Pollock oil supplementation modulates hyperlipidemia and ameliorates hepatic steatosis in mice fed a high-fat diet

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Description

Hyperlipidemia associated with obesity is closely related to the development of atherosclerosis. Both n-3 polyunsaturated fatty acids (PUFAs) and long-chain monounsaturated fatty acids (MUFAs; i.e., C20:1 and C22:1 isomers) supplementation modulate risk factors for metabolic syndrome via multiple mechanisms, including the restoration of impaired lipid metabolism. We therefore examined the effects of pollock oil, which contains a considerable amount of n-3 PUFAs as well as long-chain MUFAs, on plasma hyperlipidemia and hepatic steatosis in diet-induced obese mice. Methods Male C57BL/6J mice (24-26 g) were divided into two groups (n = 10/group) and were fed a high-fat diet containing 32% lard (control group) or 17% lard plus 15% pollock oil (experimental group) for 6 weeks. For both groups, fat comprised 60% of the total caloric intake. Results Although body and liver masses for the two groups did not differ significantly, hepatic lipids concentrations (triglycerides and total cholesterols) were lower ( P < 0.05) after pollock oil ingestion. After 2 weeks on the specified diets, plasma lipid levels (total cholesterol, LDL cholesterol, and triglycerides) significantly decreased ( P < 0.05) in the experimental group compared with the control group, although plasma HDL cholesterol levels did not differ. At the end of 6 weeks, plasma adiponectin levels increased ( P < 0.05), whereas plasma resistin and leptin levels decreased ( P < 0.05) in the experimental mice. Increased levels of long-chain MUFAs and n-3 PUFAs in plasma, liver and adipose tissue by ingesting pollock oil were possibly correlated to these favorable changes. Expression of hepatic genes involved in cholesterol metabolism ( SREBP2 , HMGCR , and ApoB ) and lipogenesis ( SREPB1c , SCD-1 , FAS , and Acac α) was suppressed in the experimental group, and may have favorably affected hyperlipidemia and hepatic steatosis induced by the high-fat diet. Conclusions We demonstrated that pollock oil supplementation effectively improved hyperlipidemia, attenuated hepatic steatosis, and downregulated the express of hepatic genes involved in cholesterol and lipid metabolism in mice with diet-induced obesity.

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Publié le 01 janvier 2011
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Langue English
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RESEARCH

OpenAccess

Pollockoilsupplementationmodulates
hyperlipidemiaandameliorateshepaticsteatosis
inmicefedahigh-fatdiet
Zhi-HongYang
*
,HirokoMiyahara,JiroTakeo,AkimasaHatanakaandMasashiKatayama

Abstract
Background:
Hyperlipidemiaassociatedwithobesityiscloselyrelatedtothedevelopmentofatherosclerosis.Both
n-3polyunsaturatedfattyacids(PUFAs)andlong-chainmonounsaturatedfattyacids(MUFAs;i.e.,C20:1andC22:1
isomers)supplementationmodulateriskfactorsformetabolicsyndromeviamultiplemechanisms,includingthe
restorationofimpairedlipidmetabolism.Wethereforeexaminedtheeffectsofpollockoil,whichcontainsa
considerableamountofn-3PUFAsaswellaslong-chainMUFAs,onplasmahyperlipidemiaandhepaticsteatosisin
diet-inducedobesemice.
Methods:
MaleC57BL/6Jmice(24-26g)weredividedintotwogroups(n=10/group)andwerefedahigh-fat
dietcontaining32%lard(controlgroup)or17%lardplus15%pollockoil(experimentalgroup)for6weeks.For
bothgroups,fatcomprised60%ofthetotalcaloricintake.
Results:
Althoughbodyandlivermassesforthetwogroupsdidnotdiffersignificantly,hepaticlipids
concentrations(triglyceridesandtotalcholesterols)werelower(
P
<0.05)afterpollockoilingestion.After2weeks
onthespecifieddiets,plasmalipidlevels(totalcholesterol,LDLcholesterol,andtriglycerides)significantly
decreased(
P
<0.05)intheexperimentalgroupcomparedwiththecontrolgroup,althoughplasmaHDL
cholesterollevelsdidnotdiffer.Attheendof6weeks,plasmaadiponectinlevelsincreased(
P
<0.05),whereas
plasmaresistinandleptinlevelsdecreased(
P
<0.05)intheexperimentalmice.Increasedlevelsoflong-chain
MUFAsandn-3PUFAsinplasma,liverandadiposetissuebyingestingpollockoilwerepossiblycorrelatedtothese
favorablechanges.Expressionofhepaticgenesinvolvedincholesterolmetabolism(
SREBP2
,
HMGCR
,and
ApoB
)and
lipogenesis(
SREPB1c
,
SCD-1
,
FAS
,and
Acac
a
)wassuppressedintheexperimentalgroup,andmayhavefavorably
affectedhyperlipidemiaandhepaticsteatosisinducedbythehigh-fatdiet.
Conclusions:
Wedemonstratedthatpollockoilsupplementationeffectivelyimprovedhyperlipidemia,attenuated
hepaticsteatosis,anddownregulatedtheexpressofhepaticgenesinvolvedincholesterolandlipidmetabolismin
micewithdiet-inducedobesity.
Keywords:
Pollockoil,n-3PUFA,MUFA,hyperlipidemia,hepaticsteatosis,adipokines

