Electronic Supplementary Material ( ESI ) for - Zhang et al. - Unknown - Unknown

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ElectronicSupplementaryMaterial(ESI)forInvestigatingobesity-alleviationeffectofEurycomalongifoliaonmicefedwithhigh-fatdietthroughmetabolomicsrevealedenhanceddecompositionandinhibitedaccumulationoflipids11111111DingkunZhang,WenZheng,XinLi,GeLiang,NanYe,YueqiuLiu,AngLi,XinLiu,Rui1111,*Zhang,JingqiuCheng,HaoYang,MengGong1LaboratoryofClinicalProteomicsandMetabolomics,InstitutesforSystemsGenetics,FrontiersScienceCenterforDisease-relatedMolecularNetwork,WestChinaHospital,SichuanUniversity,88KeyuanSouthRoad,Hi-TechZone,Chengdu610041,China*Correspondingauthors.E-mail:gongmeng@scu.edu.cnS-1

1TableofcontentsFigureS1TypicalLC-MS/MSBPIspectraofE.longifoliaaqueousextract.FigureS2TypicalLC-MS/MSTICspectraofaqueous-solublesamplesfromHFDtreatment,HFDELtreatmentgroupandcontrol.FigureS3PCAscoreplotsofPC1vs.PC2basedontheLC-MS/MSdataofaqueous-solublesamplesinplasma,liverandkidney.FigureS4PLS-DAscoreplotsofp1vs.p2basedontheLC-MS/MSdataofaqueous-solublesamplesinplasma,liverandkidney.FigureS5OPLS-DAscoreplotsofp1vs.o1basedontheLC-MS/MSdataofaqueous-solublesamplesinplasma,liverandkidney.FigureS6Theheatmapofdifferentialaqueous-solublemetabolitesinplasma.FigureS7Theheatmapofdifferentialaqueous-solublemetabolitesinliver.FigureS8Theheatmapofdifferentialaqueous-solublemetabolitesinkidney.FigureS9Volcanoplotsofalldetectedandquantifiedaqueous-solublemetabolites.FigureS10Summaryofpathwayanalysisofdifferentialaqueous-solublemetabolitesunderdifferentexperimentalconditionsinvariousorganswithMetaboAnalystR2.0.FigureS11TypicalLC-MS/MSTICspectraoflipidsfromHFDtreatment,HFDELtreatmentgroupandcontrol.FigureS12PCAscoreplotsofPC1vs.PC2basedontheLC-MS/MSdataoflipidsinplasma,liverandkidney.FigureS13PLS-DAscoreplotsofp1vs.p2basedontheLC-MS/MSdataoflipidsinplasma,liverandkidney.FigureS14OPLS-DAscoreplotsofp1vs.o1basedontheLC-MS/MSdataoflipidsinplasma,liverandkidney.FigureS15Theheatmapofdifferentiallipidsinplasma.FigureS16Theheatmapofdifferentiallipidsinliver.FigureS17Theheatmapofdifferentiallipidsinkidney.FigureS18Volcanoplotsofalldetectedandquantifiedlipids.FigureS19TypicalDESI-MSITICspectraofliversectionsincludingHFDtreatment,HFDELtreatmentgroupandcontrol.FigureS20TypicalDESI-MSITICspectraofkidneysectionsincludingHFDtreatment,HFDELtreatmentgroupandcontrol.TableS1SequencesoftheprimersforqPCRanalysis.TableS2Thecompositionofhigh-fatdiet.TableS3Summaryofaqueous-solublemetabolitevariationsindifferentconditions.TableS4Summaryoflipidvariationsindifferentconditions.S-2

2ChemicalsandmaterialsE.longifoliawaspurchasedfromPortKlangPharmacy(NegeriSelangor,Malaysia).MaturemaleBALB/cmice(5weeks)werepurchasedfromGempharmatechCo.(Nanjing,China).High-fatdiet(HFD)wasobtainedfromResearchDietsCo.(NewBrunswick,USA)with60%kcalfat.Phosphatebufferedsaline(PBS)andhematoxylinandeosin(H&E)werepurchasedfromThermoFisher(USA).Methyltert-butylether(MTBE)waspurchasedfromSigma-Aldrich(Louis,MO,USA).FormalinwasobtainedfromKelongChemicals(China).LC-MS-gradeCH3OH,formicacid,andleucineenkephalinwerepurchasedfromSigma-Aldrich.Allchemicalreagentsusedinthisexperimentwereofanalyticalgradeandusedwithoutfurtherpurification.E.longifoliaaqueousextractTheaqueousextractwaspreparedfirstlybygrinding27gofE.longifoliaintofinepowder.Thenitwasdissolvedin200mlofH2Owithvigorousstirringat80°Cfor1hinthedark.Theupperliquidwasfilteredandcollected.Then,another200mlofH2Owasaddedtotheprevioussubstrateforextraction.Aftervigorousstirringat80°Cfor1hinthedark,alltheliquidwasmixedandfreeze-dried.Theobtainedpowder(0.941g)wasstoredinarefrigeratorat4°C.ForLC-MSprofile,abovedrypowderwasre-dissolvedinH2Owithvigorousstirring.ChromatographicanalysiswasperformedusingaWatersAcquity™H-ClassUPLCsystem(WatersCorporation,oMilford,USA).Chromatographicseparationwascarriedoutat40C,usingaACQUITYUPLCHSST3column(2.1mm×100mm,1.8µm,Waters,UK)withmobilephasesA(0.1%formicacidinwater)andB(0.1%formicacidinacetonitrile).Theflowratewassetat0.4mL/min.Thegradientwassetasfollows:0-0.5min,2%B;0.5-1.5min,2%-35%B;1.5-8min,35%-95%B,thenthecolumnwaswashedwith95%Bfor2minandequilibratedwith98%mobilephaseAfor3min.MassspectrometricdetectionwascarriedonWatersSynaptG2-SiQ-TOFmassEspectrometer(WatersCorporation,Milford,USA)equippedwithanESIsource.TheMSdatawereacquiredfrom50to700Daonpositivemodewithacapillaryvoltageof3.0kVandsamplingconevoltageof40V.Ionsourcetemperaturewas150℃,conegasflowratewas50L/h,odesolvationgas(N2)flowratewas800L/handdesolvationgastemperaturewas450C.Thecollisionvoltagewassetasarampfrom10to45V.Themasswascorrectedduringacquisitionusinganexternalreference(LockSpray™)consistingofa200ng/mLsolutionofleucineenkephalininfusedataflowrateof5µL/minviaalocksprayinterface,generatingarealtime+referenceionof[M+H](m/z556.2771)toensureaccurateMSanalysis.BeforeE.longifoliaaqueousextracttreatmentonmice,thepowderwasre-dissolvedinH2Owithvigorousstirringforfinedistributionandbroughttothebodytemperatureofthemice.AnimalfeedingandE.longifoliaaqueousextracttreatmentFifteenmaturemalemiceweighingbetween22and28gwereusedinthisstudy.Themicewerehousedinstandardcages(5micepercage)atroomtemperature(22-24°C)withadequateventilation,12hlight-darkcycle,andapproximately50±5%humidity.CommercialmicepelletsS-3

