AusdemMax-Planck-InstitutfürPsychiatrie

DirektorderKlinikundChefarzt:Prof.Dr.Dr.MartinE.Keck

GhrelinandCognition

DissertationzumErwerbdesDoktorgradesderMedizin

anderMedizinischenFakultätderLudwig-Maximilians-UniversitätzuMünchen

vorgelegtvon

NicolasKunath

aus

Würzburg

Jahr

2017

2

MitGenehmigungderMedizinischenFakultätderUniversitätMünchen

Berichterstatter:Prof.Dr.AxelSteiger

Mitberichterstatter:Priv.Doz.Dr.ChristophHaffner

Prof.Dr.MichaelKiebler

Dekan:Prof.Dr.med.dent.ReinhardHickel

TagdermündlichenPrüfung:04.05.2017

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Tableofcontents

1.Formalstatements......................................................................................................................51.1Affidavit.................................................................................................................................................51.2Abbreviations......................................................................................................................................61.3Listofpublications............................................................................................................................71.3.1GhrelinagonistdoesnotfosterinsulinresistancebutimprovescognitioninanAlzheimer’sdiseasemousemodel.....................................................................................................................71.3.2Ghrelinaltersencoding-relatedbrainactivitywithoutenhancingmemoryformationinhumans.....................................................................................................................................................................7

1.4ConfirmationsofCo-Authors.........................................................................................................81.4.1Co-authorstopublication1.......................................................................................................................81.4.2Co-authorsforpublication2.....................................................................................................................8

2.Summary........................................................................................................................................92.1Zusammenfassung(deutsch).........................................................................................................92.1.1Hintergrund......................................................................................................................................................92.1.2Studie1............................................................................................................................................................112.1.3Studie2............................................................................................................................................................12

2.2Summary(English)..........................................................................................................................142.2.1Background...................................................................................................................................................142.2.2Study1.............................................................................................................................................................162.2.3Study2.............................................................................................................................................................17

3.Introduction...............................................................................................................................193.1Foodforthought:Cognitiveaspectsoffeeding......................................................................193.2Thoughtforfood:thecomplexityofenergyhomeostasis..................................................193.3Thestoryofghrelin:ahungerhormonewithatasteformemoryenhancement?....213.4Doesghrelinactasacognitiveenhancerinyoung,healthy,malehumans?...............233.5Themethodologicalchallengeofdesigningasuitableparadigmforthequestion...243.6Neurodegenerationandtheroleofinsulin,glucose–andghrelin?...............................283.7Chronicdisease,chronicadministration:Theideabehindpublication1...................29

4.Conclusions................................................................................................................................314.1Publication1:Surprisingresultsinlong-termghrelinagonisttreatment..................314.2Publication2:Whatghrelindoesnotdo..................................................................................33

5.Publication1..............................................................................................................................365.1Abstract...............................................................................................................................................375.2Introduction......................................................................................................................................385.3Results.................................................................................................................................................395.3.1Ghrelinagonistactsasalong-termcognitiveenhancerinspatiallearning......................395.3.2GhrelinagonistdoesnotsignificantlyaffectAβplaqueloadormicrogliaactivation415.3.3Long-termghrelinagonisttreatmentleadstolessweightgain,lessoverallfoodconsumption,andmoreactivity......................................................................................................................435.3.4Long-termghrelinagonisttreatmentdoesnotimpairglucosetolerance.........................465.3.5Ghrelinagonisttreatmentbeneficiallyinfluencescentralinsulinsignalingpathway..49

5.4Discussion..........................................................................................................................................515.5Methods...............................................................................................................................................545.5.1Ethicsstatement..........................................................................................................................................545.5.2Animals,diets,andtreatment................................................................................................................545.5.3Behavioralandcognitiveassessments..............................................................................................555.5.4Openfieldtest..............................................................................................................................................555.5.5Watermaze....................................................................................................................................................555.5.6Zeromaze.......................................................................................................................................................56

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5.5.7Light-dark-box.............................................................................................................................................565.5.8Immunohistochemistry............................................................................................................................575.5.9Oralglucosetolerancetest.....................................................................................................................585.5.10ProteinextractionandWesternblotting.......................................................................................585.5.11Bloodsamples............................................................................................................................................595.5.12Quantitativemagneticresonanceimaging....................................................................................605.5.13Metaboliccages.........................................................................................................................................605.5.14Activitymeasurements..........................................................................................................................605.5.15Statisticalmethods..................................................................................................................................61

5.6Acknowledgements.........................................................................................................................615.7Authorcontributions......................................................................................................................625.8AdditionalInformation..................................................................................................................62

6.Publication2..............................................................................................................................636.1Abstract...............................................................................................................................................646.2Highlights...........................................................................................................................................656.3Introduction......................................................................................................................................656.4MaterialsandMethods..................................................................................................................696.4.1Participants...................................................................................................................................................696.4.2Experimentaldesignandprocedures................................................................................................706.4.3Cognitivetesting..........................................................................................................................................726.4.4Statisticalanalysis......................................................................................................................................746.4.5fMRIdataacquisition................................................................................................................................746.4.6fMRIdataanalysis......................................................................................................................................756.4.7Restingstatepreprocessing...................................................................................................................766.4.8ROIbasedanalysis......................................................................................................................................77

6.5Results.................................................................................................................................................816.6Discussion..........................................................................................................................................846.7Supplementaldata..........................................................................................................................90

7.References..................................................................................................................................968.Acknowledgements...............................................................................................................104

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Name,Vorname

1.Formalstatements

1.1Affidavit

EidesstattlicheVersicherungKunath,NicolasIcherklärehiermitanEidesstatt,dassichdievorliegendeDissertationmitdemThema

„GhrelinandCognition“selbständigverfasst,michaußerderangegebenenkeinerweiterenHilfsmittelbedientundalleErkenntnisse,dieausdemSchrifttumganzoderannäherndübernommensind,alssolchekenntlichgemachtundnachihrerHerkunftunterBezeichnungderFundstelleeinzelnnachgewiesenhabe.IcherkläredesWeiteren,dassdiehiervorgelegteDissertationnichtingleicheroderinähnlicherFormbeieineranderenStellezurErlangungeinesakademischenGradeseingereichtwurde.

Ort,DatumUnterschriftdesDoktoranden

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1.2AbbreviationsAEBSF 4-(2-Aminoethyl)benzensulfonylfluoridANOVA AnalysisofVarianceAUC AreaunderthecurveAβ AmyloidBetaBET Brainextractiontool BOLD BloodoxygenleveldependentBOMAT BochumerMatritzentestCNS CentralNervousSystemCOG CenterofgravityCREB cAMPresponsiveelement-binding(protein)DAB 3,3'-DiaminobenzidineDCX DoublecortinDPX Distrene,Plasticiser,XyleneEGTA Ethyleneglycol-bis-N,N,N',N'-tetraaceticacidfMRI FunctionalmagneticresonanceimagingG-Protein GTPbindingproteinGH GrowthhormoneGHS-R GrowthhormonesecretagoguereceptorGI GlycemicindexGLM GenerallinearmodelIACUC InstitutionalAnimalCareandUseCommittee IBA IbandronateICV IntracerebroventricularJNK JanuskinaseMCFLIRT MotioncorrectionFMRIB'sLinearImageRegistrationToolMWT-B Mehrfachwahl-Wortschatz-IntelligenztestVersionBNIH NationalinstituteofhealthNMDA N-Methyl-D-aspartate(p-)IRS (phosphorylated)insulinreceptorsubstratePET PositronemissiontomographySAP Stress-activatedphospho(-kinase)PSD Post-synapticdensityPVT PsychomotorvigilancetaskQMR Quantitativemagnetigresonance(imaging)RDS ReversedigitspanRIPA RadioimmunoprecipitationassayROI RegionofinterestSD StandarddeviationSEM StandarderrorofthemeanTE EchotimeTgAPPSwDI TransgenicamyloidprecursorproteinSwedish-Dutch-IowaTNF TumornecrosisfactorTR RelaxationtimeTris Tris(hydroxymethyl)aminomethaneZVT Zahlenverbindungstest

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1.3Listofpublications

1.3.1GhrelinagonistdoesnotfosterinsulinresistancebutimprovescognitioninanAlzheimer’sdiseasemousemodelPublishedin: ScientificReportsDateofpublication: 19thofJune2015JournalImpactFactor: 5.578(ThomsonReuters2014)

1.3.2Ghrelinaltersencoding-relatedbrainactivitywithoutenhancingmemoryformationinhumans

Publishedin: NeuroImageDateofacceptance: 7thofJuly2016JournalImpactFactor: 5,463(ThomsonReuters2016)

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1.4ConfirmationsofCo-AuthorsAllco-authorssignedtheconfirmationpursuantto§4aParas.3and5DoctoralDegreeRegulationsforDr.med.,Dr.med.dent.andDr.rer.biol.hum.andpursuantto§7Para.4DoctoralDegreeRegulationsforDr.rernat.attheMedicalFaculty.Thesignaturesarelistedinthesection“Appendix”.

1.4.1Co-authorstopublication1ThomasvanGroen DavidB.Allison AshishKumar MoniqueDozier-Sharpe IngaKadish

1.4.2Co-authorsforpublication2NilsMüller

MatthiasTonon

BorisN.Konrad

MarcelPawlowski

AnnaKopczak

ImmanuelElbau

MartinUhr

SimoneKühn

DimitrisRepantis

KathrinOhla

TimoMüller

GuillénFernández

MatthiasTschöp

MichaelCzisch

AxelSteiger

MartinDresler

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2.Summary

2.1Zusammenfassung(deutsch)

2.1.1Hintergrund

GhrelinisteinPeptidmiteinerLängevon28Aminosäurenundbesitztinseiner

aktivenFormeinecharakteristischeAcyl-Seitenkette,diedurchdasEnzym„Ghrelin-O-

Acyltransferase“amdrittenSerin-Restangefügtwird.Eswurde1999ineiner

japanischenArbeitsgruppealsLiganddes“GrowthHormoneSecretagogue”-Rezeptors

entdeckt.DerName„Ghrelin“istsowohlAkronym(growthhormonereleaseinducing)

alsauchAnspielungaufdieproto-indo-europäischeWortwurzel“ghre”für“wachsen”.

AußerseinerRolleinderFreisetzungvonWachstumshormonistGhrelindas

einzigebislangbekannteperiphereorexigenePeptidhormonundscheintfester

BestandteilzirkadianerRhythmenderNahrungsaufnahmezusein.DerRezeptorfindet

sichanvielenOrteninSäugetierorganismenundbesitztzudemdieEigenschaft,Dimere

mitRezeptorenandererTransmittersystemewiez.B.jenesdesSerotoninsoderdes

Dopaminszubilden.DieszeigtdiebreiteRelevanzGhrelinsodervielmehrdes„Ghrelin-

Systems“,dasmannigfachaufunserenEnergiehaushaltauskognitivergenausowie

metabolischerStoßrichtungeinzuwirkenscheint–soferndieseTrennungüberhauptaus

demBlickwinkelderGhrelin-Forschungvertretbarist:Sehrbaldlegten

Forschungergebnissenahe,dassdieBedeutungdiesesPeptidsweitüberdiereine

RegulationdesEnergiehaushaltshinausgeht.

EsscheintvielmehrauchkomplexekognitiveProzessezubeeinflussen.An

Nagetiermodellenkonntekonsistentgezeigtwerden,dassGhrelindie

Gedächtnisbildungunterstützt,allenvoranindenBereichenObjekterkennung,

räumlichesLernenundaversivesGedächtnis.EswirddaheralseinemöglicheBrücke

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zwischenEnergiehaushaltundKognitiondiskutiertunderfülltdaher,bildlich

ausgedrückt,inmancherHinsichtdieRolleeines„Eichhörnchenhormons“:DieseTiere

erinnernsichinZeitendesFastensmehroderminderpräziseandieOrte,andenen

Nahrungzuvorverstecktwurde.InderTatzeigtenjüngsteProjekteanWildtierendieser

GattungeinemöglicheRelevanzGhrelinsimStoffwechseldieserTiereauf.Im

angelsächsischenSprachgebrauchistesvorallemdasBilddes„belly-brain-links“,das

dengleichenSachverhaltverdeutlichensoll.Nunverbietetsichallerdingsdiedirekte

ExtrapolationdieserDatenausNagetiermodellenundHörnchenaufdenMenschen

alleinschonwegenderungleichkomplexerenZusammenhängemenschlicherKognition.

DennochstelltsichdieFrage,obGhrelinmöglicherweiseeinenebensopositiven

EinflussaufkognitiveProzessedesMenschenhat–einSachverhaltmitmöglicher

RelevanzimVerständnissowohlderEntwicklungvonÜbergewichtalsauchAnorexie.

ZwarbestandinderArbeitsgruppeProf.AxelSteiger(PrincipalInvestigatorderStudie

2)bereitsvorStudie2einemehrjährigeErfahrungmitderGabevonGhrelinbei

menschlichenProbandenunterFragestellungenderSchlafforschungimAllgemeinen

undSchlafendokrinologieimSpeziellen.EineUntersuchungderAuswirkungenGhrelins

aufdiemenschlicheGedächtnisleistungwarjedochunseresWissensnachzuvornoch

nieerfolgt.DamitstelltStudie2dieersteStudieihrerArtdar,diediemenschliche

kognitiveLeistungsfähigkeitunterundnachGabevonAcyl-Ghrelinuntersucht.

AufgrundseinerneuroprotektivenWirkungbeineurodegenerativen

ErkrankungenkönnteGhrelininZukunfteineRollealsTherapeutikumbeiAlzheimer-

DemenzundParkinsonspielen.ArbeitenderForschungsgruppevonDr.IngaKadish

(PrincipalInvestigatorderStudie1)zeigtengareineniedrigereBelastungmitA-Beta-

PlaquesimAlzheimer-MausmodellunterchronischerBehandlungmiteinem

GhrelinagonistenbeigleichzeitigbessererkognitiverLeistungderNagetiereim

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VergleichzurPlacebo-Kontrollgruppe.Nichtabschließendgeklärtistnachwievor,über

welchenMechanismusGhrelindieseprotektiveWirkungentfaltet,einigemögliche

Hypothesenz.B.übereineBeeinflussungderSignalwegedesInsulin-Systems,werdenin

Studie1verfolgtunddiskutiert.

GleichzeitighatGhrelininsulinostatischeEigenschaftenundistdaher

möglicherweiseeinrelevanterFaktorinderPathogenesevonDiabetesmellitus.Sehroft

postuliertwurdeeindiabetogenerEffektGhrelinsdurchdieBegünstigunghoherSerum-

GlukosewertemittelsUnterdrückungderSekretionvonInsulin.AndieserStelleergibt

sicheinWiderspruchzudermittlerweilenachgewiesenenundweithinakzeptierten

pathoätiologischenVerbindungvonDiabetesundNeurodegeneration:IstGhrelinnun

wiebereitserwähntaufgrundseinerneuroprotektivenWirkungeinpotentielles

TherapeutikumfürneurodegenerativeErkrankungen?Oderbegünstigtdiechronische

GhrelingabeschlussendlichdieEntwicklungeinerdiabetogenenStoffwechsellageund

führtimGegenteilnichtnurzueinemerheblicherhöhtenDiabetesrisikosondernauch

langfristigzueinerSchädigungdesNervensystems?AndieserStellesetztStudie1an,

indemsieeinechronischeGabeeinesGhrelinagonistennichtnurimHinblickauf

kognitivesondernauchmetabolischeEffekteuntersucht.

2.1.2Studie1

AusentwicklungsgeschichtlicherPerspektivedauertenbzw.dauern

FastenperiodenseltennureinigeStunden.KrankheitenwieAlzheimer-Demenzund

DiabetessindchronischeErkrankungen,diesichüberJahreundJahrzehnteentwickeln.

NichtsdestotrotzschlossenfrühereGhrelin-StudienseltenZeiträumevonmehralszwei

WocheninihreBeobachtungenein.DaherwaresAnliegenvonStudie1,die

langfristigenEffekteeinesGhrelinagonistenaufgleichermaßenKognitionund

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Stoffwechselzubeleuchten(s.o.).DazuwurdeeinAPPSwDI-Mausmodellmiteinem

Ghrelinagonisten(LY444771)mehralsvierMonatelangbehandelt.Gleichzeitigwurde

einTeilderVersuchstieremiteigensentwickeltemFuttermiteinemhohen

glykämischenIndexgefüttert.WährenddieStudiediepositiveWirkungdesAgonisten

aufkognitiveEndpunktebestätigenkonnte,zeigtesichüberraschenderWeisekeinerlei

EinflussdesAgonistenaufdieGlukosetoleranz.KaumeinUnterschiedbestandzwischen

denunterschiedlichenFuttersorten,auchdieKombinationausGhrelinundhohem

glykämischemIndexführtezukeinerleiVerschlechterungderGlukosetoleranz.

ZudemkamdieStudieüberraschenderWeisezudemSchluss,dasseine

chronischeGabedesGhrelinagonistennichtzueinerchronischerhöhten

NahrungsaufnahmeunddamitzuchronischerGewichtszunahmebeiderVersuchstieren

führte,ohnejedocheineüberzeugendeErklärungfürdieseBeobachtungvorweisenzu

können.

2.1.3Studie2

DiezweiteStudiewardieerste,diesystematischdenEinflussvonGhrelinauf

menschlicheKognitionuntersuchte.AufgrunddervielversprechendenErgebnisseaus

NagetiermodellenlautetedieArbeitshypothesedieserStudie,dassdieEinmalgabevon

Acyl-GhrelindieGedächtnisleistunggesunderMenschenmöglicherweiseverbessert.Die

Ergebnissekonntendiesjedochnichtbestätigen.21gesundemännlicheProbanden

musstenineinemParadigmazumräumlichenLernengeschriebeneBegriffeineiner

dreidimensionalenvirtuellenUmgebungmitihrerLokalisationerlernen.

