Glucagon-like peptide-1 receptor agonist Liraglutide has anabolic bone effects in ovariectomized ...

15 Pages·2015·2.75 MB·English

RESEARCHARTICLE
Glucagon-like peptide-1 receptor agonist
Liraglutide has anabolic bone effects in
ovariectomized rats without diabetes.
NanLu¤a☯,HanxiaoSun¤b☯,JingJiaYu,XiaojingWang,DongmeiLiu,LinZhao,LihaoSun,
HongyanZhao,BeiTao*,JianminLiu*
DepartmentofEndocrineandMetabolicDiseases,Rui-jinHospital,ShanghaiJiao-tongUniversitySchoolof
Medicine,ShanghaiInstituteofEndocrineandMetabolicDiseases,ShanghaiClinicalCenterforEndocrine
andMetabolicDiseases,Shanghai200025,China
☯Theseauthorscontributedequallytothiswork.
¤a CurrentAddress:DepartmentofEndocrineandMetabolicDiseases,RenjiHospital,SchoolofMedicine,
ShanghaiJiaotongUniversity,Shanghai,China
¤b CurrentAddress:DepartmentofLaboratoryMedicine,ShanghaiTongrenHospital,ShanghaiJiaotong
UniversitySchoolofMedicine,200336,Shanghai,China
* [email protected](BT); [email protected](JML)
Abstract
OPENACCESS
Citation:LuN,SunH,YuJ,WangX,LiuD,ZhaoL, Recently,anumberofstudieshavedemonstratedthepotentialbeneficialrolefornovelanti-
etal.(2015)Glucagon-likepeptide-1receptoragonist
diabeticGLP-1receptoragonists(GLP-1RAs)intheskeletonmetabolismindiabetic
Liraglutidehasanabolicboneeffectsinovariectomized
ratswithoutdiabetes..PLoSONE10(7):e0132744. rodentsandpatients.Inthisstudy,weevaluatedtheimpactsofthesyntheticGLP-1RALira-
doi:10.1371/journal.pone.0132744 glutideonbonemassandqualityinosteoporoticratsinducedbyovariectomy(OVX)but
Editor:ChristianHolscher,UniversityofLancaster, withoutdiabetes,aswellasitseffectontheadipogenicandosteoblastogenicdifferentiation
UNITEDKINGDOM ofbonemarrowstromalcells(BMSCs).ThreemonthsaftershamsurgeryorbilateralOVX,
Received:January20,2015 eighteen5-montholdfemaleWistarratswererandomlydividedintothreegroupstoreceive
thefollowingtreatmentsfor2months:(1)Sham+normalsaline;(2)OVX+normalsaline;
Accepted:June17,2015
and(3)OVX+Liraglutide(0.6mg/day).Asrevealedbymicro-CTanalysis,Liraglutide
Published:July15,2015
improvedtrabecularvolume,thicknessandnumber,increasedBMD,andreducedtrabecu-
Copyright:©2015Luetal.Thisisanopenaccess larspacinginthefemursinOVXrats;similarresultswereobservedinthelumbarvertebrae
articledistributedunderthetermsoftheCreative
ofOVXratstreatedwithLiraglutide.FollowinginvitrotreatmentofratandhumanBMSCs
CommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany with10nMLiraglutide,therewasasignificantincreaseinthemRNAexpressionofosteo-
medium,providedtheoriginalauthorandsourceare blast-specifictranscriptionalfactorRunx2andtheosteoblastmarkersalkalinephosphatase
credited. (ALP)andcollagenα1(Col-1),butasignificantdecreaseinperoxisomeproliferator-acti-
DataAvailabilityStatement:Dataareavailablefrom vatedreceptorγ(PPARγ).Inconclusion,ourresultsindicatethattheanti-diabeticdrugLira-
Dryadathttp://dx.doi.org/10.5061/dryad.mf3rb.
glutidecanexertaboneprotectiveeffecteveninnon-diabeticosteoporoticOVXrats.This
Funding:Thisworkwassupportedby:National protectiveeffectislikelyattributabletotheimpactofLiraglutideonthelineagefatedetermi-
NatureScienceFoundationofChinaNo.81370977
nationofBMSCs.