3andwaterwereprovidedadlibitum.Theanimalswereaccustomedtothehousingconditionsfor7daysbeforestartingthetreatment.Then,themicewererandomlydividedintothreegroupsof5miceeach.After12daysoffeedingwiththeHFD(10miceintwogroups)ornormaldiet(5miceinthecontrolgroup),the20-daytreatmentwassetasfollows:controlwasgivenonlynormaldietandplainwater;HFD-treatedgroupwasgivenonlyHFDandplainwater;HFDandE.longifolia(HFDEL)-treatedgroupwasgivenHFD,plainwater,and200µlof120mg/mlE.longifoliaaqueousextractviaoralgavageeveryday(onemouseinthisgroupdiedduringtheexperiment).ThedoseofE.longifoliawasselectedtoofferoptimumbiologicalactivitybasedonpreviousresearch(1).Whilethedosagecouldfacilitateeffectivecontactandstimulationofmice;ithadnosignificanttoxicity.MiceinthecontrolgroupandHFD-treatedgroupwereparallellyfedwith200µlofPBSviaoralgavageatthesametimeeveryday.Foodweightandbodyweightwererecordedeveryday.HistologicalstudyTheliversfrommiceinthethreegroupswerefixedin10%bufferedformalinfor48h.Subsequently,thespecimensweredehydratedandinsertedinparaffinfollowedbysectioningtoathicknessof4µm.TheliversectionswerethenstainedwithH&EandobservedunderaGS3-U3-51S5M-Cmicroscope(PointGrey,Canada).PlasmametabolomesamplepreparationBloodsampleswerecollectedfromthejugularveininallexperimentalgroupsundergeneralanesthesia.AftertreatmentwithE.longifolia,bloodsampleswerecollectedinbloodcollectiontubeswithethylenediaminetetraaceticacid(EDTA)andcentrifuged(2000rpm,10min,8°C)tocollecttheplasma.Forsamplepreparation,40µlofplasma(n=6)wasmixedwith300µlCH3OHbyvortexingat1500rpmfor10s.Then,1mlofMTBEwasaddedandvortexedat1500rpmforanother20s,followedbyincubationinthedarkfor1h.Next,350µlofdoubledeionizedwaterwasaddedandvortexedat1500rpmfor20s,withsubsequentstratificationfor10minatroomtemperature.Aftercentrifugation(14,000rpm,10min,4°C),800µlofsupernatant(lipids)and250µlofsubnatant(aqueous-solublemetabolites)wereprocessedwithvacuumdryingfor12h.TissuemetabolomesamplepreparationTissuesampleswerealsoobtainedfromallexperimentalgroupsundergeneralanesthesia.AftertreatmentwithE.longifolia,theliversandkidneyswerecollectedandrinsedwithPBS.Forsamplepreparation,10mgoftheliverorkidneyfromeachsample(n=6)togetherwith6steelballsand200µlpre-cooled80%CH3OHweremixedthreetimesfortissuehomogenization(30seachtime).Aftervortexingat1500rpmfor10s,160µlCH3OHwasmixedwiththesuspensionandvortexedagainat1500rpmfor10s.Then,1mlofMTBEwasaddedandvortexedat1500S-4

4rpmforanother20s.Afterincubationinthedarkfor1h,300µlofdoubledeionizedwaterwasaddedandvortexedat1500rpmfor20s,followedbystratificationfor10minatroomtemperature.Aftercentrifugation(14,000rpm,10min,4°C),800µlofsupernatant(lipids)and250µlofsubnatant(aqueous-solublemetabolites)wereprocessedwithvacuumdryingfor12h.LC-MS/MSdetectionofaqueous-solublemetabolitesandlipidsBeforeLC-MS/MSdetection,theabovedriedaqueous-solublesampleswerere-dissolvedin750µlofHILICsolution(60%acetonitrilewith5mMammoniumformate)followedbyvortexingat1500rpmfor15minandcentrifugationat14,000rpmfor10min.The100µlsupernatantobtainedwaskeptasideforlateranalysis.Thequalitycontrol(QC)samplewasamixturecontaining30µlofeachsample.Theabovedriedlipidswerere-dissolvedin100µl50%CH2Cl2/50%CH3OHwith10mMammoniumacetatefollowedbyvortexing(1500rpm,10C,10s)andcentrifugation(14,000rpm,10min,4C).The65µlsupernatantobtainedwaskeptasideforlateranalysis.TheQCsamplewasamixturecontaining15µlofeachsample.ALC-MS/MSsystem,Qtrap5500massspectrometercoupledwithShimadzuLC-30Aliquidchromatograph(ABSCIEX,USA),wasusedfortargetedanalysisbymultiplereactionmonitoringmode.The228metabolites(99inpositivemodeand128innegativemode)and1251lipids(635inpositivemodeand616innegativemode)chosenforsuchtargetedanalysisrepresentedmajormetabolicpathwaysinobesity-alleviationeffect.Formetabolomics,HPLCconditionscontainaflowrateof300µl/minwithamobilephaseof(A)10%acetonitrileinwater(including10mMammoniumacetateand0.2%aceticacid)and(B)90%acetonitrileinwater(containing10mMammoniumacetateand0.2%aceticacid).TheACQUITYUPLCBEHAmidecolumn(2.1mm×100mm,1.7µm,Waters,UK)washeatedto40˚C.Injectionvolumewas5µl(positivemodes)or15µl(negativemodes).TheMSwascomposedofaheatingelectrosprayionization(H-ESI)source.Forpositivemode,theinterfacecapillarywaskeptat650˚Cwithasheathgasflowof50psiandauxgasflowof40psi.Thesprayvoltagewas4kV.Fornegativemode,theinterfacecapillarywaskeptat650˚Cwithasheathgasflowof50psiandauxgasflowof40psi.Thesprayvoltagewas-4kV.Forlipidomics,HPLCconditionsincludeaflowrateof500µl/minwithamobilephaseof(A)95%acetonitrileinwater(containing10mMammoniumacetate,pH=8)and(B)50%isopropanolacetonitrileinwater(including10mMammoniumacetate,pH=8).TheAcquityUPLCBEHHILICcolumn(2.1mm×100mm,1.7µm,Waters,UK)wasoperatedat35°C.Theinjectionvolumewas2µl.Forpositivemode,theMSinterfacecapillarywaskeptat500˚Cwithasheathgasflowof50psiandauxgasflowof60psi.Thesprayvoltagewas5.3kV.Fornegativemode,theMSinterfacecapillarywaskeptat500˚Cwithasheathgasflowof50psiandauxgasflowof60psi.Thesprayvoltagewas-4.5kV.S-5

