Regulation of Adipose Tissue Metabolism by the Endocannabinoid System

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Article details

Eenige R. van, Stelt M. van der, Rensen P.C.N. & Kooijman S. (2018), Regulation of Adipose Tissue Metabolism by the Endocannabinoid System, Trends in Endocrinology and Metabolism 29(5): 326-337.

Doi: 10.1016/j.tem.2018.03.001

Review

Regulation of Adipose Tissue Metabolism by the Endocannabinoid System

Robin van Eenige,

Mario van der Stelt,

Patrick C.N. Rensen,

* and Sander Kooijman

Whiteadiposetissue(WAT)storesexcessenergyastriglycerides,andbrown adipose tissue (BAT) is specialized in dissipating energy as heat. The endocannabinoidsystem(ECS) isinvolvedin a broadrange ofphysiological processes and is increasingly recognized as a key player in adipose tissue metabolism.HighECStonusinthefedstateisassociatedwithadisadvanta- geousmetabolicphenotype,andthishasledtoasearchforpharmacological strategiestoinhibit theECS. Inthisreviewwe present recentdevelopments thatcastlightontheregulationofadiposetissuemetabolismbytheECS,and wediscussnovel treatmentoptionsincludingthe modulation ofendocanna- binoidsynthesisandbreakdownenzymes.

Introduction

Since1975theprevalenceofobesityhasnearlytripled.Over1.9billionpeopleworldwideare currentlyoverweight,of whom650 million suffer fromobesity (WHOfactsheet Obesity and Overweight 2017; www.who.int/mediacentre/factsheets/fs311/en/). As a consequence, the incidenceofobesity-relateddisorderssuchastype2diabetesandcardiovasculardiseasesis rising.Interestingly,overthepastthreedecadesagreatamountofresearchhasrevealedthatthe endocannabinoidsystem(ECS)isacentralmodulatorofmetabolicphysiology,andtheECSis nowincreasinglyrecognizedasaregulatorofadiposetissuefunction.Infact,rimonabant(see Glossary), acannabinoid1receptor (CB1R)inverse agonist, wasone of thefirstdrugsthatreached theEuropeanmarketto treatobesity. Itwassuccessfulinreducingfatmassandimproving metabolichealth,butitwaswithdrawn2yearslaterowingtopsychiatricsideeffectsobservedin somepatients(reviewedin[1]).ThediscoveryofaperipheralmodeofactionofCB1Rledto renewedinterestintheECSbeingatargetforobesityandrelateddisorders[2,3].Wereviewhere recentdevelopmentsthathighlighttheroleoftheECSinadiposetissuefunction,andwediscuss alternativetreatmentoptionsthattargettheECStoimprovecardiometabolichealth.

AdiposeTissuePhysiology

RoleofWATandBATinEnergyHomeostasis

Whiteadiposetissue(WAT)haslongbeenconsideredtobeaninactiveorgan,onlycapable ofstoringenergyintheformoftriglycerides(TGs).However,itsroleasanendocrineorganand itsimportanceforwhole-bodymetabolismhasnowbeenwell-recognized.WATisresponsible forthesynthesisofvarioushormones,includingleptinandadiponectin,whicharecrucialin regulatingsatietyandinsulinsensitivity,respectively[4].WATisalsoimportantinregulating energyhomeostasisbecauseitiscapableofreleasingTG-derivedfattyacids(FAs)intothe bloodstream,whichcansubsequentlybeusedbyotherorgansasanenergysubstrateorbe packagedinTG-richlipoproteinsintheliver.

IncontrasttoWAT,themainfunctionofbrownadiposetissue(BAT)istodissipateenergy intoheat.BATislocalizedintheinterscapular,cervical,andparavertebralregionsaroundlarge

Highlights

ActivationofBATthermogenesisand WATbrowningprotectsfromadiposity andrelatedcardiometabolicdisorders.

Theendocannabinoidsystemisakey playerinadiposetissuefunction.

Endocannabinoids provide an auto- crine/paracrine negative feedback loopinadrenergicstimulationofadi- posetissue.

The development of modulators of synthesis and breakdown enzymes of endocannabinoids opens up a new window for therapeutic interventions.

1DepartmentofMedicine,Divisionof Endocrinology,andEinthoven LaboratoryforExperimentalVascular Medicine,LeidenUniversityMedical Center,Leiden,TheNetherlands

2DepartmentofMolecularPhysiology, LeidenInstituteofChemistry,Leiden University,Leiden,TheNetherlands

*Correspondence:

p.c.n.rensen@lumc.nl(P.C.N.Rensen).

326

Glossary

N-Arachidonoylphosphatidy- lethanolamine-specific

phospholipaseD(NAPE-PLD):the mainenzymeresponsiblefor anandamide(AEA)synthesis.

Brownadiposetissue(BAT):an endocrineorganspecializedin dissipatingenergythatisstoredin theformoftriglycerides(TGs)into heat.

Browning:aprocessinwhichwhite adipocytesobtainthermogenic properties.

