Expression and distribution of leptin and its receptors in the digestive tract of DIO (diet-induced obese) zebrafish.

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Annals

of

Anatomy

j o ur na l h o me pa g e :w w w . e l s e v i e r . c o m / l o c a t e / a a n a t

RESEARCH

ARTICLE

Expression

and

distribution

of

leptin

and

its

receptors

in

the

digestive

tract

of

DIO

(diet-induced

obese)

zebrafish

M.

Mania

_,

_L.

_Maruccio

_,

_F.

_Russo

_,

_F.

_Abbate

_,

_L.

_Castaldo

_,

_L.

_D’Angelo

_,

P.

de

Girolamo

_,

_M.C.

_Guerrera

_,

_C.

_Lucini

_,

_M.

_Madrigrano

_,

_M.

_Levanti

_,

A.

Germanà

a_{DepartmentofVeterinaryScience,UniversityofMessina,Italy} d_{ZebrafishNeuromorphologyLab,UniversityofMessina,Italy}

b_{DepartmentofVeterinaryMedicineandAnimalProductions,UniversityofNaplesFedericoII,Italy} c_{DepartmentofSciencesandTechnologies,UniversityofSannio,Italy}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received27October2016

Receivedinrevisedform17March2017 Accepted22March2017 Keywords: Fish Obesity Digestivetract Leptin

a

b

s

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Theexpressionandlocalizationofleptin(AandB)anditsreceptorfamilyincontrolanddiet-induced

obese(DIO)adultmalezebrafishgut,after5-weeksoverfeeding,administeringArtemianauplii,as

fat-richfood,wereinvestigated.Recently,theobeseadultzebrafishwasconsideredanexperimentalmodel

withpathophysiologicalpathwayssimilartomammalianobesity.Currently,therearenoreportsabout

leptininfishobesity,orinastateofalteredenergybalance.ByqRT-PCR,leptinAandleptinBexpression

levelsweresignificantlyhigherinDIOzebrafishgutthaninthecontrolgroup(CTRL),andthelowestlevels

ofleptinreceptormRNAappearedinDIOzebrafishgut.Thepresenceofleptinanditsreceptorproteins

intheintestinaltractwasdetectedbywesternblotanalysisinbothcontrolandDIOzebrafish.Bysingle

immunohistochemicalstaining,leptinandleptinreceptorimmunoreactiveendocrinecellswere

iden-tifiedintheintestinaltracteitherinDIOorcontrolzebrafish.Moreover,leptinimmunopositiveenteric

nervoussystemelementswereobservedinbothgroups.Bydoubleimmunohistochemicalstaining,leptin

anditsreceptorwerecolocalizedespeciallyinDIOzebrafish.Thus,ourstudyrepresentsastartingpoint

intheinvestigationofapossibleinvolvementofleptinincontrolofenergyhomeostasisincontroland

DIOzebrafish.

©2017ElsevierGmbH.Allrightsreserved.

1. Introduction

Inrecentyears,obesityhasbecomeasocialdisease,notonlydue todefectivegenes,butalsotoanunhealthylifestyle,characterized byanexcessivecalorieintakelackingphysicalactivity.This con-ditionleadstoanincreaseinadiposetissueandcorrespondingly increasedlevelsofcirculatingleptininhumans(Al-Hamodietal., 2014).Leptin,acytokine-likepeptide,wasfirstclonedinob/obmice in1994(Zhangetal.,1994),asthefactorresponsibleforthe mor-bidobesityofob/obmutantmice.Infish,leptinhasbeencloned from severalspecies: pupperfish (Takifugu rubripes) (Kurokawa etal.,2005),zebrafish(Daniorerio)(Huisingetal.,2006),atlantic salmon (Salmo salar) (Rønnestad et al., 2010), marine medaka (Oryzias latipes)(Wong etal., 2007), tilapia (Oreochromis niloti-cus)(Shpilmanetal.,2014),stripedbass(Moronesaxatilis)(Won

∗ Correspondingauthorat:ViaVeterinaria,1,Naples80137,Italy. Fax:+390812536097.

E-mailaddress:lucianna.maruccio@unina.it(L.Maruccio).

1 _{Theseauthorscontributedequallytothiswork.}

etal.,2012),Ya-fish(Schizothoraxprenanti)(Yuanetal.,2014)and red-belliedpiranha(Pygocentrusnattereri)(Volkoff,2015),among others.Inparticular,twoleptinparalogues,LepAandLepB,have beenfoundinsomefishlineages,suchaszebrafishandpufferfish, demonstratingthattheyoriginatedearlyinteleost,anevolution followingafirstduplicationeventofthewholegenome(Gorissen etal.,2009;Prokopetal.,2012).Inmammals,leptinissynthetized notonlybyadiposetissuebutalsobythedigestivetract(Zhang etal.,1994;Badoetal.,1998;Sobhanietal.,2000;Cintietal.,2000; Cammisottoetal.,2005;CammisottoandBendayan,2012;Russo etal.,2011;ParkandAhima,2014)amongothertissues.Infish, gut(Bosietal.,2004;Ronnestadetal.,2010;Liuetal.,2010;Russo etal.,2011;Varricchioetal.,2012)andliver(Huisingetal.,2006; Murashitaetal.,2008;Trombleyetal.,2012;Zhangetal.,2013)are animportantsourceofleptin.Moreover,differentstudiesreported thepresenceofleptinanditsreceptorsinseveralperipheralorgans (Kurokawaetal.,2005;Ronnestad etal.,2010;Liu etal.,2010; Copelandetal.,2011;Gongetal.,2013).

