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1、2021仓鼠的寿命有多长 仓鼠长肿瘤能活多久仓鼠的寿命有多长 仓鼠长肿瘤能活多久“支链氨基酸”延长小白鼠寿命全文CellMetabolismArticleBranched-ChainAminoAcidSupplementationPromotesSurvivalandSupportsCardiacandSkeletalMuscleMitochondrialBiogenesisinMiddle-AgedMiceGiuseppeDAntona,1,2MaurizioRagni,3AnnalisaCardile,3LauraTedesco,3,4MartaDossena,3,5FlaviaBrutt
2、ini,1,2FrancescaCaliaro,1,2GiovanniCorsetti,6RobertoBottinelli,1,2MicheleO.Carruba,3,4AlessandraValerio,3,5andEnzoNisoli3,4,*ofPhysiology,HumanPhysiologyUnitInstituteofMyologyPaviaUniversity,Pavia27100,Italy3CenterforStudyandResearchonObesity,DepartmentofPharmacology,ChemotherapyandMedicalToxicology
3、,SchoolofMedicine,MilanUniversity,Milan20129,Italy4IstitutoAuxologicoItaliano,Milan20145,Italy5PharmacologyUnit6HumanAnatomyUnitDepartmentofBiomedicalSciencesandBiotechnologies,BresciaUniversity,Brescia25123,Italy*Correspondence:enzo.nisoliunimi.itDOI10.1016/j.cmet.2010.08.0162Interuniversity1Depart
4、mentSUMMARYRecentevidencepointstoastrongrelationshipbetweenincreasedmitochondrialbiogenesisandincreasedsurvivalineukaryotes.Branched-chainaminoacids(BCAAs)havebeenshowntoextendchronologicallifespaninyeast.However,theroleoftheseaminoacidsinmitochondrialbiogenesisandlongevityinmammalsisunknown.Here,we
5、showthataBCAA-enrichedmixture(BCAAem)increasedtheaveragelifespanofmice.BCAAemsupplementationincreasedmitochondrialbiogen-esisandsirtuin1expressioninprimarycardiacandskeletalmyocytesandincardiacandskeletalmuscle,butnotinadiposetissueandliverofmiddle-agedmice,andthiswasaccompaniedbyenhancedphysicalend
6、urance.Moreover,thereac-tiveoxygenspecies(ROS)defensesystemgeneswereupregulated,andROSproductionwasreducedbyBCAAemsupplementation.AlloftheBCAAem-mediatedeffectswerestronglyattenuatedinendo-thelialnitricoxidesynthasenullmutantmice.ThesedatarevealanimportantantiagingroleofBCAAsmediatedbymitochondrialb
7、iogenesisinmammals.INTRODUCTIONAgingisanaturalprocessthataffectsmostbiologicalfunctionsandresultsinreducedresistancetostress,increasedvulnera-bilitytodiseases(includingcardiovasculardisease,cancer,diabetes,sarcopenia,osteoporosis,andkidneydisease),andincreasedprobabilityofdeath.Amongtheplethoraofbio
8、logicalphenomenaaffectedbyaging,themalfunctionofmitochondriaandthedecreaseofmitochondrialbiogenesis,togetherwithincreasedoxidativedamage,seemtoexertsomeofthemostpez-deleteriouseffectsontheorganism(Guarente,2008;LoLluchetal.,2008).Avarietyofstrategiesthatalleviateage-relateddecitsinmitochondrialbioge
9、nesisandactivity,includingcalorierestriction(CR)andmoderatephysicalexercise,promotesurvivalinmammals.Theseinterventionsincreasetheexpressionofperoxisomeproliferator-activatedreceptorgcoactivator-1a(PGC-1a,amasterregulatorofmito-chondrialbiogenesisandreactiveoxygenspeciesROSdefensesystem)andofsirtuin