Diacylglycerollipase(DAGL):the mainenzymeresponsiblefor2- arachidonoylglycerol(2-AG) synthesis.

Endocannabinoid:anendogenous lipid-basedcompoundthatbindsto cannabinoidreceptors.

Fattyacidamidehydrolase (FAAH):themainenzyme responsibleforAEAdegradation.

Insulinresistance:aconditionin whichcellsinforexampleliver, muscle,andadiposetissuefailto respondtoinsulin.

Lipoprotein:amultimolecular complexbuiltfromlipidsandprotein (s)withthepurposeoftransporting hydrophobiclipidsthroughthe aqueousplasma.

Lipoproteinlipase(LPL):aprotein boundtocapillariesofmetabolically activetissuesthathydrolyzesTGs withinlipoproteinstoliberatefatty acids(FAs)forsubsequentuptakeby thosetissues.

Monoacylglycerollipase(MAGL):

themainenzymeresponsiblefor2- AGdegradation.

Non-shiveringthermogenesis:the productionofheatwithoutshivering throughforexampleuncoupled respirationinBAT.

Rimonabant:thefirstcannabinoid receptortype1(CB1R)inverse agonistthatwasapprovedforusein humans,butwhichwaslater withdrawnowingtopsychiatricside effects.

Sympathomimetic:acompound thatmimicstheeffectofendogenous agonistsofthesympatheticnervous system.

Thermoneutrality:thetemperature atwhichananimaldoesnotneedto regulateitsbodytemperature (approximately25–26^Cforhumans and30–32^Cformice).

arteries,whereitisabletotakeupglucoseand(TG-derived)FAsfromthebloodstream.Viathe process oflipogenesis, thesenutrientsare temporarily storedin intracellularlipid droplets.

Whentheenvironmentaltemperaturedrops,BATbreaksdownthesestoredTGstoproduce heatbymitochondrialuncouplinginvolvinguncouplingprotein1(UCP-1),aprocesscalled adaptiveornon-shiveringthermogenesis(Box1).Toreplenishlipidstores,brownadipo- cytestakeupglucose(viaGLUT1andGLUT4),freeFAs(viaCD36),andTG-derivedFAsthat areliberatedbylipoproteinlipase(LPL).Importantly,adecadeagoBATwasfoundtobe presentandactiveinadulthumans,asvisualizedbyglucoseuptakein[¹⁸F]fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) scans during cold exposure[5,6].

BrowningofWAT

Prolongedcoldexposureorb3-adrenergicreceptoragonism[7]notonlystimulatesBATbut alsoleadstobrowningofWATeitherbytransdifferentiationofexistingwhiteadipocytesor bystimulationofprecursorcells todifferentiateintobrite(brown-in-white)adipocyteswithin WAT[8,9]. Therelative contributionsof thesepathwaysis amatter ofdebate, but forthe purposeofthisreviewwewillcallthesecellsbeigecells.Thesecellsarephenotypicallydifferent fromWATandBATbyhavingverylowintrinsicexpressionofBAT-specificgenes,suchas UCP-1, CIDEA, and peroxisome proliferator-activated receptor gamma coactivator 1a (PPARGC1A),butUCP-1levelscanveryrapidlybeincreaseduponsympatheticstimuli via cAMP/PKAsignaling,allowinguncoupledrespiration[10,11].Likebrownadipocytes,beige cellshavemultilocularintracellularlipiddropletsandahighmitochondrialcontent.

HumanBATConsistsofBothBeigeandBrownAdipocytes

Therehasbeendebate aboutthecellularorigin ofhumanBAT.Somestudiessuggestthat human BAT consists mostly of beige adipocytes, instead of classical brown adipocytes (reviewedin[12,13]).Forexample,Wuetal.[10]demonstratedthatexpressionofthehuman counterpartsoftheCd137,Tbx1,andTmem26genes,whicharealmostexclusivelyexpressed inmurine beigeadipocytes,arehighly expressedinhuman BAT,whereasclassical brown markersarenot.Bycontrast, amorerecent studyshowedthat humanBATdoesexpress classicalbrownadipocyte-specificmarkers[14].Thisdiscrepancymaybeexplainedbyinter-

