sign in sign up
<<
Home , Ghrelin, Thirst

181

European Journal of -19-0027 1 and Esben T Vestergaard hypopituitary patients placebo-controlled crossoverstudiesin excretion: tworandomized,double-blind, induces thirstbutdoesnotaffectsodium Acute intravenousacylghrelininfusion adrenocorticotropic (ACTH)andprolactin( gland stimulates the secretionofgrowthhormone (GH), of theGHS-Rinhypothalamus andthepituitary hormone secretagoguereceptor (GHS-R)( Acyl (AG)istheendogenousagonistforgrowth Introduction or diuresisinhumansubjects. Conclusions: increased thirst( variance fortheeffectofacylghrelininfusion Results: urinary excretionofsodiumandcreatinineweremeasured. a VisualAnalogScale(VAS).Inthesecondstudyplasmaosmolality,,copeptin,waterintake,diuresisand ghrelin (5 pmol/kg/mininthefirststudyand1 pmol/kg/minthesecondstudy).Thirstsensationwasmeasuredon two double-blindandplacebo-controlledcrossoverstudies.Thepatientsreceiveda5-hintravenousinfusionofacyl Design: hypopituitary patients. of thisclinicaltrialwastoinvestigatethedirecteffectsacylghrelinonthirstsensationandsodiumexcretionin thirst sensationhasbeenobservedasasideeffectofacylghrelinadministrationinsomehumanstudies.Theobjective is alsoorexigenicandhasrecentlybeenshowntostimulaterenalsodiumabsorptioninrodentmodels.Increased stimulates pituitarygrowthhormonerelease,andtosomedegreeadrenocorticotropicprolactin. Ghrelin Objective: Abstract Randers RegionalHospital,Randers,Denmark Hospital, Medical ResearchLaboratory,AarhusUniversity, https://doi.org/ https://eje.bioscientifica.com Clinical Study Jens Otto Lunde Jørgensen Hypopituitarypatientsonstablereplacementwithhydrocortisoneandgrowthhormonewereinvestigatedin Intheinitialstudy,acylghrelin(5 pmol/kg/min)increasedthirstsensation(time 3 Department ofPediatrics,AarhusUniversityHospital,N,Denmark, and Acylghrelin,whichistheendogenousligandforgrowthhormonesecretagoguereceptor,potently 10.1530/EJE Wedemonstratethatacylghrelininfusionincreasesthirst sensation,withoutaffectingsodiumexcretion P = 0.04) butdidnotaffecturinaryexcretionofeithersodiumorwater. -19-0027 1 , 4 , NielsMøller 1 © 2019EuropeanSociety ofEndocrinology 1 E TVestergaardandothers , 2 1 2 , Department ofDiabetesandEndocrinology,AarhusUniversity 2 , René FrydensbjergAndersen Printed inGreatBritain 1 ). Activation P = 0.003). Inthesecondstudyacylghrelin(1 pmol/kg/min)also 2 , 3 ). ).

( elevations followedbypostprandial troughs( ( AG also controls energy by inducing thirst Acyl ghrelininfusioninduces 6 4 Published byBioscientifica Ltd. ) andincreasingevidence suggests thatAGalso ) anditfluctuatesinadistinct patternwithpre-prandial Feeding anddrinkingareusually closelyconnected 4 Department ofPediatrics, 3 , SørenRittig Downloaded fromBioscientifica.com at10/01/202108:15:12PM 3 ×

treatment analysisof (2019) Endocrinology European Journalof [email protected] Email Vestergaard T E to should be addressed Correspondence 181 181 :1 , 23–30 ,

