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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
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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.
63
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
82
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-
83
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
86
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
87
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.
89
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.
90
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
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