lipids96432

ARTICLEChronicephedrineadministrationdecreasesbrownadiposetissueactivityinarandomisedcontrolledhumantrial:implicationsforobesityAndrewL.
Carey&RenataPajtak&MelissaF.
Formosa&BruceVanEvery&DavidA.
Bertovic&MitchellJ.
Anderson&NinaEikelis&GavinW.
Lambert&VictorKalff&StephenJ.
Duffy&MartinH.
Cherk&BronwynA.
KingwellReceived:21December2014/Accepted:5February2015/Publishedonline:1March2015#Springer-VerlagBerlinHeidelberg2015AbstractAims/hypothesisBrownadiposetissue(BAT)activationin-creasesenergyexpenditureandmayhavetherapeuticpoten-tialtocombatobesity.
TheprimaryactivatingandadaptivesignalforBATisviaβ-adrenergicsignalling.
WepreviouslydemonstratedthathumanBATisacutelyresponsivetooraladministrationofthesympathomimetic,ephedrine.
Hereweaimedtodeterminewhetheradaptivethermogenesiscanbeinducedviachronictreatmentwithephedrine.
MethodsTwenty-threehealthyyoungmen,recruitedfromthegeneralpublicinMelbourne,Australia,whowerenon-smokers,physicallyinactiveandnon-medicatedwithnopriorhistoryofcardiovasculardiseaseordiabeteswererecruitedforthisstudy.
Theywereassignedtoreceiveeither1.
5mgkg1day1ephedrine('active'group;n=12,age23±1years,BMI24±1kg/m2)orplacebo(n=11;22±2years,23±2kg/m2)for28daysinarandomised(computer-generat-edrandomordersequence),placebo-controlled,parallel-grouptrial.
Participantsandallinvestigatorswereblindedtotreatments.
BodycompositionwasmeasuredbeforeandaftertheinterventionbydualenergyX-rayabsorptiometry.
BATactivity,measuredvia18F-fluorodeoxyglucosepositronemis-siontomography-computedtomography,inresponsetoasin-gledoseof2.
5mg/kgephedrine,wastheprimaryoutcomemeasuretobedeterminedbeforeandafterthe28daytreat-mentperiod.
ResultsTwenty-eightindividualswererandomisedandconsentedtothestudy.
Twenty-threecompletedthetrialandonlytheseparticipantswereincludedinthefinalanalyses.
After28daysoftreatment,theactivegrouplostasignificantamountoftotalbodyfat(placebo1.
1±0.
3kg,ephedrine0.
9±0.
5kg;pcomparedwiththeplacebogroup.
Inresponsetoacuteephedrine,BATactivity(changeinmeanstandardiseduptakevalue:placebo3±7%,ephedrine22±6%)andtheincreaseinsystolicbloodpressureweresignificantlyreduced(pcomparedwithplacebo.
Conclusions/interpretationChronicephedrinetreatmentre-ducedbodyfatcontent,butthiswasnotassociatedwithanincreaseinBATactivity.
Rather,chronicephedrinesuppressedBATglucosedisposal,suggestingthatchronicephedrinetreatmentdecreased,ratherthanincreased,BATactivity.
Trialregistration:ClinicalTrials.
govNCT02236962Funding:ThisstudywasfundedbytheNationalHealthandMedicalResearchCouncilofAustraliaProgramGrantElectronicsupplementarymaterialTheonlineversionofthisarticle(doi:10.
1007/s00125-015-3543-6)containspeer-reviewedbutuneditedsupplementarymaterial,whichisavailabletoauthorisedusers.
A.
L.
Carey(*):R.
Pajtak:M.
F.
Formosa:D.
A.
Bertovic:M.
J.
Anderson:S.
J.
Duffy:B.
A.
KingwellMetabolicandVascularPhysiologyLaboratory,BakerIDIHeartandDiabetesInstitute,POBox6492,StKildaRoadCentral,Melbourne,VIC8008,Australiae-mail:andrew.
carey@bakeridi.
edu.
auB.
VanEvery:V.
Kalff:M.
H.
CherkDepartmentofNuclearMedicine,AlfredHospital,Melbourne,VIC,AustraliaD.
A.
Bertovic:S.
J.
Duffy:B.
A.
KingwellDepartmentofCardiology,AlfredHospital,Melbourne,VIC,AustraliaN.
Eikelis:G.
W.
LambertHumanNeurotransmittersLaboratory,BakerIDIHeartandDiabetesInstitute,Melbourne,VIC,AustraliaG.
W.
LambertCentralClinicalSchool,MonashUniversity,Melbourne,VIC,AustraliaDiabetologia(2015)58:1045–1054DOI10.
1007/s00125-015-3543-6(1036352)andtheOISschemefromtheVictorianStateGovernment.