Particularlyinzebrafish,leptinpresentsadifferential expres-sionpattern:leptinAisexpressedintheliverandinthegut,while http://dx.doi.org/10.1016/j.aanat.2017.03.005

leptinBis more expressedin theovary (Gorissenet al., 2009; GorissenandFlik,2014).Inaddition,althoughthe“classical”roleof leptinisconservedbetweenbothisoforms,theyarealsopleiotropic andactredundantlyorindependentlyinaleptinnetwork, allow-ingzebrafishtomaintainequilibriuminthefaceofchallengesto homeostasis(Gorissenetal.,2009;GorissenandFlik,2014; Conde-SieiraandSoengas,2017).Inspiteofthisabundantliterature,there isstillmuchtouncoverinthisrespect.

Inmammals,leptinactsthroughtheleptinreceptors(OB-Rs proteins),locatedintheplasmamembrane.Accordingtothelength oftheintracellular domain,inmammalstheOB-Rs aredivided intoonelongisoform(OB-Rb)andfourshortisoforms(OB-Ra,c, d,f)(Cintietal.,2000).Thereisalsoonesolubleisoform(OB-Re), whichlacksthetransmembranedomainandmaybeinvolvedinthe bloodleptintransport.Moreover,Rønnestadetal.(2010)reported thatthelongestisoformofleptinreceptoristheonlyformthat includesbothtransmembraneandintracellularsegments, essen-tialforsignaltransductionandconservedamongvertebrateleptin receptors.Itiswellknownthat,inmammals,leptinreceptor iso-formsshowthesameextracellulardomainandthesameaffinityfor leptin(Chenetal.,1996;Leeetal.,1996).Thesereceptorisoforms wereidentifiedbothinthecentralnervoussystemandperipheral organs(Cammisottoetal.,2010).Recently,isoformsofleptin recep-torswerealsodetectedinzebrafish(Liuetal.,2010)andotherfish species(Rønnestadetal.,2010;Prokopetal.,2012;Tinocoetal., 2012;Gongetal.,2013).

Leptininmammalsactsonthebraintoregulatefoodintake andmetabolism(Zhangetal.,1994;Pelleymounteretal.,1995). Theactionsofleptinoccuroverbothshort-andlong-term.Inthe shortterm,plasmaleptinservesassatietysignal(Ahima,2005)and, overlongperiods,dailymeanplasmaleptinconcentration commu-nicateslong-termenergystatustothebrain(Chehabetal.,1997). Lessisknownaboutleptinactiononenergyhomeostasisinfish. Certainly,ingoldfish,leptinpotentiatestheactionsofthe anorexi-genicfactorsCARTandCCKandinhibitstheactionoftheorexigenic peptidesNPYandorexinA(Volkoffetal.,2003),itincreasesfat metabolismin greensunfish(LondravilleandDuvall,2002)and actsindose-and time-dependentmanneronglucosensing and geneexpressionofneuropeptidesinvolvedinfoodintakein rain-bowtrouthypothalamusandhindbrainbyinvitrostudy(Aguilar etal.,2011).Also,zebrafishlackingafunctionalleptinreceptorhave alterationsininsulinandglucoselevels,suggestingaroleofleptin inthecontrolofglucosehomeostasis(Micheletal.,2016).

Amongstudiedfish,zebrafishisconsideredanattractivesimple vertebratemodel(D’Angeloetal.,2016a,b)foritswell-conserved organizationofcommonorgansandtissueswithmammals, includ-ingtheintestinalsystem(HarperandLawrence,2010;Wangetal., 2010;GoldsmithandJobin, 2012).Forthesereasons,inthelast decade,numerousgastrointestinalpathologieshavebeenmodeled inzebrafish(GoldsmithandJobin,2012).Thisanimalmodelwas recentlyusedtoinvestigatemetabolicparameterssuchasbody weight,adiposityandenergyexpenditure(Farberetal.,2001;Jones etal.,2008;Flynnetal.,2009;Andersonetal.,2011;Craig and Moon,2011;Sethetal.,2013),aswellasOkaetal.(2010)whoused zebrafishtocreateafishmodelfortheanalysisofdiet-induced obe-sity(DIO)underconditionsofhighfatintake.Theaimofourstudy wastoinvestigatetheexpression(byqRT-PCRandwesternblot analysis)anddistribution(bysingleanddoubleimmunostaining) ofleptin, and itsfamily receptorsin thedigestivetractof con-trol(CTRL)anddiet-inducedobesity(DIO)adultzebrafish,inorder toextendourknowledgeonleptinsysteminthisanimalmodel species.Infact,infish,thispeptideisalsoexpressedintheintestinal tract,where this hormoneis involved in theregulationof sev-eralphysiologicalprocesses,includingfoodintakeandmetabolism, amongotherfunctions(Volkoff,2015).Indetail,weevaluatedthe expressionofleptinAandleptinBinthedigestivetractofCTRLand

DIOzebrafishusingaprotocolaccordingtoGorissenetal.(2009) whoreportedaninvolvementoftheseisoformsinzebrafishfood intake.

