Dietary Enrichment with Fish Oil Prevents High Fat-Induced Metabolic Dysfunction in Skeletal ...

19 Pages·2015·2 MB·English

RESEARCHARTICLE
Dietary Enrichment with Fish Oil Prevents
High Fat-Induced Metabolic Dysfunction in
Skeletal Muscle in Mice
LisaK.Philp*,LeonieK.Heilbronn,AlenaJanovska,GaryA.Wittert
DisciplineofMedicine,UniversityofAdelaide,Adelaide,SouthAustralia,Australia
* [email protected]
Abstract
a11111
Highsaturatedfat(HF-S)dietsincreaseintramyocellularlipid,aneffectamelioratedby
omega-3fattyacidsinvitroandinvivo,thoughlittleisknownaboutsex-andmusclefiber
type-specificeffects.Wecomparedeffectsofstandardchow,HF-S,and7.5%HF-Sre-
placedwithfishoil(HF-FO)dietsonthemetabolicprofileandlipidmetabolismgeneand
proteincontentinred(soleus)andwhite(extensordigitorumlongus)musclesofmaleand
OPENACCESS
femaleC57BL/6mice(n=9-12/group).WeightgainwassimilarinHF-S-andHF-FO-fed
Citation:PhilpLK,HeilbronnLK,JanovskaA,Wittert groups.HF-Sfeedingincreasedmesentericfatmassandlipidmarker,OilRedO,inredand
GA(2015)DietaryEnrichmentwithFishOilPrevents
mixedmuscle;HF-FOincreasedinterscapularbrownfatmass.Comparedtochow,HF-S
HighFat-InducedMetabolicDysfunctioninSkeletal
andHF-FOincreasedexpressionofgenesregulatingtriacylglycerolsynthesisandfatty
MuscleinMice.PLoSONE10(2):e0117494.
doi:10.1371/journal.pone.0117494 acidtransport,HF-Ssuppressedgenesandproteinsregulatingfattyacidoxidation,where-
asHF-FOincreasedoxidativegenes,proteinsandenzymesandlipolyticgenecontent,
AcademicEditor:MariàAlemany,FacultyofBiology,
SPAIN whilstsuppressinglipogenicgenes.IncomparisontoHF-S,HF-FOfurtherincreasedfat
transporters,markersoffattyacidoxidationandmitochondrialcontent,andreducedlipo-
Received:October7,2014
genicgenes.Nodiet-by-sexinteractionswereobserved.Neitherdietinfluencedfibertype
Accepted:December24,2014
composition.However,someinteractionsbetweenmuscletypeanddietwereobserved.
Published:February6,2015
HF-Sinducedchangesintriacylglycerolsynthesisandlipogenicgenesinred,butnotwhite,
Copyright:©2015Philpetal.Thisisanopen muscle,andmitochondrialbiogenesisandoxidativegenesweresuppressedbyHF-Sand
accessarticledistributedunderthetermsofthe
increasedbyHF-FOinredmuscleonly.Inconclusion,HF-Sfeedingpromoteslipidstorage
CreativeCommonsAttributionLicense,whichpermits
inredmuscle,aneffectabrogatedbythefishoil,whichincreasesmediatorsoflipolysis,oxi-
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare dationandthermogenesiswhileinhibitinglipogenicgenes.Greaterstorageandsynthesis,
credited. andloweroxidativegenesinred,butnotwhite,musclelikelycontributetolipidaccretionen-
DataAvailabilityStatement:Allrelevantdataare counteredinredmuscle.Despiteseveralgender-dimorphicgenes,bothsexesexhibiteda
withinthepaperanditsSupportingInformationfiles. similarHF-S-inducedmetabolicandgeneexpressionprofile;likewisefishoilwassimilarly
Funding:ThisworkwasfundedbyanAustralian protectiveinbothsexes.
Post-graduateAward(http://education.gov.au/
australian-postgraduate-awards)-LKP.Thefunders
hadnoroleinstudydesign,datacollectionand
analysis,decisiontopublish,orpreparationofthe
manuscript.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
PLOSONE|DOI:10.1371/journal.pone.0117494 February6,2015 1/19
DietaryFattyAcidsDictateMuscleLipidMetabolisminMice
Introduction
Dietsrichinsaturatedfat(HF-S),particularlywhenconsumedadlibitum,increaseadiposity
inrodents[1,2].Additionally,HF-Sdietsincreasetriacylglyceroldepositioninectopicstores,
includingtheliverandskeletalmuscleinbothmice[3–5]andhumans[6–9].Wehaveprevi-
ouslyreported[10]thatinvitroexposuretopalmitate,asaturatedfattyacid,increasestriacyl-
glycerolcontentinratL6myotubes.Inhealthyleanhumans,acuteexposuretoaHF-Sdiet
increasesfattyacidoxidation[11,12].Thisadaptiveincreaseisimpairedinobesehumans[11,
13,14]andhasbeenlinkedtoincreasedintramyocellulartriacylglycerolstorage[4,5,15,16],
althoughincreasedfattyaciduptake[17,18]andreducedtriacylglycerolhydrolysis[19,20]
mayalsobeinvolved.