Background
consequenceofhyperlipidemiaareoxidizedandattract
Hyperlipidemia,amedicalconditioncharacterizedbyinflammatorymonocytes,whichdifferentiateinto
increasedbloodlevelsoflipidsincludingcholesterolandmacrophagesthattakeuptheoxidizedlipid.Oxidation
triglycerides,isacriticalcomponentofmetabolismsyn-oflow-densitylipoproteins(LDLs)inthearterialwallis
dromeaswellasapossiblepredisposingfactorforamajorandphysiologicallyrelevantmechanismforthe
atherosclerosis,aleadingcauseofdeathworldwide[1,2].pathogenesisofatherosclerosis,andthepresenceof
Lipidsthataccumulateinthearterialwallasalipid-loadedmacrophagefoamcellsinthearteryintima
isapredictorforthedevelopmentofatherosclerotic
*Correspondence:yangzh@nissui.co.jp
lesions.Acloserelationshipexistsbetweendietaryfats
CentralResearchLaboratory,TokyoInnovationCenter,NipponSuisanKaisha,
anddyslipidemia-relatedevents[3].Althoughan
Ltd.,32-3Nanakuni1ChomeHachioji,Tokyo192-0991,Japan
©2011Yangetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.

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increasedintakeofsaturatedfattyacidsispathogenicfor
Table1Fattyacidcompositionofdietaryfats(%)
coronaryheartdisease,numerousstudieshavedemon-
FattyacidLardPollockoil
stratedaprotectiveeffectofn-3polyunsaturatedfatty
C14:01.54.9
acids(PUFAs)throughavarietyofmechanisms,includ-
C16:025.49.8
ingreductionoftriglyceridesandvery-low-densitylipo-
C16:12.46.1
proteins[4].Inaddition,wehaveshownthatmarine-
C18:05.91.7
derivedlong-chainmonounsaturatedfattyacids
C18:140.614.3
(MUFAs)(i.e.,C20:1andC22:1isomers)modulate
C18:2n-610.81.3
metabolicsyndromebyrestoringimpairedglucoseand
C18:3n-31.01.1
lipidmetabolism[5].Therefore,fishoilsthatarerichin
C20:1n-90.89.1
bothn-3PUFAsandlong-chainMUFAsmayhelpalle-
C20:1n-7ND3.3
viatehypercholesterolemiaandhypertriacylglyceridemia.
C22:1n-11ND12.3
Alaskapollock(
Theragrachalcogramma
)isaNorth
C22:1n-9ND1.6
Pacificspeciesofthecodfamily,Gadidae.Pollockoil
C20:5n-30.0210.3
containsconsiderableamountsofn-3PUFAsandlong-
C22:5n-30.11.2
chainMUFAs[6].TheAlaskapollockfishingindustryis
C22:6n-30.037.9
thelargestintheUnitedStatesandoneofthelargestin
Valuescorrespondtomeanofthreeseparatesamplesprocessed
theworld.Inrecentyears,pollockfishinghasaccounted
independently.
for~30%ofallU.S.seafoodlandingsbymass[7].
ND:Notdetected.
Althoughpollockoilisusedinboththefoodandfeed
industries[8],littleisknownabouttherelationshipstudy.MaleC57BL/6Jmice(5weeksold)wereobtained
betweendietarypollockoilandhyperlipidemia.GivenfromCharlesRiverLaboratoriesJapanInc.(Yokohama,
thehealthbenefitsofn-3PUFAsandlong-chainMUFAs,Japan)andhousedatNihonBioresearchat23±1°C
weexaminedtheeffectofdietarypollockoilonhyperli-undera12/12hlight-darkcycle.Theanimalswerepro-
pidemiainmicewithdiet-induceddyslipidemia.videdfreeaccesstowaterandstandardmousechow
CRF-1(OrientalYeastCo.Ltd.,Tokyo,Japan)fora1-
Methods
weekacclimatizationperiod.
Measurementoffattyacidcompositionofdietaryoils
Afteracclimatization,micewererandomlyassignedto
CamerialardwaspurchasedfromRomiSmilfoodB.V.oneoftwogroupsforthe6-weekfeedingexperiment.The
(Heerenveen,Netherlands).Pollockoilwasobtainedcontrolgroup(n=10)wasfedahigh-fatdietcontaining
fromNipponSuisanKaisha,Ltd.(Tokyo,Japan)and32%lard(D12492RodentDietwith60kcal%Fat;Research
refinedwithsilicagelandactivatedclaysandthenDiets,Inc.,NJ,USA)andtheexperimentalgroupwasfeda
steam-distillationdeodorized.Allstandardandextracteddietsupplementedwithpollockoil(17%lardplus15%pol-
lipidswerestoredat-20°Cuntilused.Fattyacidcompo-lockoil).Tocontrolfortotalfatintake,thetotalfatcontent
sitionsofthedietaryfats(Table1)weredeterminedinbothdietscorrespondedto60%ofthecaloricintake.
aftermethylationofsampleswith14%(w/v)borontri-ThecompositionsofthedietsarelistedinTable2.Body
fluoride/methanol(SigmaChemicalCo.,St.Louis,massandfoodintakeweremonitoredthroughoutthe
USA.)at80°Cfor30min.Theresultingfattyacidstudy.Attheendoftheinterventionperiod,micewere
methylesterswerequantifiedbygaschromatography
usinganAgilent6890NNetworkGasChromatograph
System(AgilentTechnologiesJapan,Ltd.,Tokyo,Japan)
Table2Dietcompositions
equippedwithasplitinjector,FIDdetector,andfused
IngredientLarddiet(g/100gdiet)POdiet(g/100gdiet)
silicacapillarycolumn(DB-WAX,30m×0.25mmI.D.
Casein25.825.8
×0.25
μ
mfilmthickness,J&WScientific,Agilent
l-Cysteine0.40.4
Technologies).Methylesterswereidentifiedbycompari-
Maltodextrin1016.216.2
sonofretentiontimestothoseoffattyacidmethylester
Sucrose8.98.9
standards(Nu-ChekPrep,Inc.,Elysian,MN,USA).Pol-
Cellulose6.56.5
lockoilcontainsconsiderablelevelsoflong-chain
Mineralmixture1.31.3
MUFAsandn-3PUFAs(C20:1aswellasC22:1isomers
Vitaminmixture1.31.3
andn-3PUFAscombined:~45%).
Cholinebitartrate0.30.3
Soybeanoil3.23.2
Animalsanddiets
Lard3217
TheInstitutionalAnimalCareandUseCommitteeat
Pollockoil