Box1.BATPhysiology

Atthecellularlevel,brownadipocytesarecharacterizedbyahighnumberofmitochondriatoachieveahighoxidative capacity,andbymultilocularintracellularlipiddropletsinwhichfattyacidsaretemporarilystoredasTGs.Furthermore,a largenumberofnerveendingsofthesympatheticnervoussystem,spreadacrossthetissue,enablesbrownadipocytes toquicklyrespondtoacoldenvironment.Uponcoldstimuli,sympatheticoutflowtowardsBATisincreased,thereby stimulatingthereleaseofnorepinephrinebysympatheticnerveterminiintheadiposetissue.Thisbindstoandstimulates b3-adrenergicreceptors,which areGprotein-coupledreceptors,onbrown adipocytes,aprocessthatcan be mimickedbyadministrationofab3-adrenergicagonistinmice[7]andhumans[89].b3-Adrenergicactivationtriggers anintracellularsignalingcascadethatpromotesintracellularlipolysis.Specifically,theactivityofintracellularadenylyl cyclaseisactivated,resultinginariseincAMPlevels.cAMPsubsequentlyactivatesproteinkinaseA(PKA),whichthen phosphorylatesaseriesofenzymesthatarecrucialforlipolysis,namelyperilipin1,comparativegeneidentification-58 (CGI-58),hormone-sensitivelipase(HSL),andadiposetriglyceridelipase(ATGL).Phospho-HSLisresponsibleforthe hydrolysisofTGsanddiglycerides,thefirstofwhichisalsomediatedbyphospho-ATGLandistherate-limitingstepin lipolysis[90].LiberatedFAsundergob-oxidationwithinthemitochondrialmatrixandallostericallyactivateUCP-1.UCP- 1isaproteinpresentinthemitochondrialinnermembranethatuncouplesthemitochondrialelectrontransportchain fromATPsynthesisbyfacilitatingprotonleakageintothemitochondrialmatrix[91].AnincreaseinUCP-1activity thereforeresultsinheatproduction,alsoknownasuncoupledrespiration.

Importantly,thermogenicactivationofBATreducesplasmaTGandcholesterollevels,asshowninmice[7,92].

Specifically,TG-derivedFAuptakebyBATresultsinacceleratedhepaticclearanceofcholesterol-enrichedlipoprotein remnants,whichtherebyreducesthedevelopmentofdiet-inducedatherosclerosis,asreviewedbyHoekeetal.[93].

andintraindividualdifferencesbetweenBATdepotsbecauseCypessetal.[15]haveshown thatthedeeperdepotsinhumanneckBATconsistmostlyofclassical BAT,whereasmore superficialdepotsmostlycontainbeigeadipocytes.

TheEndocannabinoidSystem

The ECS is involved in a broad range of physiological processes and is an important player in energy homeostasis because it regulates appetite, nutrient partitioning, and energy expenditure (reviewed in[16–18]).ItencompassesGprotein-coupledcannabinoidreceptors,theirligands(endocan- nabinoids),andtheenzymesthatareresponsiblefortheirbiosynthesisandbreakdown.

CannabinoidReceptorTypes1and2

TheCB1R(encodedbyCNR1)wasfirstidentifiedin1990asatargetofD⁹-tetrahydrocannabi- nol(THC),theputativepsychoactiveconstituentsubstanceintheCannabisplant[19].Three yearslaterthecannabinoidreceptortype2(CB2R)wasdiscoveredviasequencehomology [20].CB1Riswidelyexpressedinthecentralnervoussystem(CNS)[19]andtoalesserextentin peripheral metabolic tissues,including WAT,BAT, liver, myocardium, and skeletal muscle [21–23].Thepresence ofthe CB1Rin adiposetissuewas first described in2003by two independentgroups[24,25],anditsexpressionishigherindifferentiatedadipocytesthanin undifferentiatedadipocytes[26],therebysuggestingadirectroleoftheECSonadiposetissue function.Onthe otherhand, theCB2R iswellknownfor itsimmune-regulatoryproperties [27,28],butitsexpressionandfunctioninothercelltypesremainsmostlyunclear.

SynthesisandBreakdownofEndocannabinoids

Thetwomostprominentendogenousligandsofthecannabinoidreceptorsare2-arachido- noylglycerol(2-AG)andanandamide(N-arachidonoylethanolamine,AEA).Theseendocanna- binoidshavethesamebackboneconsistingofthepolyunsaturatedFA(PUFA)arachidonicacid (AA).Although2-AGandAEAarebothAAderivatives,theirpathwaysofsynthesisaredistinct andareregulated bydifferentenzymes,allowingdifferentialregulationof theirlevels. More specifically,AEAissynthesizedbyhydrolysisofitsdirectprecursor,N-arachidonoylphospha- tidylethanolamine(NAPE)bytheenzymeN-arachidonoylphosphatidylethanolamine-spe- cificphospholipaseD(NAPE-PLD),whereas2-AGisprimarilyproducedbythehydrolysisof AA-containingdiacylglycerols(DAG)bydiacylglycerollipase(DAGL).DegradationofAEA and2-AGisfacilitatedby hydrolysisbyprimarilyfattyacidamidehydrolase(FAAH) and monoacylglycerollipase(MAGL),respectively.Thesebiosynthesisanddegradationpath- waysaremoreextensivelyreviewedelsewhere[29,30].

TheInvolvementoftheEndocannabinoidSystemintheControlofEnergyHomeostasis TheECShasrepeatedlybeenassociatedwithobesity.CirculatingAEAand2-AGlevelsare higherin obese individuals compared to lean individuals [31], and circulating 2-AG levels positivelycorrelate withmeasuresofobesitysuchas bodymass index(BMI)andbodyfat percentage,aswellaswithserumTGlevels[21,31–33].Moreover,circulatingendocannabi- noidlevelsinobesitypositivelycorrelatewithadversecardiacevents[34].Inlinewiththese observations,high-fatdietfeedingofmiceincreasesplasma2-AGandAEAlevelsinassocia- tionwithweightgain[35,36],andFAAHdeficiencyinmicepromotesenergystorage,ectopic lipidstorage,andinsulinresistance[37,38],whilemicedeficientforCB1Rareresistantto high-fatdiet-inducedobesity[39].Moreover,systemicblockadeofCB1Rbytheinverseagonist rimonabantreducesadiposityinmice[40]andhumans[41].