5 ). 23 –30 via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com NeoMPS, Strasbourg,France. Study2:GMP-gradehuman Synthetic human AG (Study 1: human acyl ghrelin, Preparation ofsynthetic ghrelin reported ( study anddataonmetabolismhavebeenpreviously Study 1.Thepatientsalsoparticipatedinametabolic (GCP) UnitofAarhusUniversityHospitalapproved before theonsetofenrolment.TheGoodClinicalPractice NCT00139945 (Study1)andNCT01209416 2) The protocolswereregisteredatClinicaltrials.gov Ethics CommitteeandtheDanishMedicinesAgency. protocols wereapprovedbytheCentralRegionDenmark and writteninformedconsenttoparticipate.Thestudy Helsinki Declarationandallsubjectsgavetheiroral The studieswereconductedinaccordancewiththe Subjects andmethods excretion rate. effects ofAGonwaterintake,diuresisandsodium endpoint) and secondly,(primary weinvestigated the we first assessed whether AG infusion-induced thirst stable replacementtherapywithGHandhydrocortisone, patientson randomized experimentsinhypopituitary water retention( GH andcortisolrelease,becausecausessodium effectsrelatedtoAG-induced effects ofAGfromsecondary effects onwaterorsodiumexcretion( addressed acuterenaleffectsofAG,showingno involving AGinfusions( (8 of320)volunteersparticipatinginclinicalstudies mouth’wasregisteredasanadverseeventin2.5% ‘dry have not yetbeen investigated, but increased thirst or release both Activation oftheGHS-Rpotentlystimulatesvasopressin rats stimulated by II or hypertonic saline ( polyethylene glycol-treatedhypovolemicrats( decreases waterintake( intraventricular AGadministrationdose-dependently implicated inthirstsensation( its are depolarized by AG exposure ( circumventricular (thesubfornical(SFO))and As anexample,mRNA of the GHS-R is detected in the affects fluidintake,vasopressinsecretionanddiuresis. Clinical Study In two double-blind, placebo-controlled and It remains,however, difficulttodissectthedirect Clinical effectsofAGonthirstorvasopressinsecretion 19 , in vitro 20 ). 17 , ( 18 13 ). ), and 10 ). AGisalsoantidipsogenicin 15 ). Onlyoneclinicalstudy in vivo 8 , E TVestergaardandothers 9 ) andinaneelmodel, , inrodents( 16 ). 7 ). The SFOis 11 14 ) andin ). 12 ).

All participantswereexaminedontwooccasions Study protocols a BMIof30.3 Study 2, the participants were 53 53 chronic disease.TheparticipantsofStudy1were hypertension, renaldiseaseoranyotherconcomitant participated in Study 2. None of the patients had , for stable replacementtherapywithGHandhydrocortisone menon Both studieseachincludedeighthypopituitary Subjects GCP guidelines. isotonic saline.AllprocedurescompliedwithGDPand AG; Bachem,Weil amRhein,Germany)wasdissolvedin 1 effects ( an infusionrateof1 used instudy2.Previousexperiments demonstratedthat a 5-foldlowerandmorephysiologicaldoseofAGwas by acomputerizedalgorithm. procedure wasperformedbythepharmaceuticalservices 2) forsamplingofarterializedblood.Randomization a heatedboxat65C(Study1)orinheatpad hand vein for blood sampling. The latter was placed in infusion, andonei.v. cannulawaspositioned inadorsal i.v. cannula was inserted into the antecubital region for One two randomizedanddouble-blindedinterventions. controlled crossoverstudies,eachsubjectunderwent after anovernightfast.Intwodouble-blindandplacebo- commencedat0800 Theinterventions interventions. patients emptiedtheirbladderbeforestartingtheresearch jug andglass.Water intakewasmeasuredinstudy2.The minustheremainingwaterin number ofjugservings) was measuredasamultipleof1.25 jug oftapwatercontained1.25 glass withastraw, whichmadeiteasiertodrink.Each inaclear provided inajugfromwhich,water was served during bothstudiesand study days.Tap waterwas was availableatalltimesthebedsideforsubjects oral water intake The subjectsfastedduringthetrials,butwereallowed performed inaquiet,thermoneutralindoorenvironment. weeks.Thestudieswere separated byaminimumof2 thirst Acyl ghrelininfusioninduces pmol/kg/min) orplacebo (isotonic saline‘sal’)was ± At t an effect of AG on thirst in study 1, After observing

4 years of age and had a BMI of 31.6 4 years > months. Three patients from Study 1 also 3 21 = ). 0, AG(Study1:5 ± 4.6kg/m ad libitum pmol/min/kg alsoexertedendocrine 2 Downloaded fromBioscientifica.com at10/01/202108:15:12PM . . Fresh and cool tap water L andwaterconsumption pmol/kg/min. Study2: ±

5 years of age and had 5 years L (dependingonthe 181 :1 ± 1.0kg/m 2 24 . In via freeaccess h

