THEROLEOFP ITU ITARYADENYLATECYCLASEACT IVAT ING POLYPEPT IDE(PACAP )INTHEVENTROMED IALNUCLEUSANDITS ASSOC IAT ION W ITHTHE MELANOCORT INSYSTEMINREGULAT ING ADAPT IVETHERMOGENES IS by Th e c laRa eM cM i l lan B .S c . ,Th eUn iv e r s i tyo fB r i t i shCo lumb i a,2013 THES ISSUBM ITTEDINPART IALFULF ILLMENTOF THEREQU IREMENTSFORTHEDEGREEOF MASTEROFSC IENCE IN HEALTHSC IENCES UN IVERS ITYOFNORTHERNBR IT ISHCOLUMB IA Ju ly2017 ©Th e c l aR a eM cM i l l an ,2017 ABSTRACT A c cumu l a t iono fexc e s swh i t ead ipo s et i s su einob e s i tyh a sd e t r im en t a lcon s equ en c e s fo rm e t abo l i ch e a l th .A c t iv a t iono fth e rmog en i cad ipo cy t e scon f e r sb en e f i c i a le f f e c t son m e t abo l i ch e a l thandi scon s id e r edapo t en t i a lth e r ap eu t i ct a rg e tfo rhum anob e s i ty . Ho rmon e smodu l a tingad ap t iv eth e rmog en e s i sin c lud em e l ano co r t in sandp i tu i t a ryad eny l a t e cy c l a s e a c t iv a t ingpo lyp ep t id e(PACAP ) .InPACAP/ -m i c e ,m e t abo l i smandth e rmog en i c c ap a c i tyinr e spon s etoco ldi simp a i r ed .Ihypo th e s i z edPACAPa c t sinth ev en t rom ed i a l nu c l eu softh ehypo th a l amu s ,up s t r e amo fth em e l ano co r t insy s t em ,tor egu l a t esymp a th e t i c s t imu l a t iono fth e rmog en e s i s .Toa s s e s sth i s ,PACAP+ /+andPACAP/ -m i c ew e r eco ld a c c l im a t edto4 °Candg iv end a i lyin j e c t ion so fam e l ano co r t inr e c ep to ragon i s t ,M e l ano t anI I (MT I I ) ,fo r24d ay s .Th ee f f e c to f MT I Ionth e rmog en e s i sw a sex am in edbyphy s io log i c a l , mo l e cu l a r ,andh i s to log i c a lan a ly s e s .R e su l t sshow MT I Ip a r t i a l lyr e s cu edth eimp a i r ed th e rmog en i cc ap a c i tyinPACAP/ -m i c ea scomp a r edtoPACAP+ /+m i c e ,p rov id ing ev id en c etosuppo r tou rhypo th e s i s . i i TABLEOFCONTENTS Ab s t r a c t i i T ab l eo fCon t en t s i i i L i s to fF igu r e s v L i sto fAbb r ev i a t ion s xi Chap t e rOn e Endo c r in ePhy s io logyo fOb e s i ty :En e rgyHom eo s ta s i sand Th e rmog en e s i s 1 .1 1 .2 1 .3 1 .4 1 .5 1 .6 Ob e s i tya saD i s e a s e C au s e so fOb e s i ty Wh i t eAd ipo s eT i s su ea saS to r ag eandEndo c r in eO rg an L ep t in :AK eyR egu l at o rinEn e rgyHom eo s t a s i s Th eC en t r a lM e l ano co r t inSy s t eminEn e rgyHom eo s t a s i s B rownAd ipo s eT i s su ea saPo t en t i a lTh e r ap eu t i cT a rg e tfo r Ob e s i ty Th e rmog en e s i sinB rownAd ipo s eT i s su e P i tu i t a ryAd eny l a t eCy c l a s eA c t iv a t ingPo lyp ep t id e(PACAP ) : E s s en t i a lfo rth eTh e rmog en i cR e spon s einBAT G apinL i t e r a tu r e :Conn e c t ingPACAPandth eM e l ano co r t in Sy s t em Hypo th e s i sandR e s e a r chOb j e c t iv e s R e f e r enc e s 1 .7 1 .8 1 .9 1 .10 1 .11 Chap t e rTwo Pha rma co log i ca lIn t e rv en t ionw i th M e lano tanI IPa r t ia l ly R e s cu e sth eImpa i r edTh e rmog en i cCapa c i tyo f PACAP/ -M i c e 2 .1 2 .2 2 .3 2 .4 2 .5 2 .6 In t rodu c t ion M a t e r i al sand M e thod s R e su l t s D i s cu s s ion R e f e r en c e s App end ix Chap t e rTh r e e C r ea t ingaV i ra lCon s t ru c tfo rG en e t i cIn t e rv en t ionA im eda t R e s to r ingImpa i r edTh e rmog en e s i so fPACAP/ -M i c e 3 .1 3 .2 3 .3 3 .4 3 .5 I n t rodu c t ion M a t e r i a l sand Me thod s R e su l t s D i s cu s s ion R e f e r en c e s i i i 2 3 5 8 12 14 16 21 24 25 27 37 44 57 67 80 89 91 98 108 110 118 Chap t e rFou r Con c lud ingR ema rk sfo rPACAP ’ sA s so c ia t ionw i thth e M e lano co r t inSy s t eminR egu la t ingAdap t iv eTh e rmog en e s i s 4 .1 4 .2 4 .3 Summ a ry S ign i f i c an c e R e f e r en c e s 126 129 132 iv L ISTOFF IGURES F igu r e1 .1 .F a c to r scon t r ibu t ingtoth een e rgyb a l an c eequ a t ion . 4 F igu r e1 .2 .K eyad ipok in e sandad ipo cy tok in e ss e c r e t edbywh i t ead ipo s et i s su e . TNFα,tumo rn e c ro s i sf a c to r α; MCP 1 ,mono cy t ech emo a t t r a c t an t p ro t e in1 ;IL6 ,in t e r l euk in6 ;PA I 1 ,p l a sm inog ena c t iv a to rinh ib i to r 1 . R ep r in t edf rom“Th eE f f e c t so fEx e r c i s eT r a in ingonOb e s i tyIndu c ed Dy s r egu l a t edExp r e s s iono fAd ipok in e sin Wh i t eAd ipo s eT i s su e” ,by T .S aku r a i ,J .Og a s aw a r a ,T .K i z ak i ,S .S a to ,Y .I sh ib a sh i ,M .T ak ah a sh i e ta l . ,2013 ,In t e rna t iona lJou rna lO fEndo c r ino log y ,1 28 .Copy r igh t© 2013T akuy aS aku r a ie ta l .R ep r in t edw i thp e rm i s s ionf romau tho r . 7 F igu r e1 .3 .P a thw ay sm ed i a t ingl ep t ina c t ioninth ea r cu a t enu c l eu sfo ri t sro l ein app e t i t er egu l a t ion .I l lu s t r a t ionbyD .L aV in e . MUTAGENET IX™ ,B . B eu t l e randco l l e agu e s ,2012 ,C en t e rfo rth eG en e t i c so fHo s tD e f en s e , UTSou thw e s t e rn ,D a l l a s ,TX .Copy r igh t©L abA r ch iv e s ,LLC . R ep r in t edw i thp e rm i s s ionf romau tho r . 10 F igu r e1 .4 . Mu l t ip l ep ep t id e sp rodu c edf romth es ing l ep rop ro t e in , p roop iom e l ano co r t in(POMC ) .I l lu s t r a t ionbyR .Bow en ,2006 , Co lo r adoS t a t eUn iv e r s i ty .Ob t a in edf rom h t tp : / /www .v ivo . co lo s t a t e . edu /hbook s /p a thphy s / endo c r in e /hypop i t / a c th . h tm l.R ep r in t edw i thp e rm i s s ionf romau tho r . 13 F igu r e1 .5 . Co ron a lc ro s ss e c t iono fth eb r a inshow ingth elo c a t iono fth e hypo th a l amu sandc lo s e upf iv ek eyhypo th a l am i cnu c l e iinvo lv edin th er egu l a t iono fen e rgym e t abo l i sm .PVN ;p a r av en t r i cu l a rnu c l eu s , DMN ;do r som ed i a lnu c l eu s ,LHA ;l a t e r a lhypo th a l am i ca r e a ,VMN ; v en t rom ed i a lnu c l eu s ,ARC ;a r cu a t enu c l eu s ,3V ;th i rdv en t r i c l e . Ad ap t edf rom“R ev i s i t ingth eV en t r a lM ed i a lNu c l eu so fth e Hypo th a l amu s :Th eRo l e so fSF1N eu ron sinEn e rgyHom eo s t a s i s ” ,by Y .Cho i ,T .Fu j ik aw a ,J .L e e ,A .R eu t e r ,&K .K im ,2013 ,F ron t i e r sIn N eu ro s c i en c e,7.Copy r igh t©2013Cho i ,Fu j ik aw a ,L e e ,R eu t e rand K im .Ad ap t edw i thp e rm i s s ionf romau tho r . 15 F igu r e1 .6 .H i s to log i c a lr ep r e s en t a t iond i f f e r en ti a t ing WATandBAT . WATi s un i lo cu l a rw i thl a rg el ip idd rop l e t s( im ag eda t40x ) ,wh i l eBAT con t a in smu l t i lo cu l a rl ip idd rop l e t sw i thach a r a c t e r i s t i cabund an c eo f m i to chond r i a( im ag eda t60xu s ingo i limm e r s ion ) .Im ag e sf roms tud i e s inth eG r ayl ab . 15 F igu r e1 .7 . Co ldindu c eda c t iv a t iono fad ap t iv et h e rmog en e s i sinb rownad ipo s e t i s su e .UCP1 ;un coup l ingp ro t e in1 .Ad ap t edf rom“ A c t iv a t ionand r e c ru i tm en to fb rownad ipo s et i s su ebyco ldexpo su r eandfood ing r ed i en t sinhum an s ” ,by M .S a i to ,T .Yon e sh i ro ,& M .M a t su sh i t a , 2016 ,B e s tP ra c t i c e&R e s ea r chC l in i ca lEndo c r ino log y& 17 v M e tabo l i sm,30(4 ) ,537 547 .C opy r igh t©2017E l s ev i e rB .V .Ad ap t ed w i thp e rm i s s ionf rompub l i sh e r . F igu r e1 .8 . Mo l e cu l a rm e ch an i sm so fad ap t iv eth ermog en e s i sa tth el ev e lo f b rownad ipo cy t e s .Ad ap t edf rom W ik ip ed i a ,2009 .Ob t a in edf rom h t tp s : / / en .w ik ip ed i a .o rg /w ik i /Th e rmog en es i s . 19 F igu r e1 .9 .P ropo s edp a thw ay so fn eu rop ep t id esth a tin t e r a c ttoindu c e ad ap t iv eth e rmog en e s i sinBATinr e spon s etoco ldexpo su r e .VMN ; v en t rom ed i a lnu c l eu s ,PACAP ;p i tu i t a ryad eny l a t ecy c l a s e a c t iv a t ing po lyp ep t id e , MC ;m e l ano co r t insy s t em ,SNA ;symp a th e t i cn e rv e a c t iv i ty ,BAT ;b rownad ipo s et i s su e .Im ag e sad ap t edf rom“ R ev i s i t ing th eV en t r a lM ed i a lNu c l eu so fth eHypo th a l amu s :Th eRo l e so fSF1 N eu ron sinEn e rgyHom eo s t a s i s” ,byY .Cho i ,T .Fu j ik aw a ,J .L e e ,A . R eu t e r ,&K .K im ,2013 ,F ron t i e r sInN eu ro s c i en c e,7.Copy r igh t© 2013Cho i ,Fu j ik aw a ,L e e ,R eu t e randK im .Ad ap t edw i thp e rm i s s ion f romau tho r . 23 F igu r e2 .1 . Symp a th e t i candendo c r in econ t ro lo fBATth e rmog en e s i s .βAR ; b e t a ad r en e rg i cr e c ep to r ,NE ;no r ep in eph r in e ,NP ;n a t r iu r e t i cp ep t id e , T4 ;thy rox in e ,UCP1 ;un coup l ingp ro t e in1 , WAT ;wh i t ead ipo s et i s su e . Ad ap t edf rom“A c t iv a t ionandr e c ru i tm en to fb rownad ipo s et i s su eby co ldexpo su r eandfooding r ed i en t sinhum an s ” ,by M .S a i to ,T . Yon e sh i ro ,& M .M a t su sh i t a ,2016 ,B e s tPra c t i c e&R e s ea r chC l in i ca l Endo c r ino log y& M e tabo l i sm,30(4 ) ,537 547 .Copy r igh t©2017 E l s ev i e rB .V .Ad ap t edw i thp e rm i s s ionf rompub l i sh e r . 38 F igu r e2 .2 .P ropo s edp a thw ay so fin t e r a c t ionfo rad ap t iv eth e rmog en e s i sinBAT inr e spon s etoco ldexpo su r e .VMN ;v en t rom ed i a lnu c l eu s ,PACAP ; p i tu i t a ryad eny l a t ecy c l a s ea c t iv a t ingpo lyp ep t id e , MC ;m e l ano co r t in sy s t em ,SNA ;symp a th e t i cn e rv ea c t iv i ty ,BAT ;b rownad ipo s et i s su e . Im ag e sad ap t edf rom“ R ev i s i t ingth eV en t r a lM ed i a lNu c l eu so fth e Hypo th a l amu s :TheRo l e so fSF 1N eu ron sinEn e rgyHom eo s t a s i s” ,by ron t i e r sIn Y .Cho i ,T .Fu j ik aw a ,J .L e e ,A .R eu t e r ,&K .K im ,2013 ,F N eu ro s c i en c e,7.Copy r igh t©2013Cho i ,Fu j ik aw a ,L e e ,R eu t e rand K im .Ad ap t edw i thp e rm i s s ionf romau tho r . 45 F igu r e2 .3 .S ch em a t i co fm e thodo logyov e rth ecou r s eo fth e6w e ekexp e r im en t . 48 F igu r e2 .4 .M e thodo log i c a ls t ep su s edtoan a ly z eBATs e c t ion sfo rl ip ida r e aa t 60xo i limm e r s ion .A .Im ag e sw e r econv e r t edtog r ay s c a l e .B .Con t r a s t w a sm ax im i z ed .C .S egm en t sth a tw e r enotb rownad ipo cy t e sw e r e s e l e c t iv e lyd e l e t ed( ind i c a t eda sr ed ) .D .Im ag ew a sb in a r i z edand “wh i t e ”a r e acoun t eda sl ip id . 54 v i F igu r e2 .5 .M e thodo log i c a ls t ep su s edtod e t e rm in ec e l la r e acoun t sinh i s to log i c a l s e c t ion so fgWATim ag eda t20x .A .Im ag e sw e r econv e r t edto g r ay s c a l e .B .D i f f e r en t i a lcon t r a s tw a sm ax im i z ed .C .Im ag e sw e r e b in a r i z ed .D .Ad ipo cy t e sno ten t i r e lyd i sp l ay edw i th inth ef i e ldo fv i ew w e r eex c lud edf roman a ly s e s . 56 F igu r e2 .6 . Bodym a s so fPBS WT ,PBSKO , MT I I WT ,and MT I IKOm i c e ov e rth ecou r s eo fth eexp e r im en t .T r e a tm en tw i thv eh i c l e(PBS )o r MT I Ib eg anonD ay7 .D a t aa r em e an±SEM . Twow ayANOVA ,p> 0 .05 . 59 F igu r e2 .7 .D i f f e r en c eo ff a tandl e anm a s sa sap e r c en t ag eo fto t a lm a s sin PBS WT ,PBSKO , MT I I WT ,and MT I IKOm i c ep r eandpo s t4 °C co ldch a l l eng e .D a t aa r em e an±SEM .Twow ayANOVA ,p>0 .05 . 59 e r c en tt i s su em a s sp e rto t a lbodym a s so fd i f f e r en tt i s su etyp e sin F igu r e2 .8 .P PBS WT ,PBSKO , MT I I WT ,and MT I IKOm i c ea f t e r4w e ek4 °C co ldch a l l eng e .D a t aa r em e an±SEM .Unp a i r edtt e s t s .*p<0 .05 ind i c a t e sag eno typ ee f f e c tw i th inth es am et r e a tm en t . 60 F igu r e2 .9 .A .No r ep in eph r in eindu c edth e rmog en e s i sinco lda c c l im a t edPBS WT , .M e anb a s a lm e t abo l i cr a t e PBSKO , MT I I WT ,and MT I IKOm i c e .B (B MR )andm ax im a lm e t abo l i cr a t e(MMR )o fco lda c c l im a t edPBS WT ,PBSKO , MT I I WT ,and MT I IKOm i c e .D a t aa r eexp r e s s eda s m e an±SEM .Twow ayANOVA .*p<0 .05ind i c a t e sag eno typ ee f f e c t w i th inth es am et r e a tm en t . 62 F igu r e2 .10 .R e sp i r a to ryex ch ang er a t io(RER )o fco lda c c l im a t edPBS WT , PBSKO , MT I I WT ,and MT I IKOm i c e .S t a t i s t i c a ls ign i f i c an c e(p< 0 .05 )b e tw e enPBS WTandPBSKOa t8and10m inu t e sandb e tw e en MT I I WTand MT I IKOa t2 ,4and6m inu t e s .Twow ayANOVA . 63 F igu r e2 .11 .G en eexp r e s s ioninBATo fco ld a c c l im a t edPBS WT ,PBSKO , MT I I WT ,and MT I IKOm i c e .T a rg e tg en eexp r e s s ionw a sno rm a l i z ed totwor e f e r en c eg en e s :RPL19andPP IA .D a t aa r eexp r e s s eda s± SEM .On ew ayANOVAfo re a chg en e ,p>0 .05 . 65 F igu r e2 .12 .A .Av e r ag ep e r c en tl ip ida r e a / f i e ldo fv i ewinco ld a c c l im a t edPBS WT , PBSKO , MT I I WT ,and MT I IKOm i c e .B .Av e r ag enumb e r so fnu c l e i p e rRO Iinco lda c c l im a t edPBS WT ,PBSKO , MT I I WT ,and MT I I KOm i c e .D a t aa r eexp r e s s eda s±SEM .On ew ayANOVA ,p>0 .05 . 66 F igu r e2 .13 .Numb ld a c c l im a t ed e ro fc e l l sw i th ine a chc e l la r e ab in(µm2)inco PBS WTo r MT I I WTm i c e .D a t aa r eexp r e s s eda s±SEM .Unp a i r edtt e s t s ,p>0 .05 . 68 v i i F igu r e2 .14 .R ep r e s en t a t iv eim ag e so fh em a toxy l inandeo s ins t a in edgWAT ob s e rv eda t4xand20xinco ld a c c l im a t edPBS WT(A ) ,PBSKO(B ) , MT I I WT(C ) ,and MT I IKOm i c e(D ) . 69 F igu r eS2 .1 .PCRa s s e s sm en to fPACAPg eno typ einMu smu s cu lu s. E l e c t ropho r e s i sp e r fo rm edone th id iumb rom id es t a in ed1 .5%ag a ro s e g e la t100Vfo r20m inu t e s .E a chw e l lr ep r e s en t sad i f f e r en tan im a l . (+ /+ )d eno t e sPACAPw i ld typ e ,(+ / )PACAPh e t e ro zygo t e ,and( / ) PACAPkno ckou t . 89 F igu r eS2 .2 .A s s e s sm en to fRNAin t eg r i tyo fi so l a t edMu smu s cu lu sb rown ad ipo s et i s su e(BAT )w i the l e c t ropho r e s i sonane th id iumb r om id e s t a in ed1 .5%ag a ro s eg e la t100Vfo r30m inu t e s .E a chw e l lr ep r e s en t sa d i f f e r en tBATs amp l e .Th etopb andind i c a t e sin t a c t28SrRNAandth e bo t tomb andind i c a t e sin t a c t18SrRNA . 89 F igu r e3 .1 .A .P l a sm idm apfo rp s cAAVGFP(Addg en eP l a sm id#32396 ) .Im ag e cou r t e syo fJohnT .G r ay .B .Th eCMVp romo t e rw i th inp s cAAVGFP w a sr ep l a c edw i thth eSF1p romo t e r(p s cAAVSF1GFP )w i thth ego a l o fexp r e s s ingGFPsp e c i f i c a l lyinth eVMNo fth ehypo th a l amu s ,a r eg iono fth eb r a inknowntosp e c i f i c a l lyexp r e s sSF1 .V i ru sw i l lb e d e l iv e r edtoth eb r a inv i as t e r eo t ax i cin j e c t ionin toth eVMN .Ad ap t ed w i thp e rm i s s ionf romtheau tho r .Sou r c e : h t tp s : / /www . addg en e .o rg /32396 / .A c c e s s ed M ay4 ,2017 . 99 F igu r e3 .2 .P l a sm idm apfo rpGEM®TV e c to r(P rom eg a ) .SF1in s e r ti sl ig a t edin to 104 th el a cZg en ebya t t a ch ingtoov e rh ang ingthym in enu c l eo t id e s , d i s rup t ingth el a zZg en e .A c c e s s edf rom h t tp s : / /www .p rom eg a . c a /p rodu c t s /p c r /p c rc lon ing /pg em t v e c to r sy s t em s / .Copy r igh t©2017P rom eg aCo rpo r a t ion .Im ag er ep rodu c ed w i thp e rm i s s ionf romth eco rpo r a t ion . F igu r e3 .3 . Lo c a t iono fl ambd aandb r egm aa sd e f in edbyD r .G eo rg eP ax ino s . In t e r au r a ll in eco r r e spond sw i thth ep l a c em en to fth ee a rb a r s . R ep rodu c edf rom“Th er a tb r a inins t e r eo t ax i ccoo rd in a t ion ” ,byG . P ax ino s ,&C .W a t son ,1998 ,1 s tEd i t ion ,p .11 .Copy r igh t©2017 A c ad em i cP r e s s /E l s ev i e r .R ep rodu c edw i thp e rm i s s ionf rompub l i sh e r . 107 F igu r e3 .4 .A .S t e r eo t ax i cadm in i s t r a t ion sa r eg iv enu s ingx ,y ,andzcoo rd in a t e sto asp e c i f i clo c a t ionandd ep th .B .Lo c a tiono fin j e c t ionin toth eVMNo f th ehypo th a l amu s .I l lu s t r a t ion sbyEn c ap su l aN anoS c i en c e sLLC , B r en twood ,TN .Copy r igh t©2014En c ap su l aN anoS c i en c e sLLC. Ob t a in edf romh t tp s : / /www . c lod ro som e . com / an im a lin j e c t ion / .Ad ap t ed w i thp e rm i s s ionf romEn c ap su l aNanoS c i en c e s . 107 v i i i F igu r e3 .5 . As e c t iono fnu c l eo t id e sm a t ch ingf romNCB I ’ sBLASTb e tw e enth e p s cAAV GFPp l a sm ids equ en c ep rov id edf romAddg en e(Qu e ry )and th ei so l a t edp l a sm id(Sb j c t )s equ en c edu s ing M13R ev e r s ep r im e r . Y e l lowind i c a t e sAv r I IREs i t e(5 ’CCTAGG3 ’ )andr edind i c a t e s B spE IREs i t e(5 ’TCCGGA3 ’ ) .Asm a l ls e c t iono fth ei so l a t ed p l a sm idDNAw a sun r e ad ab l e( ind i c a t edby“N ” )u s ing M13R ev e r s e p r im e r .Am a t ch ings equ en c ew a scon f i rm edu s ing M13Fo rw a rd p r im e r( r e su l t sno tshown ) . 109 F igu re3 .6 . NCB I ’ sBLASTs e a r cho fth es equ en c eob t a in edf romSF1pGEM® T V e c to r(Sb j c t )u s ingT7p r im e rr ev e a l edam a t chtoas e c t iono fth eMu s mu s cu lu ss t r a inC57BL /6 J ,ch romo som e2 ,GRCm38 .p4(Qu e ry ) :th e r eg iono fth emou s eg enom eth a thou s e sth eSF1p romo t e r .Y e l low ind i c a t e sAv r I IREs i t e(5 ’CCTAGG3 ’ )andr edind i c a t e sB spE IRE s i t e(5 ’TCCGGA3 ’ )add edtoth ePCR amp l i f i edSF1 . 111 F igu r e3 .7 .A .Co ron a lc ro s ss e c t iono fth eb r a inshow ingth elo c a t iono fth e hypo th a l amu sandc lo s eupf iv ekeyhypo th a l am i cnu c l e iinvo lv edin th er egu l a t iono fen e rgym e t abo l i sm .PVN ;p a r av en t r i cu l a rnu c l eu s , DMN ;do r som ed i a lnu c l eu s ,LHA ;l a t e r a lhypo th a l am i ca r e a ,VMN ; v en t rom ed i a lnu c l eu s ,ARC ;a r cu a t enu c l eu s ,3V ;th i rdv en t r i c l e . Ad ap t edf rom“R ev i s i t ingth eV en t r a lM ed i a lNu c l eu so fth e Hypo th a l amu s :Th eRo l e so fSF1N eu ron sinEn e rgyHom eo s t a s i s” , byY .Cho i ,T .Fu j ik aw a ,J .L e e ,A .R eu t e r ,&K .K im ,2013 ,F ron t i e r s InN eu ro s c i en c e ,7.Copy r igh t©2013Cho i ,Fu j ik aw a ,L e e ,R eu t e r andK im .Ad ap t edw i thp erm i s s ionf romau tho r .B .Do r s a lv i ewo fth e mou s eb r a in .C .V en t r a lv i ewo fth emou s eb r a in(boxind i c a t e s hypo th a l amu s ) .D .B lu edy eob s e rv edinth ehypo th a l amu s .E . An t e r io rco ron a ls e c t ionth roughth ehypo th a l amu s ;b lu edy eob s e rv ed inth e3Vandsu r rou nd inghypo th a l am i ct i s su e s .F .M idd l eco ron a l s e c t ionth roughth ehypo th a l amu s ;dy elo c a l i z eda longth e3V .G . Po s t e r io rco ron a ls e c t ionth roughth ehypo th a l amu s ;dy elo c a l i z ed fu r th e rdo r s a la longth e3V . 112 F igu r e3 .8 .M idd l eco ron a lc ro s ss e c t ionth roughth ehypo th a l amu so fMu s mu s cu lu ss t e r eo t ax i c a l lyin j e c t edw i th5xlo ad ingdy e .Ru l e rshow s 1mmin c r em en t s . 114 F igu r e3 .9 . Th eCMVp romo t e rr ep l a c edw i thth eSF1p romo t e r ,andth eGFPg en e r ep l a c edw i thth ePACAPg en e(p s cAAV SF1PACAP )w i thth ego a l o fexp r e s s ingPACAPsp e c i f i c a l lyinth eVMNo fth ehypo th a l amu s ,a r eg iono fth eb r a inknowntosp e c i f i c a l lyexp r e s sSF1 .V i ru sw i l lb e d e l iv e r edtoth eb r a inv i as t e r eo t ax i cin j e c t ionin toth eVMN .Ad ap t ed w i thp e rm i s s ionf romth eau tho r .Sou r c e : h t tp s : / /www . addg en e .o rg /32396 / .A c c e s s ed M ay4 ,2017 . 117 ix F igu r e4.1 .Hypo th e s i z edp a thw ay so fin t e r a c t ionfo rad ap t iv eth e rmog en e s i sin BATinr e spon s etoco ldexpo su r e .VMN ;v en t rom ed i a lnu c l eu s , PACAP ;p i tu i t a ryad eny l a t ecy c l a s ea c t iv a t ingpo lyp ep t id e , MC ; m e l ano co r t insy s t em ,SNA ;symp a th e t i cn e rv ea c t iv i ty ,BAT ;b rown ad ipo s et i s su e .Im ag e sad ap t edf rom“ R ev i s i t ingth eV en t r a lM ed i a l Nu c l eu so fth eHypo th a l amu s :Th eRo l e so fSF1N eu ron sinEn e rgy Hom eo s t a s i s” ,byY .Cho i ,T .Fu j ik aw a ,J .L e e ,A .R eu t e r ,&K .K im , 2013 ,F ron t i e r sInN eu ro s c i en c e,7.Copy r igh t©2013Cho i ,Fu j ik aw a , L e e ,R eu t e randK im .Ad ap t edw i thp e rm i s s ionf romau tho r . x 127 L ISTOFABBREV IAT IONS αMSH A lph aM e l ano cy t eS t imu l a t ingHo rmon e β3 AR B e t a3Ad r en e rg i cR e c ep to r 3V Th i rdV en t r i c l e Amp Amp i c i l l in ANOVA An a ly s i so fV a r i an c e ARC A r cu a t eNu c l eu s ATP Ad eno s in eT r ipho sph a t e BAT B rownAd ipo s eT i s su e BBB B loodB r a inB a r r i e r BMR B a s a lM e t abo l i cR a t e CMV Cy tom eg a lov i ru s DMN Dor som ed i a lNu c l eu s FA F a t tyA c id FFA F r e eF a t tyA c id GFP G r e enF luo r e s c en tP ro t e in GPCR GP ro t e inCoup l edRe c ep to r s gWAT Gon ad a l Wh i t eAd ipo s eT i s su e HSL Ho rmon eS en s i t iv eL ip a s e ICV In t r a c e r eb rov en t r i cu l a r ingWAT Ingu in a l Wh i t eAd ipo s eT i s su e KO Kno ckOu t LB Lu r i aB ro th LHA L a t e r a lHypo th a l am i cA r e a x i MCR M e l ano co r t inR e c ep to r MMR M ax im a lM e t abo l i cR a t e MT I I M e l ano t anI I NE No r ep in eph r in e PACAP P i tu i t a ryAd eny l a t eCy c l a s eA c t iv a t ingPo lyp ep t id e PBS Pho sph a t eBu f f e r edS a l in e PCR Po lym e r a s eCh a inR e a c t ion POMC Pro op iom e l ano co r t in PVN P a r av en t r i cu l a rNu c l eu s RE R e s t r i c t ionEn zym e RER R e sp i r a to ryEx ch ang eR a t io s cAAV9 S e l fComp l im en t a ryAd enoA s so c i a t edV i ru sS e ro typ e9 SEM S t and a rdE r ro rM e an SF1 S t e ro idog en i cF a c to r1 SNA Symp a th e t i cN e rv eA c t iv i ty UCP1 Un coup l ingP ro t e in1 VMN V en t rom ed i a lNu c l eu s WAT Wh i t eAd ipo s eT i s su e WT W i ldTyp e x i i CHAPTER1 Endo c r in ePhy s io logyo fOb e s i ty:En e rgyHom eo s ta s i sandTh e rmog en e s i s 1 1 .1OBESITYASAD ISEASE Ob e s i tyi sad i s e a s ech a r a c t e r i z edbyin c r e a s edwh i t ead ipo s et i s su e(WAT )m a s s r e su l t ingf rombo thad ipo cy t ehyp e rp l a s i aandc e l lu l a rhyp e r t rophy(G a l i c ,O akh i l l ,& S t e inb e rg ,2010 ;L e e,&Sh ao ,2014 ) .W i thch ron i cfoodabund an c eandth ech ang eto s ed en t a rywo rkp r a c t i c e sandl i f e s ty l esinmod e rnso c i e ty ,th ep r ev a l en c eo f adu l tob e s i tyh a s mo r eth andoub l edwo r ldw id es in c e1980( F inu c an ee ta l . ,2011) .I n2014 ,i tw a se s t im a t ed th a t39 %o fth ewo r ld ’ sadu l tpopu l a t ionw a s ov e rw e igh t ,d e f i n edbyth eWo r ldH e a l th O rg an i z a t ion(WHO )a sap e r sonw i thabodym a s sind ex(BM I )(bodym a s s(kg ) /h e igh t 2 (m ) )g r e a t e rth ano requ a lto25(L e e ,&Sh ao ,2014 ;WHO,2016 ;Y eo ,&H e i s l e r ,2012 ) . Ov e rw e igh tandob e s i tya f f e c t sa l lag eandso c io e conom i cg rou p sandpo s e sasub s t an t i a l e conom i cbu rd enonso c i e tyandh e a l thc a r esy s t em sdu etoin c r e a s edr i sko fd ev e lop ing ch ron i cd i s e a s esu cha sc a rd iov a s cu l a rd i s e a s e ,typ e2d i ab e t e s ,a r th r i t i s ,hyp e r t en s ion, s t rok e , andsom efo rm so fc an c e r(L id e l l ,B e t z ,&Ene rb ä ck ,2014 ; WHO ,2016 ) .Inadd i t ion ,w h i l e th ep r ev a l en c eo fob e s i tycon t inu e stor i s e ,sotoodo e sth es t igm a(L id e l le ta l . ,2014 ) . N eg a t iv es t e r eo typ e sth a tp e r son sw i thob e s i tya r el a zy ,l a ck ings e l fd i s c ip l in e ,o r unmo t iv a t edm ayt r an s l a t ein toin eq u a l i t i e sinh e a l thc a r e ,edu c a t ionandemp loym en t (C an ad i anOb e s i tyN e two rk ,2016 ) .R e s e a r chh a sshownth a tbodyw e igh ti sd e t e rm in edby bo thenv i ronm en t a landg en e t i cf a c to r s .G en e t i c a l ly ,bodyw e igh ti sapo lyg en i cf a c to r , s im i l a rtoh e igh t ,sk in ,ey e ,andh a i rco lo r ,ind i c a t ingth a tth ed ev e lopm en to fob e s i tyi s in f lu en c edbymo r eth anap e r son ’ smo t iv a t ion ,s e l fcon t ro l ,andl i f e s ty l e(H inn ey ,Vog e l ,& H eb eb r and ,2010 ) .Ap e r son ’ sg en e t i cp r ed i spo s i t iontoth ed i s e a s ec anr e su l tf rom p a thophy s io log i c a lch ang e sth a tl e adtoin c r e a s ede f f i c i en cyinen e rgym e t abo l i smandas t a t e o fch ron i cpo s i t iv een e rgyb a l an c e(Choqu e t ,& M ey r e ,2011 ;W a l l ey,2006 ) .Th e con s equ en c e so fov e rw e igh tandob e s i tyr ang ef romd e c r e a s edov e r a l lqu a l i tyo fl i f eto 2 p r em a tu r ed e a t h;ind i c a t ingth en e c e s s i tytound e r s t andth ec au s e so fob e s i tysu chth a ti tc an b ep r ev en t edandt r e a t ede f f e c t iv e ly( C en t r efo rD i s e a s eCon t ro l ,2016 ;G a l i ce ta l . ,2010; WHO ,2016) . 1 .2CAUSESOFOBES ITY Ob e s i tyi sd e t e rm in edbybo thg en e t i cm ak eupandenv i ronm en t a lf a c to r s(F a rooq i& O ’R ah i l ly ,2006;H inn ey ,Vog e l ,&H eb eb r and ,2010 ) .Th ec au s eo fob e s i tyc anb eexp l a in ed byahom eo s t a t i cimb a l an c einen e rgym e t abo l i smandimp a i r edad ipo s et i s su efun c t ion (H a rwood ,2012 ) .M e t abo l i chom eo s t a s i s i sa ch i ev edwh enen e rgyin t ak em a t ch e sen e rgy ou tpu t .En e rgyin t ak econ s i s t so ffoodcon sump t ionwh i l een e rgyexp end i tu r ein c lud e sb a s a l m e t abo l i cr a t e ,w a s t ep rodu c t ion ,phy s i c a la c t iv i ty ,andth e rmog en e s i s(F igu r e1 .1 ) ,wh i ch w i l lb eam a jo rfo cu so fth er e s e a r chinCh ap t e r s2and3andd i s cu s s edind e t a i lb e low . Wh en en e rgyin t ak ech ron i c a l lyex c e ed sen e rgyexp end i tu r e ,ad ipo s et i s su eexp and s bo thinm a s sands i z edu etoanin c r e a s einth enumb e ro fc e l l sandth es i z eo fth ead ipo cy t e s , wh i chin c r e a s ea st r ig ly c er id e sa c cumu l a t ew i th in(Fon s e c a A l an i z ,T ak ad a ,A lon soV a l e ,& L im a ,2007 ;H a rwood ,2012 ) .R e s e a r chh a sshownth a tad ipo s et i s su ei t s e l fp l ay san impo r t an tro l einth em a in t en an c eo fm e t abo li chom eo s t a s i s .Inob e s i ty , WATi s dy s fun c t ion a lw i thach ang einth ep rodu c t iono fad ipo cy t e d e r iv edf a c to r sin c lud ing ad ipok in e s(d i s cu s s edb e low ) ,f r e ef a t tya c id s(FFA s ) ,andcy tok in e sth a tcon t ro l m e t abo l i c hom eo s t a s i so fav a r i e tyo ft i s su e sonamo l e cu l a rl ev e l(G r e enb e rg ,&Ob in ,2006 ;L e e,& Sh ao ,2014 ) .I ti sth i sdy s fun c t ion a l WATth a ti sfoundtob ea s so c i a t edw i thanin c r e a s ed r i skfo rth ech ron i cd i s e a s e sl i s t edabov einob e s i ty(L e e ,&Sh ao ,2014 ) .Thu s ,r e s e a r chin to th eb io log i c a lm e ch an i sm sl ink ingad ipo cy t efun c t ionw i then e rgyhom eo s t a s i sw i l lsh ed l i gh tonth eb io log i c a lcomp l ex i t i e so fob e s i tyandm ayl e adtoinnov a t iv eth e r apeu t i c 3 En e r gy In t ak e En e r gy Exp end i tu r e Phy s i c a l A c t iv i ty Th e rmog en e s i s D i e t /F e ed ing B a s a lM e t abo l i c R a t e ���� �� � � �� �� F igu r e1 .1 .F a c to r scon t r ibu t ingtoth een e rgyb a l an c eequ a t ion . 4 t r e a tm en t sfo rth i sd i s e a s e .Fu r th e rev id en c efo rak eyro l eo fad ipo s et i s su einth e ma in t en an c eo fen e rgyhom eo s t a s i sw i l lb ed i s cu s s edh e r e . 