TheECS exertsits effects onenergy metabolism partlyviathe regulationof appetiteand hypothalamic control of energy expenditure (Box 2). However, because CB1R is also

Transdifferentiation:the differentiationofamaturesomatic cellintoadifferentmaturesomatic cell.

Uncoupledrespiration:ametabolic stateinwhichcatabolicproductsare usedforheatproductioninsteadof ATPsynthesis.

Uncouplingprotein1(UCP-1):a regulatedprotonchannelhighly expressedinbrownadipocytesand toalesserextentinbeigeadipocytes that,whenactivated,uncouples oxidativephosphorylationfromATP synthesis,resultinginheat production.

Whiteadiposetissue(WAT):an endocrineorganspecializedin storingexcessenergyinglucoseand inlipidsasTGs.

expressedinperipheralmetabolictissuesincludingBATandWAT,theECSwasalsoexpected toplayadirectroleinthesetissues,possiblyofevenmoresignificancefortheregulationof energyhomeostasisthanforthecentraleffects.Thiswouldopenpathsfordevelopingcom- poundstotargettheECSinperipheraltissuesdirectly,withoutinterferingwiththeCNS.Inthe nextsectionswediscusshowcirculatingendocannabinoidsimpactonadiposetissuefunction, andwediscusstheproductionofendocannabinoidsbyadiposetissueitselfandtheirlocal effects.

EndocannabinoidsReduceThermogenicActivityinBATandWAT

CB1RexhibitsitseffectsmainlythroughGaiproteinsthatarecapableofinhibitingadenylyl cyclase and thereby preventing intracellular cAMP production [42]. Because sympathetic stimulationofadiposetissuevianorepinephrineinducesthermogenicactivationofBATand browning of WAT via stimulation of adenylyl cyclase and subsequent cAMP production, endocannabinoidscanthuscounteracttheeffectsofnorepinephrineonthesetissues.

CB1RSignalinginWAT

TheoveralleffectofCB1RstimulationinWATistofavorfatstorage(Figure1).Severalinvitro studieshavedemonstratedthatCB1Rsignalingincreaseslipogenesis(reviewedin[43–45]) accompaniedbyelevatedLPLactivity,whichpromotestheliberationofFAsfromcirculating TG-richlipoproteinsandtheuptakeoftheseFAsbywhiteadipocytes[24].Moreover,CB1R signaling in WAT stimulates GLUT4 translocation and activates fattyacid synthase (FAS), resultinginelevatedglucoseuptakeanddenovoFAsynthesis,respectively.Inconcordance withthis,theexpressionofthenuclearreceptorperoxisomeproliferator-activatedreceptorg (PPARg),atranscriptionfactoressentialforadipocytedifferentiation,isalsoincreasedupon CB1Rsignaling[26,46].

CB1RsignalinginWATimpairsmitochondrialbiogenesisandtherebypreventsbrowningof whiteadipocytes.Specifically,CB1Rstimulationinhibitsthephosphorylationof5'-AMP-acti- vatedproteinkinase(AMPK)inculturedmurinewhiteadipocytes[47].ThisdecreaseinAMPK

Box2.HypothalamicCB1RSignalingInhibitsBrownFatThermogenicActivation

Cannabinoidreceptorsareexpressedinhypothalamicregionsinvolvedintheregulationofappetiteandenergy expenditure.Severalattemptshavebeen madetodistinguishbetweencentral anddirectperipheral effectson thermogenicBATactivity.Vertyetal.[73]andBajzeretal.[72]showedthatthermogenicBATactivityandbody weightlossinducedbyCB1Rantagonismispreventedbysympatheticdenervationofthetissue.Inaddition,Quarta etal.[94]demonstratedthatmicewithconditionalCB1Rknockoutinforebrainneuronalcellsexhibitincreased sympathetictoneandnorepinephrineturnoverinBAT,resultinginelevatedPpargc1a,Nrf1,andTfammRNAlevels comparedtowild-typemice,whichcanpromotemitochondrialbiogenesis.UCP-1levelsarehigherinBATofthese conditionalCB1Rknockoutmicethaninwild-typemice,leadingtoimprovedcoldtoleranceassociatedwithincreased oxygenconsumption[94].

TheeffectofCB1Rinthebraincanbeassignedtoparaventricularnucleus(PVN)neuronsinthehypothalamus.