(JML);NationalNatureScienceFoundationofChina
No.81370018(BT);NationalNatureScience
FoundationofChinaNo.81170804(HYZ);Shanghai
MunicipalHealthBureauProject2012-235(JML).
Thefundershadnoroleinstudydesign,data
collectionandanalysis,decisiontopublish,or
preparationofthemanuscript.
PLOSONE|DOI:10.1371/journal.pone.0132744 July15,2015 1/15
BoneEffectsofLiraglutideinNon-DiabeticRats
CompetingInterests:Theauthorshavedeclared Introduction
thatnocompetinginterestsexist.
Glucagon-likepeptide-1(GLP-1)receptoragonists(GLP-1RAs)areanewclassofanti-diabetic
medicationsthatmimictheeffectsofincretinhormones[1].GLP-1,asanincretinhormone
whichissynthesizedandsecretedfromgutLcellsinresponsetofoodintake,canstimulate
insulinreleasebypancreaticβ-cellsinaglucose-dependentmannerandsuppressglucagon
secretionfromα-cells[2].Inadditiontoitsglucose-loweringeffect,GLP-1alsodelaysgastric
emptyingandinhibitsappetite,possiblybyaffectingvagalafferentfibersandthehypothala-
mus,eventuallyleadingtoweightloss[3,4].ThesefavorableactionsofGLP-1onglucose
homeostasisaremediatedthroughGLP-1receptors.However,nativeGLP-1israpidly
degradedincirculationbytheenzymedipeptidylpeptidaseIV(DPP-IV)[5].Liraglutide,asyn-
theticGLP-1RAthatshare97%homologywiththestructureofhumanGLP-1,isresistantto
DPP-IVinactivationandpossessesamuchlongercirculatinghalf-life,therebymakingita
novelanti-diabeticdrugsuitableforonce-dailyinjection[1].
Inadditiontotheirusefulnessintreatingdiabetesandobesity[6],thepresenceofGLP-1
receptorsinvarioustissuesotherthanpancreaticβ-cellsandα-cells(i.e.,brainandheart)has
arousedagreatdealofinterestinexploringthecardiovascular,neuroprotective,renalprotec-
tiveandotherextra-pancreaticbenefitsofGLP-1RAs[7–10].Invitrostudiesshowedthatoste-
oblasticcellsexpressfunctionalreceptorsforGLP-1[11];thus,bonemightalsobeapotential
targetorganforGLP-1RAs.Indeed,continuoussubcutaneousinfusionofGLP-1orextendin-4
(anaturallyoccurringanalogueofGLP-1)for3daysinbothinsulin-resistantandtype2dia-
beticratmodelsnormalizedtheirimpairedtrabeculararchitectureandpromotedboneforma-
tionviaaninsulin-independentmechanism[12,13].GLP-1administrationcanalsorestore
bonemineraldensities(BMDs)inratswithhyperlipidemia-inducedboneloss[14].Thesefind-
ingshighlightedthepotentialuseofGLP-1RAsincombatingdiabetes-relatedbonedefects.
Bonelosscanalsoarisefromestrogendeficiency.Therefore,aninterestingquestionremains
concerningwhetherGLP-1RAscaneffectivelytreatosteoporosisinanimalmodelswithoutdia-
betes.Exendin-4wasrecentlyshowntoexhibitanosteogeniceffectinovariectomized(OVX)
rats[15];however,whetherdailyLiraglutideinjectionscaninducesimilaranabolicboneeffects
inosteoporoticrodentswithoutdiabetesisnotclear.
GLP-1receptorsarealsoexpressedonbonemarrowstromalcells(BMSCs)[16].Multipo-
tentBMSCscandifferentiateintoadipocytes,osteoblasts,chondrocytesormyocytesunderthe
controlofseveraltranscriptionfactors[17,18].Becausethereisamutuallyexclusiverelation-
shipbetweenthedifferentiationofadipocytesandosteoblastsfromtheircommonprogenitor
cells,itisnecessarytoinvestigatewhetheraGLP-1RAsuchasLiraglutidecanaffectthelineage
fatedeterminationofBMSCs.