1 .3WH ITEAD IPOSET ISSUEASASTORAGEANDENDOCR INEORGAN WAT i scomp r i s edp r edom in an t lyo fwh i t ead ipo cy t e s ;ac e l ltyp esp e c i a l i z e din s to r ingen e rgya st r ig ly c e r id e s .WAT a l socon t a in sm a c roph ag e s ,f ib rob l a s t s ,and p r e ad ipo cy t e sh e ldtog e th e rbyloo s econn e c t iv et i s su eandi sh igh lyv a s cu l a r i z edand inn e rv a t edbyth eau tonom i cn e rvou ssy s t em(Ah im a ,2006 ) .I ta c t sa sath e rm a lin su l a to rby r edu c ingh e a tlo s sth r oughth esk in ,anda sap ro t e c t iv el a y e ra roundo rg an sandmu s c l e g roup swh e r ei tso f t en simp a c ttoen su r ep rop e rfun c t ion ingo fth e s et i s su e sw i thou tth elo s s o fin t eg r i ty(H a rwood ,2012 ) . WAT h a slongb e enknownfo ri t sro l ea sanen e rgyr e s e rvo i r ,invo lv edinbo thl ip id andg lu co s em e t abo l i sm .Inp e r iod so fc a lo r i cabund an c e ,con sum edg lu co s eandl ip ids s t imu l a t ein su l inr e l e a s ef romth ep an c r e a s ,wh i ch p romo t e sup t ak eo fFFA sinth el iv e rand WATwh e r eth ey a r ecomb in e dw i thg ly c e ro ltofo rmt r ig ly c e r id e s(G a l i ce ta l . ,2010 ) . Add i t ion a l ly ,ex c e s sg lu co s ei ss to r eda st r ig ly c e r id e sinth el iv e rand WATth roughth e p ro c e s so fl ipog en e s i s(G a l i ce ta l . ,2010 ) .Du r ingen e rgysho r t ag eo rp e r iod so ff a s t ing , in su l inl ev e l sf a l lands t imu l a t el ipo ly s i sin WAT ,r e su l t inging ly c e ro landFFA sth a tsupp ly g lu con eog en i candox id a t iv ep a thw ay sinth el iv e rwh e r eg ly cog enb r e akdowno c cu r sto in c r e a s ec i r cu l a t ingg lu co s el ev e l s ,th e r e fo r esupp ly ingth eb r a inandv i t a lo rg an sw i then e rgy (G a l i ce ta l . ,2010 ;H a rwood ,2012 ;L e e ,&Sh ao ,2014 ;Vá zqu e z V e l a,To r r e s ,&Tov a r , 2008 ) . G av r i lov ae ta l .(2000 )d emon s t r a t edth ec r i t i c a limpo r t an c eo fwh i t ead ipo s et i s su ein m e t abo l i chom eo s t a s i sw i thth e i rmou s emod e lo fl ipo a t roph i cd i ab e t e s ,knowna sth eAZ IP /F1mou s el in e .Th emou s el in ew a sg en e r a t edbyt a r g e t inganad ipo cy t e sp e c i f i c 5 t r an sg en eth a texp r e s s e sadom in an t n eg a t iv ep ro t e in,knowna sAZ IP /F,r e su l t inginth e inh ib i t iono fad ipo cy t e sp e c i f i cp ro t e in sandDNAb ind ing(Mo i t r ae ta l . ,1998 ) .Th er e su l t w a sm i c eth a th adv i r tu a l lyno WAT .Th em i c edev e lop edch a r a c t e r i s t i c ss im i l a rtotho s eo f hum an sw i ths ev e r el ipo a t roph i cd i ab e t e s ,in c lud ingin su l inr e s i s t an c e ,hyp e rg ly c em i a , hyp e r l ip id em i a ,andf a t tyl iv e r s .T r an sp l an t a t iono fh e a l thy WATin toth eA Z IP /F1m i c e r ev e r s edth eph eno typ e ,andr e su l t e dinno rmog ly c em i cm i c ew i thlow e r edin su l inl ev e l s , d e c r e a s eds e rumt r i a cy lg ly c e ro l s ,d e c r e a s edh ep a t i cg lu con eog en e s i s ,andd e c r e a s edamoun t s o ff a td epo s i t edinth el iv e r .Th e s eb en e f i c i a le f f e c t sw e r ed ep end en tonth ep r e s en c eo f t r an sp l an t ed WATandt hu si tb e c am eev id en tth a tth el a cko f WATi sm e t abo l i c a l ly d e t r im en t a l(G av r i lov ae ta l . ,2000 ) .Th e s er e su l t sp rov id edacomp e l l ingp l a t fo rmfo rth e a rgum en tth a tad ipo s et i s su ep l ay sanimpo r t an tro l einm a in t a in inghom eo s t a s i sona mo l e cu l a rl ev e l,a ssugg e s t edabov e .Inf a c t ,r e s e a r chh a sshownth a t WATe f f e c t iv e ly commun i c a t e sw i thth ebodyandb r a inth roughth es e c r e t iono fg r e a t e rth an50ad ipo cy t esp e c i f i cf a c to r sth a tin c lud eho rmon e sandcy tok in e s(F igu r e1 .2 )(G a l i ce ta l . ,2010 ; H a rwood ,2012 ;L e e,&Sh ao ,2014;S aku r a ie ta l . ,2013 ) .Th e s ead ipo cy t e sp e c i f i cf a c to r s , knowna sad ipok in e s ,a c tonbo thc en t r a landp e r iph e r a lo rg an ssu cha sth el iv e r ,p an c r e a s , b r a in ,andsk e l e t a lmu s c l etocon t ro ld iv e r s em e t abo l i cp ro c e s s e sin c lud ingen e rgy exp en d i tu r e ,l ip idm e t abo l i sm ,foodin t ak e ,andin f l amm a t ion(H a rwood ,2012 ) .Th e d i s cov e ryo fad i pok in e sl edtoth enoww id e lya c c ep t edandr e cogn i z edro l eo f WATa sa m a jo rendo c r in eo rg an ,incon t r a s ttoi t se a r lyc l a s s i f i c a t iona sap a s s iv e ,m e t abo l i c a l ly i n a c t iv eo rg anth a ts imp lya c t eda sas to r ag er e s e rvo i rfo ren e rgy(G a l i ce ta l . ,2010 ; G r e enb e rg,&Ob in ,2006 ;H a rwood ,2012 ;L e e,&Sh ao ,2014 ;T r ayhu rn ,2005 ;Vá zqu e z V e l ae ta l . ,2008 ) .Inth em a jo r i tyo fc a s e so fov e rw e igh to rob e s i ty ,ad ipo s et i s su ei s ch a r a c t e r i z edbyp ro foundh i s to log i c a landb io ch em i c a lch ang e sa s so c i a t edw i th 6 F igu r e1 .2.K eyad ipok in e sandad ipo cy tok in e ss e c r e t edbywh i t ead ipo s et i s su e .TNF α,tumo r n e c ro s i sf a c to rα; MCP 1 , mono cy t ech emo a t t r a c t an tp ro t e in1 ;IL6 ,in t e r l euk in6 ;PA I1 , p l a sm inog ena c t iv a to rinh ib i to r1 .R ep r in t edf rom“Th eE f f e c t so fEx e r c i s eT r a in ingonOb e s i tyIndu c edDy s r egu l a t edExp r e s s iono fAd ipok in e sin Wh i t eAd ipo s eT i s su e” ,byT .S aku r a i ,J . Og a s aw a r a ,T .K i z ak i ,S .S a to ,Y .I sh ib a sh i ,M .T ak ah a sh ie ta l . ,2013 ,In t e rna t iona lJou rna lO f Endo c r ino log y,1 28 .Copy r igh t©2013T akuy aS aku r a ie ta l .R ep r in t edw i thp e rm i s s ionf rom au tho r . 7 dy s r egu l a t iono fad ipok in es e c r e t ion ,l ipo tox i c i ty ,andin f l amm a t ion ;a l lo fwh i chcon t r ibu t e tom e t abo l i cdy s fun c t ion(Ah im a ,2006 ;G r e enb e rg ,&Ob in ,2006 ) .A l thoughno td i r e c t l y p a r to fth er e s e a r chinth i sth e s i s ,th em e t abo l i cfun c t iono fon eo fth emo s tw e l ls tud i ed ad i pok in e s ,l ep t in ,w i l lb ed i s cu s s edh e r efo rfu r th e rb a ckg round .O fp a r t i cu l a rin t e r e s tw i l l b el ep t in ’ sa c t iononon eo fth esy s t em so fin t e r e s tfo rth i sth e s i s ;th em e l ano co r t insy s t em , d i s cu s s edind e t a i lb e low . 1 .4LEPT IN :AKEYREGULATORINENERGYHOMEOSTAS IS L ep t in,th ef i r s tad ipo cy t eho rmon etob eid en t i f i ed , i sp rodu c edm a in lybyad ipo cy t e s andc ro s s e sth eb lood b r a inb a r r i e rtob indl ep t inr e c ep to r sinth ehypo th a l amu s(Y eo ,& H e i s l e r ,2012 ) .L ep t ini sa l sop rodu c eda tlowl ev e l sbyth es tom a ch ,pl a c en t a ,bon e ,sk e l e t a l mu s c l e ,ando th e rt i s su e s ,a l thoughth et a rg e tinth e s ec a s e si sl ik e lytob elo c a landth e r e fo r e p a r a c r in eina c t ionr a th e rth anendo c r in e(T r ayhu rn ,2005 ) .Zh ange ta l .(1994 )f i r s tid en t i f i ed l ep t inwh i l es tudy ingob e s em i c ew i than a tu r a l lyo c cu r r ingg en e t i cmu t a t ioninth eobg en e (ob /o b ) .W i thou tth eobg en e ,wh i chw a s foundtocod efo rl ep t in(n am edbyH a l a a se ta l .in 1995 ) ,th em i c ew e r emo rb id lyob e s eandd i ab e t i cdu etos ev e r ehyp e rph ag i aandd e c r e a s ed en e rgyexp end i tu r e .Wh ent r e a t edw i thr egu l a rin j e c t ion so fl ep t inin toth eb r a in ,th eob /ob m i c er edu c edth e i rfoodin t ak e ,in c r e a s edth e i rm e t abo l i cr a t e ,andlo s tw e igh t(G r e enb e rg,& Ob in ,2006 ) .A l thoughan a logou sg en e t i cmu t a t ion sinhum an sa r eex t r em e lyr a r e ,the c l in i c a lp r e s en t a t iono fp a t i en t sw i thg en e t i cmu t a t ionsinth el ep t ing en eo ri t sr e c ep to ri s v e rys im i l a rtoth eph eno typ eo fth eob /obm i c eandl ep t inth e r apyi sb en e f i c i a linth e s ec a s e s (F a rooq i ,&O ’R ah i l ly ,2006 ) .Th e s es em in a lf ind ing sexh ib i t edth eimp o r t an c eo fl ep t ina sa k eyr egu l a to ro fen e rgyb a l an c e ,andin t rodu c edth eid e ath a tad ipo s et i s su ew a sanimpo r t an t endo c r in eo rg aninvo lv edinr egu l a t ingwho l ebodyen e rgym e t abo l i sm.C l a s s i cp a thw ay so f 8 l ep t ina c t ioninth eb r a inandi t se f f e c t sonf e ed ingb eh av io rh av eb e enw e l le lu c id a t edand th eb e s t d e s c r ib edp a thw ay sa r ed ep i c t edinF igu r e1 .3. B r i e f ly ,l ep t ina c t sonth em e l ano co r t inax i s(d i s cu s s edind e t a i lb e low ) ,po s i t iv e ly r egu l a t ingn eu ron sw i th inth ea r cu a t enu c l eu s(ARC )th a texp r e s sp ro op iom e l ano co r t in (POMC )andco c a in e -and amph e t am in er egu l a t edt r an s c r ip t s(CART ) .POMCth en und e rgo e spo s tt r an s l a t ion a lmod i f i c a t ionandi sp ro c e s s edin toa lph am e l ano cy t e s t imu l a t ing ho rmon e(αMSH ) ,wh i chth enb ind sm e l ano co r t in4r e c ep to r s(MC4R )toinh ib i tfoodin t ak e (How ,2016 ;Y eo ,&H e i s l e r ,2012 ) .Fu r th e rmo r e ,l ep t inn eg a t iv e lyr egu l a t e sth e MC4R an t agon i s tagou t i r e l a t edp ro t e in(AGRP )andn eu rop ep t id eY(NPY )who s eno rm a lfun c t ion s a r etoin c r e a s efoodin t ak e ,th e r e fo r efu r th e rinh ib i t ingfoodi n t ak e(How ,2016 ;T ab e t a ,& B eu t l e r ,2012 ) .A l thoughl ep t in ’ sro l einapp e t i t er egu l a t ionth roughth emodu l a t iono fth e m e l ano co r t insy s t eminth eARCh a sb e enex t en s iv e lys tud i ed ,i ti sapp a r en tth a tth eARC c anno texp l a ina l la sp e c t so fl ep t infun c t ion( B ingh ame ta l . ,2008 ) .Fo rex amp l e ,wh enl ep t in r e c ep to r sw e r esp e c i f i c a l lyd e l e t edinPOMCn eu ron s ,mou s ebodyw e igh t sin c r e a s edtoon ly 18%o fth a tob s e rv edinm i c ew i thcomp l e t el a cko fl ep t inr e c ep to r s(B a l th a s a re ta l . ,2004 ) . Th i ssugg e s t sth a tl ep t ina c t se l s ewh e r etocon t ro len e rgyhom eo s t a s i s ,andth a ti t sfun c t ionin o th e rb r a inr eg ion sshou ldb econ s id e r ed(B a l th a s a re ta l . ,2004 ) .A ssu ch ,l ep t inh a sb e en showntoenh an c een e rgyexp end i tu r ebyin c r e a s ingth e rmog en e s i s ,f a t tya c id(FA )ox id a t ion , andp l a sm ag lu co s el ev e l s(H av e l ,2002 ) .A c t inginth ev en t rom ed i a lhypo th a l amu s(VMN ) , l ep t inin c r e a s e ssymp a th e t i cn e rv ea c t iv i tybym e an so fth em e l ano co r t insy s t em ,r e su l t ingin a c t iv a t edth e rmog en e s i s( fu r th e rd i s cu s s edinCh ap t e r3 )(Bu t l e r ,&Con e ,2002 ;Hw ae ta l . , 1996 ;S a tohe ta l . ,1999 ;S c a rp a c e ,M a th eny ,Po l lo ck ,&Tum e r ,1997 ;Vo s s And r e a ee ta l . , 2007 ) .Impo r t an t ly ,th i sm e ch an i smw i l lb efu r th e rd i s cu s s edth roughou tth i sth e s i s .O th e r 9 F igu r e1 .3 .P a thw ay sm ed i a t inglep t ina c t ioninth ea r cu a t enu c l eu sfo ri t sro l einapp e t i t er egu l a t ion . I l lu s t r a t ionbyD .L aV in e . MUTAGENET IX ™ ,B .B eu t l e randco l l e agu e s ,2012 ,C en t e rfo rth e G en e t i c so fHo s tD e f en s e ,UTSou thw e s t e rn ,D a l l a s ,TX .Copy r igh t©L abA r ch iv e s ,LLC .R ep r in t ed w i thp e rm i s s ionf romau tho r . 10 fun c t ion so fl ep t inin c lud ea c t inga sam a jo rs ign a ltoth er ep rodu c t iv esy s t emanda saf a c to r inth eimmun esy s t em(F l i e r ,2004 ;H av e l ,2002 ;T r ayhu rn ,&B e a t t i e ,2001 ) . C i r cu l a t inglep t inl ev e l sin c r e a s el in e a r lyw i thw e igh tg a inandd e c r e a s ew i thw e igh t lo s so rdu r ingf a s t ing ,p ropo r t ion a ltoad ipo s et i s su em a s s.Inh e a l thyhum an s ,l ep t in p rodu c t ioni ss t imu l a t edbyad ipo cy t eg lu co s em e t abo l i sminth epo s tp r and i a ls t a t etor edu c e f e ed ingb eh av io r( Fon s e c aA l an i ze ta l . ,2007 ;M u e l l e re ta l . ,1998 ) .Du r ingf a s t ing ,bo th l ep t inandin su l ind e c r e a s ewh i chs t imu l a t e sapp e t i t e ,inh ib i t ssymp a th e t i cn e rv ea c t iv i ty , inh ib i t sth e rmog en e s i sandsupp r e s s e sth ea c t iono fr ep rodu c t iv eho rmon e s ,g row thho rmon e s andthy ro idho rmon e s(Ah im a ,2 006 ;H a rwood ,2012 ) . Wh e r e a sd e c r e a s e sinl ep t ininh ib i t foodin t ak eandfoods e ek ingb eh av io r s ,in c r e a s e sinl ep t ina s so c i a t edw i thov e r f e ed ingo r ob e s i tyapp e a rtoh av esub s t an t i a l lyl e s so fab io log i c a limp a c t(H av e l ,2002 ) .Fo rex amp l e , adm in i s t e r ingl ep t intoob e s ehum an sw i thou tl ep t ind e f i c i en cyindu c e sv a r i ab l e ,mod e s t ,o r now e igh tlo s s(H av e l ,2002 ) .D e sp i t eh av ingh ighl ev e l so fl ep t in ,wh i chin tu i t iv e lyshou ld r edu c efoodin t ak eandbodyf a t ,p eop l ew i thob e s i tym a in t a inh ighl ev e l so fad ipo s eti s su e (G a l i ce ta l . ,2010 ) .Th ep ropo s edr e a son ingfo rth i sph enom enoni sl ep t inr e s i s t an c e , p a r t i cu l a r lyinm e t abo l i c a l lya c t iv et i s su e ssu cha ssk e l e t a lmu s c l e(W e s t e r t e rp P l a t eng a , S a r i s ,Hu ck sho rn ,&C amp f i e ld ,2001 ) .Th e r e fo r e ,th em a jo rro l eo fl ep t i napp e a r sno ttob e a sar e s t r a in ttol im i ten e rgyin t ak eandob e s i ty ,bu tr a th e ranendo c r in ef a c to rth a ta l low ed phy s io log i c a lad ap t a t iontor edu c eden e rgyin t ak eint im e so fd e c r e a s ednu t r i en tav a i l ab i l i ty andr edu c edbodyf a ts to r e s(H av e l ,2002 ) .Wh e th e rl ep t inr e s i s t an c ei sth ec au s eo r con s equ en c eo fob e s i tyi sy e ttob eid en t i f i ed ,andwh i l eth em e t abo l i ce f f e c t so fl ep t inon t a rg e tt i s su e sa r erobu s t ,th ep r e c i s em e ch an i smbywh i chi ti ss e c r e t edf romth ead ipo cy t e r em a in se lu s iv e(Ah im a ,2006 ;S t ern ,Ru tkow sk i ,&S ch e r e r ,2016 ) .Fu r th e rinv e s t ig a t ion in toth ed ev e lopm en t a lo r ig in sandb io log i c a lm e ch an i sm so fl ep t ini sp e r t in en tfo rpo t en t i a l 11 th e r ap eu t i ct a rg e t ing(P e i r c e ,C a robb io ,&V id a l Pu ig ,2014;S t e rne ta l . ,2016) .A s m en t ion edabov e ,how eve r ,on eo fth ek eym ed i a to r so fl ep t ina c t ioni sth em e l ano co r t in sy s t em ,wh i chw i l lb ed i s cu s s edh e r e( Bu t l e r ,&Con e ,2002 ;Vo s sAnd r e a ee ta l . ,2007) . 1 .5THECENTRAL MELANOCORT INSYSTEMINENERGYHOMEOSTAS IS Th ec en t r a lp a thw ay sm ed i a t ingth em e l ano co r t insy s t em ’ sro l einen e rgyhom e os t a s i s i sw e l lch a r a c t e r i z ed(Con e ,1999 ;Con e ,2005 ;E l l a co t t,&Con e ,2006 ) .I td e t e c t sand in t eg r a t e sav a r i e tyo fn eu ron a l ,ho rmon a l ,andnu t r i t ion a lm e t abo l i cs ign a l ssu cha sl ep t in (d i s cu s s edabov e ) ,in su l in ,g lu co s e ,ands e ro ton intoin f lu en c een e rgym e t abo l i sm( J eong , MSH andβendo rph in ,a r eaf am i lyo fsm a l l K im ,&L e e ,2014 ) .M e l ano co r t in s ,su cha sαp ep t id e sth a to r ig in a t ef romth ePOMCp r e cu r so r(F igu r e1 .4 )(Bow en ,2006 ) . G r e enm ane ta l .(2013 )show edth a tm i c ew i thpo s tn a t a lab l a t iono fPOMCn eu ron sin t h eARCinth ehypo th a l amu sr e su l t edinanob e s eph eno typ ew i thr edu c eden e rgy exp end i tu r e ,s im i l a rtoth a tob s e rv edinl ep t in d e f i c i en tan im a lmod e l s .POMCg en ed e l e t ion andg en emu t a t ion sh av ea l soshowntob eh igh lyco r r e l a t edw i thob e s i tyd ev e lopm en tin hum an s(Dub e rne ta l . ,2008 ;Ch a l l i se ta l . ,2004 ;K rud ee ta l . ,1999 ;Y a sw en ,D i eh l , B r enn an ,&Ho chg e s chw end e r ,1999 ) .Add i t ion a l ly ,wh enPOMCi sov e r exp r e s s edinth e ARCe i th e rbyv i ru sa s so c i a t edg en ed e l iv e ryo rg en e t i cmod i f i c a t ioninl ep t ind e f i c i e n t an im a l s ,th eyw e r ep ro t e c t edf romth ed ev e lopm en to fob e s i ty(L i , Mobb s ,&S c a rp a c e ,2003 ; M i zuno ,K e l ly ,P a s in e t t i ,Rob e r t s ,& Mobb s ,2003 ) .Th e s es tud i e sd emon s t r a t eac l e a rro l eo f th ec en t r a lPOMCsy s t eminm e t abo l i cr egu l a t ion . M e l ano co r t inr e c ep to rexp r e s s ionandfun c t ioninth em amm a l i anb r a inh av eb e en w e l le lu c id a t eds in c eth e1990 ’ s(Con e ,1999 ;J eong ,K im ,&L e e ,2014 ) .Ing en e t i c a l ly a l t e r edan im a l sl a ck ingth eg en efo rm e l ano co r t inr e c ep to rtyp e s3and4(MC3Rand MC4R , r e sp e c t iv e ly ) ,anob e s eph eno typ een su e s ,p rov id ingev id en c efo rth einvo lv em en to f 12 F igu r e1 .4 .Mu l t ip l ep ep t id e sp rodu c edf romth es ing l ep ro p ro t e in ,p roop iom e l ano co r t in(POMC) . I l lu s t r a t ionbyR .Bow en ,2006,Co lo r adoS t a t eUn iv e r s i ty . Ob t a in edf rom h t tp : / /www .v ivo . co los t a t e . edu /hbook s /p a thphy s / endo c r in e /hypop i t / a c th .h tm l .R ep r in t edw i th p e rm i s s ionf romau tho r . 13 m e l ano co r t in sinth er egu l a t iono fen e rgym e t abo l i sm(Con e ,1999 ) . MC3R sand MC4R sa r e foundins ev e r a lb r a inr eg ion sinth ehypo th a l amu sknownfo rth er egu l a t iono fen e rgyin t ak e p ro c e s s e ssu cha sf e ed ing ,anden e rgyou tpu tp ro c e s s e ssu cha sth e rmog en e s i s ,in c lud ingth e p a r av en t r i cu l a rnu c l eu s(PVN ) ,th ea r cu a t enu c l eu s(ARC )(d i s cu s s ede a r l i e rint e rm so f l ep t inr egu l a t ion ) ,th ev en t rom ed i a lnu c l e u s(VMN )( th eb r a inr eg iono fin t e r e s tfo rCh ap t e r 3 ) ,th el a t e r a lhypo th a l am i ca r e a(LHA ) ,anddo r som ed i a lnu c l eu s(DMN )(F igu r e1 .5 ) (Con e ,1999 ) .Th er e s e a r chp r e s en t edinth i sth e s i sr e l a t e stoth em e l ano co r t insy s t em ’ sro l e inen e rgyou tpu tth roughth ea c t iv a t iono fth e rmog en e s i sinb rownad ipo s et i s su e(BAT ) . 1 .6BROWNAD IPOSET ISSUEASAPOTENT IALTHERAPEUT ICTARGETFOR OBES ITY B rownad ipo s et i s su e(BAT )h a sb e enw e l lch a r a c t e r i z edinrod en t sando th e rsm a l l m amm a l s(H an eye ta l . ,2002 ) .Inhum an s ,BATw a sthough ttoex i s tp r edom in an t lyinth e in t e r s c apu l a rr eg ioninin f an t son lyandw a sr ap id lylo s tpo s tn a t a l lysu chth a ton lyv e s t ig i a l amoun t sr em a in edl a t e rinl i f e(H an eye ta l . ,2002 ;N ed e rg a a rd ,B eng t s son ,&C annon ,2007 ) . In2007 ,how ev e r ,i tw a sshownth a tb rownad ipo cy t e sr e s id einth ec e rv i c a land sup r a c l av i cu l a rr eg ion sinhum anadu l t s(B a r t e l t ,&H e e r en ,2013 ;H an eye ta l . ,2002 ) . S im i l a rto WAT ,BATi sc ap ab l eo fa c cumu l a t ingands to r ingl ip id s .How ev e r ,BATi s d i s t in c tf rom WATinth a ti t sad ipo cy t e scon t a inanabund an c eo fm i to chond r i a(F igu r e1 .6 ) th a ta r een r i ch edw i thasp e c i a l i z edp ro t e inc a l l edun coup l ingp ro t e in1(UCP1 ) ,wh i ch un coup l e ssub s t r a t eox id a t ionf romad eno s in et r ipho sph a t e(ATP )p rodu c t ionsu chth a th e a t i sp rodu c ed(L id e l le ta l . ,2014 ;P e i r c ee ta l . ,2014 ) .Th i sp ro c e s so fh e a tp rodu c t ioni sth e m a jo rro l eo fBATandi sknowna sad ap t iv eth e rmog en e s i s(d i s cu s s edb e low ) . In t e r e s t ing ly ,a sar e su l to fth e rmog en i cs t imu l i ,b rown l ik ead ipo cy t e sa r eob s e rv ed insom e WATr eg ion s :ap ro c e s sknowna s“b rown ing ”o f WAT(B a r t e l t ,&H e e r en ,2013 ) . 14 PVN � L HA � � � � DMN � � VMN � � � � � � � ARC 3V F igu r e1 .5.Co ron a lc ro s ss e c t iono fth eb r a inshow ingth elo c a t iono fth ehypo th a l amu sandc lo s e up f iv ek eyhypo th a l am i cnu c l e iinvo lv edinth er egu l a t iono fen e rgy m e t ab o l i sm.PVN ;p a r av en t r i cu l a r nu c l eu s ,DMN ;do r som ed i a lnu c l eu s ,LHA ;l a t e r a lhypo th a l am i ca r e a ,VMN ;v en t rom ed i a lnu c l eu s , ARC ;a r cu a t enu c l eu s ,3V ;th i rdv en t r i c l e .Ad ap t edf rom“R ev i s i t ingth eV en t r a lM ed i a lNu c l eu so f th eHypo th a l amu s :Th eRo l e so fSF 1N eu ron sinEn e rgyHom eo s t a s i s” ,byY .Cho i ,T .Fu j ik aw a ,J . L e e ,A .R eu t e r , &K .K im ,2013 ,F ron t i e r sInN eu ro s c i en c e,7.Copy r igh t©2013Cho i ,Fu j ik aw a , L e e ,R eu t e randK im .Ad ap t edw i thp e rm i s s ionf romau tho r . W h i t eA d ipo s eT i s s u e B row nA d ipo s eT i s su e F igu r e1 .6.H i s to log i c a lr ep r e s en t a t i ond i f f e r en t i a t ingWATandBAT .WAT i sun i lo cu l a rw i thl a rg e l ip idd rop l e t s( im ag eda t40x ) ,wh i l eBATcon t a in s mu l t i lo cu l a rl ip idd rop l e t sw i thach a r a c t e r i s t i c abund an c eo fm i to chond r i a( im ag eda t60xu s ingo i limm e r s ion ).Im ag e sf roms tud i e sinth eG r ayl ab . 15 A l thoughth e s ead ipo cy t e sa r ec ap ab l eo fund e rgo ingad ap t iv eth e rmog en e s i s ,th e i rl in e ag ei s c lo s e rtoth a to fwh i t ead ipo cy t e sth anb rownad ipo cy t e s(B a r t e l t ,&H e e r en ,2013 ;G i r a l t ,& V i l l a roy a ,2013 ) .Th e s ead ipo cy t e sa r er e f e r r edtoa sb e ig e ,br i t e( b rowninwh i t e ) ,o r indu c ib l ead ipo cy t e s(G i r a l t ,&V i l l a roy a ,2013 ) .Fo rth er em a ind e ro fth i sth es i s ,th eyw i l lb e r e f e r r edtoa sb e ig ead ipo cy t e s .A l thoughth ea c t iv a t iono fBATandth eb rown ingp ro c e s sin WAT sh a r ecommonp a thw ay so findu c t ionth efo cu sh e r ew i l lb eonb rownad ipo cy t e sand BAT,wh i l ea cknow l edg ingth a to th e rth e rmog en i cad ipo cy t e sex i s tanda r ecu r r en t lyund e r in t en s es tudy(G i r a l t ,&V i l l a roy a ,2013 ) .Th er e l ev an c eo fBATw i l lb ed i s cu s s edint e rm so f en e rgym e t abo l i sm ,a sev id en c esugg e s t sth a tob e s i tym ayinp a r tb ed e t e rm in edbyr edu c ed en e rgyexp end i tu r e(F l i e r ,2004 ) . 1 .7THERMOGENES ISINBROWNAD IPOSET ISSUE Th e rmo e f f e c to rm e ch an i sm sex i s tfo rbo thh e a tandco ldd e f en s e .Th e s ein c lud e th e rmo r egu l a to ryb eh av io r stoin c r e a s eo rd e c r e a s eth eamoun to fh e a tlo s s ,cu t an eou s v a sod i l a t iono rcon s t r i c t ion ,ev apo r a t iv ecoo l ing( sw e a t ing )o rh e a tp rodu c t ion ( th e rmog en e s i s )(Mo r r i son ,2016 ) .Th e rmog en e s i s ,acompon en to fth e‘ en e rgyou t ’a sp e c to f th een e rgyb a l an c eequ a t ion( to t a lbod yen e rgy=en e rgyin–en e rgyou t ) ,i sth eb eh av io r a l andau tonom i cin c r e a s einbodyt emp e r a tu r einr e spon s etoco lds t r e s so rov e r f e ed ingto p r e s e rv een e rgyhom eo s t a s i s(Mo r r i son ,2016 ) .Uponco lds t imu l a t ioninrod en t sandhum an adu l t s ,th e rmog en e s i si sin i t i a l lym ed i a t edbysh iv e r inginsk e l e t a lmu s c l eth a tp rodu c e sh e a t th roughm e ch an i c a lwo rk(Mo r r i son ,2016 ) .A f t e rp ro long edco lds t imu l a t ion ,sh iv e r ing sub s id e swh i l enon sh iv e r ing ,o rad ap t iv eth e rmog en e s i sin c r e a s e sinb rown( andb e ig e ) ad ipo cy t e sth a ta r ea c t iv a t edbye f f e r en ts ign a l sf romth ehypo th a l amu sv i asymp a th e t i c n e rv einn e rv a t ion(F igu r e1 .7 )(B lond ine ta l . ,2014 ;L id e l le ta l . ,2014 ;P e i r c ee ta l . ,2014 ) . Th esymp a th e t i cn eu r a linpu tp romo t e sm i to chond r i aox id a t ionth a tshun t sp ro tonf lux e sin to 16 (S e eF igu r e1 . 8fo rm e ch an i sm ) F igu r e1 .7 .Co ldindu c eda c t iv a t iono fad ap t iv eth e rmog en e s i sinb rownad ipo s et i s su e .UCP1 ; un coup l ingp ro t e in1 .Ad ap t edf rom“ A c t iv a t ionandr e c ru i tm en to fb rownad ipo s et i s su ebyco ld e s t expo su r eandfooding r ed i en t sinhum an s ” ,byM .S a i to ,T .Yon e sh i ro ,& M .M a t su sh i t a ,2016 ,B P ra c t i c e &R e s ea r chC l in i ca lEndo c r ino log y& M e tabo l i sm,30(4 ) ,537 547 .Copy r igh t©2017 � E l s ev i e rB .V .Ad ap t edw i thp e rm i s s ionf rompub l i sh e r . 17 h e a tp rodu c t ionv i ath esp e c i a l i z edp ro t e inUCP 1(F igu r e1 .7 ) .Th eun iqu ec ap a c i tyto und e rgoth een e rg e t i c a l lyexp en s iv ep ro c e s so fad ap t iv eth e rmog en e s i sh a sb rough tBATin to th el im e l igh ta sapo t en t i a lth e r ap eu t i ct a rg e tinp a tho log i c a ls t a t e so fen e rgydy s fun c t ion , in c lud ingob e s i ty(Ch e ch i ,C a rp e n t i e r ,&R i ch a rd ,2013 ;L id e l le ta l . ,2014 ;S a i to ,2013 ) . No r ep in eph r in e(NE )r e l e a s edbyth epo s tg ang l ion i csymp a th e t i cn eu ron si sth e p r im a ryn eu ro t r an sm i t t e rth a ta c t iv a t e sth e rmog en e s i sinb rownad ipo cy t e sinr e spon s eto co ld(Ch e ch ie ta l . ,2013 ;L i d e l le ta l . ,2014 ;H awk ee ta l . ,2009 ) .NEb ind stoth eβ3 ad r en e rg i cGp ro t e in coup l edr e c ep to r s(GPCR s ) ,wh i cha c t iv a t e sth eGp ro t e ina lph a subun i t andpho spho ry l a t e sad eny l a t ecy c l a s e(F igu r e1 .8 ) .Th i sen zym econv e r t sATPtocy c l i c ad eno s in emonopho sp h a t e( cAMP )th a ta c t iv a t e sp ro t e ink in a s eA(PKA ) .PKAa c t son down s t r e amm e s s eng e rp ro t e in ssu cha sho rmon e s en s i t iv el ip a s e(HSL ) ,wh i chindu c e s l ipo ly s i sth a tg en e r a t e sFFA ’ s,s e rv ingtworo l e s :a c t iv a t ingUCP1wh i chi son lyfoundonth e inn e rm i to chond r i a lm emb r an eo fb rownad ipo cy t e s ,andund e rgo ingFAc a t abo l i smv i aβox id a t iontofu e lth e rmog en e s i s(Ch e ch ie ta l . ,2013 ;L id e l le ta l . ,2014 ;H awk ee ta l . ,2009 ) . A c t iv a t edUCP1un coup l e sox id a t iv epho spho ry l a t ionf romATPg en e r a t ionbybyp a s s ing ATPsyn th a s eanden ab l ingth er e f luxo fp ro ton sa c ro s sth einn e rm i to chond r i a lm emb r an e downth ep ro tong r ad i en t .I ti sth i sp ro tonl e ak ,o rd i s rup t iono fth ep ro tong r ad i en t ,th a t g en e r a t e sh e a tandr e su l t sinwho l ebodyen e rgyexp end i tu r e(Chond ron iko l ae ta l . ,2016 ; L id e l le ta l . ,2014 ) . Ev id en c efo rth ero l eo fBATinen ergyexp end i tu r eh a sb e enshownexp e r im en t a l ly inan im a landhum anmod e l s . Wh enBATi sab l a t edg en e t i c a l lyinm i c e ,ob e s i tyen su e s (Low e l le ta l . ,1993 ) .How ev e r ,wh enBATi sr eg en e r a t edinth e s et r an sg en i cm i c e ,ob e s i ty r e so lv e s(Low e l le ta l . ,1993 ) .G en e t i cov e r exp r e s s iono fUCP1inm i c eh a sa l sob e enshown tom i t ig a t eob e s i tyindu c edbyah igh f a td i e t ,p rov id ingev id en c eth a tenh an c ingth e rmog en i c 18 � ����� F igu r e1 .8.Mo l e cu l a rm e ch an i smso fad ap t iv eth e rmog en e s i sa tth el ev e lo fb rownad ipo cy t es . Ad ap t edf romW ik ip ed i a ,2009.Ob t a in edf romh t tp s : / / en .w ik ip ed i a .o rg /w ik i /Th e rmog en e s i s . 