].
Anotherpointofinterestisthepossiblegenesaffectedbymethylationandtheirimplications
foragingprocess. AlltheepigeneticchangesderivedfromOOoritsminorcompoundshavebeen
usuallystudiedonlyinspecificgenes,andseveralofthesegeneshaveshownrelationshipwiththe
aging process. In particular, many of them have been related with susceptibility to aging-related
diseasessuchasmetabolicsyndrome. Inrelationtothis,someinterventionstudieshaveindicatedthat
OOorMediterraneandietsinfluenceDNAmethylationingenesencodingenzymesinvolvedinfatty
acid metabolism. In renal patients, OO supplements altered methylation of fatty acid desaturase
(FADS)-2 CpGs and elongase (ELOVL)-5 CpGs. The methylation status of the altered CpGs in
FADS2 and ELOVL5 was associated negatively with the level of their transcripts [54]. Similarly,
inadietaryinterventionstudywithMediterraneandiettodecreaseweightofthesubjects,thosewho
lost weight showed higher levels of stearoyl CoA desaturase (SCD)-1 gene promoter methylation
afteroneyear. Furthermore,thesesubjectspresentedhigheradherencetoaMediterraneandiet[52].
Previously, it was reported that variations in SCD activity were associated with disorders such as
obesity,insulinresistance,anddiabetes[55,56],whichsuggeststhatgeneexpressionmodifications
canaffectmetabolicsyndromeonsetandprogression. Inaddition, ithasbeenreportedthatSCD1
knockdown increased the amount of SFA and decreased that of MUFA in HeLa cells membrane
phospholipidswithoutaffectingtheamountorthecompositionoffreefattyacids[57]. Thedegree
offattyacidunsaturationinmembranephospholipidsaffectsmanymembrane-associatedfunctions
which highlights the importance of SCD1. In particular, OO intake increases membrane MUFA
content[58],whichcouldbeaccountednotonlybyitshigh-MUFAcontentbutalsobyitseffecton
SCD1 gene, reinforcing the notion that Mediterranean diet effect on SCD1 may be a consequence
ofOO.
Othergenesinterestinginrelationtometabolicsyndromearethoseinvolvedincircadianrhythms.
Inwomen,ithasbeenreportedthatthepercentageofmethylationoftheclockcircadianregulatorgene
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(CLOCK)CLOCKCpGs1and8showedassociationswiththeintakeofMUFAandpolyunsaturated
fatty acids (PUFA) [59]. The circadian clock system instructs 24 h rhythmicity on gene expression
in essentially all cells [59] and it has been related to obesity and metabolic syndrome features.
Ithasbeenreportedthatpatientswithvisceralobesityandmetabolicsyndromeexhibitdisturbances
inthecircadianrhythm(chronodisruption)thatmaybeassociatedwithhigherweightincreaseand
developmentofdiabetesandatheroscleroticdisease[60,61]. Thisisinaccordancewithfindingsfrom
thesamestudysinceadifferentpatternofDNAmethylationofCLOCKandBMAL1genebetween
normal-weightandoverweightorobesesubjectswasnoted. Moreover,themethylationpatternof
differentCpGsitesofthethreegenesshowedsignificantassociationswithanthropometricparameters
suchasbodymassindexandadiposity,andwithametabolicsyndromescore[59].
Ontheotherhand,aninvitrostudywithCaco-2cellshasalsoshownpossibleanticancereffects
forEVOObymeansofthismechanism,sinceitinfluencedDNAmethylationstateatCNR1promoter
which was inversely correlated to selective and transient up-regulation of CNR1 gene, a tumor
suppressorthatencodesfortype1cannabinoidreceptor(CB1). EVOO,aphenolicEVOOextractorHT
stimulatedCB1expressionwhichwasassociatedwithreducedproliferationofthesecancercells[46].
The same authors confirmed this anticancer effect in rats where dietary EVOO supplementation
increasedCB1mRNA.Consistently,CpGmethylationofratCnr1promoter,miR23aandmiR-301a,
previously shown to be involved in the pathogenesis of colorectal cancer [62] and predicted to
target CB1 mRNA, were reduced after administration of EVOO down to ~50% of controls [46].