5StatisticalanalysisTheobtainedrawdataofaqueous-solublemetabolitesandlipidswereimportedintoMultiQuant(v2.0.3)forintegratedprocessing.Thepretreateddata,includingsampleinformation,metaboliteidentity,andpeakintensities,werethenprocessedonR(v3.5.1),wherecompoundswithmissingvalue>50%andcoefficientofvariation(CV)>20%foraqueous-solublemetabolitesandCV>30%forlipidsintheQCsampleswerefilteredandtreatedwithk-nearestneighboralgorithmformissingvalueimputation(2).Subsequently,principalcomponentanalysis(PCA),partialleastsquaresdiscriminationanalysis(PLS-DA),andorthogonalPLS-DA(OPLS-DA)wereperformedaftermediannormalizationtodiscriminateHFD-treated,HFDEL-treated,andcontrolsamples(3).Basedonp-value(p<0.1foraqueous-solublesamplesandp<0.05forlipids)intheKruskal-Wallistestandvariableimportanceintheprojection(VIP>1.0)oftheabovePLS-DAmodel,alistcontainingaqueous-solublemetabolites(TableS3)andlipids(TableS4)withcriticalcontributiontothevariancewasachieved.MetabolicpathwayanalysiswasperformedbyusingMetaboAnalystR2.0(4)accordingtothepathwaytopologyanalysistogetherwithpathwayenrichmentanalysisthroughKyotoEncyclopediaofGenesandGenomes(www.genome.jp/kegg/)databasetoidentifytherepresentativemetabolicpathways.DESI-MSIThreeliverandkidneysections(thickness:8µm)fromtheHFD-treated,HFDEL-treated,andcontrolsamplesweremountedonglassmicroscopeslidesandstoredat-80°Cpriortoanalysis.ADESI(2D,Prosolia,USA)sourcecoupledtoanMS(SynaptG2-Si,Waters,USA)wasusedforliverandkidneyimaging.TheDESI-MSIwascarriedoutinbothpositiveandnegativemodes.Inthepositivemode,thevoltagewassetas5kVfromm/z50to1200withaspatialresolutionof50µm(sprayspotdiameter)forliversamplesand80µm(sprayspotdiameter)forkidneysamples.Histologicallycompatiblesolventsystemincluded95%CH3OH/5%H2Oand0.1%formicacidataflowrateof2µl/min.Inthenegativemode,thevoltagewassetas-4.5kVfromm/z50to1200withaspatialresolutionof50µm(sprayspotdiameter)forliversamplesand80µm(sprayspotdiameter)forkidneysamples.Thehistologicallycompatiblesolventsystemincluded95%CH3OH/5%H2Oand0.1%formicacidataflowrateof2µl/min.A0.5MbarN2flowwasappliedassheathgasforelectrospraynebulization.Theliverandkidneywerescannedunderimpingingchargeddropletsona2Dmovingstageinhorizontalrowsseparatedby80μm(liver)or50μm(kidney)verticalstep.Theseexperimentswereperformedunderoptimizedexperimentalconditionswheretheinlet-to-surfacedistancewas2mm,sprayincidentanglewas60°,andspray-to-inletdistancewas5mm.LaterdataprocesswasoperatedwithHDIv1.5(Waters,USA),whichallowedtransformationofthemassspectralfiles(rawdata)intoimagefiles.Thedistributionofdifferentmetabolitesandlipidswasrecorded.WBandqPCRassaysonE.longifoliatreatedcellmodelHumanhepaticcells(L02)cellswereculturedindulbecco'smodifiedeaglemedium(DMEM,S-6

6ThermoFisher,USA)containing10%fetalbovineserum(Gibco,ThermoFisher,USA)at37˚Cwith5%CO2,whichwassubsequentlyinteractedwith0.6mg/mlE.longifoliaaqueousextract(10%ofminimumconcentrationatwhichapparentcytotoxicitywasobserved)foranother48h.SuchconcentrationwasselectedbasedonoptimumcellviabilitytogetherwithnegligibletoxicityofE.longifolia.TheexpressionofrelatedgeneswasinvestigatedbyWBandqPCRassays.ForWBassay,cellswerewashedthreetimeswithice-coldPBSandlysedinice-cold1×RIPAbuffer(CellSignalingTechnology,Beverly,Morocco)withproteaseinhibitorcocktail(10×)(RocheDiagnostics,Indianapolis,India).TheconcentrationofproteinwasdetectedbyusingBCAreagentkit(Bio-Rad,Laboratories,Richmond,CA)toreachequalamountinallsamplesfor4-15%gradientSDS-polyacrylamidegelelectrophoresis.Then,theproteinsweretransferredontopolyvinylidenedifluoride(PVDF)membranes(Bio-RadLaboratories,Richmond,CA)whichthenwereblockedwith5%skimmilkfor1hatroomtemperature.Themembraneswerereactedwith1:1000dilutionoflipoproteinlipase(LPL),branched-chainaminoacidaminotransferase1(BCAT1),fattyacidsynthase(FASN)orβ-tublinantibody(ABclonal,Wuhan,China)at4℃overnight.Afterwashingwithtrisbufferedsalinecontaining0.1%tween-20(TBST,pH7.2),themembraneswereincubatedwithHRP-conjugatedgoatanti-rabbitIgG(1:10,000dilution)(CellSignalingtechnologies).FollowinganotherwashingwithTBST,finally,themembraneswereTMstainedwithPierceECLWesternBlottingSubstrate(ThermoScientific,USA)andWBimageswereobtainedwithChemiDoc™MPimagingsystem(Bio-RadLaboratories,Hercules,Canada).ForqPCR,totalRNAfromL02cellswasextractedwithSimplyPTotalRNAExtractionkit(BioFlux,China)whiletheconcentrationandthepurityofRNAweredeterminedbyNanoDrop™OneSpectrophotometers(ThermoScientific,USA).ThecDNAwasgeneratedfrom2ugoftotalRNAwithRevertAidFirstStrandcDNASynthesiskit(ThermoScientific,USA).UsingthecDNAastemplate,themRNAexpressionsofobesityalleviation-relatedgenesweredetectedwithSYBRGreenqPCRMastermix(TsingKe,China)andtheprimersequencesusedwerelistedinTableS1.PCRamplificationwascarriedoutina96-holeformatreal-timePCRthermalcycler(Real-TimeSystem,CFXConnect,BIO-RAD,USA).ThemRNAtranscriptionlevelswerecalculatedbythe-ΔΔCt2method.References1.Putri,J.;Isa,M.;Ekram,A.;Norazlina,M.;Ahmad,S.,TheEffectsofQuassinoid-RichEurycomalongifoliaExtractonBoneTurnoverandHistomorphometryIndicesintheAndrogen-DeficientOsteoporosisRatModel.Nutrients2018,10,799-815.2.Lee,J.Y.;Styczynski,M.P.,NS-kNN:amodifiedk-nearestneighborsapproachforimputingmetabolomicsdata.Metabolomics2018,14,153-165.3.Thévenot,E.A.;Roux,A.;Xu,Y.;Ezan,E.;Junot,C.,AnalysisoftheHumanAdultUrinaryMetabolomeVariationswithAge,BodyMassIndex,andGenderbyImplementingaComprehensiveWorkflowforUnivariateandOPLSStatisticalAnalyses.JournalofProteomeResearch2015,14,3322-3335.4.Chong,J.;Jianguo,X.;Oliver,S.,MetaboAnalystR:anRpackageforflexibleandreproducibleS-7

7analysisofmetabolomicsdata.Bioinformatics2018,34,4313-4314.S-8

8TableS1SequencesoftheprimersforqPCRanalysisForwardReverseLPLTTGCCCTAAGGACCCCTGAATTGAAGTGGCAGTTAGACACAGFASNGGAGGTGGTGATAGCCGGTATTGGGTAATCCATAGAGCCCAGBCAT1GAAGTGGCGGAGACTTTTAGGTGGTCAGTAAACGTAGCTCCAGAPDHGGTCACCAGGGCTGCTTTTAACTCTTTGGAGGGATCTCGCTCCTGTableS2Thecompositionofhigh-fatdietIngredientWeight(%)Casein,lactic8L-cystine0.12Lodex105Sucrose2.88Solka-Floc2Lard9.8Soybeanoil1MineralS10026B2Cholinebitartrate0.08VitaminV10001C0.04S-9

9FigureS1TypicalLC-MS/MSbasepeakintensity(BPI)spectraofE.longifoliaaqueousextract.(a)BPIspectrawithlowsprayvoltage(2V),(b)BPIspectrawithhighsprayvoltageof(10-45kV).S-10