15
NihonBioresearchInc.(Gifu,Japan)approvedthis
POdiet:Pollockoil-supplementeddiet.

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anesthetizedwith4%sodiumpentobarbital(Dainippon
SumitomoPharma,Osaka,Japan),andbloodwascollected
byabdominalveinpuncture.Plasmawasobtainedbycen-
trifugationat1000gfor15minandstoredat-80°Cuntil
analyses.Vitalorganswereremovedandweighedaftera
shortwashincoldphosphate-bufferedsaline,pH7.4.
Mesentericwhiteadiposetissue(WAT)andLiverswere
keptat-80°Cforthefurtherlipidextractionandquantita-
tivepolymerasechainreaction(QPCR)analysis.
Lipidextractionandfattyacidanalysis
Thefattyacidcompositionsofplasma,liverandWAT
intheC57BL/6Jmiceweredeterminedasdescribed
before[5].Lipidswereextractedbyhomogenizingthe
tissuesamplesinamethanol/hexanesolution(4:1v/v)
addedwithbutylatedhydroxytoluene(BHT,50
μ
g/mL)
asanantioxidant.Thesamplesweremethylatedwith
acetylchloride,andthefattyacidmethylesterswere
separatedandquantifiedbygaschromatography.Identi-
ficationofthemethylestersweremadebycomparison
ofretentiontimesofstandardfattyacids.
Determinationofplasmalipidlevels
Bloodsamplesweretakenfromtheretro-orbitalvenous
plexusofeachmouseattheendofweeks0,2,4,and6.
Plasmaconcentrationsoftriglyceride(TG),totalcholes-
terol(TC),andhigh-densitylipoprotein(HDL)cholesterol
weremeasuredusingTriglycerolE-Test,CholesterolE-
Test,andHDL-cholesterolE-Testkits(WakoPureChe-
micalIndustries,Ltd.,Osaka,Japan),respectively.The
concentrationofLDLcholesterolwascalculatedas[LDL
cholesterol]=[TC]-[HDLcholesterol]-[TG]×0.2.
Determinationofplasmaadipokinelevels
Plasmaconcentrationsofadiponectin,resistin,andlep-
tinweredeterminedattheendofthe6-weekperiod
usingtheMouseAdiponectinELISAkit(OtsukaPhar-
maceuticalCo.,Ltd.,Tokyo,Japan),MouseResistin
ELISAkit(ShibayagiCo.Ltd.,Gunma,Japan),and
MouseLeptinELISAkit(MorinagaInstituteofBiologi-
calScience,Inc.,Yokohama,Japan),respectively.