Inanotherinvitrostudy,EVOOphenolicextractsrichinsecoiridoidspermittedHistoneH3remain
inhyperacetylatedstateatHysine18inabreastcancermodel(theHER2-geneamplifiedJIMT-1cell
line,auniquebreastcancermodel). Theresultingalterationsingeneregulationcouldreducemitotic
viability and metabolic competence of breast cancer cells, inherently refractory to HER-targeting
therapiesabinitio[53].
miRNAs are small endogenous non-coding RNAs that regulate several cellular and biologic
processesbyregulatinggeneexpression[63]thatmayepigeneticallyexplainthelong-termphenotypic
changesoftheoffspring. Someresearchershavealsostudiedthismechanismdeepenedin. Inrats,
ithasbeenreportedthatmaternalconsumptionofdifferenttypesoffatsources,includingOO,during
early pregnancy influenced miRNAs expression in both maternal and offspring tissues [64]. The
influenceofdietaryfatonmiRNAshasalsobeenreportedinhumans. InthePREDIMEDrandomized
trial,anovelassociationbetweenamicroRNAtargetsitevariantandstrokeincidencewasreported
whichismodulatedbydietintermsofdecreasingtriglyceridesandpossiblystrokeriskinrs13702C
allelecarriersafterahighly-unsaturatedfatMediterraneandietintervention[65].
5. OliveOilEffectsonProteostasis
Amultitudeofqualitycontrolmechanismshaveevolvedtopreservethestabilityandfunctionality
ofproteomesincells. Thesemechanismsfunctioninacoordinatedfashiontorestorethestructureof
misfoldedpolypeptidesortoremoveanddegradethemcompletely,thuspreventingtheaccumulation
ofdamagedcomponentsandassuringthecontinuousrenewalofintracellularproteins[1]. Protein
homeostasisorproteostasisinvolvesbothmechanismsforthestabilizationofcorrectlyfoldedproteins
(i.e., the heat shock family of proteins) and mechanisms for the degradation of proteins (i.e., the
ubiquitin-proteasome system and those including lysosomes) [66–68]. The heat shock family of
proteinsincludesseveralisoformsof70-kDaheatshockproteins(Hsp70)thatcanbeconstitutively
expressedandstress-inducedaswell.Theyarechaperonesinvolvedincrucialcellularfunctions[69–72].
WhileconstitutivelyexpressedHsp70chaperoneshavehousekeepingfunctions(e.g.,foldingofnascent
polypeptides,proteintranslocationbetweencellularcompartmentsanddegradationofunstableand
misfoldedproteins),stress-inducedHsp70spreventtheaccumulationofproteinsthathavebecome
denaturedinresponsetovariouscellularstresses(e.g.,heatstress,radiation,ischemia,heavymetals
orotherstimulithatactivatestresstranscriptionfactors). Stress-inducedsynthesisofcytosolicand
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organelle-specificchaperonesissignificantlyimpairedinaging[73]andresearchwithanimalmodels
supportsthepositiveimpactofchaperonesonlongevity[74–77].
Aging and some aging-related diseases are associated with perturbed proteostasis, and its
experimentalperturbationcanprecipitateage-associatedpathologies[1,67,78]. Thisisparticularly
notably in some age-related pathologies, such as Alzheimer’s disease, Parkinson’s disease, and
cataracts where chronic expression of unfolded, misfolded, or aggregated proteins contributes to
theirdevelopment[78]. Differentinvitrostudieshaveshowninterestingactivitiesofolivephenolic
compoundsonproteinaggregatesofthistype. HT,oleuropein,andoleuropeinaglyconeprevented
Taufibrillization,amajorfeatureinAlzheimer’sdisease(AD)pathogenesis,inaninvitroassay[79].
InTypeIIdiabetes,pancreaticamyloiddepositsofamylinareapparentlycytotoxictoβ-cells. Themain
phenoliccomponentofEVOO,oleuropeinaglycone,whenpresentduringtheaggregationofamylin,
consistentlypreventeditscytotoxicitytoRIN-5Fpancreaticβ-cells. Moreover,alackofinteraction
withthecellmembraneofamylinaggregateswasobservedwhencellsweregrowninthepresenceof
oleuropein. Theseobservationssuggestthatoleuropeinaglyconeinterfereswithamylinaggregation,
resultinginadifferentpathskippingtheformationoftoxicprefibrillaraggregates[80]. Therefore,itis
possiblethatEVOOcouldbeusedtopreventortreattheseaging-relateddiseasesoragingchanges
possibly due to some compound present in this food having an effect on a mechanism regulating
proteinhomeostasis. TherelationshipbetweenEVOOandADwillbeconsideredspecificallyinalater
section(seeSection8: OliveoilandCellularSenescence).