KeywordsAdaptivethermogenesis.
Brownfat.
Cold.
Energyexpenditure.
Ephedrine.
Noradrenaline.
Norepinephrine.
Sympathomimetic.
Type2diabetes.
Uncouplingprotein.
WhitefatAbbreviationsARAdrenergicreceptorBATBrownadiposetissueDEXADualenergyX-rayabsorptiometryNANoradrenalinePET-CTPositronemissiontomography-computerisedtomographySUVStandardiseduptakevalueUCP-1Uncouplingprotein-1WATWhiteadiposetissueIntroductionTheconclusiveidentificationoffunctionalbrown/beigeadi-posetissue(BAT)inadulthumans[1–5]anditsfunctionalimpairmentinobesity[3,6,7]hasfocussedattentiononthistissueasananti-obesitytarget.
ThisimpairmentisduetotheoxidativecapacityofBAT,which,whilerelativelylowandvariableunderbasalconditions[8–10],canincreaseunderconditionsofchronicstimulationviaaprocesscalledadaptivethermogenesis.
Chroniccoldexposureisthenaturalstimulusforadaptivethermogenesis,butmimickingcold-relatedsig-nallingpathwaysalsohasthepotentialtoelicitadaptivether-mogenesis[8].
Cold-stimulatedBATadaptivethermogenesisinhumanshasrecentlybeenreportedbyfourindependentlaboratoriesusingdifferentintermittentcoldexposureprotocols[11–13].
Prevailingviews,therefore,suggestedincreasingcoldstressbydecreasingtheambienttemperatureinhumandwellingsand/orregular,purposeful'cold-training'mayreducebodyweight,potentiallyviaincreasingBATfunctionandactivityanddecreasingbodyfat[14].
Whiletheremaybehealthben-efitsassociatedwithregularintermittentcoldexposure[15]itisunlikelytobeeffectiveforweightlossinhumans.
Prolongedcoldexposure,regardlessofintensity,willbedif-ficulttosustainand,duetocold-stimulatedhyperphagia,isunlikelytoresultinsustainedweightlossandmayactuallypromoteweightgain[16,17].
Forthepurposeofreversingobesity,identifyingpharmacologicalagentsthatincreaseBATfunctionwithoutconcomitantcentralhyperphagicsignalswouldbemosteffective[8].
Recentpharmacological(thyroxine)[18]andnutritional(capsinoidextract)[13]interventionshaveprovidedindirectevidencethatBATfunctionmaybeenhancedbychronictreat-mentwithorallybioavailableagents.
PharmacologicalstudiesinhumansinvestigatingBATthermogenesistodatehavefocussedprimarilyonsympathomimeticagents[6,19–21].
Theseagentssignalviaadrenergicreceptors(ARs),therebynotionallyreplicatingboththefacultativeandadaptivether-mogeniccentralsympatheticsignaltoBATinresponsetocoldexposure.
Werecentlyreportedthathighdosesofthesympa-thomimeticephedrinecanactivateBATinsomeleanyoungmen[6].
Thisisconsistentwiththeeffectsofacuteephedrineinmice,aspecieswherechronicadministrationofephedrineisassociatedwithadaptivethermogenesis.
ThischronicactionofephedrineinrodentBATcontrastswithothertissueswhereresponsivenesstoadrenergicagonistsisreducedwithchronicstimulation[22,23].
Inthisrespect,ephedrinereplicatessomeoftheadaptivethermogeniceffectsofchroniccoldexposureinrodents[24].
Itisunknown,however,whetherchronictreat-mentwithsympathomimeticdrugscanreplicatecold-stimulatedadaptivethermogenesisinhumans.
Whilechronicuseoftheseagentsisnotadvisedinhumansduetosignificantcardiovascularsideeffects,theyrepresentasuitabledrugclassforproof-of-conceptstudies.
Withtheadventofpositronemissiontomography-computerisedtomography(PET-CT)imagingtosemi-quantitativelyanddirectlymeasureBATac-tivity,itisnowpossibletoinvestigatethisquestion.
Accord-ingly,theaimofthepresentstudywastodeterminewhetherornotchronictreatmentwiththeorallybioavailablesympatho-mimeticephedrineincreasesBATactivityinresponsetoasingledoseofthisdrug.
MethodsTwenty-threeyoungmaleparticipants(nohistory/clinicalev-idenceofcardiovasculardiseaseandeitherdiabetes,impairedfastingglucoseorimpairedglucosetolerance,unmedicated,physicallyinactiveandnon-smokers)tookpartinthisstudy.
ThestudywasapprovedbytheAlfredHospitalEthicsCom-mitteeandperformedinaccordancewiththeDeclarationofHelsinki,SixthRevision,2008.
AConsolidatedStandardsofReportingTrials(CONSORT)checklistandflowdiagramispresentedinelectronicsupplementarymaterial(ESM)Fig.
1.
Allpatientsprovidedwritteninformedconsent.
StudydesignThisstudywasarandomised,double-blinded,placebo-controlledtrial.
Participantswererandomisedintotwogroupstoreceiveeitheractivetreatment(ephedrine1.
5mgkg1day1)orplacebo(lactose).
Dosingwasselectedbasedonpilotstudiesthatdetermined1.
5mgkg1day1tobethehighestsingledosethatcouldbesafelytakenwhileunsu-pervisedoutsidethelaboratory.
Samplesizeswerebasedonpowercalculationsdeterminedfromourpriorstudies[6,25,26].
ParticipantswererandomisedbyTheAlfredHospital1046Diabetologia(2015)58:1045–1054ClinicalTrialsPharmacystaffinblocksof4–6usingMicrosoftExcel(v2007)togeneratearandomordersequence.
Participantsvisitedthelaboratorythreetimes,alllocatedwithintheDepartmentsofCardiologyandNuclearMedicineatTheAlfredHospital.
Onvisit1,afterobtaininginformedconsentamedicalscreenandbodycompositionanalysis(dualenergyX-rayabsorptiometry[DEXA])wereconducted.
Visits2and3occurredbeforeandafterthetreatmentintervention,respectively,andincludedassessmentofBATactivity(PET-CT),whole-bodyenergyexpenditure(indirectcalorimetry)andbloodvariables(bloodglucose,NEFAandnoradrenaline[NA;norepinephrine])inresponsetoasingledoseofephed-rine(2.
5mgkg1).
Thesemeasurementsweremadeusingaprotocolpreviouslyestablishedbyus[6].
Thedayaftervisit2,participantsbegantakingasingleoraldoseofephedrine(1.
5mgkg1)orplacebobetween09:00and11:00hoursdailyfor28days.
Within2daysoftakingtheirfinaldose,partici-pantsreturnedtothelaboratory(visit3)andtheirbodycom-positionwasreassessedviaDEXA.
PreviousstudiesindicateBATactivityvariesseasonally[2,27].
WhileweconductedexperimentsbetweenMarchandOctober,participantswererandomisedinblocksoffourtosixtominimisethepotentialforseasonalvariationbetweentreatmentgroups.
Wedidnotobserveanyvariationinephedrine-stimulatedBATactivitybetweengroupsduringpre-interventionexperimentaltrials(visit2).
OutcomemeasuresTheprimaryoutcomemeasurewaschangeinBATactivity.
Secondaryoutcomemeasurescom-prisedchangesinbasalandephedrine-stimulatedenergyex-penditure,bodycomposition,circulatinghormones,lipidsandothermetabolites.
ExperimentalprotocolScreeningInitialscreeninginvolvedclinicalhistoryandex-aminationbyaphysicianandmeasurementofphysicalchar-acteristics,includingheight,weight,waist:hipratio,brachialarterybloodpressureand12-leadECG.
Afastingbloodsam-plewasdrawnformeasurementoflipidprofile(total,LDL-andHDL-cholesterolandtriacylglycerol),insulin,NA,HbA1candglucose.
AnOGTTwasthenperformed.
Briefly,partici-pantsconsumeda75gglucosesolution,afterwhichbloodglucosewasmeasuredat60and120min.
Bodycomposition(lean,boneandfatmass)wasmeasuredusingDEXA.
BATactivationtrialsBeforeandwithin24–48hafterthe4weekdrugintervention,participantsweregivenastandardisedmeal(3,180kJ;84%carbohydrate,13%protein,3%fat)toconsumetheeveningpriortoattendingthelabora-tory(at18:00–22:00hours)onbothexperimentaldays.
Laboratorytemperaturewas20–22°C.
Uponarrivalat07:30–08:00hoursafteranovernightfastandhavingabstainedfromvigorousexercise,caffeine,smokingandalco-holconsumptionforatleast2daysprior,participantsvoidedandchangedintostandardhospitalscrubsandsocks.
Theythenconsumedatelemetricpillforrecordingofcoretemper-ature(Cortemp,HQInc,Palmetto,FL,USA)andavenouscannulawasinsertedintoanantecubitalvein.
Brachialbloodpressure(PhilipsSuresignsVS3;PhilipsMedicalSystems,Andover,MA,USA)wasmeasuredevery15minandheartrate(Cortemp)wascontinuouslyrecorded.
Participantsthenrestedinasupinepositionfor2hwhilecoveredwithtwoblanketstoensurethermoneutrality.
Afterresting,energyexpenditurewasmeasuredviaindi-rectcalorimetry,abloodsamplewastakenandparticipantsthenconsumed2.