19 po t en t i a lv i ain c r e a s edUCP1a c t iv i tyi sab l etoa l t e rbodyw e igh tinm i c eandthu se l i c i t s po t en t i a la sath e r apytor egu l a t ebodyw e igh tinhum an s(Kop e ckye ta l . ,1995 ) .M i c e l a ck ingUCP1f a i ltod emon s t r a t eanob e sog en i ce f f e c ta troomt emp e r a tu r e(23 ° C) ,y e tin th e rmon eu t r a l i ty(30 °C ;th eop t im a lt emp e r a tu r ea twh i chno rm a lbodyt emp e r a tu r ec anb e m a in t a in edfo rm i c e ) ,ob e s i tyd ev e lop s(En e rb ä cke ta l . ,1997 ;F e ldm ann ,Go lo zoubov a , C annon ,&N ed e rg a a rd ,2009 ) .Th e s er e su l t sc anb eexp l a in edbyth ed em andfo rth em i c eto comp en s a t ew i thsh iv e r inga tco ld e rt emp e r a tu r e sandd emon s t r a t e sth a tamb i en tt emp e r a tu r e i sad e t e rm in an tfo rm e t abo l i cs tud i e s . Ju s ta s WATc anund e rgoad ap t iv eanddyn am i cch ang e sdu r ingp e r iod so fp ro long ed s t a rv a t iono rov e r f e ed ing ,sotooc anBATinr e spon s etoco ldo rth e rmon eu t r a l i ty(B a r t e l t ,& H e e r en ,2013 ) .Fo rex amp l e ,bo thUCP1exp r e s s ionandBATm a s sa r ein c r e a s edinm i c e hou s edatco ld e rt emp e r a tu r e swh encomp a r edtom i c ehou s eda tth e rmon eu t r a l i ty(L ime ta l . , 2012 ;Yooe ta l . ,2014 ) .Hum ans tud i e sh av ea l sod emon s t r a t edth a tBATp l ay sa phy s io log i c a l lys ign i f i c an tro l einth e rmo r egu l a t iona sob s e rv edbyin c r e a s edBATa c t iv i ty dur ingm i ldco ldexpo su r e ,andw i thth eob s e rv a t ionth a tBATa c t iv i tyi sh igh lyco r r e l a t ed w i thBM Isu chth a tBATa c t iv i tyd e c r e a s e sa sBM Iin c r e a s e s(Ch ene ta l . ,2013 ; Chond ron iko l ae ta l . ,2016 ;V i jg ene ta l . ,2011 ) .Inadd i t ion ,bo thBATa c t iv i tyandm a ss d e c l in eino ld e rind iv idu a l s ,wh i chm ayb ea s so c i a t edw i thth ea c cumu l a t iono fbodyf a t ob s e rv edw i thag ing(P f ann enb e rge ta l . ,2010 ;W ange ta l . ,2015 ;Yon e sh i roe ta l . ,2011 ) . P r e c i s em e ch an i sm so fBATa c t iv a t ionandmodu l a t ioncon t inu etob es tud i edandn e c e s s i t a t e fu r th e re lu c id a t ionb e fo r eBATc anb et a rg e t edfo rth e r ap eu t i c s .I n c r e a s ingin fo rm a t ionon som eo fth ec l a s s i cho rmon e s ,p ro t e in s ,endo c r in ef a c to r s ,andp a thw ay sr egu l a t ingBAT fun c t iona r eb e com ingknown ,in c lud ingendo c r in ep a thw ay sinvo lv ingthy ro idho rmon e s , bon emo rphog en e t i cp ro t e in s ,n a t r iu r e t i cp ep t id e s ,andth em e l ano co r t insy s t em(d i s cu s s ed 20 abov e )(Ch e ch ie ta l . ,2013 ;Koo j im ane ta l . ,2014 ;L abb ée ta l . ,2015 ) .Th eG r ayl ab ,and thu sth i sr e s e a r ch ,i sin t e r e s t edinanov e lho rmon ed e em ede s s en t i a lfo rth ead ap t iv e th e rmog en i cr e spon s eo fBATtoco ldexpo su r ec a l l edp i tu i t a ryad eny l a t ecy c l a s e a c t iv a t ing po lyp ep t id e(PACAP )andw i l lb ed i s cu s s edh e r e(Rud e ck i ,&G r ay ,2016 ) . 1 .8P ITU ITARYADENYLATECYCLASEACT IVAT INGPOLYPEPT IDE(PACAP ) : ESSENT IALFORTHETHERMOGEN ICRESPONSEINBAT D i s cov e r edin1989 ,PACAPi sw id e lyexp r e s s edinth eb r a inandp e r iph e r a lo rg an s , no t ab lyinth egon ad s ,ad r en a lg l and s ,p an c r e a s ,r e sp i r a to ryandu rog en i t a lt r a c t s ,and invo lv edinana r r ayo fphy s io l og i c a lfun c t ion sin c lud ingmodu l a t iono fimmun eand in f l amm a to ryr e spon s e s ,d i l a t iono fv e s s e l sandb ron ch i ,p sy chomo to rcon t ro l ,th es t r e s s r e spon s eanden e rgym e t abo l i sm(M iy a t ae ta l . ,1989 ;V aud rye ta l . ,2009 ;Xue ta l . ,2016 ) . R e c ep to r sa r elo c a t edinth elung s ,l iv e r ,t e s t i s ,ad r en a lg l and ,andp an c r e a t i ci s l e t sanda r e p a r t i cu l a r lyabund an tinth eb r a in ,e sp e c i a l lyinth eDMN ,PVN ,ARC ,LHA ,andVMNo f th ehypo th a l amu s(F igu r e1 .5 )( H a sh imo toe ta l . ,1996;N ak a t a ,Sh iod a ,Ok a ,M a ruy am a ,& Y ad a ,1999) .PACAPi sknownfo ri t sp l e io t rop i cb io log i c a la c t ion sandi t sro l ea sam a s t e r s t r e s sr egu l a to r(Rud e ck i ,&G r ay ,2016 ) .H e r e ,th efo cu sw i l lb eoni t sro l einth e rmog en i c s t r e s sanden e rgym e t abo l i sm . Exp r e s s edinth ep an c r e a t i cn e rv ef ib e r sandi s l e ts ,PACAPpo t en t lyaugm en t s g lu co s eindu c edin su l ins e c r e t ionbya c t ingonp an c r e a t i cb e t ac e l l sandi sthu scon s id e r eda r egu l a to ro fg lu co s em e t abo l i sm(N ak a t ae ta l . ,1999 ;N ak a t ae ta l . ,2004 ) .I ta l sop romo t e s f e ed ingb eh av io rbya c t iv a t ingn eu ron sinth eARC(d i s cu s s edabov e ) ,knowna sth e hypo th a l am i cf e ed ingc en t e r ,andindu c e sl ipo ly s i sin WAT(N ak a t a ,&Y ad a ,2007 ;Rud e ck i, &G r ay ,2016 ) .I th a sb e enshownth a tl ep t ins ign a l ingenh an c e sPACAPexp r e s s ionin s t e ro idog en i cf a c to r1(SF1 )c e l l so fth eVMNands t rongev id en c esugg e s t sth a tPACAP 21 fo rm sanimpo r t an tdown s t r e amcompon en to fth ea cu t ee f f e c t so fl ep t inonen e rgyb a l an c e . Fo rex amp l e ,PACAPmRNAl ev e l sw e r es ign i f i c an t lyr edu c edinl ep t ind e f i c i en tm i c eand w e r er e s to r edw i thexog enou sl ep t int r e a tm en t s(H awk ee ta l . ,2009 ) .Fu r th e r n eu ro an a tom i c a lm app ingi sr equ i r edtofu r th e re lu c id a t eth ein t e r a c t ion sb e tw e enl ep t inand PACAP ;how ev e r ,propo s edin t e r a c t ion sa r eshowninF igu r e1 .9 .Add i t ion a lin t e r a c t ion s invo lv edinth i sm e ch an i smw i l lb efu r th e rd i s cu s s edh e r e . Bo thph a rm a co log i c a landg en e t i cev id en c eex i s t sfo rPACAP ’ se s s en t i a lro l ein th e rmog en e s i s .A l thoughPACAPandi t sr e c ep to r sa r eh igh lyexp r e s s edinth ePVNandth e VMN ,th eVMNi sknowntob ear eg iono fth ehypo th a l amu simpo r t an tfo rth er egu l a t iono f en e rgyhom eo s t a s i sbyin t eg r a t ingb eh av io r a landm e t abo l i cm e ch an i sm s .No rm a lVMN fun c t ion sin c lud ed e c r e a s ingfoodin t ak einr e spon s etos a t i e tyandin c r e a s ingen e rgy exp end i tu r e ,a sob e s i tyen su e sw i thVMNl e s ion s(R e s che ta l . ,2011 ) . Wh enPACAPi s in j e c t edd i r e c t lyin toth eVMNo fh e a l thy ,w i ld typ em i c e ,foodin t ak ei ss ign i f i c an t ly r edu c ed ,co r ebodyt emp e r a tu r eandspon t an eou slo como to ra c t iv i tyi sin c r e a s ed ,andBAT UCP1mRNAexp r e s s ioni sin c r e a s ed(H awk ee ta l . ,2009 ;R e s che ta l . ,2011 ) .Th e s ee f f e c t s w e r eno tob s e rv edwh enPACAPw a sin j e c t edin toth ePVN ,sugg e s t ingth eVMNa sam a jo r c en t e rfo rPACAP m ed i a t edth e rmog en e s i s(R e s che ta l . ,2011 ;R e s che ta l . ,2013 ) .U s ing PACAP/ -m i c eg en e r a t edbyG r ay ,Cumm ing s ,J i r ik ,&Sh e rwood(2001 )th eG r ayl abi s exp lo r ingPACAP ’ sro l esp e c i f i c a l lyinth eVMNina c t iv a t ingBATth e rmog en e s i sandth i s w i l lb eth efo cu so fth er e s e a r chp r e s en t edinCh ap t e r3 . G en e t i c a l ly ,w i thou tth ePACAPg en e ,PACAP/ -m i c ed i sp l ayh ighmo r t a l i tyr a t es (89% )wh enr a i s eda t21 °Ca scomp a r edtotho s er a i s eda t24 °C(24% ) ,(Ad am se ta l . ,2008 ; G r aye ta l . ,2001 ;G r ay ,Y am agu ch i ,V en cov a ,&Sh e rwood ,2002 ) .Th e s em i c ea r es en s i t iv e toa4 °Cco ldch a l l eng e ,d i sp l ay ingr edu c edl ev e l so fNEandβad r en e rg i cr e c ep to r s ,and 22 C o ldExp o su r e L e p t in PACAP MC SNA � �� � VMN B row nA d ipo c y t e H e a t F igu r e1 .9.P ropo s edp a thw ay so fn eu rop ep t id e sth a tin t e r a c ttoindu c ead ap t iv eth e rmog en e s i sin BATinr e spon s etoco ldexpo su r e .VMN ;v en t rom ed i a lnu c l eu s ,PACAP ;p i tu i t a ryad eny l a t ecy c l a s e a c t iv a t ingpo lyp ep t id e , MC ;m e l ano co r t insy s t em ,SNA ;symp a th e t i cn e rv ea c t iv i ty ,BAT ;b rown ad ipo s et i s su e .Im ag e sa d ap t edf rom“R ev i s i t ingth eV en t r a lM ed i a lNu c l eu so fth eHypo th a l amu s : Th eRo l e so fSF1N eu ron sinEn e rgyHom eo s t a s i s” ,byY .Cho i ,T .Fu j ik aw a ,J .L e e ,A .R e u t e r ,& K .K im ,2013 ,F ron t i e r sInN eu ro s c i en c e,7.Copy r igh t©2013Cho i ,Fu j ik aw a ,L e e ,R eu t e randK im . Ad ap t edw i thp e rm i s s ionf romau tho r . 23 imp a i r edup r egu l a t iono fUCP1w i thco ldexpo su r e(D i an ée ta l . ,2014 ;G r aye ta l . ,2002 ) . Th e s ef ind ing sind i c a t ePACAP ’ sro l ea sac r i t i c a lho rmon er egu l a to ro fBATth e rmog en e s i s (Ad am se ta l . ,2008 ;G r aye ta l . ,2001 ;G r aye ta l . ,2002 ) . 1 .9GAPINL ITERATURE :CONNECT INGPACAPANDTHE MELANOCORT IN SYSTEM A ss t a t edabov e ,th ec en t r a lm e l ano co r t insy s t emp l ay sac r i t i c a lro l einr egu l a t ing en e rgyexp end i tu r ewh e r ebyαMSH a c t sonth e MC4Rtor egu l a t eth e rmog en e s i sv i ath e symp a th e t i cn e rvou ssy s t em(F ane ta l . ,2005 ;V augh an ,Sh r e s th a ,&B a r tn e s s ,2011 ;Vo s s And r e a ee ta l . ,2007) .A l thoughmo l e cu l a randphy s io log i c a lm e ch an i sm so fPACAP ’ s invo lv em en tinBATa c t iv a t ionr em a inun c l e a r ,som eev id en c eex i s t stosugg e s tPACAP in t e r a c t sw i thth em e l ano co r t insy s t emtoa c t iv a t eBATth e rmog en e s i s(T an id a ,Sh in t an i ,& H a sh imo to ,2011 ) .Th i spo t en t i a la s so c i a t ionandg apinth el i t e r a tu r ei sth efo cu so fth e r e s e a r chfo rCh ap t e r2 . R e s e a r chh a sshownth a tbo thPACAP/ -andm e l ano co r t in4 r e c ep to r(MC4R )/ m i c eh av eimp a i r edth e rmog en e s i sandi ti shypo th e s i z edth a tPACAPandth em e l ano co r t in sy s t ema r econn e c t edina c t iv a t ingBATth e rmog en e s i s(B e rg lunde ta l . ,2014 ;D i an ée ta l . , 2014 ;F an ,Vo s s Andr e a e ,C ao ,& Mo r r i son ,2005 ;G r aye ta l . ,2002 ;Vo s s And r e a ee ta l . , 2007 ) .I ti sc l e a rth a tBATa c t iv a t ionun equ ivo c a l lyo c cu r sbysymp a th e t i cn e rv ea c t iv i ty (SNA )in i t i a t eda tth el ev e lo fth ehypo th a l amu s ,wh e r ebyth ec en t r a lsymp a th e t i cou t f low c i r cu i t stoBATh av eb e enr ev e a l edbyin j e c t ingar e t rog r ad ev i r a lt r an sn eu ron a lt r a c tt r a c e r in toth et i s su e( B am sh ad ,Song ,&B a r tn e s s ,1999 ;B a r tn e s s ,V augh an ,&Song ,2010 ) . How ev e r ,th em e ch an i sm sbywh i chth ehypo th a l am i cnu c l e ia c t i v a t ingBATa r eno t p r e c i s e lyknown .In t e r e s t ing ly ,ah ighd eg r e eo fco lo co l i z a t iono f MC4RmRNAi sfoundon th en eu ron sa longth en eu ron a lt r a c twh e r eth ev i r a lt r a c e ri sd e t e c t ed(B a r tn e s s ,V augh an ,& 24 Song ,2010 ) . Add i t ion a l ly ,wh en MC4Ragon i s tM e l ano t anI I(MT I I )w a ss t e r eo t ax i c a l ly in j e c t edin toth eVMN ,s im i l a rr e su l t sw e r eob s e rv edtotho s ew i thph a rm a co log i c a l in j e c t ion so fPACAPin toth eVMN ;anin c r e a s einen e rgyexp end i tu r eandth e rmog en e s i s , sugg e s t ingth a tPACAPandth em e l ano co r t insy s t emm aysh a r eacommonp a thw ayin a c t iv a t ingBATth e rmog en e s i s(G av in i ,Jon e s ,&Nov ak ,2016 ) .T an id a ,Sh in t an i ,& H a sh imo to(2011 )condu c t edfu r th e rr e s e a r chin toth econn e c t ionb e tw e enPACAPandth e m e l ano co r t insy s t emwh e r eth eymon i to r edn eu r a lr e spon s einBATa soppo s edto th e rmog en i cm a rk e r s .Th eyp e r fo rm edin t r a c e r eb rov en t r i cu l a rin j e c t ion so fan MC4R an t agon i s t ,fo l low edbyPACAP ,andcomp a r edth en eu r a lr e spon s einBATtotho s ew i thou t th ean t agon i s tin j e c t ion .W i thou tth ean t agon i s t ,PACAPin j e c t ion sshow edin c r e a s edn eu r a l a c t iv i tytoBAT .How ev e r ,wh enth e MC4Ran t agon i s tw a sin j e c t edb e fo r ePACAP ,v e ry l i t t l esymp a th e t i cn e rv ea c t iv i tyw a sob s e rv eda tth el ev e lo fBAT(T an id a ,Sh in t an i ,& H a sh imo to2011 ) .Th i sp rov id e sev id en c etosugg e s tth a tPACAPa c tsup s t r e amo fth e m e l ano co r t insy s t emina c t iv a t ingBAT( a sp ropo s edinF igu r e1 .9) .F u r th e rr e s e a r chi s n e c e s s a rytosup po r tth e s er e su l t s,wh i chw i l lb ep r e s en t edinCh ap t e r2 . 1 .10HYPOTHES ISANDRESEARCHOBJECT IVES Ihypo th e s i z eth a tPACAPa c t sup s t r eamf romth em e l ano co r t insy s t emtor egu l a t e SNAtos t imu l a t ead ap t iv eth e rmog en e s i sinBAT . Mo r eov e r ,Ihypo th e s i z eth a tth eco ld s en s i t iv i tyo fPACAP/ -m i c em ayb edu etoth eab s en c eo fPACAPs ign a l ingw i th inth e VMNsp e c i f i c a l ly(F igu r e1 .9 ) . Th eob j ec t iv e so fth i sr e s e a r cha r e : 1 . Tod e t e rm in ei fth eimp a i r edth e rmog en e s i sinPACAP/ -m i c ec anb e r e s cu edph a rm a co log i c a l lyw i tham e l ano co r t inagon i s t ,M e l ano t anI I(MT I I ) , andthu sp rov id eev id en c eu s ingag en e t i cmod e lo fPACAPd e f i c i en cyfo rth e 25 conn e c t ionb e tw e enPACAPandth em e l ano co r t insy s t eminr egu l a t ing th e rmog en e s i sinBAT 2 . Toc r e a t eanad enov i r a lcon s t ru c tth a texp r e s s e sGFPund e rth er egu l a t iono f th eSF1p romo t e rtoa c ta sacon t ro lv e c to rtob eu s edins t e r eo t ax i csu rg e ry , thu sen su r ingVMN s p e c i f i cexp r e s s iono fth ev e c to r 3 . Tod ev i s eap ro to co lfo rs t e r eo t ax i cin j e c t ion swh i chr e a ch e sth eVMNinl iv e an im a l s R e su l t sob t a in edf romob j e c t iv e s2and3a c ta sp r e l im in a rys t ep sfo l low ingfu tu r eob j e c t iv e : 4 . Toc r e a t eanad enov i r a lcon s t ru c tfo rVMNsp e c i f i cexp r e s s iono fPACAPt o d e t e rm in ei fth eimp a i r edth e rmog en e s i sinPACAP/ -m i c ec anb eg en e t i c a l ly r e s cu edbyt r an sg en i cexp r e s s iono fPACAPinth eVMNon ly ,thu s d e t e rm iningth eimpo r t an c eo fVMNPACAPinr egu l a t ingth e rmog en e s i s Th ewo rki sp r es en t edins e l f con t a in edch ap t e r s .Th efo cu so fCh ap t e r2i sto d e t e rm in eth ep a thw ayconn e c t ingPACAPandth em e l ano co r t insy s t emina c t iv a t ing th e rmog en e s i sinBAT .Th i sw a sa ch i ev edbyin j e c t ingPACAP/ -m i c ew i tha ph a rm a co log i c a l MC4Ragon i s tina t t e mp ttor e s cu eimp a i r edth e rmog en e s i sinPACAP/ m i c e. M e thodo logyo fa s s e s s ingth ephy s io log i c a landb io ch em i c a lm a rk e r s fo rth e rmog en i c r e spon s ei sd i s cu s s edind e t a i linCh ap t e r2 .Ch ap t e r3fo cu s e sonth ec r e a t ionofa n ad enov i r a lcon t ro lc on s t ru c ttob eu s eda sth ein i t i a ls t epinth eg en e t i cr e in t rodu c t iono f PACAPsp e c i f i c a l lyinth eVMNtod e t e rm in ei t sro l einBATth e rmog en e s i sa c t iv a t ion .Th i s ch ap t e ra l soin c lud e sd ev e lopm en to fas t e r eo t ax i cp ro to co lfo rin j e c t iono fth ead enov i ru s in toth eVMNo fm i c etoa ch i ev eVMNsp e c i f i cexp r e s s iono fPACAP .Ch ap t e r4conn e c t s Ch ap t e r s2and3 ,p rov id ingacon c lu s ionands ign i f i c an c efo rr e s e a r chinth i sa r e a . 26 1 .11REFERENCES Abou tOb e s i t yinCanada-Canad ianOb e s i t yN e two r k.(2016 ) .Ob e s i t yn e two r k . ca.R e t r i ev ed 2Jun e2016 ,f romh t tp : / /www .ob e s i tyn e two rk . c a /ob e s i tyin c an ad a Ad am s ,B . ,G r ay ,S . ,I s a a c ,E . ,B i an co ,A . ,V id a lPu ig ,A . ,&Sh e rwood ,N .(2008 ) .F e ed ing and M e t abo l i smin M i c eL a ck ingP i tu i t a ryAd eny l a t eCy c l a s e A c t iv a t ing Po lyp ep t id e .Endo c r ino log y,149 (4 ) ,1571 1580 .h t tp : / /dx .do i .o rg /10 .1210 / en .2007 0515 Adu l tOb e s i t yFa c t s|O v e rw e igh t&Ob e s i t y|CDC.(2016 ) . 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Human Mo l e cu la rG en e t i c s, 15(R ev i ewI s su e2 ) ,R124 R130 . h t tp : / /dx .do i .o rg /10 .1093 /hmg /dd l215 W ang ,Q . ,Zh ang ,M . ,Xu ,M . ,Gu ,W . ,X i ,Y . ,&Q i ,L .e ta l .(2015 ) .B rownAd ipo s eT i s su e A c t iv a t ionI sInv e r s e lyR e l a t edtoC en t r a lOb e s i tyand M e t abo l i cP a r am e t e r sinAdu l t Hum an .PLOSONE,10(4 ) ,e0123795 .h t tp : / /dx .do i .o rg /10 .1371 / jou rn a l .pon e .01 23795 W e s t e r t e rpP l an t eng a ,M . ,S a r i s ,W . ,Hu ck sho rn ,C . ,&C amp f i e ld ,L .(2001 ) .E f f e c t so f w e ek lyadm in i s t r a t iono fp egy l a t edr e comb in an thum anOBp ro t e inonapp e t i t ep ro f i l e l inNu t r,74(4 ) ,426 34 . anden e rgym e t abo l i sminob e s em en .AmJC .(2016 ) .Who . in t.R e t r i ev ed2Jun e2016 , WHO|Con t ro l l ingth eg loba lob e s i t yep id em i c f romh t tp : / /www .who . in t /nu t r i t ion / top i c s /ob e s i ty / en / WHO|Ob e s i t y.(2016 ) .Who . in t.R e t r i ev ed2Jun e2016 ,f rom h t tp : / /www .who . in t /gho /n cd / r i sk_ f a c to r s /ob e s i ty_ t ex t / en / Xu ,Z . ,Oh t ak i ,H . ,W a t an ab e ,J . ,M iy amo to ,K . , Mu r a i ,N . ,&S a s ak i ,S .e ta l .(2016 ) . P i tu i t a ryad eny l a t ecy c l a s ea c t iv a t ingpo lyp ep t id e(PACAP )con t r ibu t e stoth e p ro l i f e r a t iono fh em a topo i e t i cp rog en i to rc e l l sinmu r in ebon em a r rowv i aPACAP sp e c i f i cr e c ep to r .S c i .R ep . ,6,22373 .h t tp : / /dx .do i .o rg /10 .1038 / s r ep22373 Y a sw en ,L . ,D i eh l ,N . ,B r enn an ,M . ,&Ho chg e s chw end e r ,U .(1999 ) .Ob e s i tyinth emou s e mod e lo fp roop iom e l ano co r t ind e f i c i en cyr e spond stop e r iph e r a lm e l ano co r t in .Na t M ed,5(9 ) ,1066 70 . Y eo ,G .&H e i s l e r ,L .(2012 ) .Un r av e l ingth eb r a inr egu l a t iono fapp e t i t e :l e s son sf rom g en e t i c s .Na tu r eN eu ro s c i en c e,15(10 ) ,1343 1349 .h t tp : / /dx .do i .o rg /10 .1038 /nn .3211 Yon e sh i ro ,T . ,A i t a ,S . ,M a t su sh i t a ,M . ,Ok am a t suOgu r a ,Y . ,K am ey a ,T . ,&K aw a i ,Y .e ta l . (2011 ) .Ag e R e l a t edD e c r e a s einCo ldA c t iv a t edB rownAd ipo s eT i s su eand e s i t y,19(9 ) ,1755 1760 . A c cumu l a t iono fBodyF a tinH e a l thyHum an s .Ob h t tp : / /dx .do i .o rg /10 .1038 /oby .2011 .125 Yoo ,H . ,Q i ao ,L . ,Bo s co ,C . ,L eong ,L . ,Ly t l e ,N . ,&F eng ,G .e ta l .(2014 ) .In t e rm i t t en tCo ld Expo su r eEnh an c e sF a tA c cumu l a t ionin M i c e .P lo sONE,9(5 ) ,e96432 . h t tp : / /dx .do i .o rg /10 .1371 / jou rn a l .pon e .0096432 Zh ang ,Y . ,P ro en c a ,R . ,M a f f e i ,M . ,B a ron e ,M . ,L eopo ld ,L . ,&F r i edm an ,J .(1994 ) . Po s i t ion a lc lon ingo fth emou s eobe s eg en eandi t shum an homo logu e .Na tu r e,372(6505 ) ,425 432 .h t tp : / /dx .do i .o rg /10 .1038 /372425 a 35 CHAPTER2 Pha rma co log i ca lIn t e rv en t ionw i th M e lano tanI IPa r t ia l lyR e s cu e sth eImpa i r ed Th e rmog en i cCapa c i tyo fPACAP/ -M i c e 36 2 .1INTRODUCT ION Ow ingtoth eth e rmog en i cc ap ab i l i t i e so fb rownandb e ig ead ipo cy t e s,th er e c en t d i s cov e ryth a tBATi sp r e s en tandfun c t ion a linadu l thum an sh a sa t t r a c t edin c r e a s ing a t t en t iontoth i st i s su e ,p a r t i cu l a r lydu etoi t sen e rgyd i s s ip a t inga c t iv i tyandpo t en t i a l p ro t e c t iv efun c t ionag a in s tbodyf a ta c cumu l a t ion(Ch ene ta l . ,2013 ;Chond ron iko l in ae ta l . , 2016 ;S a i to ,Yon e sh i ro ,& M a t su sh i t a ,2016 ) . Wh i l es ev e r a lendo c r in er egu l a to r sa r e invo lv edinth ea c t iv a t iono fBATsu cha sthy ro idho rmon e s ,h e a r tn a t r iu r e t i cp ep t id e sand l iv e rb i l ea c id s ,th eg r e a t e s tphy s io log i c a ls t imu lu si sv i asymp a th e t i ca c t iv a t ion ,wh i chi s indu c edinr e spon s etobo thf e ed ingandco ldexpo su r e(F igu r e2 .1 )(Ch e ch i ,C a rp en t i e r ,& R i ch a rd,2013 ;L abb ée ta l . ,2015 ;S a i to ,Yon e sh i ro ,& M a t su sh i t a ,2016 ) .Th efo cu so fth i s ch ap t e rw i l lb etofu r th e rund e r s t andth em e ch an i sm sbywh i chsymp a th e t i cn e rv ea c t iv i tyi s a c t iv a t edinr e spon s etoco ldexpo su r e . Co lds t r e s si sin i t i a l lyid en t i f i edbys en so ryn eu ron sonth ebodysu r f a c eth a tt r an sm i t n eu ro ch em i c a ls ign a l stoth eb r a in ,r e su l t inginin c r e a s edsymp a th e t i ca c t iv i ty(F igu r e2 .1 ) (Mo r r i son ,&N ak amu r a ,2011 ) .No r ep in eph r in e(NE )i sr e l e a s edf romabund an t lyd i s t r ibu t ed symp a th e t i cn e rv eend ing sth a tinn e rv a t eBATands t i mu l a t eb rownad ipo cy t e sv i ab ind ing p r im a r i lytoβ3ad r en e rg i cr e c ep to r s( β3AR s )( Inokum ae ta l . ,2005 ;S a i to ,Yon e sh i ro ,& M a t su sh i t a ,2016 ) .Th i st r igg e r scAMPa c t iv a t edc e l lu l a rev en t sin c lud inghyd ro ly s i so f t r ig ly c e r id e sv i aho rmon es en s i t iv el ip a s e(HSL ) ,ox id a t iono fr e su l t ingFA s,anda c t iv a t ion o fun coup l ingp ro t e in1(UCP1 ) ;th ek eym i to chond r i a lp ro t e ininvo lv edinad ap t iv e th e rmog en e s i s ,r e su l t inginh e a tp rodu c t ion(F igu r e1 .8 )( Inokum ae ta l . ,2005 ;S a i to , Yon e sh i ro ,& M a t su sh i t a ,2016 ) .Sympa th e t i ca c t iv a t iona l sor e su l t sinin c r e a s edf a t mob i l i z a t ionin WAT ,r e l e a s ingFA sth a ta r eu s edbyp e r iph e r a lt i s su e s ,su cha sBAT 37 NE WAT F igu r e2 .1 .Symp a th e t i candendo c r in econ t ro lo fBATth e rmog en e s i s .βAR ;b e t aad r en e rg i c r e c ep to r ,NE ;no r ep in eph r in e ,NP ;n a t r iu r e t i cp ep t id e ,T4 ;thy rox in e ,UCP1 ;un coup l ingp ro t e in1 , WAT ;wh i t ead ipo s et i s su e . Ad ap t edf rom“ A c t iv a t ionandr e c ru i tm en to fb rownad ipo s et i s su eby co ldexpo su r eandfooding r ed i en t sinhum an s ” ,by M .S a i to ,T .Yon e sh i ro ,& M .M a t su sh i t a , 2016 ,B e s tP ra c t i c e &R e s ea r chC l in i ca lEndo c r ino log y& M e tabo l i sm,30(4 ) ,537 547 .Copy r ight© 2017E l s ev i e rB .V .Ad ap t edw i thp e rm i s s ionf rompub l i sh e r . 38 wh e r eth eya r eu s eda sasub s t r a t efo rox id a t iv em e t abo l i sm(Chond ron iko l ae ta l . ,2016 ; S a i to ,Yon e sh i ro ,& M a t su sh i t a ,2016 ) .Ch ang e sinad ap t iv eth e rmog en e s i sc anb em e a su r ed a longth ep a thw ay sind i c a t edinF igu r e2 .1th roughth efo l l ow ing :m e a su r em en t so fSNAand NEtu rnov e ra tth el ev e lo fBAT ,th e rmog en i cg en eandp ro t e inexp r e s s iono fβ3 AR s ,HSL andUCP1inBAT ,m e a su r em en t so foxyg encon sump t ion( en e rgyexp end i tu r e )u s ing ind i r e c tc a lo r im e t ry ,co r ebodyt emp e r a tu r eandBATt emp e r a tu r e ,a sw e l la sex am in a t iono f h i s to log i c a lch ang e so fBATmo rpho logyind i c a t iv eo fth e rmog en i ca c t iv i ty(C annon ,& N ed e rg a a rd ,2010 ;C r an ee ta l . ,2014 ;Ro thw e l l ,2001 ;V i r tu e ,&V id a lPu ig ,2013 ) .Fo r ex amp l e ,m i c ehou s eda tth e rmon eu t r a l i ty( 30 °C)exh ib i tin a c t iv ead ap t iv eth e rmog en e s i s , r e su l t inginth ea c cumu la t iono fl ip id sinbo th WATandBAT(Go ldgo fe ta l . ,2014 ; H e ldm a i e r ,1975 ;V i r tu e ,&V id a lPu ig ,2013 ) .H i s to log i c a l ly , WATinan im a l shou s eda t th e rmon eu t r a l i typ r e s en t sw i thl a rg e ,l ip id f i l l edad ipo cy t e s ,wh i l eBATinan im a l shou s eda t th e rmon eu t r a l i typ r e s en t sw i thl a rg e ,mu l t i lo cu l a rl ip idd rop l e t sw i th inb rownad ipo cy t e s (Go ldgo fe ta l . ,2014 ;H e ldm a i e r ,1975 ;V i r tu e ,&V id a l Pu ig ,2013 ) .Incon t r a s t ,ad ap t iv e th e rmog en e s i si sm ax im a l lya c t iv einBATdu r ingco ldexpo su r e(4 °C ) ,r e su l t inginth e b r ea kdowno ft r ig ly c e r id e sinbo th WATandBAT ,tor e l e a s eFA sth a tfu e lth e rmog en e s i s (Chond ron iko l ae ta l . ,2016 ;Ngyu ene ta l . ,2011 ;S a i to ,Yon e sh i ro ,& M a t su sh i t a ,2016 ) . Th e r e fo r e ,th er e su l t ingh i s to log i c a lapp e a r an c eo f WATinco ld expo s edan im a l scon s i s t so f sm a l l e rwh i t ead ipo cy t e sandin c r e a s ednumb e r so fth e rmog en i c a l lya c t iv e ,b e ig ead ipo cy t e s , wh i l eBATinco ldexpo s edan im a l scon t a in sb rownad ipo cy t e sw i thsm a l l e r ,mu l t i lo cu l a r l ip idd r op l e t s(H e ldm a i e r ,1975 ;Nguy ene ta l . ,2011 ;V i r tu e ,&V id a l Pu ig ,2013 ) . A c t iv a t iono fBATbyco ldexpo su r eh a sb e enr ep e a t ed lyd emon s t r a t edinhum an s,a s v i su a l i z edw i th(18 )F f luo rod eoxyg lu co s e(FDG ) po s i t ronem i s s iontomog r aphy(PET ).In r e spon s et osho r t t e rmm i ldco ldexpo su r e(15 19 °C ) ,bo thb loodf lowandg lu co s eup t ak ein 39 hum anBATin c r e a s e s ,a sw e l la swho l ebodyen e rgyexp end i tu r e(Ch ene ta l . ,2013 ;O r av ae t a l . ,2011 ; Mu z ike ta l . ,2013 ;Ou e l l e te ta l . ,2012 ;Yon e sh i roe ta l . ,2010 ) .No t ab l y ,th e s e r e su l t sa r eob s e rv edon lyinind iv idu a l sth a tpo s s e s ss ign i f i c an tamoun t so fBAT .S tud i e s h av eshownth a tBATa c t iv i tyi sinv e r s e lyr e l a t edtoBM Iandbodyf a tp e r c en t ag esu chth a t a c t iv i tyi slow e rinob e s esub j e c t sa scomp a r edtol e anind iv idu a l s(v an M a rk enL i ch t enb e l t e ta l . ,2009 ;v an M a rk enL i ch t enb e l t ,2011 ;V i jg ene ta l . ,2011 ;W ange ta l . ,2015 ) .To d e t e rm in ei flowl ev e l so fBATa c t iv i tycou ldb ein c r e a s ed ,Yon e sh i roe ta l .