2. Materialandmethods

2.1. Animalhusbandryandexperimentaldesign:ethicsstatement Theexperimentalprotocolwasinaccordancewiththe princi-pleoutlinedintheDeclarationofHelsinkiandwiththeNational lawregardingthecareanduseoflaboratoryanimals.TheItalian MinistryofHealthhasapprovedtheexperimentalprotocol. 2.2. Zebrafishbreeding,tissuetreatment,bodymassindex(BMI) andbodyweightmeasurements

Adultmalezebrafish(n=30)wereobtainedfromthebreeding colonyattheC.I.S.S.(CentreofExperimentalIchthyopathologyof Sicily,DepartmentofVeterinaryScience,UniversityofMessina). Fishweremaintainedat28.5◦Candfedonceaday,witha14:10h L:Dcycle.Thefishweredividedintotwodietarygroups(n=15fish pergroup).Thecontrolgroupwasfedwiththeequivalentof20mg cysts/fish/day(onceaday)offreshlyhatchedArtemiafor5weeks; theDIOgroupwasfedwiththeequivalentof60mgcysts/fish/day(3 timesaday,20mgcysts/fisheachtime)offreshlyhatchedArtemia forthesameperiod.Inthisstudy,zebrafishwereseparatedin dif-ferenttanks(1zebrafishper1-ltank),accordingtotheprotocolsby Montalbanoetal.(2016).Duringtheperiodoftreatment,inorderto analyzetheincreaseinbodymassindex(BMI)levels,thestandard lengthandthebodyweightofeveryfishweremeasuredeachweek. Bodyweightwasdeterminedbyweighing fishwitha precision analyticalscale.Bodylength(asstandardlength)wasdetermined fromthetipofthemouthtothecaudalpedunclewithadigital precisioncaliper.BMIwascalculatedbydividingbodyweight(in g)forthebodylengthsquared(incm2_{), aspreviouslydescribed} inMontalbanoetal.(2016).After5weeksoftreatment,thefish werefastedfor24handthenanaesthetizedwithMS222(ethyl 3-aminobenzoatemethanesulfonate,0.2g/l;Sigma,SaintLouis,MO, USA).Successively,theanimals’headswereremovedfromtrunks andthenthetrunkswerefixedfor24hinBouin’sfixativeatroom temperature(RT),dehydratedinascendingseriesofalcoholand embeddedinparaffinwax.Finally,7–10␮m-thickcrossand sagit-talsectionswereroutinelyprocessedforlightmicroscopy(Abbate etal.,2006,2012a,b),morphometricstudyand immunohistochem-icalstaining(n=5fishpergroup)(deGirolamoandLucini,2011), whiletheintestinaltractsofotherfishwerequicklyprocessedfor theisolationofRNA,semi-quantitativereversePCR,andwestern blotanalysis(n=10fishpergroup).

2.3. qRT-PCR:leptinA,leptinBandleptinreceptor

TotalRNA wasextractedfrom thewholezebrafish intestine usingacommercialkit(TrizolReagent,Invitrogen,Carlsbad,CA, USA),accordingtomanufacturer’sinstructions.Afterprecipitation and cold ethanol washing, RNA wasdried and dissolved in an appropriatevolumeofTris-EDTAbuffer(10mMTris–HClpH8.0 and1mMEDTA-Na2).Eachsamplewastreatedwith1UofDNase Ifor1hat37◦C todigestgenomic DNA.TheRNAwas precipi-tated,washed,anddissolvedagaininthesamebuffer.RNAsolution wasquantifiedat260nm(Biomate3,ThermoElectron Corpora-tion,Waltham,MA,USA)anditspuritywasassessedbytheratioat 260/280nmreading(Magnolietal.,2012).WeusedtheHigh Capac-itycDNAarchivekit(AppliedBiosystems,FosterCity,CA,USA)and randomhexamers,and10_␮goftotalRNAtomakecDNAfollowing manufacturer’sinstructions.A1␮galiquotofthecDNAwasused todetectleptinandleptinreceptorexpressionusingqRT-PCR.

Fig.1.(a)Controlanddiet-inducedobesezebrafish.Picturesofamaleadultcontrolzebrafish(CTRL)andadiet-induceobesezebrafish(DIO).Scalebars=1cm:a–b;analysis ofBMI(b)andbodyweight(c)inCRTLandDIOzebrafish.Theresultswereobtainedusing15zebrafishpereachexperimentalgroupandareexpressedasmean±SEM. Statisticalanalyseswereperformedusingone-wayanalysisofvariance(ANOVA)tocomparetheDIOandCTRLgroupsateachtime-point.Valuesof*P≤0.05wereconsidering significant.