Determinationofhepaticlipidlevels
Totalhepaticlipidswereextractedfromliversamplesas
described[9].Extractedlipidsweredriedundervacuum
(ConcentratorPlus5305,EppendorfInc.,NY,USA)and
thendissolvedin2-propanolcontaining10%(w/w)Tri-
tonX-100.Triglycerideandtotalcholesterolconcentra-
tionsweredeterminedusingtheabove-mentioned
commercialenzymekits(Wako).

DeterminationofmRNAexpressionbyQPCR
TotalRNAwasisolatedfromliversamplesusingTRIzol
reagent(Qiagen,Valencia,CA,USA)accordingtothe

Page3of10

manufacturer

sprotocol.First-strandcDNAwasgener-
atedfromtotalRNA(1
μ
g)usingthePrimeScriptII1st
strandcDNASynthesiskit(TaKaRaBio,Otsu,Japan).
TheresultingcDNAwasusedforQPCRamplification
andspecificsequencedetectiononanAppliedBiosys-
tems7300Real-TimePCRSystem(LifeTechnologies
Ltd.,Tokyo,Japan).ThePCRcyclingparameterswere
30sat95°C;40cyclesof5sat95°C,34sat60°C;and
afinalmeltingcurveof15sat95°C,1minat60°C,15
sat95°C.Geneexpressionwasscaledtotheexpression
ofthehousekeepinggeneencoding18SribosomalRNA.
PCRreactionscontainedforwardandreverseprimers
(10
μ
Meach)andSYBRPremixExTaq(TaKaRaBio).
Thetargetedgenes,theircorrespondingproteins,and
therespectivesenseandantisensePCRprimerswere:
SREBP2
(sterolregulatoryelementbindingprotein2),
5

-TGGGCGATGAGCTGACTCT-3

and5

-ACTG-
TAGCATCTCGTCGATGT-3

;
HMGCR
(3-hydroxy-3-
methylglutaryl-coenzymeAreductase),5

-TGTTCACC
GGCAACAACAAGA-3

and5

-CCGCGTTATCGT-
CAGGATGA-3

;
ApoB
(apolipoproteinB),5

-TTGGCA
AACTGCATAGCATCC-3

and5

-TCAAATTGG-
GACTCTCCTTTAGC-3

;
ApoA
(apolipoproteinA),5

-
GGCACGTATGGCAGCAAGAT-3

and5

-CCAAG-
GAGGAGGATTCAAACTG-3

;
SREBP1c
(sterolregula-
toryelementbindingprotein1c),5

-GATGTGC
GAACTGGACACAG-3

and5

-CATAGGGGGCGT-
CAAACAG-3

;
SCD-1
(stearoyl-coenzymeAdesaturase-
1),5

-TTCTTGCGATACACTCTGGTGC-3

and5

-
CGGGATTGAATGTTCTTGTCGT-3

;
FAS
(fattyacid
synthase),5

-TTCTTGCGATACACTCTGGTGC-3

and
5

-CGGGATTGAATGTTCTTGTCGT-3

;
Acac
a
(acetyl-coenzymeAcarboxylasealpha),5

-GATGAAC-
CATCTCCGTTGGC-3

and5

-CCCAATTATGAATC
GGGAGTGC-3

.
Statisticalanalysis
Resultsareexpressedasmean±standarderrorofthe
mean.Statisticaldifferencesbetweentwogroupswere
analyzedbyStudent

s
t
-testandwereconsideredsignifi-
cantat
P
<0.05.

Results
Effectofpollockoilonbodyandorganmass
Table3listsbodyandvitalorganmassesfordiet-
inducedobeseC57BL/6Jmiceinthecontrol(lard)and
experimental(pollockoil)groups.Afterthe6-week
feedingperiod,therewerenosignificantdifferencesin
body,liver,whiteaswellasbrownadiposetissue,and
skeletonmusclemassesbetweenthetwogroups.