European Journal of Endocrinology Springs Instruments,Yellow Springs,OH,USA). glucose oxidase method (YSI 2300 STAT Plus; Yellow Instruments). point depressionmethod(Osmometer 3900,Advanced Fisher Scientific). B·R·A·H·M·S KRYPTOR CompactPlussystem(Thermo emission technique onthe resolved amplifiedcryptate Serum concentrationsofcopeptinwasmeasuredbytime- assay ( University ofSouthernDenmark)wasincorporated in the P. Bie,DepartmentofPhysiologyandPharmacology, ( using apreviouslydescribedradioimmunoassay(RIA) Sep-Pak Plus C18 cartridge (Waters, Milford, MA, USA) analyzer (RocheDiagnostics). creatinine wasanalyzedusingCREP2onaCobas6000 and wasmeasured by ISEpotentiometry wasstoredandfrozenat Biochemical analyses clamp period. period andthefromt The period from t 10 according to plasmaglucose measurements every 5.0 from t 0.6 at 120 and300 Serum concentrationsofcopeptinwasmeasuredatt was measuredinStudy2att and 300 measuredthescores. imagine’. Ablindedobserver and 100 sensation between0correspondingto‘notthirstyatall’ line (possible scores 0–100 instructed tomakeasingleverticalmarkonhorizontal 60, 120,180,240and300 Scale (VAS) att The infusionperiodslasted300 commenced. Thesodiumloadwasequaloneachoccasion. 22 Clinical Study = ). Thespecific AVP antibody(AB3096,producedby mmol/L byadjustingtheinfusionrateof20%glucose mU/kg/min, Actrapid,NovoNordisk)wasperformed 0, 120and300 Plasma osmolalitywasdeterminedusingthefreezing Serum wasstoredandfrozenat AVP wasmeasuredinplasmaafterextractiona A hyperinsulinemic–euglycemicclamp ( Plasma concentrations of arginine-vasopressin (AVP) In study2,thepatientsemptiedtheirbladderatt Thirst sensationwasdeterminedbyaVisual Analog Plasma glucosewasanalyzed bedsideusingthe 23 = 120 to300 min andtheurinewasmeasuredbyvolume. mm correspondingto‘asthirstyasyoucould ) withadetectionlimitof0.10 min, andplasmaosmolalitywasmeasured = 0, 120and300 = min instudy2. 0 to 120 min. Plasmaglucosewasclampedat min instudy2.Subjectswere mm) to indicate their current min is referred to as the basal = min instudy1andatt min. 0, 120,180and300 = E TVestergaardandothers 120 to300 − − 20°C untilanalyses. 80°C untilanalyses. pg/mL. min asthe = min. min. 120 = = 0, 0,

(2.6 Ghrelin concentrationsincreasedfrom483 Ghrelin response Results CA, USA). SigmaPlot forWindows Version 11.0,(SystatSoftware, significant. Statistical analysis was performed using and fluidintake. excretions,volumes the effectofAGandsalonurinary Student’s two-tailedpaired (‘time The interactionbetweentimeandtreatment analyzed bytwo-wayANOVA forrepeatedmeasurements. and asmedian25/75percentiles (AVP). VAS was increased inresponsetoi.v. fluidloadduringtheclamp, (77 P both studydays(1207 oral waterintakeandi.v. infusionwasalsosimilarduring vs 467 in responsetoAGandsalineinfusion(529 P increase ofVAS scoreduringAGinfusionatt ANOVA- Overall, therewasnotime VAS: Study 2 (5.9 (sal), (7.3 t =120min at infusion ANOVA- there wasatime (3.0 VAS: Study 1 AG infusion. (acylated ghrelin levels) in Study 2 ( from 49.6 to 5124 Results areexpressedasmeanandstandarderrors( Statistical analysis thirst Acyl ghrelininfusioninduces ( =0.04 = 0.73). Thediuresiswassimilar inthebasalperiod ± ± Fluid intakeanddiuresis: ± ± BaselineVAS wassimilaronbothstudydays BaselineVAS was similar onbothstudydays

20

0.5 0.7 0.7 × P ±

.0) n rmie eeae a t =300min at elevated remained and =0.004) treatment’) was consideredthe term ofinterest. mL/h (AG)vs77 P P ± L (sal), 75mL Fig. 1B mm (AG)vs3.0 mm (AG)vs2.2 mm (AG)vs3.2

= = 421 ± 0.13, but a paired t test revealed a significant 0.003. VAS score increased duringAG

9.4 pg/mL (totallevels)inStudy1( ). pg/mL atbaselineto1015.1 P valueslessthan0.05wereconsidered × treatmenteffectofAGinfusion, P ±

=

Downloaded fromBioscientifica.com at10/01/202108:15:12PM 85 ± 0.39) and the total amount of ± mm (sal), 1.1mm m (sal), 0.7mm ± × ± mL (AG)vs1179 Oralwaterintakewassimilar treatmentinteractionofAG, Lh (sal), 14mL/h

0.4 t testwasusedtoanalyze ± m (sal), 1.0mm https://eje.bioscientifica.com mm (AG)vs3.8 181 20 P 0.006) ( =0.006) :1 P ) in response to .3. Overall, =0.73). ± P ± ± .7 and =0.97) L (AG) 82mL L (sal), 79mL 34.8pg/mL ± = 52pg/mL ± 120 P 19 Fig. 1A 0.7mm =0.67). ) and min, 25 s . via freeaccess e ). . )