Table1

qRT-PCRprimers.SequencesofforwardandreverseprimersdesignedforleptinA,leptinB,leptinreceptorandß-actin.

Leptina Leptinb Leptinreceptor ␤-Actin

FORWARD 5_{-CATCATCGTCAGAATCAGGG-3} ₅_{-GATGAGCACTTCCAGATGTC-3} ₅_{-AAGTCTTCACAACGCAGGA-3} ₅_{-CACAGATCATGTTCGAGAGACC-3}

REVERSE 5_{-ATCTCGGCGTATCTGGTCAA-3} ₅_{-TGTCTATGTTGAGGCAGAGC-3} ₅_{-ATAGGCGATGGAGCACATAG-3} ₅_{-GGTCAGGATCTTCATCAGGT-3}

The primers used were designed on the mRNA sequences

published for leptin A, leptin B, leptin receptor and ß-actin

(GenBank accession numbers: leptin A NM001128576,

lep-tin B NM001030186, leptin receptor NM001113376, ␤-actin

NM131031)(Table1).

ThehomemadeTaqManprobeswerelabeledatthe5with6 FAMfluorochromesfortherhodopsin,andVICfluorochromefor ␤-actin,whilethe3endswerelabeledwiththeMinorGrowBinder (MGB)quencher. The qRT-PCR reactionswereperformed using theTaqManUniversalPCRMasterMix(AppliedBiosystems, Fos-terCity,CA,USA)using5pmolofeachprimerand9pmolofboth targetand␤-actinprobe.Theassayswereperformedintriplicate usinga 7500PCRreal-time System(AppliedBiosystems, Foster City,CA,USA). The resultswerecalculated through the 2−DCt algorithmagainst␤-actin,andexpressedasthen-folddifference comparedtoanarbitrarycalibrator,chosenasahighervaluethan Cts.

2.4. Westernblotanalysis

Proteinsfromzebrafishintestinaltractandratstomach(used aspositive control)werepooledandextractedwithRIPAbuffer (50mMTris–HClpH7.4,1%TritonX-100,0.25%Na-deoxycholate, 150mM NaCl, 1mM EDTA) and 2mM PMSF and protein cock-tail (P8340 Sigma–Aldrich, Saint Louis, MO, USA) as protein inhibitors. Samples were homogenized with Ultra-Turrax T25 (IKA-Labortechnik, Staufer, Germany) at 13,500rpm. Then the

sampleswerespunat10,000rpmfor20minat 4◦C and super-natantswerecollected.Proteinconcentrationsweredetermined withtheBio-Raddyeproteinassay.Sampleswereboiledat98◦C for10mininloadingbuffer(50mMTris–HClpH6.8,100mM ␤-mercaptaethanol,2%SDS,0.1%bluebromophenol,10%glycerol). Theproteinswereseparatedona 15%(forleptin)and 12%(for leptinreceptor)SDS-polyacrylamidegelelectrophoresiswith4% and16%stackinggelin1%Tris-glycinebuffer(0.025MTris,0.192 Mglycine,and0.1%SDS[pH8.3])inaminiproteancell(Bio-Rad, LaboratoriesIn.UK)at130Vfor2h.Theseparatedproteinswere electrotransferredontoa nitrocellulosemembranewithtransfer buffer(39mM Tris base,0.2 Mglycine, and 20%methanol [pH 8.5])inaminitransfercell(Bio-Rad)at100Vfor2hat4◦C. Mem-braneswereincubatedat4◦Cfor1hinblockingbuffercontaining 1%PBSand0.05%Tween20with5%driednon-fatmilkandthen wereprobedwithpolyclonalantibodiesinrabbitthatrecognizes leptin(Ob,A-20,sc-842,SantaCruzBiotec. Inc.,SantaCruz, CA, USA)and␤-actin(A5060,Sigma)usedasaninternalmarkerand polyclonalantibodyingoatthatrecognizeslong(Ob-R,C-20, sc-1832,SantaCruzBiotec.Inc.,SantaCruz,CA,USA)andshort(Ob-R, M-18,sc-1834,SantaCruzBiotec.Inc.,SantaCruz,CA,USA) iso-formsofleptinreceptor.Allprimaryantibodieswerediluted1:500 and incubated overnight at 4◦C. This was followed by incuba-tionwithrabbitanti-goat IgG (1:5000,Millipore,Temecula, CA, USA)andgoatanti-rabbitIgG(Sigma1:5000)for1hatRT.Signals weredetectedbychemiluminescencewiththeImmobilon West-ernChemiluminescentHRPsubstrateKit(Millipore,Temecula,CA,

Fig.2.qRT-PCR.ThebargraphshowsexpressionofleptinA,leptinB,anditsreceptormRNAinzebrafishgutoftwoexperimentalgroups.Theresultswereobtainedusing5 zebrafishpereachexperimentalgroupandareexpressedasmean±SEM.StatisticalanalysesweredonebyStudent’sttest.Valuesof*P≤0.05wereconsideredsignificant.

USA).Apre-stainedmolecular-weightladder(NovexSharp pro-teinstandard,LC5800,Invitrogen,Hilden,Germany)wasusedto determineproteinsize.