GhrelinverändertezwardieHirnaktivität(BOLDfMRI)inHirnregionen,die

bekannterWeiseeineRolleinderVerarbeitungvonwortbezogenenAssoziationen

spielenundbeeinflussteauchdieKonnektivitätinneuronalenNetzwerkenz.B.zwischen

dembeidseitigenNucleuscaudatus,demrechtenorbitofrontalenKortexundder

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beidseitigenInselrinde.ÜberraschenderweisefandsichjedochkeinEffektdesHormons

indengetestetenkognitivenDisziplinen:Arbeitsgedächtnis,BochumerMatrizentest,

Kreativität,Zahlenverbindungstest,ReaktionsgeschwindigkeitundAufmerksamkeit.

EineweitereTeilhypotheselautete,dassGhrelinmöglicherweisedazuführt,dasssich

ProbandennahrungsbezogeneBegriffebessereinprägenunddiesumsomehr,je

attraktiveroderkalorienreicherdieNahrungist.EinsolcherZusammenhangkonnte

ebenfallsnichtnachgewiesenwerden.

LetztlichscheintderEinflussvonGhrelinaufmenschlicheKognitionkomplexer

zuseinalserwartet,dochsindauchdieEinschränkungenderStudiezuerwägen:Statt

geschriebenerBegriffesolltenkünftigeStudienBildervonGegenständenund

Nahrungsmittelnverwenden,dieerfahrungsgemäßinneurokognitivenExperimenten

einehöhereSalienzbesitzen.ZudemverbessertGhrelinsehrwahrscheinlichnach

EinmalgabeauchnichtdieDenkleistunggesunderVersuchspersonen.Dochstelltdie

UntersuchungvonlängerenFastenperiodenoderchronischenGhrelingabenbeim

MenscheneineninteressantenAnsatzdar,dieAuswirkungendesHormonsaufkognitive

ProzesseamMenschenweiterzubeleuchten.

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2.2Summary(English)

2.2.1Background

Ghrelinisa28-aminoacidpeptidewithadistinctacyl-chainatitsthirdserineresidue,

addedbytheenzymeghrelin-O-acyl-transferase.Itwasdiscoveredin1999asaligandof

thegrowthhormonesecretagoguereceptorbyaJapaneseresearchgroup.Itsnameis

bothanacronym(growthhormonereleaseinducing)andanallusiontotheProto-Indo-

Europeanwordfragment“ghre”,meaning“togrow”.

Besidesitsroleingrowthhormonerelease,ithasbeenidentifiedastheonly

peripheralorexigenichormoneandappearstobeanintegralpartofcircadianrhythms

offoodintake.Thereceptoriswidelyspreadinmammalorganismsandfurtherhasthe

capacityofformingheterodimerswithothertransmittersystemssuchastheserotonin

ordopaminesystem.Thisshowsthebroadrelevanceofghrelinorratherthe“ghrelin

system”interactinginamyriadofwayswithaspectsofenergyhomeostasisbothfroma

metabolicandacognitiveperspective–ifthisseparationcanbevalidlyupheldin

ghrelinresearch.

Soonitbecameclearthatitsimportanceandimpactgoesfarbeyondthe

regulationofenergyhomeostasis.Itratherseemstoinfluenceandshapecognitive

processes,consistentlyimprovingmemoryformationindifferentrodentmodels,mainly

inthefieldsofobjectrecognition,spatiallearningandaversivememory.Itisbeing

discussedasalinkbetweenmetabolismandcognitionandthereforehasinmanyways,

metaphoricallyspeaking,theroleofa“belly-brainlink”or“squirrelhormone”:Intimes

offasting,theseanimalsremembermoreorlessaccuratelywheretheirfoodishidden.

Indeedrecentprojectsshowarelevanceofghrelininmetabolicaspectsinsciurid

hibernators.However,adirectextrapolationofdatafromrodentmodelsorsciurid

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hibernatorstohumanscannotbevalidlydoneduetothemuchmorecomplexcontextof

humancognition.Nonetheless,itisaninterestingandveryrelevantquestionwhether

ghrelinhasapositiveimpactonhumancognitiveperformanceaswellsinceitmayhelp

explainthecognitiveaspectsoffeedinginhumanswithimplicationsinunderstanding

bothobesityandanorexia.

Therehasbeenalongstandingexperienceinexperimentsinvolvingthe

administrationofghrelintohumanvolunteersevenbeforestudy2inProf.AxelSteiger’s

(principalinvestigatorstudy2)researchgroupinthecontextofsleepresearchin

generalandmorespecificallysleependocrinology.However,toourknowledge,nostudy

hadsystematicallylookedattheimpactsofghrelinonhumancognitiveperformance.

Thus,study2isthefirstofitskindlookingintothecognitiveaspectsofghrelinactionin

humansduringandaftertheadministrationofacylghrelin.

Duetoitsbeneficialimpactinneurodegenerativediseasesghrelinmightplaya

roleasatherapeuticagentinconditionssuchasAlzheimer’sandParkinson’sdisease.

ProjectsofDr.IngaKadish’s(principalinvestigatorstudy1)researchgroupeven

showedalowerA-beta-plaqueloadandbettercognitiveperformanceafterchronic

treatmentwithaghrelinagonistinanAlzheimer’sdiseasemousemodelcomparedto

controlstreatedwithplacebo.Aconclusivemechanismforthisprotectiveeffecthasnot

beenidentifiedyet,somepossiblehypothesese.g.viainfluencingsignalingpathwaysof

theinsulinsystemarepresentedanddiscussedinstudy1.

Atthesametime,ghrelinhasinsulinostaticproperties,makingitarelevant

hormonalplayerindiabetes,possiblyhelpingtoexplainthelinkbetweenboth

conditions.Ithasoftenbeenpostulatedthatghrelinmightbeadiabetogenicfactorasit

raisesserumglucoselevelsviaareductionofinsulinrelease.Thisideastandsinharsh

contrasttothewell-provenandwidelyacceptedconnectionofdiabetesand

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neurodegeneration:Isghrelinasmentionedbeforeapossibletherapeuticagentin

neurodegenerativediseasesduetoitsneuroprotectiveeffectshowninnumerous

studies?Ordoesghrelinafterallfavoradiabetogenicmetabolicsituationleadingtoa

higherriskofdevelopingdiabetesandthustoalong-termthreattotheintegrityofthe

nervoussystem?Thisistheideaofstudy1lookingintothechronicadministrationofa

ghrelinagonistnotonlywithrespecttocognitivebutalsotometaboliceffects.

2.2.2Study1

Seenfromanevolutionaryperspective,periodsoffastingwereandarerarely

short-termeventsofafewhoursanddiseasessuchasAlzheimer’sanddiabetesare

intrinsicallychronicdiseaseswithapathoetiologicalonsetofpotentiallyyearsand

decades.Nonetheless,earlystudieslookingintoghrelin’seffectsintheseconditions

hardlyevercoveredperiodsofmorethanacoupleofweeks.Thus,thefirststudyaimed

atcreatingaparadigmlookingintothelong-termeffectsofaghrelinagonistonboth

cognitiveandmetabolicendpointsbytreatinganAPPSwDImousemodelwithaghrelin

agonist(LY444771)formorethanfourmonths.

Atthesametime,someanimalswerefedwithaspecificallydevelopedhigh

glycemicindexdiet.Whileghrelin’spositiveinfluenceoncognitioninthismousemodel

couldbeconfirmedinthisstudy,itsurprisinglyshowednonegativeimpactofthe

agonistonglucosetolerancewhengiveninalong-termregimen.Therewashardlyany

differencebetweenthedifferentdiets,eventhecombinationofghrelinandahigh

glycemicindexdietdidnotleadtoasignificantdeteriorationofglucosetolerance.

Surprisingly,oneofthestudy’sconclusionswasthatchronictreatmentwithaghrelin

agonistdidnotleadtoachronicallyelevatedfoodintakeandconsequentlytoachronic

weightgain,however,withoutfindingaconvincingreasonforthisobservation.

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2.2.3Study2

Thesecondstudywasthefirstonetosystematicallylookatthehormone’seffects

onhumancognition.Withthepromisingresultsfromrodentmodelsinmind,thestudy’s

hypothesiswasthatasingleadministrationofacylghrelincouldimprovememory

formationinhealthyvolunteers.Resultshoweverdidnotshowanyimprovementof

memoryinaspatiallearningparadigminwhich21healthymalevolunteershadto

memorizewrittenwordswiththeirlocationinathree-dimensionalvirtualenvironment.

GhrelinalteredbrainactivityasmeasuredbyBOLDfMRIinbrainareasknowntobe

involvedinverbalassociationprocessingandalsoinfluencedconnectivitybetween

severalbrainregionssuchasthebilateralcaudatenucleusandtherightorbitofrontal

cortexandthebilateralinsula.Surprisingly,itdidnotaffectanyofthecognitive

disciplinestestedinthisstudy:workingmemory,fluidreasoning,creativity,mental

speed,reactiontimeandattention.

Anotherhypothesispostulatedadifferentialeffectofghrelinonthe

memorizationoffoodandnonfooditemswithabettereffectforfooditems,also

dependentontheirattractivenessandcaloricvalue.However,wedidnotseeany

significantdifferencebetweenfoodandnonfooditemsdependentonghrelin

administration,norwasthereadifferencebetweenfooditemsregardingtheircaloric

value.Afterall,ghrelin’simpactsonhumancognitionappeartobemorecomplexthan

anticipated.However,wedoseethelimitationsofourstudy:Futureprojectsshoulduse

picturesinsteadofwrittenwordsastheyusuallyhaveahighersaliencein

neurocognitiveexperiments.

Lookingattheresultsofthisstudy,asingleadministrationofghrelinmostlikely

doesnotactasacognitiveenhancerinhealthysubjects.However,itcertainlyisa

promisingapproachforfuturestudiestolookatprolongedperiodsoffasting,orevena

18

chronicadministrationofghrelininhumanvolunteersinordertofurthercharacterize

thehormone’spropertiesasaneuropeptideinhumans.

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3.Introduction

3.1Foodforthought:Cognitiveaspectsoffeeding

Regardingthepivotalroleoffoodintakeinthequesttosurviveandtoprogenerate,it

appearsevidentwhyvirtuallyallaspectsoffeedingbehaviourareintrinsicallyfinely

regulatedcognitiveprocesses.Theactoflookingforfood,thechoiceofwhattoeat,

whenandwhereaswellasthequestionsofhowtoprepareforperiodsofabsenceof

food,ofhowtocooperateforthecommongoaloffeedingorofhowtodefendone’sfood

onceitisobtainedareprobablynotonlyaspectsofcognitionbutpossibly,atleastin

part,attheoriginofwhatwedefinetodayastheabstractconceptofcognition.Or

plainly,asweputitinapreviouslypublishedbookchapter1onghrelin’sroleinmemory

relatedprocesses:Astheactofeatinginevolutionhasonlyrecentlybecomeaseasyas

toopenafridgefilledwithdelicioustreats,thereisafundamentalneedforallliving

organismstoestablishacloselinkbetweenenergyneedsandthinking,betweencraving

forfoodandbehaviour,betweenbellyandbrain.

3.2Thoughtforfood:thecomplexityofenergyhomeostasis

However,whenitcomestodefiningthedifferentelementsofthislinkthatisrathera

delicatenetworkofneuroendocrineprocesses,theroleofeachpieceinthemosaiqueis

usuallycomplexandrarelyunequivocallyclear,thinkingofthemanyimpactson

cognitionoffactorssuchasleptin,insulin,glucagoneandcortisoljusttonameafew.In

thequesttodefinetheroleofeachelement,researchersarefacingthedifficultyto

standardizetheirstudiesforallotherfactors,althoughtheymaystillbeunknowntoa

certainextent.Atthesametime,tomakesenseoftheresults,thepuzzleasawholehas

20

tobetakenintoconsideration.Evenmoresointhecaseofghrelinwhoseinteraction

withandembeddinginthesignallingsystemsofotherpeptidesinvolvedinthe

regulationofenergyhomeostasis(seebelow)isonlystartingtoemerge.

TableA:Ghrelinataglance

Discovery 1999(Kojimaetal.2)Characteristics

28aminoacids,acylation(n-octanoylation)atSerin3-residuecharacteristicforactiveform

Characterizationasanorexigen

2000(Tschöpetal.3)

Characterizationasaneuropeptiderelevantinbehaviour/cognition

From2002onwards(mostnotablyCarlinietal.,Dianoetal.4–7)

Relevanceinsleep

From2003onwards(Weikel,Rosenhagen,Steigeretal.8)

Mainplaceofproductioninmammals

Oxynticcellsofthestomach;receptorapparentlyubiquitousinmammalorganism

Receptor

Growth-hormonesecretagoguereceptor(GHS-R)

Agonistsforuseinhumans(clinicallyapproved/inphaseIII)

Pralmorelin(approved,KakenPharma,SellaPharma;GHdeficiencydiagnostic),Macimorelin(phaseIII,AeternaZentaris;GHdeficiencydiagnostic),Anamorelin(phaseIII,Helsinn,cancercachexia/anorexia,foracomprehensivelistseereview“Ghrelin”byMülleretal.9)

21

3.3Thestoryofghrelin:ahungerhormonewithatasteformemoryenhancement?

Shortlyafteritsdiscoveryasagrowthhormonesecretagogueproducedpredominantly

inthestomach2,firststudiesinrodentsattributedanorexigenicroletothepeptide,

increasingfoodintakeandweightgainwhengivenregularlyoveraperiodofafew

days3.Soonafter,rodentexperimentscouldshowaneffectonbehaviour,mainlyon

aversivememoryasdemonstratedbyCarlinietal.4ThisgroupadministeredghrelinICV

inaratmodel,themainbehaviouralread-outwasastep-downinhibitoryavoidance

task.Carliniandcolleaguescouldalsodemonstrateapositiveinfluenceofghrelinon

objectrecognitioninamousemodel10.Ghrelin’senhancingeffectsonaversivememory

andobjectrecognitionwereconfirmedbyothergroups:Goshadrouetal.observedthat

ghrelincanpreventthenegativeimpactsofanNMDA-receptorantagonistoncognition

inapassiveavoidancetask11.Atchaetal.showedbetterperformanceinobject

recognitionforaghrelinagonist12.

However,lookingatghrelin’sbehaviouraleffects,somequestionsand

inconsistenciesremain:Carlini’sstudyonobjectrecognitionusedafood-restricted

mousemodelinwhichnegativeimpactsoffoodrestrictiononmemoryperformance

werecounteractedbyghrelin.Further,onelandmarkstudybyDianoetal.observed

impairmentsinspatialmemorybutnotinaversivememoryinghrelinreceptorknock-

outmice7.Whiledifferentwaysofghrelinadministration–oral12,subcutaneous7,ICV4–

allseemtobeeffective,onlymemoryacquisitionandnotretrievalappearstobe

positivelyinfluencedbyghrelinatleastinarodentmodel6.Ontopofthat,onestudy

observedbettermemoryperformanceinGHS-R1aknock-outmice13,othersreported

impairedmemoryperformanceinneonatalchicks5aftercentralghrelinandin

correlationwithendogenousghrelininhumans14.Manyofthesepointsmaybe

22

explainedwithregardtothecaveatsofeachindividualstudydesignandmodel

organismused.However,theyclearlyshowthatghrelin’sunquestionableinfluenceon

cognitioningeneralmightreachbeyondthecomplexityofamerecognitiveenhancer

andthatallextrapolationbetweenspecieshastobedonewithutmostcare.However,

withfurtherstudiesalsoreportingapositiveeffectonspatialmemoryandevenspine

synapsedensityandlong-termpotentiation7,15,apossibleroleofghrelinasa

neurocognitiveenhancerwasincreasinglydiscussed,furthersupportedbythe

henceforthemergingroleofghrelininneurodegenerativediseases16–18suchas

Alzheimer’sandParkinson’sdisease.

Lookingbeneaththesebehaviouralresults,thetransmittersystemsand

biochemicalsignalingpathwaysinvolvedinthemediationofghrelin’sactionsforma

rathercomplexpicture.First,ghrelinhasanimpactondifferenttransmittersystems

suchasserotonin19,nitricoxide15,glutamate20anddopamine21.Second,ghrelinappears

toactviaaG-proteincoupledintracellularsignalingpathway22,leadingtochangesin

intracellularcalciumavailabilityviathesecondmessengerinsositoltrisphosphate23,

whichisarathercommonbiochemicalcascade.Further,CREBseemstobeinfluencedby

ghrelinsignaling24.Currently,thesepiecesofinformation(discussedindetailina

previouslypublishedbookchapter1)formapiecemealmosaicthatstillneedsmore

studiesinorderforustodeducearealmechanisticrelevanceandunderstandingfromit.

Keytounderstandingtheextremelywidespreadactionsoftheghrelinsystemmaybe

thecapacityoftheGHS-Rtoformheterodimerswithotherreceptorsystems25,26.

Twoofthemainneurophysiologicalcorrelatesofghrelin’simpactonmemory

formationareincreasedhippocampallongtermpotentiationandspinesynapsedensity,

bothidentifiedbyDianoandcolleagues7.Morerecentstudiesfurtherconsolidatethe

23

notionofghrelinasaneuropeptiderelevantinhippocampalmemoryformationby

showingthatghrelin’sorexigenicactionsaretoacertaindegreemediatedvianeural

pathwaysinvolvinghippocampalsubfields27,28.

Asmentionedbefore,ghrelininfluencesanumberofdifferentneuroendocrine

systems.Italsoappearstoplayaroleintheregulationofthehypothalamic-pituitary-

adrenalaxis29,thusbecomingrelevantinsleepandmentalhealth8,30–36.Thetwostudies

ofthisdissertationwerenotdesignedtoaddresstheseissuesandratherfocusedon

endpointsrelatingtomemory,cognitionaswellasenergyhomeostasis.

3.4Doesghrelinactasacognitiveenhancerinyoung,healthy,malehumans?