European Journal of Endocrinology https://eje.bioscientifica.com (in pg/mL) (0.21 (0.20/0.52) (AG) vs 0.19 (0.14/0.52) (sal), (in pg/mL)(0.21(0.20/0.52) (AG)vs0.19(0.14/0.52)(sal), osmolality P in theclampperiod(15.3 period (15.7 sodium was similar in the basalNormalized urinary in boththebasalandclampperiods,respectively. excretion bycalculatingthesodium/creatinineratio creatininesodium excretionwasnormalizedtourinary (7.5 (AG) vs6.3 similar inthebasalperiod(in (sal), P similar inboththebasal(95 ratio: (sal), affect net (479 difference intotalfluidloadanddiuresis,butAGdidnot examined if AG caused fluid retention by comparing the (152 but theincreasewassimilarontwostudydays in study2. and intravenousinfusionswasalsosimilaronbothstudydays The totalfluidintakewhichincludesbothoralwater the twostudydayswithAGandsalineinfusionin2.(D) both study1(A)and2(B).(C)Oralwaterintakewasequalon (A andB)ThirstscoreincreasedinresponsetoAGinfusion Thirst andfluidintakeduringacylghrelinsalineinfusion. Figure 1 CD A .7 ( =0.17)

= Clinical Study 0.94) andtheclampperiod(114 Plasma arginine-vasopressin, serum copeptin andplasma Plasma arginine-vasopressin, serum sodium,creatinine andsodium-to-creatinineUrinary ± ± Urinary excretionofsodium( Urinary P P

0.4 (AG)vs7.6

33 .8 ( =0.58)

= 0.27), andtheexcretionofcreatininewasalso mL/h (AG)vs173 : Arginine-vasopressin was similar at baseline Fig. 3A ± ± . (sal), 0.7

Figs 1C 3.1 (AG)vs15.2 , B and , D P C and ±

= ). . (sal), 0.7 ± 0.93) and theclampperiod ± ±

2.2 (AG)vs19.3 B Lh (sal), 18mL/h

153 2A ±

19 (AG)vs94 ± , E TVestergaardandothers mL (AG) vs 393 B . (sal), 1.5 ). μ ± mol/kg/h) (6.3

17 (AG)vs144 P .3. Urinary =0.83). μ mol/kg/h) was P P .8 and =0.88) .4. We =0.54). ± ± . (sal), 2.7 3 (sal), 13 ± 81mL ± ± 0.6 17 17

study days. the differenceintotalfluidintakeanddiuresisontwo (B) Therewasnodifferenceinfluidbalanceasestimated by saline infusioninthebasalperiodandclampperiod. infusion instudy2.(A)DiuresiswassimilarduringAGand Diuresis andfluidbalanceduringacylghrelinsaline Figure 2 association wasrecordedduring AGinfusion. vasopressin during saline infusion ( osmolality correlatedpositively withplasmaarginine- no time kg) (279.9 Plasma osmolalitywassimilaratbaseline(inmosm/ but notime (in pmol/L)(3.5 baseline beforeAGandsalinfusionbetweenstudydays there wasaslightdifferenceinserumcopeptin at effect, ANOVA- P thirst Acyl ghrelininfusioninduces B A

= 0.16), andtherewasnosignificanttime × ± treatmenteffect,ANOVA-

1.2 vs280.3 P × .9 ( =0.09 ± treatmenteffect,ANOVA-

0.5 (AG)vs3.1 Downloaded fromBioscientifica.com at10/01/202108:15:12PM i. 4A Fig. ± 1.4, P ,

