Insulin Signaling in Intestinal Stem and Progenitor Cells as ...

In addition, increased expression was observed for the Drosophila transcripts for the ... by up-regulation of the target of the brain insulin gene (tobi). NextArticleinJournal ARegulatoryNoncodingRNA,nc886,SuppressesEsophagealCancerbyInhibitingtheAKTPathwayandCellCycleProgression PreviousArticleinJournal TNFaandIL2EncodingOncolyticAdenovirusActivatesPathogenandDanger-AssociatedImmunologicalSignaling Journals Topics Information ForAuthors ForReviewers ForEditors ForLibrarians ForPublishers ForSocieties ForConferenceOrganizers ArticleProcessingCharges OpenAccessPolicy InstitutionalOpenAccessProgram EditorialProcess Awards ResearchandPublicationEthics AuthorServices Initiatives Sciforum MDPIBooks Preprints Scilit SciProfiles Encyclopedia JAMS ProceedingsSeries About SignIn/SignUp Notice Youcanmakesubmissionstootherjournals here. clear Notice Youareaccessingamachine-readablepage.Inordertobehuman-readable,pleaseinstallanRSSreader. 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FullArticleText /ajax/scifeed/subscribe share announcement question_answer thumb_up ... textsms ... NeedHelp? Support Findsupportforaspecificprobleminthesupportsectionofourwebsite. GetSupport Feedback Pleaseletusknowwhatyouthinkofourproductsandservices. GiveFeedback Information VisitourdedicatedinformationsectiontolearnmoreaboutMDPI. GetInformation clear OpenAccessArticle InsulinSignalinginIntestinalStemandProgenitorCellsasanImportantDeterminantofPhysiologicalandMetabolicTraitsinDrosophila by OlhaM.Strilbytska1,*,UlianaV.Semaniuk1,KennethB.Storey2,IhorS.Yurkevych1andOlehLushchak1 1 DepartmentofBiochemistryandBiotechnology,VasylStefanykPrecarpathianNationalUniversity,57Shevchenkastr.,76018Ivano-Frankivsk,Ukraine 2 InstituteofBiochemistry,CarletonUniversity,Ottawa,OntarioK1S5B6,Canada * Authortowhomcorrespondenceshouldbeaddressed. Cells2020,9(4),803;https://doi.org/10.3390/cells9040803 Received:3February2020 / Revised:18March2020 / Accepted:19March2020 / Published:26March2020 DownloadPDF BrowseFigures Review Reports CitationExport BibTeX EndNote RIS CiteThisPaper Abstract Theinsulin–IGF-1signaling(IIS)pathwayisconservedthroughoutmulticellularorganismsandregulatesmanytraits,includingaging,reproduction,feeding,metabolism,stressresistance,andgrowth.Here,wepresentevidenceofasurvival-sustainingroleforIISinasubsetofgutcellsinDrosophilamelanogaster,namelytheintestinalstemcells(ISCs)andprogenitorcells.UsingRNAitoknockdowntheinsulinreceptor,wefoundthatinhibitionofIISinISCsstatisticallyshortenedthelifespanofexperimentalfliescomparedwithnon-knockdowncontrols,andalsoshortenedtheirsurvivalunderstarvationormalnutritionconditions.Thesefliesalsoshoweddecreasedreproductionandfeeding,andhadloweramountsofglycogenandglucoseinthebody.Inaddition,increasedexpressionwasobservedfortheDrosophilatranscriptsfortheinsulin-likepeptidesdilp2,dilp5,anddilp6.Thismayreflectincreasedinsulinsignalinginperipheraltissuessupportedbyup-regulationofthetargetofthebraininsulingene(tobi).Incontrast,activationofIIS(viaknockdownoftheinsulinpathwayinhibitorPTEN)inintestinalstemandprogenitorcellsdecreasedflyresistancetomalnutrition,potentiallybyaffectingadipokinetichormonesignaling.Finally,PtenknockdowntoenhanceIISalsoactivatedJAK–STATsignalinginguttissuebyup-regulationofupd2,upd3,andsoc36genes,aswellasgenesencodingtheEGFreceptorligandsspitzandvein.Theseresultsclearlydemonstratethatmanipulatinginsulinlevelsmaybeusedtomodulatevariousflytraits,whichareimportantdeterminantsoforganismalsurvival. Keywords: insulinsignalingpathway;midgut;ISC;progenitorcells;lifespan;metabolism;fruitfly insulinsignalingpathway;midgut;ISC;progenitorcells;lifespan;metabolism;fruitfly 1.IntroductionTheadultDrosophilamidguthasmanysimilaritiestothemammalianintestineandisanattractivemodelforstudyingtheregulationofstemcellmaintenanceandproliferation.Moreover,maintenanceofgutintegrityasanagingmarkerisimportantforflysurvivalandcontributestodetermininglongevity[1].TheDrosophilamidgutcontainsintestinalstemcells(ISCs)thatarelocatedonthebasementmembraneoftheepithelium.ISCsundergoasymmetricdivision,whereonedaughtercellmaintainsitsstemness,whiletheotherdaughtercellbecomesanenteroblast(EB).Subsequently,anEBcandifferentiateintoanenterocyte(EC)orenteroendocrine(EE)cell[2].RegulationofISCself-renewal,proliferation,anddifferentiationhasastrongimpactonmaintainingtissuehomeostasis,andinturn,organismalaging.ThereareseveralsignalingpathwaysthatcontrolthemaintenanceandproliferationofISCs.ThecoreNotchsignalingpathwayinterplayswithothersignals,includingJNK,JAK–STAT,EGFR,Wgandinsulin–IGF-1signaling(IIS)pathwaystoregulateISCproliferation.IthasbeenreportedthatISplaysaroleintheregulationoforgansizebymodulationofISCs[3].IIScanactivateISCsthroughtwopotentialmechanisms:(a)enhanceddivisionrate,and(b)switchingtosymmetriccelldivisionfromasymmetricdivision[4].BothmechanismsleadtogrowthofISCs.Moreover,IISpositivelyregulatesISCsproliferationduringagingandregeneration[5,6].IthasbeenshownthattherateofISCproliferationdependsondietandIISactivity[7].InhibitionofIISactivitythroughinsulinreceptor(InR)loss-of-functionleadstoasignificantdecreaseintheaveragenumberofpH3+cells[8].Choiandcolleaguesshowedthatenteroblastsnon-autonomouslyregulateISCproliferationinresponsetonutritionandIIS[6].EnteroblastsinhibitISCproliferationthroughmaintenanceofE-cadherin-mediatedcell–cellinteraction[6].ThisprocessiscontrolledbytheIISpathwayinenteroblastcells.Inthepresentwork,weaskedifthemanipulationofIISinasmallgroupofgutcellsmayhaveglobaleffectsonflyphysiologyandmetabolismandlocalizedtissueeffects.WeconditionallyactivatedorinhibitedIISinISCsandEBsinadultfliesandstudiedtheirlifespan,resistancetomalnutrition,andsomemetabolites.Inaddition,gutintegrityandEGFRandJAK–STATsignalingwerealsoanalyzed.WeconcludedthatchangesinIISinbothdirectionsnegativelyaffectedflyphysiologyandmetabolism.Unexpectedly,malnutritionconditionssignificantlyenhancedsurvivalofesg/InR-RNAiflies.2.MaterialsandMethods2.1.FlyHusbandryandTransgenicFliesFourlinesofflies(escargot-Gal4UAS-GFPtub-Gal80ts,w1118,UAS-InR-RNAi,UAS-Pten-RNAi)wereculturedonstandardmolassesmedium,composedofdryyeast(5%),corn(6.1%),molasses(7.5%),nipagin(0.18%),andpropionicacid(0.4%)at18°Cforoneyear.TheGal4-UASsystem[9]wasusedtodrivespecificinterferenceRNAs.Todownregulatetheparticulargenes(InRorPten)inthepopulationofmidgutstemcells,weusedthetemperature-sensitiveescargottsdriver(esgts),whichisspecifictostemcellsandtheirimmaturedaughtercells,enteroblasts(EBs).Toreduceinsulinsignaling,aconstructUAS-InR-RNAithatknockeddowntheinsulinreceptor(InR)wasused.Conversely,IISactivationwasachievedbyknockingdownthepathwayinhibitorgenePtenthroughUAS-Pten-RNAiexpression.Fliesofthew1118linewerecrossedtoesgtogeneratetherespectivecontrolgenotypeesg/+.TheefficiencyofRNAifortheInRgenewas57%andforPtenwas71%,asdeterminedbyRT-PCR(FigureS1).Forallsubsequentexperiments(lifespan,fecundity,feeding,starvationandmalnutritionresistancetests,metabolitescontent,andgeneexpressionmeasurements),femalesofGal4lineswerematedtomalesofrespectiveUASlines.Theresultingeggs(100pervial)wereallowedtodevelopat18°C.Atthistemperature,Gal80inhibitsbindingofGal4proteintoUAS.Newlyenclosedfliesweresexed,andselectedfemaleswerekeptfortwoadditionaldaysandshiftedto29°Cfor6daystoinduceexpressionoftheUASconstructs.Geneticcrossesgeneratedthreetypesoftransgenicflylines:esg/+(controlflies),esg/InR-RNAi(IISinhibition),andesg/Pten-RNAi(IISactivation).Afterexpressioninduction,theresultingflieswereusedforallmeasurements,whichwereconductedat29°C.2.2.LifespanandFecundityApproximately100femalesofeachgenotypewereplacedin1.5Ldemographiccages.Aplasticvialfilledwith5mLofexperimentalfoodcontaining5%sucrose,5%dryyeast,1.2%agar,and0.18%nipagin(5%S+5%Y)wasattachedtothecage.Foodwaschangedeveryotherday,anddeadflieswerecounted.Datawerecollectedfromtwoindependentexperiments.After6daysofexpressioninduction,thereadoutofexperimentaldaysstartsinitiallyforthesurvivalexperiments.Foreachgenotype,tworeplicates,eachconsistingoftenrandomparingswithonefemaleandtwomaleflies,wereplacedinto5mLvialswith1mLexperimentalfoodtomeasurereproduction.Foodwaschangedeverydayandthenumberofeggslaidbyeachindividualfemalewasrecorded,aspreviouslydescribed[10,11].Thenumberofeggswascountedoveraperiodoffourdaysandduringonedayforesc/InR-RNAifliesafterinductionfor6days.2.3.FeedingFoodconsumptionbyasingleflywasmeasuredusingtheCAFEassay[10].Briefly,experimentalflieswerekeptina5µLcapillarytubefilledwithfoodcontaining5%yeastextract,5%sucrose,0.1%propionic,and0.01%phosphoricacid.Capillarieswerechangedeverydayandtheamountoffoodconsumedwasmeasuredoveraperiodoffourdays.However,fliesofesc/InR-RNAigenotypeweretestedonlyonceduetohavingaveryshortlifespan.Vialswerekeptinclosedboxeswithdistilledwateronthebottomtomaintainhighhumidityforevaporationprevention.Threenon-feedingvialsweremonitoredforchangesinvolumetocontrolforevaporation.Tenfliespergenotypeweretested.2.4.MalnutritionandStarvationForthemalnutritionexperiments,15flieswerekeptin15mLvialswith3mLofmediumcontaining1%sucrose(1%S),1%autolyzedyeast(1%AY),or0.5%ofboth(0.5%S+0.5%AY).Asolutionof0.5%agarosewasusedforstarvationassays.Thevialswerechangedevery2daysandthenumberofdeadflieswasrecordedeverydayformalnutritionexperiments,andevery6–12–6h(at9:00a.m.,3:00p.m,.and9:00p.m.)forstarvationexperiment,untilthelastflyhaddied.Survivalof45–75fliespergenotypewastested.2.5.MetabolitesGlucoseandtrehaloseinhemolymph,aswellasglucose,trehalose,andglycogeninflybodies,wasmeasuredasdescribedpreviously[12].Briefly,preweighedfliesweredecapitatedandcentrifugedtoextracthemolymph(3000×g,5min).Flybodieswerehomogenizedin50mMsodiumphosphatebuffer(pH7.0),centrifuged,andusedfordeterminationofglucoseandglycogenlevels.Measurementswereperformedusingaglucoseassaykit(LiquickCor-Glucosediagnostickit,Cormay,Poland,Cat.No.2-203).TheglycogenwasconvertedintoglucosebyincubationwithamyloglucosidasefromAspergillusniger(25°C,4h)andthenglucosewasmeasuredasabove.Thelevelsofbodyglucoseandglycogenweredisplayedasmilligrampergramofwetweight(mg/gww).Fortriglyceride(TAG)determination,flieswereweighed,homogenizedin200mMphosphatebufferedsalinecontaining0.05%TritonX100(PBST),boiled,andcentrifuged(13,000×g,10min)[13].ResultingsupernatantswereusedforTAGassaywithLiquickCor-TGdiagnostickit(Cormay,Poland).Fliesofallgenotypesweretestedinfourindependentreplicates.2.6.AnalysisofGutIntegrityTodetermineintestinalintegrity,weexaminedfliesthatconsumedfoodsupplementedwithnon-absorbablebluefooddyeE133[1].“Smurf”fliesweredefinedbyvisiblebluefooddyeseenthroughoutthebody,whichsuggestsdisruptionofgutintegrity.Intotal,150femalesofeachgenotypewereplacedintoplasticvialswithfoodsupplementedwith2.5%E133,andafter12h,thenumberof“smurf”flieswascounted.Theexperimentwasrepeatedevery5daysforupto30days(onday1,5,10,15,20,25,and30).Asthefliesofesg/InR-RNAigenotypediedquickly,fastgutintegritywasonlymeasuredonceonexperimentalday1.2.7.GeneExpressionTotalRNAfromheads,wholeflies,ordissectedgutswasextractedwithRNeasyPlusMiniKit(Qiagen,Hilden,Germany)andconvertedintocDNAwithQuantiTectReverseTranscriptionKit(Qiagen).ExpressionofgenesofinterestwasmeasuredusinganABIPrism7000instrument(AppliedBiosystems,FosterCity,CA,USA),aSensiFASTSYBRHi-ROXKit,andaQuantiTectSYBRGreenPCRKit(Qiagen)underconditionsrecommendedbythemanufacturers.Eachanalyticalandstandardreactionwasperformedinthreetechnicalreplicates.Thelevelsoftranscriptsweremeasuredusingpreviouslypublishedprimerpairs,showninTableS2[14,15].TheCtmethodwasusedwithrp49(ribosomalprotein49)asthereferencegeneforheadsandwholeflies,whilecrq(croquemort)wasthereferencegenetoassaysamplesfromthegut.2.8.StatisticalProceduresToassessstatisticallysignificantchangesinlifespanandsurvivalinresponsetoexperimentallyinducedstresses,survivalcurveswereconstructedfromthestarvation,malnutrition,andoxidativestressresistanceassays,andanalyzedwiththelog-rank(Mantel-Cox)test(PrismGraphPad,version6).DifferencesbetweengroupswereanalyzedusingANOVA,followedbyNewman–Keulspostposthoctest(PrismGraphPad,version6).3.Results3.1.LifespanandStressResistanceTheroleoftheIISpathwayinlifespanregulationisconservedacrosseukaryoticorganismsfromnematodestomammals.Themeanlifespanofcontrolfliesesg/+wasapproximately25days(range25–26days).InhibitionofIISsignalinginISCsandEBs(esc-cells)duetoInR-RNAiexpressionacceleratedthemortalityoffliesbythesecondexperimentalday(log-rank,p<0.0001;χ2=144)(Figure1A).NosignificantdifferencewasobservedinsurvivalratebetweenflieswithactivatedIISinesg-cells(esg/Pten-RNAi)andesg/+controlflies.Nutrientavailabilityisakeydeterminantoforganismalgrowthandsurvival.Theeffectsofmalnutritiononflysurvivalwerealsoevaluatedinfliesfedwithrestricteddiets(i.e.,either1%sucrose(S),1%autolyzedyeast(AY),or0.5%sucrose+0.5%AY)(Figure1).Interestingly,malnutritionprolongedthelifespanofthefliesexpressingInR-RNAiwhencomparedtothecontroldiet.AlthoughtheflieswithInR-RNAiexpressioninesg-cellslivedonly2daysonthecontroldietorduringstarvation(Figure1E),dietconditionsof1%sucrose,1%AY,or0.5%ofbothcomponentsincreasedmeanlifespanto6,7,or9days,respectively(Figure1B–D).However,resistanceofthemodifiedflies,eitherInR-RNAiorPten-RNAigenotypes,wasinallcasessignificantlylowerascomparedtotheesg/+controlphenotype.FliesthatexpressPten-RNAiinesg-cellsexhibiteddecreasedmalnutritionresistanceonadietof1%sucrose(log-rank,p=0.02;χ2=5)(Figure1B)and1%AY(8%and33%,respectively)(log-rank,p=0.01;χ2=11)(Figure1C).However,abalancedlow-caloriediet(0.5%sucroseand0.5%AY)hadnosignificantimpactonsurvivalofesg/Pten-RNAiascomparedtoesg/+fliesandsignificantlyreducedsurvivalofesg/InR-RNAifliesby56%(Figure1D).Wealsotestedthesurvivaloftransgenicfliesunderaconditionofcompletestarvation(0.5%agarosediet).Moreprecisely,thesefliesalsolivedforonly2daysundercompletestarvation(Figure1E).Theseesg/InR-RNAifliesexhibitedasignificantdecreaseinresistancetocompletestarvationby60%comparedtoesg/+controlflies(log-rank,p<0.0001;χ2=90)(Figure1E;TableS1).Moreover,weobservedasignificantdecreaseinsurvival(8%)understarvationconditionsinfliesexpressingPten-RNAiinesg-cells(log-rank,p=0.001;χ2=10)(Figure1E).3.2.FeedingandFecundityWeinvestigatedtheconsequencesofIISmodulationinstemandprogenitorcellsonfeedingandfecundityrates,whichhasaprofoundeffectonDrosophilalifespan.Controlflies(esg/+)consumedanaverageofabout1.3µLoffoodandlaidapproximately14eggsdaily.IISinhibitioninmidgutstemandprogenitorcellsdecreasedfoodconsumptionby52%(Figure2A;p<0.05)andfecundityby74%