Apeptideenhancingmemoryinfeeding-relatedprocessesinastateofhungercouldbe

seenasanevolutionaryadvantage.Inourbookchapter,weusedasquirrelthathasto

rememberinwinterwhereitsacornsarehiddenasanillustratingexample1.Although

currentstudiesindeedconfirmghrelin’srelevanceinthemetabolismofsciurid

hibernatorscaughtinthewildernessofColorado37,38,itsrelevanceformemory

processesinthisspecieshasyettobeassessed.Nontheless,wepursuedthetoacertain

extentcounterintuitiveideaofanorexigenimprovingcognitiveperformanceand

includedthehormoneinthemulticentreresearchproject“ComparingAppleswith

Oranges:ADifferentialViewonNeuroenhancement”supportedbyVolkswagen

Stiftung39lookingintothecognitiveeffectsofanumberofsubstances.Furthermore,few

studieshadandhaveaddressedtheeffectsofghrelinonhumanmemoryperformanceso

far.Althoughanumberofclinicallytestedghrelinagonistsisavailable9(seetableA),we

decidedtodesignastudyemployingthenatural,active(acylated)formofthehormone

tostayasclosetonaturalconditionsaspossible.Theproblemsarisingfromthis

24

decisionsuchasthetime-sensitiveadministration40andthehandlingofthefragile

peptidearepointedoutinthemethodspartofthesecondpublication.

FigureA:(left)Goldenmantledgroundsquirrel(Spermophiluslateralis,photo:Eborutta2003/CreativeCommonslicense)–metabolismofthissciuridhibernatorindeedappearstobeaffectedbyghrelin;(right)keytopicsinghrelinresearchandrecommendedreading

Itisimportanttonotethatthisstudydubbed“GHREEN”(ghrelinandcognitive

enhancement)didnotseektodefinetheeffectsofhungeroncognitionasthiswouldbe

farbeyondthescopeofasinglestudy.Itratheraimedatcharacterizingtheacuteeffects

ofacylghrelinoncertainaspectsofhumancognition(see“methods”study2)directlyin

theaftermathofadministration.

3.5Themethodologicalchallengeofdesigningasuitableparadigmforthequestion

AsmeasuringtheamountofacylghrelinactuallyreachingtheCNSwouldhardlybe

ethicallyjustifiedinasampleofhealthyvolunteers,wedecidedtomonitorperipheral

bioavailabilityinstead.Wewereawareofconsequentlyleavingthequestionof

Topic Publication/ReviewarticleOverview:Ghrelin Mülleretal.2015Ghrelin&Memory Kunath/Dresler2014Ghrelin&Insulin Chabotetal.2014,Tongetal.

2010,Dezaki2013Ghrelin&Sleep Steigeretal.2011Ghrelin&MentalHealth Wittekind/Kluge2015Ghrelin&Neurodegeneration Gaheteetal.2011,Baylissetal.

2013,Shietal.2016Ghrelinasabelly-brainlink Hsuetal.2016

25

interindividualdifferencesinghrelin’scentralbioavailabilitytofuturestudiesandto

accepttheuncertaintyofdifferencesinthisrespectwithinoursampleofvolunteers–

despitethegreateffortstostandardizeasmuchaspossibleforenvironmentaland

biometricvariability.Anumberofstudieshaveaddressedthequestionsofhowghrelin

crossestheblood-brainbarrier41,whatfactorsinfluencetheexistingtransport

mechanisms42andwhatcentralnetworksmediatethesubsequentresponsetoghrelin

signalling27.Infuturestudies,itwillbecrucialtodefinetheexactrelevanceofeachof

thesepathways–activebidirectionaltransport,vagalafferences,passivediffusione.g.

viacircumventricularorgans–notonlywithrespecttoghrelin’sorexigenicactionsbut

alsotootheraspectsofcognitionasfarastheycanbeseparatedfromeachother.

Furtheraddingtothecomplexityofcharacterizingghrelinsignalling,recentstudies

showthatghrelinreleaseandactionappeartoberegulatedinacircadianmannerand

influencedbythecurrentmetabolicstateoftheindividualaswellasfood

anticipation28,43–45.

Indesigningthein-fMRImemorytask,wetriedtocreateaparadigmforvisuo-

spatialmemoryinhumans,capableofmakingadifferencebetweenfood-andnon-food

itemsrealisticallyembeddedinavirtualsurroundingimitatingawalkinaneveryday

environment.Whileatthebeginning,aconsiderableeffortwasmadetousearealwalk

inalocalparkasasettingforthetask,welaterdecidedtoabandonthisideaforthesake

ofbetterstandardizationpossibilitiesinavirtualsurroundingandcreatedacustom-

builtvirtual,three-dimensionalmemorytaskbasedonthefreewarevirtualgaming

software“Sauerbraten”(see“Methods”publication2).

However,withtherecalltaskafteronedayshowingmerelyscreenshotsofthe

virtualenvironmentandwithourvolunteersoftenreportingamemorizingstrategy

employingcertainlandmarkssuchashouses,roomsorstreetsassociatedwiththeitems

26

toberecalled,ourtaskratherturnedouttobeacuedword-locationassociationtask.

Furthermore,itneedstobepointedoutthat,forabettergraphicalembeddingofitems

intothevirtualtask,weusedwrittenwordsinsteadofpictures,whichrisesthevery

validquestionofhowsalienttheitemsweusedactuallywere.

Nonetheless,withinthelimitsdescribedabove,wearecertainthatour

conclusionthatghrelindoesnotgloballyenhancecognitiveperformanceinyoung,male

humansisvalid,especiallywithBayesiananalysesbeinginfavourofthenullmodel(see

publication2).Firstofall,ourparadigmtestedalargenumberofcognitivedisciplines.

Secondly,differencesbetweentreatmentgroupswere,inmostcases,virtuallynon-

existentanddidnotevencomeclosetoastatisticaltrendorsignificance.Thirdly,

volunteersshowedanextraordinarilyhighadherencetothestudydesign,withan

overallhighmotivationtoperformwellinthecognitivetasksandalowdrop-outrate

oncethestudywasentered.

However,withanothergroup’sworkpointinginthisdirection46andwithour

study’slimitationsinmind,wedothinkthatthereisapossibilityoffindingadifference

formemoryperformanceinfood-andnon-fooditemsonceastudydesigninvolvinge.g.

moresalientstimuliisemployed.

27

Study1 Study2Drugused GhrelinagonistLY444711 NaturalactivepeptideTimeframe Long-term(months) Acute(intra-day)Organism Mousemodel(C57/BL6

APPSwDI)Young,healthy,malehumans(20-30yearsofage)

Mainquestion Long-termeffectofaghrelinagonistonAlzheimer’sdiseasepathologyandglucosehomeostasisundertheinfluenceofahigh-glycemic-indexdiet

Effectsofacuteadministrationofacylghrelinonhumandifferentdisciplinesofhumancognition

Mainparadigms Watermazetest,oralglucosetolerancetest,immunohistochemicalstainingsofbrainslices

Custom-builtfMRI-monitoredvirtualcuedlocation-wordassociationtask;cognitivetestbattery(seemethodsstudy2)

Ghrelinmonitoring Onceattheendofthestudy Constantly(hourlytoevery10-15min)

Ghrelinassayused 2-sitesandwichassay47 Radioimmunoassay

N= 36 21Institute

DepartmentofCell,DevelopmentalandIntegrativeBiology,UniversityofAlabamaatBirmingham,USA

Max-Planck-InstituteofPsychiatry,Munich,Germany

PrincipalInvestigator Dr.IngaKadish Prof.Dr.AxelSteiger/Dr.MartinDresler

Financialsupport NIHgrantsR01AG043972,P30DK056336andP30NS47466;LY444711providedatnocostbyEliLilly,Indianapolis,USA;AMIOCAstarchprovidedatnocostbyIngredionInc.,Bridgewater,NJ,USA;

Volkswagen-Stiftung,“ComparingAppleswithOranges:ADifferentialViewonNeuroen-hancement”,2011

TableB:Thetwopublicationsataglance

28

3.6Neurodegenerationandtheroleofinsulin,glucose–andghrelin?

EversincetheRotterdamstudyprovidedconvincingevidenceofadirectassociation

betweenAlzheimer’sdiseaseanddiabetes48(alinkbetweencognitivedeclineand

diabeteshadbeensuggestedlongbefore),sciencetriestofindamechanisticexplanation

forthiscorrelation.Earlystudieshadlongcontradictedtheassumptionofthebrain

beinganorganinsensitivetoinsulinsignalling49,50.Whilethehormone’sactionsinthe

braingowellbeyondmereregulatoryeffectsonmetabolism51,itisnowbecomingclear

thatcentralinsulinsignallingalsohasanimpactonperipheralglucosehomeostasis52.

Further,adeficiencyininsulinsignallingappearstobeanimportantfactorinthe

neurodegenerativecascadeleadingtotheclinicalappearanceofAlzheimer’sdisease53.

Therearetwomainreasonstobelievethatghrelinalsoplaysaroleintheinterplayof

glucosehomeostasisandneurodegeneration.First,ghrelinhasinsulinostaticproperties

whenadministeredacutely54,probablyinordertokeepglucoselevelshighinasituation

ofenergydeficiency.Second,thereisastrongbodyofevidenceofghrelinbeing

neuroprotectiveinseveralentitiesofneurodegenerativediseases16,55.Thelatterledtoa

studyperformedinIngaKadish’slabshowingthataghrelinagonistgivenchronicallyis

capableofreducingtheAβ-plaqueloadinanAlzheimer’sdiseasemousemodel17.

29

FigureA:ExampleofaW02-stainingforAβ-plaquesinaheavilyaffectedanimal(C57/BL6APPSwDImouse)instudy1(seemethodsstudy1andimmunohistochemistryresults).

3.7Chronicdisease,chronicadministration:Theideabehindpublication1

Withthisstudyinmind,weaimedatdesigningastudylookingintotheeffectsofahigh

glycemicindexdietonAlzheimer’sdiseasepathologyinthesamemousemodel–and

theimpactghrelinhasinthissetting.Ifghrelinisindeedinsulinostatic,thereisreasonto

believethatitactuallyevendeterioratesthepossiblynegativeeffectsofahighglycemic

indexdietonAlzheimer’sdiseasepathology56.This,however,wouldcontradictthe

findingsofthemanystudiesshowingthepositivepropertiesofghrelinin

30

neurodegenerativediseases(seehypothesesdescribedinpublication1).Atthesame

time,neithertheeffectsofghrelinagonistadministrationoninsulinsecretionand

glucosetolerancenortheresultingimpactonAlzheimer’sdiseasepathologyhad

previouslybeenaddressedinalong-termtreatmentmodel.Thisissurprisingas

Alzheimer’saswellasotherneurodegenerativediseasesareintrinsicallychronic

diseasesandthus,anytherapeuticapproachisnecessarilyalong-termapproach.

Highglycemicindexdiet ControldietProtein(%kcalfrom) 20.8 18.8Carbohydrate(%kcalfrom) 60.2 63.9Fat(%kcalfrom) 19.0 17.2Caloricdensity(kcal/g) 3.4 3.8Maincarbohydrateingredients AMIOCAwaxymaize

starch,maltodextrinCornstarch,maltodextrin

TableC:TogetherwithspecialistsfromIngredionInc.andHarlan/Teklad,acustomresearchhighglycemicindexdietwasdeveloped–withtheamountofcaloriesstemmingfromeachmacronutrientaswellascaloricdensitybeingclosetothestandardcontroldiet(seemethodsstudy1).

31

4.Conclusions

4.1Publication1:Surprisingresultsinlong-termghrelinagonisttreatment

Inoursetting,ahighglycemicindexdietdidnotdeteriorateperformanceinawater

mazetaskafterfourmonthsoftreatmentnordiditworsenimmunohistochemical

parametersinanAβ-plaquemousemodelcomparedtoothergroupsonadifferentdiet.

Thismaybeinpartbecauseourmicewerestillnotoldenoughwhentheywere

sacrificedtoshowadetrimentaleffectinthesemeasurescausedbytheirsugarydiet

(seemethodsstudy1).Neitherdidweseeanimprovementinimmunohistochemical

parametersafterghrelinagonisttreatmentasinapreviousstudywithadifferent

design17.

Thereare,however,twoimportantmessagescontainedintheresultsofthis

study:First,thecognitiveenhancementseenafterlong-termghrelinagonisttreatment

appearstoberobust,thatisconsistentoverbothstudiesinthesamemousemodel17,18

andindependentfromthefeedingregimenused.

Second,thelong-termeffectsofghrelinagonisttreatmentdiffergreatlyfromthe

short-termeffectsofghrelin(agonist)treatmentinmeasuressuchasfoodintake,body

weightdevelopment,bodycompositionand,aboveall,glucosetolerance(seeresultsand

discussionstudy1).Theeffectsinweightdevelopmentandbodycomposition–a

glucosetolerancetestwasnotperformedatthattime–seeninDr.Kadish’sprevious

studywiththesamemousemodelbutdifferentdietswerenotasradicalasinthestudy

presentedinthisdissertation.Nonetheless,theweightgaineffectsthatwerethusfar

regardedastypicalofghrelin(agonists)couldnotbeobservedeither.

32

Althoughthepossibilitiesofextrapolatingtheresultsofourstudytoothermouse

modelsorevenothermammalorganismsareverylimited–averyspecificmousemodel

withaveryspecificdietandaveryspecificghrelinagonistwereused–itshouldmake

ussensitivetopossiblybigdifferencesintheimpactsofghrelinonmammalorganisms

dependingonwhetheritisgiveacutelyoronalong-termfeedingregimen(see

discussionstudy1).

Howdoweexplaintheimprovementsincognitiveperformanceifnodifferences

inimmunohistochemicalendpointscouldbedetected?Althoughfindingaprecise

answertothisquestionresemblesthesearchforthenotoriousneedleinahaystack,the

positiveeffectsofghrelinagonisttreatmentonglucosetolerancehintinthedirectionof

insulinsignallingasapossibleendpointtolookat.Thedatawefoundinthisrespect–a

lowerexpressionofp-IRS-1Ser636,whichhasbeenshowntobeassociatedwithboth

Alzheimer’sdiseaseanddiabetes57,afterghrelinagonisttreatment–isfarfromrobust

andfailstofullyexplainthebehaviouralchangeswesawinoursampleofmice.

Nonetheless,itislittlesurprisingandopensupanewpathwayofthinkingwhenit

comestoexplainingghrelin’sbeneficialeffectsonbothglucosetoleranceandcognitive

performance.

Oneimportantfindingthatmaybeseenasaconfounderinthisstudyisthe

higherlevelofactivityduringtheactiveperiodinmicetreatedwiththeghrelinagonist

(seefigure3ofpublication1).Therefore,onemightregardthepositiveeffectsofthe

ghrelinagonistoncognitiveparametersasamereconsequenceofthealreadyknown

positiveeffectsofexerciseoncognitiveperformancebothinrodentsandhumans58,59.

Althoughweregardthisasavalidlimitationtotheinterpretationofourresults,wedo

notthinkthatexercisealonecanexplainourfindingsregardingfoodintakeandweight

33

gainafterghrelinagonisttreatment.Nonetheless,especiallyinfuturelong-termstudies,

thispossibleconfounderhastobetakenseriously.

4.2Publication2:Whatghrelindoesnotdo

Theresultofthesecondstudyare,atleastasfarasbehaviouralparadigmsare

concerned,negative.Thecognitiveenhancingeffectswehypothesizedbutcouldnot

detectappeartobeeitherutterlyabsentor,morelikely,limitedtotasksinvolvingmore

salientstimulirelevanttoapersoninastateofhunger46.ThefMRIresultsappeartobe

morerobustbutalackofbehaviouralresultsmakesanyinterpretationrelatively

difficult.

Despitetheoverallnegativeresultsofthisstudy,anumberofconclusionsand

ideasforfuturestudiescanbedrawnfromit.Firstofall,astudywithasimilardesign

butmoresalientstimulicouldlookintothedifferenceofmemoryperformanceforfood

andnon-fooditems.Althoughourstudyfailedtoshowsignificantdifferences,thisstill

remainsapromisingapproachwhichcouldhelptofurtherdefinetheroleghrelinhasto

playinthe“belly-brain-link”describedabove.

Second,withthefMRIBOLDsignalessentiallyrelyingonavascularresponseand

withthevascularcontributionstothedevelopmentofAlzheimer’sandotherformsof

dementia60,itshouldbeaskedtowhatextentghrelincouldbeabletoselectively

improvebloodflow61,oxygenationandconsequentlymetabolicintegrityinbrainareas

relevanttomemoryformation.Dependingonthemodelorganismandthetechnique

used,astudyaddressingthisquestionmayfindinterestingresultsinashorttermas

wellasalong-termparadigm.

34

Further,alsothinkingoftheresultsfromstudy1,thelong-termeffectsofghrelin

anditsagonistsonglucosehomeostasis,weightdevelopmentandcognitionneedtobe

thoroughlydefined.Assomeagonistswillprobablysoonbeusedinlargersamplesof

patientsforchronicconditionssuchascancercachexia62,itwouldbebothrelatively

simpleandhighlyimportanttoincludetheseendpointsinlarge-scaleclinicaltrials.

Asthecurrentevidencesuggeststhatghrelin(agonists)havebothshort-term

andlong-termcognitiveenhancingeffectsatleastinrodents,itshouldbeaskedwhat

effectismorerobustandreliable.Towhatextentarethecognitiveenhancingeffectsof

theearlystudieswithghrelin,oftengivenICV4duetoastateofarousalmoreorless

independentofthesubstanceadministered?Inpart,thisquestionisansweredbythe

efficacyofotherroutesofadministration(seeabove).Arethelong-termresults

replicableinother(non-pathological)mousemodels,ratsandpossiblyevenprimatesor

aretheyratherrestrictedtoaveryspecificsetting?