Therefore,thecurrentstudywasperformedinOVXratswithoutdiabetestoascertainthat
theboneeffectsofthisanti-diabeticdrugcouldalsobedemonstratedinosteoporoticratsina
mannerthatwasindependentofitsbloodglucose-loweringeffects.Additionally,weinvesti-
gatedtheregulatoryactionsofLiraglutideonosteoblastogenesisandadipogenesisinratand
humanBMSCs.
MaterialsandMethods
Animalsandtreatment
Eighteen6-week-oldfemaleWistarratswereusedinthisstudy.TheratswerebredinShanghai
SlacLaboratory(SlacLaboratoryAnimalCo.Ltd,Shanghai,China)andraisedinShanghai
Jiao-TongUniversity.Theanimalswerehousedindividuallyincagesinanair-conditioned
environmentwithstabletemperature(22°C±2°C),humidity(50%to70%)anda12‐hour
PLOSONE|DOI:10.1371/journal.pone.0132744 July15,2015 2/15
BoneEffectsofLiraglutideinNon-DiabeticRats
light‐darkcycle(lightsonat8:00A.M.),andprovidedaccesstoastandardchowdietandwater
adlibitum.TheuseoflaboratoryanimalsinthisstudywasapprovedbytheAnimalCareCom-
mitteeoftheShanghaiJiao-TongUniversityandperformedunderacceptedethicalguidelines.
Aftera14dayacclimatizationperiod,therats(8-weeks-old)wereanesthetizedviaanintra-
peritonealinjectionof5%chloralhydrate(8ml/kg)andunderwenteitherashamsurgeryor
bilateralovariectomy.Threemonthsafterthesurgery,therats(5monthsofage)wereran-
domlydividedintothreegroupstoreceivethefollowingtreatmentfor2months:(1)Sham
+normalsaline;(2)OVX+normalsaline;and(3)OVX+Liraglutide.RatsintheOVX+Lira-
glutidegroupweregivenoncedailys.c.injectionsofLiraglutide(NovoNordiskA/S,Denmark)
atadoseof0.6mg/dayat8A.M.Normalsalinewasusedasthevehicleandwassubcutaneously
injectedintothecontrolratsdaily.
Thebodyweightandfedstatebloodglucoselevelsweremonitoredweekly.Priortoeuthana-
sia,anintra-peritonealglucosetolerancetest(IPGTT)wasperformedasfollows:afterovernight
fasting,theanimalsweregivenaninjectionofglucosesolution(2g/kgbodyweight).Bloodsam-
pleswascollectedfromthecaudalveinimmediatelypriortoglucoseadministrationandthen
15min,30min,60min,and120minafterforthemeasurementofglucoselevelsusingaglucose
reagentstripandglucometer(One-TouchUltra,Lifescan).Attheendoftheexperiment,therats
wereeuthanizedbyetherinhalation.Afterwards,thefemursandthefifthlumbarvertebraewere
harvestedandcarefullystrippedofsofttissueattachmentsat4°Ctoavoidanydamagetothe
bones.Therightfemursandthelumbarvertebraewerestoredin4%paraformaldehydein0.1M
sodiumphosphatebuffer(PBS,pH7.4)forthemicro-CTevaluation.
Micro-CTevaluation
Bonesamplesstoredin4%paraformaldehydefor24hoursweredehydratedwith70%ethyl
alcoholfor48hours,thenpositionedinacustomjigwith40%ethylalcoholandmonitored
usingamicro-CTscannerata14μmresolution(GEExploreLocusSPSpecimenScanner,
GECareCo.,London).Themineralizedtissuesweredifferentiallysegmentedusingaglobal
thresholdingprocedure[19].Briefly,forthetrabecularboneanalysisthefemurswerescanned
from1.0mmbelowthelowerendofthegrowthplateandatthemid-portionofthelumbar
vertebrae.Forthecorticalboneanalysis,themid-portionofthefemurswasscanned.Thefol-
lowingmorphometricparametersfromthebinarizedvolumesofinterestwereanalyzedviathe
ExploreMicroViewversion2.2(GEHealthcare):bonevolumeratio(BV/TV),trabecularnum-
ber(Tb.N),trabecularthickness(Tb.Th),trabecularseparation(Tb.Sp)andcorticalthickness.