10FigureS2TypicalLC-MS/MSTICspectraofaqueous-solublesamplesfromHFDtreatment,HFDELtreatmentgroupandcontrol.HFDtreatment:fedwithhigh-fatdiet;HFDELtreatment:fedwithhigh-fatdietand120mg/mlE.longifolia;control:fedwithnormaldiet.Pos:LC-MS/MSdetectionwithpositivemode,neg:LC-MS/MSdetectionwithnegativemode.S-11

11FigureS3PCAscoreplotsofPC1vs.PC2basedontheLC-MS/MSdataofaqueous-solublesamplesinplasma,liverandkidney.(a)ControlgroupvsHFD-treatedgroupinplasma,(b)controlgroupvsHFDEL-treatedgroupinplasma,(c)HFD-treatedgroupvsHFDEL-treatedgroupinplasma,(d)controlgroupvsHFD-treatedgroupinliver,(e)controlgroupvsHFDEL-treatedgroupinliver,(f)HFD-treatedgroupvsHFDEL-treatedgroupinliver,(g)controlgroupvsHFD-treatedgroupinkidney,(h)controlgroupvsHFDEL-treatedgroupinkidney,(i)HFD-treatedgroupvsHFDEL-treatedgroupinkidney.HFD:high-fatdiet,HFDEL:HFD&E.longifolia.S-12

12FigureS4PLS-DAscoreplotsofp1vs.p2basedontheLC-MS/MSdataofaqueous-solublesamplesinplasma,liverandkidney.(a)ControlgroupvsHFD-treatedgroupinplasma,(b)controlgroupvsHFDEL-treatedgroupinplasma,(c)HFD-treatedgroupvsHFDEL-treatedgroupinplasma,(d)controlgroupvsHFD-treatedgroupinliver,(e)controlgroupvsHFDEL-treatedgroupinliver,(f)HFD-treatedgroupvsHFDEL-treatedgroupinliver,(g)controlgroupvsHFD-treatedgroupinkidney,(h)controlgroupvsHFDEL-treatedgroupinkidney,(i)HFD-treatedgroupvsHFDEL-treatedgroupinkidney.HFD:high-fatdiet,HFDEL:HFD&E.22longifolia.Permutationtest:pR=0.01,pQ=0.01.S-13

13FigureS5OPLS-DAscoreplotsofp1vs.o1basedontheLC-MS/MSdataofaqueous-solublesamplesinplasma,liverandkidney.(a)ControlgroupvsHFD-treatedgroupinplasma,(b)controlgroupvsHFDEL-treatedgroupinplasma,(c)HFD-treatedgroupvsHFDEL-treatedgroupinplasma,(d)controlgroupvsHFD-treatedgroupinliver,(e)controlgroupvsHFDEL-treatedgroupinliver,(f)HFD-treatedgroupvsHFDEL-treatedgroupinliver,(g)controlgroupvsHFD-treatedgroupinkidney,(h)controlgroupvsHFDEL-treatedgroupinkidney,(i)HFD-treatedgroupvsHFDEL-treatedgroupinkidney.HFD:high-fatdiet,HFDEL:HFD&E.22longifolia.Permutationtest:pR=0.01,pQ=0.01.S-14

14FigureS6Theheatmapofdifferentialaqueous-solublemetabolitesinplasma.Thecolorscaleillustratestherelativeabundancesacrossthesamples.Blueindicatesmetabolitesthatweresignificantlydownregulated,whileredindicatessignificantlyupregulatedmetabolites.S-15

15FigureS7Theheatmapofdifferentialaqueous-solublemetabolitesinliver.Thecolorscaleillustratestherelativeabundancesacrossthesamples.Blueindicatesmetabolitesthatweresignificantlydownregulated,whileredindicatessignificantlyupregulatedmetabolites.S-16

16FigureS8Theheatmapofdifferentialaqueous-solublemetabolitesinkidney.Thecolorscaleillustratestherelativeabundancesacrossthesamples.Blueindicatesmetabolitesthatweresignificantlydownregulated,whileredindicatessignificantlyupregulatedmetabolites.S-17

17FigureS9Volcanoplotsofalldetectedandquantifiedaqueous-solublemetabolites.Plotsillustratetheoverallpatternofmetabolitesandtheirrelativeabundancesinplasma,liverandkidney.(a)ControlgroupvsHFD-treatedgroupinplasma,(b)controlgroupvsHFDEL-treatedgroupinplasma,(c)HFD-treatedgroupvsHFDEL-treatedgroupinplasma,(d)controlgroupvsHFD-treatedgroupinliver,(e)controlgroupvsHFDEL-treatedgroupinliver,(f)HFD-treatedgroupvsHFDEL-treatedgroupinliver,(g)controlgroupvsHFD-treatedgroupinkidney,(h)controlgroupvsHFDEL-treatedgroupinkidney,(i)HFD-treatedgroupvsHFDEL-treatedgroupinkidney.HFD:high-fatdiet,HFDEL:HFD&E.longifolia.Thex-axiswasthelog2(foldchange)oftherelativeabundanceofeachgroup.They-axisrepresents-log(p-value)betweengroups.pvalue≤0.05,foldchange>2or<0.5.S-18

18TableS3Summaryofaqueous-solublemetabolitevariationsindifferentconditionsVIPMolecularPlasmaLiverKidneyCompoundp-valuevalueweightHFDEL-conHFD-conHFDEL-conHFD-conHFDEL-conHFD-conAminoacidsAcetylcarnitine1.1120.009203.2356-0.6-0.8-1.3-0.85-Aminovalericacid1.0110.005117.0789790.31.1Arginine1.0050.027174.2010.70.3Asparagine1.0210.024132.11790.80.3Carnitine1.0940.010161.1989-0.7-0.6-0.5-0.5Citrulline1.0140.024175.1857-0.5-0.5Deoxycarnitine1.1090.010145.1995-0.8-0.90.5-0.2Glutamine1.2110.015146.14450.80.2Glycine1.0220.00575.032028-0.4-0.6Homoserine1.1640.005119.1192-0.20.6Hydroxyproline1.0020.005131.1299-0.2-0.6-0.2-0.4Leucine1.0070.003131.17290.30.8Lysine1.1120.009146.1876-0.10.4Methionine1.1390.010149.2113-0.10.53-Nitro-tyrosine1.0070.056226.0589751.50.5N-AcetylGlycine1.3510.017117.0425951.5-0.10.7-0.40.4-0.5Oxidizedglutathione1.1110.006612.1519780.50.7Phenylalanine1.0400.003165.18910.20.60.40.3Proline1.0710.018115.1305-0.30.8-0.10.3Sarcosine1.1690.02889.047676-0.70.3Taurine1.1200.009125.0146640.90.9Threonine1.1500.005119.1192-0.30.5Tryptophan1.0720.006204.22520.40.80.10.2Tyrosine1.1380.011181.1885-0.60.5Valine1.1180.008117.14630.21.10.00.3OrganicacidsAdipicacid1.1560.010146.057907-0.80.2Chenodeoxycholate1.3670.016391.2853700.9-0.1D-LeucicAcid1.0530.003132.0786440.40.6GlutaricAcid1.2300.015132.0422520.31.4Guanidinoacetate1.2380.003116.046547-0.8-1.5-0.5-0.53-Hydroxybenzoicacid1.2240.015138.0316931.90.72-Hydroxyisovaleric1.0480.005118.0629960.60.90.20.50.00.4acid4-Hydroxyphenylpyruvic1.0920.006180.042252-1.4-0.8-0.40.2acidHyppuricacid1.1420.005179.058243-3.5-2.43-Indoxylsulfate1.0830.004213.009583-1.2-1.7-0.9-1.4Isovalericacid1.1940.027102.068077-0.20.2α-Ketoglutaricacid1.1710.023146.0215300.90.3S-19