And,asalastpoint,itneedstobeaskedwhatrelevancestudiesusing

supraphysiologicallyhighlevelsofghrelincanhaveineverydaylife–bothforhumans

aswellasothermiceandothermammals.Whatnaturalsecretionpatterndoesghrelin

showinlong-termsettingsinvolvingsituationsofexercise,differentfoodcompositions

anddifferentrhythmsoffoodintakeandsleep?Currentstudieslookingatthecircadian

characterofghrelinsecretionarealreadyfollowingthistrainofthought28.Arethere

differencesbetweensmallandlargeanimalsorbetweensmallandlargespecimensof

thesamespecies,alsothinkingofghrelin’scharacterasagrowthhormone

secretagogue?Whatexactlyhappenstoghrelinandinsulinsignalling(andessentially

thenutrientsandsubstancesforwhoseregulationourbodiessynthesizethese

hormonessuchasglucose,proteinsandfattyacids)attheverymomentthebodyadapts

35

tolongerperiodsoffastingor,almostmorerelevantly,toprolongedperiodsof

overeating?

Howcanwedefinethecourseofanorexianervosa63,bulimianervosa64and

obesity65intermsofghrelin(andinsulin)signalling9andpossiblydevelop

pharmaceuticalwaystopreventthepatientfromcrossinga“pointofnoreturn”?And,

withallthequestionsaskedinthelastparagraph:Whatexactlyiscognitioninthe

differentsettingsandhowisitaffected?Withregardtothecomplexityoftheghrelin

system,verbalaccuracyamongstresearchers,acleardifferentiationofwhataspectof

cognitionisexaminedinrelationtowhatexactpartofghrelinsignallingwillbecrucial

infutureghrelinresearch.Withghrelinanditsagoniststillbeingatarelativelyearly

stageofwidespreadclinicaluse,westillhaveachancetobetterunderstandghrelin’s

roleandrelevanceinhumanandotherorganismsaswellasitsdelicateinteractions

withotherhormonalsystemsbeforewetrytointerferewithittherapeutically.

36

5.Publication1

GhrelinagonistdoesnotfosterinsulinresistancebutimprovescognitioninanAlzheimer’sdiseasemousemodelNicolasKunath(1)(3),ThomasvanGroen(1),DavidB.Allison(2),AshishKumar(1),MoniqueDozier-Sharpe(1),IngaKadish(1)(1)DepartmentofCell,DevelopmentalandIntegrativeBiology,UniversityofAlabamaatBirmingham,BirminghamAL,USA(2)OfficeofEnergetics;NutritionObesityResearchCenter;DepartmentofNutritionSciences,UniversityofAlabamaatBirmingham,Birmingham,AL,USA(3)DepartmentofClinicalResearch,Max-Planck-InstituteofPsychiatry,Munich,Germany

37

5.1Abstract

Theorexigenichormoneghrelin,apotentialantagonistoftheinsulinsystem,ensures

sufficientserumglucoseintimesoffasting.Intheracefornewtherapeuticsfordiabetes,

onefocusofstudyhasbeenantagonizingtheghrelinsysteminordertoimproveglucose

tolerance.Weprovideevidenceforadifferentialroleofaghrelinagonistonglucose

homeostasisinanAlzheimer’sdiseasemousemodelfedahigh–glycemicindexdietasa

constantchallengeforglucosehomeostasis.Theghrelinagonistimpairedglucose

toleranceimmediatelyafteradministrationbutnotinthelongterm.Atthesametime,

theghrelinagonistimprovedspatiallearninginthemice,raisedtheiractivitylevels,and

reducedtheirbodyweightandfatmass.Immunoassayresultsshowedabeneficial

impactoflong-termtreatmentoninsulinsignalingpathwaysinhippocampaltissue.The

presentresultssuggestthatghrelinmightimprovecognitioninAlzheimer’sdiseaseviaa

centralnervoussystemmechanisminvolvinginsulinsignaling.

SupportedinpartbyNIHgrantsR01AG043972,P30DK056336andP30NS47466.The

opinionsexpressedarethoseoftheauthorsanddonotnecessarilyrepresentthoseofthe

NIHoranyotherorganization.

38

5.2Introduction

Eversincethediscoveryofghrelinasaligandofthegrowthhormonesecretagogue

receptorin19992,ourunderstandingoftheversatileroleofghrelininmammalshas

constantlyexpanded.Thecharacterizationofghrelinhasspanneditsactionsasan

orexigenichormoneleadingtoweightgainandadiposityinrodents3,66,tothe

stimulationofappetiteinhumans67,itsimpactsoncognitiveprocessesinrodents4,7and

humans46,68,69,anditsroleasaneuroprotectiveagentinneurodegenerative

diseases8,16,17,70,71.Ghrelin’sinvolvementinglucosemetabolismbecameapparentvery

early72,73,withevidenceforadifferentialroleofdes-acylghrelin74,75.Recently,many

groupshavefocusedontheinteractionsofghrelinwiththeinsulinsystemin

humans54,69.Antagonizingtheinsulinostaticghrelinsystemhasrepeatedlybeen

suggestedasanovelmechanismbywhichtoimproveglucosehomeostasisinhumans.

However,toourknowledge,noneofthestudiesoftheinteractionsofghrelinwith

glucosehomeostasishaveaddressedthelong-termimpactofghrelinadministrationon

amammal.

Ourgroupshowedpreviouslythatadministrationofaghrelinagonistleadsto

improvedcognitionandimprovedmarkersofpathologyinanAlzheimer’sdisease

mousemodel,evenintheabsenceofcaloricrestriction17.Thepathophysiological

correlationsbetweenAlzheimer’sdisease,impairedglucosemetabolism,anddiabetes

arewellestablished76–78,andelevatedserumglucoselevelshavebeenshowntobean

independentriskfactorfordementiainhumans79.Inthepresentstudy,therefore,we

aimedtoinvestigatethelong-termeffectsofaghrelinagonistgivenfor4monthson

Alzheimer’sdiseasepathology,cognition,andmetabolisminthesamemousemodelfed

ahigh–glycemicindex(GI)dietasaconstantchallengeforglucosehomeostasis.We

39

hypothesizedtoseeeither(i)adetrimentaleffectofghrelinagonisttreatmentin

combinationwiththisdietoncognitiveandmetabolicendpointsowingtointerference

withinsulinsignalingandconsequentlyhigheroverallbloodglucoselevelsor(ii)a

protectiveeffectasseeninourpreviousstudyviaathusfarunknownmechanism.

5.3Results

5.3.1Ghrelinagonistactsasalong-termcognitiveenhancerinspatiallearning

Othergroupshavepreviouslyreportedincreasedlevelsofanxietyinneonatalchicks

andratsintheopenfieldtestafterghrelinadministration4,5.Inseveralpreliminarytests

weperformedtoexcludeanyaprioridifferencesbetweengroups,wedidnotobserve

anystatisticallysignificantdifferencesbetweengroupsincategoriessuchasanxietyor

explorationactivity(openfield,zeromaze,dark-light-box;seemethods;datanot

shown).Wealsodidnotdetectanysignificantgroupdifferencesinperformanceinan

objectrecognitiontask,whichhadbeenobservedtobeimprovedbyshort-termghrelin

treatmentbyanotherresearchgroup10.

40

Figure1:Ghrelin-agonist-treatedanimalsperformedbetterinawatermazetest.Theyshowedafaster

learningcurvethandidthegroupfedahigh-GIdietalone.Intra-daydifferencesbetweenhigh-GIandhigh-GI

+ghrelinagonistgroupsweresignificantforday3((a),one-wayANOVAfollowedbypost-hocTukey’s

multiplecomparisonstest,p=0.026),anArea-Under-The-Curve(AUC)-comparisonforthegraphsin(a)

revealedthatghrelinagonisttreatedanimalsshowedastrongtendencytoperformbetterovertheentire

experiment((c),p=0.061,one-wayANOVA/Tukey’s).Duringprobetrials(timetofirstentryinthecorrect

quadrant),thedifferencebetweenhigh-GIandhigh-GI+ghrelinagonistweresignificantattendencylevel

only((b),p=0.096forhighGIvs.highGI+ghrelinagonist,p=0.054forhighGI+ghrelinagonistvs.controls,

one-wayANOVA/Tukey’s).BarsindicateSEM.

41

Amongthethreestudygroups(thegroupfedahigh-GIdiet,thegroupfedahigh-GIplus

ghrelinagonist,andthecontrolgroup,whichwasfedanAIN-93Gpurifieddiet),the

groupfedahigh-GIdietplusghrelinagonistshowedthebestmemoryperformancein

thewatermaze(figure1).Bothinitslearningdynamicsinthecourseofthetestdays

andinitsperformanceintheprobetrial,thisgroupoutperformedtheothergroups.

However,thegroupfedahigh-GIdietwasnotimpairedinitscognitiveperformance

comparedwiththecontrolgroupasweoriginallyhypothesized.

5.3.2GhrelinagonistdoesnotsignificantlyaffectAβplaqueloadormicrogliaactivation

InapreviousstudywereportedapositiveinfluenceofghrelinonAlzheimer’sdisease

pathologymarkerssuchasAβplaqueload(humanAβ4-10;seemethods)andactivated

microglia17.Inthecurrentstudy,however,wedidnotobserveanysignificant

differencesbetweenthetreatmentgroupsineitheroftheseimmunohistochemical

endpointsinthestratumoriensanddentategyrusofthedorsalhippocampalarea

(figures2,(a)and(b)).Becausetheolfactoryepitheliumhasbeenshowntobeinvolved

atanearlystageinAlzheimer’sdisease80,weincludedtheolfactorybulbinour

immunohistochemicalmeasurements.Microgliaactivationintheolfactorybulbwasless

inthegroupfedahigh-GIdietplusghrelinagonistthaninthegroupfedahigh-GIdiet

alone(p=0.057,figure2,(c)).TheAβplaqueloadintheolfactorybulb,however,didnot

differsignificantlybetweenthesegroupsasmeasuredinagrayscaledensityassessment

(figure2,(c);seemethods).Otherresearchgroupshavereportedanincreasednumber

ofdoublecortin(DCX)-positivecellsafterghrelintreatmentinthehippocampusof2-

month-old5XFADmice81.Wedidnotobserveanysignificantdifferencesbetween

groupsinDCX-positivecellcountinthedentategyrus(datanotshown).

42

Figure2:

Neithermarkersforactivatedmicroglia(IBA,toprow)norforAβ-load(W02,lowerrow)weresignificantly

differentafterlong-termghrelinagonisttreatmentinthedentategyrus(a)andstratumoriens(b).Onlythe

levelofactivatedmicrogliaintheolfactorybulbofghrelin-agonist-treatedanimalsshowedatendencytobe

lowerthaninanimalsfedthehigh-GIdietalone((c),Kruskal-Wallistest,followedbypost-hocDunn’s

multiplecomparisonstest,p=0.057).BarsindicateSEM.

43

5.3.3Long-termghrelinagonisttreatmentleadstolessweightgain,lessoverallfoodconsumption,andmoreactivity

Ghrelinanditsagonistsleadtoovereatingandobesitywhenfoodintakeisunlimited3,82.

Interestingly,thegroupfedahigh-GIdietplusghrelinagonistdidnotgainasmuch

weightasdidtheothertreatmentgroups(figure3,(a)).Onlyweightgaininthetwo

groupsnottreatedwiththeagonistwashighlysignificant(figure3,(a),p=0.009for

high-GIvs.highGI+ghrelinagonistgroup,p=0.015forcontrolsvs.high-GI+ghrelin

agonistgroup,ANOVA/Tukey’s).Ofnote,theincreaseinfatmasswasparticularlylowin

thegroupfedahigh-GIdietplusghrelinagonist(figure3,(b)).

Becausethefoodconsumptionofagonist-treatedanimalswaslimitedtothe

averageamountconsumedbythegroupfedthehigh-GIdietalone(seemethods),

overeatingtriggeredbytheghrelinagonistwasnotpossibleinthisgroup.Weobserved

astrongfeedingresponseinouranimalsaftertheadministrationoftheghrelinagonist;

however,theattempttoquantifythisresponseinCLAMSmetaboliccagemeasurements

failed.Themicedidnottoleratetheprocedure,mainlybecauseofanaccidentallyshifted

dark-nightcycle.Asaproofofconcept,wehaveincludedCLAMSdatafromprevious

studieswithC57/BL6micethatclearlyshowtheimmediatefeedingresponseafterthe

administrationofthesameagonistLY444711(figure3,(e)and(f)).

Interestingly,dailyrecordingoffoodintakeinthegroupfedahigh-GIdietplus

ghrelinagonistover8weeksshowedthattheanimalsdidnotconsumethefullamount

offoodgiventothemdaily(figure3,(g)).Thisfindingandtheoverallelevatedactivity

levelsinagonist-treatedanimalscomparedwiththosefedthehigh-GIdietalone

(p<0.001forhigh-GI/controlsvs.ghrelinagonisttreatedgroup,ANOVA/Tukey’s,figure

3,(d))canexplainthelesserweightgaininthistreatmentgroup.

44

45

Figure3:

Overaperiodof3months((a)-(c),comparetimepoints“week8”and“week21”ofthestudy),animalsnot

treatedwiththeghrelinagonistgainedsignificantlymoreweightthanghrelinagonisttreatedanimals((a),

p=0.009forhigh-GIgroupvs.ghrelinagonistgroup,p=0.015forcontrolsvs.ghrelinagonistgroup,one-way

ANOVA/Tukey’s).Thesamegroupsshowedatendencytogainmorefatmass((b),p=0.062forhighGIvs.

ghrelinagonistgroup,p=0.069forcontrolsvs.ghrelinagonistgroup,one-wayANOVA/Tukey’s)thanghrelin

agonisttreatedanimals.Thehigh-GIgroupgainedsignificantlymoreleanmassthantheghrelin-agonist

treatedgroup((c),p=0.048),thecontrolsshowedatendency((c),p=0.069,one-wayANOVA/Tukey’s).

Activitylevelsduringthemice’sactiveperiod(measurementstakeninweek21)werehigheringhrelin-

agonist-treatedanimalsthaninthehigh-GIandcontroldietgroups((d),p<0.001forbothcomparisons,one-

wayANOVA/Tukey’s).Immediatelyafteradministration,theghrelinagonistledtosignificantlyhigherfood

intakeduringthe2subsequenthours((e),p=0.045forAUCbetweengrayarrowsin(f),dataforasampleof

12-month-oldC57/BL6micefromadifferentstudy,t-testforunpairedsamples).However,cumulativefood

intakeasmeasuredforanentiredayhardlyeverreachedthemaximumoffoodassignedtoghrelin-agonist-

treatedanimalsasindicatedbythegraylines((g),daysrefertotheperiodwhilefoodintakewasrecorded).

BarsindicateSEM.

46

5.3.4Long-termghrelinagonisttreatmentdoesnotimpairglucosetolerance

Inordertocharacterizetheimpactsofahigh-GIdietandlong-termghrelinagonist

treatmentonglucosemetabolism,weperformedanoralglucosetolerancetestafter3

and4monthsoftreatment.Baselineglucoselevelsafter6hoursoffasting(seemethods)

didnotdiffersignificantlybetweenthegroupsineithertest(figure4,(a)).

Acomparisonoftheareaunderthetimecurve(AUC)forbothhigh-GIgroupsas

wellasthecontrolsduringthefirsttest,whichwasperformedshortlybeforethedaily

administrationoftheghrelinagonist,didnotrevealanydifferences.Thissuggeststhat

neitherthehigh-GIdietonitsownnorincombinationwithlong-termghrelinagonist

treatmentimpairedglucosetolerance(figure4,(b)).Inordertoclarifytheghrelin

agonist’sshort-termeffectsonglucosehomeostasis,inthesecondglucosetolerancetest,

wetreatedanimalswiththeghrelinagonistimmediatelybeforeadministeringthe

glucoseload.Inthisexperiment,theagonist-treatedanimalsshowedasignificantly

higherAUCthanduringthefirsttest(p=0.010,t-testforpairedsamples,figure4,(b)),

whereasthemeanAUCfortheothergroupsdidnotchangesignificantly.Therewere

alsonosignificantdifferencesinthesecondtestbetweengroups.Thisresultillustrates

thedifferentialeffectoftheghrelinagonistonshort-termandlong-termglucose

homeostasis.

Weexpectedtoseeoveralllowerendogenousacylghrelinlevelsafterlong-term

treatmentwithaghrelinagonist,hypothesizingthatartificiallyhighghrelinlevelsovera

longperiodoftimewouldleadtoadown-regulationofendogenousproductionofthe

activepeptide.However,bothserumacylanddesacylghrelinlevelsasmeasuredaftera

6-hourfastweresignificantlyhigherinthegroupfedahigh-GIdietplusghrelinagonist

thaninthegroupfedthehigh-GIdietalone(figure4,(f)and(h)).Across-reactivityin

theassaybetweenghrelinagonistandendogenousghrelincannotbeexcludedwith

47

absolutecertaintybutappearsbothhighlyunlikelyandprobablyinsignificantbecause

thelastadministrationoftheagonisttookplace24hoursbeforethebloodsampleswere

taken.Insulinlevelsmeasuredatthesametimedidnotdiffersignificantlybetween

groups(figure4,(g)).

Itcouldbespeculatedthatthelong-termghrelinagonisttreatmentledtoalower

amountofghrelinreceptorsinperipheraltissues,requiringhighercirculatingactive

ghrelinlevelsforthesamemetaboliceffects.However,thedifferentialanalysisof

peripheraltissuesforendpointsrelevanttoinsulinandghrelinsignalingwasbeyondthe

scopeofthisproject.Possiblefutureresultsofcurrentlyongoingmeasurementswillbe

discussedinaseparatepublication.

48

49

Figure4:

Baselinebloodglucoselevelsafterasixhoursfastingperioddidnotdiffersignificantlybetweentreatment

groups((a),one-wayANOVA/Tukey’s).Overall,theresultsofanoralglucosetolerancetestwerenot

influencedbylong-termghrelinagonisttreatment(AUC=areaunderthecurve,(b)-(e)).Inthesecondtest,

animalsfromthehighGI+ghrelingroupweretreatedwiththeghrelinagonistimmediatelybeforethe

glucosetolerancetestandshowedsignificantlyhigherbloodglucoselevelsthaninthefirsttest(p=0.010,t-

testforpairedsamples,(b)and(d)).Bothserumacyl((f),p=0.020,Kruskal-Wallis/Dunn’s)anddesacyl

ghrelin((h),p=0.020,ANOVA/Tukey’s)levelsmeasuredaftera6-hourfastingperiodweresignificantly

higherinanimalstreatedwiththeghrelinagonist.Therewerenosignificantdifferencesinseruminsulin

levelsinthesamesamples((g),one-wayANOVA/Tukey’s4.7).BarsindicateSEM.