5mgkg1ephedrinehydrochlorideinagelatinecapsulewithwater.
Bloodsamplesweretakenat15,30,60and90minafterdrugingestionforsubsequentanalyses(describedbelow).
ParticipantswereinjectedwithanFDGtracerforBATglucoseuptakeassessmentviaPET-CT60minafterdrugingestion.
Energyexpenditurewasagainmeasuredviaindirectcalorimetry60–90minafterdruginges-tionbecausethistimewaspredictedtocorrespondtopeakplasmaNAconcentrationsandBATactivity[6,19].
IndirectcalorimetryEnergyexpenditurewasmeasuredwithaParvoMedicsTrueOne2400metabolicanalyser(ParvoMedicsInc,EastSandy,UT,USA).
Mixedexpiredgasesweremeasuredafter10-minequilibration.
Energyex-penditureandrespiratoryexchangeratiowerecalculatedandaveragedover20min[6].
PET-CTimagingPET-CTimagingandanalyseswerecon-ductedaspreviouslydescribed[6].
PET-CTvariablesresultedinaneffectiveradiationdoseofcompassalargertissueregion.
Wedidnot,however,observeincreasedsupraclavicularadiposeSUVmaxabovebasallevelsinthemajorityofparticipantsinresponsetoacuteephedrine.
Inlightofthisfinding,andgiventheprolongedinterventionalnatureofthisstudy,weconducteda'fixedvolume'analysis[35].
FromthreeconsecutivePETimageslices(4mmthick-ness)withinthesupraclavicularregion,SUVmeanfromre-gionsofinterestof100mm2weredeterminedonleftandrightsides.
Thesedataare,therefore,reportedasSUVmeanper2,400mm3.
SUVmaxandSUVmeanaredeterminedinunitsofg/ml,howeverneitherourmethodofreconstructionanddataanalysisnortheformulatoquantifythesevariablesarequanti-tative.
Dataare,therefore,presentedsimplyas'SUVmax'and'SUVmean'withoutunits.
BiochemicalanalysesWhererequired,plasmawascentri-fugedandfrozenforanalyses.
Plasmawasmeasuredforglu-cose,totalcholesterol,HDL-cholesterol,LDL-cholesterol,tri-acylglycerol,insulin,HbA1candNAasdescribed[6].
PlasmaNEFAlevelsweremeasuredusingacommerciallyavailablekit(WacoDiagnostics,Richmond,VA,USA).
StatisticalanalysesPhysicalcharacteristicsbetweengroupsandchangeinbodycompositionbetweengroupsasaresultoftreatmentswerecomparedusingunpairedtwo-tailedStu-dent'sttests.
Todetermineeffectsofacuteephedrinetreat-mentonenergyexpenditure,thechangeinresponsetotheacuteephedrinetreatmentwasdeterminedbothbefore(Pre)andafter(Post)thechronictreatmentineachgroup.
ThechangeinthisvaluefromPretoPostwasthendeterminedandthischangewascomparedbetweenthetwotreatmentgroupsusinganunpairedtwo-tailedStudent'sttest.
Thesameanalysiswasappliedforcomparisonofhaemodynamic,coretemperatureandcirculatingfactordata,howeverthechangeinresponsetoacuteephedrinetreatmentforeachgroupandtime-pointwasdeterminedbysubtractingthebasalvaluefromthemeanofvaluesobtainedbetween60–90minaftertheacuteephedrinedose.
Aspreviouslyreportedbyus[6]andothers[19],thepeakinacutephysiologicalresponsestooralephedrinevariesbetweenindividualsbutoccurswithinthisperiod.
EffectsonBATactivityweredeterminedasdescribedaboveforbodycompositiondata,howeversincePET-CTdatawerenotnormallydistributed,anon-parametricMann–WhitneyUtestwasusedtocomparebetweengroups.
AnalyseswereconductedusingSPSS(v15)andMicrosoftExcel.
Resultsareexpressedasmean±SEM,andresultswerecon-sideredsignificantwhenp≤0.
05.
ResultsBaselineparticipantcharacteristicsarepresentedinTable1.
Groupswerenotstatisticallydifferentforallcriteria.
Table2showsbodycompositionatbaselineandinresponsetothe28-dayintervention.
Thechangeintotalmass(changeplacebo1.
1±0.
3kg;ephedrine0.
9±0.
5kg),bodyfatmass(changepla-cebo0.
5±0.
2kg;ephedrine1.
1±0.
3kg),percentbodyfat(changeplacebo0.
4±0.
2%;ephedrine1.
2±0.
3%)andesti-matedvisceraladiposetissuemass(changeplacebo6.
4±19.
1g;ephedrine134±43g)betweengroupswereallsignif-icantlyreducedafterchronicephedrinetreatmentonly(Table2;p0.