Evidenceforthiswasfoundbyablationofsingle-minded1(Sim1)neurons,thataccountforthemajorityofthePVN neurons,whichcausesobesitybyinducinghyperphagiaandreducingenergyexpenditure[95].Similarly,removing CB1R-dependentinhibitionofglutamatereleaseintheseneuronsbySim1-specificCB1Rdeficiencyleadstoincreased overallenergyexpenditurethatisindependentoffoodintakeandtohigherthermogenesisonahigh-fatdiet[96].These findingsdemonstratetheimportanceoftheECSinthebrainforregulatingBATactivity.

TheeffectsofCB1Ronappetitemaybemediatedbothcentrallyandperipherally.Ontheonehand,stimulationofthe ventromedialhypothalamusbyanandamidedirectlyincreasesfoodintake[97],andtheECSisthoughttostimulatefood rewardandpalatability(reviewedin[98]).Ontheother,adipose-specificCB1Rknockoutmicehaveareduceddaily caloricintake,suggestingthepresenceofaperipheralmechanismthatinfluencesappetite,possiblymediatedvia retrogradesignalingfromadiposetissuetothebrain[59].

activityis accompaniedbydecreasedexpressionofPpargc1a, encodingPGC-1a,andby reducedexpressionofnuclearrespiratoryfactor-1(Nrf1)andmitochondrialDNAtranscription factorA (Tfam), genes encodingfactorsthat are consideredto be importantregulatorsof mitochondrialbiogenesisandthermogenesis[47–50].SimilareffectsofCB1Rstimulationon mitochondrialbiogenesishavebeenobservedinculturedhumansubcutaneousandvisceral adipocytes[47].

Conversely,pharmacologicalblockadeofCB1RinawhiteadipocytecelllineincreasesUCP-1 expression,stimulatesphosphorylationofAMPK,andpromotesexpressionofPGC-1a[51].

Thesechangesingeneexpressionareaccompaniedbyinductionofmitochondrialbiogenesis, togetherresultinginincreasedoxygenconsumption[51].Inaddition,subcutaneous,butnot visceral,adipocytesofCB1RknockoutmiceshowhigherUCP-1andPGC-1aexpression, higheroxygenconsumption,andelevatedmitochondrialbiogenesiscomparedtoadipocytes derived from wild-type control mice, indicating sensitization towards a brown phenotype [26,52].ThisalsoindicatesthatCB1Rsignalingcanhavedifferentialeffectsindifferentadipose tissuedepots,andthismaybeofimportanceintheevaluationofCB1Rmodulators[26].

Emergingdatapoint towardsan interaction betweenthe ECSand insulinsignaling thatis independentofweightgain(reviewedin[53]).PeripheralCB1Rinhibitionbyaninverseagonist improvesinsulinsensitivity[54].Somestudieshavesuggestedthattheseeffectsaremediated viaanincreaseinadiponectinlevels,anadipokineknownforitsinsulin-sensitizingeffects[55].

Forexample,adiponectinisdownregulateduponCB1Rstimulation,asshownin3T3-F442A [26]or3T3-L1[56]cells,aswellasinculturedmatureadipocytesderivedfromhumanomental adiposetissue[57],andthisisreversedbyCB1Rblockade[26,54,56–58].Inaddition,adipose- specific CB1R knockout mice have higher plasma adiponectin levels than controls [59].

Moreover, CB1R antagonism is ineffective in increasing insulin sensitivity in adiponectin ECs

Brown adipose ssue White adipose ssue

Thermogenesis De novo lipogenesis

Adipocyte differen a on Fa y acid uptake Mitochondrial biogenesis Mitochondrial

biogenesis

Cold-induced browning CNS

Fa y acid uptake

Regula on of endocrine signaling (e.g., glucocor coids and thyroid hormone)

Figure1.SchematicOverviewoftheOverallEffectsofEndocannabinoidsonWhiteandBrownAdipose Tissue.Circulatingendocannabinoids(ECs)favorfatstorageinwhiteandbrownadiposetissue.Theseeffectsare mediatedinpartbydirectbindingofECstothecannabinoidreceptor1(CB1R)onthewhiteandbrownadipocytes.In addition,someoftheeffectsofECsaremediatedviathecentralnervoussystem(CNS),andsomemaybemediatedvia regulationofendocrinesignalingactingonnon-adiposetissues.

receptorknockoutmice[60].Ontheotherhand,theinsulin-sensitizingeffectofrimonabantis notaffectedindiet-inducedobeseadiponectinknockoutmice[61]andonlypartiallyreducedin adiponectinknockoutob/obmice[62].Althoughtheeffectsofadiponectinoninsulinsensitivity havebeenwellestablishedinmice[63],suchapotentialroleofadiponectininhumanshasnot beenfirmlyestablished becausenoevidencewasfoundforanassociationbetweenadipo- nectinlevels andinsulinsensitivity ina Mendelian randomizationstudy [64]. Analternative mechanismexplainingtheincreasedinsulinsensitivityuponrimonabanttreatmentisashiftin macrophagephenotypefrom aproinflammatoryM1toan anti-inflammatoryM2phenotype [65].