Dietarysupplementationwithfishoilhasmultiple,well-establishedhealthbenefits,includ-
ingreducingcirculatingtriacylglycerolandananti-inflammatoryaction[21,22].Inmice,re-
placing1.5%ofacornoilbasedhighfatdiet(35%lipid;n-6PUFA-rich)withdocosahexaenoic
acid(DHA)derivative,α-ethylDHAethylester,reducedintramyocellulartriacylglycerolby
morethanhalf[23]andlikewise,incorporating3.6%eicosapentaenoicacid(EPA)ethylester
intoa45%highfatdietelicitedintramyocellulartriacylglycerolloweringeffects.Mechanistically
severalpathwaysmaybeinvolved.Ahighfatdiet(35.5%energyfromfat)ofpredominantly
fishoilincreasedmusclefattyacidtransporter,Fat/cd36,mRNAinmice,comparedtothose
consuminganisocaloriclard-basedhighfatdiet[24].Supplementingacafeteriahighfatdiet
(62%energyfromfat(45%saturatedfat,noEPAandDHA))withEPAethylester(1g/kg),re-
ducedacetylCoAcarboxylaseβ(Acc-β)mRNAcontentinratmuscle,whichmaysimulta-
neouslyresultinsuppressionoflipogenesisandenhancedβ-oxidationinmyocytes[25].While
replacing15%offatinhighfatdiet(35.2%fat(mainlyn-6PUFA-richcornoil))withn-3
PUFAconcentrate(3.29:1DHA:EPA)inmicepromotedefficientβ-oxidationoffattyacids
withinskeletalmusclemitochondria[26].Whethern-3PUFAsfromnaturalfishoilabrogate
intramyocellularlipidaccumulationinaHF-Ssettingisunclearandthepathwaysinvolvedre-
quireclarification.Additionally,thevastmajorityofpaststudieshaveexaminedonlytheeffect
ofdietaryn-3PUFAsupplementationineitherwhiteglycolyticskeletalmuscles[24]ormuscles
ofmixedfibertype[25–30]thatarereliantonglucoseorafuelmixtureastheirmainsubstrate
[31].Redfibersareoxidativeandrelyheavilyonlipidsasfuel[31–33].Theextenttowhichthe
additionofn-3PUFAstoaHF-Sdietmodifieslipidmetabolismintheseredfibersremainsto
bedetermined,althoughincreasedCpt1bandUcp3mRNAhavebeenreportedinthemouseso-
leusinresponsetoEPAethylesterenrichmentofahighfatdiet[34].Paststudieswerealsocon-
ductedsolelyinmalerodentsandthuscannotanswerwhethertherearesex-relateddifferences
inthisresponse.Especiallyasinrats[35,36],highfat-fedmalesarereportedtobelessefficient
atpromotingadiposetissuedeposition,arelessproficientatamplifyingmuscleoxidativecapac-
ityandinsteadexhibitgreaterhepatictriacylglycerolcontentandfattyacidoxidationrate,
whencomparedtohighfat-fedfemales[36].Theaimofthisstudywasthereforetodetermine
thedifferentialeffectsoffeedingasaturatedfat-richdiet(HF-S)oraHF-Sdietwith7.5%of
fattyacidsreplacedwithn-3PUFAsfromfishoil(HF-FO)inmaleandfemalemiceonbody
compositionandpathwaysofskeletalmusclelipidmetabolisminboth“white”fast-twitchgly-
colyticandoxidative-glycolyticand“red”slow-twitchoxidativemuscles,byquantifyingthehis-
tologicalchangesinmusclelipidcontentandoxidativecapacity(SDH,NADH-TR)andthe
mRNAcontentofkeygenesinvolvedinfattyacidtransport(Fat/cd36,Fabp ,Fatp1,Fatp4),li-
pm
pogenesisandtriacylglycerolstorageandhydrolysis(Srebf1,Insig1,Dgat1,Scd1,Hsl),andfatty
aciddisposal(Pdk4,Ampkα1,Ampkα2,Acc-β,Cpt1b,Ucp3,Pgc1α,Pparα)usingrealtime
quantitativePCRandtheabundanceofkeyproteinsinvolvedinmitochondrialoxidationby
Westernblotanalysis(PGC1α,PPARα,CPT1b,OXPHOSComplexI-V).
PLOSONE|DOI:10.1371/journal.pone.0117494 February6,2015 2/19
DietaryFattyAcidsDictateMuscleLipidMetabolisminMice
MaterialsandMethods
EthicsStatement
AllprocedureswereapprovedbytheUniversityofAdelaideandInstituteofMedicalandVet-
erinaryScienceAnimalEthicsCommittees,andUniversityofAdelaideguidelinesfortheuse
andcareoflaboratoryanimalswerefollowed(approvalnumber:M-027–2007).Allanimals
wereprovidedwithenvironmentalenrichmentthroughoutthedietaryinterventionandall
proceduresthereafterwereperformedunderisoflurane-inducedanaesthesiato
minimisesuffering.