Cellculture
TheeffectsofLiraglutideonosteoblastogenicandadipogenicdifferentiationinvitrowere
determinedbyculturingratandhumanBMSCsindifferentdifferentiationmediawithorwith-
out10nMLiraglutide.
RatBMSCswerederivedfrom4-week-oldmaleWistarrats,andbothadipogenesisand
osteoblastogenesiswereinducedaspreviouslydescribed[20].PrimaryhumanBMSCswere
giftsfromtheShanghaiKeyLaboratoryofOrthopedicImplant.ThehumanBMSCswere
donatedbypatientswithwritteninformedconsent,andthisexperimentwasapprovedbythe
IndependentEthicsCommitteeofShanghaiNinthPeople’sHospitalaffiliatedwithShanghai
JiaoTongUniversitySchoolofMedicine(SJTUSM)[21].Foradipocytedifferentiation,human
BMSCswereinitiatedwithanadipogenicinductionmedium(AIM):DMEM-lowglucose
mediasupplementedwith15%FBS,10μMinsulin,1μMdexamethasone,0.5mM3-isobutyl-
1-methylxan-thine(IBMX),and200μMindomethacininthepresenceorabsenceofLiraglu-
tide.Thereafter,theAIMwasreplacedwithadipogenicmaintenancemedium(AMM)
PLOSONE|DOI:10.1371/journal.pone.0132744 July15,2015 3/15
BoneEffectsofLiraglutideinNon-DiabeticRats
consistingofDMEM-lowglucose,15%FBS,and10μMinsulininthepresenceorabsenceof
Liraglutidefor1day,andthenthecellswereswitchedbacktoAIM.Alternateincubationswith
thetwodifferentmediawererepeatedevery3days[16].Forosteoblastogenicdifferentiation,
humanBMSCswereculturedinDMEM-lowglucosemediasupplementedwith15%FBS,100
nMdexamethasone,10mM2-glycerolphosphateand0.05mMascorbicacid[22,23].
LipiddropletswithinthedifferentiatedadipocytesfromratandhumanBMSCswere
observedusingamodifiedOilRedOstainingmethod[16]after14and17daysofadipogenic
differentiation,respectively.Briefly,thecellswerewashedtwicewithPBS,andthenfixedfor
5minwith4%formaldehyde.FixedcellswereincubatedwithOilRedOfor30minatroom
temperature,followedbythreewasheswithPBS.OilRedOwaspreparedbydiluting60mLof
stocksolution(0.5gin100mLofisopropanol)in40mLofdistilledwaterandfilteredthrough
coarsefilterpaperpriortouse.AfterstainingwiththeOilRedO,thecellswerewashedand
photographed.Thedyeretainedbythecellswaselutedbyincubationwithisopropanolfor
15minutesandquantifiedbyabsorbancemeasurementsat510nm(Safire2,TECAN).One
weekafterosteoblastogenicinduction,alkalinephosphatase(ALP)stainingwasaccomplished
withtheuseofanALPstainingkit(Renbao,Shanghai,China)[24].Alizarinredstainingwas
performedforthevisualizationofcalciumdeposits[25]after21daysofosteoblastogenesis.
Cellswereincubatedwith2%alizarinred(pH4.2)for10minutes,thensubsequentlywashed
withdistilledwaterandphotographed.Calciumdepositswereextractedwith1mLof0.1N
sodiumhydroxide,andtheopticaldensitywasrecordedat548nm(Safire2,TECAN).
Real-timePCR
TotalRNAwasextractedfromthecellsdescribedabovewiththeTRIzolreagent(Invitrogen).
Then,theextractedRNAwasreversed-transcribedusingtheReverseTranscriptionSystem(Pro-
mega,Madison,WI,USA).ThePCRprimersequencesareprovidedinTable1.PCRamplifica-
tionwasperformedusingSYBRPremixExTaq(TAKARA,Japan)inaLightCyclerinstrument
(Roche).ThethermalcyclingconditionsforthePCRreactionswere95°Cfor30seconds,fol-
lowedby40cyclesof95°Cfor10seconds,60°Cfor20seconds,and95°Cfor15secondsand
endingwith60°Cfor15seconds.Allreactionswereperformedintriplicate.ThemRNAexpres-
sionlevelswerenormalizedtotheendogenouscontrolGAPDH.Therelativeexpressionlevelsof
controlandLiraglutidetreatedsamplesatdifferenttimepointsoverday0(0d)controlsample
werecalculatedwiththe2-ΔΔCtmethod,where-ΔΔCt=ΔCt -ΔCt .
treatment 0dcontrol
Statistics
Dataarepresentedasthemean±SEM.StatisticalanalyseswereperformedwithSPSSsoftware
(version18.0).VariablesweretestedfornormalityusingtheKolmogorov-SmirnovZstatistic.