Interestingly,theinteractionbetweentheECSandinsulinsignalingmaybereciprocalbecause insulintreatmentofwhiteadipocytesresultedinaloweringof2-AGandAEAlevels,together with increased mRNA expression of ECS degradation enzymes and a decrease in ECS synthesis enzymes [26]. Similarly, following hyperinsulinemia, FAAH gene expression was elevated in subcutaneous adipose tissue of lean but not of obese individuals, whereas CNR1geneexpressionwasnotaltered[66].

CB2RSignalinginWAT

EvidenceforaroleofCB2RinWATsignalingisscarce,andismostlylinkedtotheregulationof inflammation. CB2R is expressed inWAT [67] and its expression is increased inobesity, especiallywithinthemacrophage-enrichedstromalvascularfraction[68].Importantly,CB2R signalinginimmunecellsinthistissuemaybelinkedtoadiposephysiology.Forexample,CB2R stimulationpromotestype2Thelpercell(Th2)polarizationandinterleukin-4secretion,resulting inbrowningofadipocytesinWAT[69].Inaddition,associationscouldbemadebetweenCB2R geneticvariantsandBMI[69],andtreatmentofobesemicewithaCB2Ragonistloweredfood intake,reducedbodyweight,andincreasedlipolysis,evidencedbyhigheradiposetriglyceride lipase(ATGL)proteinexpressionandreducedadipocytecellsize[70].Althoughthesestudies suggest a beneficial role for CB2R in the regulation of adipose tissuemetabolism, more researchwillbenecessarytofirmlyestablishthisrelationshipaswellastherelativecontribution ofCB2RversusCB1Rsignalingto metabolichomeostasisbecause mostdatapointinthe directionofunfavorablemetaboliceffectsofECSsignalingmediatedviaCB1R.

CB1RSignalinginBAT

ComparedtoWAT,evidencefortheeffectsofendocannabinoidsonBATremainsscant.CB1R inverseagonismbyrimonabantstimulatesthermogenesisinbrownT37iadipocytesinvitro, evidencedbyincreasedoxygenconsumptiontogetherwithelevatedUCP-1expressionand glycerolreleaseasameasureofintracellularlipolysis[22].Stimulationofthesebrownadipo- cyteswiththeCB1Rinverseagonistincombinationwithnorepinephrineledtoasynergistic increaseinintracellularphospho-HSLlevels,indicatingthatCB1Rsignalingisalsocoupledto theadrenergicpathwayinBAT[22].EvidencefortheeffectsofCB1Rantagonismonthermo- genicactivationofBATinvivoisalsoavailable.CB1Rblockadebyrimonabantortheperipheral antagonistAM6545reversesdiet-inducedobesity[71]andstimulatesuptakeandcombustion ofVLDL-TG-derivedFAbyBAT,aneffectpreservedatthermoneutrality[22].CB1Rblockade alsoincreasesBATUCP-1expression[22,72].Consistently,rimonabanttreatmentincreases energyexpenditureand BATtemperature[72,73],andHsiaoet al.[74] demonstratedthat chronicperipheralinhibitionofCB1RbyBPR0912inducedUCP-1expressioninBATofmice andelevatedcorebodytemperature,indicatingincreasedthermogenicactivityofBAT.Impor- tantly,BATcanbevisualizedinratsusingradioactivePETligandswithhighaffinityforCB1R, therebydemonstratingitsdensepresenceinthetissueinvivo[75].

Inall,thereisapositiveassociationbetweenCB1RblockadeandBATthermogenicactivity.

Conversely,serumendocannabinoidlevelsarehigherintheSouthAsianpopulation[76],who areknowntohavereducedBATvolumeandactivity,increasedvisceraladiposetissuedepots, andanelevatedriskformetabolism-relateddiseasessuchascardiovasculardiseasesandtype 2 diabetes, compared to the white Caucasian population [77,78]. Further studies will be necessarytodemonstrateacausalrelationshipbetweenhighECStonusandreducedBAT activity.

EndocannabinoidsAreProducedandBrokenDownbyAdiposeTissueItself Surprisinglylittleisknownabouttheoriginandregulationoflocalandcirculatingendocanna- binoidlevels[79].However,althoughthemainorgansresponsibleforplasmaendocannabinoid levelshavenotyetbeenidentified,therearestrongindicationsthatadiposetissuecontributes to the pool of circulating endocannabinoids [80]. In fact, endocannabinoids produced by adiposetissuemayinducelocalautocrineandparacrinenegativefeedbackloopsinresponse toadrenergicstimulation.

EndocannabinoidSynthesisandDegradationinWAT

Recently,Krottetal.[81]reportedthatcoldoradrenergicreceptoragonismnotonlyincrease expressionofUcp1andPpargc1ainWAT,butalsoofCn1r,whichgoestogetherwithelevated levelsofAEAand2-AGandoftheirsynthesisenzymesinWAT[81].Althoughtheintracellular signalingcascadeleadingtoenhancedexpressionofthesegenesremainstobeidentified,the latterfinding suggeststhatthe ECSmaybeactivatedto dampenthe effectsofadrenergic signalinginanautocrine/paracrinefashion.EndocannabinoidssecretedbyWATwilldecrease adenylylcyclaseactivity,asdiscussedbefore,therebypreventingadrenergic-inducedbrown- ingofWAT[81](Figure2,KeyFigure).