(2013 )expo s ed sub j e c t sw i thlowo rund e t e c t ab l el ev e l so fBATtor ep e a t edco ldexpo su r eov e r6w e ek s . R e su l t sind i c a t edbo thanin c r e a s einBATa c t iv i tyandad e c r e a s einbodyf a tm a s s (Yon e sh i roe ta l . ,2013 ) .Impo r t an t ly ,th ech ang einbodyf a tm a s sw a sinv e r s e lyco r r e l a t ed w i thBATa c t iv i ty ,sugg e s t inganimpo r t an tin t e r a c t ionb e tw e enBATr e c ru i tm en tandbody f a tr edu c t ion(Yon e sh i roe ta l . ,2013 ) .T ak ingth el i t e r a tu r ein tocon s id e r a t ion ,i ti sth e r e fo r e con c e iv ab l eth a tBATm ayb eap rom i s ingt a rg e tfo rcomb a t t ingob e s i tyandr e l a t ed m e t abo l i cd i so rd e r sinhum an s ,byr e s to r ingBATa c t iv i tytoth eupp e rr ang eo f phy s io log i c a l lyno rm a ll ev e l s .Ino rd e rtod e t e rm in ee f f e c t iv eth e r ap eu t i ct a rg e t s ,how ev e r , phy s io log i c a lm e ch an i sm sandp a thw ay sr egu l a t ingBATa c t iv a t ionth roughSNAmu s tb e fu r th e re lu c id a t ed . A sd i s cu s sedinCh ap t e r1 ,th em e ch an i smo fin t e r e s tandfo cu so fth i sch ap t e ri sth e po t en t i a lconn e c t ionb e tw e enPACAPandth em e l ano co r t insy s t eminth ea c t iv a t iono fSNA inBATth e rmog en e s i s .Th eev id en c ed emon s t r a t ingbo thPACAPandth em e l ano co r t in sy s t em ’ sin vo lv em en tinBATa c t iv a t ionin t rodu c edinCh ap t e r1w i l lb efu r th e rd i s cu s s ed h e r e . A sar e su l to fPACAP ’ sp l e io t rop i cb io log i c a la c t ion sa san eu rop ep t id eandi t sro l ea s am a s t e rs t r e s sr egu l a to r ,PACAP/ -m i c eh av eb e enshowntoh av es ev e r a lsymp a th e t ic a l ly 40 con t ro l l edabno rm a l i t i e sin c lud ingr e sp i r a to rycon t ro ld e f e c t s ,imp a i r edcoun t e r r egu l a to ry r e spon s etohypog ly c a em i a ,andimp a i r edth e rmog en e s i s(Ad am se ta l . ,2008 ;Cumm ing s , P end l ebu ry ,Sh e rwood ,& W i l son ,2004 ;D i anée ta l . ,2014 ;G r ay ,Cumm ing s ,J i r ik ,& Sh e rwood ,2001 ;G r ay ,Y am agu ch i ,V en cov a ,&Sh e rwood ,2002 ) .Con s equ en t ly ,PACAP/ -m i c ed i sp l ay edh ighr a t e so fmo r t a l i ty ,p a r t i cu l a r lyine a r lypo s tn a t a ll i f e(G r aye ta l . , 2001 ;G r aye ta l . ,2002 ) .No t ab ly ,m i c eth a td idno tsu rv iv eexh ib i t e ds ev e r e lyd ep l e t ed ad ipo s et i s su eandadd i t ion a ldy s fun c t ion sinl ip idandc a rbohyd r a t em e t abo l i sm ,ab s en tin tho s eth a td idsu rv iv e ,ind i c a t ingth ecomp l ex i tyo fth ePACAP/ -ph eno typ e(G r aye ta l . , 2011 ) .In t e r e s t ing ly ,th emo r t a l i tyr a t eo fPACAP/ -pup sd e c r e a s eds ign i f i c an t lywh enm i c e w e r er a i s eda t24 °Cr a th e rth an21 °C ,ind i c a t ingat emp e r a tu r es en s i t iv eph eno typ e(G r aye t a l . ,2002 ) .Th i st emp e r a tu r e s en s i t iv eph eno typ ew a sfu r th e rsuppo r t edbyth er edu c ed th e rmog en i cc ap a c i tyd i sp l ay edinadu l tPACAP/ -m i c ea scomp a r edtoPACAP+ /+m i c e du r inga4 °Cco ldch a l l eng e ,a sind i c a t edbyr edu c edl ev e l so fNEandexp r e s s iono fβ3 AR s , imp a i r edup r egu l a t iono fUCP1p ro t e in ,andr edu c edoxyg encon sump t iondu r ingNE indu c edth e rmog en e s i s( D i anée ta l . ,2 014 ;G r aye ta l . ,2002 ) .Fu r th e rev id en c efo rPACAP ’ s ro l einad ap t iv eth e rmog en e s i so r ig in a t ef romph a rm a co log i c a ls tud i e sth a tin c lud eth e in j e c t iono fPACAPin toth eth i rdc e r eb r a lv en t r i c l eo fPACAP/ -m i c e ,in t r a th e c a l adm in i s t r a t ion so fPACAPinh e a l thyr a t s ,andVMNin j e c t ion so fPACAPinw i ld typ em i c e ; a l lo fwh i chr e su l t edinin c r e a s edBATth e rmog en e s i s ,ind i c a t edbyin c r e a s edSNAtoBAT andNE s e c r e t ingn eu ro endo c r in ec e l l s ,in c r e a s edco r ebodyt emp e r a tu r e ,andup r egu l a t ed UCP1mRNAexp r e s s ion( Ing lo t t ,F a rnh am ,&P i low sky ,2011 ;R e s che ta l . ,2011 ;R e s che t a l . ,2013 ;T an id ae ta l . ,2010 ) .Con c lu s iv e ly ,PACAPi sd e em ede s s en t i a lfo rth eth e rmog en i c r e spon s einBAT . 41 S im i l a r lytoPACAP ,th ec en t r a lm e l ano co r t insy s t ema l sop l ay sac r i t i c a lro l ein en e rgyhom eo s t a s i sinbo than im a l sandhum an s .Inp a r t i cu l a r ,th em e l ano co r t in4r e c ep to r (MC4R )s ign a l ingp a thw ayin f lu en c e sbo then e rgyin t ak eanden e rgyexp end i tu r e(d i s cu s s ed inCh ap t e r1 ) ,in c lud ingth e rmog en e s i sw i thb ind ingo fth em e l ano co r t inp ep t id e ,αMSH (B e rg lunde ta l . ,2014 ;F an ,Vo s s And r e a e ,C ao ,& Mo r r i son ,2005 ) .A sar e su l t , MC4R/ m i c eexh ib i tin c r e a s edad ipo s i tyandw e igh tg a indu etobo thin c r e a s edfoodin t ak eand d e c r e a s eden e rgyexp end i tu r ev i ar edu c edphy s i c a la c t iv i tyandth e rmo g en e s i s(B e rg lunde t a l . ,2014 ;Bu t l e r&Con e ,2003 ;Bu t l e re ta l . ,2001 ;Con e ,1999 ) .Add i t ion a l ly , MC4R/ m i c eexh ib i tad e f e c tinBATUCP1up r egu l a t ioninr e spon s etoco ldexpo su r e ,a scomp a r ed to MC4R+ /+m i c e(B e rg lunde ta l . ,2014 ;Vo s s And r e a ee ta l . ,2007 ) .Tom im i cth e b io log i c a la c t iono fαMSH ,M e l ano t anI I(MT I I ) ,asyn th e t i c MC3 /4Ragon i s t ,w a sd e s ign ed andsyn th e s i z edin1989byA l Ob e id ie ta l .B e c au s eth ea c t iv i tyo f MC3R sinen e rgy hom eo s t a s i si smu chmo r esub t l eth an MC4R s ,th efo cu sh e rew i l lb eon MC4R s(G i r a rd e t& Bu t l e r ,2014 ;T ao ,2010 ) .Uponbo thp e r iph e r a landc en t r a l( in t r a c e r eb rov en t r i cu l a r( ICV ) ) d e l iv e ryo f MT I I ,th e rmog en e s i sw a sin c r e a s edin MC4R/ -and+ /+m i c e ,w i ld typ er a t sand w i ld typ eh am s t e r s ,a sob s e rv edbyin c r e a s edoxyg encon sump t ionandSNAtoBATth rough NEtu rnov e r ,in c r e a s edBATt emp e r a tu r e sandUCP1mRNAexp r e s s ion ,andd e c r e a s ed w e igh tandad ipo s i tyth rough WATl ipo ly s i sind i e tindu c edob e s eph eno typ e s(B r i toe ta l . , 2007 ;En r io r ie ta l . ,2011 ;F an , Mo r r i son ,C ao ,&Yu ,2007 ;G av in i ,Jon e s ,&Nov ak ,2016 ; G l av a se ta l . ,2007 ;H ayn e se ta l . ,1999 ;L i ,Zh eng ,W i l s ey ,&S c a rp a c e ,2004 ;Sk ib i ck a& G r i l l ,2009 ;Songe ta l . ,2008 ;S t r ad e re ta l . ,2007 ;W i l l i am se ta l . ,2003 ;Y a sud a ,M a s ak i , S ak a t a ,&Yo sh im a t su ,2004) .Conv in c ing ly ,ands im i l a r lytoPACAP ,th em e l ano co r t in sy s t emi sa l sod e em ede s s en t i a lfo rth eth e rmog en i cr e spon s einBAT . 42 Bo thPACAPand MCR sa r eco lo c a l i z edinth eVMN ;on eo fth ek eyhypo th a l am i c nu c l e ithough ttob einvo lv edina c t iv a t iono fBATth e rmog en e s i s( in t rodu c edinCh ap t e r1 andd i s cu s s edind e t a i linCh ap t e r3 )(B a r tn e s s ,V augh an ,&Song ,2010 ;H awk ee ta l . ,2009 ; N ak a t ae ta l . ,1999 ) .Upons t e r eo t ax i c MT I Iin j e c t ion sin toth eVMN ,s im i l a rr e su l t sw e r e ob s e rv edtotho s ew i thph a rm a co log i c a lin j e c t ion so fPACAPin toth eVMN :anin c r e a s ein en e rgyexp end i tu r eandth e rmog en e s i s(G av in i ,Jon e s ,&Nov ak ,2016 ) .Th e s er e su l t ssugg e s t th a tPACAPandth em e l ano co r t insy s t emsh a r eacommonp a thw ayina c t iv a t ingBAT th e rmog en e s i s ,how ev e r ;wh i l econ s id e r ab l eamoun t so fl i t e r a tu r eex i s tfo rth ec ru c i a lro l eo f PACAPandth em e l ano co r t insy s t eminBATth e rmog en e s i sind iv idu a l ly ,v e ryl i t t l eev id en c e ex i s t sconn e c t ingth etwo .O fth el im i t edex i s t ingev id en c efo rth econn e c t ionb e tw e en PACAPandth em e l ano co r t insy s t eminen e rgyin ta k e ,av a i l ab l el i t e r a tu r esugg e s t sth a t PACAPa c t sup s t r e amf romth em e l ano co r t ind ep end en tp a thw aytoinh ib i tfoodin t ak e(Dü r r e ta l . ,2007 ;I emo lo ,F e r r agud ,Co t ton e ,&S ab ino ,2015 ; Moun i ene ta l . ,2008 ) .S im i l a r ly ,th e so l ep i e c eo fl i t e r a tu r eth a tex i s t sfo rth econn e c t ionb e tw e enPACAPandth em e l ano co r t in sy s t eminen e rgye xp end i tu r ea l sosugg e s t sth a tPACAPa c t sup s t r e amf romth em e l ano co r t in sy s t emina c t iv a t ingBATth e rmog en e s i s(T an id a ,Sh in t an i ,&H a sh imo to ,2011 ) .A s d e s c r ib edinCh ap t e r1 ,r e s e a r ch e r si l lu s t r a t edth i sconn e c t ionbyp e r fo rm ingICVin j e c t ion s o fa MC4Ran t agon i s tfo l low edbyPACAP ,andcomp a r edth en eu r a lr e spon s einBATto tho s ew i thou tth ean t agon i s tin j e c t ion(T an id a ,Sh in t an i ,&H a sh imo to ,2011 ) .W i thou tth e an t agon i s t ,PACAPin j e c t ion sshow edin c r e a s edn eu r a la c t iv i tytoBAT .How ev e r ,wh enth e MC4Ran t agon i s tw a sin j e c t edp r io rtoPACAP ,n eg l ig ib l esymp a th e t i cn e rv ea c t iv i tyw a s ob s e rv eda tth el ev e lo fBAT ,sugg e s t ingth a tPACAPa c t sup s t r e amo fth em e l ano co r t in sy s t emina c t iv a t ingBAT(T an id a ,Sh in t an i ,&H a sh imo to2011 ) .Fu r th e rr e s e a r chi s 43 n e c e s s a rytosuppo r t(o rr e fu t e )th e s er e su l t s ,wh i chh a sb e encondu c t edh e r einth eG r ayl ab andd e s c r ib edinth i sch ap t e r . Hypo th e s i sandA im s W ehypo the s i z eth a tPACAPa c t sup s t r e amf romth em e l ano co r t insy s t emtor egu l a t e SNAtos t imu l a t ead ap t iv eth e rmog en e s i sinBAT(F igu r e2 .2 ) . Th eob j e c t iv e so fth i sch ap t e ra r e : • Tod e t e rm in ei fth eimp a i r edth e rmog en e s i sinPACAP/ -m i c ec anb e r e s cu edph a rm a co lo g i c a l lyw i tham e l ano co r t inagon i s t ,M e l ano t anI I(MT I I ) , andthu sp rov id eev id en c eu s ingag en e t i cmod e lo fPACAPd e f i c i en cyfo rth e conn e c t ionb e tw e enPACAPandth em e l ano co r t insy s t eminr egu l a t ing th e rmog en e s i sinBAT Toadd r e s sth ehypo th e s i sanda im s,th e rmog en i cc ap a c i tyw a sev a lu a t edth rough phy s io log i c a l(bodyw e igh tandcompo s i t ion ,m e t abo l i cr a t e(op enc i r cu i tc a lo r im e t ry ) ,and r e sp i r a to ryex ch ang er a t io(RER ) ) ,mo l e cu l a r( th e rmog en i cg en eexp r e s s ion ) ,and h i s to log i c a l( l ip ida r e aandc e l lcoun t )an a ly s e s .Comp l e t em a t e r i a l sandm e thod sa r e d e s c r ib edind e t a i lb e low . 2 .2 MATER IALSAND METHODS An ima l s Adu l t ,f em a l em i c e( ag ed25mon th s )w e r eg en e r a t edf romab r e ed ingco lonyo f PACAPnu l lm i c ea tth eUn iv e r s i tyo fNo r th e rnB r i t i shCo lum b i a .M i c eus edinth i ss tudy w e r ed e r iv edf romth ePACAPnu l ll in eg en e r a t edbyG r aye ta l .(2001 )andw e r eo fm ix ed g en e t i cb a ckg round(C57 BL/6 129 / Sv J) .G eno typ e sw e r ed e t e rm in edp r io rtoexp e r im en t s v i aane s t ab l i sh edpo lym e r a s ech a inr e a c t ion(PCR )g eno typ ingr e a c t ion ,d e s c r ib edb e low (G r aye ta l . ,2002 ) .B r i e f ly ,pup sw e r ee a rc l ipp edino rd e rtop rov id eat i s su es amp l efo r 44 � C o ldExp o su r e PACAP MC SNA � �� � VMN B row nA d ipo c y t e He a t F igu r e2 .2 .P ropo s edp a thw ay so fin t e r a c t ionfo rad ap t iv eth e rmog en e s i sinBATinr e spon s etoco ld expo su r e .VMN ;v en t rom ed i a lnu c l eu s ,PACAP ;p i tu i t a ryad eny l a t ecy c l a s e a c t iv a t ingpo lyp ep t id e , MC ;m e l ano co r t insy s t em ,SNA ;symp a th e t i cn e rv ea c t iv i ty ,BAT ;b rownad ipo s et i s su e .Im ag e s a d ap t edf rom“R ev i s i t ingth eV en t r a lM ed i a lNu c l eu so fth eHypo th a l amu s :Th eRo l e so fSF1 N eu ron sinEn e rgyHom eo s t a s i s” ,byY .Cho i ,T .Fu j ik aw a ,J .L e e ,A .R eu t e r , &K .K im ,2013 , F ron t i e r sInN eu ro s c i en c e,7.Copy r igh t©2013Cho i ,Fu j ik aw a ,L e e ,R eu t e randK im .Ad ap t edw i th p e rm i s s ionf romau tho r . 45 DNAex t r a c t iona sw e l la sfo ran im a lid en t i f i c a t ion .G enom i cDNAw a sex t r a c t edf romth e e a rc l ipt i s su es amp l eu s ingaso lu t iono f10%Ch e l ex(B io R adL abo r a to r i e s ,In c . ,H e r cu l e s , CA )w i thp ro t e in a s eKand10%Tw e en20 .T h er e a c t ionw a sin cub a t eda t50 °Cfo r45 m inu t e s ,fo l low edby15m inu t e sa t95 ° Ctoin a c t iv a t eth ep ro t e in a s eK .Th eso lu t ionw a s m ix edandth eCh e l exw a sa l low edtos e t t l etoth ebo t tomo fth etub e .G enom i cDNA(2µl ) w a sth enadd edtoa20µ lPCRr e a c t ioncon t a in ing0 .25µlT aqpo lym e r a s e(Th e rmoF i sh e r S c i en t i f i c ,W a l th am ,M a s s .) ,1 0× N ewEng l andBio l ab sPCRbu f f e r ,10µM d eoxynu c l eo s id e t r ipho sph a t e s(dNTP s ) ,and10µMo fth r e ep r im e r s :5 ′ MP1(5′ATGTGTAGCGG AGCA AGGCTGG3′ ) ,5′ MP2(5 ’GAACACGAGTGATGACTGGTCAGTC3 ’ ) ,andPA1(5′ CACTCGCACGGCATCTTCACAGATAG3′ ) .Th er e a c t ioncondi t ion sw e r e : d en a tu r a t iona t94 °Cfo r5m info l low edby94 °Cfo r30s e c ;ann e a l inga t67 °Cfo r30s e c ; ex t en s iona t7 2 °Cfo r30s e cfo r32cyc l e sandalongex t en s iona t72 ° Cfo r7m in .PCR r e a c t ionp rodu c t sw e r erunona ne th id iumb rom id es t a in ed1 .5%ag a ro s eg e landv i su a l i z ed und e ru l t r av io l e t( UV)l igh t .Th eg eno typ e so fth em i c ew e r ec l a s s i f i edbyv i su a l i z edb and s : th ew i ldtyp ea l l e l ep rodu c edab ando fapp rox im a t e ly550b a s ep a i r(bp) ,wh e r e a sth e kno ckou ta l l e l ep rodu c edab ando fapp rox im a t e ly950bp( App end ixF igu r eS2 .1 ). W a t e r , r a th e rth ang enom i cDNA,s e rv eda san eg a t iv econ t ro lfo rPCRr e a c t ion s . U s ingfourcoho r t s ,ato t a lo f32m i c ew e r eg en e r a t edfo rth i ss tudy .PACAP+ /+ (n=16 )andPACAP/ -(n=16 )l i t t e rm a t em i c ew e r ehou s edinp a i r s ,w i thon ePACAP+ /+ andon ePACAP/ -p e rc ag ew i thas t and a rd i z edamoun to fn e s t ingm a t e r i a l(1squ a r e / c ag e ) andco rnc obb edd ingtocon t ro lfo rth eamoun to fb edd ingm a t e r i a lth a tcou ldb eu s eda sa th e rmog en i csub s t r a t e .Th roughou tth eexp e r im en t ,th em i c ew e r eexpo s edtoano rm a l12h l igh t :12hd a rkcy c l e( l igh t son0700–1900h )andh adf r e ea c c e s stow a t e rands t and a r d rod en tchowd i e t(L abD i e t5001 ,L abD i e t ,In c . ,B r en twood ,L edu c ,AB ,C an ad a ; 46 m e t abo l i z ab l een e rgy3 .02k c a l /g ) .M i c ew e r ew e igh ed tw i c ew e ek lyandc ag e sch ang ed tw i c ew e ek ly .Th ec a r eandt r e a tm en to fth em i c ew a sina c co rd an c ew i thth egu id e l in e so f th eC an ad i anCoun c i lonAn im a lC a r e ,andpro to co l sw e r eapp rov edbyth eUn iv e r s i tyo f No r th e rnB r i t i shCo lumb i a ’ sAn im a lC a r eandU s eComm i t t e e . Co ldA c c l ima t ionand M e lano tanI It r ea tm en t F em a l em i c e(PACAP+ /+(n=16 ) ,PACAP/ -(n=16 ) )w e r ea c c l im a t edto4 ° Cby in i t i a l lyb e ingmov edinth e i rhom ec ag e sf rom24 °Cto18 °Cfo r7d ay sandth ento4 °Cfo r 30d ay s(F igu r e2 .3) .Th i st im e f r am ew a ss e l e c t eda sfu l la c c l im a t iontoco ldandm ax im a l indu c t iono fad ap t iv eth e rmog en e s i sr equ i r e s3 4w e ek sa t4 °C(C annon&N ed e rg a a rd ,2010 ; M ey e re ta l . ,2010 ;Uk rop e ce ta l . ,2006 ) . A f t e r7d ay so fa c c l im a t ionin4 °C ,th em i c ew e r eg iv end a i lyin t r ap e r i ton e a l( IP ) in j e c t ion s( a l t e rn a t ingin j e c t ions i t eonl e f tandr igh ts id e sev e ry3d ay s )o fe i th e rpho sph a t e bu f f e r eds a l in e(PBS )a sth ev eh i c l e(1xPBS ,0 .12m l )o rM e l ano t anI I(MT I I ,0 .4mg /kgin 0 .12m l1xPBS)fo r23d ay s .PACAP+ /+(n=9 )andPACAP/ -m i c e(n=9 )r e c e iv edPBS,and PACAP+ /+(n=7 )andPACAP/ -m i c e(n=7)r e c e iv ed MT I I .Du r ingth ed ayo fc a lo r im e t ry , in j e c t i on sw e r eno tg iv en .Th roughou tth i sp ap e r ,PACAP+ /+m i c ew i l lb er e f e r r edtoa s w i ld typ e(WT )andPACAP/ -a skno ckou t(KO ) .Thu s ,fou rt r e a tm en tg roup sex i s tandw i l l b er e f e r r edtoa sth efo l low ing :PBS WT ,PBSKO , MT I I WTand MT I IKO . BodyCompo s i t ion Bodycompo s i t ionw a sm e a su r edp r e-andpo s t t r e a tm en tu s ingt im e dom a innu c l e a r m agn e t i cr e son an c e(TDNMR) .Un an a e s th e t i s edm i c ew e r eind iv idu a l lyp l a c edina r e s t r a in e rw i tha c c e s stooxyg enandth enp l a c edinth em in i sp e cmqTD NMRm a ch in efo r twom inu t e swh i l em e a su r em en t so fto t a lbodyf a t ,f lu id ,andl e anm a s s w e r eob t a in ed (m in i sp e cmq ,TD NMR ,B ruk e r ,CA ) .Th ef a tandl e anm a s s e sw e r ec a l cu l a t eda sa 47 F igu r e2 .3 .S ch em a t i co fm e thodo logyov e rth ecou r s eo fth e6w e ekexp e r im en t . 48 p er c en t ag eo fto t a lbodym a s s ,andth ep r e -andpo s t t r e a tm en tf a tandl e anm a s sp e r c en t ag e s w e r ec a l cu l a t ed . Th e rmog en i cCapa c i tyth roughOx yg enCon sump t ion Ond ay21o ft r e a tm en t ,oxyg encon sump t iono fth em i c ew a sind iv idu a l lym e a su r ed byop enc i r cu i tc a l o r im e t ry(Oxym axm a ch in e ,Co lumbu sIn s t rum en t s ,Co lumbu s ,OH , USA) .Th es ing l emou s eOxym axhou s ingch amb e rw a sequ i l ib r a t edfo rtwohou r sb e fo r e b a s a loxyg encon sump t ionw a sm e a su r edinth ean im a l s .An im a l sw e r ean e s th e t i s edw i th sod iump en tob a rb i t a l( 5060mg /kg ,ip ) .On c eap l an eo fan e s th e s i aw a sr e a ch ed ,th e in t e r s c apu l a rr eg ionw a ssh av edandd i s in f e c t edw i th90%i sop ropy la l coho l .Ac annu l aw a s l lowfo rth eadm in i s t r a t iono fNE in s e r t edsub cu t an eou s lyov e rth ein t e r s c apu l a rBATtoa l a t e rinth eexp e r i m en twh i l eavo id ingop en ingth ech amb e r .Th em i c ew e r eth enp l a c edin to th eO xym axch amb e rfo r10m inu t e stoa l lowfo requ i l ib r a t iono fth em a ch in e .Th ean im a l s th ensp en tapp rox im a t e ly15m inu t e sinth ech amb e rtoa l lowfo rb a s a lm e t abo l i cr a t e(BMR ) (d e f in edb e low )tob er e co rd ed .A f t e roxyg encon sump t ionr e ad ing sw e r es t ab l e ,NE ( 1mg /kg ,s c )w a sin j e c t edov e rth eBATo fth em i c ev i ath ec a nnu l aandoxyg encon sump t ion w a sm e a su r edfo rapp rox im a t e lyon ehou run t i lth em ax im a lm e t abo l i cr a t e(MMR )o c cu r r ed , a sd e t e rm in edbyap e akinoxyg encon sump t ionfo l low edbyas t e adyd e c l in einoxyg en con sump t ion .BMRw a sd e f in eda sth em e ano fth ef iv elow e s toxyg encon sump t ionv a lu e s p r io rtoth eNEin j e c t ion . MMRw a sd e f in eda sth em e ano fth ef iv eh igh e s toxyg en co n sump t ionv a lu e sa f t e rNEadm in i s t r a t ion .Oxyg encon sump t ionv a lu e sw e r er e co rd ed ev e ry10s e cond sandw e r eth enc a l cu l a t edandr epo r t eda sanav e r ag er e ad ingov e r2 m inu t e s ,b eg inn ing10m inu t e sp r io rtoNEin j e c t ionandend ingapp rox im a t e ly34m inu t e s pos tNEin j e c t ion .Oxyg encon sump t ionv a lu e sf romth eOxym axso f tw a r ew e r eno rm a l i z ed toe a chan im a l ’ sp r e t r e a tm en tl e anm a s s(m l /m in /kgl e anm a s s ) .R e sp i r a to ryex ch ang er a t io 49 (RER )i sac a l cu l a t edr a t iob e tw e enth evo lum eo fc a rbond iox id e(VCO rodu c edand 2)p oxyg en(VO2)u s ed(VCO ndr ep r e s en t sam e a su r eo fwh i chm a c ronu t r i en t( f a to r 2/VO 2)a c a rbohyd r a t e )i sox id i z edfo rfu e lu t i l i z a t ion(G av in i ,Jon e s ,&Nov ak ,2016 ) .A sw i th oxyg encon sump t ionv a lu e ,RERv a lu e sw e r er e co rd edev e ry10s e cond sandw e r eth en c a l cu l a t edandr epo r t eda sanav e r ag er e ad ingov e r2m inu t e s ,b eg inn ing10m inu t e sp r io rto NEin j e c t ionandend ingapp rox im a t e ly34m inu t e spo s tNEin j e c t ion . Po s tmo r t emAna ly s i s Uponcomp l e t iono fth e23d ayt r e a tm en t,th enon f a s t edm i c ew e r ew e igh edand d e ep lyan e s th e t i z edw i thi so f lu r an e .B loodw a sco l l e c t edin toh ep a r in i z edtub e sbyc a rd i a c pun c tu r e( app rox .0 .7 1 .0m l /mou s e )andspundowna t3 ,000rpmfo r10m inu t e sandp l a sm a co l l e c t ed .Th em i c ew e r eth eneu th an i z edw i thi so f lu r an eandc e rv i c a ld i s lo c a t ion .T i s su e s w e igh edandco l l e c t edin c lud edBAT ,ingu in a landgon ad a lwh i t ead ipo s et i s su e( ingWAT , gWAT ) ,l iv e randp an c r e a s .Th e s et i s su e s ,inadd i t iontosk e l e t a lmu s c l e ,sm a l lin t e s t in e , k idn ey ,h e a r tandlung sw e r ef l a shf ro z eninl iqu idn i t rog enands to r edin80°C .T i s su e s f ix edin10%fo rm a l inin c lud edBAT ,ingWAT ,gWAT ,sk e l e t a lmu s c l e ,k idn ey ,p an c r e a s , andad r en a lg l and s .Th eb r a inw a sco l l e c t ed ,f ro z enands to r eda t80 °C . RNAEx t ra c t ionandcDNAg en e ra t ion RNAex t r a c t ion sw e r ep e r fo rm edonfro z ens amp l e so fBAT ,ingWATandgWAT . T i s su e s(0 .051g )w e r ehomog en i z edinTR I zo l(L i f eT e chno log i e s )andRNAw a sex t r a c t ed a c co rd ingtoth em anu f a c tu r e r ’ sp ro to co l ,u s ingth eadd i t ion a lop t ion a lp ro c e s sfo rs amp l e s w i thh ighf a tcon t en tp rov id ed .TURBODN a s e(L i f eT e chno log i e s )w a su s edina c co rd an c e w i thth em anu f a c tu r e r ’ sp ro to co ltor emov eDNAcon t am in a t ion .RNAcon c en t r a t ionand pu r i tyw e r eev a lu a t edth roughUVl igh tab so rb an c em e a su r em en t su s ingsp e c t ropho tom e t ry (N anod ropND 1000 ;Th e rmoS c i en t i f i c ,Ro ck fo rd ,IL ,USA ) .Ab so rb an c el ev e l so fth e 50 s amp l ea t260nmw e r eu s eda sasu r rog a t em e a su r eo fRNAcon c en t r a t ion( th ew av e l eng tha t wh i chnu c l eo t id e sab so rbUVl igh t ).Th er a t ioo fab so rb an c ea t260nmand280nm( th e w av e l eng tha twh i chp ro t e inab so rb sUVl igh t)w a su s edtoa s s e s sth epu r i tyo fRNA ,wh e r e ar a t ioo f~2 .0i sg en e r a l lya c c ep t eda s“pu r e ”fo rRNA .A sas e cond a rym e a su r eo fnu c l e i c a c idpu r i ty ,th er a t ioo fab so rb an c ea t260nmand230nmw a su s edtoid en t i fyth ep r e s en c eo f con t am in an t sth a tab so rba t230nm ,su cha sph eno lp r e s en tinth eTR I zo lr e ag en tu s eddu r ing RNAex t r a c t ion s .260 /230v a lu e scommon lyr ang eb e tw e en2 .02 .2fo r“pu r e ”nu c l e i ca c id . RNAin t eg r i tyw a sa s s e s s edbyob s e rv ingin t a c t ,b r igh tandd i s t in c t18Sand28SrRNAb ands one th id iumb rom id es t a in ed1 .5%ag a ro s eg e l s(App end ixF igu r eS2 .2 ) .RNA(500ng )w a s r ev e r s et r an s c r ib edin tocDNA(Sup e r s c r ip tI I I ,Inv i t rog en )w i thab l endo fo l igo (dT )and r andomh ex am e rp r im e r sa c co rd ingtoth em anu f a c tu r e r ’ sp ro to co l . G en eExp r e s sion BATth e rmog en i cg en eexp r e s s ion(β3AR ,HSL ,andUCP1 ) ,on econ t ro lg en e(β1AR ) ,ands ixr e f e r en c eg en e s(18S ,TBP ,GAPDH ,PGK1 ,RPL19 ,andPP IA )w e r ea s s e s s ed u s ingqu an t i t a t iv er e a lt im epo lym e r a s ech a inr e a c t ion(qPCR ) .15µ lr e a c t ion sw e r ec a r r i ed ou tu s inganiQ5th e rmo cy c l e r(B ioR adL abo r a to r i e s )andcon t a in edfo rw a rdandr ev e r s e p r im e r s(1µ M ;S igm a) ,nu c l e a s e f r e eH ,1 /10cDNA(1 .5µ l ) ,andSYBRG r e enSup e rm ix 2O (1X ) .P r im e r su s edinth i sexp e r im en th adb e enp r ev iou s lyop t im i z edinth eG r ayl ab(b y D a emonC l in e )andcon fo rm edtoth eM in imumIn fo rm a t ionfo rPub l i c a t iono fQu an t i t a t iv e R e a lt im ePCRExp e r im en t s(M IQE s )gu id e l in e s(Bu s t ine ta l . ,2009 ) .Am e a su r eo f r e f e r en c eg en es t ab i l i ty(M )a c ro s sg eno typ e sandt r e a tm en t sw a sd e t e rm in edw i thB iog a z e l l e (B iog a z e l l eqB a s eP lu s2 .4 ,Zw i jn a a rd e,B e lg ium ) wh e r er e f e r en c eg en e sw i th M<0 .5w e r e a c c ep t ed .T wor e f e r en c eg en e s(RPL19andPP IA )w e r es e l e c t edfo re a chg en eexp r e s s ion 51 an a ly s i s( M=0 .26) .Fo ldch ang eing en eexp r e s s ionw a sc a l cu l a t edfo re a chg en e ,ine a ch g roup ,a scomp a r edtoexp r e s s ionf romPBS WTan im a l s . H i s to logy Fo rm a l inf ix edt i s su e sw e r es en tfo rh i s to log i c a ls e c t ion ing(W ax I tH i s to log i c a l S e rv i c e s ,UBC ,V an couv e r ,C an ad a ) :BAT ,ingWAT ,andgWAT(PBS WT(n=4 ) ,PBSKO (n=4 ) , MT I I WT( n=4 ) , MT I IKO(n=4 ) ) .T i s su e sw e r ep a r a f f inemb edd ed ,s e c t ion ed(5µm th i ck ) ,ands t a in edw i thh em a toxy l inandeo s info rv i ew ingonal igh tm i c ro s cop e(O lympu s BX61) .An a ly s e sw e r ecomp l e t edfo rBATandgWATincon jun c t ionw i thanund e rg r adu a t e honou r sth es i s( inco l l abo r a t ionw i th M a egh anFo r s t e r ) . H i s to log i ca lM i c rog raph s H i s to log i c a lan a ly s e sp e r fo rm edinth i ss tudyw e r er e c en t lyd ev e lop edbyUNBC G r ayl abm emb e r sandh av eno tb e enp r ev iou s lydo cum ent edinou rr e s e a r ch .Th e r e fo r e , con t ro lan im a l sw i thw e l l do cum en t edd i f f e r en c e sinth eh i s to log i c a lp r e s en t a t iono fBAT w e r ein i t i a l lyan a ly z ed( th e rmon eu t r a l(30 °C )v e r su sco lds t r e s s ed(4 °C )an im a l s ;r e su l t sno t showno rd i s cu s s ed ) .R e su l t su s ingth i sn ew lyd ev e lop edp ro to co lw e r econ s i s t en tw i ththo s e do cum en t edinth el i t e r a tu r e ,thu sen su r ingth ev a l id i tyo fth ep ro to co lfo rexp e r im en t a l an a ly s e s . T enm i c rog r aph so fBATw e r et ak en(O lympu sBX61 )p e rmou s e(n=4m i c ep e r t r e a tm en t )a t60xm agn i f i c a t ionu s ingano i limm e r s ionob j e c t iv ew i th inth r e et i s sues e c t ion s (3t i s su es e c t ion s / s l id e ) .Ti s su es e c t ion su s edfo rth em i c rog r aph sw e r es e l e c t edr andom ly , how ev e r ,c a r ew a st ak entoavo idf i e ldo fv i ew sth a tcon t a in edl a rg eb loodv e s s e l s ,d am ag ed c e l l s ,o rh e t e rog en e i tyamongc e l ltyp e stoen su r ep rop e rr ep r e s en t a t iono fad ipo cy t e s . Th r e em i c rog r aph so fgWATw e r et ak en(O lympu sBX61 )p e rmou s e(n=4m i c ep e r t r e a tm en t )a t20xm agn i f i c a t ion .Inco ld a c c l im a t edPBSWT and MT I IWTm i c e , 52 m i c rog r aph sw e r et ak enu s ingth es am em e thodo logyfo rBATw i thr e sp e c ttor andom s e l e c t iond e s c r ib edabov e .Incon t r a s t ,homog enou sgWATa r e a sw e r es e l e c t iv e lycho s enin co ld a c c l im a t edPBSKOand MT I IKOm i c edu etoah ighd eg r e eo fun exp e c t edb e ig e ad ipo cy t e sw i th inth egWATs e c t ion s ,wh i chl e f tv e ryf ewa r e a sw i thl a rg eenoug hr eg ion so f wh i t ead ipo cy t e s . BATL ip idA r ea C e l lS en sSo f tw a r e(O lympu s )w a su s edina l lp r ep a r a t ionandan a ly s i so fl ip ida r e a m e a su r em en t so fBAT .Im ag e sw e r econv e r t edf romb r igh tf i e ldtog r ay s c a l e(F igu r e2 .4A ) , andcon t r a s tw a sm ax im i z edb e fo r ean a l y s e stod i s t ingu i shb l a ckf romwh i t e(F igu r e2 .4B ) . Ing r ay s c a l ead ipo cy t eh i s to logy ,l ip ida r e aandin t r a c e l lu l a rsp a c e sapp e a r ed“wh i t e , ”wh i l e c e l lm emb r an e s ,nu c l e i ,andb loodc e l l sapp e a r eda sd i f f e r en tsh ad e so f“g r ey . ”M i c rog r aph s w e r efu r th e red i t edbys e l e c t iv e lyd e l e t inga r e a sth a tw e r eno thomog en eou sr ep r e s en t a t iv e s o fb rownad ipo s ec e l l s ,su cha sl a rg ein t r a c e l lu l a rsp a c e s ,b loodv e s s e l s ,o re ry th ro cy t e s (F igu r e2 .4C ) .D e l e t inganob j e c tf romth er eg iono fin t e r e s t(RO I )a l sor emov edth a ta r e a f romth eto t a lRO Ifo rfu tu r ea r e ap e r c en t ag ec a l cu l a t ion s .Am anu a lin t en s i tyth r e sho ldw a s s e tth a tb e s tr ep r e s en t edth e“g r ey ”r eg ion s ,wh i chr ep r e s en t ednon l ip ida r e a so fe a cho fth e c ro s ss e c t ion(g r aych ann e l :m in=0 ,m ax=172 ) .Th e s e“g r ey ”a r e a sw e r equ an t i f i eda f t e r s egm en t ingth em i c rog r aph sin tob in a rypho to s(F igu r e2 .4D ) ,andth enm e a su r inga r e afo r e a chco lou r(wh i t eo rg r ey ) .R e su l t sw e r eno rm a l i z edtoth e i rr e sp e c t iv eRO Ia r e ap e rpho to ( ex c lud ingd e l e t edr eg ion s )byconv e r t ingtop e r c en t ag e s ,andsub t r a c t edf rom100tog iv e to t a ll ip id( “wh i t e ” )a r e ap e rm i c rog r aph . BATC e l lCoun t C e l lcoun tp e rf i e ldo fv i ewinBATw a sp e r fo rm edbyind iv idu a l lycoun t ingth e numb e ro fnu c l e iine a chim ag e ,p rov id ingasu r rog a t em e a su r eo fov e r a l lc e l ls i z eb e tw e en 53 F igu r e2 .4 .M e thodo log i c a ls t ep su s edtoan a ly z eBATs e c t ion sfo rl ip ida r e aa t60xo i limm e r s ion . A .Im ag e sw e r econv e r t edtog r ay s c a l e .B .Con t r a s tw a sm ax im i z ed .C .S egm en t sth a tw e r eno t b rownad ipo cy t e sw e r es e l e c tiv e lyd e l e t ed( ind i c a t eda sr ed) .D .Im ag ew a sb in a r i z edand“wh i t e ” a r e acoun t eda sl ip id . 