Animalsanddiets
Specificpathogen-free6-week-oldmaleandfemaleC57BL/6JmicewerepurchasedfromLabo-
ratoryAnimalServices,UniversityofAdelaide(Adelaide,Australia).Micewerehousedinindi-
vidualcagesinananimalholdingroomwithfixedphotoperiod(12:12hrlight/darkcycle)and
temperature(24.5°C).Onarrival,miceunderwentanacclimatizationperiodof2wks,during
whichtheywereprovidedstandardrodentchowdietandwateradlibitum.Followingtheaccli-
matizationperiod,micewererandomlyassignedtooneofthreediets,fedeitherastandard
chow((control(CON);AIN-93G),16.1MJ/kg,15.91%energyfromfat,25.08%energyfrom
protein,58.48%energyfromcarbohydrate),highsaturatedfat((HF-S;SF07–066),21.8MJ/kg,
59.60%energyfromfat(richinsaturatedfat),18.53%energyfromprotein,21.20%energy
fromcarbohydrate),orHF-Sfishoilenriched((HF-FO;SF07–067),21.8MJ/kg,59.60%energy
fromfat(7.5%ofHF-Sreplacedwithn-3PUFAs(%asfed)),18.53%energyfromprotein,
21.27%energyfromcarbohydrate)diet.DietsweremanufacturedbySpecialtyFeedsPtyLtd
(GlenForrest,Australia)andn-3PUFAsintheHF-FOdietwereprovidedasHiDHA25N
tunaoil(26%DHA,6%EPA)(kindlydonatedbyNu-megaIngredientsPtyLtd(Nathan,Aus-
tralia)).Bothhighfatdietswerestoredat-20°C,whilsttheHF-FOdietwasalsostoredinali-
quotsundernitrogengastoavoidoxidation.Miceweremaintainedontheirrespectivedietsfor
aperiodof11or14wks,seeExperimentalProtocolsandTissueCollection,duringwhichfood
andwaterwereprovideddailyadlibitum.
ExperimentalProtocolsandTissueCollection
Cohort1:Miceweremaintainedontheirrespectivedietsfor14wks(±4d).Bodyweight(g)
wasmeasuredthriceweeklyandatthetimepointsofarrival(6wks-of-age)andpost-mortem
(20wks-of-age)(n=CON(male)=10,CON(female)=12,HF-S(male)=11,HF-S(female)=
11,HF-FO(male)=9,HF-FO(female)=11).Bodyweightuponarrival(Table1;P=0.16
(CONvs.HF-S),P=1.0(CONvs.HF-FO),P=0.99(HF-Svs.HF-FO)andduringtheacclima-
tizationperiod(datanotshown;P=0.24(CONvs.HF-S),P=0.42(CONvs.HF-FO),P=1.0
(HF-Svs.HF-FO))wassimilarinalldietarygroups.Foodintakewasmeasureddaily,from
whichcumulativeenergyintake(MJ)wascalculated.
Following14wksdiet,whilstinthefed-state,miceunderwentnon-recoverysurgeryforthe
excisionofskeletalmuscle.Surgerieswerescheduledandperformedtominimisetemporal
variation.Anaesthesiawasinducedusingamixtureofoxygen(0.5L/min),nitrousoxide
(0.5L/min)and2%isoflurane(Forthane,AbbottAustralasiaPtyLtd(Kurnell,Australia))and
maintainedusing1.5%isoflurane,0.4L/minoxygenand0.4L/minnitrousoxide.Thewhole
“white”extensordigitorumlongus(EDL)and“red”soleus(SOL)muscleswerethenrapidly
dissected,snapfrozeninliquidnitrogenandstoredunderliquidnitrogenvapourphasestorage
untilsubsequentanalysesofmRNAcontentandproteinabundance.Followingskeletalmuscle
surgery,acardiacpuncturewasperformedandbloodwascollectedinMicrovetteCB300tubes
PLOSONE|DOI:10.1371/journal.pone.0117494 February6,2015 3/19
DietaryFattyAcidsDictateMuscleLipidMetabolisminMice
Table1.Bodyweight-relatedandplasmabiochemicalparameters.
Male Female Stat
CON HF-S HF-FO CON HF-S HF-FO
StartWeight(g)† 22.0±0.6 22.5±0.2 22.6±0.3 17.9±0.2 18.9±0.3 18.3±0.3 S
FinalWeight(g)† 28.0±0.8 31.1±0.7* 31.8±0.9* 21.9±0.3 26.0±0.8* 27.6±0.6* D,S
WeightGain(g)† 6.05±0.8 8.63±0.6* 9.25±0.9* 3.99±0.3 7.14±0.7* 9.25±0.6* D,S
EnergyIntake(MJ) 4.36±0.1 5.45±0.1§ 5.08±0.2§ 3.94±0.1 5.50±0.2§ 5.49±0.1§ DxS
PooledAT(g) 2.48±0.2 3.00±0.3* 3.19±0.5* 1.64±0.1 2.64±0.4* 2.92±0.3* D
SubcutaneousAT(mg/g) 35.0±2.4 40.5±4.8 41.5±6.3 34.0±2.1 44.9±5.4 47.3±6.0 N/S
MesentericAT(mg/g) 20.0±1.0 18.7±0.6 15.6±1.1# 17.9±0.6 21.7±2.2 18.4±0.8# D
PerirenalAT†(mg/g) 8.2±0.7 9.3±1.1 11.5±2.0* 5.9±0.4 7.8±1.0 9.9±1.0* D,S
BrownAT(mg/g) 4.7±0.4 3.8±0.3 5.2±0.5# 4.4±0.4 3.8±0.2 5.5±0.4# D
PerigonadalAT(mg/g) 20.0±1.3 22.3±2.4 23.8±4.0 13.4±1.4 18.6±2.3 22.4±2.9* D
PlasmaGlucose(mM)† 8.04±0.6 7.98±0.5 6.84±0.7 7.34±0.6 5.96±0.5 6.64±0.5 S
PlasmaInsulin(pM) 63.8±12 109.7±29 59.7±14 71.9±14 114.2±26 70.7±18 N/S
PlasmaTG(mM) 0.83±0.1 0.62±0.0 0.53±0.1*# 0.72±0.1 0.79±0.1 0.55±0.1*# D
Measuredinmaleandfemalemicefedcontrol(CON),highsaturatedfat(HF-S)andhighfatfishoilenriched(HF-FO)dietsfor14wks(Cohort1).