FurtherevidencesuggestingthatWATproducesendocannabinoidsisprovidedbythefinding thatobesemicehavehigherplasma2-AGandAEAlevels,havehigheradiposetissue2-AG levels,andalsoshowalteredexpressionofECsynthesisanddegradationenzymesinadipose tissue[35].Similarly,Engelietal.[82]showedinsubcutaneousadiposetissueofobesehumans increasedexpressionofDAGLA,butnotofNAPEPLD,whichare2-AGandAEAsynthesis enzymes,respectively.TheyalsoobservedareductioningeneexpressionofFAAHandMGLL that encode the AEA- and 2-AG-degrading enzymes, respectively [82]. Interestingly, the inhibitoryeffects ofleptinonlipogenesis havebeenshown to bemediatedviaareduction inAEAsynthesisinWAT ofmice[83].Together thesedatasuggesta rolefor WATinthe productionofendocannabinoids.

Endocannabinoid-relatedcompoundsderivedfromadipocytesmayalsoplayaroleinbrown- ing. For example, Geurts et al. [84] recently showed that conditional adipocyte-specific Napepldknockoutmicehave higherbodyfat mass despiteequal food intake.In addition, thesemicehave decreased expressionofbrowning markersinWAT and exhibit impaired adaptationtocoldexposurecomparedtowild-typemice[84].Surprisingly,AEAlevelsinWAT of these knockout mice were not different from wild-type mice. The effects of Napepld knockoutonbrowningwerepossiblyexplainedbyreducedprostaglandinE2(PGE2)levels, anotherproductderivedfromAA.Furthermore,decreasedproductionofotherN-acylethanol- amines (NAEs) in the adipose tissue of these knockout mice altered the gut microbiota composition,thereby suggestingtheexistenceofanadiposetissueto gutmicrobiotaaxis.

Becausetheplasmaendocannabinoid levelswere notreported intheseNapeplddeficient mice,theextenttowhichWATisresponsibleforplasmaendocannabinoidlevelsremainstobe determined.

KeyFigure

Hypothetical Model Showing How Endocannabinoids (ECs) Inhibit Norepinephrine (NE)-Induced Activation of Brown Adipose Tissue (BAT) Thermogenesis and White Adipose Tissue (WAT) Browning

SympatheƟc nerve

β3-Adrenergic receptor Adipocyte

Adenylyl cyclase

CB1R

NegaƟve feedback loop

CB1R

EC synthesis ECs

DAGLα NAPE-PLD UCP-1

PGC-1α Thermogenesis

Mitochondrial biogenesis

UCP-1 UCP-1

Protein kinase A

CREB

Gαs Gαi

ATP cAMP

ECs

ECs

FA Lipolysis TG

NE NE NE

NE NE NE NENENE NENENE

NE NE NENE NE

NENE NE

H⁺ H⁺

(Seefigurelegendatthebottomofthenextpage.) Figure2.Uponcoldexposure,sympatheticnerveendingsintheadiposetissuereleaseNEthatbindstoandstimulatesb-adrenergicreceptorsonadipocytes.

Stimulationofthesereceptorsactivatesadenylylcyclase,resultinginariseincAMPlevelsandsubsequentactivationofproteinkinaseA(PKA).PKAenhances intracellularlipolysis,resultingintheliberationoffattyacids(FAs)fromtriglycerides(TGs).Inaddition,PKAphosphorylatesCREBwhichinitiates,amongothers, transcriptionalupregulationofuncouplingprotein-1(UCP-1)andPGC-1a,resultinginincreaseduncoupledrespirationandmitochondrialbiogenesis,respectively.

EndocannabinoidSynthesisandDegradationinBAT

DataontheroleofBATintheproductionofendocannabinoidsarelimited.Krottetal.[81]

reportedthatBATstimulationviab3-adrenergicreceptoragonistCL316,243increasesthe levelsofAEAand2-AGinBAT,accompaniedbyelevatedexpressionofDaglaandasuggested increase inthe bioavailabilityof AEAprecursors, because they showedthat BATNapepld expressiondecreasesuponacuteCL316,243stimulation.TheyalsoshowedthatCnr1gene expressioniselevateduponb3-adrenergicreceptorstimulationinprimarybrownadipocytes [81]. This suggeststhe presenceof a similar autocrine and paracrine negative regulatory feedbackloop inBAT as in WAT(Figure2),which may control norepinephrine-stimulated thermogenicBATactivity,therebypreventingexcessheatproduction.