10、1(SIRT1,amemberofthesirtuinfamilylinkedtolifespanextension,enhancedmitochondrialbiogenesis,anddecreasedROSproduction),thusreducingoxidativedamageinmetabolicallyactivetissuesofmiceandhumans(Civitareseetal.,2007;Nisolietal.,2005;Ristowetal.,2009).TheCReffectsonmitochondrialbiogenesisaredue,atleastinpa
11、rt,toinductionofendothelialnitricoxidesynthase(eNOS)expression(Nisolietal.,2005).Indeed,eNOSnullmutant(eNOS/)micearecharacterizedbyareducedlifespan(Lietal.,2004),duetoage-relateddiseases(Cooketal.,2003),andbyareducedmitochondrialbiogenesis(Nisolietal.,2003,2004)andSIRT1expression(Nisolietal.,2005).A
12、lthoughCRhasbenecialeffectsinhumans(Heilponnetal.,2006),suchadietaryregimenisunlikelytobewidelyadoptedintheelderly.Assuch,manyresearchershavefocusedonthedevelopmentofCRmimeticcompoundsprovidingsomeofthebenetsofdietaryrestrictionwithoutreductionincaloricintake(Ingrametal.,2004).Suchattemptshavebeenon
13、lypartiallysuccessfulinexperimentalmodelsuptonowandarenotimmi-nentlyfeasibleforhumans.Recentintriguingresultsindicatethataminoacidsleucine,isoleucine,andvalineextendchronologicallifespaninSaccharo-mycescerevisiae(Alversetal.,2009),thusidentifyingpanched-chainaminoacids(BCAAs)aspotentialcandidatesinp
14、romotingsurvival.Weinvestigatedwhetherlong-termdietarysupplemen-tationwithaspecicBCAA-enrichedmixture(BCAAem)thatimprovesage-relateddisordersinanimalsandhumans(Pansar-asaetal.,2008;Solerteetal.,2008a)alsopromotesmicesurvival.362CellMetabolism12,362372,October6,20102010ElsevierInc.CellMetabolismBranc
15、hed-ChainAminoAcidsandSurvivalFigure1.BCAAemSupplementationIncreasesAverageSurvivalofMaleMiceIndependentlyofAdiposeTissueChanges(AandB)Kaplan-MeiersurvivalcurvesforWT(A)andeNOS/mice(B),withPvaluescalculatedusingthelogranktest.AsignicantextensioninaveragesurvivalwasobservedinBCAAem-supplementedWTmice
16、(c2=8.154;p=0.0043versusuntreatedWTmice;hazardratio,0.50;95%CIofratio,0.2499to0.7727),whilediffer-encesbetweenBCAAem-supplementedanduntreatedeNOS/micedidnotreachthesignif-icance(c2=1.646;p=0.1995;hazardratio,0.7257;95%CIofratio,0.4179to1.200).(C)ComparativesurvivalcharacteristicsofeNOS/andWTmice.Old
17、est10%arethemeanlifespanofthelongest-living10%ofanimalswithinagenotype(p0.05forBCAAemeffectinbothmicestrain).(D)BodyweightofmaleWTmicetreated(closedcircles)ornot(opencircles)withBCAAem,housedindividually(n=30animalseachgroup).Timescaleshowsthetreatmentperiodsstartingfrom9-month-oldmice.(E)Adiposity,
18、expressedaspercentageofweightofvisceralandsubcutaneousfatwhenuntreatedmice(opencolumns)weregivenavalueof100.(F)Foodintake(g/day)ofuntreated(opencircles)andBCAAem-supplemented(closedcircles)mice.Timescaleshowsthetreatmentperiodsstartingfrom9-month-oldmice.Datain(D)(F)representmeanSEM.SeealsoFigureS1.