54 t r e a tm en tg roup s .Toin c r e a s ecoun t inga c cu r a cy ,a4 s egm en tg r idw a sl a idov e re a chim ag e (M i c ro so f tPow e rpo in t ) .Nu c l e icoun t so fe a chqu a r t e rs egm en to fapho tow e r eadd ed tog e th e rtog iv eon ecoun tp e rim ag e .Av e r ag enumb e ro fc e l l sinaf i e ldo fv i eww a sth en c a l cu l a t ed . gWATC e l lA r eaB inn ing Tod e t e rm in eth ed i s t r ibu t iono fd i f f e r en twh i t ead ipo cy t es i z e singWAT ,C e l lS en s So f tw a r e(O lympu s )w a su s edtom e a su r eth ea r e ao fwh i t ead ipo cy t e sinim ag e st ak enu s ing a20xob j e c t iv e .Im ag e sw e r ep r ep a r edfo ran a ly s i sbyconv e r tingth emtog r ay s c a l e(F igu r e 2 .5A)fo l low edbym ax im i z ingcon t r a c tu s ingad i f f e r en t i a lcon t r a s tenh an c em en tf i l t e r(B and W id th=35 , Enh an c em en t=100 ,Ov e r f low=0 .05% )(F igu r e2 .5B) .Sub s equ en t ly ,im ag e s w e r eb in a r i z edtos ep a r a t el ip id(wh i t e )f romnonl ip ida r e a s(g r ey )(F igu r e2 .5C )(G r ey ch ann e l :m in=0 ,m ax=213 ) .C e l lbound a r i e sw e r eth ens p l i tau tom a t i c a l lybyth eC e l lS en s s o f tw a r e ,fo l low edbym anu a lsp l i t t ingfo rc e l l sth a tcon t a in edv e ryth inc e l lm emb r an e s t a in ingandw e r ethu sm i s s edbyau tom a t edsp l i t t ing .C e l l sa longth eou t e rm i c rog r aphedg e s th a td idno td i sp l ayth een t i r ec e l lc i r cu m f e r en c ew e r ed e l e t ed(F igu r e2 .5D) .Anav e r ag eo f th el a rg e s tandsm a l l e s tc e l la r e a sa c ro s sm i c rog r aph sw a sc a l cu l a t edtod e t e rm in ear ang efo r ad ipo s ec e l ls i z eb inn ingan a ly s i s(M in :80 ax :2000 µm2) .Ob j e c ts i z e stool a rg eo rtoo µm2, M sm a l ltor ep r e s en tad ipo cy t e sw e r eex c lud eda sth eyl ik e lyr ep r e s en ta r t i f a c t so fg r a in in e s so r l a rg ein t r a c e l lu l a rsp a c e sw i th inam i c rog r a ph .A r e ad a t aw a sexpo r t edtoE x c e landso r t ed f roml a rg e s ttosm a l l e s t .C e l la r e a sw e r eso r t edin tob in sb a s edonc e l la r e a( i nµm2:80 499 , 500999 ,1000 1499 ,15002000 ) .On lyth egWATo fco ld a c c l im a t edPBSand MT I It r e a t ed PACAP WTm i c ew e r es t a t i s t i c a l lyan a ly z eddu etoth eun exp e c t edob s e rv a t iono fan imm en s eamoun to fb e ig ead ipo cy t e singWATo fco ld a c c l im a t edPBSand MT I It r e a t ed PACAPKOm i c e . 55 F igur e2 .5 .M e thodo log i c a ls t ep su s edto d e t e rm in ec e l la r e acoun t sinh i s to log i c a ls e c t ion so fgWAT im ag eda t20x .A .Im ag e sw e r econv e r t edtog r ay s c a l e .B .D i f f e r en t i a lcon t r a s tw a sm ax im i z ed .C . Im ag e sw e r eb in a r i z ed .D .Ad ipo cy t e sno ten t i r e lyd i sp l ay edw ith inth ef i e ldo fv i eww e r eex c lud ed f roman a ly s e s . 56 S ta t i s t i ca lAna ly s e s R e su l t sw e r eexp r e s s eda sg roupm e an±s t and a rde r ro rm e an(SEM ) .Twow ay ANOVAw a su s edtod e t e rm in em e and i f f e r en c e sb e tw e ent r e a tm en tg roup s( co ld a c c l im a t ed PBS WT ,PBSKO , MT I I WT ,and MT I IKOm i c e ) ,wh e r ein t e r a c t ion sb e tw e entwo ind ep end en tv a r i ab l e sm ayin f lu en c eth ed ep end en tv a r i ab l e .Th eind ep end en tv a r i ab l e sw e r e t im eandt r e a tm en tg roup ,andth ed ep end en tv a r i ab l ew a son eo fth efo l low ing :bodym a s s , bodycompo s i t ion ,oxyg encon sump t ion ,o rRER .On e w ayANOVAw i thTuk ey ’ st e s tfo r p a i rw i s ecomp a r i son sw a su s edfo rcomp a r i son sb e tw e ena l lfou rt r e a tm en tg roupm e an sand w e r ep e r fo rm edfo rg en eexp r e s s ion ,BATl ip ida r e a ,andBATc e l lcoun t .Unp a i r edtt e s t s w e r eus edtoan a ly z em e and i f f e r en c e sb e tw e ent i s su em a s s e sfo rco ld a c c l im a t edPBS WT andco ld a c c l im a t edPBSKOm i c e ,a sw e l la sco ld a c c l im a t edPBS WTandco ld a c c l im a t ed MT I IKOm i c e .Unp a i r edtt e s t sw e r ea l sou s edfo rth enumb e ro fingWATc e l l sw i th ine a ch c e l ls i z eb inb e tw e enco ld a c c l im a t edPBS WTandco ld a c c l im a t ed MT I I WTm i c e .Th e d e t e c t iono fou t l i e r sw a sd e t e rm in edu s ingG rubb ’ st e s t .R e su l t sw e r econ s id e r eds t a t i s t i c a l ly s ign i f i c an tu s ingana lph ao f0 .05 .S t a t i s t i c a lan a ly s e sw e r ep e r fo rm edu s ingG r aphP adP r i sm So f tw a r e ,v e r s ion6 .0b . 2 .3RESULTS Ana ly s e sB e tw e enD i f f e r en tG eno typ e sandT r ea tm en t s Th i ss tudyex am in edth ee f f e c tandin t e r a c t iono fbo thPACAPg eno typ eand m e l ano co r t inagon i s tt r e a tm en tonth e rmog en i cc ap a c i tyinm i c eandthu s ,a sd e s c r ib ed abov e ,w ep r e s en th er er e su l t sfo r4g roup so fco ld a c c l im a t edan im a l s :PBS WT ,PBSKO , MT I I WTand MT I IKO.Comp a r i son sb e tw e enco lda c c l im a t ed MT I I WTandPBSKOa r e no tr epo r t ed ,a sth eya r eno tphy s io log i c a l lyr e l ev an t ;co ld a c c l im a t ed MT I IW Tan im a l s w e r eu s edtod e t e rm in eth ee f f e c t so f MT I Ion WTan im a l s( i fany )toen su r ep rop e ran a ly s i s 57 o f MT I IKOan im a l s .A l thoughnos ign i f i c an c ew a sob s e rv edinan yr e su l tb e tw e enco ld a c c l im a t edPBS WTand MT I I WT ,th et r end sth a td idex i s tw i l lb ed i s c u s s edandsugg e s t th a t ,a sexp e c t ed MT I Im ayh av eh adsom ephy s io log i c a le f f e c tonco ld a c c l im a t ed WT an im a l s ,andthu sa l low edu stocon f id en t lya s s e s sth eco ld a c c l im a t ed MT I IKOan im a l s . Comp a r i son sb e tw e enco lda c c l im a t edPBSKOand MT I IKOm i c e(o rb e tw e enco ld a c c l im a t ed MT I I WTand MT I IKOm i c e )w e r ea l sono tth efo cu so fth e s ean a ly s e s(bu tw i l l b ed i s cu s s edwh e r eapp rop r i a t e )a sw ea im edtod e t e rm in ei f MT I IKOm i c ew e r er e s cu ed b a cktophy s io log i c a lno rm a ll ev e l s( a scomp a r edtoco ld a c c l im a t edP BS WTan im a l s ) . Th e r e fo r e ,chang e sinth ed i f f e r en c e sb e tw e enco lda c c l im a t edPBS WTandPBSKOm i c e v e r su sco lda c c l im a t edPBS WTand MT I IKOm i c ew i l lb eth efo cu so fth ean a ly s e s . MT I IT r ea tm en t Cau s edNoS ign i f i can tE f f e c tonBody W e igh to rBodyCompos i t ion Nos ign i f i c an td i f f e r en c einbodyw e igh to rbodycompo s i t ionw a sob s e rv edb e tw e en co ld a c c l im a t edPBS WT ,PBSKO , MT I I WTo r MT I IKOan im a l s( two w ayANOVA ,p> 0 .05 )(F igu r e2 .6 ,F igu r e2 .7 ) . MT I IPa r t ia l lyR e s cu e sD im in i sh edgWATD epo tinCo ldA cc l ima t edPACAPKOM i c e A sexp e c t edf romp r ev iou s lypub l i sh edr e su l t s ,th e r ew a sas ign i f i c an td e c r e a s ein gWATm a s sinco lda c c l im a t edPBSKOm i c ecomp a r edtoco ld a c c l im a t edcon t ro lm i c e , PBS WT(unp a i r edtt e s t ,p<0 .05 )(F igu r e2 .8 )( Ad am se ta l . ,2008;D i anée ta l . ,2014 ) .Th i s s ign i f i c an c ew a sno tob s e rv edb e tw e enco ld a c c l im a t edPBS WTand MT I IKO(unp a i r edt t e s t ,p>0 .05 ) .S im i l a rt r end sw e r eob s e rv edinbo thingWATandBAT ,bu tnos ign i f i c an c e w a sid en t i f i ed(F igu r e2 .8 ) . 58 F igu r e2 .6.Body m as so fPBS WT ,PBSKO , MT I I WT ,and MT I IKOm i c eov e rth ecou r s eo fth e exp e r im en t .T r e a tm en tw i thv eh i c l e(PBS )o r MT I Ib eg anonD ay7 .D a t aa r em e an±SEM . Twow ay ANOVA ,p>0 .05 . F igu r e2 .7.D i f f e r en c eo ff a tandl e an m a s sa sap e r c en t ag eo fto t a l ma s sinPBS WT ,PBSKO , MT I I WT ,and MT I IKO m i c ep r eandpo s t4 °Cco ldch a l l eng e .D a t aa r em e an±SEM .Twow ayANOVA , p>0 .05. 59 F igu r e2 .8.P e r c en tt i s su em a s sp e rto t a lbody m a s so fd i ff e r en tt i s su etyp e sinPBS WT ,PBSKO , MT I I WT ,and MT I IKO m i c eaf t e r4 w e ek4 °Cco ldch a l l eng e .D a t aa r em e an±SEM .Unp a i r edtt e s t s .*p<0 .05ind i c a t e sag eno typ ee f f e c tw i th inth es am et r e a tm en t . 60 MT I IPa r t ia l lyR e s cu e sImpa i r edTh e rmog en i cCapa c i tyo fCo ldA c c l ima t edPACAP KOM i c e Tot e s tth ehypo th e s isth a tPACAPa c t sup s t r e amo fth em e l ano co r t insy s t em ,th e e f f e c to fth em e l ano co r t inagon i s t , MT I I(0 .4mg /kg ,23d ay s )onth e rmog en e s i sinco ld a c c l im a t edPACAP WTandPACAPKOm i c ew a sev a lu a t edf i r s tth roughoxyg en con sump t iona sam e a su r em en to fth e rmo g en i cc ap a c i ty .A l thoughnos ign i f i c an c ew a s ob s e rv edb e tw e enco lda c c l im a t edPBS WTandPBSKOo rco ld a c c l im a t edPBS WTand MT I IKO ,at r endshow edco lda c c l im a t edPBS WTa ch i ev ingh igh e roxyg encon sump t ion v a lu e sth anco lda c c l im a t edPBSKO ,wh i l eco ld a c c l im a t ed MT I IKOv a lu e st r end edh igh e r th anco ld a c c l im a t edPBSKO( two w ayANOVA ,p>0 .05 )(F igu r e2 .9A ) .Add i t ion a l ly , wh i l enod i f f e r en c ew a sob s e rv edinBMRb e tw e ena l lfou rg roup s( twow ayANOVA ,p> 0 .05 ) ,a sp r ev iou s lyr epo r t edbyth eG r ayl ab ,t h e r ew a sas ign i f i c an td i f f e r en c ein MMR b e tw e enco ld a c c l im a t edPBS WTandPBSKOm i c e( two w ayANOVA ,p<0 .05 )(F igu r e 2 .9B )(D i anée ta l . ,2014 ) .Th i ss ign i f i c an c ew a slo s tb e tw e enco ld a c c l im a t edPBS WTand MT I IKOm i c e( twow ayANOVA ,p>0 .05 )(F igur e2 .9B ) . MT I IPa r t ia l lyR e s cu e s Impa i r edFu e lU sag einCo ld A c c l ima t edPACAPKOM i c e R e sp i r a to ryex ch ang er a t io(RER ) ,am e a su r eo fwh i chm a c ronu t r i en t( f a to r c a rbohyd r a t e )i sm e t abo l i z edfo rfu e lu t i l i z a t ion ,w a sc a l cu l a t edf romOxym axd a t aa sar a t io ofVCO oVO ndshow edth a t ,a sexp e c t ed ,th eadm in i s t r a t iono fNEindu c edad e c r e a s e 2t 2a inRERinbo thco ld a c c l im a t edPBS WTand MT I I WTm i c e ,ind i c a t ingp r e f e r en t i a l ox id a t iono fl ip id sov e rc a rbohyd r a t e sfo l low ingad r en e rg i cs t imu l a t ion(F igu r e2 .10 ) . Pr ev iou s ly ,th eG r ayl abh a sshownth a tco ld a c c l im a t edPACAPKOm i c edono texp e r i en c e th i ssh i f tinRERw i thad r en e rg i cs t imu l a t ion :af ind ingth a tw a sr ep e a t edinth i ss tudy ,wh e r e RERinco lda c c l im a t edPBSKOm i c er em a in eds t e adya t~0 .85a f t e rth eNEin j e c t ion . 61 A . B . F igu r e2 .9.A .No r ep in eph r ineindu c edth e rmog en e s i sinco ld a c c l im a t edPBS WT ,PBSKO , MT I I WT ,and MT I IKO m i c e .B .M e anb a s a lm e t abo l i cr a t e(BMR )and m ax ima lm e t abo l i cr a t e(MMR ) o fco lda c c l im a t edPBS WT ,PBSKO , MT I I WT ,and MT I IKOm i c e.D a t aa r eexp r e s s eda sm e an± SEM .Twow ayANOVA .*p<0 .05ind i c a t e sag eno typ ee f f e c tw i th inth es am et r e a tm en t . 62 F igu r e2 .10.R e sp i r a toryex ch ang er a t io(RER )o fco ld a c c l im a t edPBS WT ,PBSKO , MT I I WT ,and MT I IKO m i c e .S t a t i s t i c a ls ign i f i c an c e(p<0 .05 )b e tw e enPBS WTandPBSKOa t8and10m inu t e s andb e tw e en MT I I WTand MT I IKOa t2 ,4and6 m inu t e s .Twow ayANOVA . 63 In t e r e s t ing ly ,co ld a c c l im a t ed MT I IKOm i c eshow edad e c r e a s einRERr ep r e s en t ing ap r e f e r en c etow a rdl ip idu t i l i z a t ionfo l low ingad r en e rg i cs t imu l a t ion ,a sob s e rv edinco ld a c c l im a t edPBS WTm i c e(F igu r e2 .10 ) .S t a t i s t i c a ls ign i f i c an c ew a sexh ib i t edb e tw e enco ld a c c l im a t edPBS WTandPBSKOa t8and10m inu t e sandb e tw e enco ld a c c l im a t edMT I I WTand MT I IKOa t2 , 4and6m inu t e s( two w ayANOVA ,p<0 .05 ) . NoD i f f e r en c einTh e rmog en i cG en eExp r e s s ionwa sOb s e rv edinBATB e tw e enCo ldA c c l ima t edPBSo r MT I IT r ea t edPACAP WTo rPACAPKO M i c e Nos ign i f i c an td i f f e r en c ew a sob s e rv edb e tw e enco lda c c l im a t edPBS WT ,PBSKO , AR s ,HSLandUCP1 MTI I WT ,o r MT I IKOm i c efo rth e rmog en i cg en eexp r e s s iono fβ3 (on e w ayANOVA ,p>0 .05 )(F igu r e2 .11 ) .β1ARexp r e s s ion ,u s eda sacon t ro ltoen su r e MT I Ie f f e c t sw e r elo c a l i z edtop a thw ay sinvo lv edinth e rmog en e s i s ,w a sa l sono t s ign i f i c an t lyd i f f e r en tb e tw e eng roup s(on e w ayANOVA ,p>0 .05 )(F igu r e2 .11 ) . NoS ign i f i can tD i f f e r en c eB e tw e enL ip idA r eao rC e l lS i z einBATo fCo ldA c c l ima t ed MT I I WTand MT I IKO M i c e A l thoughnos ign i f i c an td i f f e r en c einl ip ida r e ab e tw e enco lda c c l im a t edPAC AP WT o rPACAPKOm i c et r e a t edw i the i th e rPBSo r MT I Iw a sob s e rv ed(on e w ayANOVA , Tuk ey ’ sT e s t ,p>0 .05 )(F igu r e2 .12A ) ,at r endind i c a t edth a tbo thco lda c c l im a t ed MT I I WT and MT I IKOan im a l sd i sp l ay edg r e a t e rl ip ida r e ap e rRO Icomp a r edtoco ld a c c l im a t edPBS WTandPBSKOm i c e . Inag r e em en tw i thth e s er e su l t s ,nos ign i f i c an td i f f e r en c einth enumb e ro fc e l l sp e r f i e ldo fv i ewb e tw e enco ld a c c l im a t edPACAP WTo rPACAPKOm i c et r e a t edw i the i th e r PBSo r MT I Iw a sob s e rv ed(on ew ayANOVA ,Tuk ey ’ sT e s t ,p>0 .05 )(F igu r e2 .12B ) . How ev e r ,at r endind i c a t edth a tbo thco lda c c l im a t ed MT I I WTand MT I IKOan im a l s d i sp l ay edf ew e rnumb e r so fc e l l sp e rRO Icomp a r edtoco ld a c c l im a t edPBS WTandPBS 64 mRNAe xp r e s s ion( f o ldc h an g e ) F igu r e2 .11.G en eexp r e s s ioninBATo fco lda c c l im a t edPBS W T ,PBSKO , MT I I WT ,and MT I I KOm i c e .T a rg e tg en eexp r e s s ionw a sno rm a l i z edtotwor e f e r en c eg en e s :RPL19andPP IA .D a t aa r e exp r e s s eda s±SEM.On e w ayANOVAfo re a chg en e ,p>0 .05 . 65 A . B . F igu r e2 .12.A .Av e r ag ep e r c en tl ip ida r e a / f i e ldofv i ewinco ld a c c l im a t edPBS WT ,PBSKO , MT I I WT ,and MT I IKO m i c e .B .Av e r ag enumb e r so fnu c l e ip e rRO Iinco lda c c l im a t edPBS WT ,PBS KO , MT I I WT ,and MT I IKOm i c e.D a t aa r eexp r e s s eda s ±SEM.On e w ayANOVA ,p>0 .05 . 66 KOm i c e . MT I I WT M i c eD i sp lay In c r ea s edNumb e ro fSma l l Wh i t eAd ipo cy t e sCompa r edto PBS WT M i c e Th e r ew a snos ign i f i c an td i f f e r en c eb e tw e enth enumb e ro fwh i t ead ipo cy t e sw i th in e a chc e l ls i z eb inb e tw e enco ld a c c l im a t edPACAP WTan im a l st r e a t edw i thPBSo r MT I I (unp a i r edt t e s t ,p>0 .05 )(F igu r e2 .13 ) .How ev e r ,at r endshow edco lda c c l im a t ed MT I I WT m i c eh adh igh e rnumb e r so fsm a l lc e l l s(80900µm )th antho s einco lda c c l im a t edPBS WT m i c e .Th ean a ly s e sw e r eno tcomp l e t edfo rco lda c c l im a t edPBSKOo r MT I IKOan im a l s du etoun exp e c t edh e t e rog en e i tyo fad ipo cy t etyp e s(b rowno rb e ig ead ipo cy t e s )w i th inth e gWATo fco lda c c l im a t edPACAPKOan im a l s(d i s cu s s edb e low ) . Co ldA c c l ima t edPACAPKO M i c eHav eEx t en s iv eB rown ing W i th inTh e i rgWAT D epo t Co lda c c l im a t edPBS WTand MT I I WTd i sp l ay e dhomog en eou sgWATcompo s ed o fch a r a c t e r i s t i cun i lo cu l a rwh i t ead ipo cy t e sw i thi so l a t ed ,sm a l lr eg ion so fm i ldb rown ing (F igu r e2 .14A ,C ) .Incon t r a s t ,h i s to log i c a ls e c t ion so fgWATinco ld a c c l im a t edPBSKO and MT I IKOr ev e a l edex t en s iv emu l t i lo cu l a rad ip o cy t e s(b e ig eo rb rown )w i th inth e WAT s e c t ion s(F igu r e2 .14B ,D ) .B rown ingw a ssoex t en s iv eingWATs amp l e so fa l lco ld a c c l im a t edPACAPKOm i c eex am in ed(n=7 ) ,th a ti tw a sno tpo s s ib l etoob t a inth r e e im ag e scomp r i s edp r edom in an t lyo fun i lo cu l a r ,wh i t ead ipo cy t e s . 2 .4D ISCUSS ION Inr e spon s etoco ldexpo su r e ,l i t e r a tu r eshow sPACAPKOm i c eexh ib i timp a i r ed th e rmog en i cc ap a c i tycomp a r edtoPACAP WTm i c e ,ind i c a t edbybo thphy s io log i c a land mo l e cu l a rd e t r im en t s(Ad am se ta l . ,2008 ;D i anée ta l . ,2014 ;G r aye ta l . ,2001 ;G r aye ta l . , 2002 ) .Phy s io log i c a limp a i rm en t sp r ev iou s lyob s e rv edinco lda c c l im a t edPACAPKOm i c e 67 2 F igu r e2 .13.N umb e ro fc e l l sw i th ine a chc e l la r e ab in(µm )inco ld a c c l im a t edPBS WTo r MT I I WT m i c e.D a t aa r eexp r e s s eda s±SEM.Unp a i r edt t e s t s ,p>0 .05 . 68 PBS WT( 4x ) PBS WT( 20x ) PBSKO(4x ) PBSKO(20x ) MT I I WT(4x ) MT I I WT(20x ) MT I IKO( 4x ) MT I IKO( 20x ) A . B . C . D . 200µm 50µm F igu r e2 .14.R ep r e s en t a t iv eim ag e so fh em a toxy l inandeo s ins t a in edgWATob s e rv eda t4xand20x inco ld a c c l im a t edPBS WT(A ) ,PBSKO(B ) , MT I I WT(C ) ,and MT I IKOm i c e (D ) . 69 anddo cum en t edi nth epub l i sh edl i t e r a tu r ein c lud e :d e c r e a s edad ap t iv eth e rmog en e s i sin r e spon s etoco lds t r e s sa sm e a su r edbyd e c r e a s edNEindu c edoxyg encon sump t ion ,an in ab i l i tytou t i l i z el ip ida sth ep r im a ryfu e lsou r c einr e spon s etosymp a th e t i cs t imu l a t ion ,and d e c r e a s edwh i t ead ipo s et i s su em a s scomp a r edtoPACAP WTm i c e(D i an ée ta l . ,2014 ;G r ay e ta l . ,2002 ) .No t ab ly ,th e s er e su l t sh av eb e enr ep l i c a t edh e r einco ld a c c l im a t edPACAPKO m i c e(PBSKOm i c e ) ,a l thoughno ttoth es am ed eg r e ea sind i c a t edinp r ev iou s lypub l i sh ed s tud i e s ,po s s ib lydu etoth ed i f f e r en tg en e t i cb a ckg roundo fth ePACAPnu l lmou s el in eu s ed b e tw e ens tud i e s(d i s cu s s edb e low )(F igu r e2 .9 ,2 .10 ,and2 .8 ,r e sp e c t iv e ly ) .Fu r th e rmo r e ,in th i ss tudy ,a l lth r e eob s e rv edphy s io log i c a limp a i rm en t sw e r ep a r t i a l lyr e s cu edinco ld a c c l im a t edPACAPKOm i c et r e a t edw i th MT I I(MT I IKOm i c e ) ,a sind i c a t edby in t e rm ed i a t eph eno typ e sinth i sg roupcomp a r edtoco ld a c c l im a t edPBS t r e a t edPACAP WT (PBS WTm i c e )(F igu r e2 .8 ,2 .9 ,and2 .10 ) .E a cho fth e s ef ind ing sw i l lb ed i s cu s s edh e r ein d e t a i l . Impo r t an t ly ,NE indu c edoxyg encon sump t ioni sam e a su r eo fad ap t iv e th e rmog en e s i sinBAT ,ap ro c e s sth a ti simp e r a t iv einm a in t a in inghom eo s t a s i so fbody t emp e r a tu r einr e spon s etoco ldexpo su r eo rf e ed ing(C annon&N ed e rg a a rd ,2004 ; N ed e rg a a rd&L indb e rg ,1979 ) . Wh enBATi sab s en to rfun c t ion a l lyd e f i c i en t ,on e m e ch an i smbywh i chth ebodya t t emp t stocon t ro lbodyt emp e r a tu r ei sv i ask e l e t a lmu s c l e ox id a t ionth roughsh iv e r ingo rin c r e a s edphy s i c a la c t iv i ty(G rubb&Fo lk ,197 6 ;S ch a e f f e r , V i l l a r in ,&L ind s t ed t ,2003 ;Sh io t a& M a sum i ,1988 ) .Th e r e fo r e ,ino rd e rtoen su r eth e m e a su r em en t so fNEindu c edoxyg encon sump t ionr e f l e c t edth eth e rmog en i cc ap a c i tyo f BATtop e r fo rmad ap t iv eth e rmog en e s i s ,r a th e rth anin c r e a s edmu s c l ea c tiv i ty ,oxyg en con sump t ionm e a su r em en t sw e r ep e r fo rm edonan e s th e t i z edan im a l s .Th elow e roxyg en con sump t ionv a lu e sob s e rv edinco ld a c c l im a t edPBSKOm i c ea scomp a r edtoco ld 70 a c c l im a t edPBS WTm i c e(F igu r e2 .9 )th e r e fo r econ f i rm simp a i r edBATa c t iv i tyinP ACAP KOm i c e :ar e su l tp r ev iou s lyob s e rv ed(D i an ee ta l . ,2014 ) .In t e r e s t ing ly ,NEindu c edoxyg en con sump t ionm e a su r em en t sin c r e a s edinco ld a c c l im a t ed MT I IKOm i c ea sth eyd i sp l ay ed oxyg encon sump t ionl ev e l sh igh e rth antho s eo fco ld a c c l im a t edPBSKOm i c e .A l though , co ld a c c l im a t ed MT I IKOm i c ed idno texh ib i timp rov em en t sinad ap t iv eth e rmog en e s i sto l ev e l sob s e rv eding en e t i c a l lyun a l t e r edco ld a c c l im a t edPBS WTm i c e ,anin t e rm ed i a t e ph eno typ ew a sob s e rv ed(F igu r e2 .9 ) .Th i sin t e rm ed i a t eph eno typ ew a so b s e rv edov e rt im e , a sind i c a t edinth eoxyg encon sump t ioncu rv e(F igu r e2 .9A ) ,andw a sfu r th e rsuppo r t edw i th th ec a l cu l a t ed MMR(F igu r e2 .9B ) ,sugg e s t ingap a r t i a lr e s cu eo fth eimp a i r edBATa c t iv i ty inco ld a c c l im a t edPACAPKOm i c ew i th MT I It r e a tm en t . Ap r ev iou s lypub l i sh eds tudyshow edin c r e a s edspon t an eou slo como to ra c t iv i tyin PACAPKOm i c ecomp a r edtocon t ro l WTm i c e ,wh i l eo th e rs tud i e sshow edin c r e a s ed sk e l e t a lmu s c l ea c t iv i tyandox id a t iv econ sump t ionc ap a c i tyinco ld a c c l im a t edan im a l sw i th ab s en tBAT(G rubb&Fo lk ,1976 ;H a sh imo toe ta l . ,2001 ; Mo l l i c ae ta l . ,2005 ;S ch a e f f e r , V i l l a r in ,&L ind s t ed t ,2003 ;Sh io t a& M a sum i ,1988 ;S imony ane ta l . ,2001 ) .In c r e a s ed sk e l e t a lmu s c l ea c t iv i tyw a sob s e rv edbyin c r e a s edmu s c l et emp e r a tu r ewh e r ec a lo r i e sw e r e p r e sum ab lyd i s s ip a t eda sh e a ten e rgy ,andin c r e a s edNEtu rnov e rtosk e l e t a lmu s c l e ind i c a t ingin c r e a s edsymp a th e t i cn e rv ea c t iv i tytosk e l e t a lmu s c l e(Nogu e i r a se ta l . ,2007 ) . Th e r e fo r e ,in c r e a s edsk e l e t a lmu s c l ea c t iv i tym ayb eam e ch an i smbywh i chPACAPKO m i c ead ap ttosu rv iv eco ldexpo su r ewh ena f f ron t edw i thimp a i r edad ap t iv eth e rmog en e s i s (G rubb&Fo lk ,1976 ;H a sh imo toe ta l . ,2001 ; Mo l l i c ae ta l . ,2005 ;S ch a e f f e r ,V i l l a r in ,& L ind s t ed t ,2003 ;Sh io t a& M a sum i ,1988 ;S imony ane ta l . ,2001 ) . A l t e r ingfu e lu s ag ei sano th e rm e thodo fad ap t a t ionth a ti sr equ i r edfo rar e spon s eto env i ronm en t a ls t r e s so r ssu cha sco ldexpo su r e(H am ane ta l . ,2002 ) .Fu e lsou r c e sin c lud e 71 c a rbohyd r a t e s ,p ro t e in ,andl ip id s(H am ane ta l . ,2002 ) .Du r ingp ro long edco ldexpo su r e , mu s c l econ t r a c t ion sr e c e iv eth em a jo r i tyo fth e i rfu e lf romc a rbohyd r a t e sw i tham ino r con t r ibu t ionf romp ro t e in ,wh e r e a sad ap t iv eth e rmog en e s i sinBATi sfu e l edbyFFA sd e r iv ed f romth eb r e akdowno fin t r a c e l lu l a ro rc i r cu l a t ingt r ig ly c e r id e s(F igu r e1 . 8 )(B ab ae ta l . , 2012 ;H am ane ta l . ,2002 ;Ou e l l e te ta l . ,2012 ) .B e c au s eth eox id a t iono fth e s e m a c ronu t r i en t sp rodu c ed i f f e r en tqu an t i t i e so fCO2,c a l cu l a t ingth er a t iob e tw e enth evo lum e o fCO2andth evo lum eo fO RER )a sm e a su r edbyind i r e c tc a lo r im e t ryi sanind i c a to ro f 2( wh i chsub s t r a t ei sp r e f e r en t i a l lyox id i z edfo rfu e lu t i l i z a t ion(V i r tu e&V id a lPu ig ,2013 ) . RER sr ang ef rom0 .7(pu r el ip idox id a t ion )to1 .0(pu r ec a rbohyd r a t eox id a t ion )(G av in i , Jon e s ,&Nov ak ,2016 ;V i r tu e&V id a l Pu ig ,2013 ) .No rm a l ly ,a sob s e rv edinco ld a c c l im a t edPBS WTcon t ro lm i c e ,RERd rop stow a rd sl ip idu t i l i z a t ion(0 .7 )uponNE s t imu l a t ion ,a sad r en e rg i cs t imu l a t ionp romo t e sl ipo ly s i so ft r ig ly c e r id e stoFFA sin WAT andBAT ,p rov id ingsub s t r a t efo rad ap t iv eth e rmog en e s i sinBAT(F igu r e2 .10 )(D i an ee ta l . , 2014 ) .A sp r ev iou s lypub l i sh edandshownh e r einth e s er e su l t s ,co ld a c c l im a t edPBSKO m i c ef a i l edtod i sp l ayad ropinRERinr e spon s etoNEandin s t e add i sp l ay edacon s i s t en t RERov e rt im ew i thv a lu e sc lo s e rtotho s eind i c a t iv eo fc a rbohyd r a t eu s e(F igu r e2 .10 ) (D i an ee ta l . ,2014 ;N ed e rg a a rd&L indb e rg ,1979 ;Ou e l l e te ta l . ,2012 ) .Th i sa l t e r edRER d i sp l ay edinco lda c c l im a t edPBSKOm i c ecou ldind i c a t eady s fun c t ioninl ip idmob i l i z a t ion f romad ipo s et i s su e ,po s s ib lydu etoad e c r e a s edab i l i tytor e spondtoNE ,wh i chi sknownto indu c el ipo ly s i sin WATandBAT(Chond ron iko l ae ta l . ,2016 ;S a i to ,Yon e sh i ro ,& M a t su sh i t a ,2016 ) . In t e r e s t ing ly ,RERo fco ld a c c l im a t ed MT I IKOm i c efo l low edas im i l a rt r endtoth a t o fco ld a c c l im a t edPBS WTm i c e ,sugg e s t ingap a r t i a lr e s cu eo fth ea l t e r edfu e lu s ag e ob s e rv edinPACAPKOm i c ew i th MT I It r e a tm en t(F igu r e2 .10 ) .Fu r th e rmo r e ,co ld 72 a c c l im a t ed MT I I WTm i c ed i sp l ay edat r endinRERth a td e c r e a s edb eyondv a lu e sob s e rv ed inco ld a c c l im a t edPBS WTm i c e(F igu r e2 .10 ) ;ar e su l tob s e rv edp r ev iou s lyinth el i t e r a tu r e inr a t st r e a t edw i th MT I Ia tth el ev e lo fth eVMN(du r ingbo ths t e adyandin c r e a s edphy s i c a l a c t iv i ty )andcon s i s t en tw i thth em e l ano co r t insy s t em ’ sro l einin c r e a s ingl ip idmob i l i z a t ion (G av in i ,Jon e s ,&Nov ak ,2016 ;Nogu e i r a se ta l . ,2007 ) .Th i sob s e rv a t ionw a sfu r th e r suppo r t edinth eh i s to log i c a lex am in a t iono fgWATwh e r eat r endind i c a t edco ld a c c l im a t ed MT I I WTm i c ed i sp l ay ed h igh e rnumb e r so fsm a l lc e l l s(80 900µm )th antho s einco ld a c c l im a t edPBS WTm i c e(F igu r e2 .13 ) ,sugg e s t ingin c r e a s edl ip idmob i l i z a t ionw i th MT I I t r e a tm en t . PBSKOm i c eh av eb e enshownp r ev iou s lytod i sp l ayd e c r e a s ed WATm a s s ;at r end ob s e rv edh e r einingWAT(nons ign i f i c an t )andgWAT( s ign i f i c an t )(F igu r e2 .8 )(D i an ée t a l . ,2014 ) .Th er edu c ed WATd epo t sinco ld a c c l im a t edPACAPKOm i c em ayb er e l a t edto th eimp a i r edNE indu c edoxyg encon sump t ionob s e rv edinco ld a c c l im a t edPACAPKO m i c e .Inamou s emod e lw i ths ev e r e lyr edu c ed WATm a s s( l ipo a t roph icA Z IP /F1 , d i s cu s s edinCh ap t e r1 ) ,m i c et r e a t edw i thaβ3agon i s td idno td i sp l ayanin c r e a s einoxyg en con sump t ion ,sugg e s t ingak eyd r iv e rfo rNE indu c edth e rmog en e s i sinth eav a i l ab i l i tyo f fu e ls to r e sf romad ipo cy t e s(G av r i lov a ,M a r cu s S amu e l s ,&R ei tm an ,2000 ) .Th e r e fo r e , imp a i rm en t sin WATm ayl e adtoimp a i rm en to fad ap t iv eth e rmog en e s i sinBATo fPACAP KOm i c e ,a sFFA sr e l e a s edf romt r ig ly c e r id es to r e sin WATa r eak eysub s t r a t efo rl ip id ox id a t ioninad ap t iv eth e rmog en e s i s( Chond ron iko l ae ta l . ,2016 ;S a i to ,Yon e sh i ro ,& M a t su sh i t a ,2016) .Suppo r t ingth ein t e rm ed i a t eph eno typ eo fco ld a c c l im a t ed MT I IKOm i c e d i s cu s s edabov e ,t r e a tm en to fco ld a c c l im a t edPACAPKOm i c ew i th MT I Ip a r t i a l lyr e s cu ed th ed e c r e a s edad ipo s et i s su em a s so fco ld a c c l im a t edPACAPKOm i c ecomp a r edtoco lda c c l im a t edPACAP WTm i c e(F igu r e2 .8 ) . 