(Figure2B;p<0.05)ascomparedtocontrol.IISpathwayactivationthroughPten-RNAiexpressioninesg-cellsresultedin43%higherfoodintakeascomparedtoesg/+flies(Figure2A;p<0.05).Moreover,thesefliesshoweda23%increaseinthedailyproductionofeggs(Figure2B;p<0.05).3.3.MetabolismInsulinsignalingplaysakeyroleinmaintainingmetabolichomeostasis.TodeterminewhethermodulationoftheIISpathwayinstemandprogenitorcellshasglobaleffectsonDrosophilametabolism,wemeasuredglucoseandglycogenlevelsinthewholebodyofflies.WefoundthatInRknockdowninISCsdecreasedthelevelofwhole-bodyglucoseby20%versuscontrols(Figure3A;p<0.05).Interestingly,trehaloselevelswithinhemolymphorinthebodywerenotaffectedbyIISmodulationinesg-cells(notshown).ExaminationofstoredfuelreservesrevealedthatInR-RNAi-expressingfliesinesg-cellscontainedsignificantlylowerglycogenlevels,inthiscasea35%decreaseascomparedtocontrols(Figure3B;p<0.05).However,therewerenoeffectsofIISmodulationinISCsonTAGstorage(FigureS2).3.4.ExpressionofDILPGenesDrosophilainsulin-likepeptides(DILPs)regulateglucosemetabolism[16],howeveracleardefinitionofthefunctionsofthevariousDILPshasnotyetbeenachieved.ThemanipulationsofIISinesg-cellsinthisstudyaffectedthesteady-statetranscriptlevelsofdilp2,dilp5,anddilp6(Figure4).Significantlyhigherrelativeexpressionofdilp2inflyheadswasfoundwhenIISwasinhibitedinISCs(by77%)(Figure4A;p<0.05).Asimilarresultwasobservedfordilp5transcriptsinflyheads,witha50%increaseindilp5expressionlevelsinInR-RNAi-expressingesg-cells(Figure4C;p<0.05).Interestingly,bothactivationandinhibitionofIISinesg-cellsledtohigherwhole-bodydilp6transcriptlevels,reachingnearly2-foldincreases(Figure4D;p<0.05).ExpressionofInR-RNAiandPten-RNAiinesg-cellsdidnotaffecttherelativeexpressionofdilp3(Figure4B).3.5.ExpressionofGenesRelatedToGlucagon-LikeSignalingandMetabolismTheadipokinetichormone(AKH)playsanimportantroleinDrosophilametabolismandservesasadeterminantofcarbohydrateandlipidlevels[17].Wefounda2-foldincreaseofakhtranscriptlevelsinwholeDrosophilabodywhenIISwasactivatedinISCsofPten-RNAiflies(Figure4E;p<0.05).Thetargetofbraininsulintobiisregulatedbybothinsulinandglucagonsignaling,anditsincreasedmRNAlevelmightcauseadecreaseinglycogencontent[18].InR-RNAiexpressioninISCsledtoa3-foldincreaseintobitranscriptlevelsinwholeDrosophilabodies(Figure4F;p<0.05).Wealsoobserveda1.5-foldincreaseintobitranscriptlevelswhenIISwasactivatedinesg-cellsexpressingPten-RNAi(Figure4F;p<0.05).DrosophilaPEPCKisinvolvedingluconeogenesisandglycerogenesis[19].Bothgeneticmanipulationsinesg-cellshadnoimpactonpepcktranscriptlevelsinwholeDrosophilabodies(Figure4G).Wealsodidnotobserveanysignificantchangesin4ebptranscriptswhenIISwasmodulatedinesg-cells(Figure4H).3.6.GutTissue-SpecificEffectsSincestemandprogenitorcellsareimportantformaintenanceofguttissuehomeostasis,weevaluatedgutintegrityandJAK–STATandEGFRsignalingpathways,whichareinvolvedinregulationofISCproliferationandEBdifferentiation[8,20].Measurementsofgutintegritywiththesmurfassay(dyediffusiontoothertissuesasaresultofgutdamage)[1]showedthatperturbationofIISdidnotaffecttissueintegrity.Inallcases,incidenceof“smurf”flieswaslowerthan7%ofthetotalflies(FigureS3).Wefoundthattranscriptlevelsofthecytokine-inducinggene,upd2,were4-foldhigherinthegutoffliesexpressingPten-RNAiinesg-cells(Figure5A;p<0.05).Similarly,transcriptlevelsofupd3alsoincreased3-foldinthegutoftheseflies(Figure5B;p<0.05).Wealsomeasuredthetranscriptlevelofsoc36,whichisatargetforJAK–STATsignaling,andfounda50%increaseinsoc36transcriptlevelsinthegutofPten-RNAi-expressingflies(Figure5C;p<0.05).NochangeintranscriptlevelsofthesethreegenesoccurredinthegutoffliesexpressingInR-RNAiinesg-cells.Hence,upd2,upd3,andsoc36displayedsimilartrendsinrelativeexpressionwithinthegroupsoftransgenicesgflies.GenetranscriptstoEGFRligands,namelyspi(Spitz),krn(Keren),andvn(Vein),werealsomeasuredintheflygut.Ourdatademonstratedsignificantincreasesinspiandvntranscriptlevels(~2.4fold)whenIISwasactivatedinesg-cells(Figure5D,F;p<0.05).Moreover,weobserved2-foldhigherrelativevnexpressioninfliesexpressingInR-RNAiinesg-cells(Figure5F;p<0.05).However,modulationofIISactivitydidnotaffectkrntranscriptlevels(Figure5E).4.DiscussionTheIISpathwayregulatesdiversephysiologicalprocessesinmulticellularorganisms,includinggrowth,reproduction,metabolism,andlongevity[16].Reducedexpressionoftheinsulinreceptor[21]andPtenoverexpression[22]bothhavebeenshowntoextendDrosophilalifespan.Similareffectswereobservedwheninsulin-producingcellswereablated[16].TheseresultssuggestanimportantroleofIISinaging.Inaddition,loss-of-functiongeneticstudiesinCaenorhabditiseleganshaveshownthatthenematodeinsulinreceptor,daf-2,prolongedlifespan[23].Inmice,deletionofInRinwhiteadiposetissuealsoincreasedlongevity[24].WeusedInR-RNAiexpressiontoinhibitIISinfliesandPten-RNAiexpressiontofunctionallyactivateIISbydecreasingPTENphosphatasemRNAlevelsinstemandprogenitorcellsoffruitflygut(Figure6).Table1summarizesallresultsofthisstudyinordertocompareactivationversusinhibitionofIIS.OurstudyrevealedthatInRknockdowninstemandprogenitorcellscauseddetrimentaleffectsonflysurvival,supportingthehypothesisthatIISplaysanimportantroleinthenormalfunctionsofstemcells.SinceIISplaysdifferentrolesindifferenttissues,theresultingphenotypesfrommodulatedIISactivitycanbedifferent.Weproposethataffectingtheinsulinreceptorinstemandprogenitorcellsmayhavewholeorganismaleffects.Whentheinsulinreceptorisknockeddown,stemcellssignaltoothercelltypesinthegut,suchasenterocytesandenteroendocrinecells.Signalingtothesecellsmayreducenutrientuptakeinthegut,suggestingstarvation.Inhibitionofstemcellactivitymayalsoinducelossoftissueintegrity[8].Importantly,manipulationsofIISinbothcelltypesdidnotinducedamagetoguttissue.Thesmurfassayshowednochangeinthepercentageofflieswithdamaged(leaky)gutsincontrolversusexperimentalgroups(FigureS3).AnabsenceofeffectsongutintegritywheninsulinsignalingwasdisruptedmightbeexplainedbychangesinJAK–STATandEGFRsignaling.Theunpaired(upd)2and3proteinsactivatetheJAK–STATpathwayactivityinISCs[20].Toevaluatethemechanismsinvolvedinmaintenanceofguthomeostasis,wetestedthepossibleinvolvementoftheJAK–STATandEGFRpathways.ThetwopathwaysregulateISCproliferation[21](Figure6).Upd2andupd3expressioninenterocytes,enteroblasts,orstemcellsintheDrosophilagutleadtoenhancedISCproliferation,whichinturncauseshyperplasia[21].EGFRsignalingisnecessaryforISCproliferation[22].WetestedtherelativeexpressionofgenesencodingligandsforJAK–STAT(upd2,upd3)anditstargetgene(soc36),aswellasligandsforEGFreceptor(spi,krn,vn)signalingpathways.Ourdatademonstrateahighlysignificantincreaseinupd2andupd3transcriptswhenIISwasactivatedasaresultofPTENsuppression,indicatingthatIIScanactivateJAK–STATsignalinginthegut.Moreover,increasedrelativeexpressionofsoc36inPten-RNAiexpressingfliesconfirmstheinterdependencebetweenIISandJAK–STAT.TheEGFRpathwayactivitymaybeactivatedintheadultintestinebytheligandsVein(Vn),Spitz(Spi),andKeren(Krn),whichareabletobindtotheEGFreceptor[25].KerenisproducedbyECs[26],butneitherinhibitionnoractivationofIISinISCsaffectedkrnexpression.Furthermore,IISactivationincreasedspi,whichwasdetectedinEBs,aswellasvnexpression,whichwasdetectedinbothECsandvisceralmuscle[26].ThesedatasuggestthatmanipulationsofinsulinsignalinginstemandprogenitorcellsaffectothercelltypesandinvolvesignalingspecificallylinkedtoEGFRandJAK–STAT.ManipulationswithIISinmidgutstemandprogenitorcellsaffectedflylifespan,aswellassensitivitytostarvationandmalnutrition.Weobservedrapidmortalitywithin2daysinflieswithInRknockdowninstemandprogenitorcells.OurresultsareconsistentwiththestudyofBiteauandcolleagues,whichshowedanimpactofIISonstemandprogenitorcells,shorteninglifespanwhenIISwasinhibitedinbothcelltypes[8].However,bycontrast,ourdatademonstratedgreatlyshortenedlifespanonlywhenIISwasinhibitedinISCs.Moreover,thesetransgenicflieswereshort-livedundercompletestarvation.Theseeffectsmaybeexplainedbyareducedgutsize,whichhasbeenpreviouslyshowntobeduetoadecreaseininsulinsignaling[3].Reducedgutsizealsoexplainsthedecreaseinfeedingbymorethan50%,aswellasthereductioninbodyfuelreservesofglucoseandglycogen.Inaddition,thesefliesshowedincreasedtranscriptionofdilp2,5,and6withincreasedinsulinsignalingtoperipheraltissues,asindicatedbytheinductionoftobi.Wealsoobservedanincreaseindilp6transcriptlevelsinflieswithactivatedIISinesg-cells.Mutationsofdilp6significantlyreducedflyfecundity[27],suggestingthatup-regulateddilp6canincreaseflyfecundity[28].Inaddition,increasedfooduptakemaycontributetoincreasedfecunditybysupplyingmorenutrientsforeggproduction.ThismaybebecausetheesgdrivermightbeexpressedintheotherDrosophilatissues.Indeed,accordingtoFlyAtlas,theesgdriverisalsoexpressedinflytestis.Consequently,therearesomepotentialcontributionsfromothercellsandtissuestothesystemicassaysperformed.Onemorecontributortotheseresultsmaybetheup-regulationofthePEPCKtranscript,animportantenzymeformaintainingthebalancebetweencatabolicandanabolicprocesses[29].InductionofpepcktranscriptioncouldbestimulatedbythestronginductionofAKHsignalingasaresultofthe2-foldincreaseinakhtranscriptlevels.Interestingly,malnutritionpartiallyrescuedthelongevityphenotypeinflieswithdecreasedIISinstemandprogenitorcells.Underanormaldiet,themeanlifespanofflieswithinhibitedIISwasabouttwodays,howeverdietwithonly1%sucroseenabledfliestoliveupto6days.Inaddition,fliesfedthelowproteindiet(1%AY)lived7daysandthosefedlow-carbohydratelow-proteinfood(0.5%sucroseand0.5%AY)livedupto9days(Figure7).TheseresultsclearlyshowthatIISplaysakeyroleinmediatingtheeffectofdietaryregimensonoverallphysiology.Differentmechanismsandpathwaysareinvolvedindetectingtheintracellularandextracellularlevelsofmacronutrients,includingtheTORsignalingpathway,whichhasasignificantimpactonlifespan[30,31].Moreover,wehavepreviouslydemonstratedthatTORsignalinginhibition,whichactsinastronginteractionwithIIS,inDrosophilaISCs-EBscanhaveimportantdeleteriousconsequencesforregenerativecapacity,whichinturndecreasesresistancetomalnutrition[32].However,ourresultsshowedthatTORsignalingactivationthroughrhebexpressioninDrosophilaISCsandEBsincreaseslifeexpectancyunderconditionsofmalnutrition(1S,1Y),whichsuggestedthatactivationofTORsignalingcanoffsetmalnutritionasastressfulagent[15].ContrastingphenotypesforIISactivationandinhibitioninesgcellswereobservedforfecundityandfeeding.BothparameterswerereducedasaresultofdecreasedIIS,butwereincreasedwhenIISwasactivated.However,inthecaseofdecreasedmalnutrition(1%sucrose)resistanceandenhanceddilp6transcriptlevels,wefoundthesameeffectsundereitherIISactivationorinhibition.Manyparameters,suchaslifespan,bodyglucose,andglycogenlevels,transcriptsofdilp2,dilp5,andtobiwereaffectedonlybyareductionofIIS.However,gut-specificincreasesinmRNAtranscriptlevelsofupd2,upd3,soc36,spi,andkrnwereseenonlyinflieswithactivatedIISinesgcells(Table1).Thus,currentdataprovideevidenceaboutthesignificanceofcooperationbetweenmultiplesignalingpathways,includingJAK–STAT,downstreamofEGFRandinsulinsignalingforregulationofstemcellmaintenanceandactivity.5.ConclusionsAltogether,theworkdescribedhererevealstheimportanceofinsulinsignalinginmidgutstemandprogenitorcells.Activationorinhibitionoftheinsulinsignalingpathwayinfluencedflyphysiologyandmetabolism,andaffectedtranscriptionofgenesinvolvedintheregulationofmetabolism,guthomeostasis,andsurvival,bothinthewholebodyandinthegutitself.Theseresultsclearlydemonstratethepossibilitytomodulatevariousflytraitsthroughinteractionswithinsulinsignalinginthegutstemandprogenitorcells. SupplementaryMaterialsThefollowingareavailableonlineathttps://www.mdpi.com/2073-4409/9/4/803/s1,FigureS1:RelativeInRandPtenexpressioninflieswithIISinhibition/activationinwholeDrosophilabody,FigureS2:AmountofTAGofflieswithIISmodulatedinstemandprogenitorcells,FigureS3:Numberof“smurf”flies,anindicatorofgutintegrity,incontrol,InR-RNAi-andPten-RNAi-expressingflies,TableS1:Statisticalanalysisoflifespanandstressresistanceoftheexperimentalflieswithinhibited/activatedIISinISCsandEBs,TableS2:PrimerpairsusedforqRT-PCR.AuthorContributionsConceptualization,O.L.andO.M.S.;methodology,O.L.;validation,O.L.andK.B.S.;formalanalysis,O.M.S.;investigation,O.M.S.,U.V.S.,andI.S.Y.;datacuration,O.L.;writing—originaldraftpreparation,O.M.S.;writing—reviewandediting,O.L.andK.B.S.;visualization,O.M.S.;supervisionandprojectadministration,O.L.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.FundingThisworkwaspartiallysupportedbyadiscoverygrantfromtheNaturalSciencesandEngineeringResearchCouncilofCanada(#6793)toKennethB.Storey.TheauthorswouldliketoacknowledgetheUnitedStatesDepartmentofDefense,DefenseThreatReductionAgency(DTRA),andBiologicalThreatReductionProgram(BTRP)fortheirassistanceandfinancialsupportinthepublicationofthispaper.WhileDTRA/BTRPdidnotsupporttheresearchdescribedinthispublication,theprogramsupportedthemanuscriptpublicationeffort.ThecontentofthispublicationistheresponsibilityoftheauthorsanddonotnecessarilyreflecttheviewsofDTRAortheUnitedStatesGovernment.AcknowledgmentsTheauthorsarethankfultoLuisDaSilva(MetabiotaInc.,USA)forhismentorshipindevelopingthismanuscript.ConflictsofInterestTheauthorsdeclarenoconflictofinterest.ReferencesRera,M.;Clark,R.I.;Walker,D.W.IntestinalbarrierdysfunctionlinksmetabolicandinflammatorymarkersofagingtodeathinDrosophila.Proc.Natl.Acad.Sci.USA2012,109,21528–21533.[GoogleScholar][CrossRef][PubMed]Ohlstein,B.;Spradling,A.MultipotentDrosophilaintestinalstemcellsspecifydaughtercellfatesbydifferentialnotchsignaling.Science2007,315,988–992.[GoogleScholar][CrossRef][PubMed]O’Brien,L.E.;Soliman,S.S.;Li,X.;Bilder,D.Alteredmodesofstemcelldivisiondriveadaptiveintestinalgrowth.Cell2011,147,147–603.[GoogleScholar][CrossRef]Zeng,X.;Han,L.;Singh,S.R.;Liu,H.;Neumüller,R.A.;Yan,D.;Hu,Y.;Liu,Y.;Liu,W.;Lin,X.;etal.Genome-wideRNAiscreenidentifiesnetworksinvolvedinintestinalstemcellregulationinDrosophila.CellRep.2015,10,1226–1238.[GoogleScholar][CrossRef][PubMed]Amcheslavsky,A.;Jiang,J.;Ip,Y.T.Tissuedamage-inducedintestinalstemcelldivisioninDrosophila.CellStemCell2009,4,49–61.[GoogleScholar][CrossRef][PubMed]Choi,N.H.;Lucchetta,E.;Ohlstein,B.NonautonomousregulationofDrosophilamidgutstemcellproliferationbytheinsulin-signalingpathway.Proc.Natl.Acad.Sci.USA2011,108,18702–18707.[GoogleScholar][CrossRef]McLeod,C.J.;Wang,L.;Wong,C.;Jones,D.L.Stemcelldynamicsinresponsetonutrientavailability.Curr.Biol.2010,20,2100–2105.[GoogleScholar][CrossRef]Biteau,B.;Karpac,J.;Supoyo,S.;Degennaro,M.;Lehmann,R.;Jasper,H.LifespanextensionbypreservingproliferativehomeostasisinDrosophila.PLoSGenet.2010,6,e1001159.