Resultsaremean±SEMof9–12animalspergroup.Adiposetissue,AT;TG,triacylglycerol.
Statistics:Effectofdiet(D):
*P(cid:1)0.05,vsCON;
#P(cid:1)0.05,comparedtoHF-S.Effectofsex(S):
†P(cid:1)0.05malevsfemale.Diet*sexinteraction(DxS):
§P(cid:1)0.05,comparedtoCONofsamegender.N/S,notsignificant.
doi:10.1371/journal.pone.0117494.t001
treatedwithEDTAdipotassiumsalt(Sarstedt(Nümbrecht,Europe)).Post-mortem,adipose
tissuefromthepooledposteriorandanteriorsubcutaneous(dorso-lumbar,inguinal,gluteal,
whiteinterscapular,subscapular,axillo-toracicandsuperficialcervicaldepots);visceralmesen-
teric;pooledperirenalandretroperitoneal;pooledbrowndeepcervicalandinterscapular;and
perigonadal(periovariac,females;epididymal,males)depotswasdissected,snapfrozeninliq-
uidnitrogenandevaluatedforweight.Adiposetissuedepotweightsareexpressedrelativeto
bodyweight,toallowcomparisonacrosssexes.
Cohort2:Miceweremaintainedontheirrespectivedietsfor11wks(±7d).Following11
wksdiet,whilstinthefed-state,miceunderwentnon-recoverysurgeryfortheexcisionofskele-
talmuscle,withanaesthesiainducedandmaintainedasdescribedabove.ThewholeEDL(un-
dividedfromthetibialismuscle),andsoleus(undividedfromthegastrocnemiusandplantaris
muscles)werethenrapidlydissected,followedbyexcisionofthewholeundividedquadriceps
muscles.MusclegroupswereembeddedatrestingtensioninTissue-TekOCT(SakuraFinetek
CoLtd(Tokyo,Japan))andgentlyfrozen.OCT-embeddedmuscleswerestoredunderliquid
nitrogenvaporphasestorageuntilsubsequenthistologicalanalysesofmusclelipid,fibertype
compositionandoxidativecapacity.
PlasmaBiochemistry
Plasmaglucoseandtriacylglycerol(mM)concentrationsweremeasuredinduplicateusing
Gluco-quantGlucose/HKandTGGPO-PAPkits(RocheDiagnostics(Mannheim,Germany)),
respectively,ontheCOBASBioautomatedanalysissystem(RocheDiagnosticsAustraliaPty
Ltd(CastleHill,Australia)).Plasmainsulinconcentrations(pM)weremeasuredbyDRG
PLOSONE|DOI:10.1371/journal.pone.0117494 February6,2015 4/19
DietaryFattyAcidsDictateMuscleLipidMetabolisminMice
UltrasensitiveMouseInsulinELISA(DRGInstruments(Marburg,Germany))asper
manufacturer’sinstructions.
mRNAExpressionAnalysesusingtheGenomeLabGeXPGenetic
AnalysisSystem
TotalRNAfromwholeEDLandsoleusmuscleswasisolatedusingTRIzolreagent(Invitrogen
AustraliaPtyLtd(MountWaverley,Australia)).RNAqualityandconcentrationwereevaluat-
ed(NanoDrop1000Spectrophotometer,ThermoScientificInc(Wilmington,USA)).RNAwas
treatedusingDNaseI(InvitrogenAustraliaPtyLtd).cDNAwasgeneratedbyreversetran-
scriptionaccordingtomanufacturer’sinstructions(GenomeLabGeXPGeneticAnalysisSys-
tem,BeckmanCoulterInc),resultingincDNAofgene-specificsequenceswithaflanking
universalsequence.Forwardandreverseprimersweredesignedtoamplifyasectionofthepro-
teincodingsequenceandtobepositionedindifferentexonsasdeterminedbyEntrezNucleo-
tide(NationalCenterforBiotechnologyInformation(Bethesda,USA))andEnsembl
(EuropeanBioinformaticsInstitute/WellcomeTrustSangerInstitute(Cambridge,United
Kingdom)),respectively.PrimersweredesignedusingGenomeLabGeXPeXpressProfilersoft-
ware(ver.10.0BeckmanCoulterInc(Fullerton,USA))togenerateanamplifiedproductwitha
genefragmentlengthbetween137–356nucleotidesandseparationsizeofatleast6nucleotides.
FinallyprimersequencesweresubmittedtoBLAST(BasicLocalAlignmentSearchTool,Na-
tionalCenterforBiotechnologyInformation(Bethesda,USA)).Auniversalsequencewasthen
addedtobothforwardandreversedesignedprimersequences,generatingchimericprimers.
Resultantforwardandreverseprimers(GeneWorksPtyLtd(Hindmarsh,Australia))arelisted
intableform(SupportingInformation—S1Table).Primerswereoptimizedinsingletandmul-
tiplexreactions,accordingtomanufacturer’sinstructions(GenomeLabGeXPGeneticAnalysis
System,BeckmanCoulterInc).MultiplexPCRwasthenperformedontheEppendorfMaster-
cyclerGradient(EppendorfSouthPacificPtyLtd(NorthRyde,Australia)).Negativecontrols
(notemplate,noreversetranscriptase)wereruninparallel.Fluorescently-labeledPCRprod-
uctswereseparated,detected,quantifiedandanalyzedinduplicateusingtheGenomeLab
GeXPGeneticAnalysissystemandGenomeLabGeXPeXpressProfilersoftware.Output
mRNAcontentswerethennormalizedtotheaveragemRNAcontentof3housekeepinggenes,
TATA-bindingprotein,RNAPolymerase2candlargeribosomalproteinP0.