Different compounds or extracts from EVOO have been related with an increase of some
components of the chaperones family or up-stream factors regulating them. Treatment with an
EVOOsecoiridoid-richphenolicextractmarkedlyup-regulatedtheexpressionoftheHSPA6(Hsp70B’)
gene,whichisstrictlyinduciblewithnodetectablebasalexpression[81,82]. Indeed,HSPA6protein
induction is a sensitive biomarker of cellular stress that appears transiently in response to heat
stress, whereas levels of HSPA1A (Hsp72), which was also induced by an EVOO secoiridoid-rich
phenolicextract,persistfordays[83]. ThesecoiridoidsalsoenhancedtheexpressionoftheHSPA1L
gene (Hsp70-hom or Hsp70t), which encodes a constitutively expressed, non-inducible cytosolic
proteinthatishighlyabundantintestis[84,85],andofHSPA2(Hsp70-2),aconstitutivelyexpressed
genethatisalsoexpressedathighlevelsintestis[86]. Treatmentwithtyrosolexpandedlongevity
of several mutant strains of C. elegans and such effect has been related with the master regulator
of the heat-shock response, the transcription factor HSF-1 [87]. The importance of the activation
degreeofthistranscriptionfactorforlongevityandthermotoleranceincreaseinnematodeshasbeen
previously reported [88,89]. In mammalian cells, deacetylation of HSF-1 by SIRT1 potentiates the
transactivation of heat-shock genes such as Hsp70, whereas down-regulation of SIRT1 attenuates
theheat-shockresponse[90]. SIRT1interactswithFOXO1,ananti-agingtranscriptionfactorwhich
is negatively regulated by Akt [91,92]. OO could have effect on proteostasis by means of SIRT1
activation. Inskeletalmusclecells, treatmentwitholeicacid(OA)increasedintracellularlevelsof
cyclicadenosinemonophosphate(cAMP)thatturnedonproteinkinaseAactivity. Thisresultedin
SIRT1phosphorylationatSer-434andelevationofitscatalyticdeacetylaseactivity[93].
In relation to proteasome, continuous treatment with oleuropein has shown to increase
proteasome-mediated degradation rates in early passage human embryonic fibroblasts cultures,
possiblythroughconformationalchangesoftheproteasome.Asconsequence,adecreaseintheamount
of oxidized proteins was also observed. In addition, oleuropein-treated cells retained proteasome
functionduringreplicativesenescenceandculturesexhibitedadelayintheappearanceofsenescence
morphology and their life span was extended by approximately 15% [94] which confirmed the
importanceofproteasomefunctioninagingprocesses. Furthermore,thisindicatesthatcertainextracts
from olive leaf may be beneficial with respect to proteasome activity. The possibility exists that
oleuropein derivatives found in greater concentration in EVOO might exert similar effects, which
remainsforfurtherinvestigation. Indeed,therearemorecompoundsshowntoinfluenceexpressionof
factorsimplicatedincellsignalingpathwaysthatcontaincomponentsoftheproteasome-ubiquitin
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system. Regardingautophagy,ithasbeenreportedthatEVOOfeedingincreasedmRNAlevelsofthe
authophagymarkerLC3withrespecttootherdietaryfatsources(SOandfishoil)in24monthsoldrat
gums. Inthesamestudy,levelsofmRNAwerealsomeasuredinratsaged6months,butLC3change
wasonlyclearlynotedinoldanimals[95]. Inturn,dietarysupplementationwithEVOOdecreased
mRNAslevelsoftheautophagymarkersLC3andBeclin1inmuscleofatransgenicmousemodelof
amyotrophiclateralsclerosis,amongothereffects[96]. Theseresultsareconsistentwiththeideathat
VOOtargetsotherprocessesthatpreventproteindamageinthistissueand,consequently,lessprotein
removalisneeded.
Research focused on the effects of different compounds on cell signaling cascades has also
suppliedinterestingresultsconcerningautophagycontrol. Inparticular, themammaliantargetof
rapamycin (mTOR) have spurred extraordinary interest after the demonstration that constant or
intermittent administration of its inhibitor, rapamycin, can increase the lifespan of middle-aged
mice[97,98]anddelaysmultipleaspectsofaginginmice[99]. Inmostofcases, theactionsofOO
compounds on mTOR-dependent signaling were accompanied by higher surveillance and stress
resistance. Part of these effects also could be caused by effects on other processes regardless of
autophagy,asmanyantioxidantdefensesystemssharepartofthesesignalingcascades[100]. Since
mTORrouteisspecificallyinvolvedinnutrientsensing,anotherwell-establishedhallmarkofaging,
theeffectofOOcomponentsonthemTORkinasewillbedescribedinthenextsection(seeSection6:
OliveoilandNutrientSensingPathways).