Groupcomparisonswereevaluatedwiththeone-wayANOVAtest.Homogeneityofvariance
wasassessedusingLevene'stest(Tb.N,Tb.Th,corticalBMDandTb.BMDofthefemurand
Tb.Thofthelumbarvertebraehadequalvariance;BV/TV,Tb.Sp,andcortical.Thofthefemur
andBV/TV,Tb.N,Tb.Sp,andTb.BMDofthelumbarvertebraehadheterogeneousvariance).
TheLSDtestwasusedwhenequalvariancewasassumed;Tamhane'sT2testwasusedwhen
equalvariancewasnotassumed.P<0.05wasconsideredstatisticallysignificant.
Results
LiraglutidepreventedbonelossinducedbyOVX
Asshowninthedifferentdimensionviews,thebonemassandbonecorticalthicknesswere
decreasedandthetrabecularmicro-architecturewasmarkedlyimpairedinOVXrats
PLOSONE|DOI:10.1371/journal.pone.0132744 July15,2015 4/15
BoneEffectsofLiraglutideinNon-DiabeticRats
Table1. Primerssequencesusedinthestudy.
Gene primersequences
Ratprimers
CTCAACTACATGGTCTACATGT(Forward)
GAPDH
CTTCCCATTCTCAGCCTTGACT(Reverse)
ACTGGCGGTGCAACAAGA(Forward)
Runx2
TAGTTCTCATCATTCCCGGC(Reverse)
CCAAAAACTCAACACCAACG(Forward)
ALP
TCCATCTCCAGCCGTGTCT(Reverse)
TGTCCCAACCCCCAAAAA(Forward)
Col-Ⅰ
ATGACTTCTGCGTCTGGTGAT(Reverse)
TGACCCAATGGTTGCTGATT(Forward)
PPARγ
GCCTGTTGTAGAGTTGGGTTT(Reverse)
Humanprimers
GAAGTGAAGGTCGGAGTC(Forward)
GAPDH
GAAGATGGTGATGGGATTTC(Reverse)
CTACCACCCCGCTGTCTTC(Forward)
Runx2
CAGAGGTGGCAGTGTCATCA(Reverse)
CCCCGTGGCAACTCTATCTTT(Forward)
ALP
GATGGCAGTGAAGGGCTTCTT(Reverse)
TTGACCCAGAAAGCGATTCC(Forward)
PPARγ
AAAGTTGGTGGGCCAGAATG(Reverse)
doi:10.1371/journal.pone.0132744.t001
comparedtosham-operatedrats.ThesedecreasesweresignificantlyimprovedbyLiraglutide
treatment(Figs1Aand2A).
LiraglutidetreatmentofOVXratsinducedasignificantimprovementintrabecularthick-
nessby36%(P=0.023),trabecularnumberby20%(P=0.039),andtrabecularBMDby4%
(P=0.001)comparedtotheirOVXlittermatestreatedwithvehicleinthefifthlumbarvertebra.
Animprovementintrabecularvolume(BV/TV)anddecreaseintrabecularspacingwere
alsoobserved,althoughthedifferencesbetweengroupsdidnotreachstatisticalsignificance
(Fig1B).TheseanaboliceffectsofLiraglutidewerealsoobservedinthelongbones.Analysisof
therightfemurusingmicro-CTdisplayedanincreaseinBV/TVby27%(P=0.049),cortical
thicknessby5%(P=0.020),corticalBMDby1%(P=0.008),trabecularthicknessby11%
(P=0.033),trabecularnumberby24%(P=0.002),andtrabecularBMDby2%(P=0.015),
andadecreaseintrabecularspacingby42%(P=0.032)intheLiraglutide-treatedOVXrats
(Fig2B).AsshowninFig1BandFig2B,mostboneindicesintheOVX+Liraglutidegroup
werestillsignificantlyworsethanthoseoftheSHAMrats.