73 Co l l e c t iv e ly ,th e s ephy s io log i c a lr e su l t sshowanin t e rm ed i a t eph eno typ ed i sp l ay edin co ld a c c l im a t edPACAPKOm i c eupona c t iv a t iono fth em e l ano co r t insy s t emw i th MT I I . A c t iv a t iono fth em e l ano co r t insy s t emu s ingth i sapp ro a chd idno tfu l lyr e s to r eth eimp a i r ed phy s io log i c a lr e spon s e so fth eco lda c c l im a t edPACAPKOm i c etono rm a l( a sm e a su r edin con t ro l ,co ld a c c l im a t edPBS WTm i c e )bu tp rov id e sp r e l im in a ryev id en c eth a tsuppo r t sth e hypo th e s i sth a tPACAPa c t sup s t r e amo fth em e l ano co r t insy s t eminr egu l a t ingSNAto s t imu l a t eBATth e rmog en e s i sinr e spon s etoco ldexpo su r e .Fu r th e rmo r e ,th e s eob s e rv a t ion s p rov id eg en e t i csuppo r tfo rth es tudyd e s c r ib edabov eandinCh ap t e r1wh e r eT an id a , Sh in t an i ,&H a sh imo to(2011 )u s edapha rma co log i ca lin t e rv en t ionshow ingth a tPACAP s ign a l ingtoBATdo e sno to c cu rwh ena MC4Ran t agon i s ti su s ed ,thu ssugg e s t ingPACAP a c t sup s t r e amf romth em e l ano co r t insy s t emina c t iv a t ingSNAtos t imu l a t eth e rmog en e s i s . Mo l e cu l a ran a ly s e so fth e rmog en i cg en eexp r e s s ionw e r ep e r fo rm edino rd e rtor e fu t e o rsuppo r tth ep r e l im in a ryphy s io log i c a limp rov em en t sinth e rmog en e s i sob s e rv edinco ld a c c l im a t edPACAPKOm i c ew i th MT I It r e a tm en t .How ev e r ,o fth e28m i c eth a tun d e rw en t Oxym axexp e r im en t s ,13d i ede i th e rdu r ingo rsho r t lya f t e rth eexp e r im en t ,po s s ib lydu eto th ein ab i l i tytoe f f e c t iv e lyr egu l a t ebodyt emp e r a tu r ea f t e rp ro long edan e s th e s i a .A sa con s equ en c eo fth ea cu t ee f f e c t so fNEs t imu l a t iononBAT ,p a r t i cu l a r lyonth eg en e s invo lv edinth em e ch an i smo fad ap t iv eth e rmog en e s i s ,on lyt i s su e sf roman im a l sth a tl iv ed un t i lth ed ayo fd i s s e c t ionw e r eu s edfo rqPCRth e rmog en i cg en eexp r e s s ionan a ly s i s (C annon&N ed e rg a a rd ,2004 ) .Du etoth elows amp l es i z e s(p a r t i cu l a r lyinco ld a c c l im a t ed PBSKO(n=3 )and MT I IKO(n=4 )m i c e ) ,l a rg es t and a rde r ro r(p a r t i cu l a r lyinco lda c c l im a t ed MT I IKOm i c e ) ,andin con s i s t en tr e su l t scomp a r edtop r ev iou s lypub l i sh ed l i t e r a tu r e ,i tw a scon c lud edth a tadd i t ion a ls amp l e sw e r en e c e s s a ryino rd e rtocon f id en t ly comp a r er e su l t so fg en eexp r e s s ionan a ly s i sb e tw e eng roup s(F igu r e2 .11 ) . 74 H i s to log i c a lan a ly s e so fBATandgWATw e r ea l sop e r fo rm edtop rov id efu r th e r ev id en c einsuppo r to roppo s i t iono fth ein t e rm ed i a t eph eno typ ed i sp l ay edinco ld a c c l im a t ed PACAPKOm i c eupona c t iv a t iono fth em e l ano co r t insy s t emw i th MT I I.A l thoughno s ign i f i c an c ew a sob s e rv ed ,h i s to log i c a lan a ly s e so fBATr ev e a l edat r endwh e r ebo thco ld a c c l im a t ed MT I I WTand MT I IKOexh ib i t edg r e a t e rl ip ida r e aandd e c r e a s ednumb e ro f c e l l scomp a r edtoco ld a c c l im a t edPBS WTandPBSKOm i c e(F igu r e2 .12 ) .In i t i a l ly ,th i s r e su l tapp e a r edtob econ t r ad i c to rytop r ed i c t edr e su l t s ,wh e r es t imu l a t ingad ap t iv e th e rmog en e s i sw i th MT I It r e a tm en twou ldb eexp e c t edtod e c r e a s el ip ids to r e sinBAT . How ev e r ,uponfu r th e rinv e s t ig a t iona sd e s c r ib edabov e ,th i sob s e rv a t ionm ayb eexp l a in ed fo rco ld a c c l im a t ed MT I I WTm i c ebyth ea l t e rn a t iv eu s eo fl ip id sf rom WATtofu e l ad ap t iv eth e rmog en e s i sinr e spon s eto MT I It r e a tm en t .Th i sexp l an a t ionw a ssuppo r t edby th ein c r e a s ednumb e ro fsm a l lc e l l s(80 900µm )ingWATo fco lda c c l im a t ed MT I I WTm i c e comp a r edtoco ld a c c l im a t edPBS WTm i c e ,con s i s t en tw i thp r ev iou sl i t e r a tu r e(F igu r e2 .13 ) (Q i ane ta l . ,2013 ) .Th ewh i t ead ipo cy t e sa r e a singWATo fco ld a c c l im a t ed MT I IKOm i c e , how ev e r ,w e r eno tan a ly z eddu eth eex t en s iv eb rown ingth a tw a sob s e rv ed(F igu r e2 .14 ) . C l a s s i c a l ly ,gWATcon t a in shomog en eou swh i t ead ipo cy t e sandfun c t ion sa sam a jo r l ip ids to r ag ed epo tanda sin su l a t ionfo ro rg an s(G i r a l t ,&V i l l a r roy a ,2013 ;v anB e eke ta l . , 2015 ) .A l t e rn a t iv e ly ,th eh e t e rog en e i tyiningWAT(no tan a ly z edh e r e )h a sb e enw e l l do cum en t ed ,wh e r eb e ig ead ipo cy t e sapp e a rw i th inth e WATd epo tinr e spon s etoco ld expo su r e( B a r t e l t ,&H e e r en ,2013 ;Sh ab a l in ae tal . ,2013 ) .Th i ss e l e c t iv eb rown ingin WAT i sthough ttoo c cu rw i th insub cu t an eou s WATd epo t s( su cha singWAT ) ,a sth eya r ec lo s e rto th esu r f a c eo fth ebodyandth e r e fo r eexp e c t edtolo s eh e a tf a s t e rth anin t e rn a l WATd epo t s ( su cha sgWAT )( B a r t e l t ,&H e e ren ,2013 ) .In t e r e s t ing ly ,gWATinbo thco ld a c c l im a t ed PBSKOand MT I IKOm i c ed i sp l ay edex t en s iv eb rown ing ,sugg e s t ingth a tinr e spon s eto 75 imp a i r edth e rmog en e s i sdu r ingco ldexpo su r e ,y e tano th e rm e thodo fad ap t ionm ayex i s t ,in add i t iontoin c r e a s edsk e l e t a lmu s c l ea c t iv i tyanda l t e r edfu e lu s ag ed i s c u s s edabov e . No t ab ly ,th eex t en to fth eob s e rv edb rown ingingWATinco ld a c c l im a t edPACAPKOm i c e h a sno tb e enp r ev iou s lydo cum en t edinth el i t e r a tu r e .B rown ingingWATh a s ,how ev e r ,b e en ob s e rv eduponc en t r a ladm in i s t r a t ion so fthy ro idho rmon e sinth eVMN ,IPin j e c t ion so faβ3 agon i s t ,ands t imu l a t iono fn a t r iu r e t i cp ep t id e sr e c ep to r s(K ime ta l . ,2016 ;M a r t in e z S an ch e z e ta l . ,2016 ;N e in a s te ta l . ,2015 ;W e in e re ta l . ,2016 ) .In t e r e s t ing ly ,th e s ea r eknown r egu l a to r so fBATth e rmog en e s i sd e s c r ib edinF igu r e2 .1 . A l thoughno tqu an t i f i ed ,gWATinco lda c c l im a t ed MT I IKOm i c eapp e a r edto con t a inf ew e rb e ig ead ipo cy t e scomp a r edtoco ld a c c l im a t edPBSKOm i c e ,sugg e s t ingl e s s comp en s a t ionbyb rown ing WATinco l da c c l im a t edPACAPKOm i c ew i th MT I It r e a tm en t (F igu r e2 .13 ) .A l thoughth i sf ind ingoni t sowndo e sno tp rov ideenoughev id en c etosuppo r t th ep a r t i a lr e s cu eo fimp a i r edth e rmog en e s i sinco ld a c c l im a t edPACAPKOm i c ew i th MT I I t r e a tm en t ,i tdo e sp rov id ean o th e rp i e c eo fev id en c etocon t r ibu t etoth ephy s io log i c a lr e su l t s d i s cu s s edabov eth a tsugg e s tPACAPa c t sup s t r e amf romth em e l ano co r t insy s t emin r egu l a t ingSNAtos t imu l a t ead ap t iv eth e rmog en e s i sinr e spon s etoco ldexpo su r e .Fu tu r e qu an t i f i c a t ion so fth eamoun to f“b rown ing ”th roughmo l e cu l a ran a ly s e scou ldfu r th e r suppo r to rr e fu t eth ep a r t i a lr e s cu eo fth eimp a i r edad ap t iv eth e rmog en e s i sinco ld a c c l im a t ed PACAPKOm i c et r e a t edw i th MT I Id i s cu s s edabov e . Impo r t an t ly ,th eg en e t i cb a ckg roundo fth em i c eshou ldb econ s id e r edwh en r e f e r en c ingp r ev iou sl i t e r a tu r etoen ab l eapp rop r i a t ein t e rp r e t a t iono fth eph eno typ i c exp r e s s iono fth esp e c i f i cg en e s( a sm en t ion edabov e ) .Th em i c eu s edinth e s eexp e r im en t s w e r eo fm ix edg en e t i cb a ckg round(C57BL /6and129/ Sv J ) ,wh e r e a sm i c eu s edp r ev iou s lyin th eG r ayl abw e r eo fpu r eg en e t i cb a ckg round(b a ck c ro s s edon toaC57BL /6b a ckg round ) 76 (D i an ée ta l . ,2014 ;G r aye ta l . ,2002 ) .A sar e su l to fex t en s iv einb r e ed ing ,pu r eg en e t i c b a ckg round sa r eg en e t i c a l lyun i fo rm ,wh i cha l low sfo ramo r ea c cu r a t eev a lu a t iono fth e ob s e rv edph eno typ e( r e su l t ingf romg en e t i cm an ipu l a t ion )bye l im in a t ingph eno typ i c v a r i a t ion sc au s edbym ix edg en e t i cb a ckg round s(Yo sh ik i& Mo r iw ak i ,2006 ) .A ssu ch ,m i c e f rompu r eg en e t i cb a ckg round sa r etyp i c a l lyth ego lds t and a rdu s edint r an sg en i cr e s e a r ch , wh i chi swhyth eyw e r ein i t i a l lyu s edinth eG r ayl ab .How ev e r ,du etoth ecomp l ex i tyo fth e PACAPKOph eno typ eth a tr e su l t edinh ighmo r t a l i tyr a t e s ,an im a lc a r eandf in an c i a l con c e rn sw e r econ s id e r ed :b e c au s eal a rg enumb e ro fan im a l sw e r er equ i r edtop rov id e su f f i c i en tan im a lnumb e r sfo rrobu s texp e r im en t sandan a ly s e s ,th ed e c i s iontou s em i c ew i th am ix edg en e t i cb a ckg roundino rd e rtoimp rov ePACAPKOpupsu rv iv a landr edu c eco lony numb e r sw a sm ad e . An im a l sonapu r eg en e t i cb a ckg roundo f t end i sp l ayamo r es ev e r eph eno typ e comp a r edtotho s eo fm ix edg en e t i cb a ckg roundth a td i sp l ayg r e a t e rv a r i ab i l i tyinth e ph eno typ e :ar e su l tth a tw a sob s e rv edh e r ewh encomp a r ingr e su l t sd e r iv edinC57BL /6 129 /Sv JPACAPKOm i c etop r ev iou sl i t e r a tu r e ,th a tu s edpu r eC57BL /6PACAPKOm i c e (Do e t s chm an ,2009 ;Yo sh ik i& Mo r iw ak i ,2006 ) .Th i sph enom enoni sp a r t i cu l a r lyp r ev a l en t inm i c ew i tht a rg e t edmu t a t ion s(KOan im a l s )andm ayb edu etomod i f i e rg en e sth a t in f lu en c eg en eexp r e s s ionth roughsupp r e s s iono renh an c em en t ,a l t e r a t iono fDNA t r an s c r ip t ion ,andep ig en e t i ce f f e c t ssu cha sDNAm e thy l a t ion(Do e t s chm an ,2009 ;J a ck son L abo r a to ry ,2006 ;Yo sh ik i& Mo r iw ak i ,2006 ) .Th e r e fo r e ,PACAPKOm i c eo fm ix ed g en e t i cb a ckg roundu s edh e r em ayh av ecomp en s a to ryphy s io log i c a lm e ch an i sm sinr e spon s e toco ldexpo su r ewh encomp a r edtog en e t i c a l lypu r ePACAPKOm i c e ,l e ad ingtoal e s s s ev e r eph eno typ e . 77 Fu r th e rmo r e ,con t rov e r syex i s t sa stowh e th e r MT I Ii sc ap ab l eo fc ro s s ingth eb lood br a inb a r r i e r(BBB ) ,andi ti simp e r a t iv etot ak eth i sin tocon s id e r a t iona sw esugg e s tPACAP in t e r a c t sw i thth em e l ano co r t insy s t emc en t ra l l yinth eVMNo fth ehypo th a l amu s(wh e r e bo thPACAPandm e l ano co r t in sa r eexp r e s s ed )wh enou rm e thodo log i c a lapp ro a chcon s i s t ed o fp e r iph e ra lMT I Iin j e c t ion s(G l av a se ta l . ,2007 ;K ime ta l . ,2000 ; Mod ie ta l . ,2015 ; P igg in se ta l . ,1996 ;T r iv ed ie ta l . ,2003 ) .No t ab ly ,inadd i t iontor e c ep to r slo c a t edinth e b r a in , MC4RmRNA sh av eb e end e t e c t edinp e r iph e r a lt i s su e sin c lud ingsk e l e t a lmu s c l eand ad ipo s et i s su e ,sugg e s t ingbo thc en t r a landp e r iph e r a la c t iono fth em e l ano co r t insy s t em (Bo s ton&Con e ,1996 ;Ch agnone ta l . ,1997 ;Hogg a rde ta l . ,2004 ; Moun t joye ta l . ,2003 ) . In t e r e s t ing ly ,wh e th e radm in i s t e r edc en t r a l lyo rp e r iph e r a l ly , MT I Ie l i c i t sinh ib i to rye f f e c t s onfoodin t ak eandex c i t a to rye f f e c t sonen e rgyexp end i tu r e ,sugg e s t ingth a te i th e rb r a in p en e t r a t ioni sno tr equ i r edfo r MC4Ragon i s t ssu cha s MT I Itosupp r e s sfoodin t ak eand in c r e a s een e rgyexp end i tu r e ,o r ,m o r el ik e ly ,th a tth eBBBi sl e aky ,a l low ing MC4Ragon i s t s toc ro s sa tc e r t a ina r e a sanda c tonc en t r a lsy s t em s(G av in i ,Jon e s ,&Nov ak ,2016 ;G l av a se t a l . ,2007 ;H ag an ,Bo lon ,&K e en e ,2012 ;K i ev i te ta l . ,2012 ;T r iv ed ie ta l . ,2003 ) .Tofu r th e r e lu c id a t ehypo th a l am i cp a thw ay sb e tw e enPACAPandth em e l ano co r t insy s t emina c t iv a t ing BATth e rmog en e s i s ,th e r e fo r e ,c en t r a ladm in i s t r a t iono f MT I Iinco lda c c l im a t edPACAP KOm i c ecou ldb ep e r fo rm ed .Th i s ,how ev e r ,f i r s tr e l i e sonth eknow l edg eo fwh i ch hypo th a l am i cb r a inr eg ionPACAPa c t stor egu l a t eth e rmog en e s i s .Th i si sth em a jo rfo cu so f Ch ap t e r3andw i l lb ed i s cu s s edind e t a i l . Incon c lu s ion ,a c t iv a t iono fth em e l ano co r t insy s t emw i thp e r iph e r a lin j e c t ion so f MT I Ip a r t i a l lyr e s cu e sth eimp a i r edth e rmog en es i so fco ld a c c l im a t edPACAPKOm i c e , p rov id ingp r e l im in a ryev id en c etosuppo r tth ehypo th e s i sth a tPACAPa c t sup s t r e amf romth e m e l ano co r t insy s t eminr egu l a t ingSNAtoBATtoindu c ead ap t iv eth e rmog en e s i sdu r ing 78 co ldexpo su r e .Fu tu r ed i r e c t ion so fth i sp ro j e c tin c lud ein c r e a s ings amp l es i z etomo r e robu s t lym e a su r eg en eandp ro t e inexp r e s s iono fth e rmog en i cp ro t e in sinBAT ,ingWAT ,and gWATd epo t s .Add i t ion a l ly ,fu r th e rh i s to log i c a lan a ly s e so fad ipo s et i s su e stoid en t i fy ch a r a c t e r i s t i c so fth e rmog en i c a l l ya c t iv ead ipo s et i s su e ,su cha sm e a su r ingl ip idcon t en tand b rown inginingWATandgWATd epo t sw i l lb ep e r fo rm ed .Th e s ean a ly s e sw i l lh e lpsuppo r t o rr e fu t eth ep a r t i a lphy s io log i c a lr e s cu eo fad ap t iv eth e rmog en e s i sw i th MT I It r e a tm en to f co ld a c c l im a t edPACAPKOm i c ed e s c r ib edh e r e . 79 2 .5REFERENCES Ad am s ,B . ,G r ay ,S . ,I s a a c ,E . ,B i an co ,A . ,V id a lPu ig ,A . ,&Sh e rwood ,N .(2008 ) .F e ed ing and M e t abo l i smin M i c eL a ck ingP i tu i t a ryAd eny l a t eCy c l a s e A c t iv a t ing Po lyp ep t id e .Endo c r ino log y,149 ( 4 ) ,15711580 .h t tp : / /dx .do i .o rg /10 .1210 / en .2007 0515 A lOb e id i ,F . ,C a s t ru c c i ,A . ,H ad l ey ,M . ,&H ruby ,V .(1989 ) .Po t en tandp ro long eda c t ing cy c l i cl a c t aman a log so f. a lph a . m e l ano t rop in :d e s ignb a s edonmo l e cu l a r rna lO fM ed i c ina lCh em i s t r y ,32(12 ) ,2555 2561 . dyn am i c s .Jou h t tp : / /dx .do i .o rg /10 .1021 / jm00132 a010 B ab a ,S . ,J a c en e ,H . ,Eng l e s ,J . ,Hond a ,H . ,& W ah l ,R .(2010 ) .CTHoun s f i e ldUn i t so f B rownAd ipo s eT i s su eIn c r e a s ew i thA c t iv a t ion :P r e c l in i c a landC l in i c a l S tud i e s .Jou rna lO fNu c l ea rM ed i c in e ,51(2 ) ,246 250 . h t tp : / /dx .do i .o rg /10 .2967 / jnum ed .109 .068775 B a r tn e s s ,T . ,V augh an ,C . ,&Song ,C .(2010 ) .Symp a th e t i cands en so ryinn e rv a t iono fb rown ad ipo s et i s su e .In tJOb e sR e la tM e tabD i so rd ,34,S36 S42 . h t tp : / /dx .do i .o rg /10 .1038 / i jo .2010 .182 B e rg lund ,E . ,L iu ,T . ,Kong ,X . ,Sohn ,J . ,Vong ,L . ,&D eng ,Z .e ta l .(2014 ) .M e l ano co r t in4 r e c ep to r sinau tonom i cn eu ron sr egu l a t eth e rmog en e s i sandg ly c em i a .Na tu r e N eu ro s c i en c e,17(7 ) ,911 913 .h t tp : / /dx .do i .o rg /10 .1038 /nn .3737 Bo s ton ,B . ,&Con e ,R .(1996) .Ch a r a c t e r i z a t iono fm e l ano co r t inr e c ep to rsub typ eexp r e s s ion inmu r in ead ipo s et i s su e sandinth e3T3 L1c e l ll in e .Endo c r ino log y,137(5 ) ,2043 2050 .h t tp : / /dx .do i .o rg /10 .1210 / endo .137 .5 .8612546 B r i to ,M . ,B r i to ,N . ,B a ro ,D . ,Song ,C . ,&B a r tn e s s ,T .(2007 ) .D i f f e r en t i a lA c t iv a t iono fth e Symp a th e t i cInn e rv a t iono fAd ipo s eT i s su e sby M e l ano co r t inR e c ep to r S t imu l a t ion .Endo c r ino log y,148 (11 ) ,5339 5347 .h t tp : / /dx .do i .o rg /10 .1210 / en .2007 0621 Bu s t in ,S . ,B en e s ,V . ,G a r son ,J . ,H e l l em an s ,J . ,Hugg e t t ,J . ,&Kub i s t a ,M .e ta l .(2009 ) .Th e M IQEGu id e l in e s :M in imumIn fo rm a t ionfo rPub l i c a t iono fQu an t i t a t iv eR e a lT im e PCRExp e r im en t s .C l in i ca lCh em i s t r y,55(4 ) ,611 622 . h t tp : / /dx .do i .o rg /10 .1373 / c l in ch em .2008 .112797 Bu t l e r ,A .&Con e ,R .(2003 ) .Kno ckou tS tud ie sD e f in ingD i f f e r en tRo l e sfo rM e l ano co r t in R e c ep to r sinEn e rgyHom eo s t a s i s .Anna l sO fTh eN ewYo r kA cad em yO f ,994(1 ) ,240 245 .h t tp : / /dx .do i .o rg /10 .1111 / j .17496632 .2003 . tb03186 .x S c i en c e s Bu t l e r ,A . ,M a rk s ,D . ,F an ,W . ,Kuhn ,C . ,B a r to lom e ,M . ,&Con e ,R .(2001 ) .M e l ano co r t in4 r e c ep to ri sr equ i r edfo ra cu t ehom eo s t a t i cr e spon s e stoin c r e a s edd i e t a ryf a t .Na tu r e N eu ro s c i en c e,4(6 ) ,605 611 .h t tp : / /dx .do i .o rg /10 .1038 /88423 C annon ,B . ,&N ed e rg a a rd ,J .(2004 ) .B rownAd ipo s eT i s su e :Fun c t ionandPhy s io log ic a l y s io log i ca lR e v i ew s,84(1 ) ,277 359 . 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Mou s ePh enom eR e s e a r ch :Imp l i c a t ion so fG en e t i c B a ckg round .ILARJou rna l ,47 (2 ) ,94 102 .h t tp : / /dx .do i .o rg /10 .1093 / i l a r .47 .2 .94 88 2 .6APPEND IX 950bp 550bp F igu r eS2 .1.PCRas s e s sm en to fPACAPg eno typ einMu smu s cu lu s.E l e c t ropho r e s i sp e r fo rm edon e th id iumb rom id es t a in ed1 .5%ag a ro s eg e la t100Vfo r2 0m inu t e s .E a chw e l lr ep r e s en t sad i f f e r en t an im a l .(+ /+ )d eno t e sPACAPw i ld typ e ,(+ / )PACAPh e t e ro zygo t e ,and( / )PACAPkno ckou t . W e l l s 28S 18S W e l l s 28S 18S F igu reS2 .2 .A s s e s sm en to fRNAin t eg r i tyo fi so l a t edMu smu s cu lu sb rownad ipo s et i s su e(BAT ) w i the l e c t ropho r e s i sonane th id iumb rom id es t a in ed1 .5%ag a ro s eg e la t100Vfo r30 m inu t e s .E a ch w e l lr ep r e s en t sad i f f e r en tBATs amp l e .Th etopb andind i c a t e sin t a c t28SrRNAandth ebo t tomb and ind i c a t e sin t a c t18SrRNA . 89 CHAPTER3 C r ea t ingaV i ra lCon s t ru c tandD e l iv e ry M e thodfo rG en e t i cIn t e rv en t ionA im eda t R e s to r ingImpa i r edTh e rmog en e s i so fPACAP/ -M i c e 90 3 .1INTRODUCT ION A sd i s cu s s edind e t a i linCh ap t e r2 ,PACAPi se s s en t i a lfo rth eno rm a lth e rmog en i c r e spon s eo fBATa sind i c a t edbylo s sandg a ino ffun c t ionexp e r im en t su s ingbo thg en e t i c andph a rm a co log i c a lin t e rv en t ion s( Ad am se ta l . ,2008 ;Cumm ing s ,P end l ebu ry ,Sh e rwood , &W i l son ,2004 ;D i anée ta l . ,2014 ;G r aye ta l . ,2001 ;G r aye ta l . ,2002 ;Ing lo t t ,F a rnh am ,& P i low sky ,2011 ;R e s che ta l . ,2011 ;R e s che ta l . ,2013 ;T an id ae ta l . ,2010 ) .Th em e ch an i sm s bywh i chPACAPr egu l a t e sbodyt emp e r a tu r eandthu sen e rgym e t abo l i sm ,how ev e r ,r em a in unknown .B e c au s eth eg r e a t e s tphy s io log i c a ls t imu lu so fad ap t iv eth e rmog en e s i sinBATi s symp a th e t i cn e rv ea c t iv a t ionin i t i a t eda tth el ev e lo fth ehypo th a l amu s(d i s cu s s edb e low ) ,th e ev id en c efo rPACAP ’ sc en t r a lro l einth eb r a inw i l lb er ev i ew edh e r e ,w i thth efu tu r ea imo f e lu c id a t ingth esp e c i f i chypo th a l am i cm e ch an i sm sbywh i chPACAPa c t stor egu l a t e th e rmog en e s i s(B am sh ad ,Song ,&B a r tn e s s ,1999 ;B a r tn e s s ,V aughn ,&Song ,2010 ;Ch e ch i , C a rp en t i e r ,&R i ch a rd,2013 ;L abb ée ta l . ,2015 ;S a i to ,Yon e sh i ro ,& M a t su sh i t a ,2016 ) . B r i e f lyin t rodu c edinCh ap t e r1 ,th ehypo th a l amu si scompo s edo fs ev e r a ld i s t in c t nu c l e i ,in c lud ingth eARC(d i s cu s s edinCh ap t e r1 ) ,DMN ,VMN ,LHAandPVN(F igu r e 1 .5 ) ,andco l l e c t iv e lyin t eg r a t e sbo thc en t r a landp e r iph e r a ls i gn a l so fbodyen e rgys t a tu sto r egu l a t efoodin t ak eanden e rgyexp end i tu r etoapp rop r i a t e lym a in t a inbodyw e igh tand en e rgyhom eo s t a s i s(R e a ,2015 ) . Wh i l ePACAPandPACAPr e c ep to r sa r eexp r e s s edine a ch o fth enu c l e in am edabov e ,s ign i f i c an t lyh igh e rexp r e s s ionl ev e l so c cu rinth eVMNandth e PVN(A r imu r ae ta l . ,1991 ;D a s ,V ih l en ,&L eg r ad i ,2006 ;H ann ib a le ta l . ,2002 ;H a sh imo to e ta l . ,1996 ;Jo l iv e le ta l . ,2009 ;M a suoe ta l . ,1993 ;S eg a le ta l . ,2005 ;Sh ew a rd ,Lu t z ,Copp , &H a rm a r ,1998 ) .A l thoughth efun c t ion a lro l eo fc e r t a inhypo th a l am i cnu c l e ia r ew e l l known( su cha sapp e t i t er egu l a t ioninth eARC ) ,o th e r sh av eov e r l app ingandev enoppo s ing fun c t ion s ,m ak ingi tch a l l eng ingtod i s c e rnth ep a thw ay sr egu l a t ingen e rgyhom eo s t a s i s .Fo r 91 ex amp l e ,d i s inh ib i t ingn eu ron so fth eDMNo rth eLHAw i thaGABAr e c ep to ran t agon i s t r e su l t edinin c r e a s edth e rmog en e s i sa sm e a su r edbyin c r e a s edco r eandBATt emp e r a tu r e , sugg e s t ingth a ta c t iv a t iono fth e s enu c l e ir e su l t sinin c r e a s eden e rgyexp end i tu r e(C ao ,F an , & Mo r r i son ,2004 ;C e r r i ,& Mo r r i son ,2005 ;D iM i c co ,&Z a r e t sky ,2006 ; Mo r r i son ,& N ak amu r a ,2011 ;Z a r e t sk a i a ,Z a r e t sky ,Sh ekh a r ,&D iM i c co ,2000 ) .Add i t ion a l ly ,d i s rup t ion o fl ep t inr e c ep to r sinth eDMNb lo ck edth e rmog en i cr e spon s e stop e r iph e r a ll ep t in ,r e su l t ing inob e s i ty(Dodde ta l . ,2014 ;En r io r ie ta l . ,2011 ) .Conv e r s e ly ,how ev e r ,l e s ion stoth eDMN o rLHAr e su l t edinhypoph ag i aandr edu c edbodyw e igh t ,sugg e s t ingth a tth ero l eo fth e s e nu c l e iinen e rgyexp end i tu r em ayb einh ib i to ryr a th e rth anex c i t a to ry ,an dth a tmu l t ip l e p a thw ay sa r einvo lv edinr egu l a t ingen e rgyhom eo s t a s i s ;aph enom enonth a tw i l lb efu r th e r d i s cu s s edb e low(B e l l ing e r ,&B e rn a rd i s ,2002 ;Yo sh id a ,K emn i t z ,&B r ay ,1983 ) . O th e rhypo th a l am i cnu c l e ia r ea l soimp l i c a t edinr egu l a t ingen e rgyhom eo s t a s i s . A c t iv a t iono fth em e l ano co r t insy s t emv i ain j e c t ion so fth esyn th e t i cagon i s t MT I Iin tobo th th ePVNandth eVMNr e su l t edinin c r e a s edBATt emp e r a tu r e sv i ath e rmog en e s i s(G av in i , Jon e s ,&Nov ak ,2016 ;Songe ta l . ,2008 ) .Add i t ion a l ly ,bo thho rmo n a lande l e c t r i c a l s t imu l a t iono fth ePVNandth eVMNr e su l t edind e c r e a s edfoodin t ak e ,wh i l elo s so f fun c t iono fe i th e rinth e s enu c l e i(v i afun c t ion a ld i s rup t ion sbytox in s ,o re l e c t ro ly t i co r ch em i c a ll e s ion s )r e su l t edinhyp e rph ag i aandin c r e a s edbodyw e igh t ,l e ad ingtoob e s i ty (Cho i ,&D a l lm an ,1999 ;L e ibow i t z ,H amm e r ,&Ch ang ,1981 ;P e rk in se ta l . ,1981 ;R e s che t a l . ,2011 ;R e s che ta l . ,2013 ;S ab a t i e r ,L eng ,& M en z i e s ,2013 ) .Bo thnu c l e ia r e ,th e r e fo r e , common lya s so c i a t edw i ths a t i e tyanda r eknowntob eimpo r t an tnu c l e ifo rth er egu l a t iono f en e rgym e t abo l i sm(P e rk in se ta l . ,1981 ;S ab a t i e r ,L eng ,& M en z i e s ,2013 ;S eg a le ta l . , 2005 ) . 