Effectofpollockoilonplasmalipidlevels
Comparedtocontrol,totalcholesterolplasmalevelsfor
theexperimentalgroupwerereducedby27%(
P
<0.01),

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Table3Foodintake,bodymass,andvitalorganmassesTable5Fattyacidcompositioninplasma(%)
LarddietPOdietFattyacidLarddietPOdiet
Foodintake(g/day)2.5±0.032.3±0.0414:00.20±0.020.29±0.02
Initialbodymass(g)25.5±0.425.5±0.516:013.95±0.3311.24±1.17
Finalbodymass(g)32.7±1.631.6±2.418:08.68±0.246.80±0.86
Organmasses(mg/gbodymass)SAF22.84±0.4718.33±2.03
Liver33.1±0.733.6±0.712:123.68±0.9024.71±1.84
EpididymalWAT50.9±3.045.2±3.916:10.69±0.100.79±0.08
MesentericWAT15.4±0.913.7±1.118:19.23±0.377.77±0.52*
Brownadiposetissue3.3±0.53.6±0.320:1n-90.18±0.010.24±0.02*
Skeletalmuscle5.9±0.45.9±0.320:1n-7ND0.22±0.02***
Eachvaluerepresentsthemean±SE(n=10).POdiet:Pollockoil-
22:1n-11ND0.17±0.02**
supplementeddiet;WAT:whiteadiposetissue.
22:1n-9ND0.01±0
MUFA33.77±0.9733.91±2.42
17%(
P
<0.01),and30%(
P
<0.001)attheendofweeks
18:2n-617.04±0.2510.81±1.13**
2,4,and6,respectively(Table4).Pollockoilintakealso
18:3n-60.26±0.010.08±0.01***
significantlyreducedplasmaLDLcholesterollevelsby
20:2n-60.13±00.07±0.01*
33%(
P
<0.05),23%(
P
<0.01),and38%(
P
<0.001),
20:3n-60.89±0.070.29±0.04***
andreducedplasmatriglyceridelevelsby40%(
P
<
20:4n-614.03±0.573.61±0.36***
0.01),50%(
P
<0.01),and46%(
P
<0.01)attheendof
22:4n-60.02±00.02±0
weeks2,4,and6,respectively.Nosignificantdifferences
n-6PUFA32.37±0.3814.87±1.54***
inplasmaHDLcholesterollevelsweredetected.
18:3n-30.18±0.020.16±0.02
20:5n-30.40±0.036.32±0.81**
Effectofpollockoilonfattyacidcompositionsofplasma,
22:5n-30.32±0.074.71±1.76**
liverandWAT
22:6n-35.11±0.1111.62±2.04**
Plasma,liverandmesentericWATfattyacidcomposi-
n-3PUFA6.01±0.1122.85±2.18**
tionsinthecontrolandpollockoilgroupareshownin
Eachvaluerepresentsthemean±SE(n=10).ND:Notdetected;POdiet:
Table5andTable6.Althoughtotalsaturatedfattyacid
Pollockoil-supplementeddiet;SAF:saturatedfattyacids;MUFA:
levelsdidnotdifferbetweenthecontrolandexperimen-
monounsaturatedfattyacids;PUFA:polyunsaturatedfattyacids;*
P
<0.05;**
P
<0.01;***
P
<0.001.
talgroup,pollockoilingestionmarkedly(
P
<0.05)
increasedlong-chainMUFA(i.e.,C20:1andC22:1iso-
merscombined)levels3-,1.2-and5-foldinplasma,liverandWAT,respectively.Intakeofpollockoilalso
significantly(
P
<0.05)increasedtotaln-3PUFAlevels
3-,2-and7-foldinplasma,liverandWAT,respectively.
Table4Plasmalevelsoftotalcholesterol,LDL
Incontrast,totaln-6PUFAlevelsweresignificantly(
P
<
cholesterol,HDLcholesterol,andtriglycerides
0.05)decreasedby50%,31%and14%inplasma,liver
Lipids(mg/dL)Week0Week2Week4Week6
andWAT,respectivelyinthepollockoilgroupascom-
Total
paredtothecontrol.
cholesterol
Larddiet84.7±7.1154.5±10.5158.1±4.5161.5±4.3
Effectofpollockoilonplasmaadipocytokinelevels
POdiet80.8±6.0112.9±3.7**130.8±5.7**113.1±2.7***
Todetermineifpollockoilcouldrelievetheadipokine
LDL
dysregulationcausedbyexcessivetotalcaloricintake,
cholesterol
wemeasuredtheadipokineplasmalevels.Intakeofpol-
Larddiet39.1±7.083.5±9.387.7±3.478.5±3.5
lockoilincreasedtheplasmaconcentrationofadiponec-
POdiet38.1±5.656.1±3.1*67.4±5.4**56.6±2.5***
tinby15%(
P
<0.05)intheexperimentalgroup
HLDcholesterol
comparedtothecontrolgroup(Figure1A).Plasma
Larddiet34.6±2.054.4±2.851.4±2.059.2±2.5
resistinandleptinconcentrationswerereducedby14%
POdiet30.6±2.447.3±1.853.7±2.743.7±3.4
(
P
<0.05)and41%(
P
<0.05),respectively(Figure1B
Triglycerides
and1C).
Larddiet56.4±4.480.0±10.195.7±12.9118.7±19.8
POdiet60.6±4.947.7±6.7**48.2±6.8**63.9±5.7**
Effectofpollockoilonhepaticsteatosis
Becauseobesitycantriggerhepaticsteatosis,whichis
Eachvaluerepresentsthemean±SE(n=10).POdiet:Pollockoil-
supplementeddiet;LDL:low-densitylipoprotein;HDL:high-density
associatedwithhyperlipidemia,wemeasuredthelevels
lipoprotein;*
P
<0.05;**
P
<0.01;***
P
<0.001.
ofhepaticlipidstodetermineifpollockoilsuppressed