Specificitywasdeterminedbypre-absorptionofprimary anti-bodieswiththeirrelativecontrolpeptidesbeforewesternblotting. 2.5. Singleimmunohistochemistry

Crossandsagittalsectionswerestainedbytheavidin-biotin immunohistochemicaltechnique.In thespecificstep,polyclonal antibodiesraisedinrabbitagainstleptin,andpolyclonal antibod-iesraisedingoatagainstlongandshortisoformofleptinreceptor wereused.Theantibodieswerediluted1:300andappliedon sec-tionsovernightat4◦C.Theothercomponentsoftheimmunological reactionwerecontainedintheVectastainEliteABCkitfrom Vec-tor Laboratories Inc (PK 6101 for rabbit primary antibody and PK6105for goatprimary antibodies,Burlingame, CA,USA).The final stainingwasperformedusinga solutionof 10mg of3-3 -diaminobenzidinetetrahydrochloride(DAB,SigmaChemicalCo.)in 15mlofa0.5MTrisbuffer,pH7.6,containing1.5mlof0.03%H2O2. Controlswereobtainedbysubstitutingtheprimaryantiserumwith PBSornormalseruminthespecificstep,oralternatively,by absorb-ingeachprimaryantiserumwithanexcessoftherelativeblocking peptide(Ob,A-20P,sc-842P;Ob-R,C-20,sc1832P;Ob-R,M-18, sc-1834PSantaCruz,Biotec.Inc.,SantaCruz,CA,USA).

2.6. Immunopositivecellscount

Inorder toobtainquantitativedata,20 consecutivesections wereobtainedfromtheproximalanddistalintestineofDIOand CTRLzebrafish.Fivenon-adjacentsectionsfromeachseriesof sec-tionswereimmunolabelled asdescribed abovetodetect leptin immunopositivecells. Slides wereobservedusing amicroscope LeicaDMRA2(Leica,Wetzlar,Germany)equippedwithaDC300F digitalcameraandthenumberofleptinimmunopositivecellswas calculatedusingImageJprogram.

2.7. Doubleimmunostaining

Doubleimmunohistochemicalstainingusingtwoprimary anti-seraraisedindifferentspecieswasperformed(Gattaetal.,2014). Sagittalsectionsweredewaxed,rehydrated,rinsedinPBSand incu-batedfor30minatRTwithnormalrabbitserum(S-5000Vector) 1:5.Successively,blockingofendogenous biotinwasperformed (Avidin-Biotinblockingkit, SP-2001, Vector).Then, thesections were incubated with the first primary antibody raised against long-orshortisoformofleptinreceptor(1:50)overnightat4◦C. Successively,thesectionswerewashedwithPBSandincubated withbiotinylated secondaryrabbitanti-goatantibody(1:20 BA-5000Vector)for2hatRT.AfterrinsinginPBS,thesectionswere incubatedwithStreptavidin TexasRed® conjugate(1:50;S-872, LifeTechnologiesEurope,Monza,Italy)for2hatRTindarkhumid chamber.AfterrinsinginPBS,thesectionswereincubatedwith normaldonkeyserum(1:5;017-000-121,JacksonImmunoresearch LaboratoriesInc.,Suffolk,UK)for2hatRTindarkhumidchamber. Afterwards,thesectionswereincubatedwiththesecondprimary antibodyinrabbitagainstleptin(1:50)overnightat4◦Cindark humidchamber.AfterrinsinginPBS,thesectionswereincubated AlexaFluor® 488-conjugatedaffinipuredonkeyanti-rabbit(1:50, 711-545-152,Jackson)for2hatRTindarkhumidchamber.

Finally,thesectionswerewashedwithPBS,mountedwith glyc-erinedilutedwithPBS1:1andobservedonaconfocalmicroscope (LeicaDM6000BbyLeica,Wetzlar,Germany)equippedwithaLeica DFC450Cdigitalcamera(Leica,Wetzlar,Germany).Pictureswere acquiredthroughLeicaLASAFsoftwarepoweredbyMetamorph®.

2.8. Statisticalanalysis

Theassayswerecarriedoutintriplicate.Allexperimentaldata arereportedasmean±SEM.StatisticalanalysesonBMIandbody weight data were performed by one-way analysis of variance (ANOVA)andanysignificantdifferencewasdeterminedata signif-icancelevelof0.05viatheapplicationofaTukey’stest.Statistical analysesonqRT-PCRdatawereperformedbytheunpaired Stu-dent’sttest.Pvalueslowerthan0.05wereconsideredsignificant (seeMontalbanoetal.,2016).

Immunopositive cell count data were analyzed by one-way analysisofvariance(ANOVA)and anysignificantdifferencewas determinedatasignificancelevelof0.05viatheapplicationofa Tukey’stest.TheanalyseswerecarriedoutusingStatisticaversion 7.0(StatsoftInc.,Tulsa,OK).