92 Insuppo r to fth el i t e r a tu r eth a tind i c a t e sPACAPi sinvo lv edinth er egu l a t iono f en e rgym e t abo l i sm(d e t a i l edinCh ap t e r2 ) ,s i t esp e c i f i cPACAPin j e c t ion sin toth ePVNand th eVMNs ign i f i c an t lyd e c r e a s edfoodin t ak e ,wh i chw a sr ev e r s edw i thaPACAPr e c ep to r an t agon i s t(R e s che ta l . ,2013 ) .No t ab ly ,how ev e r ,PACAPin j e c t ion sin toth eVMNr e su l t ed ins ign i f i c an t lyin c r e a s edco r ebodyt emp e r a tu r eandspon t an eou slo como to ra c t iv i ty , wh e r e a sPVNin j e c t ion so fPACAPh adnoe f f e c tonen e rgyexp end i tu r e(R e s che ta l . ,2013 ) . Th i ssugg e s t sth a td e sp i t es im i l a rr egu l a t iono fen e rgyin t ak euponPACAPs t imu l a t ioninth e PVNandVMN ,th e s enu c l e ia r ed i f f e r en t i a l lyr e spon s iv etoPACAPint e rm so fen e rgy exp end i tu r e(R e s che ta l . ,2013 ) .Fu r th e rmo r e ,incon t r a s ttoin c r e a s edBATt emp e r a tu r e s ob s e rv edinan im a l sr e c e iv ingin j e c t ion so fan MC4Ragon i s t(MT I I )inth ePVN ,th e r e in t rod u c t iono f MC4Rexp r e s s ioninth ePVNo f MC4RKOm i c ed idno tr e su l tinin c r e a s ed th e rmog en e s i s(B a l th a s a re ta l . ,2005 ;Songe ta l . ,2008 ) .No t ab ly , MC4RKOm i c ed i sp l aya mo rb id lyob e s eph eno typ e ,r e su l t ingf romin c r e a s edapp e t i t eandd e c r e a s eden e rgy exp end i tu r e(B a l th a s a re ta l . ,2005 ) .Th er e s to r a t iono f MC4Rexp r e s s ioninth ePVNo f MC4RKOm i c ep r ev en t ed60%o fth eob e s i ty ,wh e r eth ein c r e a s edfoodin t ak ew a s comp l e t e lyr e s cu ed ,wh i l eth er edu c eden e rgyexp end i tu r ew a sno t(B a l th a s a re ta l . ,2005 ; M add en ,& Mo r r i son ,2008 ) .Th i ssugg e s t sth a t ,s im i l a rtoth ed i f f e r en t i a lr e spon s eto PACAPinth ePVNandVMNd i s cu s s edabov e , MC4R sinth ePVNcon t ro lfoodin t ak e wh i l e MC4R se l s ewh e r econ t ro len e rgyexp end i tu r e(B a l th a s a re ta l . ,2005 ) .Po t en t i a l ly ,th i s r egu l a t iono fen e rgyexp end i tu r eby MC4R sm ayo c cu rinth eVMN ,po s s ib lyund e rth e r egu l a t iono fPACAP( th efo cu so fCh ap t e r2 ) . Wh i l eth eVMNexh ib i t sam a jo rro l einapp e t i t er egu l a t ion ,i ti sa l sor e cogn i z edfo r i t sro l einen e rgyexp end i tu r e ,in c lud in gth e rmog en e s i s ,andcon t inu e stob eas i t eo fin t e r e s t fo rbodyw e igh tr egu l a t ion(Cho ie ta l . ,2013 ;Lop e z ,&T en a S emp e r e ,2017 ;M a r t in e zd e 93 Mo r en t ine ta l . ,2014 ;P e rk in se ta l . ,1981 ; Wh i t t l ee ta l . ,2012 ) .I tcon t a in snum e rou s r e c ep to rtyp e sknowntor egu l a t een e rgym e t abo l i sm ,in c lud ingh ighexp r e s s iono fl ep t inand PACAPr e c ep to r s(B ingh ame ta l . ,2008 ;Dh i l lone ta l . ,2006 ;H awk ee ta l . ,2009 ;S eg a le t a l . ,2005 ) .A sd i s cu s s edinCh ap t e r1 ,l ep t in ’ sfun c t ioninen e rgym e t abo l i smi sno tso l e lyin ap p e t i t er egu l a t ioninth eARC :l ep t ina l sor egu l a t e sen e rgyexp end i tu r em a in lyv i ath eVMN (B ingh ame ta l . ,2008 ) .A ssu ch ,wh enl ep t inr e c ep to r sw e r ekno ck edou tinth eVMNon ly , m i c eexh ib i t eds ign i f i c an t lyin c r e a s edad ipo s em a s sonalowf a td i e t ,andob e s i tyonah igh f a td i e t(B ingh ame ta l . ,2008 ) .L ep t infun c t ioni sa l soconn e c t edtoth a to fPACAP ,wh e r eby l ep t in d e f i c i en tm i c eexh ib i t eds ign i f i c an t lyr edu c edPACAPmRNAl ev e l sth a tw e r e r e s to r edw i thexog enou sl ep t int r e a tm en t s(H awk ee ta l . ,2009 ) .A dd i t ion a l ly ,l ep t ina c t sby m e an so fs t e ro idog en i cf a c to r1(SF1 ) ,wh e r el ep t ind epo l a r i z a t ionr e su l t sinin c r e a s edf i r ing r a t eo fSF1n eu ron sinth eVMN(Dh i l lone ta l . ,2006 ;H awk ee ta l . ,2009 ) .Th eimpo r t an c e o fSF1inr egu l a t ingg en eexp r e s s ioninth eVMNw i l lb ed i s cu s s edh e r e . SF1i sat r an s c r ip t ion a lf a c to rr equ i r edbo thfo rt e rm in a ld i f f e r en t i a t iono fth eVMN a sw e l la sen e rgym e t abo l i smp ro c e s s e s(Cho ie ta l . ,2013 ) .No t ab ly ,o fa l lth eg en e s exp r e s s edinth eVMN ,SF1i sth eon lyg en eth a ti ssp e c i f i c a l lyandex c lu s iv e lyexp r e s s edin th eVMN(Cho ie ta l . ,2013 ) .SF1exp r e s s iona l soo c cu r sinth ep i tu i t a ryg l and ,ad r en a l g l and sandth et e s t e s ,bu tinnoo th e rb r a inr eg ion( Ik ed ae ta l . ,1995 ;Ing r ah ame ta l . ,1994 ; P a rk e r ,&S ch imm e r ,1997 ) .InSF1KOan im a l s ,imp a i r edd ev e lopm en to fth eVMNen su ed wh i l eo th e rhypo th a l am i cnu c l e ir em a in edin t a c t ,u l t im a t e lyr e su l t inginh ighd e a thr a t e s l ik e lya t t r ibu t ab l etoad r en a lin su f f i c i en cy(D e l lov ad ee ta l . ,2000 ;Ik ed ae ta l . ,1995 ;Luo , Ik ed a ,&P a rk e r ,199 4 ) .Toc i r cumv en tth e s ed e a th s ,h e a l thyad r en a lg l and sw e r et r an sp l an t ed in toSF1KOm i c e ,r e su l t inginacomp l e t er e s cu eo fth ead r en a lph eno typ ewh i l ea l low ing fo rth ean a ly s i so fp r edom in an t lyVMNSF1KOe f f e c t s(M a jd i ce ta l . ,2002 ) .In t e r e s t ing ly , 94 ad r en a l t r an sp l an t edSF1KOm i c eexh ib i t edm a s s iv eob e s i tycomp a r edto WT s ,sugg e s t ing th a tth eexp r e s s iono fSF1inth eVMNi sc r i t i c a linr egu l a t ingbodyw e igh t(M a jd i ce ta l . , 2002 ) .How ev e r ,th eob e s eph eno typ em ayh av er e su l t eda sacon s equ en c eo fth eg r o s s ly imp a i r edVMNs t ru c tu r e ,th einh e r en ts e cond a rye f f e c t sf romth et r an sp l an t a t iono ra comb in a t iono fbo th ,r a th e rth anth eab s en c eo fSF1s ign a l inginth eVMN .Th e r e fo r e ,mou s e mod e l sw e r ed ev e lop edu s ingth eC r et r an sg en ewh e r eSF1w a sd e l e t edpo s tn at a l lytoa l low fo rp rop e rd ev e lopm en to fth eVMNandthu savo idcon found ingd ev e lopm en ts id ee f f e c t s (B ingh am ,V e rm a Ku rv a r i ,P a r ad a ,&P a rk e r ,2006 ;K ime ta l . ,2011 ) .In t e r e s t ing ly ,th e s e VMNsp e c i f i cSF1KOan im a l scon t inu edtoexh ib i tanob e s eph eno type ,w i thimp a i r ed th e rmog en e s i sandd e c r e a s edl ep t inr e c ep to rexp r e s s ioninth eVMN(K ime ta l . ,2011 ) . Co l l e c t iv e ly ,th e s er e su l t sp rov id ecomp e l l ingev id en c eth a tSF1exp r e s s ioninth eVMNi s imp e r a t iv efo rr egu l a t ingen e rgyhom eo s t a s i s ,in c lud ingth e rmog en e s i s . A l tog e th e r ,acon s id e r ab l eamoun to fev id en c econn e c t sPACAPandth eVMNin th e i re s s en t i a lro l e sina c t iv a t ingth e rmog en e s i s .Th ef in a lp i e c eo fev id en c etocon s id e ri sth e hypo th a l am i cinn e rv a t iontoBAT ,a sth i si sth es i t eo fad ap t iv eth e rmog e n e s i s(C annon ,& N ed e rg a a rd ,2004 ) . N eu ron a lp a thw ay sm ed i a t inghypo th a l am i cr egu l a t iono fad ap t iv eth e rmog en e s i sin BATh av eb e ene lu c id a t edw i thth eu s eo fr e t rog r ad ev i r a lt r a c e r s . Wh ensu chv i ru s e sa r e in j e c t edin toBAT ,th eyin f e c tands imu l t an eou s lyl ab e ln eu ron s( th roughexp r e s s iono fa mo l e cu l a rl ab e l )inar e t rog r ad ef a sh ion ,su chth a tth een t i r ee f f e r en tch a ini sl ab e l edf romth e p e r iph e r a lt i s su etoth eb r a inr eg ion sf romwh i chth es ign a lo r ig in a t ed .In t e r e s t ing ly , r e t rog r ad ev i r a lt r a c ings tud i e sinBATh av eshownconn e c t ion stoth ePVN ,LHA ,DMN , andARC ,bu tno tth eVMN(B am sh ad ,Song ,&B a r tn e s s ,1999 ;B a r tn e s s ,V aughn ,&Song , 2010 ;O ld f i e lde ta l . ,2002 ;Ryue ta l . ,2015 ) .S im i l a r ly ,du r ingsho r tt e rmco ldexpo su r e 95 ( fou rhou r s ) ,v i r a l ly in f e c t edn eu ron sw e r eob s e rv edinth ePVN ,LHAandDMN ,bu tag a in , no tth eVMN(C anoe ta l . ,2003 ) .Th econ sp i cuou sab s en c eo fv i r a lt r a c inginth eVMNi sa t odd sw i thfun c t ion a lev id en c ed i s cu s s edabov eth a tc l e a r lyd emon s t r a t e saro l efo rth eVMN inen e rgye xp end i tu r e .Th i sm ayb edu etoth el im i t a t ion so fv i r a lt r a c ings tud i e s ,wh i cha r e g en e r a l lyl im i t edtoth r e esyn ap t i cr e l ay s(S t e f an id i s ,W i edm ann ,Ad l e r ,&O ld f i e ld ,2014 ) . Th e r e fo r e ,i fth eVMNi sth ein i t i a lnu c l e iinwh i chth e rmog en e s i si sa c t iv a t ed ,f romwh i ch n eu ron sp ro j e c ttoo th e rnu c l e i ,th eVMNm ayb eb eyondth eex t en to fp en e t r a t iono fth e r e t rog r ad ev i r a lt r a c e r sinth eCNS .A l t e rn a t iv e ly ,fun c t ion a ls tud i e sth a timp l i c a t eth eVMN m aya c tu a l lyb einvo lv ingn e a rbyr eg ion s ,r e su l t ingincomp rom is edan a ly s i s .Fo rex amp l e , ch em i c a ll e s ion so fth eVMNl ik e lyd am ag e ssu r round ingr eg ion s ,wh i l em i c ro in j e c t ion sin to th eVMNm ayr e su l tinl e ak ag eou t s id eth et a rg e t eda r e a(Cho ie ta l . ,2013 ) .How ev e r , b e c au s eSF1i sex c lu s iv e lyexp r e s s edinth eVMN ,th eev id en c ed i s cu s s edabov efo rSF1 ’ s , andthu sth eVMN ’ s ,ro l einen e rgym e t abo l i smi sindub i t ab l e(B ingh ame ta l . ,2008 ) . N ev e r th e l e s s ,a l thoughcomp l exandpo t en t i a l lycon t r a s t ingev id en c eex i s t sfo rth e hypo th a l am i cr eg ion sr egu l a t ingen e rgyexp end i tu r e ,th ek eyp l ay e r sr em a incon s i s t en tand in c lud el ep t inandth em e l ano co r t insy s t em .Add i t ion a l ly ,w ep ropo s ePACAPtob eak ey n eu ro ch em i c a lr egu l a to ro fen e rgym e t abo l i sminth eVMN . B e c au s em a in t en an c eo fen e rgyhom eo s t a s i si sfund am en t a lfo rl i f e ,i ti sl ik e lyth a t hypo th a l am i cn eu ron sov e r l ap ,commun i c a t ingbo thd i r e c t lyandind i r e c t lyw i the a cho th e r andthu sp rov id ingr edund an tp ro c e s s e stom a in t a inen e rgyhom eo s t a s i s(Cho ie ta l . ,2013 ) . A ssu ch ,bo thPACAPandl ep t ininn e rv a t ionton e rv et e rm in a l sinth ePVNh av eb e enshown too r ig in a t eino th e rhypo th a l am i cnu c l e i ,w i thm a jo rp ro j e c t ion sin i t i a t edinth eVMN(D a s , V ih l en ,&L eg r ad i ,2007 ;E lmqu i s te ta l . ,1998 ) .W i thimm en s er e s e a r chev id en c e con s id e r ed ,i ti sr e a son ab l etosugg e s tth a thypo th a l am i cs ign a l ingr egu l a t ingBAT 96 th e rmog en e s i sl ik e lyin c lud e smu l t ip l ehypo th a l am i cnu c l e iandth a tth ecomp l e t en e two rk s o fn eu r a lp a thw ay sinvo lv edr em a intob ee lu c id a t ed .G iv enth efun c t ion a lev id en c eo fth e VMN ’ sro l einth e rmog en e s i s ,th eh ighexp r e s s iono fPACAPinth eVMN ,th ed a t af romth e PACAPKOmou s emod e lth a tshow simp a i r edth e rmog en e s i sinth eab s en c eo fPACAP ,and th eev id en c ef romph a rm a co log i c a ls tud i e sth a tshowin j e c t ion so fPACAPinth eVMN in c r e a s e sSNAtoBAT ,w econ c lud eth a tth eVMNi salog i c a landv a l idt a rg e ttofu r th e r e lu c id a t ePACAP ’ sro l einth e rmog en e s i s . Ino rd e rtot a rg e tth eVMN ,l im i t a t ion sinth ecu r r en tl i t e r a tu r en e edtob eov e r com e . A l thoughin t r a c e r eb rov en t r i cu l a randin t r a th e c a lin j e c t ion so fPACAPh av er e su l t edin in c r e a s edBATa c t iv i ty ,th e s ein j e c t ion sdono td i f f e r en t i a t eb e tw e enth ehypo th a l am i c r eg ion ssp e c i f i c a l lyinvo lv edinBATa c t iv a t ion ,a sin j e c t edPACAPl ik e lysp r e ad stoto v a r iou ssu r round ingb r a inr eg ion s( Ing lo t t ,F a rnh am ,&P i low sky ,2011 ;T an id ae ta l . ,2010 ) . Add i t ion a l ly ,ind i f f e r en t i a t ingPACAP ’ sro l einth ePVNandVMN ,R e s che ta l .(2013 ) p rov id edr e su l t sth a tw e r eon lyco l l e c t edov e ra24 hou rp e r iod .Th e r e fo r e ,inadd i t ionto r ep l i c a t ingr e su l t stoen su r ea c cu r a cy ,a s s e s s ingth elong t e rmfun c t iono fPACAPinth e VMNwou ldexp andth el i t e r a tu r e ,p a r t i cu l a r lyine lu c id a t ingPACAP ’ sro l eina c t iv a t ing ad ap t iv eth e rmog en e s i sdu r ingco ldch a l l eng e s ,a sfu l la c c l im a t iontoco ldandm ax im a l indu c t iono fad ap t iv eth e rmog en e s i sr equ i r e s3 4w e ek sa t4 °C(C annon&N ed e rg a a rd ,2010 ; M ey e re ta l . ,2010 ;Uk rop e ce ta l . ,2006 ).A ssu ch ,infu tu r e ,anSF1 d r iv enexp r e s s ionv e c to r hou s ingPACAPw i l lb ein t rodu c edbyv i r a ld e l iv e ryu s ings t e r eo t ax i cin j e c t ion sd i r e c t lyin to th eVMNo fPACAPKOm i c etoa s s e s sth ee f f e c to fg en e t i cr e in t rodu c t iono fPACAP sp e c i f i c a l lyinth eVMNo fan im a l sl a ck ingPACAP .Th eu s eo fanad eno a s so c i a t edv i ru s s e ro typ e9(AAV9 )w i l la l lowfo rr ap idon s e to ft r an sdu c t iono fth eb r a int i s su e sinth e ab s en c eo fimmun er e spon s e s ,a sw e l la slongt e rmexp r e s s ion ,andth eu s eo facon s t ru c t 97 wh e r ePACAPexp r e s s ioni fd r iv enbyth eSF1p romo t e rw i l len su r ePACAPi son ly exp r e s s edinVMNin f e c t edn eu ron sth a tsp e c i f i c a l lyexp r e s sSF1(D ay a ,&B e rn s ,2008 ; Z in c a r e l l ie ta l . ,2008 ) .Comp l e t em e thodologyw i l lb ed i s cu s s edb e low . Hypo th e s i sandA im s Ihypo th e s i z eth a tth eco lds en s i t iv i tyo fPACAP/ -m i c em ayb edu etoth eab s en c e o fPACAPs ign a l ingw i th inth eVMNsp e c i f i c a l ly . Du etoth ecomp l ex i tyandlongt e rmn a tu r eo fth i sp ro j e c t ,myinvo lv em en tandthu sth e ob j e c t iv e so fth i sch ap t e ra r e : • Toc r e a t eanad enov i r a lcon s t ru c tth a texp r e s s e sGFPund e rth er egu l a t iono f th eSF1p romo t e rtoa c ta sacon t ro lv e c to r ,thu sen su r ingVMN sp e c i f i c exp r e s s iono fth ev e c to r • D ev e lopap ro to co lfo rs t e r eo t ax i cin j e c t iono fth ev e c to rwh i chr e a ch e sth e VMNinl iv ean im a l s Fu tu r ed i r e c t ion sw i l lb ed i s cu s s edind e t a i lb e low( s e e3 .4D i s cu s s ion ) ;how ev e r ,top rov id e fu r th e rund e r s t and ingth eya r es t a t edh e r e : • Toc r e a t eanad enov i r a lcon s t ru c tfo rVMNsp e c i f i cexp r e s s iono fPACAPt o d e t e rm in ei fth eimp a i r edth e rmog en e s i sinPACAP/ -m i c ec anb eg en e t i c a l ly r e s cu edbyt r an sg en i cexp r e s s iono fPACAPinth eVMNon ly ,thu s d e t e rm iningth eimpo r t an c eo fVMNPACAPinr egu l a t ingth e rmog en e s i s 3 .2 MATER IALSAND METHODS AAVP la sm idV e c to rBa ckbon e Th es e l fcomp l im en t a ry( s c )p s cAAV GFPp l a sm id(F igu r e3 .1A )( c r e a t edbyJohnT . G r ay ,Addg en eP l a sm id#32396 )w a scho s ena sth eexp r e s s ionv e c to rb a ckbon efo rl a rg e- 98 A . B . SF 1 F igu r e3 .1 .A .P l a sm id m apfo rp s cAAVGFP(Addg en eP l a smid#32396 ) .Im ag ecou r t e syo fJohnT . G r ay .B .Th eCMVp romo t e rw i th inp s cAAVGFPw a sr ep l a c edw i thth eSF1p romo t e r(p s cAAVSF1GFP )w i thth ego a lo fexp r e s s ingGFPsp e c i f i c a l lyinth eVMNo fth ehypo th a l amu s ,ar eg iono f th eb r a inknowntosp e c i f i c a l lye xp r e s sSF1 .V i ru sw i l lb ed e l iv e r edtoth eb r a inv i as t e r eo t ax i c in j e c t ionin toth eVMN .Ad ap t edw i thp e rm i s s ionf romth eau tho r .Sou r c e :� h t tp s : / /www . addg en e .o rg /32396 /.A c c e s s ed M ay4 ,2017 . 99 s c a l ep a ck ag ingandp rodu c t iono fAAV9a im eda tt r an s f e c t ingc e l l sinth eVMN .Toen su r e sp e c i f i cVMNt r an s f e c t ion ,th eub iqu i tou sp romo t e rinp s cAAV GFP ,cy tom eg a lov i ru s (CMV ) ,w a sr ep l a c edw i thth eVMN sp e c i f i cSF1p romo t e r(F igu r e3 .1B )f romi so l a t ed mou s eDNAp r io rtoAAV9p a ck ag ing .Toen su r ead enov i r a lexp r e s s iono fGFPund e rth e SF1p romo t e rupons t e r eo t ax i cin j e c t ion sinth eVMN ,th er eg iono fth eSF1g en echo s enfo r i so l a t ionandamp l i f i c a t ionw a s400bplongw i thth ein c lu s iono fup s t r e amt r an s c r ip t ion f a c to rb ind ings i t e sn e c e s s a ryfo rSF1p romo t e rfun c t ion( Wood son ,C r aw fo rd ,S adov sky ,& M i lb r and t ,1997 ) .D e t a i l edm e thodo logyi sd i s cu s s edb e low . I so la t ingth ep s cAAV GFPP la sm id Th ep s cAAVGFPp l a sm idw a spu r ch a s edf romAddg en eandr e c e iv edp a ck ag ed w i th inE s ch e r i ch iaco l i( E .co l i)b a c t e r i a( t r an s fo rm edinS tb l2comp e t en tc e l l s )inanag a r s t ab .B a c t e r i aw i th inth eag a rs t abw e r es t r e ak edon toLu r i ab ro th(LB )ag a rp l a t e scon t a in ing amp i c i l l in(Amp )(100µg /m l )(p r ep a r eda c co rd ingtoAddg en ep ro to co l )andin cub a t ed ov e rn igh t(18hou r s )a t30 °C ,ina c co rd an c ew i thth eb a c t e r i a lg row thcond i t ion s .Th r e e i so l a t eds ing l eb a c t e r i aco lon i e sw e r ep i ck edf romth ep l a t e sande a chg rownin2m lo fLBAmpl iqu idcu l tu r e(p r ep a r eda c co rd ingtoAddg en ep ro to co l )inash ak ingin cub a to r(300 rpm )fo r12hou r sa t30 °C ,wh i chs e rv eda ss t a r t e rcu l tu r e sfo rs c a l edp rodu c t iono fth i slow copyp l a sm id .An eg a t iv econ t ro lth a tcon t a in edLBm ed i aandAmpw i thou tanyb a c t e r ia w a sin c lud edinth eabov es t ep sa sanind i c a to ro fnonsp e c i f i cb a c t e r i a lg row th .C e l l sf rom e a chs t a r t e rcu l tu r ew e r ep e l l e t edbyc en t r i fug a t ion ,r e su sp end edin1m lo ff r e shLB Amp m ed i aandadd edto100m lLBAmpm ed i a ,wh i chs e rv eda sas e cond a rycu l tur e .On lyon e s t a r t e rcu l tu r er e su l t edinp e l l e t edc e l l sandw a sth e r e fo r eu s ed ,wh i l eth eo th e rtwow e r e d i s c a rd ed .Th es e cond a rycu l tu r ew a sp l a c edinash ak ingin cub a to r(300rpm )fo r12hou r sa t 30 °Ctoa l lowfo rg row th .C e l l sw e r ep e l l e t ed6x1m lin4s ep a r a t eepp endo r ftub e s(1 .5m l ) 100 a c co rd ingtoth em anu f a c tu r e r ’ sp ro to co lfo rth eQ IAp r epSp in M in ip r epK i t(Q i ag en , H i ld en ,G e rm any ) .R e su sp en s ionandly s i sbu f f e r(P1andP2 )vo lum e su s edw e r e1 .6xtho s e d i r e c t edtoen su r ecomp l e t er in s ingo fth ec e l l sf romth er em a in ingLBm ed i aa sw e l la s comp l e t ec e l lly s i s .N eu t r a l i z ingbu f f e r(N3 )w a su s eda tavo lum e1 .6xth a td i r e c t edbyth e m anu f a c tu r e r ’ sp ro to co landuponadd i t ion ,tub e sw e r einv e r t edfo r3m inu t e stoa l lowfo r comp l e t eDNAr en a tu r a t ionandp r e c ip i t a t iono fp ro t e in sandl ip id s .Th eop t ion a lw a shs t ep w a su s edtor emov eendonu c l e a s e sp r e s en tinth eb a c t e r i as t r a in(S tb l2 ) .Toin c r e a s ey i e ld , th ee lu t edDNAf romon eepp endo r ftub ew a su s edsub s equ en t lytoe lu t eth en ex tina s equ en t i a lm ann e r .A l lo th e rs t ep sw e r efo l low eda c co rd ingtoQ IAp r epSp in M in ip r epK i t p ro to co l s(Q i ag en ,H i ld en ,G e rm any ) .DNAcon c en t r a t ionandpu r i tyw a sa s s e s s edth rough UVl igh tab so rb an c em e a su r em en t su s ingsp e c t ropho tom e t ry(N anod ropND1000 ;Th e rmo S c i en t i f i c ,Ro ck fo rd ,IL ) .A snu c l eo t id e sab so rbUVl igh ta taw av e l eng tho f260nm , ab so rb an c el ev e l so fth es amp l ea t260nmw e r eu s eda sasu r rog a t em e a su r eo fDNA con c en t r a t ion .Th er a t ioo fab so rb an c ea t260nmand280nm( th ew av e l eng tha twh i ch p ro t e inab so rb sUVl igh t )w a su s edtoa s s e s sth epu r i tyo fDNA ,wh e r ear a t ioo f~1 .8i s g en e r a l lya c c ep t eda s“pu r e ”fo rDNA .A sas e cond a rym e a su r eo fnu c l e i ca c idpu r i ty ,th e r a t ioo fab so rb an c ea t260nmand230nmw a su s edtoid en t i fyth ep r e s en c eo fcon t am in an t s th a tab so rba t230 nm .260 /230v a lu e scommon lyr ang eb e tw e en2 .0 2 .2fo r“pu r e ”nu c l e i c a c id .Tocon f i rmin t eg r i tyo fth ev e c to r ’ ss equ en c e( i . e .th eab s en c eo fmu t a t ion so r d e l e t ion s ) ,ad i agno s t i cr e s t r i c t iond ig e s tw a sp e r fo rm eda c co rd ingtom anu f a c tu r e r ’ s p ro to co l s(N ewEng l andB ioL ab s ,Ip sw i ch ,M a s s a chu s e t t s ) .Th er e a c t ionin c lud edp l a sm id DNA(0 .5µg ) ,H ind I I IandE coR I(10un i t so fen zym ee a ch ) ,andN ewEng l andB ioL ab s (NEB )Bu f f e r2 .1 .D ig e s t ionp rodu c t sw e r erunonane th id iumb rom id es t a in ed1 .5%ag a ro s e g e lfo r30minu t e sa t100Vandv i su a l i z edund e rUVl igh t .Exp e c t edb ands i z e sw e r e3714bp 101 and655bp .Tofu r th e rcon f i rmth ep l a sm idDNAw a sf r e eo ft r an s c r ip t ion a le r ro r s s equ en c ing ,s equ en c ingw a sp e r fo rm edu s ing M13Fo rw a rdandR ev e r s ep r im e r s(App l i ed B io sy s t em s3130x ,UNBCG en e t i c sL ab ) . I so la t ingth eSF1P romo t e r M e thodo logyd e s c r ib edinth i ss e c t ionw a scomp l e t edincon jun c t ionw i than und e rg r adu a t ehonou r sth e s i ss tud en t( inco l l abo r a t ionw i thA a ronG e rmu th ) . SF1DNAE x t ra c t ion C57BL /6 WTm i c ew e r eeu th an i zed(110mg /kgeu th any l )andb r a in sw e r eco l l e c t ed andimm ed i a t e lyp l a c edin80 °C ,en su r ingr ap idf r e e z ing .Th ehypo th a l amu sw a sd i s s e c t ed f romth ef ro z enb r a in(~40mg ) ,homog en i z edinTR I zo l(L i f eT e chno log i e s ) ,andDNAw a s i so l a t eda c co rd ingtoth em anu f a c tu r e r ’ sp ro to co l .DNAcon c en t r a t ionandpu r i tyw e r e a s s e s s edbysp e c t ropho tom e t rya sd e t a i l edabov e(N anod ropND 1000 ;Th e rmoS c i en t i f i c , Ro ck fo rd ,IL ) . SF1P r im e rD e s ignandAmp l i f i ca t ionw i thPCR P r im e r sw e r ed e s ign edfo rPCRamp l i f i c a t iono fth e400bpr eg iono fSF1w i tha t l e a s t20comp l im en t a rynu c l eo t id e s .Tof a c i l i t a t efu tu r ec lon ing ,REs i t e s ,m a t ch ingtho s eon e i th e rend so fth eCMVp romo t e rr eg ionw i th inp s cAAV GFP ,w e r eadd edtoe a chs id eend o fth eSF1p romo t e rr eg ion ,w i tha5bpov e rh angtof a c i l i t a t eREd ig e s t ion .REs i t e sadd ed w e r esp e c i f i ctoAv r I I(5 ’CCTAAG3 ’ )andB spE I(5 ’TCCGGA3 ’ ) .C a r ew a st ak ento en su r es im i l a rGCcon t en tandm e l t ingpo in t so fth ep r im e r s .PCRcond i t ion sw e r ea s fo l low s :in i t i a ld en a tu r a t iona t98 °C(30s e cond s ) ,fo l low edby35cy c l e so fd en a tu r a t ion (98 °Cfo r10s e cond s ) ,ann e a l ing(67 °Cfo r30s e cond s ) ,andex t en s ion(72 °Cfo r30 s e cond s ) .Af in a lex t en s ionw a sp e r fo rm eda t72 °Cfo r5m inu t e s .PCRp rodu c t sw e r erunon 102 a ne th id iumb rom id es t a in ed1 .5%ag a ro s eg e lfo r40m inu t e sa t100Vandv i su a l i z edund e r UVl igh tw i thanexp e c t edb ands i z eo f400bp . TAC lon ing PCRamp l i f i edSF1w a sl ig a t edin topGEM® TV e c to r(F igu r e3 .2 )u s ing2XR ap id L ig a t ionBu f f e randT4DNAL ig a s ea c co rd ingtom anu f a c tu r e r ’ sp ro to co l(P rom eg a , M ad i son ,W I ) ,wh i cha l low edfo rb lu ewh i t es c r e en ings e l e c t ion .Ana l iquo to fth el ig a t ion r e a c t ion(2 .5µ l )w a sadd edto50µ lo f MAXE f f i c i en cyS tb l2Comp e t en tC e l l s( Inv i t rog en , C ar l sb ad ,CA )andp l a t edonLB Ampp l a t e sa c co rd ingtoAddg en e ’ s“B ac t e r i a l T r an s fo rm a t ion ”p ro to co l s ,andin cub a t edov e rn igh ta t37 °C .R e su l t ingwh i t eco lon i e sw e r e p i ck edin to10m lo fLB Ampm ed i aandin cub a t edov e rn igh ta t37 °Cinash ak ingin cub a to r (225rpm ) .Co lonyPCRfo rth eSF1p romo t e rw a sp e r fo rm edonmu l t ip l ec o lon i e sand su c c e s s fu l lyc lon edp l a sm id s( a sd e t e rm in edbyb r igh t400bpb and sonth er e su l t ingPCR g e l )w e r ei so l a t edu s ing W i z a rd®P lu sSV M in ip r ep sDNAPu r i f i c a t ionSy s t em(P rom eg a , M ad i son ,W I ) .G ly c e ro ls to ck so fth el ig a t edSF1f r agm en tandpGEM®TVe c to r( fu r th e r r e f e r r edtoh e r ea sth eSF1 pGEM® TV e c to r )w e r ec r e a t edu s ingov e rn igh tcu l tu r eand g ly c e ro la c co rd ingtoAddg en ep ro to co l s .Ex t r a c t edp l a sm id sw e r es c r e en edfo rco r r e c t p l a c em en to fth eSF1p romo t e rf r agm en tbyp e r fo rm ingadoub l eREd ig e s ta c co rd ingto m anu f a c tu r e r sp ro to co l s(Th e rmoS c i en t i f i c ,Ro ck fo rd ,IL ) .Th er e a c t ioncon t a in ed :p l a sm id DNA(1µg ) ,Av r I IandB spE I(10un i t so fen zym ee a ch )and1XTh e rmoS c i en t i f i cT ango Bu f f e r .P rodu c t sw e r erunonane th id iumb rom id es t a in ed1 .5%ag a ro s eg e lfo r30m inu t e sa t 100Vandv i su a l i z edund e rUVl igh t .Exp e c t edb ands i z e sw e r e2600bpand400bp .To fu r th e rcon f i rmth eSF1 pGEM®Tp l a sm idw a sf r e eo ft r an s c r ip t ion a le r ro r s ,i tw a s s equ en c edu s ingT7andSP6p r im e r s(App l i edB io sy s t em s3130x ,UNBCG en e t i c sL ab ) . 103 F igu r e3 .2 .P l a sm id m apfo rpGEM® TV e c to r(P rom eg a ) .SF1in s e r ti sl ig a t edin toth el a cZg en eby a t t a ch ingtoov e rh ang ingthym in enu c l eo t id e s ,d i s rup t ingth el a cZg en e .A c c e s s edf rom h t tp s : / /www .p rom eg a . c a /p rodu c t s /p c r /p c rc lon ing /pg em t v e c to rsy s t em s / .Copy r igh t©2017 P rom eg aCo rpo r a t ion .Im ag er ep rodu c edw i thp e rm i s s ionf romth eco rpo r a t ion . 104 R ep la c ingth eCMVP romo t e rw i thth eSF1P romo t e rinth ep s cAAVGFPV e c to r Th ei so l a t edp s cAAVGFPp l a sm id(15µg )andi so l a t edSF1pGEM®Tp l a sm id (5µg )w e r es en ttoV ig en eB io s c i en c e sIn c .(Ro ckv i l l e , MD )wh e r eSF1w a sc lon edin toth e p s cAAVGFPv e c to rtor ep l a c eth eCMVp romo t e r(F igu r e3 .1B )u s ingAv r I IandB spE IRE d ig e s t ionandl ig a t ionr e a c t ion s .Th en ewp l a sm idw a sn am edp s cAAV SF1GFP . P rodu c t iono fAAV9Exp r e s s ingp s cAAVSF1GFP AAV9p a ck ag ingo fth eexp r e s s ionv e c to r(p s cAAVSF1GFP )andl a rg es c a l e p rodu c t iono fth ev i ru sAAV9 SF1GFPw a sp e r fo rm ed( con c en t r a t ion2 .24x1014g enom e copy /m l )(V ig en eB io s c i en c e s ,In c . ,Ro ckv i l l e , MD ) .Th i sv i ru sw i l lb eu s eda sacon t r o l v i ru sfo rth i sp ro j e c t ,exp r e s s ingGFPund e rth econ t ro lo fth eSF1p romo t e rinsp e c i f i cb r a in r eg ion sin f e c t edw i thth ev i ru sth roughs t e r eo t ax i cin j e c t ion s(d e s c r ib edb e low ) . D ev i s ingaS t e r eo tax i cP ro to co lfo rIn j e c t ion sin toth eVMN S t e r eo t ax i csurg e ryi san ewp ro c edu r einth eG r ayl abandth e r e fo r er equ i r edt r a in ing andd ev e lopm en to fexp e r im en t a lp ro c edu r e s .S t e r eo t ax i cwo rk shop sw e r eh e ldund e rth e d i r e c t iono fSh e l l ey M cE r l an e(DVM ) ,S en io rC l in i c a lV e t e r in a r i anandH e ado fT r a in ingfo r An im a lC a r eS e rv i c e sa tth eUn iv e r s i tyo fB r i t i shCo lumb i a ,andLyd i aT ro c(RVTand RLAT )f romth eUn iv e r s i tyo fNo r th e rnB r i t i shCo lumb i a ,b e fo r ep e r fo rm ingsu rg e r i e s ind ep end en t ly . Po s i t ion ingth e Mou s einth eS t e r eo ta x i cF ram e M i c ew e r ew e igh ed ,an e s th e t i z ed w i thsod iump en tob a rb i t a l(90mg /kg ,IP ) ,and p l a c edinas ep a r a t ec ag eun t i lap l an eo fan e s th e s i aw a sr e a ch ed ,a sob s e rv edbyd e ep , r egu l a rb r e a th ingandnow i thd r aw a luponato ep in ch .Fu rw a ssh av edf romth esu rg i c a ls i t e , w i thc a r et ak enno ttosh av eth ewh i sk e r s ,andd i s in f e c t edw i th70%a l coho l .Ey elub r i c an t w a sp l a c edov e rth eco rn e a so fbo they e s .E a rb a r so fth es t e r eo t ax i cf r am e(S to e t l ing , Wood 105 D a l e ,I l l ino i s )w e r eg en t lyp l a c edinth eex t e rn a laud i to ryc an a lw i th inth ebu l l ainbo the a r s andt i gh t en edin top l a c e .Co r r e c tpo s i t ion ingw a sd e t e rm in edbysmoo thro t a t ion a lmov em en t o fth esnou tupanddown ,w i thl i t t l eo rnol a t e r a lmov em en t .E a rb a rm e a su r em en t sw e r e con s i s t en tone a chs id esu chth a tth emou s ew a sc en t e r ed .In c i so r sw e r eth enp l a c edov e rth e in c i so rb a randth ean e s th e t i ccon ew a sf i t t edsnugg lyov e rth eno s e .Oxyg enf lowr a t ew a s k ep ta t0 .8L /m inwh i l ei so f lu r an ew a sad ju s t edth roughou ttom a in t a inasu rg i c a ll ev e lo f an e s th e t i c(no rm a l lym a in t a in eda t2% ) .Th roughou tth ep ro c edu r e ,th ean im a lw a sp l a c edon ah e a t ingp adcon t ro l l edbyath e rmom e t e rp l a c edinth er e c tum ,m e a su r ingbodyt emp e r a tu r e con t inuou s lyandad ju s t ingth eh e a t ingp adt emp e r a tu r ea c co rd ing ly(Rod en tW a rm e r , S to e t l ing , WoodD a l e ,I l l ino i s ) .Alo c a lan e s th e t i c(bupiv a c a in e ,2 .5mg /m l ;no tex c e ed ing 8mg /kg )w a sin j e c t edsub cu t an eou s lya longth ep l ann edin c i s ions i t ea sal in eb lo ck . S t e r eo ta x i cSu rg e r y S t e r eo t ax i ccoo rd in a t e spub l i sh edfo rth eVMNandu s edinth i sexp e r im en tw e r e :1 .46mmpo s t e r io rf romb r egm a ,±0 . 39mml a t e r a l lyf romm id l in eand5 .50mmb e lowth e su r f a c eo fth eb r a in(A l l enIn s t i tu t efo rB r a inS c i en c e ,2016 ) .As t ab l ep l an eo fan e s th e s i a w a sen su r edp r io rtocomm en c ingsu rg e ry .Anin c i s ionw a sth enm ad esu chth a tl ambd aand b r egm aw e r eexpo s edonthesku l l(F igu r e3 .3 ) .Th esk inw a sg en t lypu l l edb a ckandth esku l l w a sc l e an edu s ingco t tont ipp edapp l i c a to r s .Ad r i l lw i tha1mmd r i l lb i tw a sa t t a ch edtoth e s t e r eo t a c t i ca rm .Th et ipo fth ed r i l lb i tw a spo s i t ion edonl ambd aandth eh e igh tw a s r e co rd e d .Th et ipo fth ed r i l lb i tw a sth enpo s i t ion edonb r egm a(F igu r e3 .4A )wh e r eth e h e igh tw a sr e co rd edag a in .Th ein c i so rb a rw a sr a i s edo rlow e r edtol ev e lth esku l lsu chth a t th eh e igh to fl ambd aandb r egm aw e r eth es am e .Th ed r i l lw a smov edinth epo s t e r io r d i r e c t ionaw ayf romb r egm a1 .46mm ,andtoth er igh to fth em id l in e0 .39mmun t i lth e app rop r i a t er e ad ing sw e r ea l ign edonth eV e rn i e rs c a l eo fth es t e r eo t ax i cf r am e .A tth e 106 F igu r e3 .3.Lo c a t iono fl ambd aandb r egm aa sd e f in edbyD r .G eo rg eP ax ino s .In t e r au r a ll in e co r r e spond sw i thth ep l a c em en to fth ee a rb a r s .R ep rodu c edf rom“ Th er a tb r a inins t e r eo t ax i c coo rd in a t ion ” ,byG .P ax ino s , &C .W a t son ,1998 ,1 s tEd i t ion ,p .11 .Copy r igh t©2017A c ad em i c P r e s s /E l s ev i e r .R ep rodu c edw i thp e rm i s s ionf rompub l i sh e r . A . B . F igu r e3 .4.A .S t e r eo t ax i cadm in i s t r a t ion sa r eg iv enu s ingx ,y ,andzcoo rd in a t e stoasp e c i f i c lo c a t ionandd ep th .B .Lo c a t iono fin j e c t ionin toth eVMNo fth ehypo th a l amu s .I l lu s t r a t ion sby En c ap su l aN anoS c i en c e sLLC ,B r en twood ,TN .Copy r igh t©2014En c ap su l aN anoS c i en c e sLLC. Ob t a in edf romh t tp s : / /www . c lod ro som e . com / an im a lin j e c t ion / .Ad ap t edw i thp e rm i s s ionf rom En c ap su l aN anoS c i en c e s . 