The effects of fatty acids and other minor compounds from EVOO on the unfolded protein
response(UPR),aspecificpathwayaimedtorestoreproteostaisinendoplasmicreticulum(ER),have
beenalsoextensivelystudied. Thispathwaymainlyoperatesbyattenuatingproteinsynthesisand
importtoER,andbyactivatingacascadeoftranscriptionfactorsthatregulategenesencodingforER
chaperones,componentsoftheER-associatedproteindegradationsystem(i.e.,ubiquitin-proteasome
components),andcomponentsoftheautophagymachinery[101,102]. Asinothercellcompartments,
unfoldedormisfoldedproteinscanaccumulateintheERlumen,aggregateandhencebecometoxic
anddetrimentaltocellsurvival[101]. FacingthesesituationscollectivelyreferredasERstress,cells
haveevolvedsystemstodetect,eliminateandavoidfurtheraccumulationofunfoldedormisfolded
proteins. ERstressseemstooccurincorrelationwithsenescence,leadingtoactivationofUPR[101].
Actually,somechronicdiseaseshavebeenassociatedwithapoptosisornecrosisderivedfromERstress
includingdiabetesmellitus[103]ordiabeticnephropathy[104].
Two invitro studies have shown that, in comparison to SFA, MUFA as present in OO could
preventordecreaseapoptosisand/ornecrosisderivedfromERstress[103,104]. InINS-1Eβ-cells,
combination of oleate with palmitate prevents apoptosis and viability decrease that is otherwise
notedwhencellsaretreatedwithpalmitatealone. Inaddition,oleatealsopreventstheincreaseof
UPRmarkerassociatedtopalmitate,whichsuggestedthatoleatepreventER-stressandthus,UPR
isnotneeded. Ontheotherhand,mRNAexpressionoftheERchaperones(Bip,Pdi,Calnexinand
Grp94)wasnotalteredbyfattyacidsinthismodel[103]. ThissuggeststhatoleateprotectsINS-1E
β-cellsfrompalmitate-inducedapoptosisbythesuppressionofERstresswhichwasindependenton
chaperoneactivation. Theanalysisofthedatafromdifferentstudiesdoesnothighlightanydifferences
regarding the cell type [101]. The activation of the UPR seems to occur in all types of senescence,
whatevertheinducerissuccessivereplications[105,106],oncogeneactivation[107],DNA-damaging
agents[107,108],oroxidativestress[105]. However,theprecisesignatureofERstressvariesaccording
tothecontext[101]. Inthesamesense,analysisofSCD1knockdownmodelsthatleadtoanincreaseof
SFAandadecreaseofMUFAhavebeenassociatedtoseveralmarkersofUPRactivationindifferent
cell cultures [57]. In other study carried out at a cellular level, such intervention has shown to
increasepalmitate-inducedERstressandapoptosis,whereasoverexpressionofSCD2thatincreased
desaturationofpalmitatetoMUFA,attenuatedpalmitate-inducedERstressandapoptosis[109].
Accordingtothesefindings,adecreaseinmembraneMUFAwouldinduceERstressand,asa
consequence,UPRwouldbeactivatedtorestorehomeostasis. Onthecontrary,anincreaseofMUFA
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makescellslesssusceptibletoSFA-inducedERstressandapoptosis.OOhasbeenrelatedtoanincrease
ofMUFAmembranecontent[58].Thus,basedonthefattyacidsprofileinOO,thisfoodshoulddecrease
accumulationofmisfoldedandunfoldedproteinsinERandpossiblyinotherorganelles,although
the mechanisms under this effect need to be explained. Similarly, there are also invitro evidences
foreffectsofotherminorcompoundsonthissense. AnEVOOsecoiridoid-richextractupregulated
thetranscriptionalexpressionoftheDNAJA4andDNAJC3genes,whichencodestheER-localized
DNAJfamilyofproteins[110]. TheinducedtranscriptionofDNAJgenesispartoftheUPR[111–114].
TheabilityofsecoiridoidsfromEVOOtoupregulateasetofgenesinvolvedintheERstressresponse
toaccumulationofunfoldedproteinsmayappeartoconflictwiththedemonstratedabilityofthese
compoundstostronglyinhibitthegrowthofhighlyaggressivebreastcancercells[53,115]. However,
theUPRisthemajorprotectiveandcompensatorymechanismthatenablescellstosurviveduring
ERstress. Secoiridoidspromotecelldeath-UPRbranchsignalingbyimpedingthealleviationofER
stress[116].