19Lactate1.0970.00789.024422-0.7-0.4-0.30.0Malate1.1760.015132.006973-0.10.3Malonicacid1.2400.010104.0109561.80.7Oxalicacid1.0910.01189.995308-0.9-0.6-0.30.1Propionate1.1610.01073.029503-0.30.84-Pyridoxicacid1.1260.017183.0531621.10.01.10.1AminesAllantoin1.0180.008158.043991-0.2-0.90.0-0.6Serotonin1.0030.040176.094955-0.5-0.2Trimethylamine-N-oxide1.0330.00875.068413-2.7-2.2AlkalinesAcetylcholine1.1410.003146.117554-0.7-1.1Betaine1.0610.003118.1537-0.6-1.1-0.7-1.8Choline1.0030.034104.10699-0.1-0.3Guanosine1.1120.031283.0916750.20.7Indole1.0040.006117.14790.50.8Pipecolate1.0560.011128.0717010.0-0.3CarbohydratesFructose1.2810.016180.15590.2-0.1Galactose1.2670.015180.15590.2-0.1Glucosamine1.0390.024179.1711-0.20.0Glucose1.1660.027180.15590.2-0.1Lactose1.2380.018342.2965-0.6-0.1Fattyacids12-HETE1.2320.016320.46632.90.313-HODE1.0300.039296.44488-0.10.5-0.70.0Linolenicacid1.0240.015278.2245791.20.80.20.6AlcoholsInositol1.0720.007180.1559-0.7-1.00.1-0.3NucleotidesCytidine1.0190.036243.0855260.2-0.6Inosine1.4480.009268.22610.1-0.11-Methyladenosine1.2000.018281.26790.4-0.112-HETE:12-hydroxyeicosatetraenoicacid,13-HODE:13-hydroxyoctadecadienoicacid.HFD:high-fatdiet,HFDEL:HFD&E.longifolia,con:control,fedwithnormaldiet.S-20

20FigureS10Summaryofpathwayanalysisofdifferentialaqueous-solublemetabolitesunderdifferentexperimentalconditionsinvariousorganswithMetaboAnalystR2.0.(a)Plasma,(b)liver,(c)kidney.Thenodecolorwasbasedonitspvalueandthenoderadiuswasdeterminedbasedontheirpathwayimpactvalues.SamplesweredividedintoHFDtreatment,HFDELtreatmentgroupandcontrol.HFDtreatment:fedwithhigh-fatdiet;HFDELtreatment:fedwithhigh-fatdietand120mg/mlE.longifolia;control:fedwithnormaldiet.A:phenylalanine,tyrosineandtryptophanbiosynthesis,B:glycine,serineandthreoninemetabolism,C:ubiquinoneandotherterpenoid-quinonebiosynthesis,D:argininebiosynthesis,E:arginineandprolinemetabolism,F:citratecycle,G:galactosemetabolism,H:phenylalaninemetabolism,I:taurineandhypotaurinemetabolism,J:starchandsucrosemetabolism,K:biotinmetabolism,L:alpha-linolenicacidmetabolism,M:alanine,aspartateandglutamatemetabolism,N:pyruvatemetabolism,O:glutathionemetabolism,P:pentosephosphatepathway,Q:glycerolipidmetabolism,R:glycerophospholipidmetabolism,S:tryptophanmetabolism,T:starchandsucrosemetabolism,U:nicotinateandnicotinamidemetabolism,V:fructoseandmannosemetabolism.ThedatawereobtainedfromtheOPLS-DAmodel.S-21

21S-22

22FigureS11TypicalLC-MS/MSTICspectraoflipidsfromHFDtreatment,HFDELtreatmentgroupandcontrol.HFDtreatment:fedwithhigh-fatdiet;HFDELtreatment:fedwithhigh-fatdietand120mg/mlE.longifolia;control:fedwithnormaldiet.Pos:LC-MS/MSdetectionwithpositivemode,neg:LC-MS/MSdetectionwithnegativemode.S-23

23FigureS12PCAscoreplotsofPC1vs.PC2basedontheLC-MS/MSdataoflipidsinplasma,liverandkidney.(a)ControlgroupvsHFD-treatedgroupinplasma,(b)controlgroupvsHFDEL-treatedgroupinplasma,(c)HFD-treatedgroupvsHFDEL-treatedgroupinplasma,(d)controlgroupvsHFD-treatedgroupinliver,(e)controlgroupvsHFDEL-treatedgroupinliver,(f)HFD-treatedgroupvsHFDEL-treatedgroupinliver,(g)controlgroupvsHFD-treatedgroupinkidney,(h)controlgroupvsHFDEL-treatedgroupinkidney,(i)HFD-treatedgroupvsHFDEL-treatedgroupinkidney.HFD:high-fatdiet,HFDEL:HFD&E.longifolia.S-24

24FigureS13PLS-DAscoreplotsofp1vs.p2basedontheLC-MS/MSdataoflipidsinplasma,liverandkidney.(a)ControlgroupvsHFD-treatedgroupinplasma,(b)controlgroupvsHFDEL-treatedgroupinplasma,(c)HFD-treatedgroupvsHFDEL-treatedgroupinplasma,(d)controlgroupvsHFD-treatedgroupinliver,(e)controlgroupvsHFDEL-treatedgroupinliver,(f)HFD-treatedgroupvsHFDEL-treatedgroupinliver,(g)controlgroupvsHFD-treatedgroupinkidney,(h)controlgroupvsHFDEL-treatedgroupinkidney,(i)HFD-treatedgroupvsHFDEL-treatedgroupinkidney.HFD:high-fatdiet,HFDEL:HFD&E.longifolia.Permutation22test:pR=0.01,pQ=0.01.S-25

25FigureS14OPLS-DAscoreplotsofp1vs.o1basedontheLC-MS/MSdataoflipidsinplasma,liverandkidney.(a)ControlgroupvsHFD-treatedgroupinplasma,(b)controlgroupvsHFDEL-treatedgroupinplasma,(c)HFD-treatedgroupvsHFDEL-treatedgroupinplasma,(d)controlgroupvsHFD-treatedgroupinliver,(e)controlgroupvsHFDEL-treatedgroupinliver,(f)HFD-treatedgroupvsHFDEL-treatedgroupinliver,(g)controlgroupvsHFD-treatedgroupinkidney,(h)controlgroupvsHFDEL-treatedgroupinkidney,(i)HFD-treatedgroupvsHFDEL-treatedgroupinkidney.HFD:high-fatdiet,HFDEL:HFD&E.longifolia.Permutation22test:pR=0.01,pQ=0.01.S-26

26FigureS15Theheatmapofdifferentiallipidsinplasma.Thecolorscaleillustratestherelativeabundancesacrossthesamples.Blueindicatesmetabolitesthatweresignificantlydownregulated,whileredindicatessignificantlyupregulatedmetabolites.S-27

27FigureS16Theheatmapofdifferentiallipidsinliver.Thecolorscaleillustratestherelativeabundancesacrossthesamples.Blueindicatesmetabolitesthatweresignificantlydownregulated,whileredindicatessignificantlyupregulatedmetabolites.S-28

28FigureS17Theheatmapofdifferentiallipidsinkidney.Thecolorscaleillustratestherelativeabundancesacrossthesamples.Blueindicatesmetabolitesthatweresignificantlydownregulated,whileredindicatessignificantlyupregulatedmetabolites.S-29