5.3.5Ghrelinagonisttreatmentbeneficiallyinfluencescentralinsulinsignalingpathway

Becauseotherauthorssuggestedaninvolvementofthetumornecrosisfactorα(TNF-

α)/c-Junn-terminalkinase(JNK)pathwayinAlzheimer’sdiseasetriggeredbyAβ-

oligomers57,wemeasuredTNF-α,pSAP-JNK,andphosphorylatedinsulinreceptor

substrate1(p-IRSSer636)aswellassynaptophysinandPSD-95assynapticmarkersin

hippocampalbraintissuefromthegroupsfedthehigh-GIdiet(figure5).Wefounda

significantdifferenceinp-IRSlevelsbetweenthegroups(fig.5,(a),p=0.039,

nonparametricKolmogorov-Smirnovtest),indicatingapossibleinteractionoflong-term

ghrelinagonisttreatmentwithcentralinsulinsignaling.

Therewasamoderatenegativecorrelationinalinearregressionanalysis

betweenbehavioralresultsandp-IRSlevelsforbothgroups(r=-0.41);however,this

correlationwasnotsignificant(p=0.175,datanotshown).Wedidnotobserveany

50

differencesinstructuralsynapticmarkers,neitherpresynaptically(synaptophysin)nor

postsynaptically(PSD-95).BecausetherewerenogroupdifferencesinTNF-αorJNK-

levels,wecouldnotreproducetheTNF-α/JNKinterrelationsinAlzheimer’sdiseasein

ourmice.

51

Figure5:

AnimalstreatedwithaghrelinagonistshowedasignificantlyloweramountofphosphorylatedIRS(pIRS

Ser636),whichhasbeenshowntobeassociatedwithimpairedglucosetolerance((a),p=0.039,

nonparametricKolmogorov-Smirnovtest).However,wedidnotdetectanysignificantdifferencesin

hippocampaltissuebetweenthehighGIandhighGI+ghrelinagonistgroupsforsynaptophysin(c),

pSAP/JNK(d),TNF-α(e)orPSD-95(f).BarsindicateSEM.

5.4Discussion

Type2diabetesandAlzheimer’stypedementiaarechronicdiseases;consequently,all

symptomatictreatmentsareintrinsicallylong-term.However,moststudiesofthe

interactionsofghrelinandinsulin,whichpartlyaimedtoderivenoveltherapeutic

pathwaysindiabetes,havelookedatfairlyshorttimeframesofhours,days,orweeks83–

85.Inourstudy,wechoselong-termghrelinagonistadministrationinordertomodelthe

impactsoftherapeuticallyinfluencingthissysteminamammaloveraperiodofseveral

months.First,wecouldreproducethepreviouslyknowncognitive-enhancingeffectsof

ghrelinandghrelinagonists8,andatthesametimeweshowedthatthiseffectisseen

evenundertheinfluenceofahigh-GIdietdespitetheghrelinagonist’sshort-term

insulinostaticeffect.Thecognitive-enhancingeffectswereseeninthewatermazetest

(figure1),whichismainlyahippocampus-dependentspatiallearningtask86.This

findingunderlinestherelevanceofthisghrelinagonist’scognitiveeffectsinthe

Alzheimer’stypeofdementia,whichmostprominentlyaffectshippocampalbrainareas

andfunctions.

52

Mostinterestingly,wecouldshowalong-termeffectofghrelinagonisttreatmenton

metabolismthatdifferedfromitsshort-termactionsonfoodconsumption,weight

development,andglucosetolerance.Atthesametime,weobservedthewell-known

short-termorexigenicandinsulinostaticeffectsofthisendogenouspeptide.These

findingsindicateadifferentialmetabolicroleoftheghrelinsysteminshort-termand

long-termtreatmentandcallforafurtherdifferentiationofghrelin’slongtermroleon

glucosehomeostasis,e.g.byincludingglucoseclamptechniquesinalong-termstudy

design.Further,theobservationsinmetabolicendpointsweremadeusingtheghrelin

agonistincombinationwithahighglycemicindexdiet.Towhatextenttheresults

presentedinthismanuscriptdependonthisspecificcombinationandtowhatextent

theyarealsovalidforacombinationofanormaldietwithaghrelinagonistwillbe

addressedinfutureandongoingstudies.

Givenghrelin’sdifferentialinteractionswithinsulinsignaling,possiblyalsovia

mTORC1-dependentpathways84,87,88,wehypothesizedapotentiallyprotectiveeffectof

ghrelinagonisttreatmentoninsulinsignalinginthecentralnervoussystem.Inagonist-

treatedanimals,wefoundalowerexpressionofp-IRS-1Ser636,whichhasbeenshown

tobeassociatedwithbothperipheralinsulinresistance89,obesity90andAlzheimer’s

disease57.Wethereforespeculatethatghrelinandinsulinsignalinginthecentral

nervoussystemare,toanextent,synergistic.Ontheonehand,thehormonereduces

peripheralglucoseuptakeinperiodsoffasting,whereasontheotherhanditimproves

oratleastdoesnotreduceglucoseuptakeinthecentralnervoussysteminsituationsof

energydeficiency91.

Alimitationoftheinterpretationofthepresentresultsisthatthedataarebasedona

mousemodelforAlzheimer’sdiseaseundertheinfluenceofaveryspecifichigh-GIdiet.

53

Thelattermightexplainwhywecouldnotreplicatetheimmunohistochemistryresults

ofourpreviousstudy17.Allextrapolationofthesefindingstootheranimalmodelsmust

bedonewithcare.Furthermore,wedidnotobserveanystructuraldifferencesin

immunohistochemicalmarkersforAβplaqueloadorcentralnervoussystem

inflammationorinsynapticmarkers,whichessentiallyleavesthetaskofidentifyingan

immediatecorrelateofcognitiveenhancementbyghrelintofuturestudies.

Thepresentfindingsdosuggestthatanynewtherapeuticapproachesinboth

diabetesandneurodegenerativediseasesthatarebasedonamanipulationoftheghrelin

systemmustbeaddressedwithutmostcare.Counteractingghrelinsignalingforbetter

glucosecontrolorenhancingghrelinsignalinginthecentralnervoussystemfor

neuroprotectionandcognitiveenhancementaretwotemptingtherapeuticpathwaysin

neuroscienceandendocrinology.However,bothhavetowithstandlong-termtesting

andthepotentiallycontrastingeffectsofghrelinandghrelinagonistsinperipheral

tissuesandinthebrain.

Figure6:

Timelineofthestudy

54

5.5Methods

5.5.1Ethicsstatement

AllanimalprotocolswereapprovedbytheUniversityofAlabamaatBirmingham

InstitutionalAnimalCareandUseCommittee(IACUC).Allmethodswerecarriedoutin

accordancewiththeapprovedguidelinesandprotocols.

5.5.2Animals,diets,andtreatment

Thestudytimelineisshowninfigure6.Atotalof36maleTgAPPSwDI(humanAPPwith

Swedish,Dutch,andIowamutationsonaC57BL/6background)wereraisedunder

equaldietaryconditionsfor2months.At10weeksofage,theanimalsweredividedinto

threegroupsof12animalseachandreceivedadietconsistingof60%ofkcalin

carbohydrateswithequalamountsofmaltodextrinandsucrosepluseitherwaxymaize

starch(high-GIdietgroups)orAIN-93Gpurifieddiet(controls).Fordetaileddiet

composition,seethesupplementarymaterial.Duringthefirstweekofdietary

acclimatization,allanimalsreceiveda45-mgsucrosepelletdaily.Afterthat,thegroup

fedthehigh-GIdietplusghrelinagonistreceiveda45-mgsucrosepelletcontaining

1.66%ghrelinagonist92(LY444711;EliLilly,Indianapolis,IN)everyday(30mg/kg/day,

paralleltoourpreviousstudy17,dosedeterminedaccordingtopreviousworkby

Giddingsetal.2008,abstractaddedtosupplement);theothergroupscontinuedtobe

treatedwithsucrosepelletsasplacebos.Treatmenttookplacedailyatthesametime

between2:00and4:00pmduringtheanimals’lightcycleandcontinueduntilthe

animalsweresacrificed(treatmentperiod:week11untilweek30).Staffwatchedall

animalstakeandeatthepelletsandnotedthedayswhenthepelletwasnotconsumed.

55

Thiswasonlythecaseforfewanimalsduringdietaryacclimatization.Duringthe

treatmentperiodallanimalsatethepellets.Theamountoffoodconsumedbyallgroups

wasmeasuredevery2weeksandthethresholdoffoodrestrictionfortheghrelin-

agonist-treatedgroupwassetattheaverageleveloffoodconsumptionofthegroupfed

thehigh-GIdietalone.

5.5.3Behavioralandcognitiveassessments

Allbehavioralandcognitiveassessmentstookplacebetweenweeks22and24(seefig.

6).Allteststookplaceduringthelightcycle.Feedingtimeswerenotchanged

throughouttheassessments.

5.5.4Openfieldtest

Themazeconsistedofa42by42cm2arenawithclearsides(20cmhigh).Theanimal

wasplacedinthearenaandobservedfor4minuteswithacamera-driventracker

system(Ethovision9.5,Noldus,TheNetherlands).Thearenawassubdividedintothe

opencenterareaandthesides.Thesystemrecordedthepositionoftheanimalat5

frames/s.

5.5.5Watermaze

Thewatermazeapparatusandprocedureweredescribedindetailbefore93.Briefly,we

usedablueplasticpool,120cmindiameter,andasee-throughroundplatform,10cmin

diameter,located0.5cmbelowthewatersurface.Duringdays1through5ofthetesting

period,themiceweretrainedtofindthehiddenplatform,whichwaskeptinaconstant

56

positionthroughoutthese5days.Threetrialswererunperday;allstartingpositions

wereusedequallyinapseudo-randomorder.Themiceweregiven60stofindthe

platformand10stostayontheplatform.Ifthemousedidnotfindtheplatforminthe

assignedtime,itwasmanuallyputontotheplatform.Theinter-trialintervalduring

whichthemousewasplacedinatowel-beddeddryingcagelasted1minute.Learningof

thetaskwasevaluatedbyrecordingthelatencytimetofindtheplatform.Attheendof

thefourtrialsonday5ofthetestingperiod,themiceweretestedina60-sprobetrial

withnoescapeplatformpresent.Micethathadlearnedtheplatformposition

predominantlysearchedinthe“correct”quadrantofthepoolduringtheprobetrialor

enteredthecorrectquadrantfaster.Trialswererecordedbyusingacamera-driven

trackersystem(Ethovision9.5,Noldus,TheNetherlands).

5.5.6Zeromaze

Forthezeromazetest,weusedaroundmazewithadiameterof61cmdesignedfor

mice(SDInstruments,SanDiego,CA).Atthebeginningofthetrial,allmicewereplaced

onthesameopenpartfacinginthesamedirection.Velocity,distancesmoved,andtime

spentintheopenandclosedpartswererecordedfor4minutesbyusingacamera-

driventrackersystem(Ethovision9.5,Noldus,TheNetherlands).

5.5.7Light-dark-box

Weusedacustom-builtplasticlight-darkbox(46.5cmlength,22cmwidth,28x22cm

lightpart,18.5x22cmdarkpart).Timespentinthelightanddarkpartsaswellasthe

numberofentriesintothedarkpartwererecordedfor5minutesbyusingacamera-

57

driventrackersystem(Ethovision9.5,Noldus,TheNetherlands).Micewereplacedin

thelightpartoftheboxfacingawayfromtheentrancetothedarkpart.

5.5.8Immunohistochemistry

Animalsweresacrificedatweek30forimmunohistochemical,Westernblot,andELISA

analyses.Micewereanesthetizedwithketamine/xylazine(100/10mg/kg)andperfused

withcoldsaline.Thebrainswereremovedandcutinhalfsagittally,andtheright

hemisphereofthebrainwasplacedin4%paraformaldehydeovernight.Theleft

hemispherewasdissectedintofourpieces(rostralcortex,caudalcortex,hippocampus,

andmidbrain/brainstem)andstoredfrozenat-80oCforproteinanalysis(ELISA,

Westernblot).Therighthalfandtheintactwholebrainsfrom12animals,4pergroup,

wereputin30%sucroseforcryoprotection,and30-μmthickcoronalsectionswerecut

onafreezing-slidingmicrotome.

Sectionsfrom29brainswerestainedforAβwiththeW0-2antibody(humanAβ4-10;

1:2000;TheGeneticsCompany,Schlieren,Switzerland).Anotherseriesofsectionsfrom

thesame29brainswasstainedforIba-1(1:1000;Wako,Richmond,VA)asamarkerfor

activatedmicroglia.ForAβstaining,sectionswerepretreatedfor30minutesin85°C

sodiumcitratesolution(pH=6.5).Followingincubationwiththeprimaryantibodyin

TBS-Tovernightatroomtemperature,tissueswererinsedthreetimesandincubated

withtheappropriatebiotinylatedsecondaryantibodyfor2hoursatroomtemperature.

Sectionswereagainrinsedthreetimesandputfor2hourswiththetertiaryantibody,

extraAvidin-peroxidase.Afteranotherthreerinses,metal-enhancedDABstainingwas

usedforvisualization.Foreachantibody,allsectionswereprocessedinonestaining

tray.Allslideswereair-dried,clearedinxylene,andcoverslippedwithDPX.

58

ImageJsoftware(NIHopensource;http://imagej.nih.gov/ij/)wasusedtoanalyzethe

areaoccupiedbyAβandglialreactivityinstratumoriensofthedorsalhippocampusand

inthedorsaldentategyrus.Imagesoftheappropriatebrainareaswereacquiredwithan

OlympusDP70digitalcamera.Allimageswereacquiredinonesessiontoavoidchanges

inlightlevels.ImageJmeasurementswereperformedbyascientistwhowasblindtothe

studydesign.Fewimageshadtobeexcludedduetostaining/tissuepreparation

problems(seefig.2).

5.5.9Oralglucosetolerancetest

Inordertoavoidaprioridifferencesinbaselinebloodglucoselevels,micehadnoaccess

tofoodforaperiodofsixhoursbeforetheglucosetolerancetest.Fortheoralglucose

tolerancetest,300µlofasolutionof16.7gglucosein100mlofpurifiedwaterwas

administereddirectlyintothemice’sstomachviagavageneedles.Bloodsampleswere

takenfromtailveinsandimmediatelymeasuredwiththeTRUE2Gobloodmeasurement

system94foronebaselinetimepointandthenafter17,34,60,and90min.Themice

wereplacedinaplasticretainersystemduringtheprocedure.Onemousewasexcluded

fromtheanalysisbecauseitdidnottoleratethegavageprocess.

5.5.10ProteinextractionandWesternblotting

ForELISAandWesternblots,braintissuewashomogenizedinRIPA(150mMNaCl,

0.1%SDS,0.5%sodiumdeoxycholate,1%NP-40,50mMTris,pH8,20mMNaF,2mM

EGTA,0.5%levamisole,1mMNaVO4)plusproteaseinhibitorcocktail(p2714Sigma-

Aldrich,StLouis,MO)byuseofthefasthomogenizationprocessMinilys®(Precellys,

59

Bertin,France).AfterproteinestimationwiththeBradfordmethod95,sampleswere

dilutedtoanappropriateconcentration.

ForWesternblotting,p-IRSSer636antibody(SantaCruzBiotechnology,Dallas,TX),

synaptophysinantibodycloneSVP-38(Sigma-Aldrich,StLouis,MO),pSAPK/JNK

Thr183/Tyr185(CellSignalingTechnologies,Danvers,MA)andPSD-95antibody

(Upstate/Millipore,Billerica,MA)wereused.Afterelectrophoresisandtransferto

nitrocellulose,sampleswereincubatedwiththeprimaryantibodyovernightandwere

thenincubatedwiththesuitablesecondaryantibodyfor90minutes.Formeasuring

TNF-alpha,acommercialELISAkitwasused(EMTNFA,ThermoScientific,Rockford,IL).

5.5.11Bloodsamples

Bloodsamplesweretakenaftera6-hourfastingperiodviaintracardialpuncturefrom

theleftventricleshortlybeforetheanimalswereperfused.Samplesof250µlofblood

werecollectedinchilledEDTAtubes(Becton,DickinsonandCompany,FranklinLakes,

NJ)thatwereprefilledwith5µlof200mMAEBSFstockyieldingafinalconcentrationof

4mMAEBSF.Sampleswerecentrifugedfor20minutesat17000rpmand4°Candthe

plasmacollectedwasimmediatelyacidifiedwith200µlof1MHClper1mlofplasma.

pHwasadjustedaccordinglybeforeELISAmeasurementsforinsulinandghrelin.Acyl

ghrelinanddes-acylghrelinwasmeasuredwithacustom-built2-sitesandwichELISA47.

ForthemeasurementofinsulinacommerciallyavailableELISAkitwasused(EZRMI-

13K,EMD-Millipore,Billerica,MA).

60

5.5.12Quantitativemagneticresonanceimaging

Invivobodycomposition(totalbodyfatandleantissue)ofmicewasdeterminedby

usinganEchoMRI™3-in-1quantitativemagneticresonance(QMR)machine(Echo

MedicalSystems,Houston,TX).Asystemtestwasperformedbyusingaknownfat

standardbeforethemeasurementsweretaken.Micewereweighedandthenplaced

intoaclearholdingtubecappedwithastopperthatrestrictedverticalmovementbut

allowedconstantairflow.Thetubewasinsertedintothemachineandthemousewas

scannedbyusingNormalPrecisionmode.

5.5.13Metaboliccages

Twenty-four-hourpatternsoffoodintake,energyexpenditure(indirectcalorimetry),

andphysicalactivityweremeasuredbyusingCLAMS(ColumbusInstrumentsInc.,

Columbus,OH).Thisinstrumentalsoenforcedthefeedingregimensinanautomated,

computer-controlledmanner.Bodyweightwasmonitoredweekly.