ProteincontentanalysesbyWesternBlot
WholeEDLandsoleusmuscleswereweighedandhomogenisedinice-coldlysisbufferasde-
scribedpreviously[37]andproteinconcentrationdeterminedbybicinchoninicacidprotein
assay(PierceBiotechnology(Rockford,USA)).FormeasuringtheabundanceofPPARα,oxi-
dativephosphorylation(OXPHOS)complexesI-V,peroxisomeproliferativeactivatedreceptor
γcoactivator1α(PGC1α),CPT1bandβ-tubulinproteins,16–20μgofmuscleproteinwassub-
jectedtoSDS-PAGEusingprecast10%Bis-Trisgelsor4–12%Bis-Trisgels(Bio-RadLaborato-
riesPtyLtd(Gladesville,Australia))andtransferredtoPVDFmembranes.Membraneswere
incubatedovernightinprimaryantibody;PPARα(Abcam(Cambridge,UK)),1:500;OXPHOS
proteins(MitoSciences(Eugene,USA)),1:250;PGC1α(Abcam),1:1000;CPT1b(AlphaDiag-
nosticInternationalInc(SanAntonio,USA)),1:1000;andβ-Tubulin(CellSignalingTechnolo-
gy(Danvers,USA)),1:2000.Boundprimaryantibodiesweredetectedwithsheepanti-rabbit
(1:2500,ChemiconInternational(Billerica,USA))orgoatanti-mousealkaline-phosphatase
linkedantibody(1:2000,Millipore(Billerica,USA)).Membranesweredevelopedwithchemi-
fluoresencesubstrate(ECF),scannedbyTyphoonImager(GEHealthcareBio-Sciences(Rydal-
mere,Australia))andwerequantifiedusingImageQuantsoftware(MolecularDynamics).
PLOSONE|DOI:10.1371/journal.pone.0117494 February6,2015 5/19
DietaryFattyAcidsDictateMuscleLipidMetabolisminMice
MuscleHistology
OCT-embeddedmusclegroups(theEDLandtibialisgroup,andthesoleus,gastrocnemiusand
plantarisgroup,andthequadricepsmusclesgroup)werecutto4,9or10μmthickcross-sec-
tionsat-20°C.Sectionswerestainedwithhaematoxylinandeosintoconfirmnormalcellular
morphology(datanotshown)orwithOilRedO,amarkerofintramyocellularlipidcontent.
Serialsectionswerestainedformyofibrillarmyosinadenosinetriphosphatase(mATPase),fol-
lowingalkaline(pH10.4)andacidic(pH4.1,pH4.3)preincubations,toevaluatemusclefiber
type[38].Sectionswerestainedforsuccinicdehydrogenase(SDH)andNADHtetrazoliumre-
ductase(NADH-TR)toconfirmmusclefibertypeandevaluateoxidativecapacity.Following
allstaining,slideswerescannedusingtheNanoZoomerDigitalPathologyscanner(Hamama-
tsuPhotonicsK.K.(HamamatsuCity,Japan)).ThesoleusandEDLmuscleswereanalyzedin
duplicatetodeterminemusclefibertypeandoxidativecapacityinpredominantlyredand
whitemuscles,respectively,withthesame75musclefibersineachscannedimageassessedfor
allstains.Imageswerescoredwhilstblindedtodietarygroupandsex.
TheactivityofmATPasewasjudgedonasubjectivebasisbyoneobserverandmusclefibers
weregivenascorefrom1–5basedontheintensityofstaining(1=light,2=light-moderate,3
=moderate,4=moderate-dark,5=dark).Adefinedschemewasusedtoclassifyfibertype,
basedonscoresgiventomusclefibersstainedformATPase[38].Fibertypecomposition(%)
wascalculatedastheproportionofeachfibertyperelativetothetotalnumberoffibersscored.
SDHandNADH-TRstainingintensitywereassessedbymeanpixeldensityusingImageJ
software(ver.1.42q,NationalInstituteofHealth(Bethesda,USA))fromgrayscaleimagesman-
uallytracedatcellperimeters.ThesamefibersusedtoassesstheactivityofbothSDHand
NADH-TRwerealsousedtodeterminemusclefibertypebymATPasestaining,allowingclas-
sificationofoxidativeactivityrespectivetofibertype.Cellareaofeachmusclefibertypewas
alsoassessed.Theareaofeachfibertypewascalculatedrelativetothetotalareameasured,pro-
vidingtherelativeareaoccupiedbyeachmusclefibertypewithintheEDLandsoleusmuscles.
Resultsarepresentedasthemeancross-sectionalcellareaofclassifiedfibersandthe%areaoc-
cupiedbyeachmusclefibertype.