29FigureS18Volcanoplotsofalldetectedandquantifiedlipids.Plotsillustratetheoverallpatternofmetabolitesandtheirrelativeabundancesinplasma,liverandkidney.(a)ControlgroupvsHFD-treatedgroupinplasma,(b)controlgroupvsHFDEL-treatedgroupinplasma,(c)HFD-treatedgroupvsHFDEL-treatedgroupinplasma,(d)controlgroupvsHFD-treatedgroupinliver,(e)controlgroupvsHFDEL-treatedgroupinliver,(f)HFD-treatedgroupvsHFDEL-treatedgroupinliver,(g)controlgroupvsHFD-treatedgroupinkidney,(h)controlgroupvsHFDEL-treatedgroupinkidney,(i)HFD-treatedgroupvsHFDEL-treatedgroupinkidney.HFD:high-fatdiet,HFDEL:HFD&E.longifolia.Thex-axiswasthelog2(foldchange)oftherelativeabundanceofeachgroup.They-axisrepresents-log(p-value)betweengroups.pvalue≤0.05,foldchange>2or<0.5.S-30

30TableS4SummaryoflipidvariationsindifferentconditionsMolecularPlasmaLiverKidneyCompoundVIPvaluep-valueweightHFDEL-conHFD-conHFDEL-conHFD-conHFDEL-conHFD-conPhospholipidsPC(14:0/18:2)1.013729.53090.018-0.4-0.3PC(14:0/20:4)1.645753.53090.0030.60.20.80.6PC(14:0/22:4)1.513781.56220.0181.00.5PC(16:0/18:0)1.276761.59350.0180.40.3PC(16:0/18:1)1.291759.57780.0110.20.3PC(16:0/18:2)1.200757.56220.042-0.2-0.1PC(16:0/20:3)1.020783.57780.011-0.8-0.8-0.5-0.5PC(16:0/20:4)1.129781.56220.0070.40.3PC(16:0/20:5)1.120779.54650.007-2.0-1.8PC(16:0/22:4)1.102809.59350.0111.11.1PC(16:0/22:5)1.109807.57780.0080.80.8PC(16:0/22:6)1.044805.56220.003-0.4-0.2-0.3-0.2-0.3-0.2PC(16:1/18:1)1.698757.56220.021-0.10.30.10.3PC(16:1/18:2)1.131755.54650.042-0.10.2-0.5-0.3PC(18:0/14:0)1.085733.56220.0031.01.70.60.6PC(18:0/16:1)1.152759.57780.0030.71.4PC(18:0/18:0)1.419789.62480.0260.30.4PC(18:0/18:1)1.054787.60910.0030.50.80.30.5PC(18:0/18:2)1.223785.59350.0050.10.3PC(18:0/18:3)1.098783.57780.0030.81.2PC(18:0/20:3)1.029811.60910.009-0.7-0.8PC(18:0/20:4)1.154809.59350.0080.60.5PC(18:0/20:5)1.128807.57780.010-1.9-1.8PC(18:0/22:4)1.115837.62480.0081.21.01.31.3PC(18:0/22:6)1.292833.59350.011-0.3-0.1PC(18:1/16:1)1.144757.56220.0050.40.80.30.5PC(18:1/18:1)1.109785.59350.0030.91.50.50.7PC(18:1/18:2)1.324783.57780.0040.30.90.00.3PC(18:1/18:3)1.400781.56220.0110.10.9PC(18:1/20:1)1.318813.62480.0080.10.50.30.50.10.4PC(18:1/20:2)1.181811.60910.0030.71.30.40.6PC(18:1/20:3)1.300809.59350.024-0.40.0-0.8-0.6-0.10.2PC(18:1/20:4)1.741807.57780.0110.80.8PC(18:1/20:5)1.127805.56220.011-1.7-1.7PC(18:1/22:4)1.078835.60910.0111.51.6PC(18:1/22:5)1.151833.59350.003-0.9-0.4-0.9-0.7PC(18:1/22:6)1.841831.57780.0060.10.3PC(18:2/18:2)1.676781.56220.010-0.50.2-0.5-0.2-0.4-0.1PC(18:2/18:3)1.591779.54650.017-0.90.0-0.8-0.6PC(18:2/20:1)1.510811.60910.015-0.6-0.1-0.5-0.3-0.50.0S-31

31PC(18:2/20:2)1.373809.59350.0080.20.8-0.3-0.1PC(18:2/20:3)1.250807.57780.015-0.8-0.3-0.8-0.4-0.6-0.4PC(18:2/20:4)1.307805.56220.0030.30.7PC(18:2/20:5)1.149803.54650.011-2.2-1.8PC(18:2/22:4)1.092833.59350.0111.00.9PC(18:2/22:6)1.206829.56220.003-0.9-0.4-0.6-0.3-0.4-0.3PC(20:0/18:1)1.209815.64040.020-0.5-0.1PC(20:0/20:5)1.121835.60910.010-2.6-2.3PC(20:0/22:6)1.036861.62480.009-1.1-0.9PE(14:0/18:2)1.123687.48390.009-1.2-0.4PE(14:0/22:6)1.031735.48390.005-1.1-0.7PE(16:0/14:0)1.299663.48390.0100.40.8PE(16:0/16:0)1.533691.51520.0110.00.80.30.5PE(16:0/16:1)2.724689.49960.011-0.20.1PE(16:0/18:1)1.424717.53090.014-0.30.4PE(16:0/18:2)1.425715.51520.016-0.8-0.1-0.4-0.1PE(16:0/18:3)1.077713.49960.010-1.1-0.4-0.8-0.9-0.7-0.3PE(16:0/20:3)1.065741.53090.009-0.6-0.5PE(16:0/20:4)1.426739.51520.0270.11.2PE(16:0/20:5)1.118737.49960.008-0.11.4-2.2-2.1PE(16:0/22:4)1.118767.54650.0081.51.31.01.0PE(18:0/16:0)1.331719.54650.0070.20.9PE(18:0/18:1)1.364745.56220.0070.31.20.00.2PE(18:0/18:2)1.500743.54650.0110.10.9PE(18:0/18:3)1.495741.53090.016-0.30.8-0.4-0.2PE(18:0/20:3)1.075769.56220.011-0.2-1.4-1.3-0.7PE(18:0/22:6)1.290791.54650.010-0.8-0.6PE(18:1/18:1)1.471743.54650.0070.31.50.10.4PE(18:1/18:2)1.643741.53090.007-0.31.00.00.3PE(18:1/18:3)1.597739.51520.009-0.41.2PE(18:1/20:1)1.417771.57780.0110.00.80.30.50.50.7PE(18:1/20:2)1.241769.56220.0030.61.40.30.5PE(18:1/20:3)1.459767.54650.031-0.50.1PE(18:1/20:4)1.021765.53090.0090.30.3PE(18:1/20:5)1.121763.51520.004-0.10.9-2.2-2.0PE(18:1/22:4)1.089793.56220.0060.71.41.41.21.41.4PE(18:2/18:2)1.627739.51520.018-1.20.2-0.5-0.1PE(18:2/18:3)1.370737.49960.018-1.80.0-0.7-0.4PE(18:2/20:1)1.477769.56220.038-0.80.1-0.30.0PE(18:2/20:2)1.343767.54650.0080.31.4PE(18:2/20:3)1.280765.53090.034-0.40.5-0.5-0.1PE(18:2/20:4)1.291763.51520.0110.10.7PE(18:2/20:5)1.074761.49960.011-2.0-2.0PE(18:2/22:4)1.107791.54650.0101.61.4S-32