19、Here,wedemonstratedthattheBCAAemsupplementationincreasedaveragelifespanofmalemice,andthiswasaccompa-niedbyincreasedmitochondrialbiogenesisandSIRT1expres-sionbothincardiacandskeletalmuscles,unlikeadiposetissuesandliverofmiddle-agedmice.Further,themuscleROSdefensesystemgeneswereupregulatedbyBCAAemsupp
20、lementation,resultingindecreasedindicesofoxidativedamage.RESULTSAverageLifeSpanIsExtendedbyBCAAemSupplementationinMiceDifferentaminoacidmixtureshavebeenusedtoimprovemeta-bolicproleandrestoreproteincontentofdefectivetissues,inparticularofskeletalmuscles,inagedsubjects(Dillonetal.,2009;Volpietal.,1998
21、).BCAAemsupplementationhasbeenre-portedtoimproveage-relateddisorderssuchassarcopenia,insulinresistance,type2diabetes,andcardiovasculardysfunc-tion(Solerteetal.,2008a,2008b).Recently,isoleucine,threonine,andvalineorextrasupplementalleucinewereshowntoextendchronologicallifespaninS.cerevisiae(Alverseta
22、l.,2009).Thus,weinvestigatedtheeffectsofBCAAemonmousesurvivalandaskedwhethereNOSexpressionandactivitymightbeinvolvedintheantiagingeffectsofBCAAs.Nine-month-oldmaleWT(F2HypidB6.129S2)miceweresupplementedwithBCAAem(1.5mg/gbodyweight/dayindrinkingwater,contributing6kcal/kg/day,correspondingto$1%dailyca
23、loricintake)aspreviouslydescribed(Pellegrinoetal.,2005).Nostatisticaldiffer-enceindailydrinkingvolumeswasfoundbetweenuntreated(7.01.0ml,n=10animals)andBCAAem-supplementedWTmice(6.52.0ml,n=10animals).At$700daysofage,thesurvivalcurvesofthesupplementedanduntreatedWTgroupsbegantodivergeandremainedsepara
24、tedbyameanintervalof70days(Figure1A).Themedianlifespanwas774daysforalloftheuntreatedcontrols,avaluelowerthanthoserecentlydescribedineitherparentalstrain(Yuanetal.,2009)butcompa-rabletovaluesreportedbyothers(Contietal.,2006),asopposedto869daysforallofthesupplementedWTmice(12%increase),withoutsignican
25、tchangeofmaximallifespan(Figures1Aand1C).Notably,thebodyweight,adiposity,andfoodintakeofsup-plementedanduntreatedWTmicewerenotstatisticallydifferent(Figures1D1F).TheseresultssuggestthattheeffectofBCAAemonmicesurvivalisindependentofadiposetissuereduction.Conversely,thesurvivalcurvesofthesupplementeda
26、nduntreatedeNOS/micedidnotstatisticallydivergethroughouttheentireperiodoftreatment(Figures1Band1C).Nostatisticaldifferenceinfoodintake(datanotshown)anddailydrinkingvolumeswasfoundbetweenuntreated(6.91.0ml,n=10animals)andBCAAem-supplemented(6.51.5ml,n=10animals)eNOS/mice.TheacuteBCAAemsupplementation
27、transientlyincreasedcirculatingBCAAlevelsineNOS/,similartowhatobservedinWTmice(FigureS1),suggestingthatBCAAintestinalabsorptionorrst-passmetabolismaresimilarinthesemousestrains.ThesendingssuggestedthateNOS-mediatedphenomenamightcontributetotheextensionofaveragelifespanbyBCAAem.HormonalProleinBCAAem-