107 in t e r s e c t iono fth e s ecoo rd in a t e s ,inth eXandYp l an e ,aho l einth esku l lw a sm ad eby d r i l l in ginsm a l lin c r em en t ssu chth a tth eund e r ly ingb r a inw a sno td am ag ed .Th eh e igh to f th etopo fth eb r a inw a sth enm e a su r edonth eV e rn i e rs c a l e .Th ed r i l lw a sr ep l a c edw i tha H am i l tonsy r ing e(70001KH ,H am i l tonRobo t i c s ,Sw i t z e r l and )w i tha25g aug en e ed l e lo ad edw i th1µ lo f5xlo ad ingdy e .F romth esu r f a c eo fth eb r a in ,th en e ed l ew a slow e r ed 5 .50mminth eZp l an ea tar a t eo f1mm /m inu t e(F igu r e3 .4B ) .Dy ew a sin j e c t edv e rys low ly a tar a t eo f200n l /m inu t e ,andth enth en e ed l ew a sl e f tinp l a c efo r5m inu t e stoa l lowth e f lu idtob ead so rb edb e fo r er emov ingth en e ed l ei ta tar a t eo f1mm /m in . Po s tOp e ra t i v eP ro c edu r e M i c ew e r eeu th an i z edw i thi so f lu r an eandc e rv i c a ld i s lo c a t ion .Th eb r a inw a s r emov edimm ed i a t e lyandp l a c edin10%fo rm a l info r24hou r s .Th ef ix edb r a inw a s d i s s e c t edandth ehypo th a l amu sv i ew edund e ras t e r eom i c ro s cop e( O lympu sSXZ7)to v i su a l i z eth elo c a t ionandtod e t e rm in ea c cu r a cyo fth ein j e c t ion .Im ag e sw e r et ak enw i than In f in i ty1c am e r aandIn f in i tyC ap tu r eSo f tw a r e(Lum en e r a ,O t t aw a ,ON ) . 3 .3RESULTS p s cAAVGFPP la sm idwa sSu c c e s s fu l lyI so la t ed ,F r e eo fT ran s c r ip t ionE r ro r s Tocon f i rmth a tth eb a c t e r i acon t a in ingth elow copyp s cAAV GFPp l a sm idh adb e en g rownupsu c c e s s fu l lyw i thnot r an s c r ip t ion a le r ro r s ,th ep l a sm idDNAw a ss equ e n c edu s ing M13 Fo rw a rdandR ev e r s ep r im e rs .U s ingth eN a t ion a lC en t e rfo rB io t e chno logy In fo rm a t ion ’ s(NCB I )B a s i cLo c a lA l ignm en tS e a r chToo l(BLAST ) ,s equ en c ingr e su l t so f th ei so l a t edp s cAAV GFPp l a sm idind i c a t edm a t ch ingnu c l eo t id es equ en c e stoth a to fth e pu r ch a s edp l a sm id(Addg en eP l a sm id#32396 ) ,ind i c a t ingnot r an s c r ip t ione r ro r s(F igu r e 3 .5 ) . 108 F igu r e3 .5.As e c t iono fnu c l eo t id e sm a t ch ingf romNCB I ’ sBLASTb e tw e enth ep s cAAVGFP p l a sm ids equ en c ep rov id edf romAddg en e(Qu e ry )andth ei so l a t edp l a sm id(Sb j c t )s equ en c edu s ing M13R ev e r s ep r im e r .Y e l lowind i c a t e sAv r I IREs i t e(5 ’CCTAGG3 ’ )andr edind i c a t e sB spE IRE s i t e(5 ’TCCGGA3 ’ ) .Asm a l ls e c t iono fth ei so l a t edp l a sm idDNAw a sun r e ad ab l e( ind i c a t edby “N ” )u s ing M13R ev e r s ep r im e r .A m a t ch ings equ en c ew a scon f i rm edu s ing M13Fo rw a rdp r im e r ( r e su l t sno tshown ) . 109 SF1wa sSu c c e s s fu l lyL iga t edw i thth epGEM®TV e c to r Tocon f i rmth a tSF1w a ssu c c e s s fu l lyl ig a t edin toth epGEM®TV e c to rw i thno t r an s c r ip t ion a le r ro r s ,b a c t e r i a lp l a sm idDNAw a ss equ en c edu s ingT7andSP6p r im e rs . U s ingNCB I ’ sBLAST ,th eDNAs equ en c eo fth el ig a t edSF1pGEM®TV e c to rm a t ch ed nu c l eo t id es equ en c e stoth a to fMu smu s cu lu ss t r a inC57BL /6 J ,ch romo som e2 ,G enom e R e f e r en c eCon so r t ium Mou s eR e f e r en c e38p a t ch4(GRCm38 .p4 ) ,wh i chco r r e spond stoth e s equ en c eo fSF1inth emou s eg enom e(F igu r e3 .6 ) . S t e r eo ta c t i cIn j e c t edDy ewa sLo ca l i z edinth eHypo tha lamu s Tocon f i rmth es t e r eo t ax i cin j e c t iono fth edy esu c c e s s fu l lyt a rg e t edth eVMNo fth e hypo th a l amu s ,th eb r a inw a sc rud e lyc ro s s s e c t ion edandv i su a l i z edund e ras t e r eo m i c ro s cop e(O lympu sSXZ7) .Im ag e sshow edb lu edy ev i s ib l einth eth i rdv en t r i c l e(3V )o f th ehypo th a l amu sandsu r round inga r e a s ,in c lud ingth eVMN(F igu r e3 .7 ) .How ev e r ,dy e app e a r edfu r th e rdo r s a linth ehypo th a l amu sth anan t i c ip a t ed(F igu r e3 .7 ) .Th edy ew a sno t ob s e rv edinanyr eg ion sou t s id eo fth ehypo th a l amu ssu cha sth eb a s a lfo r eb r a in( r e su l t sno t . shown ) 3 .4D ISCUSS ION C en t r a landv i t a ltoth i sp ro j e c tw a sen su r ingth a tth econ s t ru c t sfo rth eVMNsp e c i f i c ad eno a s so c i a t edv i ru s e sw e r ep rop e r lyd e s ign edandc r e a t edsu chth a tfu tu r ev i r a lin j e c t ion s in toth eVMNv i as t e r eo t ax i csu rg e ryw i l lp rov es a f eande f f e c t iv e .Th ec lon ings t r a t egy d ev e lop edfo rth i sp ro j e c tu t i l i z edanexp r e s s ionv e c to rc a l l edp s cAAV GFPa sab a ckbon e . Th i sv e c to rc anb ep a ck ag edinanad enoa s so c i a t edv i ru ss e ro typ e9 ,su chth a tin f e c t edc e l l s exp r e s sGFPund e rcon t ro lo fth eCMVp romo t e r .B e c au s et h eCMVp romo t e rwou ldd r iv e g en eexp r e s s ionina l lb r a int i s su e s ,CMVw a sr ep l a c edw i thSF1 ,wh i chwh enin j e c t edin to th eb r a inw i l ld r iv eg en eexp r e s s ioninc e l l so fth eVMNon ly .U s ingth eSF1g en er eg ionth a t 110 � F igu r e3 .6.NCB I ’ sBLASTs e a r cho fth es equ en c eob t a in edf romSF1pGEM®TV e c to r(Sb j c t ) u s ingT7p r im err ev e a l eda m a t chtoas e c t iono fth eMu smu s cu lu ss t r a inC57BL /6 J ,ch romo som e2 , GRCm38 .p4(Qu e ry ):th er eg iono fth e mou s eg enom eth a thou s e sth eSF1p romo t e r .Y e l low ind i c a t e sAv r I IREs i t e(5 ’CCTAGG3 ’ )andr edind i c a t e sB spE IREs i t e(5 ’TCCGGA3 ’ )add edto th ePCR amp l i f i edSF1 . 111 A PVN � L HA � � � � DMN � � VMN � � � � � � � ARC 3V � B C D � E F G � F igu r e3 .7 .A .Co ron a lc ro s ss e c t iono fth eb r a inshow ingth elo c a t iono fth ehypo th a l amu sandc lo s eupf iv ek eyhypo th a l am i cnu c l e iinvo lv edinth er egu l a t iono fen e rgy m e t abo l i sm .PVN ; p a r av en t r i cu l a rnu c l eu s ,DMN ;do r som ed i a lnu c l eu s ,LHA ;l a t e r a lhypo th a l am i ca r e a ,VMN ; v en t rom ed i a lnu c l eu s ,ARC ;a r cu a t enu c l eu s ,3V ;th i rdv en t r i c l e .Ad ap t edf rom“R ev i s i t ingth e V en t r a lM ed i a lNu c l eu so fth eHypo th a l amu s :Th eRo l e so fSF1N eu ron sinEn e rgyHom eos t a s i s ” , byY .Cho i ,T .Fu j ik aw a ,J .L e e ,A .R eu t e r , &K .K im ,2013 ,F ron t i e r sInN eu ro s c i en c e,7.Copy r igh t ©2013Cho i ,Fu j ik aw a ,L e e ,R eu t e randK im .Ad ap t edw i thp e rm i s s ionf romau tho r .B .Do r s a lv i ew o fth e mou s eb r a in .C .V en t r a lv i ewo fth e mou s eb ra in(boxind i c a t e shypo th a l amu s ) .D .B lu edy e ob s e rv edinth ehypo th a l amu s .E .An t e r io rco ron a ls e c t ionth roughth ehypo th a l amu s ;b lu edy e ob s e rv edinth e3Vandsu r round inghypo th a l am i ct i s su e s .F .M idd l eco ron a ls e c t ionth roughth e hypo th a l amu s ;dy elo c al i z eda longth e3V .G . Po s t e r io rco ron a ls e c t ionth roughth ehypo th a l amu s ; dy elo c a l i z edfu r th e rdo r s a la longth e3V . 112 in c lud eda l ln e c e s s a ryt r an s c r ip t ionf a c to r sfo rSF1p romo t e ra c t iv i tyw i l la l lowfo rth e exp r e s s iono fGFP( con t ro l )o rPACAP ,sp e c i f i c a l lyinth eVMNo fAAV9 in j e c t edm i c e .In o rd e rtop rov id eV ig en ew i thth ecompon en t sr equ i r edtog en e r a t eAAV9exp r e s s ingth e con t ro lp l a sm id(AAV9 SF1GFP ) ,th ep s cAAVGFPandSF1pGEM® Tv e c to r sw e r e p rodu c edandV ig en esu c c e s s fu l lyc lon edSF1in toth ep s cAAV GFPv e c to randp a ck ag ed th i scon s t ru c tin toAAV9su chth a tw eh av eth eAAV9 SF1GFPcon t ro lv i ru stot e s tVMN sp e c i f i cg en ed e l iv e ry . P r io rtocomm en c ings t e r eo t ax i cin j e c t ion sw i thth econ t ro lv i ru sexp r e s s ingGFP (AAV9 SF1GFP ) ,w ein s t e adin j e c t eddy econ t a in ingb romoph eno lb lu etov i su a l i z ei fth e pub l i sh edVMNcoo rd in a t e s( an t e r io r /po s t e r io r1 .46mmf romb r egm a ;m ed i a l / l a t e r a l ,± 0 .39mmf romm id l in e ;anddo r s a l /v en t r a l ,5 .50mmf romsu r f a c eo fth eb r a in(A l l enIn s t i tu t e fo rB r a inS c i en c e ,2016 ) )w e r einf a c ta l low ingt a rg e t edd e l iv e rytoth eVMN .F romth e ob s e rv edfo rm a l in f ix edhypo th a l am i cs e c t ion s ,dy ew a sob s e rv edsup e r io rtoth ean t i c ip a t ed lo c a t ionandth e r e fo r ei spo t en t i a l lyno tr e a ch ingth eVMN(F igu r e3 .7 ) .W ethu scon c lud e th a tth ecoo rd in a t efo rth eZ p l an ei slow e rth an5 .50mmf romth esu r f a c eo fth eb r a in ,and fu tu r ein j e c t ion sw i l lu s eth ecoo rd in a t eo f5 .70mmf romth esu r f a c eo fth eb r a ininhop e so f a ch i ev ingin j e c t ion sc lo s e rtoth eb a s eo fth ehypo th a l amu s ,sp e c i f i c a l lyinth eVMN .Th i s ch ang em ayapp e a rcon s e rv a t iv e( Δ0 .2mm ) ,bu tp r e c i s ioninth ehypo th a l amu si simp e r a t iv e a sth ed i s t an c eb e tw e enth etopandbo t tomo fth e3Vi son ly1mm(F igu r e3 .8 ) . A l thoughth ego a lo fth es t e r eo t ax i cin j e c t ion si stor e a chth eVMNa sth i swou ld r e su l tinth eg r e a t e s tin f e c t iono ft a rg e tc e l l s ,in j e c t ion sin toth e3Vi sa l soanop t iona sou r exp r e s s ionv e c to ri sd r iv enbyth eSF1p romo t e rwh i chi sex c lu s iv e lyexp r e s s edinth eVMN (Cho ie ta l . ,2013 ) .Th e r e fo r e ,i fin j e c t ion sw e r em ad ein toth e3V( al a rg e rt a rg e tth anth e VMN )o rno ten t i r e lyinth eVMN ,w ec anb econ f id en tth a tGFP ,andinfu tu r ePACAP ,w i l l 113 F igu r e3 .8 .M idd l eco ron a lc ro s ss e c t ionth roughth ehypo th a l amu so f Mu smu s cu lu ss t e r eo t ax i c a l ly in j e c t edw i th5xlo ad ingdy e .Ru l e rshow s1mmin c r em en t s . 114 on lyb eexp r e s s edinth eVMN .Add i t ion a lin j e c t ion sw i l lb econdu c t edtoen su r elo c a t ion a c cu r a cyp r io rtocomm en c ingexp e r im en t su s ingth econ t ro lv i ru s . Fu tu r eD i r e c t ion s P e r fo rm ingag en e t i cin t e rv en t iontoa s s e ssi fth eimp a i r edth e rmog en i cc ap a c i ty ob s e rv edinPACAPKOc anb er e s cu edw i thVMN sp e c i f i cin j e c t ion so fPACAPi sbo tha comp l exandlong t e rmp ro j e c tinth eG r ayl ab .A sp a r to fth i sth e s i s ,p r e l im in a rywo rkh a s b e encomp l e t edsu chth a tth ep ro j e c tm ayp rog r e s s .H e r e ,anad enov i r a lcon s t ru c tth a t exp r e s s e sGFPund e rth er egu l a t iono fth eSF1p romo t e rh a sb e enc r e a t ed ,andas t e r eo t ax i c p ro to co lfo rth ein j e c t iono fth i sv i ru sh a sb e end ev e lop ed .Th er em a ind e ro fth i sp ro j e c tw i l l b ecomp l e t edbyth eG r ayl ab . Th elong t e rma imo fth i sp ro j e c ti stog en e t i c a l lyr e in t rodu c ePACAPinPACAPKO m i c einth en eu ron so fth eVMNon ly ,anda s s e s si fth eimp a i r edth e rmog en e s i si simp rov ed (o rr e s cu ed ) .Todoso ,th econ t ro lv i ru sd i s cu s s edabov e(AAV9 SF1GFP )w i l lb eu s edfo r twor e a son s :f i r s t ly ,tocon f i rmth ee f f i c a cyo fth eICV VMNin j e c t ion s ,ands e cond ly ,to b eh av ea sacon t ro lfo rcomp a r i son sb e tw e enm i c ein j e c t edw i thth eexp e r im en t a lv i ru s (AAV9 SF1PACAP )(d i s cu s s edb e low ) . Tod e t e rm in ee f f i c a cyo fth eICVVMNin j e c t ion s ,AAV9SF1GFPin j e c t edan im a l b r a in sw i l lb es e c t ion ed ,andth elo c a t iono fGFPw i l lb ea s s e s s edu s ingane s t ab l i sh ed immunoh i s to ch em i s t ryp ro to co linth eG r ayl ab ,wh e r eap r im a ryan t ibodyb ind stoGFP , fo l low edbys e cond a ryan t ibodyb in d ingtoamp l i fyth es ign a lsu chth a tth elo c a t iono fGFP exp r e s s ionc anb ev i su a l i z edund e raf luo r e s c en tm i c ro s cop e . On c eth elo c a t iono fGFP exp r e s s ioni sd e t e rm in edinth eVMN ,exp e r im en t a lin j e c t ion sm aycomm en c e .F i r s t , how ev e r ,con s t ru c t iono fth eexp e r im en t a lv i ru s ,AAV9 SF1PACAPi sr equ i r ed ,wh e r e PACAPw i l lr ep l a c eGFPinth econ t ro lv i ru s ,AAV9SF1GFP . 115 Th ep s cAAVSF1GFPexp r e s s ionv e c to rw i l lb eu s edfo rcon s t ru c t iono fth e exp e r im en t a lv i ru s .PCRamp l i f i c a t iono fPACAPf romhypo th a l am i ccDNAw i l lb el ig a t ed w i thth epGEM®TV e c to r(F igu r e3 .2 )ands en ttoV ig en e ,a longw i thi so l a t edp s cAAVSF1GFPp l a sm idwh e r ePACAPw i l lr ep l a c eGFP(F igu r e3 .9 ) ,andl a rg es c a l eAAV9 p a ck ag ingo fth eexp r e s s ionv e c to rw i l lb ep rodu c ed .Th i sv i ru sw i l lb en am ed :AAV9 SF1PACAP . Toa s s e s swh e th e rth eco lds en s i t iv i tyo fPACAP/ -m i cei sdu etoth eab s en c eo f PACAPs ign a lingw i th inth eVMNsp e c i f i c a l ly ,phy s io log i c a landb io ch em i c a la s s e s sm en t s o fth e rmog en i cm a rk e r ss im i l a rtotho s einCh ap t e r2w i l lb eu s edt od e t e rm in ei fth ePACAP / -ph eno typ ec anb er e s cu edw i thg en e t i cr e in s e r t iono fPACAPinth eVMN .Th e r e fo r e , a s ep t i csu rg i c a lt e chn iqu ew i l lb ep e r fo rm edino rd e rtoavo idin f e c t ion ,andan im a l sw i l lb e r e cov e r edpo s t su rg e rytoa l lowfo rv i r a lt r an s f e c t iono fc e l l sinth eVMNa sw e l la sfo rco ld a c c l im a t ionp r io rtoth e rmog en i ca s s e s sm en t s . Incon c lu s ion ,fu r th e re lu c id a t iono fth ecomp l exp a thw ay sa s so c i a t edw i thPACAPin th eVMNandth e i rro l e sinr egu l a t ingad ap t iv eth e rmog en e s i sw i l lcon t r ibu t etoth epoo lo f know l edg eav a i l ab l efo rp a thw ay scon t ro l l ingen e rgyexp end i tu r e .B e c au s een e rgy exp end i tu r econ t r ibu t e simm en s e lytowho l ebodyen e rgyhom eo s t a s i s ,th i sknow l edg em ay con t r ibu t etoth ed ev e lopm en to fs a f eande f f e c t iv eth e r ap i e sfo rm e t abo l i cd i s o rd e r s , in c lud ingob e s i ty ,andw i l lb efu r th e rd i s cu s s edinCh ap t e r4 . 116 SF 1 PACAP F igu r e3 .9.Th eCMVp romo t e rr ep l a c edw i thth eSF1p romo t e r ,andth eGFPg en er ep l a c edw i thth e PACAPg en e(p s cAAVSF1PACAP )w i thth ego a lo fexp r e s s ingPACAPsp e c i f i c a l lyi nth eVMNo f th ehypo th a l amu s ,ar eg iono fth eb r a inknowntosp e c i f i c a l lyexp r e s sSF1 .V i ru sw i l lb ed e l iv e r edto th eb r a inv i as t e r eo t ax i cin j e c t ionin toth eVMN .Ad ap t edw i thp e rm i s s ionf romth eau tho r .Sou r c e :� h t tp s : / /www . addg en e .o rg /32396 /.A c c e s s ed M ay4 ,2017 . 117 3 .5REFERENCES Ad am s ,B . ,G r ay ,S . ,I s a a c ,E . ,B i an co ,A . ,V id a lPu ig ,A . ,&Sh e rwood ,N .(2008 ) .F e ed ing and M e t abo l i smin M i c eL a ck ingP i tu i t a ryAd eny l a t eCy c l a s e A c t iv a t ing Po lyp ep t id e .Endo c r ino log y,149 (4 ) ,1571 1580 .h t tp : / /dx . do i .o rg /10 .1210 / en .20070515 Addg en e :p s cAAVGFP.(2017 ) .Addg en e .o rg.R e t r i ev ed4 M ay2017 ,f rom h t tp s : / /www . addg en e .o rg /32396 / A l l enIn s t i tu t efo rB r a inS c i en c e .(2016 ) .In j e c t ion ss i t e sands t e r eo ta x i ccoo rd ina t e sfo r an t e rog rad ep ro j e c tom e(b ra inw id e )(1 s ted . ,p .112 ) .S e a t t l e :A l l enIn s t i tu t efo rB r a in S c i en c e .R e t r i ev edf romh t tp : / /h e lp .b r a in m ap .o rg /d i sp l ay /mou s e conn e c t iv i ty /Do cum en t a t ion An ima lIn j e c t ion–C lod ro som e :L ipo soma lC lod rona t e.(2017 ) .C lod ro som e . com.R e t r i ev ed 4M ay2017 ,f romh t tp s : / /www . c lod ro som e . com / an im a l in j e c t ion / A r imu r a ,A . ,Somogyv á r iV igh ,A . ,M iy a t a ,A . ,M i zuno ,K . ,Coy ,D . ,&K i t ad a ,C .(1991 ) . 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Theob j e c t iv e so fth i sr e s e a r chw e r e : 1 . Tod e t e rm in ei fth eimp a i r edth e rmog en e s i sinPACAP/ -m i c ecou ldb e r e s cu edph a rm a co log i c a l lyw i tham e l ano co r t inagon i s t ,M e l ano t anI I(MT I I ) , andthu sp rov id eev id en c eu s ingag en e t i cmod e lo fPACAPd e f i c i en cyfo rth e conn e c t ionb e tw e enPACAPandth em e l ano co r t insy s t eminr egu l a t ing th e rmog en e s i sinBAT r e a t eanad enov i r a lcon s t ru c tth a texp r e s s e sGFPund e rth er egu l a t iono f 2 . Toc th eSF1p romo t e rtoa c ta sacon t ro lv e c to rtob eu s edins t e r eo t ax i csu rg e ry , thu sen su r ingVMN sp e c i f i cexp r e s s iono fth ev e c to r 3 . Tod ev i s eap ro to co lfo rs t e r eo t ax i cin j e c t ion sth a tr e a ch edth eVMNinl iv e an im a l s Ob j e c t iv e s2and3w e r ed ev i s eda sp r e l im in a rys t ep sfo rth efo l low ingfu tu r eob j e c t iv e : r e a t eanad enov i r a lcon s t ru c tfo rV MNsp e c i f i cexp r e s s iono fPACAPto 4 . Toc d e t e rm in ei fth eimp a i r edth e rmog en e s i sinPACAP/ -m i c ec anb eg en e t i c a l ly r e s cu edbyt r an sg en i cexp r e s s iono fPACAPinth eVMNon ly ,thu s d e t e rm in ingth eimpo r t an c eo fVMNPACAPinr egu l a t ingth e rmog en e s i s 126 � C o ldExp o su r e PACAP MC SNA � �� � VMN B row nA d ipo c y t e H e a t F igu r e4.1 .Hypo th e s i z edp a thw ay so fin t e r a c t ionfo rad ap t iv eth e rmog en e s i sinBATinr e spon s eto co ldexpo su r e .VMN ;v en t rom ed i a lnu c l eu s ,PACAP ;p i tu i t a ryad eny l a t ecy c l a s e a c t iv a t ing po lyp ep t id e , MC ;m e l ano co r t insy s t em ,SNA ;symp a th e t i cn e rv ea c t iv i ty ,BAT ;b rownad ipo s et i s su e . Im ag e sa d ap t edf rom“R ev i s i t ingth eV en t r a lM ed i a lNu c l eu so fth eHypo th a l amu s :Th eRo l e so fSF1N eu ron sinEn e rgyHom eo s t a s i s” ,byY .Cho i ,T .Fu j ik aw a ,J .L e e ,A .R eu t e r , &K .K im ,2013 , F ron t i e r sInN eu ro s c ien c e ,7.Copy r igh t©2013Cho i ,Fu j ik aw a ,L e e ,R eu t e randK im .Ad ap t edw i th p e rm i s s ionf romau tho r . � � � � � � � � � � � � � � � � � � � � 127 Ch ap t e r2add r e s s edth ehypo th e s i sandr e s e a r cha im1th roughth eev a lu a t iono f th e rmog en i cc ap a c i tyo fco ld a c c l im a t ed MT I It r e a t edPA CAP/ -m i c e ,u s ingphy s io log i c a l (bodyw e igh tandcompo s i t ion ,m e t abo l i cr a t e(op enc i r cu i tc a lo r im e t ry ) ,andr e sp i r a to ry ex ch ang er a t io(RER ) ) ,mo l e cu l a r( th e rmog en i cg en eexp r e s s ion ) ,andh i s to log i c a l( l ip ida r e a andc e l lcoun t )an a ly s e s .R e su l t sind i c a t eth a tth rougha c t iv a t iono fth em e l ano co r t insy s t em w i thp e r iph e r a lin j e c t ion so fth e MC4Ragon i s t MT I I ,th eimp a i r edth e rmog en e s i so fco lda c c l im a t edPACAP/ -m i c ew a sp a r t i a l lyr e s cu ed .Th i sf ind ingp rov id e sp r e l im in a ry ev id en c etosuppo r tth ehypo th e s i sth a tPACAPa c t sup s t r e amf romth em e l ano co r t insy s t em inr egu l a t ingSNAtoBATtoindu c ead ap t iv eth e rmog en e s i sdu r ingco ldexpo su r e .Th i s p r e l im in a ryev id en c esuppo r t sth eav a i l ab l e ,bu tl im i t ed ,l i t e r a tu r ew i thth eu s eo fag en e t i c mod e lo fPACAP/ -m i c eandth e r e fo r eadd ss ign i f i c an tv a lu etoth ecu r r en tpoo lo fr e s e a r ch . Fu tu r ean a ly s e sw i l lh e lpsuppo r to rr e fu t eth ep a r t i a lphy s io log i c a lr e s cu eo fad ap t iv e th e rmog en e s i sw i th MT I It r e a tm en to fco ld a c c l im a t edPACAP/ -m i c eandw i l lin c lud e : in c r e a s ings amp l es i z etomo r erobu s t lym e a su r eg en eandp ro t e inexp r e s s iono fth e rmog en i c p ro t e in sinBAT ,ingWAT ,andgWATd epo t s ,andp e r fo rm ingfu r th e rh i s to log i c a lan a ly s e s o fad ipo s et i s su e stoid en t i fych a r a c t e r i s t i c so fth e rmog en i c a l lya c t iv ead ipo s et i s su e( su cha s m e a su r ingl ip idcon t en tandb rown inginingWATandgWATd epo t s ) . Ch ap t e r3add r e s s edth ehypo th e s i sth roughr e s e a r cha im2and3 ,wh i chw e r ev i t a l p r e l im in a rys t ep sfo rth efu tu r er e s e a r cha im4 .A im2en su r edth a tth econ s t ru c t sfo rth e VMNsp e c i f i cad eno a s so c i a t edv i ru s e sw e r ep rop e r lyd e s ign edandc r e a t edsu chth a tfu tu r e v i r a lin j e c t ion sin toth eVMNv i as t e r eo t ax i csu rg e ryw i l lp rov es a f eande f f e c t iv e .A im3 fu r th e ren su r eds a f eande f f e c t iv es t e r eo t ax i csu rg e ryth roughth ed ev e lopm en to fapp rop r i a t e s t e r eo t ax i cin j e c t ionp ro to co l s .P ro to co l sw e r ed ev i s edth roughth ea t t end an c eo fs t e r eo t ax i c su rg e rywo rk shop s ,a sw e l la sth roughv i su a l i z a t iono fth eb r a inpo s t in j e c t ion sto 128 p r e l im in a r i lya s s e s sth ee f f i c a cyo fth eICV VMNin j e c t ion s .Th eov e r a r ch ingfu tu r ea imo f th i sp ro j e c ti stog en e t i c a l lyr e in t rodu c ePACAPinPACAP/ -m i c einn eu ron so fth eVMN on ly ,anda s s e s si fth eimp a i r edth e rmog en e s i si simp rov ed(o rr e s cu ed ) .Ch ang e sin th e rmog en i cc ap a c i tyw i l lb em e a su r edv i aphy s io log i c a landb io ch em i c a la s s e s sm en t so f th e rmog en i cm a rk e r su s edinCh ap t e r2 . 4.2S IGN IF ICANCE Ob e s i tyi sbo thad eb i l i t a t ingd i s e a s ea sw e l la sar i skf a c to rfo ranumb e ro fo th e r ch ron i cm e t abo l i cd i s e a s e s ;th econ s equ en c e so fwh i chr ang ef romd e c r e a s edqu a l i tyo fl i f e top r em a tu r ed e a th( L id e l l ,B e t z, &En e rbä ck ,2014 ; WHO ,2016 ) .R ep e r cu s s ion so fth e d i s e a s ea r eh igh ,bo tha tth eind iv idu a ll ev e lw i thp a t i en tsu f f e r ing ,a sw e l la sth eso c i e t a l l ev e lw i thin c r e a s ede conom i cbu rd en(L id e l le ta l . ,2014 ,U zog a r a ,2017 ) .Top rov id e app rop r i a t ep r ev en t ionandm an ag em en tfo rth i sd i s e a s e ,th em e ch an i sm so fa c t ionr e su l t ing inth ed i s e a s emu s tin i t i a l lyb eund e r s tood .W i thob e s i ty ,th ed i s rup t edm e ch an i smin qu e s t ioni sen e rgyhom eo s t a s i s ,con t ro l l edbo thbyen e rgyin t ak eanden e rgyexp end i tu r e (F a rooq i&O ’R ah i l ly ,2006 ;H inn ey ,Vog e l ,&H eb eb r and ,2010 ) .D e sp i t el i f e s ty l e in t e rv en t ion sth a tsugg e s td e c r e a s ingfoodin t ak eandin c r e a s ingphy s i c a la c t iv i ty ,th e p r ev a l en c eo fadu l tob e s i tycon t inu e stor i s e(G a l i ce ta l . ,2010; WHO ,2016 )Th e r e fo r e ,i t h a sb e com eev id en tth a tfu r th e rexp lo r a t ionin toth ep a thophy s io logyo fth ed i s e a s ei s n e c e s s a rytop r ev en tandm an ag eth i sd i s e a s ee f f e c t iv e ly(G a l i ce ta l . ,2010; WHO ,2016 ) . H e r e ,th er e s e a r chw a sfo cu s edonen e rgyexpend i tu r e ,wh i chi scomp r i s edo fm anysy s t em s andm e ch an i sm s ,on eo fwh i chi sad ap t iv eth e rmog en e s i sinBAT(B lond ine ta l . ,2014 ; L id e l le ta l . ,2014 ;P e i r c ee ta l . ,2014 ) .Th eg r e a t e s tphy s io log i c a ls t imu lu sr egu l a t ing ad ap t iv eth e rmog en e s i si sv i aSNAwh e r eth er e l e a s eo fNEa c t sd i r e c t lya tth el ev e lo f b rownad ipo cy t e stog en e r a t eh e a tinth ebodyv i ath eth e rmog en i cp ro t e in ,UCP1(L id e l le t 129 a l . ,2014 ;P e i r c ee ta l . ,2014 ;S a i to ,Yon e sh i ro ,& M a t su sh i t a ,2016 ) .Fu r th e rmo r e ,a s p ropo s edinth i sth e s i s ,i ti shypo th e s i z edth a tPACAPandth em e l ano co r t insy s t ema c tinth e VMNtor egu l a t eSNAth a td r iv e sad ap t iv eth e rmog en e s i s(D i anée ta l . ,2014 ;G r aye ta l . , 2002 ;T an id a ,Sh in t an i ,&H a sh imo to ,2011 ) .U l t im a t e ly ,und e r s t and ingth ero l eo fPACAP inth eVMNandi t sa s so c i a t ionw i thth em e l ano co r t insy s t eminr egu l a t ingSNAtos t imu l a t e ad ap t iv eth e rmog en e s i sinBATw i l lp rov id efu r th e rknow l edg er eg a rd ingon eo fth em any p a thw ay sr egu l a t ingen e rgyexp end i tu r e .B e c au s em e t abo l i cd i s e a s e s ,in c lud ingob e s i ty ,a r e ex amp l e so fdy s r egu l a t eden e rgyhom eo s t a s i s ,d e t e rm in inghowandwh e r eno rm a l phy s io logygo e saw ry(p a thophy s io logy )w i l lpo t en t i a l lyp rov id eth e r ap eu t i ct a rg e t stoh e lp co r r e c tth e s ed e t r im en t a ld i s e a s e s ,e i th e rth roughl i f e s ty l ein t e rv en t ion s ,ph a rm a co log i c a l in t e rv en t ion s ,o rbo th . Fo rex amp l e ,b e c au s ea c t iv a t iono fth esymp a th e t i cn e rvou ssy s t em(SNS )h a sb e en showntoin c r e a s eBATa c t iv a t ionandth e rmog en e s i s ,an t i ob e s i typh a rm a c eu t i c a l ssu cha s s ibu t r am in eandeph ed r aw e r ed ev e lop edtos t imu l a t eth eSNS(S a i to ,Yon e sh i ro ,& M a t su sh i t a ,2016 ) .How ev e r ,r e s e a r chh a sshownth a ta c t iv a t ingth eSNSa lon eth roughth e u s eo fth e s eph a rm a c eu t i c a l si sno tanapp rop r i a t eop t ionfo rw e igh tlo s s ,a sth eya r eknown toc au s eund e s i r ab l eandd ang e rou ss id ee f f e c t sin c lud ingh ep a to tox i c i ty ,c a rd iov a s cu l a r comp l i c a t ion s ,andsudd end e a th(Y en ,&Ew a ld ,2012 ) .A ssu ch ,fu r th e rr e s e a r chin to p a r t i cu l a rm e ch an i sm so fen e rgym e t abo l i smw i l la l lowfo rth eop t im i z a t iono fth e th e rmog en i cc ap a c i tyo fBATw i thou ts id ee f f e c t sd e r iv edw i thSNSa c t iv a t ion .Po t en t i a l ly , th i sm ayb eth roughs e l e c t iv es t imu l a t iono fth eSNStoBAT ,o th e rmod e so fBATa c t iv a t ion b eyondth eSNS ,o ro th e rav enu e so fen e rgyhom eo s t a s i sa l tog e th e r .A l tog e th e r , und e r s t and ingth eb io log i c a lm e ch an i sm sth a tcon t r ibu t etoth eh e a l thc r i s i so fob e s i tyi s c r i t i c a lind ev e lop ingin t e rv en t ion stor edu c ei t sp r ev a l en c e . 130 L a s t ly ,r eg a rd l e s sa stowh e th e rapp rop r i a t el i f e s ty l ein t e rv en t ion so rth e r ap eu t i c t a rg e t sa r ed e r iv edf romth i sa r e ao fr e s e a r ch ,th e r ei ss t i l la nab so lu t e lyc l e a rb en e f i t :th a ti s , p rov id ingev id en c eth a tth ed i s e a s eo fob e s i tyinvo lv e smo r eth ans imp lyin c r e a s ingon e ’ s s e l f con t ro l ,e a t ingl e s s ,andex e r c i s ingmo r e .H e lp ingtoe l im in a t eth i ss t igm abyp rov id ing phy s io log i c a lev id en c eth a ten e rgym e t abo l i smi sd e ep lys e ed edinho rmon a lcon t ro lth rough th ebody ’ sm e t abo l i csy s t em si sanin con t rov e r t ib l eadv an c em en tr e su l t ingf romth i sa r e ao f r e s e a r ch . 131 4.3REFERENCES B lond in ,D . ,L abb é ,S . ,Pho en ix ,S . ,Gu é r in ,B . ,Tu r co t t e ,É . ,&R i ch a rd ,D .e ta l .(2014 ) . 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