LiraglutideaffectedbodyweightandbloodglucoselevelsinOVXrats
AsshowninFig3A,theOVXratswereheavierthantheirshamlittermates.DailyLiraglutide
administrationfor8weeksinducedsignificantweightlossbeginningatthe4thweek.After8
weeksoftreatment,thebodyweightofOVX+Liraglutideratsweresignificantlylowerthan
theOVX+normalsalinegroup,withan8.4%differencebetweengroups(268.5±17.1gvs
293.3±11g;P=0.014).
TheOVXandsham-operatedratshadidenticalfedstatebloodglucoselevelsandsimilar
glucosetolerancerevealedbyIPGTT(Fig3B–3D).Interestingly,Liraglutideimprovedglucose
toleranceintheOVXrats(Fig3Cand3D).
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BoneEffectsofLiraglutideinNon-DiabeticRats
Fig1.EffectsofLiraglutideonthebonemicro-architectureofthevertebrainOVXrats.(A)
Representativemicro-CTimagesofthe5thlumbarvertebra.Theupperrowpresentsthecoronalview,axial
viewandsagittalviewinthedifferentgroups,whilethelowerrowprovidesathree-dimensionalvisualization
ofthetrabecularmicro-architecture.(B)Thetrabecularmicro-architectureparametersofthe5thlumbar
vertebrawereshownas:BV/TV,Tb.Th,Tb.N,trabecularBMD,andTb.Sp.Eight-week-oldfemaleWistarrats
weresubjectedtoashamovariectomy(SHAM,blackbar)orovariectomysurgery(OVX);then,3monthsafter
surgerywhentheratshadestablishedosteoporosis,vehicle(OVX,whitebar)orLiraglutide(OVX+L,redbar)
wereadministeredfor8weeks.Valuesareexpressedasthemean±SE;n=6ratspergroup.*,P<0.05;**,
P<0.01,***,P<0.001.
doi:10.1371/journal.pone.0132744.g001
Liraglutideinhibitedadipogenesisandenhancedosteoblastogenesisin
ratandhumanBMSCs
WehypothesizedthatthebeneficialboneeffectsofLiraglutidewerepossiblyattributabletoits
impactsonadipogenesisandosteoblastogenesisofratBMSCs.AsshowninFig4A,Liraglutide
significantlyinhibitedtheformationofadipocytes.OilredOstainingdemonstratedthepres-
enceofnumerousintracellularlipiddropletsinthecontrol,whilesignificantlysmallerorfewer
lipiddropletswereobservedintheLiraglutide-treatedcells.TheOilRedOincellswasquanti-
fiedafterdecoloring,andasignificantdecreasewasobservedintheLiraglutidetreatedgroup
(Fig4B).Theseeffectsweresupportedbythereal-timePCRanalysis,whichshowedthatthe
expressionoftheadipocyte-specifictranscriptionfactorperoxisomeproliferator-activated
receptorγ(PPARγ)wassignificantlydown-regulatedfollowingLiraglutidetreatment(Fig4F).
Conversely,therewasanobviousincreaseinALPstainingwhenLiraglutidewasaddedfor7
daystoculturesofratBMSCsundergoinginducedosteoblastogenicdifferentiation(Fig4C).
Real-timePCRshowedthatLiraglutidefullypromotedanincreaseintheexpressionofRunx2,
atranscriptionfactorthatisessentialforosteoblastdifferentiation.Thesephenotypicchanges
werealsoaccompaniedbytheelevatedexpressionofkeymarkersforosteoblastogenesis,such
asALPandCol-1(Fig4G,4Hand4I).