StatisticalAnalysis
Alldataarepresentedasmean±SEM.Two-wayANOVA,withpairwisecomparisons(Bonfer-
ronipost-hoc),wasusedtodeterminetheeffectofdiet(CON,HF-S,HF-FO),muscle(EDL,so-
leus),sex(male,female)andtheirinteractiononparameters.InCohort2analyses,astherewas
nosignificantinteractionofsexanddiet,toenhancestatisticalpower,datafrommale(n=3
pergroup)andfemale(n=2–3pergroup)micewerecombined.One-wayANOVAs,with
Bonferronipost-hocanalysis,werethereforeusedtodeterminetheeffectofdietintheEDL
andsoleusmusclesonmusclefibertype-specificparameters.Statisticswereperformedusing
StatisticalPackageforSocialScientistsver.17.0.0(SPSSInc(Chicago,USA)).P<0.05wascon-
sideredstatisticallysignificant.
Results
Effectofdietaryfatcompositiononadiposetissuedistributionand
plasmabiochemistry(Table1)
ComparedtoCON,HF-S-andHF-FO-fedmicegainedmoreweight(effectofdiet:P(cid:1)0.001),
buttherewerenodifferencesbetweenthetwohighfatdietgroups.Irrespectiveofdiet,males
gainedmoreweightthanfemales(effectofsex:P(cid:1)0.05).ComparedtoCON,cumulativeener-
gyintakewashigherinthetwohighfatdietgroups(effectofdiet:P<0.005),buttherewasno
PLOSONE|DOI:10.1371/journal.pone.0117494 February6,2015 6/19
DietaryFattyAcidsDictateMuscleLipidMetabolisminMice
differencebetweenhighfatdietsandnoeffectofsex.ComparedtoCON,HF-SandHF-FOin-
creasedpooledadiposetissuemasssimilarly,inbothsexes(effectofdiet:P(cid:1)0.05,P(cid:1)0.01).
However,fatstorageindistinctadiposetissuedepotswasaffectedbydietaryfattyacidcompo-
sition.ComparedtoCON,HF-FOincreasedpooledperirenalandretroperitonealadiposetis-
sue(effectofdiet:P(cid:1)0.005),irrespectiveofsex,andgonadalperiovariacadiposetissue(effect
ofdiet:P(cid:1)0.05)infemalemice.Inmales,gonadaldepot,epididymaladiposetissue,wasun-
changedbydiet;similarlysubcutaneousfatmasswasnotdiet-dependent.ComparedtoHF-S,
HF-FOdecreasedmesentericadiposetissue(effectofdiet:P(cid:1)0.05)andincreasedbrownadi-
posetissue(effectofdiet:P(cid:1)0.05),inbothsexes.Otherthanhigherpooledperirenalandretro-
peritonealadiposetissueinmales(effectofsex:P(cid:1)0.05),theadiposetissuedepotsmeasured
werenotsexuallydimorphic.ComparedtoCONandHF-S,HF-FOloweredplasmatriacylgly-
cerol(effectofdiet:P(cid:1)0.005,P(cid:1)0.05),irrespectiveofsex.Neitherhighfatdietinfluencedplas-
maglucosenorinsulinconcentrations,irrespectiveofsex.Irrespectiveofdiet,glucoselevels
werehigherinmalesthanfemales(effectofsex:P(cid:1)0.05).
Effectofdietaryfatcompositiononskeletalmusclelipidcontentinwhole
muscleandmusclefibertypedistribution(Fig.1)
IncreasedOilRedOstaining,amarkerofintramyocellularlipid,wasapparentinsoleus(pre-
dominatelyred)andquadriceps(mixedfibertype)ofHF-S,butnotCONandHF-FO,mice
(Fig.1A).NeitherhighfatdietinfluencedOilRedOstainingintheEDL(predominantlywhite
muscle).PercentagemusclefibertypecompositionintheEDLandsoleusmuscleswasnot
influencedbydiet(Fig.1B),norwasthecross-sectionalareaofeachmusclefibertype(datanot
shown).
EffectofdietaryfatcompositiononmusclefattyacidtransportermRNA
content(Table2)
ComparedtoCON,bothhighfatdietsincreasedFat/cd36andFatp1mRNA(effectofdiet:
Fat/Cd36:P(cid:1)0.001;Fatp1:HF-SP(cid:1)0.005;HF-FOP(cid:1)0.001),butHF-FOaloneincreasedFatp4
mRNA(effectofdiet:P(cid:1)0.02),irrespectiveofmuscletype.ComparedtoHF-S,HF-FOin-
creasedFat/Cd36mRNA(effectofdiet:P(cid:1)0.001),irrespectiveofmuscletype.Allfattyacid
transportersexhibitedgreatermRNAcontentinthesoleusmuscle(effectofmusclefibretype:
P(cid:1)0.001).ExceptforhigherFatp1mRNAinmalescomparedtofemalesintheEDLonly
(muscle(cid:3)sexinteraction:P(cid:1)0.001),therewerenoothersexdifferencesinfattyacidtransporter
mRNAcontent.
EffectofdietaryfatcompositiononthemRNAcontentofgenes
influencingfattyacidstorageandlipogenesis(Table3)
ComparedtoCON,HF-SincreasedSrebf1andInsig1mRNAs(effectofdiet:P(cid:1)0.02),irrespec-
tiveofmusclefibertype,andincreasedDgat1anddecreasedScd1mRNAinthesoleusmuscle
alone(diet(cid:3)muscleinteraction:P(cid:1)0.05;P(cid:1)0.005).ComparedtoCON,HF-FOincreased
Dgat1mRNAinbothmuscles(diet(cid:3)muscleinteraction:P(cid:1)0.001).ComparedtobothCON
andHF-S,HF-FOdecreasedScd1mRNAinbothmuscles(diet(cid:3)muscleinteraction:P(cid:1)0.02)
andincreasedHslmRNA(effectofdiet:P(cid:1)0.001,P(cid:1)0.05),irrespectiveoffibertype.Com-
paredtoHF-Salone,HF-FOloweredthemRNAcontentofInsig1,irrespectiveofmuscletype
(effectofdiet:P(cid:1)0.001)andincreasedDgat1mRNAinthesoleusmuscle(diet(cid:3)muscleinterac-
tion:P(cid:1)0.001).