32PE(18:2/22:6)1.013787.51520.009-1.0-0.6PE(O-16:0/16:1)1.338675.52030.0160.11.0PE(O-16:0/18:1)1.190703.55160.0030.61.30.50.7PE(O-16:0/18:2)1.364701.53590.037-0.50.1PE(O-16:0/18:3)1.183699.52030.005-1.4-0.6PE(O-16:0/22:4)1.140753.56720.0170.80.51.11.2PE(O-16:0/22:5)1.220751.55160.0070.20.8PE(O-18:0/16:1)1.267703.55160.0130.10.8-0.7-0.40.20.6PE(O-18:0/18:1)1.029731.58290.0041.01.50.60.8PE(O-18:0/18:2)1.319729.56720.0110.10.6PE(O-18:0/18:3)1.561727.55160.018-0.70.0PE(O-18:0/20:1)1.032759.61420.0080.40.8PE(O-18:0/20:4)1.346753.56720.0320.70.4PE(O-18:0/20:5)1.036751.55160.0420.60.4PE(O-18:0/22:4)1.601781.59850.0041.71.11.91.7PE(P-16:0/18:1)1.190701.53590.0230.10.7PE(P-16:0/18:3)1.183697.50460.003-1.6-0.9PE(P-16:0/20:1)1.065729.56720.0170.90.9PE(P-16:0/20:2)1.002727.55160.0201.00.8PE(P-16:0/20:5)1.122721.50460.007-2.9-2.8PE(P-16:0/22:4)1.167751.55160.0271.00.51.31.1PE(P-16:0/22:6)1.556747.52030.020-0.30.1PE(P-18:0/18:2)1.083727.55160.021-0.30.20.0-0.5PE(P-18:0/18:3)1.255725.53590.041-1.1-0.4PE(P-18:0/20:3)1.371753.56720.0181.00.60.70.6PE(P-18:0/20:4)1.287751.55160.0101.00.60.60.5PE(P-18:0/20:5)1.111749.53590.007-2.1-2.0PE(P-18:0/22:4)1.287779.58290.0061.71.21.81.7PE(P-18:0/22:6)1.342775.55160.024-0.10.2-0.20.8PG(14:0/18:1)1.042720.49410.007-0.7-0.60.60.8PG(14:0/18:2)1.184718.47850.007-1.0-0.6PG(14:0/20:1)1.307748.52540.0080.40.6PG(14:0/20:2)1.081746.50980.0110.50.6PG(14:0/20:3)1.064744.49410.0100.80.7PG(16:0/14:0)1.046694.47850.0111.21.2PG(16:0/16:0)1.099722.50980.0110.80.8PG(16:0/16:1)1.164720.49410.008-1.2-0.81.61.8PG(16:0/18:0)1.014750.54110.0110.40.4PG(16:0/18:2)1.074746.50980.0070.60.7PG(16:0/18:3)1.157744.49410.011-0.8-0.50.80.9PG(16:0/20:3)1.295772.52540.0060.60.5PG(16:0/20:4)1.191770.50980.0071.31.1PG(16:0/22:4)1.077798.54110.0112.42.3PG(16:0/22:6)1.163794.50980.0111.00.7S-33

33PG(18:0/14:0)1.103722.50980.0081.41.4PG(18:0/18:2)1.050774.54110.0031.42.0PG(18:0/18:3)1.207772.52540.0030.81.8PG(18:0/20:1)1.109804.5880.0100.70.8PG(18:0/20:2)1.035802.57240.0110.70.6PG(18:0/20:3)1.109800.55670.0100.70.6PG(18:0/20:4)1.234798.54110.0052.01.8PG(18:0/22:4)1.102826.57240.0081.51.32.42.3PG(18:0/22:6)1.085822.54110.0170.60.3PG(18:1/18:1)1.062774.54110.0101.42.40.91.1PG(18:1/18:2)1.253772.52540.0031.72.90.80.8PG(18:1/18:3)1.327770.50980.0050.82.30.70.7PG(18:1/20:1)1.198802.57240.0060.61.40.60.8PG(18:1/20:2)1.082800.55670.0110.60.6PG(18:1/20:3)1.125798.54110.0090.60.5PG(18:1/20:4)1.139796.52540.0102.22.1PG(18:1/20:5)1.064794.50980.007-1.5-0.9PG(18:1/22:4)1.075824.55670.0131.61.22.82.8PG(18:1/22:6)1.148820.52540.0111.21.3PG(18:2/18:2)1.380770.50980.0040.72.0PG(18:2/18:3)1.413768.49410.0090.31.8-0.7-0.30.0-0.2PG(18:2/20:1)1.150800.55670.0110.31.00.10.5PG(18:2/20:3)1.546796.52540.015-0.2-0.6PG(18:2/20:4)1.796794.50980.0260.40.1PG(18:2/22:6)1.170818.50980.011-0.3-0.9LysophospholipidsLPC(16:0)1.068495.33250.017-0.2-0.1LPC(18:1)1.017521.34810.0050.30.6LPC(18:2)1.065519.33250.0160.10.4-0.10.5LPE(20:0)1.522509.34810.020-0.7-0.1LPG(16:0)1.126484.28010.0031.62.5LPG(18:1)1.001510.29580.0051.01.5LPG(18:2)1.037508.28010.0031.21.8LPG(20:0)1.434540.34270.0200.2-0.4LPG(20:4)1.098532.28010.0071.71.52.12.2LPG(22:4)1.127560.31140.0112.32.2LPG(22:6)1.013556.28010.009-0.10.10.50.7GlycerolipidsTAG54:1-FA16:01.0480.0031.41.9888.8146TAG54:1-FA20:11.1890.0461.00.8TAG54:3-FA16:11.119884.78330.0040.71.2TAG54:4-FA20:11.2260.0080.51.0TAG54:4-FA20:41.572882.76760.0080.40.8TAG54:4-FA22:41.3540.0071.00.7S-34

34TAG54:5-FA16:11.0700.0160.40.8TAG54:5-FA18:01.0350.0130.61.0TAG54:5-FA18:11.0660.0051.01.4TAG54:5-FA18:21.066880.7520.0030.91.3TAG54:5-FA18:31.1930.0030.91.4TAG54:5-FA20:41.6100.0080.50.9TAG54:5-FA20:51.0990.020-0.9-0.6TAG54:6-FA18:11.4270.0100.30.8TAG54:6-FA18:21.2620.0040.51.0TAG54:6-FA18:31.140878.73630.0060.61.1TAG54:6-FA20:51.1270.015-0.9-0.8TAG54:6-FA22:51.0540.0450.10.5TAG54:7-FA18:31.4000.0210.20.8TAG54:7-FA20:51.0810.007-1.6-1.2876.7207TAG54:7-FA22:51.0280.005-0.7-1.2TAG54:7-FA22:61.0050.011-1.3-1.3TAG54:8-FA22:61.064874.7050.011-1.2-1.1TAG56:10-FA18:21.137898.7050.0060.50.9TAG56:1-FA16:01.274916.84590.0090.51.1TAG56:2-FA16:01.297914.83020.0150.30.8TAG56:3-FA16:01.406912.81460.0140.10.6TAG56:4-FA18:21.0100.0050.1-0.3910.7989TAG56:4-FA22:41.0430.0080.50.3TAG56:5-FA20:11.0620.0041.11.5TAG56:5-FA20:41.015908.78330.0130.70.6TAG56:5-FA22:51.0800.0091.51.30.40.0TAG56:7-FA18:11.1000.0090.30.6904.752TAG56:7-FA22:41.0490.0131.11.0TAG56:8-FA22:61.120902.73630.032-0.6-0.5TAG56:9-FA20:51.0980.013-1.3-1.5900.7207TAG56:9-FA22:61.0560.011-1.2-1.2TAG57:3-FA18:21.192926.83020.038-0.5-0.7TAG58:10-FA20:41.7020.0070.80.4926.7363TAG58:10-FA20:51.1900.012-0.3-0.7TAG58:2-FA18:11.024942.86150.0041.41.9TAG58:3-FA18:11.099940.84590.0031.21.7TAG58:5-FA18:11.032936.81460.0101.91.7TAG58:6-FA16:01.5870.0170.90.5934.7989TAG58:6-FA22:51.1270.0072.21.8TAG58:8-FA20:41.024930.76760.0101.41.1TAG58:9-FA20:41.028954.76760.0072.21.91.51.3TAG60:10-FA22:51.064954.76760.0102.01.7TAG60:11-FA22:51.103952.7520.0081.41.1-0.1-1.4TAG60:12-FA22:61.040950.73630.016-0.8-0.8S-35