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Table6Fattyacidcompositioninliverandmesenteric
WAT(%)
FattyacidLiver
MesentericWAT
LarddietPOdietLarddietPOdiet
14:00.29±0.010.30±0.011.03±0.031.85±0.03*
16:021.47±0.1320.18±0.24***20.43±0.1119.43±0.23**
18:08.09±0.1310.06±0.21***4.39±0.115.02±0.15**
SAF29.85±0.1530.55±0.1725.85±0.1526.29±0.22
12:17.37±0.297.87±0.282.11±0.101.98±0.09
16:11.46±0.051.25±0.07*4.05±0.213.94±0.17
18:122.55±0.3513.58±0.47***45.71±0.1537.42±0.33***
20:1n-90.51±0.020.60±0.02**0.85±0.033.12±0.13***
20:1n-7ND0.41±0.01***0.15±01.55±0.05***
22:1n-11ND0.19±0.01***0.05±01.37±0.08***
22:1n-90.06±00.06±0.010.04±00.25±0.01**
MUFA32.05±0.3223.86±0.51***52.95±0.2349.63±0.51**
18:2n-614.13±0.2811.89±0.16***14.47±0.1712.70±0.28***
18:3n-60.29±0.010.10±0.03***0.07±00.06±0.01
20:2n-60.22±00.12±0***0.27±00.17±0.03*
20:3n-60.86±0.040.47±0.03***0.12±00.06±0***
20:4n-69.31±0.154.66±0.09***0.24±0.010.15±0***
22:4n-60.31±0.010.03±0***0.07±00.03±0***
n-6PUFA25.11±0.1817.26±0.18***15.23±0.1713.17±0.28***
18:3n-30.45±0.020.56±0.03**0.07±00.96±0.04***
20:5n-30.29±0.014.84±0.17***0.10±00.73±0.05***
22:5n-30.51±0.011.66±0.07***0.11±00.44±0.01***
22:6n-36.32±0.1215.36±0.26***0.18±01.42±0.04***
n-3PUFA7.55±0.1222.42±0.18***0.45±0.013.54±0.12***
Eachvaluerepresentsthemean±SE(n=10).ND:Notdetected;WAT:white
aacdiidpso;sMeUtiFssA:uem;oPnOoduinest:atPuorlaltoecdkfoaitl-tsyuapcpildes;mPeUntFeAd:pdioelyt;uSnAsaFt:usraattuerdatfeadttfyatatcyids;
*
P
<0.05;**
P
<0.01;***
P
<0.001.
hepaticlipidaccumulation.Totalhepaticlipid,triglycer-
ide,andtotalcholesterollevelswerereducedby21%(
P
<0.01),40%(
P
<0.001),and12%(
P
<0.05),respec-
tively,intheexperimentalgroupcomparedwiththe
controlgroup(Figure2).
EffectofpollockoilonmRNAexpressionofgenes
involvedinhepaticlipidmetabolism
Thepollockoil-supplementeddietledtoa66%reduction
(
P
<0.001)inmRNAexpressionof
SREBP2
,agenethat
encodesatranscriptionfactorinvolvedmainlyinregula-
tionofcholesterolsynthesis(Figure3A).Italsoreduced
expressionofthecholesterogenicgene
HMGCR
by51%
(
P
<0.05)andof
ApoB
by23%(
P
<0.05),althoughithad
nosignificanteffectonexpressionof
ApoA
.Furthermore,
intakeofpollockoilcauseda24%(
P
<0.05)reductionin
mRNAexpressionof
SREBP1c
,whichencodesforthe
lipogenictranscriptionfactorSREBP1c,andalsoreduced
expressionofthedownstreamlipogenicgenes
SCD-1
,
FAS
,and
Acac
a
by68%(
P
<0.001),69%(
P
<0.001),and
33%(
P
<0.05),respectively(Figure3B).