PLOSONE|DOI:10.1371/journal.pone.0117494 February6,2015 7/19
DietaryFattyAcidsDictateMuscleLipidMetabolisminMice
Fig1.MuscleLipidContentandFiberTypeComposition.(A)OilRedOstaining,amarkerofintramyocellularlipid,ofthewholeextensordigitorum
longus(EDL),soleus(SOL)andquadriceps(rectusfemoris(RECTFEM),vastuslateralis(VASTLAT),vastusintermedius(VASTINT)andvastusmedialis
(VASTMED))musclesand(B)musclefibertypecomposition(%)oftheEDLandSOLmusclesofmicefedacontrol(CON),highsaturatedfat(HF-S)orhigh
fatfishoilenriched(HF-FO)dietfor11wks(Cohort2).Scalebarsrepresent100μm.
doi:10.1371/journal.pone.0117494.g001
PLOSONE|DOI:10.1371/journal.pone.0117494 February6,2015 8/19
DietaryFattyAcidsDictateMuscleLipidMetabolisminMice
Table2.FattyacidtransportermRNAcontent.
Male Female Stat
CON HF-S HF-FO CON HF-S HF-FO
Fat/Cd36 EDL 100±6 116±8* 133±5*# 95±7 105±5* 137±7*# D,M
SOL+ 100±3 121±4* 146±7*# 94±5 115±6* 139±7*#
Fabp EDL 100±3 107±4 100±3 106±8 98±4 107±5 M
pm
SOL+ 100±3 96±2 110±5 101±6 100±7 113±8
Fatp1 EDL‡ 100±8 137±8* 145±9* 81±10 102±7* 113±13* D,MxS
SOL+ 100±6 114±8* 129±6* 93±9 112±12* 141±9*
Fatp4 EDL 100±6 99±9 119±13* 107±12 93±7 108±8* D,M
SOL+ 100±5 128±10 150±6* 117±12 142±20 142±14*
Fattyacidtranslocase(Fat/Cd36),Fattyacidbindingprotein(Fabp )andFattyacidtransportproteins1(Fatp1)and4(Fatp4)inextensordigitorum
pm
longus(EDL)andsoleus(SOL)musclesofmaleandfemalemicefedcontrol(CON),highsaturatedfat(HF-S)orhighfatfishoilenriched(HF-FO)diets
for14wks(Cohort1).
mRNAcontentsareexpressedasapercentageofthevalueofmaleanimalsunderCONdiet.Resultsaremean±SEMof9–12animalspergroup.
Statistics:Two-wayANOVA:Effectofdiet(D):
*P(cid:1)0.05,vsCON;
#P(cid:1)0.05,comparedtoHF-S.Effectofmuscletype(M):
+P(cid:1)0.05EDLvsSOL.Muscle*sexinteraction(MxS):
‡P(cid:1)0.05,malevsfemale(EDL).
doi:10.1371/journal.pone.0117494.t002
Table3.Lipogenesis,triacylglcerolsynthesisandstoragegenesmRNAcontent.
Male Female Stat
CON HF-S HF-FO CON HF-S HF-FO
Srebf1 EDL† 100±5 143±10* 104±11 78±8 103±8* 104±9 D,MxS
SOL 100±10+ 99±11*+ 92±16+ 90±11 108±13* 141±27
Insig1 EDL 100±8 128±9* 89±9# 109±15 97±5* 81±5# D
SOL 100±8 147±10* 104±9# 112±12 148±18* 107±16#
Dgat1‡ EDL 100±15 167±23 197±41* 113±19 178±29 209±22* S,DxM
SOL 100±15 162±17*^ 224±39*#^ 117±16 197±34*^ 300±33*#^
Scd1 EDL 100±8 86±8 56±3*# 109±17 77±4 84±7*# DxMMxS
SOL† 100±11^ 84±6*^ 32±2*# 149±13^ 110±19*^ 40±5*#
Hsl EDL 100±7 128±8 142±14*# 103±12 117±12 135±11*# D,M
SOL+ 100±7 117±7 144±13*# 107±12 124±14 150±13*#
Sterolregulatoryelementbindingtranscriptionfactor1(Srebf1),insulininducedgene1(Insig1),diacylglycerolacyltransferase1(Dgat1),stearoyl-
CoenzymeAdesaturase1(Scd1)andhormonesensitivelipase(Hsl)inextensordigitorumlongus(EDL)andsoleus(SOL)musclesofmaleandfemale
micefedcontrol(CON),highsaturatedfat(HF-S)orhighfatfishoilenriched(HF-FO)dietsfor14wks(Cohort1).
mRNAcontentsareexpressedasapercentageofthevalueofmaleanimalsunderCONdiet.Resultsaremean±SEMof9–12animalspergroup.