[GoogleScholar][CrossRef]Brand,A.H.;Perrimon,N.Targetedgeneexpressionasameansofalteringcellfatesandgeneratingdominantphenotypes.Development1993,118,401–415.[GoogleScholar]Lushchak,O.V.;Gospodaryov,D.V.;Rovenko,B.M.;Glovyak,A.D.;Yurkevych,I.S.;Klyuba,V.P.;Scherbij,M.V.;Lushchak,V.I.BalancebetweenmacronutrientsaffectslifespanandfunctionalsenescenceinfruitflyDrosophilamelanogaster.J.Gerontol.ABiol.Sci.Med.Sci.2012,67,118–125.[GoogleScholar][CrossRef]Lushchak,O.V.;Gospodaryov,D.V.;Rovenko,B.M.;Yurkevych,I.S.;Perkhulyn,N.V.;Lushchak,V.I.SpecificdietarycarbohydratesdifferentiallyinfluencethelifespanandfecundityofDrosophilamelanogaster.J.Gerontol.ABiol.Sci.Med.Sci.2014,69,3–12.[GoogleScholar][CrossRef][PubMed]Rovenko,B.M.;Kubrak,O.I.;Gospodaryov,D.V.;Perkhulyn,N.V.;Yurkevych,I.S.;Sanz,A.;Lushchak,O.V.;Lushchak,V.I.HighsucroseconsumptionpromotesobesitywhereasitslowconsumptioninducesoxidativestressinDrosophilamelanogaster.J.InsectPhysiol.2015,7,42–54.[GoogleScholar][CrossRef][PubMed]Rovenko,B.M.;Perkhulyn,N.V.;Lushchak,O.;Storey,J.M.;Storey,K.B.;Lushchak,V.I.Molybdatepartlymimicsinsulin-promotedmetaboliceffectsinDrosophilamelanogaster.Comp.Biochem.Physiol.CToxicol.Pharmacol.2014,165,76–82.[GoogleScholar][CrossRef][PubMed]Lushchak,O.V.;Carlsson,M.A.;Nässel,D.R.Foododorstriggeranendocrineresponsethataffectsfoodingestionandmetabolism.Cell.Mol.LifeSci.2015,72,3143–3155.[GoogleScholar][CrossRef][PubMed]Strilbytska,O.M.;Semaniuk,U.V.;Storey,K.B.;Edgar,B.A.;Lushchak,O.V.ActivationoftheTor/Mycsignalingaxisinintestinalstemandprogenitorcellsaffectslongevity,stressresistanceandmetabolisminDrosophila.Comp.Biochem.Physiol.BBiochem.Mol.Biol.2017,203,92–99.[GoogleScholar][CrossRef][PubMed]Broughton,S.J.;Piper,M.D.;Ikeya,T.;Bass,T.M.;Jacobson,J.;Driege,Y.;Martinez,P.;Hafen,E.;Withers,D.J.;Leevers,S.J.;etal.Longerlifespan,alteredmetabolism,andstressresistanceinDrosophilafromablationofcellsmakinginsulin-likeligands.Proc.Natl.Acad.Sci.USA2005,102,310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Figure1. Survivalcurvesfortheexperimentalflieswithinsulin–IGF-1signaling(IIS)manipulatedinstemandprogenitorcellsundervariousdietaryconditions:A—standardmedium(5%S+5%Y);B—lowcarbohydratediet(1%S);C—lowproteindiet(1%AY);D—balancedmalnutritiondiet(0.5%S+0.5%AY);E—completestarvation(0.5%agarose).Thereadoutoftheexperimentaldaysstartsinitiallyaftersixdaysofexpressioninductionat29°C.Eachcurverepresentsthepercentageofindividualsalive.Thegenotypeswerecomparedusinglog-ranktest(seeTableS1forcompletestatistics). Figure1. Survivalcurvesfortheexperimentalflieswithinsulin–IGF-1signaling(IIS)manipulatedinstemandprogenitorcellsundervariousdietaryconditions:A—standardmedium(5%S+5%Y);B—lowcarbohydratediet(1%S);C—lowproteindiet(1%AY);D—balancedmalnutritiondiet(0.5%S+0.5%AY);E—completestarvation(0.5%agarose).Thereadoutoftheexperimentaldaysstartsinitiallyaftersixdaysofexpressioninductionat29°C.Eachcurverepresentsthepercentageofindividualsalive.Thegenotypeswerecomparedusinglog-ranktest(seeTableS1forcompletestatistics). Figure2. Dailyfoodconsumption(A)andnumberofeggslaiddailyperfemale(B)forflieswithIIS,eitheractivatedorinhibitedinesg-cells.Resultsarepresentedasmean±SEM,with10–20fliestestedpergenotype.Theexperimentsstartedaftersixdaysofexpressioninductionat29°Candwereperformedoverfourdays.Duetotheirreducedlifespans,esc/InR-RNAigenotypefliesweretestedones;thus,barspresentresultsofsingle-daymeasurements.GroupcomparisonswereperformedusingANOVAfollowedbyNewman–Keulsposthoctest.Asterisksindicatesignificantdifferencefromcontrolflies(esg/+)(p<0.05);hashsignsshowsignificantdifferencefromflieswithinhibitedIIS(esg/InR-RNAi). Figure2. Dailyfoodconsumption(A)andnumberofeggslaiddailyperfemale(B)forflieswithIIS,eitheractivatedorinhibitedinesg-cells.Resultsarepresentedasmean±SEM,with10–20fliestestedpergenotype.Theexperimentsstartedaftersixdaysofexpressioninductionat29°Candwereperformedoverfourdays.Duetotheirreducedlifespans,esc/InR-RNAigenotypefliesweretestedones;thus,barspresentresultsofsingle-daymeasurements.GroupcomparisonswereperformedusingANOVAfollowedbyNewman–Keulsposthoctest.Asterisksindicatesignificantdifferencefromcontrolflies(esg/+)(p<0.05);hashsignsshowsignificantdifferencefromflieswithinhibitedIIS(esg/InR-RNAi). Figure3. Bodyglucosecontents(A)andglycogenamounts(B)inflieswithIISmodulatedinstemandprogenitorcells.Flieswereusedaftersixdaysofexpressioninductionbyflytransferto29°C.Resultsrepresentthemean±SEMof4or5replicatespergenotype.GroupcomparisonswereperformedusingANOVAfollowedbyNewman–Keulsposthoctest.Asterisksindicatesignificantdifferencefromthecontrolflies(esg/+)(p<0.05);hashsignshowssignificantdifferencefromflieswithinhibitedIIS(esg/InR-RNAi). Figure3. Bodyglucosecontents(A)andglycogenamounts(B)inflieswithIISmodulatedinstemandprogenitorcells.Flieswereusedaftersixdaysofexpressioninductionbyflytransferto29°C.Resultsrepresentthemean±SEMof4or5replicatespergenotype.GroupcomparisonswereperformedusingANOVAfollowedbyNewman–Keulsposthoctest.Asterisksindicatesignificantdifferencefromthecontrolflies(esg/+)(p<0.05);hashsignshowssignificantdifferencefromflieswithinhibitedIIS(esg/InR-RNAi). Figure4. ThemRNAlevelsfordilp2(A),dilp3(B),anddilp5(C)inflyheadsanddilp6(D)fromwholeflybodiesandtranscriptlevelsofgenesrelatedtoglucagon-likesignalingandmetabolism:akh(E),tobi(F),pepck(G),and4ebp(H)fromwholeflybodies.Parametersweremeasuredinflieskeptat29°CforsixdaysforRNAiexpressioninduction.Dataaremeanvaluesfor4independentmeasurements(±SEM).GroupcomparisonswereperformedusingANOVAfollowedbyNewman–Keulsposthoctest.Asterisksindicatesignificantdifferencefromthecontrolflies(esg/+)(p<0.05);hashsignsshowsignificantdifferencefromflieswithinhibitedIIS(esg/InR-RNAi). Figure4. ThemRNAlevelsfordilp2(A),dilp3(B),anddilp5(C)inflyheadsanddilp6(D)fromwholeflybodiesandtranscriptlevelsofgenesrelatedtoglucagon-likesignalingandmetabolism:akh(E),tobi(F),pepck(G),and4ebp(H)fromwholeflybodies.Parametersweremeasuredinflieskeptat29°CforsixdaysforRNAiexpressioninduction.Dataaremeanvaluesfor4independentmeasurements(±SEM).GroupcomparisonswereperformedusingANOVAfollowedbyNewman–Keulsposthoctest.Asterisksindicatesignificantdifferencefromthecontrolflies(esg/+)(p<0.05);hashsignsshowsignificantdifferencefromflieswithinhibitedIIS(esg/InR-RNAi). Figure5. ExpressionofgenesrelatedtoJAK–STATandligandsforEGFRinthegut.Transcriptionallevelsofcytokine-inducinggenesupd2(A),upd3(B),andthedownstreamtargetsoc36(C)weremeasured,aswellasthoseforspi(D),krn(E),andvn(F).Fliesweretestedaftersixdaysafterexpressioninductionat29°C.Datarepresentmeanvaluesfor4independentmeasurements(±SEM).GroupcomparisonswereperformedusingANOVAfollowedbyNewman–Keulsposthoctest.Asterisksindicatesignificantdifferencefromthecontrolflies(esg/+)(p<0.05);hashsignsshowsignificantdifferencefromflieswithinhibitedIIS(esg/InR-RNAi). Figure5. ExpressionofgenesrelatedtoJAK–STATandligandsforEGFRinthegut.Transcriptionallevelsofcytokine-inducinggenesupd2(A),upd3(B),andthedownstreamtargetsoc36(C)weremeasured,aswellasthoseforspi(D),krn(E),andvn(F).Fliesweretestedaftersixdaysafterexpressioninductionat29°C.Datarepresentmeanvaluesfor4independentmeasurements(±SEM).GroupcomparisonswereperformedusingANOVAfollowedbyNewman–Keulsposthoctest.Asterisksindicatesignificantdifferencefromthecontrolflies(esg/+)(p<0.05);hashsignsshowsignificantdifferencefromflieswithinhibitedIIS(esg/InR-RNAi). Figure6. ThedigestivetractofDrosophilamelanogasterandnicheintestinalstemcell(ISC)ofmidgutepithelium.IISwasmanipulatedinISCsandEBsintheDrosophilamidgut,andregulatedISCproliferationandEBdifferentiation.ECsproducesignalingligandsUpd2andUpd3forJAK–STATactivationinISCs,EBs,andvisceralmuscles.JAK–STATinvisceralmusclesactivatesVeinsecretion,whichtogetherwithSpitzandKerenmaybindEGFRinISCs.JAK–STAT,EGFR,andlocalIISregulateISCproliferationandEBdifferentiation. Figure6. ThedigestivetractofDrosophilamelanogasterandnicheintestinalstemcell(ISC)ofmidgutepithelium.IISwasmanipulatedinISCsandEBsintheDrosophilamidgut,andregulatedISCproliferationandEBdifferentiation.ECsproducesignalingligandsUpd2andUpd3forJAK–STATactivationinISCs,EBs,andvisceralmuscles.JAK–STATinvisceralmusclesactivatesVeinsecretion,whichtogetherwithSpitzandKerenmaybindEGFRinISCs.JAK–STAT,EGFR,andlocalIISregulateISCproliferationandEBdifferentiation. Figure7. MeanlifespanofflieswithinhibitionofIISinISCs(esg/InR-RNAi)underdifferentdietaryregimens.Asterisksshowasignificantdifferencefromthestandarddietarycondition(5%S+5%Y)withp<0.05. Figure7. MeanlifespanofflieswithinhibitionofIISinISCs(esg/InR-RNAi)underdifferentdietaryregimens.Asterisksshowasignificantdifferencefromthestandarddietarycondition(5%S+5%Y)withp<0.05. Table1. SummaryofISmodulationinISCsandEBsontheresponsesofflyphysiological,metabolic,andgeneexpressionparameters. Table1. SummaryofISmodulationinISCsandEBsontheresponsesofflyphysiological,metabolic,andgeneexpressionparameters. ParametersInhibitionActivationLifespanStarvationresistanceMalnutritionresistance1%sucroseMalnutritionresistance1%autolyzedyeastMalnutritionresistance0.5%Sand0.5%AYFeeding,FecundityBodyglucose,glycogendilp2,dilp5,tobidilp6akhupd2,upd3,soc36spivn ©2020bytheauthors.LicenseeMDPI,Basel,Switzerland.ThisarticleisanopenaccessarticledistributedunderthetermsandconditionsoftheCreativeCommonsAttribution(CCBY)license(http://creativecommons.org/licenses/by/4.0/). Cells, EISSN2073-4409, PublishedbyMDPI Disclaimer Thestatements,opinionsanddatacontainedinthejournalCellsaresolely thoseoftheindividualauthorsandcontributorsandnotofthepublisherandtheeditor(s). MDPIstaysneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. 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