= B 0.77), andtherewas and ± 181 Fig. 5 . (sal), 0.5 :1 P C .6 Plasma =0.76. ), whereas no ). Bychance, x treatment P P =0.04), =0.91. 26 via freeaccess European Journal of Endocrinology clamp periods. the sodium-to-creatinineratio (C)duringthebasalor impact onsodiumexcretion(A), creatinineexcretion(B)or saline infusioninstudy2.Acylghrelindidnothavean Urine excretionofsodiumandcreatinineduringAG Figure 3 C B A Clinical Study E TVestergaardandothers compared tosalineinfusion. vasopressin (A),serumcopeptin (B)orplasmaosmolality(C)as saline infusioninstudy2.AGdid notaffectplasma Plasma vasopressinandosmolalityduringacylghrelin Figure 4 thirst Acyl ghrelininfusioninduces C B A Downloaded fromBioscientifica.com at10/01/202108:15:12PM https://eje.bioscientifica.com 181 :1 27 via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com balances and systemic AVP and copeptin levels. The effect lower AGdoseandalsomeasured electrolyteandfluid independently ofGHandACTH. InStudy2,weuseda We clearly documented that AG infusion induced thirst and 5 1) in which we measured thirst at baseline and after 2 ( but hasonlybeenrecordedasasideeffectinsometrials previously beenstudiedasanendpointinclinicalstudies, thirst duringsomeofthestudydays.Thirsthasnot effects andpharmacokineticsofAGreportedincreased earlier blinded AG infusion studies focusing on metabolic stimulates thirst.Healthysubjectsparticipatinginour actions ( size ofameal( and theamountofwaterintakeisassociatedwith sodium excretioninhumans. vasopressin, serumcopeptin,waterintake,diuresisor infusion acutelyinducesthirst,butdoesnotaffectplasma AG. We document for the first time that intravenous AG us toinvestigateGH-andACTH-independenteffects of The inclusionofpatientswithhypopituitarismallowed diuresis andsodiumexcretionrateinhumansubjects. effects of intravenous AG infusion on thirst, water intake, The aimofthepresentstudywastoinvestigate Discussion abrogated byAGinfusion. between osmolalityandvasopressin,buttheassociationwas 2. Duringsalineinfusion,therewasapositiveassociation concentration duringacylghrelinandsalineinfusioninstudy Correlation analysesofplasmaosmolalityandvasopressin Figure 5 15 Clinical Study ). Thispromptedustoperformapilotstudy(Study Eating anddrinkingaretemporallycloselyconnected h of AG infusion in hypopituitary patients ( h of AG infusion in hypopituitary 4 , 5 ), butithasnotbeenestablishedifAGalso 6 ). AGpossessespowerfulorexigenic E TVestergaardandothers 19 ). address the dipsogenic effect of AG after interventions, address thedipsogeniceffectofAGafterinterventions, comparable withourclinicalprotocols,becausethey the publishedstudiesinanimalmodelsarenotdirectly effects ofAGinanimals( be investigated. in thesemolecularmechanisms,butthisremainsto unraveled ( these changesandtheneuronalmechanismshavebeen it hasbeendocumentedthatthirstsensationprecedes response toosmolarityandfluidchanges,butrecently, Traditionally, thirsthasbeenconsideredtoevolvein ghrelin-induced thirstisvasopressinindependent. plasma vasopressinandosmolalitysuggestingthat infusion abrogatedthepositiveassociationbetween copeptin, plasma osmolality or bloodpressure. Ghrelin impact significantlyonsystemicarginine-vasopressin, and suggestsadose-dependenteffectofAG.AGdidnot of thelower AG doseon thirst inStudy 2 wassmaller to ourknowledge,notbeeninvestigatedpreviously( ( and toalesserextentalsoACTHprolactinrelease species differenceremainstobestudied. angiotensin IIinjections( dipsogenic effectsofAGinratsafterhypovolemiaand combination ( to hypertonicsalineandangiotensinIIalonein thatAGdecreasedwaterintakeafterexposure observed intake duringdifferentdipsogenicconditionsinratsand humans. Mietlicki effects ( II injection. In rats, AG exposure had no dipsogenic hypovolemia, hypertonicsalineinfusionandangiotensin example waterdeprivation,pharmacologicallyinduced which areusuallyassociatedwithincreasedthirst produced at thebasolateral membrane inthe distal tubular GHS-R mRNA( Several peripheraltissuesincluding thekidneyexpress excretion inpatientswith chronicheartfailure( arterial pressure,butdoesnot changediuresisorsodium sympathetic activityinhealthysubjects( AG onsystemicvasopressinlevelsinhumans. and animalmodels,becausewedidnotrecordeffects of in rats( ( AG alsoactivateshypothalamicneuropeptideY(NPY) AG andGHS-Rstimulatevasopressinsecretion( Animal modelsand thirst Acyl ghrelininfusioninduces 24 3 ), buttheeffectofAGonvasopressininhumanshas, , A fewstudieshaveinvestigatedthepossibledipsogenic AG administrationcauseshypotensionanddecreases AG administrationpotentlyinducesGHsecretion 25 ) that has been shown to induce vasopressin release ) thathasbeenshowntoinducevasopressinrelease 26 12 ). Ourdatacontrastwith ), which is in line with our observations in ), whichisinlinewithourobservations 9 ). We hypothesizethatAGcouldplayarole 12 28 ). Hashimoto ) andbindsyntheticGHS ( et al. in vitro studiedtheeffectofAGonwater Downloaded fromBioscientifica.com at10/01/202108:15:12PM 11 10 ). Thereasonforthisapparent , experimentssuggestthat 11 t al. et , 12 181 in vitro ). Theprotocolsof alsoreportanti- :1 27 ) anddecreases experiments 29 per se ). AGis 28 15 16 13 for via freeaccess ). ). ).