05forall(groupscomparedusinganunpairedStudent'sttest)1048Diabetologia(2015)58:1045–1054treatedgroupbutwasunchangedintheplacebogroupwhenmeasuredaschangeinSUVmax(Fig.
1a;meanchangepla-cebo3±6%;ephedrine13±7%,p=0.
03)orSUVmean(Fig.
1b;meanchangeplacebo3±7%;ephedrine22±6%;p=0.
01).
SUVmaxinWATwashalfthatofBATandwasunchangedinbothgroupsinresponsetoacuteephedrinebe-forechronictreatment(placebopre0.
48±0.
02,placebopost0.
45±0.
02;ephedrinepre0.
45±0.
02,ephedrinepost0.
43±0.
03).
Basalenergyexpendituredidnotchangeasaresultofchronicephedrinetreatment(Table3).
Theincreaseinenergyexpenditureinresponsetoacuteephedrinewasunchangedafterthechronictreatmentperiodbetweengroups(Table3).
Therespiratoryexchangeratiowasnotaffectedbyeitheracuteorchronicephedrinetreatment(Table3).
Thechangeinsystolicbloodpressure,heartrate,coretem-perature,bloodglucose,plasmaNAandplasmaNEFAsinresponsetotheacutedoseofephedrineareshowninFig.
2bothbefore(Pre)andafter(Post)the28-daychronicephedrineintervention.
Comparedwithplacebo,inthechronicephed-rinetreatmentgroupthechangefrompre-topost-interventionwassignificantlylessforsystolicbloodpressure(pcompositionCharacteristicPlaceboEphedrinePrePostChangePrePostChangeTotalmass(kg)74.
3±2.
275.
4±2.
31.
1±0.
380.
0±2.
879.
1±2.
90.
9±0.
5aLeanmass(kg)54.
6±1.
855.
1±1.
90.
56±0.
355.
9±1.
356.
2±1.
40.
24±0.
3Fatmass(kg)16.
8±2.
117.
3±2.
10.
50±0.
220.
9±2.
319.
8±2.
31.
1±0.
3aFatmass(%totalmass)23.
2±2.
623.
6±2.
50.
4±0.
226.
6±2.
225.
4±2.
21.
2±0.
3aeVAT(g)493±109500±1166.
4±19.
1512±73378±95134±43aBonemineralcontent(g)2,915±952,917±951.
6±943,150±943,132±9418±8Valuesaremean±SEMapcomparedwithplacebo(thepre–postchangewascomparedbetweentreatmentgroupsusinganunpairedStudent'sttest)eVAT,estimatedvisceraladiposetissueab0PrePostPrePostPrePostPrePost0.
51.
01.
52.
02.
5*00.
51.
01.
52.
0*SUVmaxSUVmean(per2,400mm3)Fig.
1(a)SUVmaxand(b)SUVmeanvaluesplottedforeachindividualinsupraclavicularbrown/beigeadiposetissueinresponsetotreatmentwithasingledoseofephedrine(2.
5mgkg1),before(pre)andafter(post)28daystreatmentwithplaceboorephedrine(1.
5mgkg1day1).
SUVmaxgroupmeanvalues:placebopre(opencircles)0.
98±0.
09;post(opensquares)0.
96±0.
08;ephedrinepre(closedcircles)0.
98±0.
13;post(closedsquares)0.
80±0.
07.
SUVmeangroupmeanvalues:placebopre0.
71±0.
06;post0.
70±0.
05;ephedrinepre0.
76±0.
10;post0.
55±0.
04.
*pcomparedbetweentreatmentgroupsusinganon-para-metricMann–WhitneyUtest)Diabetologia(2015)58:1045–10541049withsympatheticactivation,whichmayleadtobluntedsym-patheticresponsivenessincertaintissues[36].
Ourfindingsmay,therefore,contributetoanexplanationfortheobservedreductioninBATfunctioninobeseindividuals.
BATbiopsiesandothermeasureswerenotpossibletodi-rectlymeasureBATfunctionorBATsympatheticnerveactiv-ity.
Nevertheless,areductionintheincreaseinsystolicbloodpressureandbloodglucose,andatrendtowardsabluntingofTable3IndirectcalorimetryCharacteristicPlaceboChronicephedrinePrePostChangePrePostChangeBasalenergyexpenditure(kJh1)287±17285±82±14286±16287±100.
4±15Ephedrine-stimulatedenergyexpenditure(kJh1)326±14322±104±13340±11323±1116±11BasalRER0.
84±0.
020.
82±0.
010.
02±0.
020.
84±0.
020.
81±0.
010.
03±0.
01AcuteephedrineRER0.
82±0.
010.
82±0.
010.
01±0.
020.
85±0.
040.
84±0.
020.
02±0.