35TAG44:1-FA18:11.049748.65810.0240.41.0TAG45:0-FA16:01.213764.68940.0181.31.0TAG46:0-FA14:01.261778.7050.0150.00.60.12.5TAG46:1-FA16:01.030776.68940.0190.40.8TAG46:2-FA16:11.1520.0310.12.01.61.2TAG46:2-FA18:11.112774.67370.0050.61.20.01.6TAG46:2-FA18:21.0550.0090.40.80.02.0TAG46:3-FA18:11.1030.0031.01.7772.6581TAG46:3-FA18:21.0940.0050.61.2TAG46:4-FA18:21.411770.64240.021-0.20.5TAG48:1-FA18:01.474804.72070.0270.90.5TAG48:3-FA18:11.240800.68940.0071.41.1TAG48:4-FA18:11.074798.67370.0170.70.4TAG49:1-FA18:11.061818.73630.0360.30.6TAG49:3-FA16:11.957814.7050.027-0.9-0.5-0.20.7TAG50:3-FA14:01.0990.0200.50.9828.7207TAG50:3-FA18:31.0880.0230.40.8TAG50:4-FA18:11.0170.0360.61.0TAG50:4-FA18:21.398826.7050.0490.10.6TAG50:4-FA20:41.1520.0311.00.6TAG50:5-FA20:41.197824.68940.0181.20.81.10.3TAG51:1-FA18:01.003846.76760.0110.90.8TAG51:2-FA16:11.3490.0230.20.8844.752TAG51:2-FA17:01.0570.0050.81.2TAG51:3-FA16:11.0750.0080.50.9TAG51:3-FA18:21.525842.73630.0090.31.0TAG51:3-FA18:31.0950.0170.41.2TAG51:4-FA18:21.5050.0480.00.5TAG51:4-FA18:31.449840.72070.0160.20.8TAG51:4-FA20:41.1710.0171.10.6TAG51:5-FA18:32.070838.7050.005-0.50.4TAG52:1-FA20:01.018860.78330.0100.71.1TAG52:2-FA20:11.056858.76760.0060.50.9TAG52:3-FA16:01.0270.0080.12.4856.752TAG52:3-FA18:31.0010.0080.21.6TAG52:4-FA18:11.1250.0130.11.0TAG52:4-FA18:21.2000.0120.01.4TAG52:4-FA18:31.067854.73630.0100.21.6TAG52:4-FA20:01.1150.029-0.11.5TAG52:4-FA20:31.0150.0230.21.4TAG52:5-FA18:11.0820.0200.40.8TAG52:5-FA18:31.3270.0130.20.9852.7207TAG52:5-FA20:41.1270.0481.00.8TAG52:5-FA20:51.0280.011-1.3-1.1S-36

36TAG52:5-FA22:51.0990.0360.30.70.70.4TAG52:6-FA18:11.1640.0050.71.2850.705TAG52:6-FA20:51.0270.008-1.7-1.2TAG53:0-FA16:01.011876.81460.032-0.7-0.6TAG53:1-FA17:01.0290.0470.80.80.10.9TAG53:1-FA18:01.199874.79890.0081.21.0-0.21.0TAG53:1-FA18:11.2430.0310.60.40.00.7TAG53:2-FA16:01.0220.0030.91.3TAG53:2-FA17:01.010872.78330.0041.41.9TAG53:2-FA18:11.0130.0041.31.8TAG53:3-FA16:01.2430.0050.51.0TAG53:3-FA17:01.190870.76760.0031.01.5TAG53:3-FA18:21.0120.0060.81.2TAG53:4-FA17:01.2100.0100.40.9868.752TAG53:4-FA18:21.3030.0030.40.8TAG53:5-FA20:41.184866.73630.0241.00.9DAG(14:0/18:2)1.2880.0310.80.5564.4754DAG(14:0/22:6)1.0030.046-0.7-1.8DAG(16:0/18:1)1.0990.0180.00.61.01.5DAG(16:0/18:2)1.2660.023-1.1-0.2DAG(16:0/20:3)1.333594.52230.0300.0-0.4DAG(16:0/20:4)1.0690.0061.42.4DAG(16:0/22:6)1.0840.029-0.2-1.0DAG(18:1/18:2)1.1250.033-1.3-0.4618.5223DAG(18:1/20:4)1.0530.0100.50.90.30.7DAG(18:2/20:5)1.5990.0240.0-0.9638.491DAG(18:2/22:4)1.7670.0400.2-0.8SphingolipidsSM(14:0)1.060674.53630.0111.41.5SM(16:0)1.264702.56760.0080.10.80.10.1SM(20:0)1.038758.63020.0070.50.6SM(22:1)1.021784.64580.0100.20.40.60.6SM(26:0)1.114842.72410.024-0.5-0.2-0.5-0.3Cer(14:0)1.117747.71050.0080.20.6Cer(20:1)1.475341.2930.0400.1-0.40.90.5Cer(22:0)1.220859.83570.0100.70.6DCer(16:0)1.308539.52770.0140.21.2DCer(18:0)1.128567.5590.0031.01.81.31.1DCer(20:0)1.130623.62160.0150.90.9DCer(20:1)1.410593.59030.0160.40.8DCer(22:0)1.007623.62160.0120.40.80.80.7HCer(16:0)1.033699.56490.0150.60.50.60.5HCer(18:0)1.153727.59620.0101.00.8HCer(20:0)1.023755.62750.0071.41.20.70.6S-37

37HCer(20:1)1.200749.59620.0071.31.0HCer(22:0)1.088783.65880.0110.70.6HCer(26:1)1.457837.70580.015-0.30.8PC:phosphatidylcholine,PE:phosphatidylethanolamines,PG:phosphatidylglycerols,LPC:lysoPC,LPE:lysoPE,LPG:lysoPG,TAG:triacylglycerols,DAG:diacylglycerols,SM:sphingomyelin,Cer:ceramides,DCer:dihydroceramides,HCer:hexosylceramides.S-38

38FigureS19TypicalDESI-MSITICspectraofliversectionsincludingHFDtreatment,HFDELtreatmentgroupandcontrol.Pos:LC-MS/MSdetectionwithpositivemode,neg:LC-MS/MSdetectionwithnegativemode.FigureS20TypicalDESI-MSITICspectraofkidneysectionsincludingHFDtreatment,HFDELtreatmentgroupandcontrol.Pos:LC-MS/MSdetectionwithpositivemode,neg:LC-MS/MSdetectionwithnegativemode.S-39