European Journal of Endocrinology clinical implicationsremain tobefurthercharacterized. diuresis orsodiumexcretion. Thephysiologicaland subjects withoutaffectingwaterintake, hypopituitary and wasonstablereplacement withdesmopressin. than onepatientinStudy1hadcentraldiabetesinsipidus the interpretationofresults.Finally, itshould beadded inStudy1isalsoalimitation in during theinterventions period fromtheeveningbeforeinfusionstudyand water intakeassessmentinallsubjectsduringthefasting results, atypeIIerrorcannotberuledout.Fourth,lack of the samplesizewasquitesmallandforinsignificant state may not apply to non-fasting conditions. Third, thirst sensationinfastingsubjectsthepostabsorptive of above thephysiologicalrange.Second,observations limitations alsoapply. First,AGlevelswereelevated investigation ofthedirecteffectsAG,butcertain rate inhumans. not playasignificantroleindiuresisorsodiumexcretion rodents ( physiological importantregulatorofsodiumexcretionin in thestudybyKemp natriuretic effectoftheAGantagonist[d-Lys-3]-GHRP-6 studying GH-andcortisol-independent effects ofAG.The patients constituteanattractive AG-induced GHandACTHsecretion,hypopituitary thick ascendinglimbofthekidney( of theNa rats ( retention isawell-knowneffectofGHadministrationin elevated systemicAGlevelsandeffects.Ofnote,sodium possibility thatintra-renalAGinfusionsalsoresultsin subcutaneous administration( of AGonnatriuresis. between humans and rats may exist regarding the effect of proteinintheirurine( proteinuria andasrodentsexcreteconsiderableamounts ( causes increased apical E through adenylylcyclase,cAMP, proteinkinaseAand E The GHS-Rco-localizeswiththeepithelialsodiumchannel without affectingtheglomerularfiltrationrate(GFR)( and sodiumexcretionwhencomparedtobaselinelevels and intra-renal infusion of AG decreases arterial pressure The GHS-Rislocalizedonthecorticalcollectingduct, for electrolytehomeostasisandsystemicbloodpressure. cells andinthecollectingducts( 31 Na Clinical Study C inratkidney( ). E The strengthofourstudyisthatthedesignallows Systemic AGlevelsincreaseevenbylow-dose In conclusion,AGadministration increasesthirstin 34 Na ) and humans ( C-mediated sodiumreabsorptiondependson 31 + ), but our study indicates that AG , K + , 2Cl 31 ) andintra-renalAGinfusionsignals − et al. cotransporter in the medullary cotransporterinthemedullary Na 18 C expression and antinatriuresis 32 stronglypointstowardAGasa ) involving increased activity ), species-specificdifferences 30 33 E TVestergaardandothers ), whichareimportant ), anditremainsa in vivo 35 ). To obviatethe modelfor per se does 28 ).

References excellent technicalassistance. for acknowledged are Knudsen J Mrs. and Buus L Mrs. Hornemann, E Mrs. Acknowledgements grant Health ResearchFundofCentralDenmarkRegion. fellow research and Foundation Moller A.P. the Fonden, Riisfort postdoctoral from grants and Sciences) a (Medical Research Independent for by Council Danish the from supported (11-105283) was study The Funding be could that interest of conflict perceived asprejudicingtheimpartialityofthisstudy. no is there that declare authors The Declaration ofinterest

thirst Acyl ghrelininfusioninduces 11 10 4 9 8 7 6 2 1 5 3 Hashimoto H, Otsubo H,Fujihara H, Suzuki H,Ohbuchi T, Kozaka T, Fujii Y&Ando M.Centraleffectsofvariousligands Zimmerman CA, Lin YC,Leib DE,Guo L,Huey EL,Daly GE, Verbalis JG. Howdoesthe brainsenseosmolality? Cottrell GT&Ferguson AV.Pulman KJ, Fry WM, Thesubfornical controlof Fitzsimons TJ &LeMagnen J.Eatingasaregulatory Howard AD, Feighner SD,Cully DF, Arena JP, Liberator PA, Kojima M, Hosoda H,Date Y, Nakazato M, Matsuo H&Kangawa K. Cummings DE, Purnell JQ,Frayo RS,Schmidova K,Wisse BE & Wren AM, Frost GS,Murphy KG, Seal LJ,Cohen MA,Brynes AE, Takaya K, Ariyasu H,Kanamoto N,Iwakura H,Yoshimoto A, (https://doi.org/10.1007/s12576-009-0062-6) hypovolemia inrats. potently inhibitswaterintakeinduced byangiotensinIIand Yokoyama T, Takei Y &Ueta Y. Centrallyadministeredghrelin jeb.00146) Experimental Biology on drinkingbehaviorineelsacclimatedtoseawater. (https://doi.org/10.1038/nature18950) consequences ofeatinganddrinking. Chen Y &Knight ZA.Thirstneuronsanticipatethehomeostatic org/10.1681/ASN.2007070825) American SocietyofNephrology JNEUROSCI.3218-05.2006) of Neuroscience organ: acentraltargetforcirculating feedingsignals. 1969 intherat. doi.org/10.2337/diabetes.50.8.1714) . Ghrelin isagrowth-hormone-releasingacylatedpeptidefrom in mealinitiationhumans. Weigle DS. Apreprandialriseinplasmaghrelinlevels suggests arole jcem.86.12.8111) and Metabolism increases foodintakeinhumans. Dhillo WS, Ghatei MA&Bloom SR.Ghrelinenhancesappetiteand doi.org/10.1210/jcem.85.12.7167) Clinical EndocrinologyandMetabolism strongly stimulatesgrowthhormonereleaseinhumans. Harada M, Mori K,Komatsu Y, Usui T, Shimatsu A org/10.1126/science.273.5277.974) growth hormonerelease. et al. Rosenblum CI, Hamelin M,Hreniuk DL,Palyha OC,Anderson J A in pituitary andhypothalamusthatfunctionsin Areceptorinpituitary 67 273–283. Nature 2006 2001 1999 (https://doi.org/10.1037/h0026772) 2003 Journal ofComparativeandPhysiologicalPsychology Journal 26 Journal ofPhysiologicalSciences Journal 86 402 2022–2030. 5992. Science 206 656–660. Downloaded fromBioscientifica.com at10/01/202108:15:12PM 687–692. 2007 Diabetes (https://doi.org/10.1210/ 1996 Journal ofClinicalEndocrinologyJournal 18 (https://doi.org/10.1523/ https://eje.bioscientifica.com (https://doi.org/10.1038/45230) 2000 Nature 3056–3059. 273 2001 (https://doi.org/10.1242/ 974–977. 85 181 2016 50 4908–4911. 1714–1719. :1 Journal ofthe Journal et al. 537 (https://doi. 2010 Journal of Journal (https://doi. Journal Journal 680–684. Ghrelin Journal of Journal 60 (https:// (https:// 19–25. 29 via freeaccess