3. Results

Thisstudyreportsthepresenceandexpressionofleptinand itsreceptorsfamilyin thedigestivetractofDIOzebrafish com-paredwiththesameparametersobtainedinCTRLzebrafish.The picturesofamaleadultCTRLzebrafish(Fig.1a)andofDIOzebrafish (Fig.1a)demonstrated that,underamacroscopicpoint ofview, theabdomenwidthoftheDIOzebrafishisduetoalargervisceral

Fig.3.Westernblottinganalysis.BlotA:leptinimmunopositivebandsof∼16kDain zebrafishintestine(a),ratstomach(b);blotB:Ob-Rlongisoformimmunopositive bandsof∼120kDainzebrafishintestine(a),ratstomach(b);blotC:Ob-Rshort isoformimmunopositivebandsof∼100kDainzebrafishintestine(a),ratstomach (b).(m):Marker;(a)zebrafishintestine;(b)ratstomach;(c)pre-absorption.The resultswereobtainedusing5zebrafishpereachexperimentalgroup.

fatdepotcomparedtoCTRLgroup,consistentlytopreviousresults (Montalbanoetal.,2016).

3.1. BMIandbodyweightmeasurements

NormalizedBMI(Fig.1b)andnormalizedbodyweight(Fig.1c) showedastatisticallysignificant(*P≤0.05)increaseinDIOgroup ateverytimepointanalyzed.Attheend oftheexperiment,the increaseinBMIwasgreaterthan45%inDIOzebrafis(1.47±0.07), comparedtothestartingconditions.Aninitialsubstantialincrease inBMIinthefirst2–3weeksisfollowedbyaminorincreaseinfat accumulationand theachievementofa“plateau”,characterized bynofurtherBMIincrease.Conversely,controlsshowamodest decreaseinBMI(approximately−10%),withrespecttothe start-ingconditions(finalvalue0.94±0.10),thusmaintainingtheirBMI quiteconstantthroughthewholeexperimentalperiod.Similarly towhat happens forBMI,weightgain inDIOzebrafishis more than40%(1.46±0.05),whileinCTRLzebrafishthereisamodest decreaseinweightgain(0.93±0.12;approximately−10%).InDIO zebrafish,aninitialsubstantialincreaseinbodyweightinthefirst 2–3weeksisfollowedbyaminorincreaseinfataccumulationand theachievementofa“plateau”,characterizedbynofurtherweight increase.

3.2. qRT-PCR:leptinA,leptinBandleptinreceptor

Intheintestinaltract,theexpressionlevelsofleptinAand lep-tinB(Fig.2)weresignificantly(*P≤0.05)higherinDIOzebrafish (leptinA0.80±0.07;leptinB,60±0.11)thanintheCTRLgroup (leptinA0.62±0.08;leptinB0.50±0.01).Onthecontrary,leptin

receptormRNAwasdownregulatedintheintestinaltractofDIO group(0.08±0.04)comparedtoCTRLgroup(0.17±0.07)(Fig.2).

3.3. Proteinexpression

Western blot analysis was carried out on homogenates of intestinaltractofDIOandCTRLzebrafish.Theresultsofwestern blotanalysisshowedthepresenceofleptin(∼16kDaband)inthe intestinaltractofbothexperimentalgroups(Fig.3A).The pres-enceofOb-Rlongisoformimmunoreactivebandofabout120kDa (Fig.3B)andOb-Rshortisoformimmunoreactivebandofabout 100kDa(Fig.3C)inzebrafishintestinaltractofbothexperimental groupsweredetected.

Thespecificityoftheresponsewasconfirmedbypre-incubation ofleptin,Ob-RlongisoformandOB-shortisoformantibodieswith their respective blocking peptide. There was no expression of leptin,Ob-RlongandOb-Rshortisoformproteinsinthese prepa-rations,whereasthepresenceoftheseproteinswasdetectedinrat stomachhomogenatethatwasusedaspositivecontrol(Fig.3).

3.4. Singleimmunohistochemistry

Noreactionwasobservedincontrolsperformedbysubstituting theprimaryantibodieswithPBSornormalserum,orbyadsorbing eachprimaryantiserumwithanexcesstheirhomologuesantigens. Leptinandleptinreceptorsimmunoreactivity(IR)wasobserved inepithelial cellsof intestinalmucosalfolds(Figs.4–6)of both experimentalgroups.Consideringtheparticularmorphologyofthe

Fig.4. Singleimmunohistochemistry.CrosssectionofDIOandCTRLzebrafishintestinaltract:numerousleptinimmunoreactiveendocrinecells(arrows)inepithelialmucosa ofproximalintestinaltractofDIOzebrafish(a,a1_{detailofa);rareleptinimmunoreactiveendocrinecellsinepithelialmucosaofproximalintestinaltractofCTRLzebrafish}

(b,b1_{detailofb);someleptinimmunoreactiveendocrinecells(arrows)inepithelialmucosaofdistalintestinaltractofDIOzebrafish(c,c}1_{detailofc);asingleleptin}

immunopositivecellinthedistalintestinaltractofCTRLzebrafish(d,d1_{detailofd).Theresultswereobtainedusing5zebrafishpereachexperimentalgroup.}

Fig.5.Singleimmunohistochemistry.SagittalandcrosssectionsofDIOandCTRL zebrafishintestinal tract:numerousleptinimmunoreactiveendocrinecells in epithelialmucosaofproximalintestinaltractofDIOzebrafish(a,a1_detailofa);

leptinimmunopositivefibersincircularmusclelayer(*)andneurons(**)in myen-tericganglia(b,c).Theresultswereobtainedusing5zebrafishpereachexperimental group.