5.5.14Activitymeasurements

Additionalactivitymeasurementsoveraperiodoffiveconsecutivelightanddarkcycles

wereperformedatweek21byusingacustom-builtinfrared-basedbeam-breaking

systemthatrecordedhorizontalandverticalmovements.Micewereplacedinthe

systemintheirhomecageswithreducedbeddinginordertonotdisruptthecontinuous

infraredmeasurements.Onlydatarecordedondays2to4wereincludedintheanalysis.

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5.5.15Statisticalmethods

AlldatasetsweretestedforGaussiandistributionusingaD’Agostino&Pearsonomnibus

normalitytest.Wheneveranormal(Gaussian)distributioncouldbevalidlyassumed,a

one-wayANOVA,thenapost-hocTukey’stestformultiplecomparisonswasusedtotest

forsignificantdifferencesbetweengroups(referredtoas“ANOVA/Tukey’s”).

NonparametricsamplesweretestedusingtheKruskal-WallistestandDunn’stestfor

multiplecomparisonsasapost-hoctest(referredtoas“Kruskal-Wallis/Dunn’s”).

Wheneveronlytwogroupswereinvolvedinthemeasurements,differencesweretested

usingat-testforpaired/unpairedsamplesinparametricdistributionsoraKolmogorov-

Smirnovtestfornonparametricdistributions.Beingawareofthenesteddataproblem96,

weonlycomparedvaluesonthesamelevelofanalysistodecreasethelikelihoodof

type-1errors.AllanalyseswereperformedwithGraphPadPrismsoftwareversion6.05

(GraphPadSoftware,Inc.,LaJolla,CA).

5.6Acknowledgements

WeparticularlythankourstudentsRebeccaWhiteandWilliamMcGilberryfortheir

dedicatedhelpwitheverydaylabwork.WealsothankDr.DanielL.Smith,Rachel

Brewer,andNathanMiyasakifortheirvaluablecriticalinputandconstructiveideas.

TheghrelinagonistLY444711waskindlyprovidedatnocostbyEliLilly,Indianapolis,

IN.AMIOCAwaxymaizestarchwasprovidedatnocostbyIngredionInc.,Bridgewater,

NJ.

62

5.7Authorcontributions

N.K.andI.K.areresponsiblefortheidea,conceptanddesignofthestudy,supportedby

thecriticalinputofT.v.G.I.K.performedallimmunohistochemicalmeasurementsin

collaborationwithT.v.G.N.K.performedallbehavioralassessments,underassistance

andsupervisionofT.v.G.andA.K.M.D.S.wasresponsibleforthelong-termfeeding

regimen,animalhealthandbothoralglucosetolerancetests,assistedbyN.K.A.K.was

responsibleforWesternBlots,inparticularqualitymanagementanddataaggregation.

D.B.A.substantiallyguidedandsupportedtheconceptionandwritingofthefinal

publicationmanuscript.Allauthorsreviewedthemanuscript.

5.8AdditionalInformation

Allauthorsdeclarenocompetingfinancialinterests.

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6.Publication2

Ghrelinaltersencoding-relatedbrainactivitywithoutenhancingmemory

formationinhumans

KunathN1*,MüllerNCJ2*,TononM1,KonradBN2,PawlowskiM1,KopczakA1,ElbauI1,

UhrM1,KühnS3,RepantisD4,OhlaK5,MüllerTD6,7,FernándezG2,TschöpM6,7,Czisch

M1,SteigerA1,DreslerM1,2

1MaxPlanckInstituteofPsychiatry,Munich,Germany2DondersInstituteforBrain,CognitionandBehaviour,RadboudUniversityMedicalCentre,Nijmegen,The

Netherlands3MaxPlanckInstituteforHumanDevelopment,Berlin,Germany4Charité–UniversitätsmedizinBerlin,DepartmentofPsychiatryandPsychotherapy,CBF,Berlin,Germany5GermanInstituteforHumanNutrition,Potsdam-Rehbrücke,Germany6InstituteforDiabetesandObesity,HelmholtzZentrumMünchen,Munich,Germany7DepartmentofMedicine,TechnischeUniversitätMünchen,Munich,Germany

*Theseauthorscontributedequally

64

6.1Abstract

Ghrelinregulatesenergyhomeostasisinvariousspeciesandenhancesmemoryin

rodentmodels.Inhumans,theroleofghrelinincognitiveprocesseshasyettobe

characterized.Hereweshowinadouble-blindrandomizedcrossoverdesignthatacute

administrationofghrelinaltersencoding-relatedbrainactivity,howeverdoesnot

enhancememoryformationinhumans.Twenty-onehealthyyoungmaleparticipants

hadtomemorizefood-andnon-food-relatedwordspresentedonabackgroundofa

virtualnavigationalroutewhileundergoingfMRIrecordings.Afteracuteghrelin

administration,weobserveddecreasedpost-encodingrestingstatefMRIconnectivity

betweenthecaudatenucleusandtheinsula,amygdala,andorbitofrontalcortex.In

addition,brainactivityrelatedtosubsequentmemoryperformancewasmodulatedby

ghrelin.Onthenextday,however,nodifferenceswerefoundinfreewordrecallorcued

location-wordassociationrecallbetweenconditions;andghrelin’seffectsonbrain

activityorfunctionalconnectivitywereunrelatedtomemoryperformance.Further,

ghrelinhadnoeffectonacognitivetestbatterycomprisingtestsforworkingmemory,

fluidreasoning,creativity,mentalspeed,andattention.Inconclusion,incontrastto

studieswithanimalmodels,wedidnotfindanyevidenceforthepotentialofghrelin

actingasashort-termcognitiveenhancerinhumans.

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6.2Highlights

- Effectsofghrelinonmemoryforfood-relatedwords-locationassociationswere

tested.

- Functionalconnectivityduringpost-encodingrestwasalteredafterghrelin

injection.

- Acuteghrelinadministrationhadnobehavioraleffectsonlong-termmemory

retention.

- Acuteghrelinadministrationhadnobehavioraleffectsonseveralothercognitive

tasks.

- Ghrelin’seffectsonmemorymarkedlydifferbetweenanimalmodelsandhuman

subjects.

6.3Introduction

Theorexigenicpeptideghrelinisinvolvedinappetiteregulation3,9,butalsoinfluencesa

numberofcognitivefunctionsinrodentmodels,suchasfearlearning,objectrecognition

andspatiallearning1,4,10,97.Thehippocampusappearstobeacentralstructurein

ghrelin’seffectsonmemory,withthepeptideleadingtoalowerthresholdforlongterm

potentiationinthedentategyrusandtoanincreaseinhippocampalspinesynapse

density7.Inanimalmodelsofneurodegenerativediseasesandage-relatedmemory

decline,ghrelinappearstoexertaneuroprotectiveeffect16–18.

Duetoitsdualroleinappetiteregulationandhippocampus-relatedmemory

formation,anevolutionaryroleofghrelininforagingprocesseswassuggested:ghrelin

mightsupportlearningoffood-associatedlocations98,99.Inhumans,effectsofghrelinon

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appetite-andmemory-relatedbrainregionshavebeenreported46,68,69,however,the

specificroleofghrelininhumancognitionisyettobedefined1,8.Studiesonthe

associationbetweenghrelinserumlevelsandcognitivefunctioninhealthyand

pathologicalaginghavebeenrathercontradictorysofar14,71,100,101.Alsoforyounger

humansubjects,inconclusiveresultshavebeenreportedfortheroleofghrelinin

memoryprocessing:memoryforfood-comparedtonon-food-relatedpictureswas

enhancedafteradministrationofghrelininasimplerecognitionparadigm46,whereas

nocturnalghrelinadministrationhadnopositiveeffectonsleep-relatedconsolidationof

asimplemotorlearningtask102.Effectsofghrelinonmorecomplexcognitiveprocesses

includingencodingorconsolidationofhippocampus-dependentmemoriesofspatialor

verbalinformationhavenotbeenstudiedyet.

Inthisstudy,21healthyyoungmaleparticipantsperformedtwosubsequentruns

ofaspatial-verballearningtaskwhileundergoingfunctionalmagneticresonance

imaging(fMRI).Theyhadtolearnfoodandnon-foodwordspresentedonthe

backgroundofaspatialnavigationenvironment(figure1).Aftereachrun,acylghrelin

orplacebowasadministeredinadouble-blind,randomized,placebo-controlledwithin-

subjectdesign,therebytestingghrelineffectsonbothpureconsolidation(pre-injection

encodingrun)andencoding(post-injectionencodingrun)processes.Memory

performancewastestedonedaylaterinbothalocation-independentfreerecalltaskand

acuedlocation-wordassociationrecalltaskusingscreenshotsofthepotentialword

presentationlocationsasspatialcues.Immediatelybeforeandaftertheencodingruns,

participantsunderwentarestingstatefMRIscan.

Ourhypothesiswasthatghrelinwouldenhancebothmemoryencodingand

consolidation,particularlyforfood-relatedinformationassociatedwithspatiallocations.

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Wefurtherhypothesizedthatthesememory-enhancingeffectswouldbereflectedby

specificactivationchangesinappetite-andmemory-relatedbrainregionssuchasthe

orbitofrontalcortex,insula,nucleuscaudatus,nucleusaccumbens,amygdala,and

hippocampus,bothintask-relatedandrestingstatefMRI.Inaddition,weexploratively

testedtheeffectsofghrelinonacognitivetestbatteryincludingworkingmemory,fluid

reasoning,creativity,mentalspeedandattentiontasks.

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Figure1:Overviewofthetestprotocol.All21participantsperformedthescheduletwiceinadouble-blind,

randomized,placebo-controlledwithin-subjectdesign.Onehourafterastandardizedlunch,twoencoding

runswereperformedunderfMRIconditions,withintravenousghrelin(orplacebo)administrationshortly

beforethesecondrun.Beforethefirstandafterthesecondrun,aneyes-closedrestingstatescan(rs-fMRI)

wasrecorded.Immediatelyaftertheinside-fMRIsessions,aseconddoseofghrelin(orplacebo)wasgivenand

participantsunderwentacognitivetestbattery.Memoryperformancewastestedonedayafterencoding

withfreewordrecallandcuedlocation-wordassociationrecall.

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6.4MaterialsandMethods

6.4.1Participants

Twenty-onemale,healthy,right-handedvolunteersattheageof23±3years(mean±SD,

range:20-30)yearsandwithabodyweightof72±7kg(range:60-80kg)participatedin

ourstudy.Theirhealthstatuswasconfirmedwithamedicalscreeningincluding

psychiatricinterview;bloodscreening(fullbloodcount,ureaandelectrolytes,liver

functionparameters,thyroidfunctionparameters,inflammatorymarkers);urine

screeningforinfectionsanddrugs;comprehensivequestionnairecoveringeatingand

sleepinghabitsandintakeofalcoholandcaffeine,andassessmentofverbalcompetence

viaastandardizedGermanvocabularytest(MWT-B103).

Exclusioncriteriawereasfollows:1)irregulareatingpatternsordietary

restraintsincludingvegetarian/vegan/lactose-freeornon-Westerndiet;2)historyofor

ongoinginflammatory,degenerative,neoplastic,endocrine,metabolic,cardiovascular,

neurologicalorpsychiatricdiseaseorseriousinjuries;3)historyoforongoingdrug

abuse;4)irregularchronobiologicalrhythmincludingshiftworkorlate-nightwork;5)

ferromagneticobjectsinsidethebody,claustrophobiaorotherconditionsthatarenot

compatiblewithfMRIprocedures;6)non-righthandednessaccordingtotheEdinburgh

HandednessInventory;7)non-nativeGermanlanguageuse.Foraperiodofoneweek

beforethefirsttestblockanduntilthelasttestblock,participantswereaskedtostickto

athree-meals-a-dayrhythm.Duringtestblocks,participantswereaskedtocompletely

refrainfromcaffeineandalcoholconsumption.Ethicalapprovalwasgrantedbythe

ethicscommitteeoftheUniversityofMunich.Accordingly,allparticipantsgavewritten

informedconsent.

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6.4.2Experimentaldesignandprocedures

Participantsweretestedinarandomized,placebo-controlled,within-subjectcrossover

design.Allparticipantscompletedtwotwothree-daytestblocks(eachconsistingofa

pre-test,alearningtrialandare-test;seefigure1),whichwereabouttwoweeks(14±4

days)apart.Thenightsinbetweenthetestdayswerespentathome.Onpre-testdays,

weexplainedthegeneralprocedureofthemainlearningtrialstoourparticipantsin

ordertoavoidunnecessarydelaysparticularlyafterthetime-sensitiveadministrationof

ghrelin.

Duringthemaintestday,participantsarrivedatourinstituteat09.00a.m.with

nopreviousbreakfast.Rightafterarrival,astandardvenouscannula(18Gor21G,

B.Braun,Germany)wasinsertedintoanantecubitalvein.Viathiscannula,5mlofblood

weretakenevery60min,duringthein-scanlearningsessionandduringthecognitive

testbattery,abloodsamplewastakenevery15min(figure1).Thebloodwasfirstfilled

intotubescontaining150µgofAprotinin/150µgEDTAandputoniceforamaximumof

60minbeforecentrifugationandfreezingoftheserumsamples.Inordertopreventthe

bloodinthecannulafromclotting,participantsreceivedaconstantinfusionofNaCl

0,9%(B.Braun,Germany)with400I.U./500mlNaClofhighmolecularweightheparin

(Ratiopharm,Germany)atacontrolledspeedof50-70mlperhour,reachingatotalof

500–700mlpertestday.Serumghrelinlevelsweremeasuredviaradioimmunoassay

bytheMaxPlanckInstituteofPsychiatryclinicalchemistrycoreunit(GhrelinactiveRIA

kit,DRGInstrumentsGmbH,Marburg,Germany).

Volunteersreceivedastandardbreakfastoftwowheatrolls,butterandjam,a

smallsausageand200mloforangejuice(intotalapprox.520kcal/2200kJ,proteins11g,

fat21g,carbohydrates70g)rightafterintravenouscatheterization,andastandardlunch

ofturkeysteakwithmushroomsauce,boiledriceandvegetablesplusachocolate

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puddingasadesert(intotalapprox.550kcal/2300kJ,proteins27g,fat13g,

carbohydrates80g)between12.00and12.30p.m.Waterwasofferedadlibitumto

participantsthroughouttheentiretestday.Allparticipantsreportedsufficientsatiety

levelsafterlunch.Beforethebeginningofthein-MRIlearningsessionsataround1.00

p.m.,a45minsbreakwastakenbeginningatthestartoflunch.Thetimebetween

breakfastandlunchwasfilledwithamovie.Alltrainingsandtestswereperformedin

thesameroomssupervisedbythesamelabpersonell.

Beforethesecondencodingsession,participantsreceivedasemi-bolusof100µg

acylghrelin(Bachem,Switzerland)dilutedin5mlaquaadinjectabilia(B.Braun,

Germany)oraplaceboof5mlNaCl0,9%(B.Braun,Germany).Theghrelindose,

representingaquantityinthemiddleofthespectrumgiveninpreviousstudies32,40,was

givenoveraperiodof2-3min,injecting1mlofthesolutionevery30-45sec.Toavoid

losinganyghrelininthebloodwithdrawalsystem,thevolumeofthetubeswas

measuredinadvanceandpre-filledwithghrelinsolutionbeforethe30-45secinjection

intervalswerestartedandflushedwithseveralmillilitersofsalinerightaftertheghrelin

injection.Therewasadelayofabout10minfromtheendoftheinjectionperioduntil

thebeginningofthesecondencodingsessioninordertoensureasufficientcentral

bioavailabilityduringthelearningprocess.Duetoacylghrelin’sshorthalf-lifetimeof

about8-12min104,105,afterthesecondrestingstatescanweinjectedanother100µgof

ghrelinintravenouslytoensureapproximatelythesameamountofghrelinbeing

measurablyavailableintheparticipant’sorganismduringthesubsequentcognitivetest

battery(seealsosupplementalfigureS1).Ghrelinorplacebowasadministered

consistentlywithintestdays,i.e.participantsreceivedeithertwoghrelinortwoplacebo

injectionsonagiventestday.

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Noneoftheparticipantsreportedanyadverseeffectsofghrelinadministration

suchasnausea,vomiting,headache,dizzinessorworse.Assomeofthesesideeffects

havebeenreportedinaprevioussystematicstudyonghrelin’spharmacological

propertiesinhumans67,wesuspectthatpossiblyadministrationasasemi-bolusmaybe

beneficial.Althoughourcognitivetestbatterydidnotincludee.g.explicithungerratings

asasubjectiveindicatorofghrelinefficacy,participantswereweabletoindicatetheir

assumptionaboutreceivingghrelinwithrelativelyhighacuityonavisualanaloguescale

(74+/-22vs.26+/-24intheghrelinvs.placebocondition,respectively).

6.4.3Cognitivetesting

Forpreparationofthelearningtask,participantstrainedontwothree-dimensional

virtualtracksbeforeeverytestday.Similarsimulationsofspatialnavigationhavebeen

successfullyusedinfMRIstudiesofspatialandgridcell-likeprocessesinthehuman

medialtemporallobebefore(Doelleretal.,Nature2010;Kunzetal.,Science2015).

Everyparticipanthadtowalkthesevirtualtracks(Sauerbraten/Cube2,

sauerbraten.org)markedbyblackboxesfourtimes,oncewiththehelpofatest

assistant,onceonhisownandtwicecountingblackboxes.Theseboxeswereplaced

exactlywherescreenshotsweretakenofthetrackandwherethewordstobelearned

thenextdaywouldappearduringthelearningsessions.Screenshotsweretakenin

approximatelythesamevirtualdistanceandpresentedintheorderofthetrack.The

numberofboxescountedbythesubjectwerenotedandcomparedtotheactualnumber

placedonthetrackinordertocontroltrainingcompliance.