04Whole-bodyenergyexpenditureandRERmeasuredbefore(basal)and70–90minafter(ephedrine-stimulated)ingestionofasingledoseofephedrine(2.
5mgkg1),measuredbefore(pre)andafter(post)28daystreatmentwitheitherplaceboorephedrine(1.
5mgkg1day1).
ChangerepresentsthedifferencebetweenpreandpostValuesaremean±SEM.
(Thepre–postchangewascomparedbetweentreatmentgroupsusinganunpairedStudent'sttest)RER,respiratoryexchangeratioPrePostPrePostPrePostPrePostPrePostPrePostPrePostPrePostPrePostPrePostPrePostPrePost0510152025303540ΔSBP(mmHg)a*05101520ΔHR(bpm)b00.
10.
20.
30.
40.
5ΔCoretemperature(°C)c-0.
200.
20.
40.
60.
81.
0ΔBloodglucose(mmol/l)d**02004006008001,0001,200ΔPlasmaNA(pmol/l)e020406080100120140ΔPlasmaNEFA(pmol/l)fFig.
2Changes(Δ)in(a)systolicbloodpressure(SBP),(b)heartrate(HR),(c)coretemperature,(d)bloodglucose,(e)plasmaNA(p=0.
06forchangefrompre-topost-treatmentbetweengroups)and(f)NEFAfromimmediatelypriortoingestionofasingledoseofephedrine(2.
5mg/kg)tothemeanofvaluestakenbetween60and90minafteringestionofthedoseofephedrine,before(pre)andafter(post)28daystreatmentwithplaceboorephedrine(1.
5mgkg1day1).
Openbars,placebo;closedbars,ephedrine.
*pcomparedbetweentreatmentgroupsusinganunpairedStudent'sttest)1050Diabetologia(2015)58:1045–1054theriseinplasmaNAinresponsetoacuteephedrineinthechronicephedrinetreatmentgroupsupportabluntingofsym-patheticresponsivenesstothisprotocol.
Thesympathomimet-icactionofephedrineisbasedonincreasedendogenousNAreleasefromsympatheticnerveterminalsandblockadeofitsreuptake,therebyincreasingandprolongingsynapticNAcon-centrations,spilloverintocirculationandsubsequentexposuretocells[37].
Thus,themechanismbywhichchronicephed-rinetreatmentdownregulatesadiposeadrenergicsensitivitytoacuteephedrineinhumansislikelytobeduetodirectdown-regulationofβ-ARontargettissuesand/oralteredsynapticNArelease/reuptakeviasynapticNAtransporters.
BluntingtheriseinplasmaNAresponsetoacuteephedrineinthechronicephedrinegroupindicatesanalterationinthesynapticregulationofNAreleaseand/orreuptake.
Further,inrodents,chronicβARstimula-tionwithisoproterenoldecreasesβ-ARsensitivityandreceptordensity[38],thereforebothmechanismsareprobablyinvolved.
Inmanytissues,stimulationofβ-ARsresultsinbothshort-term(hours)andchronic(days)downregulationofreceptorsensitivityanddensity,respectively[23,39].
Rodentstudies,however,indicatethatBATβ3-ARareunaffected,thusallowingadaptivethermogenesistooccurinresponsetoper-sistentstimulation[22,23].
Accordingly,chronicephedrinetreatmentincreasesuncouplingprotein-1(UCP-1)andBATactivityinresponsetoacuteephedrinetreatmentinmice[24],andsimilarresultshavebeenreportedwithβ3-ARagonisttreatmentinadultdogs,whichreportedlyhavefunctionallysimilarBATtoadulthumans[40].
Itisunknownwhichβ-ARsareprimarilyresponsibleforhumanBATthermogenesis,however,whileexpressed[4],β3-ARcontentislikelytobelow[23]andtohavelittle[41,42]orno[43]involvementinhumanthermogenesis.
Thepresentdata,therefore,supportthenotionthathumanBATisfunctionallydistinctfromthatofsmallrodentsandotherlargermammalssuchasdogs.
HumanBAThasrecentlybeenreportedtobecomposedpredominate-lyofbeigeratherthanclassicbrownadipocytes[29,31–33],andarecentinvitrostudyhighlightedthathumanandmouseBATcellsexpressUCP-1inanopposingmannerinresponsetoall-transretinoicacidtreatment[44].
Moreover,chronicephedrinetreatmentmimicsadaptivethermogenesisinmice[24]andnostudyhasreportedareductioninfacultativeoradaptivethermogenesisinclassicbrownorbeigeadiposede-potsinmiceinresponsetosympatheticstimuli.
Therefore,itislikelyhumanBATrequiresalternatestimulitobecomere-sponsivetoadrenergic/sympatheticinputs,andthismaycon-tributetothefailureofβ3-ARagoniststoincreaseBATac-tivityinhumans[45].