6. OliveOilandNutrientSensingPathways
Aging is characterizedby a deterioration ofhomeostatic maintenanceover time, leading toa
functionaldeclinewithincreasedriskfordiseaseanddeath.Thisprocessismetabolicallycharacterized
byinsulinresistance,changesinbodycomposition,andphysiologicaldeclinesinsexsteroids,growth
hormone(GH)andinsulin-likegrowthfactor-1(IGF-1)[117]. Thislatteralterationisindicativeofa
deregulationinanutrientsensingrouteduringaging,becausetheintracellularsignalingpathway
ofIGF-1issharedwiththatofinsulin,whichinformscellsofthepresenceoftheglucose,beingthen
regardedasinsulin/IGF-1signaling(IIS)[1]. Geneticmanipulationscarriedoutindifferentanimal
models as worms, flies and mice in order to down-regulate this route have resulted in extended
lifespan [118] but, paradoxically, levels of GH and IGF-1 show a decline during both normal and
prematureaging[119]. Thisapparentparadoxcanbeexplainedonthebasisofadefenseresponse
elicited by the organism in order to minimize cell growth and metabolism in a systemic damage
scene[120].
SincehighlevelsofIGF-1areoftenassociatedwithdifferentkindsofcancer,adefensiveresponse
againstdamagemayhavetheriskofbecomingdeleteriousandaggravatingaging. Inaccordance,
lowperipheralIGF-1levelsareassociatedwithincreasedriskofnumerouspathologicconditionsin
aginghumans,includingtype2diabetesmellitus,cardiovasculardiseases,sarcopenia,osteoporosis,
andfrailty[117,121]. Manyresearchgroupshavethustriedtopalliatethesedeleteriousalterations
usingnaturalsubstancessuchasOOanditsderivatives. Inthisway,Gonzálezandcolleagues[122]
firstpostulatedtheantidiabeticeffectofoleuropeinandHTinananimalmodelofalloxan-induced
diabetesmellitus. Similarstudieshavebeenperformedbyothergroupsthatstronglysupportthis
protectiveeffect. Al-AzzawieandAlhamdani[123]used3months-oldNewZealandmalerabbits
rendereddiabeticbyalloxantreatmentandagroupoftheseanimalsweregivenadailyoraldoseof
20mg/kgofoleuropeinfor16weeks. Bloodglucoselevelsweredecreasedinoleuropein-treatedgroup
incomparisonwiththediabeticcontrols. Anincreaseinoxidativestresswasshowninalldiabetic
rabbits but was decreased significantly by oleuropein after 16 weeks of treatment. In accordance,
enzymaticandnon-enzymaticantioxidantswerediminishedafterdiabetesinduction,butoleuropein
treatmentrevertedthiseffect,supportingthatoleuropeinattenuatedoxidativestressandenhanced
body´sownantioxidantdefenses.
Hamdenandpartners[124]testedthehypoglucemicandantioxidanteffectsofpurifiedHTand
WW-PE,bothinalloxan-induceddiabeticratstreatedfortwomonthsandthroughinvitromodels.
Phenolicswereextractedfromolivemillwastetoseparatetwofractions: oneofthemenrichedin
monomericphenolics(F1)andtheotheroneenrichedinoligomericandpolymericphenolics(F2).
Glucoselevelswerediminishedwithallthetreatmentswhereashepaticglycogenlevelswereincreased
withrespectthediabeticgroup. Animprovementofantioxidantdefenseandadecreaseinhepatic
andrenalindexoftoxicitywereobservedinthepresenceoftheOOphenolicsincomparisonwith
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thediabeticnonsupplementedgroup. Thehistologicalanalysesrevealedthereductionoffattycysts
inliver, thedecreaseoffattyinfiltrationinkidney, andtheimprovementofvasculardegenerative
damageandnumberofβ-cellsinpancreas,beingtheseobservationsindicativeoftissueprotection
againstdeleteriousdiabeticalterations. Invitroassayswerecarriedoutinparallelinperfusedpancreas
ofnon-diabeticratswithdifferentconcentrationsofglucoseplusF1, F2orHTtreatment. Ofnote,
whenpancreatictissuewasincubatedwithhighlevelsofglucosecombinedwithmonomericphenols
orHT,theorganincreasedinsulinsecretion,whichwasotherwiserepressedwhenmaintainedwith
high-glucosewithoutadditionaltreatment. Followingasimilarapproach, anotherresearchgroup
tested the antidiabetic and antioxidant activity of oleuropein and HT at 8 and 16 mg/kg of body
weightinalloxan-induceddiabeticWistarrats[125]. Resultsindicatedthatpolyphenolsadministration
significantlydecreasedbloodglucoseandincreasedhepaticglycogenlevels. Asreportedpreviously,
polyphenols attenuated oxidative stress and restored antioxidant defense systems that had been
down-regulated in diabetic rats. In addition, these beneficial effects followed a dose dependent
response,withthehighestconcentrationsofpolyphenolsshowingthemostprominenteffects.