28、SupplementedMiceMetabolicandendocrinealterationsareinvolvedinregulationofagingandantiagingprocesses(RussellandKahn,2007).BCAAemsupplementationfor3monthsdidnotchangetheplasmalevelsCellMetabolism12,362372,October6,20102010ElsevierInc.363CellMetabolismBranched-ChainAminoAcidsandSurvivalFigure2.BCAAemPr
29、omotesMitochondrialBiogenesisinHL-1Cardiomyocytes(AandB)PGC-1a,NRF-1,Tfam,andb-F1-ATPase(A)andCOXIVandCytC(B)mRNAanalyzedbymeansofquantitativeRT-PCR.Relativeexpressionvaluesoftheuntreatedcells(openbars)weretakenas1.0(n=5experiments;*p<0.001).(C)MitochondrialDNAamountanalyzedbymeansofquantitativeP
30、CR.Relativeunitsareexpressedincomparisontothoseofuntreatedcells(openbars)takenas1.0(n=5experiments;*p<0.001).(DandE)Citratesynthaseactivity(D)andATPlevels(E).Valuesareexpressedasfoldchangeversusuntreatedcells(openbars)takenas1.0(n=5experiments;*p<0.001).(F)Copper/zincsuperoxidedismutase(SOD1),
31、manganeseSOD(SOD2),catalase,andglutathioneperoxidase(GPx1)mRNAanalyzedbymeansofquantitativeRT-PCR.Relativeexpressionvaluesoftheuntreatedcells(openbars)weretakenas1.0(n=5experiments;*p<0.001).(G)SIRT1mRNAandproteinlevels.ArepresentativeimmunoblotisshownforSIRT1protein,andnumbersbelowtheblotsareval
32、uesofSIRT1densitometricanalysisreferredtoGAPDH.Valuesmeasuredintheuntreatedcells(Ctrl)aretakenas1.0(n=5experiments;*p<0.001).BCAAemwasusedat13concen-tration.AlldatarepresentmeanSEM.SeealsoFigureS2.ofinsulin(untreated,1.850.07ng/ml;treated,1.900.05ng/ml;n=14animals/group),insulin-likegrowthfactor-
33、1(IGF-1)(untreated,30736ng/ml;treated,31925ng/ml;n=14animals/group),andtestosterone(untreated,359.7nmol/l;treated,35.214nmol/l;n=8animals/group)inmiddle-agedWTmice.Althoughnotstatisticallychanged,growthhormone(GH)levelswereincreasedbyBCAAem(untreated,21.38.5ng/ml;treated,39.111.1ng/ml;n=10animals/gr
34、oup).ThisandresultsdescribedbelowonmusclebersizedonotruleoutthatGHmayplaysomeroleinBCAAemeffects,eveniflackofdifferencesinIGF-1alongwithunalteredinsulinlevelswouldsupporttheconclu-sionthatGHlevelswerenotaffectedbysupplementation.MitochondrialBiogenesisIsIncreasedbyBCAAeminaTissue-SpecicMannerBoostin
35、gmitochondrialactivitymayprovideatleastsomeoftheprocessesthatincreaselifespaninyeast,Caenorhabditisele-gans,Drosophilamelanogaster,andmammals(Bauretal.,2006;Bonawitzetal.,2007;Lagougeetal.,2006;Linetal.,2002;Zidetal.,2009).Moreover,ahigherpooloffunctionalmito-chondriamayleadtoreducedROSproduction,wi
36、thameliora-tionofage-relatedtissuedamages(Barja,2007).Thus,weaimedtoverifytheeffectsofBCAAemincomparisontosingleaminoacidsorotheraminoacidmixtures(TableS1)onmitochondrialbiogenesismarkers(i.e.,mitochondrialDNAmtDNAamountandPGC-1amRNAlevels)inHL-1adultcardiomyocytes.TheBCAAem(mixture#3)wasmoreeffecti
37、vethanothermixturesindose-dependentlypromotingmitochondrialbiogenesis(FigureS2).Withfurtherinvestigation,treatmentofHL-1cellswithBCAAemincreasedthemRNAsencodingPGC-1a,nuclearrespiratoryfactor-1(NRF-1),mtDNAtranscriptionfactorA(Tfam),andbsubunitofthemitochondrialH+-ATPsynthase(b-F1-ATPase)(Figure2A).