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BoneEffectsofLiraglutideinNon-DiabeticRats
Fig2.EffectsofLiraglutidetreatmentonthebonemicro-architectureofthefemurinOVXrats.(A)
Representativemicro-CTimagesoftherightfemur.(B)Thetrabecularmicro-architectureparametersofthe
distalendofthefemurwereshownas:BV/TV,Tb.Th,Tb.N,Tb.Sp,andtrabecularBMD.Thecortical
parameterswerealsoexhibitedasthecorticalBMDandcorticalwallthickness.Eight-week-oldfemaleWistar
ratsweresubjectedtoashamovariectomy(SHAM,blackbar)orovariectomysurgery(OVX);then,3months
aftersurgerywhentheratshadestablishedosteoporosis,vehicle(OVX,whitebar)orLiraglutide(OVX+L,red
bar)wereadministeredfor8weeks.Valuesareexpressedasthemean±SE;n=6ratspergroup.*,P<0.05;
**,P<0.01;***,P<0.001.
doi:10.1371/journal.pone.0132744.g002
Fig3.EffectsofLiraglutideonbodyweightandbloodglucoselevelsinOVXrats.Thebodyweight(A)
andrandomfedstatebloodglucoselevels(B)weremonitoredweeklyinvehicle-treatedsham-operated
Wistarrats(SHAM),vehicle-treatedOVXrats(OVX)andLiraglutide-treatedOVXrats(OVX+L)during8
weeksoftreatmentwithLiraglutide.Priortoeuthanasia,anintraperitonealglucosetolerancetest(IPGTT)
wasperformed.Theglucoselevelandtheareaundercurve(AUC)oftheglucosetoleranceinIPGTT(C,D)
werealsoassessed.Valuesareexpressedasthemean±SE;n=6ratspergroup.*,P<0.05;**,P<0.01,
***,P<0.001;OVXvs.OVX+L.
doi:10.1371/journal.pone.0132744.g003
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BoneEffectsofLiraglutideinNon-DiabeticRats
Fig4.EffectsofLiraglutideonadipogenesisandosteoblastogenesisofratBMSCs.PrimaryratBMSCs
wereharvestedfromthefemurandtibiabonemarrowfrom4-week-oldmaleWistarrats.After3passages,the
ratBMSCswereinducedtodifferentiateintoadipocytesorosteoblastsinthepresenceorabsenceof10nM
Liraglutide.(A)Following14daysoftreatmentafteradipogenicinducement,thecultureswerefixedandstained
withOilredO,thenvisualizedandphotographed.(B)TheamountofOilRedOinthecellswasquantified.(C)
ALPstaining,whichindicatestheearlystageofosteoblastogenesis,and(D)Alizarinredstaining,which
representsthemineralizationlevelsoflaterstagesofosteoblastogenesis,wasperformedattheindicatedtime-
points.(E)Alizarinreddyewasanalyzed.(F)PPARγ,acrucialgenepromotingadipogenesis,wasquantified
usingreal-timePCRafter14daysofadipogenesis.(G.H.I)Theexpressionpatternsofosteoblastogenic
differentiationmarkers(Runx2,ALP,Col-1)ofratBMSCsinresponseto10nMLiraglutidewereassessed
usingreal-timePCR.Relativeexpressionlevelsofeachgenewerecalculatedusingthe2-ΔΔCtmethod.The
expressionofGAPDHmRNAwasusedforreference.Valuesareexpressedasthemean±SE.*,P<0.05;**,
P<0.01;***,P<0.001.
doi:10.1371/journal.pone.0132744.g004
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BoneEffectsofLiraglutideinNon-DiabeticRats
Alizarinstainingwasperformedafter21daysofosteoblastogenicculturetoassessmineral
depositionasalatemarkerofmatureosteoblastfunction;however,wefailedtoobserveclearly
enhancedalizarindepositionfollowingLiraglutidetreatment(Fig4Dand4E).
Finally,weexaminedwhetherLiraglutideexertedsimilaractionsonhumanBMSCs.The
attenuatedOilredOstaining(Fig5A)anddecreasedPPARγexpression(Fig5B)suggested
thatLiraglutidealsoinhibitedhumanBMSCadipogenesis.Moreover,inagreementwithprevi-
ousresultsobservedinratBMSCs,Liraglutidesimilarlyenhancedosteoblastogenicprocesses
inhumanBMSCsbasedonenhancedALPstaining(Fig5C)andanincreasedexpressionpat-
ternofRunx2andALPduringtheearlystagesofosteoblastdifferentiation(Fig5D).