Micerequireallβ-ARsforfullfunctionofclassicBAT,butarepredominantlydependantontheβ3-AR[22,23,46].
SincehumanBATisnotgenerallyaswelladaptedasmurineBATitmayrequireproliferativeandadipogenicstimulationtoprecedebrowningtomaximiseadaptivethermogenesis.
Theseprocessesmaybemoredependentonβ1-ARthanβ3-ARsignalling[23,47],thuspotentiallyexplainingadesensitisationratherthanadaptivephenomenonmorerepre-sentativeoftissuesthathighlyexpressβ1/2-ARs.
Fromanobesitytherapeuticperspective,infutureitwould,therefore,beprudenttoconsiderhumanBATasgenotypicallyandphe-notypicallydistinctfrommurineBAT.
Additionally,alteredsympatheticfunctionhasbeenimplicatedasbothacauseandconsequenceofobesity[36]andweightlossreversesdysregulatedbasalandglucose-stimulatedsympatheticre-sponsiveness[48,49].
NeitherreducedBATactivitynorsym-patheticover-activityhavebeencausativelylinkedtoobesity.
Since,however,BATactivityisreducedandbasalsympathet-icnervoussystemactivityisincreasedinobesity,thepresentdataconnectingchronicsympatheticover-stimulationtore-ducedBATactivitystrengthensthehypothesisthattheseob-servationsarelinked.
Inourpreviousstudy,upperthoracicBATactivity,mea-suredasSUVmax,inresponsetoacuteephedrinewas~2.
0,whichwas~1.
0higherthantheacuteresponsetoplacebo[6].
Inthecurrentstudy,baselinevaluesforSUVmaxinresponsetoacuteephedrinewerelowerthoseofourpreviousstudyat~1.
0.
Nevertheless,chronicephedrinetreatmentreducedSUVmaxcomparedwithplacebo.
ConsideringthatSUVmaxofdeepupperthoracicBATissignificantlyhigherthansub-cutaneousWATdepots(~100%and>2SDsinthepresentstudyandourpreviouslyreportedbasallevels[6]),itisclearlydistinctinhavinghighermetabolicactivity.
Accordingly,hu-manupperthoracicBATisnowcharacterisedasadistinctbrown/beigefatdepotand,therefore,mayfluctuateinoxida-tivecapacity.
Nevertheless,thisabsolutelevelofBATactivitycontributesonlyasmallportionofwhole-bodyenergyexpen-diture[8].
Whilestatisticallysignificant,thesmallreductionobservedherewouldbeunlikelytocontributemeaningfullytowhole-bodyenergyexpenditureunderthepresentexperimen-talconditions.
However,otherphysiologicalstimulithatacti-vateBATmayresultingreaterdifferencesoflargerabsolutemagnitude,thereforeitisnotyetpossibletoconclusivelyevaluatethephysiologicalrelevanceofthesefindings.
Con-versely,sincewedidnotobserveanincreaseinactivityaboveapredeterminedbasallevel,measurementofbasalBATactiv-itybeforeandafterthepresentinterventionwouldhavebeenoflimitedvalue.
Nevertheless,whileethicalconsiderationsrelatedtoradiationexposurepreventedadditionalbasalPET-CTscansinthecurrentstudy,futureinterventionstudiesshouldaimtostudythefunctionalrelevanceofBATunderunstimulatedconditions.
Thelowproportionofparticipantsforwhomweobservedacuteephedrine-stimulatedactivityapproachingthatofourpreviousstudy[6]couldsimplyberelatedtoonlythesmallestand/orleanestindividualsbeingresponsive;theleangroupinourpriorstudy(BMI21kg/m2,bodymass66kg,bodyfatcontent17%)wassignificantlyDiabetologia(2015)58:1045–10541051lighterwithlowerbodyfatcontentthanthatofthepresentcohort(~24kg/m2,76kg,25%).
Chronicephedrinetreatmentresultedinasignificantlossofbodyfat,particularlyinthevisceralcompartment,withnolossofleanmassorbonemineralcontent.
Thishighlydesirableoutcomewasnotattributabletoanyalterationinthermogenicfunctionsinceneitherbasalnorephedrine-stimulatedenergyexpenditurechangedinresponsetotreatment.
Thelikelyex-planationforthelossofbodymassandfatisanincreaseinenergyexpenditureincombinationwithdecreasedenergyin-take.
Ephedrinehasasystemichalf-lifeof~4handsingleephedrinedosesof1–2.
5mgkg1increaseenergyexpenditureby~10–15%forseveralhourspost-treatment,putativelymainlyviaincreasedmusclethermogenesis[19].