Statistics:Two-wayANOVA:Effectofdiet(D):
*P(cid:1)0.05,vsCON;
#P(cid:1)0.05,comparedtoHF-S.Effectofmuscletype(M):
+P(cid:1)0.05EDLvsSOL.Effectofsex(S):
‡P(cid:1)0.05,malevsfemale.Muscle*sexinteraction(MxS):
†P(cid:1)0.05,malevsfemale(ofsamemuscle).Diet*muscle(DxM):
^P(cid:1)0.05,EDLvsSOL(ofsamediet).
doi:10.1371/journal.pone.0117494.t003
PLOSONE|DOI:10.1371/journal.pone.0117494 February6,2015 9/19
DietaryFattyAcidsDictateMuscleLipidMetabolisminMice
Table4.FattyAcidUtilisationGenemRNAContent.
Male Female Stat
CON HF-S HF-FO CON HF-S HF-FO
Pdk4 EDL 100±18 167±13* 206±25* 131±27 160±20* 192±19* D,M
SOL+ 100±14 176±5* 234±13* 118±20 204±28* 241±24*
Ampk1 EDL 100±7 97±8 101±8 107±12 97±6 128±11 M
SOL+ 100±7 116±7 118±5 122±8 111±6 110±6
Ampk2 EDL† 100±5 106±3* 91±2# 85±5 94±29* 85±7# DMxS
SOL+ 100±3 103±4* 93±3# 95±5 106±9* 92±5#
Acc- EDL 100±7 110±9 111±10 140±40 104±6 104±12 M
SOL+ 100±8 122±7 139±6 134±28 147±33 154±27
Cpt1b‡ EDL 100±6 94±6 115±4*# 65±5 88±3 104±10*# DxMS
SOL+ 100±4 111±5 146±4*# 85±4 89±10 116±9*#
Pgc1‡ EDL 100±10 88±6 97±5 78±6 79±9 94±13 DxMS
SOL 100±11^ 68±9* 185±33*#^ 87±10^ 44±7* 144±16*#^
Ppar EDL 100±5 84±6* 90±6# 105±15 92±11* 95±12# D,M
SOL+ 100±9 78±7* 102±7# 104±10 87±6* 100±8#
Ucp3 EDL 100±15 116±7 163±18* 78±14 105±12* 136±13* DxM
SOL 100±19^ 196±16 283±25* 111±27^ 248±34* 255±24*
Pyruvatedehydrogenasekinase4(Pdk4),AMP-activatedproteinkinasecatalyticsubunitsα1(Ampkα1)andα2(Ampkα2),acetyl-CoAcarboxylase-
(Acc-),carnitinepalmitoyltransferase1b(Cpt1b),peroxisomeproliferativeactivatedreceptorcoactivator1(Pgc1),peroxisomeproliferatoractivator
receptorα(Pparα)anduncouplingprotein3(Ucp3)inextensordigitorumlongus(EDL)andsoleus(SOL)musclesofmaleandfemalemicefedcontrol
(CON),highsaturatedfat(HF-S)orhighfatfishoilenriched(HF-FO)dietsfor14wks(Cohort1).
mRNAcontentsareexpressedasapercentageofthevalueofmaleanimalsunderCONdiet.Resultsaremean±SEMof9–12animalspergroup.
Statistics:Two-wayANOVA:Effectofdiet(D):
*P(cid:1)0.05,vsCON;
#P(cid:1)0.05,comparedtoHF-S.Effectofmuscletype(M):
+P(cid:1)0.05EDLvsSOL.Effectofsex(S):
‡P(cid:1)0.05,malevsfemale.Muscle*sexinteraction(MxS):
†P(cid:1)0.05,malevsfemale(ofsamemuscle).Diet*muscle(DxM):
^P(cid:1)0.05,EDLvsSOL(ofsamediet).
doi:10.1371/journal.pone.0117494.t004
mRNAcontentsofgenesinvolvedinlipogenesis,lipolysisandtriacylglycerolmetabolism
weremostlyhigherinthesoleusthantheEDL,butthiswassex-anddiet-dependent.Srebf1
(malemice),Hsl(irrespectiveofdietorsex),Dgat1(HF-S,HF-FO(diet(cid:3)sex)andScd1(CON,
HF-S(diet(cid:3)muscle),maleandfemale(sex(cid:3)muscle))exhibitedgreatermRNAinsoleuscom-
paredtoEDLmuscle.Sex-dependenteffectswereobservedforSrebf1(inEDL)whichexhibited
greatermRNAinmales,andScd1(insoleus)andDgat1(irrespectiveofdietormuscle)which
exhibitedgreatermRNAinfemales.
EffectofdietaryfatcompositiononfattyacidoxidationgenemRNA
content(Table4)
ComparedtoCON,HF-SdecreasedPgc1αmRNAinthesoleusmuscle(diet(cid:3)muscleinterac-
tion:P(cid:1)0.001)andPparαmRNAinbothmuscles(effectofdietP(cid:1)0.05)andincreased
Ampkα2mRNAirrespectiveofmusclefibertype(effectofdietP(cid:1)0.05).ComparedtoCON,
HF-FOincreasedCpt1b,Ucp3,Pgc1αandPdk4mRNAinbothmuscles(diet(cid:3)muscleinterac-
tion:P(cid:1)0.05;P(cid:1)0.001;P(cid:1)0.001;andeffectofdiet:P(cid:1)0.001).ComparedtoHF-S,HF-FO
PLOSONE|DOI:10.1371/journal.pone.0117494 February6,2015 10/19