European Journal of Endocrinology https://eje.bioscientifica.com

24 23 22 21 20 19 18 17 16 15 14 13 12 Clinical Study Cowley MA, Smith RG,Diano S,Tschop M, Pronchuk N,Grove KL, Bie P &Sandgaard NCF. Determinants ofthenatriuresisafteracute, Emmeluth C, Drummer C,Gerzer R&Bie P. Natriuresisinconscious Vestergaard ET, Buhl M,Gjedsted J,Madsen M,Jessen N,Nielsen S, Vestergaard ET, Jessen N,Møller N&Jørgensen JOL.Acylghrelin Vestergaard ET, Gormsen LC,Jessen N,Lund S,Hansen TK,Moller N Ikkos D, Luft R&Gemzell C-A.Theeffectofhumangrowth Biglieri EG, Watlington CO & Forsham PH.Sodiumretentionwith Nagaya N, Miyatake K,Uematsu M,Oya H,Shimizu W, Hosoda H, Garin MC, Burns CM,Kaul S&Cappola AR.Clinicalreview:the Ishizaki S, Murase T, Sugimura Y, Kakiya S,Yokoi H, Tachikawa K, Korbonits M, Little JA,Forsling ML,Tringali G, Costa A, Mietlicki EG, Nowak EL&Daniels D.Theeffectofghrelinonwater Strasburger CJ, Bidlingmaier M,Esterman M,Heiman ML (https://doi.org/10.1152/ajpregu.2000.278.1.R1) Regulatory, IntegrativeandComparative Physiology slow sodiumloadinginconsciousdogs. 403–411. blockade. dogs causedbyincreasedcarotid[Na+]duringangiotensinIIand 468–477. men. metabolic effectsofintraarterialghrelininfusioninhealthyyoung Gaylinn BD, Liu J,Thorner MO,Moller N srep42706) fatty acids. induces insulinresistanceindependentlyofGH,,andfree 57 resistance andlipolysisindependentofGH-signaling. & Jorgensen JO.Ghrelininfusioninhumansinducesacuteinsulin hormone inman. org/10.1210/jcem-21-4-361) Endocrinology andMetabolism human growthhormoneanditssubfractions. 5854–5859. failure. and hormonaleffectsofghrelininfusioninpatientswithchronic Kojima M, Nakanishi N,Mori H&Kangawa K.Hemodynamic,renal, org/10.1210/jc.2012-4247) Endocrinology andMetabolism human experiencewithGhrelinAdministration. 1589–1593. vasopressin releaseinconsciousrats. Arima H, Miura Y&Oiso Y. Roleofghrelinintheregulation 2826.1999.00353.x) Neuroendocrinology hormone releasefromacuterathypothalamicexplants. hormone secretagoguesandneuropeptideYonhypothalamic Navarra P, Trainer PJ &Grossman AB.Theeffectofgrowth 37–43. intake duringdipsogenicconditions. 3205–3210. Journal ofClinicalEndocrinologyandMetabolism Journal (https://doi.org/10.1016/j.physbeh.2008.08.004) (https://doi.org/10.1111/j.1748-1716.1994.tb09760.x) (https://doi.org/10.1210/jc.2010-1995) Scientific Reports (https://doi.org/10.1210/jcem.86.12.8115) (https://doi.org/10.1210/endo.143.5.8804) Journal ofClinicalEndocrinologyand Metabolism Journal (https://doi.org/10.2337/db08-0025) 1999 Acta Endocrinologica Acta PhysiologicaScandinavica 11 2017 521–528. 1961 2013 7 42706. 21 98 Endocrinology Physiology andBehavior (https://doi.org/10.1046/j.1365- 361–370. 1826–1837. E TVestergaardandothers 1959 American Journal ofPhysiology American Journal et al. (https://doi.org/10.1038/ XXXII Journal ofClinical Journal Acuteperipheral 2000 (https://doi. Journal ofClinical Journal 2002 (https://doi. 341–361. 1994 2011 278 Diabetes Journal of Journal 143 151 et al. R1–R10. 96 2001 2009