Onthetestday,thespatio-verballearningtaskconsistedoftwoencodingruns

with50wordseach(25food-related,25non-food-related),inordertotestghrelin

effectsonbothconsolidation(firstrun)andencoding(secondrun).Allwordswere

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commonGermannouns(notethatinfigure1,nounsareshowninEnglishforbetter

understandingonly);encodingdifficultywasmatchedbetweenlistsandtestedinpilot

trialsindifferentsubjects.Thewordswerepresentedonscreenshotsofthetwotracks

thevolunteershadwalkedthedaybeforeintheorderoftheblackboxes,imitatingthe

verysamevirtualwalks.Screenshotswerepresentedinblocksofeightimagesfor

2500mseach,separatedbyajittered(2500–5000ms)fixationcross.Eachencoding

blockwasstartedwithabriefinstructionandcontained4-7screenshotswithwordsand

1-4emptyscreenshotsinpseudo-randomorder.Foreachencodingrun,insum,50

wordswereplacedon80screenshots(i.e.including30word-freescreenshots).In

betweentheencodingblocks,therewasarestblock(fixationcross)of17.5seconds,

duringwhichparticipantshadbeeninstructednottorehearse.

Participants´memorywastestedonthefollowingdayinatwo-stepsretrieval

test.First,afreerecallsessionof7minwasheldinwhichparticipantswereaskedto

writedownonablanksheetanyofthewordstheystillrememberedfromanyofthetwo

tracksfromthepreviousdaywithoutanycueing.Inasecondstep,emptyscreenshotsof

thetracksusedthedaybeforewerepresentedviatheprogramE-Prime.Each

screenshotwaspresentedforadurationof3secfollowedbya30secresponsetime

(blackscreen)inwhichparticipantsweresupposedtowritedownusingacomputer

keyboardwhatitemtheythinkwasplacedonthescreenshotofthisparticularlocation.

Allofthe2x80screenshotswerepresentedduringtheE-Primesessionregardlessof

whetherawordhadbeenshownonthemornot.

Acognitivetestbatteryofabout60minimmediatelyfollowedthein-fMRI

learningsessionandsubsequentsecondghrelinadministration.Itcompriseda

nonverbalfluidreasoningtest(BOMAT,10minversion106),aworkingmemorytask

(reversedigitspan107),acreativitytask(alternativeuses108),aperceptualspeedtest

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(trailmakingtaskZVT109),andtestsforreactiontimesandpsychomotorvigilance

(PVT110).

6.4.4Statisticalanalysis

Forfreewordrecall,cuedlocation-wordassociationrecall,andacombinedscore

includingallwordscorrectlyrecalledduringfreeorcuedrecallindependentofposition,

repeatedmeasuresANOVAswereperformed,eachcomprisingthefactorscondition

(ghrelinorplacebo),time(consolidationvs.encoding),andstimulus(foodvs.non-food

items)forthespatial-verballearningtask.Forthecognitivetestbattery,arepeated

measuresANOVAwiththefactorcondition(ghrelinorplacebo)wasperformed.All

behavioraldatawasanalyzedusingIBMSPSSStatisticsVersion22(IBM,Armonk,NY),

anαofp<.05wasconsideredsignificant.Separatepowercalculationsforconditionmain

effects,condition×timeinteractions,andcondition×stimulusinteractionswere

performedforeachfreerecall,cuedlocation-wordassociationrecall,andacombined

scoreofthesewithG*Power3111,assumingmediumeffectsizesoff=.25.Wefurther

performedBayesianrepeatedmeasuresANOVAswithdefaultpriorscalesforthe

free/cuedrecallcombinedscoreandthecognitivetestbatteryusingJASPVersion0.7.5.6

(jasp-stats.org).

6.4.5fMRIdataacquisition

Whole-brain functional imageswereacquiredona3T (GEDiscoveryMR750) scanner

usinga2Dgradientechoplanarimagesequence.Forboththetaskandtherestingstate

scansweusedarepetitiontime(TR)of2.5s,anechotime(TE)of30msandaflipangle

of90°.Fortherestingstatescansweacquired34interleavedsliceswithafieldofview

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(FOV) of 24 cm x 24 cm, a matrix size of 64 x 64, resulting in an in-plane spatial

resolutionof3.75mm,andaslicethicknessof3mmandaslicegapof1mm.Intotal192

volumeswereacquired.Forthelearningsessionscansweacquired42interleavedslices

withaFOVof24cmx24cm,amatrixsizeof96x96,resulting inan in-planespatial

resolutionof1.875mm,andaslicethicknessof2mmandaslicegapof0.5mm.Intotal

weacquired312volumes.

6.4.6fMRIdataanalysis

Preprocessing:AllfMRIanalyseswereconductedusingtheFMRIBSoftwareLibrary

(FSL)version6.0112.Forpreprocessing,thefunctionalimageswerecorrectedforeffects

ofheadmotionusingMCFLIRTandthebrainwasextractedusingBET.Slicetime

correctionwasdoneusingFourier-spacetime-seriesphase-shifting.Forspatial

smoothingweusedaGaussiankernelwithfullwidthhalfmaximumof6mm.Thewhole

4DVolumewasnormalizedbymultiplicationbyasinglefactor.Toremovetemporal

driftsinthedataweappliedhighpassfilterwithasigmaof50s.4Dummyvolumeswere

acquiredanddiscarded.

Task-basedanalyses:Allthedifferenttask-basedanalysisusedahierarchical

generallinearmodel(GLM)approachwiththreelevels:arunlevel,asubjectleveland

finallyagrouplevel.Onthefirstlevelwemodeledtheeventsduringeachindividualrun:

stimulusonsetsaswellasfixationeffectsweremodeled.Thestimuluseventsweresplit

intolaterrememberedandlaterforgottenitemstobecontrastedinasubsequent

memoryanalysis.Onthesecondlevelthedataofthefourruns(encoding1and2inthe

ghrelinorplaceboconditions)werecombinedusingafixedeffectmodel.This

combinationwaseitherdonebyaveragingallruns(taskmaineffect),onlycontrasting

thesecondrunplaceboversusghrelin(drugmaineffect)orcontrastingthesecond

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versusthefirstrunacrossthedays(interactionrunxdrug).Thenweusedamixedeffect

modeltocombinetheresultsonthesubjectleveltocreatethegroupstatistics.Nextto

theregressorsofinterestallfirstlevelGLMscontainednuisanceregressorsforthewhite

matterandcerebrospinalfluidsignal(1each,compartmentswereestimatedusingthe

segmentationtoolofFSLfast),and24motionparameters(3parametersforrotation,3

fortranslation,6derivativesofthese,12squaresofallofthese).AllGLMcontrastswere

correctedformultiplecomparisonsusingclustersdeterminedbyZ>2.3anda

(corrected)clustersignificancethresholdofp<0.05.Ofnote,whileithasrecentlybeen

stressedthatsomeclustercorrectionmethodsleadtoinflatedfalsepositiverates,FSL

FLAMEasusedherewasreportedtobelargelyexemptfromtheseproblems(Eklundet

al.,2015).

Foranalyzingthedesigninablockfashion,wemodeledtheonsetanddurationof

theblocksandcontrastedencodingblockswithbaselinefixationblocks.Toinvestigate

whetherghrelinmodulatestheBOLDresponseassociatedwiththeviewingoffoodvs.

non-foodwordsweusedregressorsfortheonsetsoffoodandno-fooditemsand

contrastedthemwithinrunandacrossruns.Toassessthetask-relatedbrainactivity

associatedtosuccessfulmemoryformationweperformedasubsequentmemory

analysisusingthelaterremembereditems(eitherinthefreeorthecuedrecall)and

contrastedthemwiththelaterforgottenitems,independentofthetypeofitem(foodor

non-food).

6.4.7Restingstatepreprocessing

Fortherestingstatedataweappliedthesamepreprocessingasforthetaskscans

exceptthatweremovedtwoadditionalvolumesatthestart.FortheROI-based

connectivityanalysisweusedICA-AROMA113,anICAbaseddenoisingmethodthatfilters

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outnoisecomponentsfromthedata,alsoweregressedtheglobalsignaloutasitwould

confoundROItoROIcorrelationestimates.ForthedualregressionapproachICA-

AROMAisnotnecessaryasthenoisecomponentsendupinseparateICAcomponents.

Dual Regression analysis: To investigate ghrelin-induced changes in resting state

networksweuseddualregression114.Sinceweweremost interestedinchangesofthe

defaultmodenetworkandthesaliencenetwork,weusedthe20dimensionalICAresults

ofBrainMap115,116ascomponentstoregressagainst.Thesespatialmapswerethenused

to generate subject specificmaps and time serieswith dual regression117. The spatial

mapswere then compared between the conditions using the randomize permutation

testimplementedinFSL.

Asacontrolanalysiswerepeatedthedualregression,butthistimeinsteadofusing

the established networks of BrainMap we used Melodic to estimate independent

componentsontherestingstatedataitself.TohaveanunbiasedestimateweusedFSL

MelodictoestimatetheICsduringpost-encodingrestintheplaceboconditionandthen

regressed those components against post-encoding rest in the drug and the placebo

condition. The number of dimensions of the ICA was estimated using the Laplace

approximationtotheBayesianevidenceofthemodelorder.

6.4.8ROIbasedanalysis

Foranalyzingwhetherghrelininducedchangesinfunctionalconnectivitynotona

networklevelbutonasmallerscale,weconductedanROIbasedrestingstateanalysis.

TheROIswerebasedonpreviousstudies46,68andincludedtheamygdala,hippocampus,

caudatenucleus,nucleusaccumbens,insulaandtheorbitofrontalcortex.Wecreatedthe

ROIsfromtheHarvardOxfordCorticalandsubcorticalatlasincludedinFSL.Foreach

regionweextractedthetimeseriesforeachvoxel.Betweenregionscorrelationswere

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calculatedbycorrelatingthemeantimeseriesperregion.Thecorrelationofeachregion

withtherestofthebrainwascalculatedbycorrelatingthemeantimeseriesoftheROI

withthemeantimeseriesoftherestofthebrain.Totestdifferencesforsignificancewe

usedapermutationtest.

Figure2:Ghrelinadministrationdidnotleadtoimprovedmemoryencodingorconsolidationforanyofthe

outcomemeasures.Combinedscorerepresentsitemsthatwerecorrectlyrecalledinfreerecallofwordsor

cuedrecalloflocation-wordassociations.BarsindicateSEM.

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Figure3:Comparingbothrestingstatescans(onebefore,oneafterghrelinapplication),wefounddecreased

functionalconnectivityofthebilateralcaudatenucleuswiththebilateralinsulaandrightorbitofrontal

cortex,andoftherightcaudatenucleuswiththerightamygdalaintheghrelincondition.Significanteffects

onanFDR-correctedp<.05levelareindicatedbyanasterix.

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Figure4:Performanceinnoneofthetestsusedinourcognitivetestbatterywasinfluencedbyghrelin

administration.Resultsinaworkingmemorytask(reversedigitspan),afluidreasoningtest(BOMAT

matrices),acreativitytask(alternativeuses),amentalspeedtest(trailmaking),areactiontimetask

(psychomotorvigilancetask,PVT:meanreactiontimesofthefastest10reactionsinms)andanattention

task(PVT:numberofmissesdefinedasreactiontimeover355ms)werenotdifferentbetweenconditions(all

F<2.13,p>0.16).BarsindicateSEM.

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6.5Results

Incontrast toourhypothesesandabodyofanimalresearch,wedidnot findany

positiveeffectsofghrelinadministrationonaspatial-verballearningtask(figure2).As

we injected ghrelin/placebo between two subsequent learning runs, we aimed to

differentiate between potential ghrelin effects on pure consolidation processes (first

run, before ghrelin application) and encoding processes (second run, after ghrelin

application). Given that previous findings show bettermemory performance for food

versusnonfood items inphysiological statesofhunger118 andafter ghrelin46,weused

food and non-food items as stimuli. In a repeated measures ANOVA comprising the

factors condition (ghrelin vs. placebo), time (consolidation vs. encoding) and stimulus

(food vs. non-food),we observed no significantmain effect of condition on freeword

recall (F1,20=.356, p=.558, η2=.017), cued location-word association recall (F1,20=.014,

p=0.906,η2=.001)oracombinedscorecomprisingallwordsrememberedinbothfree

and cued recall (F1,20=.271, p=.608, η2=.013). We further observed no significant

condition × time interaction, condition × stimulus interaction, or condition × time ×

stimulus interaction foranyof theoutcomemeasures (allF<1.08,p>.311,η2<.051;see

figure 2 and supplemental table T1). Given our sample size and within-subject

correlations of test scores, medium-sized main effects of ghrelin and medium-sized

condition×stimulus interactionswouldhavebeendetectedwith>95%probability for

eachfreerecall,cuedrecalloracombinedscoreofthese.Medium-sizedcondition×time

interactionswouldhavebeendetectedwith>90%probability for freerecall,andwith

>95% probability for cued recall or the combined score. Bayesian analyses of the

combinedscorewere in favorof theNullmodel(conditionBF10=0.25;condition×time

interaction BF10=0.33). Since memory was nominally even worse under ghrelin as

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comparedtoplacebo,positiveeffectsofghrelinontheperformedmemorytaskscanbe

excludedwithconsiderableconfidence.

Totesttheeffectsofghrelinonaneurobiologicallevel,wefirstanalyzedthe

interactionofcondition(ghrelinvs.placebo)andtime(consolidationvs.encodingrun)

ontask-relatedfMRIBOLDresponseforthecontrastbetweenencodingvs.restblocks.

Wefoundtherightoccipitalcortex,rightlingualgyrusandrightfusiformgyrustobe

moreactivatedintheghrelinascomparedtotheplacebocondition(seesupplemental

figureS2/supplementaltableT2),however,effectsinneitheroftheseregionswere

relatedtomemoryperformance(allp>.2).Tofurthertestwhetherghrelinaffectedthe

task-relatedfMRIBOLDresponseassociatedwithsuccessfulmemoryformation,we

conductedasubsequentmemoryanalysisandthentestedwhethertheactivationwas

modulatedbyghrelin.Contrastingallcorrectlyremembereditemswiththeforgotten

onespersubjectacrossallsessionsrevealedactivationinregionsknowntoberelated

withsubsequentmemoryforwordsandverbalassociations119suchastheleft

intraparietalsulcus,bilateralfusiformgyrus,leftparahippocampalgyrus,andleft

superiorfrontalgyrus,anddeactivationsintherightfrontalpoleandrightlateral

occipitalcortex(seesupplementalfigureS3/supplementaltableT3),whichiscongruent

withourdesignemployingwordspresentedinfrontofscenesofavirtualroute.Ina

nextstep,wetestedifghrelinmodulatesthissubsequentmemoryeffectbycontrasting

ghrelinandplaceboconditions.Wefoundincreasedactivationoftheleftintraparietal

sulcus,bilateraloccipitalcortexandprecuneusanddecreasedactivationintheleft

frontalpoleunderghrelin(figureS3/tableT3).Again,however,thesedifferences

betweenghrelinandplaceboconditionsinthesubsequentmemoryeffectdidnot

correlatewithmemoryperformance(allp>.4).InanadditionalanalysisofthefMRI

BOLDresponseassociatedwiththeviewingoffoodstimuli,wefoundalteredencoding-

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relatedbrainprocessingforfoodwordsascomparedtonon-foodwordsinthe

precuneus,occipitalcortexandleftsuperiorfrontalgyrus(seesupplementalfigureS4).

However,wedidnotfindanyenhancingormodulatingeffectofghrelinonthe

behavioralorneurobiologicaleffectsofstimulustype,i.e.foodvs.non-fooditems.

To test whether ghrelin modulated brain activation during rest, we first

performedan independentcomponentanalysis(ICA)withsubsequentdualregression

onthefMRIrestingstatedatainordertosearchforghrelin-induceddifferencesinlarge-

scale functional brain networks. Setting the focus on memory- and appetite-related

changes, we restricted our analysis to the default mode network and the salience

network.A comparisonof functional connectivitywithin thesenetworksdidnot yield

anysignificantdifferencesbetweenconditions.

InadditiontotheICAdualregressionapproach,wealsoperformedaconnectivity

analysisofthefMRIrestingstatedatabetweenthefollowingregionsofinterest(ROI)of

each hemisphere based on previous literature46,68: hippocampus, amygdala,

orbitofrontalcortex(OFC),insula,caudatenucleus,andnucleusaccumbens.Inthepost-

as compared to pre-encoding resting state, we found a reduction of functional

connectivity of the bilateral caudate nucleus with the right orbitofrontal cortex and

bilateral insula, andbetween the right caudate nucleus and the right amygdala under

ghrelincomparedtoplacebo(allpFDR<.05;seefigure3).

We did not detect any influence of ghrelin on other cognitive domains.

Performances in a working memory task (reverse digit span), a fluid reasoning test

(BOMAT matrices), a creativity task (alternative uses), a mental speed test (trail

making),andareactiontimeandattentiontask(psychomotorvigilance)didnotdiffer

significantly under the influence of ghrelin vs. placebo (all p>0.160; figure 4). All

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Bayesian analyses of the cognitive test batterywere in favor of the Nullmodel (BF10

between0.3and0.8).

Throughoutbothtestdays,bloodsampleswerefirsttakenhourly,thenevery15

minutes(figure1).Intheghrelincondition,serumghrelinlevelsbothduringthe

encodingblockandduringthecognitivetestbattery(seesupplementalfigureS1)were

markedlyhigherthanbaseline,demonstratingthatparticipantsperformedallcognitive

tasksunderstrongghrelininfluenceintheghrelincondition.

6.6Discussion

Besidesitsroleinmetabolicprocesses,accumulatingevidencefromanimalmodels

pointstoanenhancingroleofghrelinonfearlearning,objectrecognitionandspatial

memory,inparticularwhengivenbeforetheencodingphaseofmemoryformation1.On

thisbackground,thecentralghrelinreceptorhasbeenproposedasatargetforcognitive

enhancementinterventionsalsoinhumans12.Incontrasttoanimalresearch,however,

evidenceforaroleofghrelininhumanmemoryissparse.Memoryforfood-compared

tonon-food-relatedpictureswasenhancedafteradministrationofghrelininanitem

recognitionmemoryparadigm46,whereasnocturnalghrelinadministrationhadno

positiveeffectonsleep-relatedconsolidationofamotorsequencelearningtask102.