Basedonthepresentdataindicatingnochangeinbasalorephedrine-stimulatedenergyexpenditureinresponsetochronicephed-rinetreatment,tolerancedoesnotdevelopwithrespecttothewhole-bodythermogenicresponse.
Suchshiftsinenergybal-ancemaybecompensatedbyincreasedgutandcentralorexigenicsignalsand,therefore,energyintake[50];however,ephedrineisalsoanappetitesuppressant[41].
Wecanonlyspeculateastothemajorcontributingfactorsincebodycom-positionwasnotaprimaryoutcomemeasureandfooddiaries(whicharenotparticularlyreliable)werenotrecorded.
Whileacutetreatmentwith2.
5mgkg1ephedrinedidnotresultinincreasedBATactivityinthemajorityofparticipantsinthisstudy,andsincewepreviouslyreportedthisdosewouldlikelybetheminimumnecessarytoactivateBATinleanadults[6],ideallywewouldhaveadministeredatleastthisdosedailyinthepresentstudy.
Duetothecardiovascularactivationinducedbythishighdose,however,weoptedtotreatwithalowerdose,hypothesisingthatthenecessaryadap-tiveadrenergicsignallingeventswouldoccurinfattissuewhetherornotsignificantthermogenesiswasinduced.
Re-gardless,basedonourobservations,ahigherdosewouldlike-lyhaveonlyresultedingreatersuppressionofbasalBATFDGuptake.
Itisworthconsideringthattheresponsetocoldexposureafterthechronicephedrineinterventionmaydiffertothatofacutehigh-doseephedrine.
Coldexposurewillacti-vateBATtoagreaterextentviaphysiologicalpathwaysthatmayresultindifferingsignallingand/orsubstratepreference.
Thispossibilityremainstobeinvestigated.
BATfunctionmeasuredviaFDGPETposesanumberoftechnicallimitations,includingassessmentofglucoseuptakewhenlipidsmaybethepredominantBATsubstrateforther-mogenesis.
Itisconceivablethatinourstudiesofacutehigh-doseephedrinetreatmentglucoseisnotamajorBATsub-strate.
Thisis,however,unlikely,sincechroniccoldexposurestudiesreportsimilarresultswhetherusingPETimagingwithFDG[12,13]oranacetatetracertomeasurewholetissuemetabolism[11].
Nevertheless,thispossibilityalsoremainstobestudiedviaatechniquethatassesseswholetissueenergyexpenditure.
Inthepresentstudy,weprovideevidencethat,contrarytoourhypothesisandincontrasttomice,chronictreatmentwithasympathomimeticdecreasesbasalBATactivityandlikelythermogenicresponsivenesstoadrenergicstimuli.
Thisfind-ingrepresentsanimportantdifferencebetweenmouseandhumanBATandthedifferingfunctionofhumanBATshouldbecarefullyconsideredwhenassessingfutureBAT-dependanttherapeutictargetsforobesity.
Importantly,whilereducedBATactivityhasyettobecausativelylinkedtoobesity,ourdatasuggestthattheelevatedsympatheticactivityobservedinobesitymayperpetuateweightgainbyfurtherreducingBATfunction.
AcknowledgementsTheauthorsthankC.
Despott(DepartmentofNu-clearMedicine,AlfredHospital,Melbourne,VIC,Australia)andS.
Phil-lips(HumanNeurotransmittersLaboratory,BakerIDIHeartandDiabetesInstitute,Melbourne,VIC,Australia)fortechnicalassistance,andtheresearchparticipantsfortheirtimeandinterestinourstudy.
FundingThisstudywassupportedbyaNationalHealthandMedicalResearchCouncilofAustraliaProgramGrant(1036352)andtheOISschemefromtheVictorianStateGovernment.
BAKholdsaNationalHealthandMedicalResearchCouncilSeniorPrincipalResearchFellow-ship(1059454).
GWLholdsaNationalHealthandMedicalResearchCouncilSeniorPrincipalResearchFellowship(1042492).
ContributionstatementALC,RP,MFF,BVE,DAB,MJA,VK,SJD,MHCandBAKwereresponsiblefortheconceptionanddesignofthestudyandexperiments.
ALC,RP,MFF,BVE,DAB,MJA,NE,GWL,VK,SJD,MHCandBAKacquiredthedata.
ALC,RP,MFF,BVE,NE,GWL,VK,SJD,MHCandBAKanalysedandinterpretedthedata.
ALC,RP,MFF,BVE,DAB,MJA,NE,GWL,VK,SJD,MHCandBAKdraftedandcriticallyrevisedthemanuscript.
Allauthorsapprovedthisversionofthemanuscript.
ALCistheguarantorofthiswork.
DualityofintereststatementTheauthorsdeclarethatthereisnodu-alityofinterestassociatedwiththismanuscript.
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