2008

86 96

Accepted 10May2019 Revised versionreceived26April2019 Received 10January2019

thirst Acyl ghrelininfusioninduces 32 35 30 29 28 27 26 25 34 33 31 Yabuki A, Taharaguchi S, Ichii O,Kojima M,Nishi Y, Mifune H, Papotti M, Ghe C,Cassoni P, Catapano F, Deghenghi R,Ghigo E Kemp BA, Howell NL,Gray JT, Keller SR,Nass RM&Padia SH. Krapalis AF, Reiter J,Machleidt F, Iwen KA,Dodt C,Lehnert H& Willoughby JO &Blessing WW. NeuropeptideYinjectedinto Coiro V, Saccani-Jotti G,Rubino P, Manfredi G,Melani A& Dimke H, Flyvbjerg A,Bourgeois S,Thomsen K,Frøkiær J, Lees P, Lockett MF&Roberts CN.Someeffectsofgrowthhormone Veldhuis JD, Reynolds GA,Iranmanesh A&Bowers CY. Twenty- Bell ME. Albuminuriainthenormalmalerat. Kemp BA, Howell NL,Gildea JJ,Keller SR&Padia SH.Intrarenal jcem.85.10.6846) and Metabolism in peripheralhumantissues. & Muccioli G.Growthhormonesecretagoguebindingsites (https://doi.org/10.1161/HYPERTENSIONAHA.110.166413) sodium reabsorptioninnormalrats. Intrarenal ghrelininfusionstimulatesdistalnephron-dependent R1305–R1312. Regulatory, IntegrativeandComparativePhysiology vasomotor toneinhealthyhumans. Sayk F. Ghrelinmodulatesbaroreflex-regulationofsympathetic org/10.1016/0304-3940(87)90068-1) unanesthetized rat. the supraopticnucleuscausessecretionofvasopressinin humans. Chiodera P. Effectsofghrelinoncirculating neuropeptideYlevelsin S0896-6273(03)00063-1) homeostasis. demonstrates anovelhypothalamiccircuit regulatingenergy The distributionandmechanismofactionghrelinintheCNS ajprenal.00276.2006) ofNKCC2. increases phosphorylation administration inducesantidiureticandantinatriureticeffects Houillier P, Nielsen S&Frische S.Acutegrowthhormone (https://doi.org/10.1113/jphysiol.1964.sp007385) on waterdiuresisinrats. 93 GH . pulsatile, nycthemeralandentropic(feedback-regulated)modesof four hourcontinuousghrelininfusionaugmentsphysiologically sp003040) 1933 (https://doi.org/10.1038/ki.2013.187) a cAMP-dependentpathway. ghrelin receptorsregulateENaC-dependentsodiumreabsorptionby 0165-6) Biology localization ofghrelininrodentkidneys. Kamimura R, Matsumoto M&Suzuki S.Immunohistochemical 3597–3603. 79 2006 191–193. Neuro EndocrinologyLetters 126 Journal ofClinicalEndocrinologyandMetabolism Journal (https://doi.org/10.1152/ajpregu.00663.2011) 2000 (https://doi.org/10.1210/jc.2008-0620) 2007 231–238. (https://doi.org/10.1113/jphysiol.1933. Neuroscience Letters 2003 85 292 3803–3807. Journal ofPhysiology Journal F723–F735. 37 Downloaded fromBioscientifica.com at10/01/202108:15:12PM (https://doi.org/10.1007/s00418-006- 649–661. Journal ofClinicalEndocrinology Journal International 2006 (https://doi.org/10.1210/ American Journal ofPhysiology American Journal Hypertension 1987 American Journal ofPhysiology.American Journal (https://doi.org/10.1152/ (https://doi.org/10.1016/ 27 Histochemistry andCell Histochemistry 181 755–757. 75 1964 17–22. :1 Journal ofPhysiology Journal 2012 2013 2011 171 302 84 397–402. 57 (https://doi. 501–508. 633–639.

2008 30 via freeaccess