38、MitochondrialgenetargetsofPGC-1ainvolvedinoxidativephosphorylationwerealsoupregulatedbyBCAAem(Figure2B).Theseeffectsongeneexpressiontrans-latedintoa2.6-foldincreaseinmtDNAcontent(Figure2C),a2-foldincreaseincitratesynthaseactivity(Figure2D),anda1.5-foldincreaseinATPamount(Figure2E).Moreover,thegeneso
39、ftheROSdefensesystem,includingcopper/zincsuper-oxidedismutase(SOD1),manganesesuperoxidedismutase(SOD2),catalase,andglutathioneperoxidase(GPx1),whoseexpressionisincreasedbyPGC-1aactivation(St-Pierreetal.,2006),wereupregulatedbyBCAAemsupplementation(Figure2F).Notably,SIRT1mRNAandproteinlevelswereincre
40、asedaswell(Figure2G).Next,tovalidatetheseobservationsinvivo,weexaminedtheeffectsofBCAAemsupplementationinmiddle-agedmice.GeneexpressionofmitochondrialtranscriptionalregulatorsandSIRT1aswellascitratesynthaseactivityweremarkedlyincreasedbyprolonged(3months)supplementationinheart,diaphragm,soleus,andti
41、bialismuscles,unlikewhite(WAT)andpownadiposetissue(BAT)andliver(Figures3A3CandS3AS3F).Correspondingly,theexpressionofgenesoftheROSdefensesystemwasincreasedbyBCAAemincardiacandskeletalmuscles(Figure3D),unlikeadiposetissuesandliver(datanotshown).Theseeffectswereabsentinyounganimals.Interestingly,BCAAe
42、msupplementationtoexercisedmicefurtherstrengthenedthemitochondrialbiogenesis(Figure3A3C)364CellMetabolism12,362372,October6,20102010ElsevierInc.CellMetabolismBranched-ChainAminoAcidsandSurvivalFigure3.BCAAemSupplementationPromotesMitochondrialBiogenesisandIncreasestheROSDefenseSysteminCardiacandSkel
43、etalMusclesofSedentaryandExercise-TrainedMiddle-AgedMice(AandB)PGC-1a,NRF-1,andTfam(A)andSIRT1(B)mRNAlevelsanalyzedbymeansofquantitativeRT-PCR.Relativeexpressionvaluesofuntreated(openbars)sedentarymiceweretakenas1.0(n=6experiments;*p<0.05,*p<0.01versuscorrespondinguntreatedanimals;yp<0.05ve
44、rsuscorrespondingsedentaryanimals).(C)Citratesynthaseactivity.Valuesinuntreated(openbars)sedentarymicetakenas1.0(n=3experiments;*p<0.05,*p<0.01versuscorrespondinguntreatedanimals;yp<0.05versuscorrespondingsedentaryanimals).(D)SOD1,SOD2,catalase,andGPx1mRNAlevelsanalyzedbymeansofquantitative
45、RT-PCR.Relativeexpressionvaluesofuntreated(openbars)sedentarymiceweretakenas1.0(n=6experiments;*p<0.05,*p<0.01versuscorrespondinguntreatedanimals;yp<0.05versuscorrespondingsedentaryanimals).AlldatarepresentmeanSEM.SeealsoFigureS3.andROSdefensesystemincardiacandskeletalmuscles(Figure3D).Inad
46、dition,electronmicroscopyanalysisconrmedincreasedmitochondrialmassincardiacandskeletalmuscleofsedentaryandtrainedmice(Figure4)followingBCAAemsupplementation.BCAAemPreservesMuscleMorphologyandImprovesFunctionalCapacityItiswellknownthatagingcausesagraduallossofskeletalmuscleefciency,andseverallinesofe
47、videncesuggestmito-chondrialinvolvementinthiscondition(Marzettietal.,2009).Here,weobservedthatalthoughweightandmusclemassrelativetothebodyweightofvastus,gastrocnemius,andtibialismusclewereunchangedorminimallydecreasedinthemiddle-agedcomparedtoadultWTanimals(datanotshown),thebercross-sectionalareasof
48、thesemusclesweresignicantlydecreasedinmiddle-agedmice(Figure5A)andwererestoredtothelevelsofadultmicebyBCAAemsupplementation(Figure5A).Consistently,BCAAemsupplementationimprovedwholeendurancecapacityintreadmilltestsofsedentaryandtrainedmice(Figure5B).Moreover,inmotorcoordinationtestsonarotatingrod,BC
49、AAem-supplementedmiceperformedbetterthanuntreatedanimals(Figure5C),particularlyafterexercisetraining.CellMetabolism12,362372,October6,20102010ElsevierInc.365CellMetabolismBranched-ChainAminoAcidsandSurvivalBCAAem-supplementedanimalsshowedareducedmitochon-drialH2O2release(anindexofmitochondrialsuperoxideanionproduction),decreasedmitochondrialROSproduction(asas-sessedbythebasal/totalaconitaseactivityratio,asensitivemarkerofoxidativestress),andincreasedcapacitytoeliminatesuperoxidesatthemitochondriallevel(sinceS