Discussion
Themajorfindingsofthecurrentstudywerethattheanti-diabeticdrugLiraglutideimproved
bonequalityandincreasedBMDinosteoporoticOVXrats.
Recently,thepotentialbeneficialroleofnovelanti-diabeticGLP-1RAsinskeletonmetabo-
lismindiabeticrodentsandpatientshasbecomeahottopic[26–28].Hyperglycemiccondi-
tionscouldcompromiseosteoblastogenesisandosteoclastogenesisandevenimpairembryonic
skeletondevelopment[29,30];thus,theanabolicboneeffectsofGLP-1RAsobservedindia-
beticmodelsinpreclinicalandclinicalstudiesmayhavebeenasecondaryoutcomeofthe
improvementofglucosecontrol.Toaddresswhethertheseeffectscouldbereplicatedinnon-
diabeticosteoporoticrodents,wetreatedOVXratswithoutdiabeteswithLiraglutide.Liraglu-
tideimprovedthetrabecularvolume,thicknessandnumber,increasedBMD,andreduced
trabecularspacinginthefemurintheseosteoporoticrats.Similareffectswereobservedinthe
lumbarvertebraeofOVXratstreatedwithLiraglutide.Thesefindingssuggestedthattheosteo-
genicboneeffectsofGLP-1RAscouldbeappliedtonon-diabeticosteoporoticrodents.Itwas
noteworthythatalthoughLiraglutidetreatmentinOVXratsimprovedBMDandbonequality,
itstillcouldnotrestoretheseindicesbacktothoseoftheSHAMrats.Wealsoexploredthepos-
sibleboneeffectofLiraglutideonnon-diabetic-non-osteoporoticshamrats.Asillustratedin
S1Fig,nosignificantenhancementofhistomorphometricparameterswasobservedinthe
bone-soundanimalsafter8weeksoftreatmentofLiraglutide.Furtherstudiesareneededto
investigatewhethertheboneeffectsofLiraglutideareonlyobservedinosteoporoticrodents,
butnotinnormalrodents.
Inthecurrentstudy,wefoundthatOVXratswithoutdiabetesdemonstratedimprovedglu-
cosetoleranceaftertreatmentwithLiraglutide.Thisfindingechoestheresultsreportedfroma
mousestudyshowingthat6weeksoftreatmentwithLiraglutideinnormalglycemicmice
resultedinincreasedinsulinsensitivityandβcellfunction,accompaniedbyreducedβcell
mass[31].The8.4%lowerbodyweightintheLiraglutide-treatedOVXratscomparedwiththe
vehicle-treatedratsattheendofourstudymightbeanadditionalfactorresponsibleforthis
change.However,wehypothesizethattheboneeffectsofLiraglutideoccurindependentofthe
bloodglucose-loweringeffect.Apparently,thepoorbonequalitydisplayedinOVXratswasa
directconsequenceoftheovariectomy.Thereductioninbonequalitywasnotcausedbyhyper-
glycemiabecauseOVXratsandshamratshadsimilarfedstateglucoseandglucosetolerance
levelsinourstudy.Furthermore,humanstudiessuchastheActiontoControlCardiovascular
RiskinDiabetes(ACCORD)trial,whichwasthefirstrandomizedtrialtoevaluatetheeffectof
intensiveglycemiatherapyonfracturesandfalls,demonstratedthatintensiveglycemiacontrol
hadaneutraleffectontheoccurrenceofnon-spinefractures,fallsandBMDduringthe3.8
yearsoffollow-up[32].Thus,improvementofglucosecontroldoesnotnecessarilyrelatetoa
betteroutcomefortheskeleton.Accordingly,theboneeffectsofLiraglutideinnon-diabetic
rodentsarenotderivedfromitsanti-diabeticefficacy.
PLOSONE|DOI:10.1371/journal.pone.0132744 July15,2015 9/15
BoneEffectsofLiraglutideinNon-DiabeticRats
Fig5.EffectsofLiraglutideonadipogenesisandosteoblastogenesisofhumanBMSCs.Primaryhuman
BMSCswereinducedtodifferentiateintoadipocytesorosteoblastsinthepresenceorabsenceof10nM
PLOSONE|DOI:10.1371/journal.pone.0132744 July15,2015 10/15