Page5of10

Discussion
Peoplewithobesityhaveanincreasedriskofcardiovas-
culardisease,whichisamajorcauseoftheirincreased
mortality.Althoughmultiplefactorsarethoughttocon-
tributetotheseelevatedrisks,onemaindeterminantis
theadverseeffectofobesityonlipoproteinlevels[10].
Forthestudyreportedherein,weexaminedtheeffectof
pollockoilondyslipidemiaindiet-inducedobesemice,
andshowedthatinclusionofpollockoilinahigh-fat
dietsignificantlydecreasedplasmalevelsoftotaland
LDLcholesterolandtriglyceride.
Toidentifypossiblemechanismsunderlyingthis
reduction,wemeasuredplasmaadipokinelevels.Adipo-
nectin,anadipocyte-derivedhormone,isbelievedto
playanimportantroleinregulatinghyperglycemia,
hyperlipidemia,andendothelialdysfunctioninhumans,
allofwhichprobablycontributetocertainmarkedly
increasedrisksassociatedwithobesity-relateddisorders,
e.g.,atherosclerosisanddiabetes[11].Adiponectinlevels
correlatenegativelywithserumtriglyceridelevelsin
non-diabeticsubjectsandtype-2diabetics,andhypoadi-
ponectinemiaisassociatedwithsmallerLDLparticle
size,indicatingalinkbetweenadiponectinanddyslipi-
demia[12-15].Furthermore,
invitro
dataindicatethat
adiponectinisananti-inflammatoryandanti-prolifera-
tivemediatorthatcanmodulateatherosclerosisprogres-
sion[16-18].Conversely,increasedlevelsof
proinflammatoryadipokines(e.g.,tumornecrosisfactor
a
,resistin,monocytechemotacticprotein1,andinter-
leukin8)havebeenassociatedwithincreasedserum
lipids(e.g.,triglycerides,andtotalandLDLcholesterol),
whichincreasemonocyterecruitmentandadherenceto
arterialwalls,causingwallremodeling[19-22].Inaddi-
tion,leptin,thefirstadipocytokinediscovered,hasbeen
associatedwithdevelopmentofobesity,as
ob
/
ob
leptin-
deficientmicearemarkedlyobese[23].However,most
obesehumanshaveincreasedbloodleptinconcentra-
tions,likelyreflectingresistancetotheaction(s)ofleptin
[24].Leptinisalsoassociatedwithincreasedinsulin
resistance,whichcancausehyperlipidemia[25].There-
fore,thedecreasedplasmalipidconcentrationsobserved
followingpollockoilingestionwerepossiblyassociated
withelevatedplasmaadiponectinlevelsandreduced
plasmaproinflammatoryadipokinelevels.Asendogen-
ousligandsforperoxisomeproliferator-activatedrecep-
tors,n-3PUFAsregulatetheexpressionofgenes
encodingkeyproteinsinvolvedinmetabolism[26,27].
Intakeoffishoilenrichedinn-3PUFAshasbeen
reportedtoincreaseplasmaadiponectinlevelsand
lowerproinflammatoryadipokinelevels[28,29].Further-
more,wehaveshownthatadietrichinlong-chain
MUFAsmodulatesadipokineprofiles[5].Fattyacid
compositionanalysesrevealedthatpollockoilingestion

hYtatnp:g//
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Page6of10

Figure1
Effectofpollockoilonplasmaadipokinelevelsinmicefedahigh-fatdiet
.Plasmalevelsofadiponectin(A),resistin(B),and
leptin(C)inC57BL/6Jmicefedfor6weekswithadietcontaining32%lard(larddiet)or17%lardplus15%pollockoil(POdiet).Valuesare
mean±SE(n=10).*
P
<0.05.

hYtatnp:g//
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ds
w
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o
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rld
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Page7of10

Figure2
Effectofpollockoilonhepaticsteatosisinmicefedahigh-fatdiet
.Totalhepaticlipid(A),triglyceride(B),andtotalcholesterol(C)
levelsinC57BL/6Jmicefedfor6weekswithadietcontaining32%lard(larddiet)or17%lardplus15%pollockoil(POdiet).Valuesaremean±
SE(n=10).*
P
<0.05;**
P
<0.01;***
P
<0.001.

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Page8of10

Figure3
Effectofpollockoilontranscriptionofgenesrelatedtohepaticcholesterolandlipidmetabolism
.mRNAlevelsforgenes
involvedincholesterol(A)andlipid(B)metabolisminC57BL/6Jmicefedfor6weekswithadietcontaining32%lard(larddiet)or17%lard
plus15%pollockoil(POdiet).Valuesaremean±SE(n=10).*
P
<0.05;***
P
<0.001.