Scalebars=50␮m:a;10␮m:a1_{;10␮m:b;10␮m:c.}

immunopositivecells(elongatedinshapeandgenerallybipolar), wehereinrefertothemasendocrinecells.

Leptin immunoreactive endocrine cells were frequently observedinthemucosalfoldsofproximalintestinaltract(Figs. 4a,b;5a)andmorerarelyindistalintestinaltract(Fig.4c,d)both inDIO(Fig.4a,c)andCTRLzebrafish(Fig.4b,d).Inaddition,leptin immunopositivefibersandneuronsinthemyentericplexusesand fibersincircularandlongitudinalmusclelayersweredetectedin DIOandCTRLzebrafishintestine(Fig.4).

Ob-Rlongisoform(Fig.6b,c)andOb-Rshortisoform(Fig.6a,d) immunopositivefinegranuleswerelocatedinendocrinecellsof epithelialmucosaofintestinaltractbothinDIO(Fig.6a,c,d)thanin CTRLzebrafish(Fig.6b).

3.5. Immunopositivecellscount

Thenumberofleptinimmunopositivecellswasincreasedina statisticallysignificantmanner(*P≤0.05)inDIOzebrafishintestine (51.0±1.7)comparedtothatofCTRLzebrafish(7.0±1.5)(Fig.7).

3.6. Doubleimmunostaining

In DIO zebrafish intestinal tract, double immunostaining showed leptin immunoreactivity coexisted with OB-R long (Fig. 8a–c)and -shortisoform receptors(Fig.9a,c)especially in endocrinecellsofepithelialmucosalfolds.InCTRLzebrafish,leptin immunopositiveepithelialcellsdidnotco-localizewithOB-Rlong (Fig.8d–f)and-short(Fig.9e,f)isoformreceptors.

Fig.6.Singleimmunohistochemistry.SagittalandcrosssectionsofDIOandCTRL zebrafishintestinaltract:numerousleptinreceptorlong(b,c)andshort(a,d)isoform immunopositivecellsintheepithelialliningofDIOzebrafish(a,c,d)andCTRL(b) intestinaltract.Theresultswereobtainedusing5zebrafishpereachexperimental group.

Scalebars=30␮m:a;50␮m:b;10␮m:c;10␮m:d.

4. Discussion

Inthisstudy,wedemonstratedtheexpressionandlocalization ofleptinanditsreceptorinthegutofanobesezebrafishmodelin responsetoahigh-fatdiet.Recentlyithasbeendemonstratedthat inthisexperimentalmodelmanypathophysiologicalconditionsare sharedwithmammalianobesity(Okaetal.,2010;Montalbanoetal., 2016).Itiswellknownthatleptinplaysanessentialroleinthe reg-ulationoffoodintakeandcontrolofbodyweight,bothinfishand inmammals(Volkoffetal.,2005;Kloketal.,2007;Volkoff,2015); however,howleptinexpressioninthedigestivetractisregulated bynutritionalstatusinzebrafishhasnotbeendemonstratedsofar. Forthesereasons,thisstudywasundertakeninordertobetter elu-cidatetheinvolvementofleptinintheregulationoffoodintake inzebrafishunderpathophysiologicalconditions.Recentstudies onstomach-lessandstomach-containingfishhavereportedthe expressionandlocalizationofseveralneuropeptides(Gambardella etal.,2010;Micaleetal.,2010,2012;Russoetal.,2011;Varricchio etal.,2012;D’Angeloetal.,2016a,b).Inagreementwiththose stud-ies,ourresultsdescribedthemajorityofleptinimmunoreactive endocrinecellsintheproximalportionoftheintestinaltract.The presenceofleptinimmunoreactivecellsgraduallydecreasesfrom theproximaltothedistalportionofzebrafishintestinaltract,which

Fig.7. Cellcountgraph.BargraphshowingleptinimmunopositivecellcountsinDIOandCTRLzebrafishintestinaltract.Immunopositivecellcountdatawereanalyzedby one-wayanalysisofvariance(ANOVA)andanysignificantdifferencewasdeterminedatasignificancelevelof0.05viatheapplicationofaTukey’stest.Theanalyseswere carriedoutusingStatisticaversion7.0(Statsoftinc.,Tulsa,OK).

