Brain-derived neurotrophic factor regulates energy balance downstream of melanocortin-4 receptor

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1 Brin-erive neurotrophi ftor regultes energy lne ownstrem of melnoortin-4 reeptor Boji Xu 1,5,Evn H Gouling 2,Keling Zng 1,Dvi Cepoi 3,Roger D Cone 3,Kevin R Jones 4,Lurene H Teott 2 & Louis F Reihrt 1 The melnoortin-4 reeptor (MC4R) is ritilly involve in regulting energy lne, n oesity hs een oserve in mie with muttions in the gene for rin-erive neurotrophi ftor (BDNF). Here we report tht BDNF is expresse t high levels in the ventromeil hypothlmus (VMH) where its expression is regulte y nutritionl stte n y MC4R signling. In ition, similr to MC4R mutnts, mouse mutnts tht expresses the BDNF reeptor TrkB t qurter of the norml mount showe hyperphgi n exessive weight gin on higher-ft iets. Furthermore, BDNF infusion into the rin suppresse the hyperphgi n exessive weight gin oserve on higher-ft iets in mie with efiient MC4R signling. These results show tht MC4R signling ontrols BDNF expression in the VMH n support the hypothesis tht BDNF is n importnt effetor through whih MC4R signling ontrols energy lne. Neurotrophins re fmily of struturlly relte growth ftors, inluing rin-erive neurotrophi ftor (BDNF), tht exert mny of their effets on neurons through Trk reeptor tyrosine kinses. Among these, BDNF n its reeptor TrkB re the most wiely n unntly expresse in the rin. BDNF hs een shown to regulte neuronl evelopment n to moulte synpti plstiity 1.Oesity phenotypes hve een oserve in BDNF heterozygous mie n in mie in whih the BDNF gene hs een elete in exittory neurons in the rin 2 4.These mutnts lso show hypertivity, hyperleptinemi, hyperinsulinemi, hyperglyemi n inrese liner growth 3,4.Moreover, oth entrl n peripherl ministrtion of BDNF erese foo intke, inrese energy expeniture n meliorte hyperinsulinemi n hyperglyemi in ieti / mie 5 8.However, the mens y whih BDNF lters energy lne n the reltionship of TrkB signling to the mjor pthwys previously shown to e involve in the regultion of energy lne remin unknown. The sttus of energy lne is ommunite to the hypothlmus through neuronl n hormonl signls. Severl hypothlmi nulei re involve in the regultion of energy lne, inluing the ventromeil (VMH), prventriulr (PVN) n rute (ARC) hypothlmi nulei n the lterl hypothlmi re (LH). Although the oservtion tht ilterl VMH lesions proue hyperphgi n oesity hs implite this region in the regultion of energy lne 9 11, the neurl mehnisms through whih the VMH funtions to influene energy lne re not ler. One of the mjor signls tht serves s monitor of energy lne is leptin, polypeptie generte in ipoytes 12. One of the min trgets of leptin in the hypothlmus is the ARC, region ontining t lest two istint popultions of neurons One popultion of neurons expresses two orexigeni polypepties, neuropeptie Y (NPY) n gouti-relte protein (AgRP), wheres the other expresses the norexigeni polypepties proopiomelnoortin (POMC) n oine n mphetmine regulte trnsript (CART). Leptin suppresses foo intke n inreses energy expeniture t lest prtilly y positively regulting the suset of neurons expressing POMC n CART n y negtively regulting the suset of those expressing NPY n AgRP. Other metoli signls prt from leptin regulte the POMC-expressing neurons in the ARC; these inlue gluose, insulin, the gut peptie PYY 3 36 n serotonin These melnoortin-expressing neurons thus seem to serve s one of the onuits for signls relevnt to energy lne 21. Two melnoortin reeptors, MC4R n melnoortin 3 reeptor (MC3R), re expresse in the rin n ply ritil role in the ontrol of energy lne, espeilly the MC4R. In the gouti lethl yellow mutnt (A y ), etopi expression of the gouti protein ( homologue of AgRP) uses hyperphgi n oesity 22,23.The phenotype of the MC4R / null mutnt is lmost ientil to tht of the A y mutnt, n onsistent with this, the gouti protein hs een shown to e n ntgonist of