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significantlyincreasedlong-chainMUFAandn-3PUFA
levelsinplasmaandvitalorgans.Therefore,thefavor-
ablechangesintheplasmaadipokineprofilemightbe
attributedtoacombinedeffectofn-3PUFAsandlong-
chainMUFAsfoundabundantlyinpollockoil.
Ingestionofpollockoilalsodecreasedhepaticlipid
levelsintheexperimentalgroup,indicatingthatpollock
oilinhibitedhepaticsteatosistriggeredbyobesity.The
liveristhemostimportantorganinenergymetabolism,
andisvitaltotheproductionandcatabolismofplasma
lipoproteinsandendogenouslipids[30,31].The
improvementinfattyfiltrationintheliversofpollock
oil-fedmicewaspossiblyrelatedtothedecreased
plasmalipidlevels.Tocharacterizehowpollockoil
decreasedhepaticlipidaccumulation,wealsodeter-
minedthehepaticmRNAlevelsofgenesrelatedtolipid
metabolism.Intheliver,sterolregulatoryelement-bind-
ingproteins(SREBPs)arekeytranscriptionfactorsthat
regulatethelevelsoflipidsproducedforexportintothe
plasmaaslipoproteinsandintothebileasmicelles.The
SREBPfamilyincludesSREBP1a,1c,and2[32].
SREBP1cregulatestranscriptionofgenesinvolvedin
fattyacidmetabolism(e.g.,
SCD-1
,
FAS
,and
Acac
a
),
andSREBP2regulatestranscriptionofcholesterol-
relatedgenes(e.g.,
HMGCR
,whichencodestherate-
limitingenzymeofcholesterolbiosynthesis).Increased
SREBPactivitycausescholesterolandfattyacidaccumu-
lationanddownregulatestheSREBP-cleavage-activating
protein(SCAP)/SREBPpathwaybyfeedbackinhibition
[33].Ourdatashowthat,concomitantwithdownregula-
tionofSREBP1candSREBP2mRNA,intakeofpollock
oilsuppressedtranscriptionofgenestargetedbySREBP.
Furthermore,pollockoilintakeinhibitedhepatic
ApoB
transcription.ApolipoproteinBiscentralinlipoprotein
metabolism[34],servingasastructuralandfunctional
componentoftriglyceride-richvery-low-densitylipopro-
teinsandtheirmetabolicproducts,e.g.,intermediate-
densitylipoproteinandLDL.Downregulationof
ApoB
expressionmaythereforedecreasecirculatingLDLcho-
lesterollevels.Notably,pollockoilingestiondidnot
altermRNAexpressionof
ApoA
,thestructuralcompo-
nentofHDL,whichmayreflectthefactthatplasma
HDLcholesterollevelswerethesameinthecontroland
experimentalgroups.Collectively,theobserved
decreasesinmRNAlevelssuggestthattheimprove-
mentsinhepaticlipidlevelsandassociatedhyperlipide-
miafoundintheexperimentalgroupmaybepartially
associatedwithdecreased
denovo
cholesterol,lipid,and
ApoBsynthesis.
Conclusions
Ourstudyshowedthatingestionofpollockoilamelio-
ratedhypercholesterolemiaandhypertriacylglyceridemia
indiet-inducedobesemice.Thehypolipidemiceffectof

Page9of10

pollockoilwaspossiblyrelatedtoanincreaseinplasma
adiponectinconcentrationsandadecreaseintheplasma
levelsofproinflammatoryadipokines.Downregulationof
mRNAexpressionoflipogenicgenesandgenesinvolved
incholesterolmetabolismpositivelyaffectedhepatic
lipidaccumulation,whichlikelyledtoanimproved
plasmalipidprofile.
Listofabbreviations
Acac
α
:acetyl-coenzymeAcarboxylasealpha;ApoA:apolipoproteinA;ApoB:
apolipoproteinB;FAS:fattyacidsynthase;HDL:high-densitylipoprotein;
HMGCR:3-hydroxy-3-methylglutaryl-coenzymeAreductase;LDL:low-density
lipoprotein;MUFA:monounsaturatedfattyacids;PUFA:polyunsaturatedfatty
acids;QPCR:quantitativepolymerasechainreaction;SCD-1:stearoyl-
coenzymeAdesaturase-1;SREBP:sterolregulatoryelementbindingprotein;
WAT:whiteadiposetissue.
Acknowledgements
WethankMr.NobushigeDoisakiandMs.KiyomiFurihatainNipponSuisan
Kaisha,Ltd.fortechnicalassistance.
Authors

contributions
ZHYparticipatedintheplanningofthestudy,dataanalysis,andmanuscript
preparation.HMparticipatedinexperimentalwork.JT,AHandMK
participatedintheplanningandorganizationofthestudy.Allauthorsread
andapprovedthefinalmanuscript.
Competinginterests
Theauthorsdeclarethattheyhavenocompetinginterests.
Received:21September2011Accepted:25October2011
Published:25October2011
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