Fig.8. Doubleimmunostaining.SagittalsectionofDIOandCTRLzebrafishintestinaltract:co-localizationofleptin(Lep)(green)andleptinreceptorlongisoform(OB-R-lf) (red)immunopositiveendocrinecellsinintestinaltractepitheliumofDIOzebrafish(a–c),andCTRLzebrafish(d–f).Theresultswereobtainedusing5zebrafishpereach experimentalgroup.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)

Scalebars=40␮m:a–f;20␮m:a1_,b1_d1_,e1_.

isconsistentwithresultsreportedingoldfishandrainbowtroutgut (Russoetal.,2011;Varricchioetal.,2012).Moreover,forthefirst time,wehavedemonstratedthecolocalizationofleptinandleptin receptorisoforms(longandshort)usingconfocallasermicroscopy inasubpopulationofenteroendocrinecellsinDIOzebrafish.These findingsinthisteleostsubstantiatethepreviousresultsobtainedin

DIOratexperimentalmodel(ZhangandScarpace,2006)andallow ustospeculate that aleptinsignalingdisruptionin a zebrafish modelofdiet-induced obesityis similartothedisruption caus-inghyperleptinemiaandleptinresistanceinmammals,including humans(Londravilleetal.,2014;ParkandAhima,2015;Ritzeetal., 2016).

Fig.9. Doubleimmunostaining.SagittalsectionofDIOandCTRLzebrafishintestine:co-localizationofleptin(Lep)(green)andleptinreceptorshortisoform(OB-R-sf) (red)immunopositiveendocrinecellsinintestinaltractepitheliumofDIOzebrafish(a–c),andCTRLzebrafish(d–f).Theresultswereobtainedusing5zebrafishpereach experimentalgroup.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)

Scalebars=40␮m:a–f;20␮m:a1_,b1_d1_,e1_.

Thehighernumberofleptinimmunoreactivecellsobservedin theproximalintestineandtheincreaseincellsexpressingleptin proteininDIOzebrafishgutcouldberelatedtoanhigh-fatdiet.

Inaddition,theleptindistributionintheentericnervoussystem alongtheintestinaltractofDIOandCTRLzebrafishcouldsuggestan involvementofthisproteinintheregulationofmotilityand absorp-tionofnutrientsaspreviouslydescribedinmammals(Guilmeau etal.,2004;Yarandietal.,2011)actingthroughthevagalafferent pathwayinresponsetoexcessivefoodintake(Atteleetal.,2002). Theactionofneuropeptidesonthemotilityandabsorptionhas beenalsoreportedinothervertebratespecies(Arcamoneetal., 2014;Varricchioetal.,2014).

The presence of leptin short and long isoform receptors in zebrafishintestinaltractcouldsuggestapossiblelocalactionofthis peptide,inagreementwithpreviousdata(Atteleetal.,2002;Pinto etal.,2004;Cammisottoetal.,2010).Moreoverourfindingscould supportapossibleroleofleptininregulatingzebrafishdigestive processandinformingthebrainaboutenergyandnutrientinput, throughsignalingviatheenteroendocrinesystem(Gambardella etal.,2010;Varricchioetal.,2012).Ourresultsobtainedby qRT-PCRreportanoverexpressionofleptins(Aand B)mRNAinthe intestinaltractofDIOgroupcompared tocontrolgroup.InDIO zebrafishleptinAlevelswerehigherthanleptinBones accord-ingtoGorissenetal.(2009).TheoverexpressionofleptinsmRNA couldberelatedtotheincreasednumberofcellsexpressingleptin proteinafterahigh-fatdiet.Adownregulationofleptinreceptor mRNAinintestinaltractofDIOzebrafishwasdemonstrated, sug-gestingthatleptincouldbeinvolvedinthealterationofappetite regulationinobesity(Copelandetal.,2011).Moreover,allthese

dataaddfurtherevidencetothehypothesisthatleptincouldbe anupstreamfactorinthemechanismunderlyingchangesin feed-ingandexploratorybehaviorassociatedwithfoodintake(Attele etal., 2002).In conclusion,a variationof leptinsystem expres-sionlevels and immunohistochemicallocalization in thegutin responsetoahigh-fatdietinDIOzebrafishwasreportedforthe firsttime.Therefore,thisstudycouldbeconsideredasastarting pointtoaddfurtherknowledgeonthepleiotropicroleofthis hor-moneinfish.Althoughleptinisinvolvedinfoodintakeorobesity infish(Huisingetal.,2006;Okaetal.,2010;Volkoff,2015),among otherfunctions,Micheletal.(2016)showedthatzebrafishleptin isnot onlyrequiredforfoodintakebutalsoin glucose homeo-stasis,asinmammals,suggestingasglucosehomeostasisappears tobeconservedacrossvertebrates.Nevertheless,furtherstudies areneededinordertobetterclarifytheplethoraoffunctions car-riedoutbyleptinsysteminboth larvaland adultlife stagesin fish,consideringthat,althoughthetertiarystructureishighly con-servedamong vertebrates,thegenomeduplicationeventmight haveaddedmultipleregulationrolestoleptinsystemcompared toothervertebrates(Gorissenetal.,2009;Copelandetal.,2011; Conde-SieiraandSoengas,2017).

Acknowledgements

TheauthorswouldliketothankDr.AshaSeth(WellcomeTrust SangerInstitute, GenomeCampus,Cambridge,UK)forkindand criticalrevisionofthemanuscript.Thispaperwassupportedby IAOgrant(Prof.A.Germanà).

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttp://dx.doi.org/10.1016/j.aanat.2017.03. 005.

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