More recently, de Bock and collaborators carried out a 30-week randomized, double-blinded,
placebocontrolled,crossovertrial[126]inordertotesttheeffectsofsupplementationwithanoliveleaf
extract(OLE)inmiddle-agedoverweightmen,whoaremorepronetobeinsulinresistant. Participants
receivedcapsuleswithOLEorplacebofor12weeks,andtheychangedthetreatmentafter6weeks
of washout period. Interestingly, OLE supplementation improved insulin sensitivity by 15% and
thepancreaticβ-cellsfunctionby28%. Thisisconsistentwiththedecreasedlevelsofglucoseand
insulinreachedinblood. Higherlevelsofinterleukin-6andIGFBP-1and-2wereobserved, while
liverfunction,lipidprofileandcarotidintimamediathicknessdidnotshowanychangesamongOLE
vs. placebo participants. Of note, this study demonstrated that two aspects of glucose regulation,
pancreaticβ-cellfunctionandinsulinsensitivity,wereimprovedinjust12weeksoftreatment.
BesidestheIIS,changesinothernutrient-sensingsystemsarealsoassociatedwithagingprocess.
OneofbestcharacterizedsystemsisthemTORkinasepathway,whichplaysaroleinsensinghigh
aminoacidconcentrationsintheorganism[1]. Geneticdown-regulationofoneofthecomplexeswhich
constitute the mTOR kinase, mTORC1, extends longevity in yeast, worms and flies [127]. Studies
carriedoutbyHarrisonetal.[128]inmiceusingrapamycin,awell-knowninhibitorofmTOR,have
shownextendedlongevityandareconsideredasthemostrobustchemicalinterventiontoincrease
lifespan in mammals [98]. Conversely, the abnormal activation of this serine/threonine kinase is
associatedwithadecreaseinlifespanand,furthermore,isfrequentlyobservedindiseasesassociated
withaging,suchascancer[128,129],Alzheimer´sdisease[130,131]anddiabetes[132].
SeveralresearchgroupshavetesteddifferentkindofsubstancesabletoinhibitmTORkinase,
considered as a good target for the treatment of cancer [133]. Evidences from independent
studies carried out with cell cultures have indicated that compounds contained in EVOO could
target differentially mTOR signaling cascades. On the one hand, HT was shown to activate the
mTOR/p70S6-kinasepathwaysinculturedhumanretinalpigmentepithelialcellsfromtheARPE-19
cell line [100], and a gene expression study reported that OA increased mTOR pathway genes
expression in preadipocytes cultures [134]. On the other hand, oleocanthal, which exhibits potent
anticancerandneuroprotectiveactivities,inhibitedtheenzymaticactivityofmTORinotherinvitro
assay,andtheproposalwasmadethatbeneficialactivityofoleocanthalmightbedue,atleastinpart,
tomTORinhibition[135].InhibitoryeffectsonmTORactivitybydifferentconcentrationsofoleocanthal
wereclearlyobserved,whichledtoantiproliferativeeffectsinMCF-7,T47DandMDA-MB-231breast
cancercelllines,withthelattercellsbeingthemostaffected. Howeverpoorantiproliferativeeffects
wereshownincolorectal(Caco-2)andcervical(HeLa)cancercelllines. Thiscouldbeduetothefact
thatmTORishighlyexpressedinbreastcancercelllines. Inaddition,analysesofHGF-inducedmTOR
phosphorylationwerecarriedoutinMDA-MB-231cells,eitherintheabsenceorinthepresenceof
oleocanthal,andtheresultsindicatedthatthissecoiridoidwasabletosuppressmTORphosphorylation
bymorethan60%withoutalteringitstotallevels. Insum,theseevidencesstronglysupporttheidea