InWATandBATthisnegativefeedbackloopisofinterestforthetreatmentofcardiometabolic disorders.Targetingthe ECSby modulatingthe biosynthesisand/or degradation ofendo- cannabinoids,forexamplebytheuseofDAGLinhibitors[85],mayincreaseitsthermogenic activityinBAT,therebyimprovingmetabolichealth,withoutsufferingfromsideeffectsrelatedto the inverseagonistic natureof most CB1R blockers [86].In addition,a combination of a sympathomimeticorcoldexposurewithlocalreductionofECStonuscouldbeofinterestasa newtherapeuticstrategybecause theseinterventionsactsynergisticallyandthereforemay havegreaterpotentialthanasingleintervention.

EndocannabinoidsInhibitNorepinephrineReleaseinPeripheralPresynapticNerves

Finally,there isevidenceforanadditionaleffectofendocannabinoids secretedfromadipo- cytes.CB1RispresentinsympatheticterminalsofnervesinnervatingWATandBAT.CB1R signaling inthesesympathetic nerveterminals directly inhibits norepinephrinerelease [87], possibly by activating inwardly rectifying K⁺ channel conductance and inhibiting N-type voltage-dependent Ca²⁺channel conductance [88]. Of note, high-fat diet feeding of mice decreasesnorepinephrinecontentinadiposetissue,whichcanbereversedbypharmacologi- calCB1Rblockade[54].Todate,theroleofadipocyte-derivedendocannabinoidsinregulating sympatheticoutflowremainsarelativelyunexploredterrain,butcouldalsopartiallyexplainthe metabolicbenefitsofCB1Rblockerssuchasrimonabant.

ConcludingRemarks

TheECSisanimportantplayerinenergymetabolism,andweareonlystartingtounravelits complexinteractionsthatarenotonlycontext-dependentbutpossiblyarealsomediatedvia othertissuesandendocrinesignaling.ThenetinhibitoryeffectoftheECSonthermogenesis, andthenetstimulatoryeffectonadiposity,makestheECSanattractivetargetforthetreatment ofobesityandobesity-relateddisorderssuchastype2diabetesandcardiovasculardiseases, althoughmanyquestionsonhowtotargettheECSneedtobeaddressedfirst(seeOutstanding Questions).PharmaceuticaltargetingofCB1Rhasprovedto beeffectiveinreducingbody massandimprovingoverallmetabolichealth,asshownbytheRimonabantinObesity(RIO) trials. However, caution is needed regarding psychiatric side-effects. The withdrawal of rimonabantresultedindiscontinuation of numeroustrials involvingCB1R antagonists. The elucidationoftheimportanceofperipheralCB1RsignalingledtorenewedinterestinCB1Rasa therapeutictargetand thedevelopment ofperipherally restrictedantagonists. These com- poundshavebeenshowntobeeffectiveinrodents,andmaythereforeprovetobeusefulto

Simultaneously,adrenergicstimulationofadipocytespromotesgeneexpressionlevelsofenzymesinvolvedintheproductionofECs.TheseECsmaysubsequentlyact inanegativefeedbackloop(i)toinhibitNEreleasebythesympatheticnervesbybindingtothecannabinoid1receptor(CB1R)onnerveterminals,and(ii)toinhibit adenylylcyclasebybindingtoCB1Ronadipocytes.

OutstandingQuestions

Whichorgan(s)is(are)thesourceof circulatingendocannabinoids?

Arecirculatingendocannabinoidlevels representative for endocannabinoid signalinginadipose(orother)tissues?

Why are (circulating) endocannabi- noidsincreasedinobesity?

Isitpossibletotherapeuticallylower the endocannabinoid tonus without affectingtheCNS,forexamplethrough peripherally restricted CB1R antagonists?

IsCB2Ragonismapotentialstrategy toreduceinflammationandpromote metabolichealth?

Isloweringtheendocannabinoidtonus inadiposetissuesastrategytostimu- latethermogenesisandinduceweight lossinhumans?

Can wepharmacologically modulate local endocannabinoid production andbreakdowninadiposetissues?

safelylowerECStonusinhumansinthefuture.Furthermore,CB2Ragonismisapotential strategytoreduceinflammationandpromotemetabolichealthifsupportedbyfutureresearch.

As an alternative, compounds that inhibit the production or stimulate the degradation of endocannabinoidsinadiposetissuemayprovidenoveltoolsto evenmoresafelymodulate the ECS tonus. So far, a couple of inhibitors of endocannabinoid production have been developed andwere proven to beeffectivein vitro andto some extentin vivo inrodents [85].Long-termstudiesevaluatingthepotentialmetabolicbenefitsinmetabolicallychallenged rodentswillbenecessarytoprovideproof-of-concept.

Acknowledgments

ThisworkwassupportedbytheDutchHeartFoundation(Dekkergrant2017T016toS.K.),theEuropeanFoundationfor theStudyofDiabetes(EFSDRisingStarFellowshipProgramtoS.K.),andtheNetherlandsCardioVascularResearch Initiative:theDutchHeartFoundation,DutchFederationofUniversityMedicalCenters,theNetherlandsOrganisationfor HealthResearchandDevelopment,andtheRoyalNetherlandsAcademyofSciencesENERGISEproject‘Targeting energymetabolismtocombatcardiovasculardisease’(CVON2014-2).

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