Effectsofghrelinonmorecomplexcognitiveprocessesincludingencodingor

consolidationofhippocampus-dependentmemoriesofspatialorverbalinformation

havenotbeenstudiedyet.

Manyofthecognitiveenhancingeffectsofghrelininrodentswereobservedin

hippocampus-dependentspatiallearningtaskssuchasthewatermaze17ortheplus

maze7.Duetoitsdualroleinappetiteandmemoryregulation,ghrelinhasbeen

85

suggestedtoenhancespatialmemoryforfood-associatedlocations,possiblysupporting

evolutionaryfunctionsrelatedtoforaging98,99.Ourspatiallearningtaskwasdesignedto

associateappetitiveandnon-appetitiveverbalmaterialwithabackgroundofa

naturalisticenvironmentbasedonathree-dimensionalnavigationalcomputergame,

therebytestingthisforagingfunctionhypothesis.Incontrasttobothanimalresearch

andourhypothesis,wedidnotobserveanyenhancingeffectsofghrelinadministration

oneithertheencodingorconsolidationphaseofaspatial-verbalassociationtask.This

wastrueforbothfoodandnon-foodrelateditems,andbothforfreeandspatiallycued

recall.Asalllearnedstimulihadtoberecalledonedayafterencoding,theseeffectsare

independentfrompotentiallymodulatingeffectsofghrelinonretrieval.

Ontheneurobiologicallevel,ghrelinincreasedactivityintherightoccipital

cortex,rightlingualgyrusandrightfusiformgyrusduringencoding(seesupplemental

figureS2/supplementaltableT2),howeverthiseffectwasunrelatedtomemory

performance.Ghrelinalsomodulatedthesubsequentmemoryeffectintheleft

intraparietalsulcus,bilateraloccipitalcortex,precuneus,andleftfrontalpole.This

suggeststhatsuccessfulmemoryformationwasachieveddifferentlyunderghrelinas

comparedtoplacebo,howeverwithoutanyeffectonovertbehavioralmemory

performance.

Duringpost-encodingrest,ghrelinadministrationledtodecreasedfunctional

connectivityofthecaudatenucleuswiththeamygdala,insulaandorbitofrontalcortex

(seefigure3).Generally,ghrelin’sinteractionwithdopaminergicbraincircuitsiswell

established,andanegativeassociationoftheconnectivityofthesebrainregionswith

ghrelinlevelshasbeendemonstratedfortask-relatedfMRIdatabefore:Obese

individuals,whoareknowntoexhibitdecreasedghrelinlevels120,showincreased

connectivityofthecaudatenucleuswiththeamygdala,insula,andprefrontalregions

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duringpresentationofappetizingpictures121.However,duetothelackofbehavioral

ghrelineffectsonencodingorconsolidationinourstudy,thesefunctionalconnectivity

changesareunlikelytoberelatedtomemoryprocesses.

Previousstudiesfoundghrelineffectsonpleasantnessratingsoffooditemsthat

mimickedfasting68.Inaddition,viewingfooditemsversuscontrolincreasedghrelin

release122andactivatedrewardandmemoryregionssuchasorbitofrontalcortex,

nucleusaccumbens,amygdala,insula,hippocampusandthecaudatenucleus68-46.

Enhancingeffectsofghrelinonrecognitionoffoodpictures46mightthereforebe

mediatedbyenhancedrewardprocessingrelatedtofoodstimuli123–125.Inourstudy,we

foundbetterfreerecallperformanceonthebehaviorallevelandalteredencoding-

relatedbrainprocessingforfoodwordsascomparedtonon-foodwordsonthe

neurobiologicallevel(seesupplementaltableT1andfigureS4).However,wedidnot

findanyenhancingormodulatingeffectofghrelinonthebehavioralorneurobiological

effectsofstimulustype,possiblydueitsabstractionlevelorsalience:foodnamesin

contrasttopicturesoffood.Insteadofprofitingfromtheintrinsicallyrewardingeffects

ofappetizingstimuli,participantsmighthaveutilizedthefoodcategoryasacuethat

helpedtoprimefoodwords,thusleadingtobetterfreerecallincontrasttonon-food

wordsthatdidnotformasinglecongruentcategory.Thisinterpretationissupportedby

thefactthatnosignificantdifferencebetweenfoodandnon-foodstimuliwasfoundfor

cuedrecall.

Ghrelin’sroleinmemoryprocessesmightthusberestrictedtosimpletaskswith

aclearappetitivecomponentthatactivatestherewardsystem.Incontrast,itdoesnot

increasememoryperformanceformoreabstractornon-appetitiveinformation.A

generalmemoryenhancingeffectofghrelinonhumanmemorywouldalsobe

inconsistentwithearlierfindingsthatonlyrecognitionoffoodpicturesbutnotscenes

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profitedfromghrelinadministration46.Inanimalstudies,memorytasksgenerally

involveappetitivestimuliorotherhighlysalientcomponentssuchasfearinorderto

motivatetheanimalstoperformthetask,whichmightleadtoperformanceenhancing

effectsinabroaderrangeofmemorytasksinanimalmodels.

Baselineghrelinlevelsafteranovernightfastaswellasghrelinlevels

immediatelybeforetheadministrationofthefirstdoseofghrelinvariedconsiderably,

despitematchingofourstudyparticipantsregardingage/weightandthorough

standardizationofalltestmeals,possiblyduetofactorswedidnotstandardizeforin

ourstudysuchasourparticipants’exactbodycomposition126,127.However,

hyperghrelinemiaachievedafterintravenousadministrationofghrelininourstudy

reachedconsiderablybeyondtherangeofendogenousghrelinlevels(supplemental

figureS1),therebyclearlyovercompensatinginter-individualdifferencesin

anthropometricandmetabolicparameters.Cognitivelymodulatingeffectsofghrelin

reportedinotherstudieswereachievedindifferentmetabolicstates,acrosssexesand

differentagegroupsonthebasisonsimilarlysupraphysiologicallevelsofghrelin46,68.

Nonetheless,futurestudiesneedtoaddressthequestionofsusceptibilitytoexogenous

ghrelinadministration,e.g.bydefiningrelevantmetabolicpredictors,inordertodiscern

thesubtleeffectsofghrelinoncentralnervousprocesseswhichhavebeenshownto

dependonmetabolicstateinratmodels128–130.Asfoodavailabilityseemstoplayan

importantrolewhenmeasuringcognitiveeffectsofthepeptide131,132,westrictly

standardizedfoodintakeduringtestdays.Further,ordereffectscanbeaconcernin

within-subjectcrossoverdesigns,sinceimprovementsincognitivetasksfromfirstto

secondsessionmightoccurandinteractwiththedrug.Includingtheorderofplacebovs.

ghrelininjectionsasabetweensubjectfactorintotherepeatedmeasuresANOVA,

88

however,wedidnotfindanyorder×drugconditioninteractioneffectsonencodingor

consolidationasassessedbyeitherfreeorcuedrecall(allF<.2,p>.6).

Itisimportanttonotethatrecallwastestedonedayaftermemoryacquisition.

Whileearlystudiesonghrelin’sroleinmemoryformationandcognitionalmost

exclusivelylookedatshort-termprocesses(Carlinietal.,2002;Dianoetal.,2006),

recentevidencesuggeststhatrobustfindingsthatarealsoindependentfromarousal

effectsbyacuteadministrationarefoundinlong-termtreatmentstudies(Dhurandharet

al.,2013;Kunathetal.,2015)andlikelydependonneurogeniceffects(Cahilletal.,2014;

Kentetal.,2015;Hornsbyetal.,2016).

Afurthercrucialaspectintheinterpretationofthelackofbehavioraleffectsis

thepossibilitythati.v.ghrelindidnotreachthosebrainregionsrelevantforlearning

andmemory.Inanimalmodels,divergentfindingssuggestthattheremaybedifferences

betweenspeciesconcerningtheamountofghrelincrossingtheblood-brainbarrierand

therelevantbindingsites7,133,134.Wecanpresentonlyindirectindicatorsastowhat

extentactiveghrelinactuallycrossedtheblood-brainbarrierandbecameavailableto

learning-relatedbrainregions.Whereasweobservedamygdalaconnectivitytobe

modulatedbyghrelinduringpost-encodingrestingstate,wedidnotfindhippocampal

activitytobeaffectedbyghrelinduringeithertaskorrest.Futurestudiesinhumans

involvingtechnologiessuchasMR-spectroscopy,PET-MRIorthemeasurementof

cerebrospinalfluidlevelsmaydrawaclearerpictureofhowandwhereexactlycentrally

availableghrelinmodulatesbrainmetabolism.Givenghrelin’sconsiderableinteractions

withglucosehomeostasis54,135,136,suchstudiesshouldalsoconsiderthepossibilitythat

indirecteffectsmediatedbysystemicallyhigherorlowerglucoselevelsmadeavailable

forbrainmetabolismmaybemoreimportantthantheactualdirectbindingofghrelinto

theGHS-R1aitself.

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Theaimofthisstudywastodrawamorecomprehensivepictureofghrelin’s

short-termeffectsonhumanmemoryandgeneralcognitiveperformance.Aswe

observednoimprovementinanycognitivedomaintestedinourtrial,weconcludethat

ghrelindoesnotgenerallyactasashort-termcognitiveenhancerinhumans.Differences

inthefMRIsubsequentmemoryeffectsuggestthatsuccessfulmemoryformationmight

havebeenachieveddifferentlyunderghrelin,howeverwithoutanyeffectonovert

behavioralmemoryperformance.Itwillhavetobetestedifthislackofbehavioraleffect

inhumanswillalsoholdforinformationwithstrongerappetitivevalenceorfear/stress

componentsandunderalong-termperspective.Wesuggestthatfuturestudiesaiming

attransferringthepromisingdataonghrelin’smemoryeffectsinrodentsonhuman

samplesshouldmakeaclear-cutdifferentiationofghrelin’sshort-termactionsasan

orexigenicneuropeptidepossiblymodulatingcertaincognitivefunctionssuchasfood

preferenceandappetitivebehavior46,122,137,138anditspotentialneuroprotectiveeffects

inlong-termorpathologicalmodels16,17,70,atthesametimethoroughlytakinginto

accountaspectsofsusceptibilityanddosage.

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6.7Supplementaldata

freewordrecall cuedassociationrecall combinedscore

drug F1,20=.356,p=.558,η2=.017 F1,20=.014,p=.906,η2=.001 F1,20=.271,p=.608,η2=.013

time F1,20=6.415,p=.020,η2=.243

F1,20=3.237,p=.087,η2=.139

F1,20=8.700,p=.008,η2=.303

stimulus F1,20=8.273,p=.009,η2=.293

F1,20=.816,p=.377,η2=.039 F1,20=3.720,p=.068,η2=.157

drug×time F1,20=.059,p=.811,η2=.003 F1,20=.018,p.896=,η2=.001 F1,20=.023,p=.882,η2=.001

drug×stimulus F1,20=.828,p=.374,η2=.040 F1,20=.028,p=.868,η2=.001 F1,20=.545,p=.469,η2=.027

time×stimulus F1,20=.247,p=.625,η2=.012 F1,20=1.850,p=.189,η2=.085

F1,20=.070,p=.794,η2=.003

drug×time×stimulus F1,20=.589,p=.452,η2=.029 F1,20=1.079,p=.311,η2=.051

F1,20=.328,p=.573,η2=.016

SupplementalTableT1:Forthethreedifferentoutcomemeasuresfreewordrecall,cuedlocation-word

associationrecall,andacombinedscoreofthesetwo,repeatedmeasuresANOVAscomprisingthefactors

drug(ghrelinvs.placebo),time(consolidationvs.encoding)andstimulus(foodvs.non-food)didnotreveal

anysignificantmaineffectofdrugandnosignificantinteractionofdrugwithanyoftheotherfactors.The

analysesdidrevealsignificantmaineffectsoftime,probablyduetomoreinterferenceinthesecondas

comparedtothefirstencodingrun.Further,theanalysesdidrevealasignificantstimuluseffectforfree

recall,suggestingthatparticipantscouldutilizethefoodcategoryasacuethathelpedtorecallfoodwords.

Non-foodworddidnotstemfromasinglecongruentcategory,hencenocategoricalcuecouldbeutilizedfor

these.Thisinterpretationissupportedbythefactthatnosignificantstimuluseffectwasfoundforcued

location-wordassociationrecall.

91

Voxels P Zmax Z-maxX Z-maxY Z-maxZ Z-COGX Z-COGY Z-COGZ

645 0.000938 3.49 32 -82 24 25.8 -78 34.1

408 0.0195 3.66 24 -60 -10 24.1 -70.6 -6.67

SupplementalTableT2:Clustershowingasignificantinteractionofcondition(ghrelinvs.placebo)andtime

(consolidationvs.encoding)inthepositivecontrastbetweenencodingvs.baselineblocks.Effectsarecluster-

correctedatp<0.05withZ>2.3.Foreachsignificantcluster,thenumberofvoxels,thep-value,themaximum

z-value,MNIspacecoordinatesofthemaximumz-valuevoxel,andcoordinatesofthecenterofgravity(COG)

aregiven.

92

Subsequentmemoryeffect,positivecontrast:

Voxels P Zmax Z-maxX Z-maxY Z-maxZ Z-COGX Z-COGY Z-COGZ

1815 3.57E-10 3.71 -40 -50 62 -28.1 -60.9 52.7

817 2.06E-05 3.48 -34 -30 -20 -46.9 -58.6 -2.7

812 2.20E-05 3.95 10 10 66 -1.61 11.6 61.7

671 0.000141 3.4 -28 -2 64 -41.8 4.76 43.2

347 0.0181 3.48 50 -42 -20 47.5 -50.1 -11.8

Subsequentmemoryeffect,negativecontrast:

Voxels P Zmax Z-maxX Z-maxY Z-maxZ Z-COGX Z-COGY Z-COGZ

803 2.47E-05 3.47 54 -60 42 50.6 -59.6 42.8

422 0.00534 3.82 46 46 -8 44.3 49.5 -4.82

346 0.0184 3.35 14 70 18 16.6 62.5 25.4

337 0.0215 3.62 4 -48 22 5.51 -47 26.9

Ghrelinmodulationofthesubsequentmemoryeffect,positivecontrast:

Voxels P Zmax Z-maxX Z-maxY Z-maxZ Z-COGX Z-COGY Z-COGZ

1824 4.65E-10 3.75 20 -88 16 15.9 -72.1 22.7

611 0.000381 3.75 -36 -48 70 -37.7 -49.6 63.2

442 0.00446 3.37 -34 -84 16 -31.1 -80.3 19.2

Ghrelinmodulationofthesubsequentmemoryeffect,negativecontrast:

Voxels P Zmax Z-maxX Z-maxY Z-maxZ Z-COGX Z-COGY Z-COGZ

406 0.00781 3.47 -20 58 8 -15 64 5.27

SupplementalTableT3:Clustershowingasignificantsubsequentmemoryeffect(wordsrememberedvs.

wordsforgottenafter24h,combinedscorecomprisingallwordsrecalledinfreeorcuedrecall),anda

significantmodulationbyghrelinofthesignificantsubsequentmemoryeffect.Effectsarecluster-correctedat

p<0.05withZ>2.3.Foreachsignificantcluster,thenumberofvoxels,thep-value,themaximumz-value,MNI

spacecoordinatesofthemaximumz-valuevoxel,andcoordinatesofthecenterofgravity(COG)aregiven.

93

SupplementalFigureS1:a)Groupaverage.Serumghrelinlevelsweresignificantlyhigher(p<0.0001each)

bothafterthefirstandafterthesecondinjectionthanatbaseline(„beforeinj1“,averagedvaluesofsamples

takenbeforethefirstinjection).BarsindicateSEM.b)Serumacylghrelinlevelsrosesharplyafterthefirst

injection,thentookashortdipduetoghrelin’sshorthalf-lifetimeandroseagainafterthesecondinjection

beforevanishingtowardstheendofeachtestday.BothduringthesecondlearningphaseinsidetheMRI

scannerandduringthecognitivetestbattery,supraphysiologicalserumacylghrelinlevelscouldbe

measuredinallparticipants.

SupplementalFigureS2:Interactionofcondition(ghrelinvs.placebo)andtime(consolidationvs.encoding)

inthecontrastbetweenencodingvs.baselineblocks.Effectsarecluster-correctedatp<0.05withZ>2.3.See

supplementaltableT2andtextfordetails.

94

SupplementalFigureS3,top:Subsequentmemoryanalysisofwordsrememberedvs.wordsforgottenafter

24h(combinedscorecomprisingallwordsrecalledinfreeorcuedrecall).Bottom:Subsequentmemoryeffect

asmodulatedbyghrelin.Effectsarecluster-correctedatp<0.05withZ>2.3.SeesupplementaltableT3and

textfordetails.

95

SupplementalFigureS4:Brainactivationrelatedtothepresentationoffoodvs.non-foodwords(main

effect).Effectsarecluster-correctedatp<0.05withZ>2.3.Seetextfordetails.

96

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8.Acknowledgements

ParticularthankstoDr.IngaKadishandDr.MartinDreslerwhowerebothoutstanding

supervisors

ThankstoDr.ThomasvanGroenforhismarvellousintroductiontobehavioural

experimentsinmice

ThankstoProf.AxelSteigerforhismorethanvaluableinputasanexperienced

researcher

ThankstoProf.FlorianHolsboerforgivingmetheopportunitytoworkandpublishat

hisinstitute

ThankstoAshishKumarandMatthiasTononwhosupportedtheprojectsnotonlyas

greatcolleaguesbutalsoasfriends

Thankstoalldoctorsinourteam,especiallyAnnaKopczakandherbabysonwho

supportedtheprojectevenindifficulttimes

SpecialthankstoSinjaHegerforherincrediblepatienceinsustainingfrequentperiods

ofstress-inducedgrumpiness