MC4R, ut not MC3R Hyperphgi resulting from norml feeing response to ietry ft proly ontriutes signifintly to oesity in the MC4R / mie 27. Here we report tht MC4R- n TrkB-meite signling re ouple in the regultion of energy lne. Our t show tht signling through the MC4R ontrols BDNF expression in the VMH, supporting moel in whih BDNF funtions s n importnt effetor through whih MC4R signling regultes energy lne. 1 Howr Hughes Meil Institute, 2 Center for Neuroiology n Psyhitry, University of Cliforni, Sn Frniso, Cliforni 94143, USA. 3 Vollum Institute, Oregon Helth n Siene University, Portln, Oregon 97201, USA. 4 Deprtment of Moleulr, Cellulr n Developmentl Biology, University of Coloro, Bouler, Coloro 80309, USA. 5 Present ress: Deprtment of Phrmology, Georgetown University Meil Center, SE402 Meil/Dentl Builing, 3900 Reservoir R. NW, Wshington, DC 20057, USA. Corresponene shoul e resse to L.F.R. (lfr@gl.usf.eu) or B.X. (x3@georgetown.eu). 736 VOLUME 6 NUMBER 7 JULY 2003 NATURE NEUROSCIENCE

2 RESULTS BDNF in the VMH is n norexigeni ftor Expression of BDNF ws exmine in the hypothlmus y using BDNF lz mouse strin in whih the reporter gene for β-gltosise, lz, is trgete to the BDNF lous 28. BDNF is moestly expresse in some ells of the orsomeil hypothlmus (DMH), ut not in the ARC (Fig. 1). Only smll numer of ells express BDNF in the LH. Consistent with in situ t 3, BDNF is highly expresse in the VMH. Sine ilterl VMH lesions proue hyperphgi n oesity 9 11, this expression pttern suggests tht BDNF in the VMH my suppress foo intke n therefore my e regulte y feeing sttus. To test this, we exmine BDNF expression in fooeprive wil-type (WT) C57BL/6 mie with in situ hyriiztion. Foo eprivtion hs een shown to inrese expression of orexigeni neuropepties while eresing expression of norexigeni neuropepties 13,15,29.Foo eprivtion speifilly reue the level of BDNF mrna y 60% in the VMH (Fig. 1 ). In ontrst, expression ws not ffete in the neoortex (Fig. 1). Coexpression of TrkB n neuropepties in the hypothlmus BDNF might regulte energy lne through iret regultion of neuropepties known to ontrol energy lne. If so, the TrkB reeptor shoul e oexpresse with these neuropepties. We exmine TrkB expression in the hypothlmus y using ult trkb lz /+ mie in whih tu lz reporter gene is inserte into trkb lous 30,repitulting the expression pttern of TrkB. Sine the ARC n LH re two importnt rin regions in the ontrol of feeing, we exmine oexpression of TrkB n neuropepties in these two nulei. There ws little oexpression of TrkB with melnin-onentrting hormone (MCH) or orexin in the LH n lmost no expression of TrkB in neurons tht express CART or NPY in the ARC (Fig. 2 ). Therefore, it is unlikely tht BDNF iretly moultes CART/POMC- n NPY/AgRP-expressing neurons in the ARC or MCH- n orexin-expressing neurons in the LH. Figure 1 Energy sttus regultes levels of BDNF in the VMH. () Expression of BDNF in the hypothlmus ws exmine y using n ult BDNF lz /+ mouse. Brin setions were stine for β-gltosise with X-gl stining n ounterstine with nuler fst re. BDNF is highly expresse in the VMH. Ar, rute nuleus; DMH, orsomeil hypothlmus; f, fornix; LH, lterl hypothlmus; mt, mmmillothlmi trt; VMH, ventromeil hypothlmus; ZI, zon inert. Sle r, 100 µm. (,) Representtive rin setions of BDNF in situ hyriiztion show tht BDNF expression is speifilly reue in the VMH of foo-eprive mie. The in situ proe ws 35 S-lele ntisense RNA omplementry to the oing region of BDNF trnsripts. Arrows inite the lotion of the VMH. () The optil ensity of BDNF in situ signls ws mesure with NIH imge softwre in two setions of eh niml. Eh group ws ompose of eight femle C57BL/6J wil-type mie t 9 weeks of ge. One group of mie h free ess to mouse how, n the other group ws eprive of foo for 48 h. The neoortex in the sme setion ws use s ontrol. **P < 0.01 (twotile Stuent s t-test). kinse t pproximtely qurter of the norml mount in norml pttern throughout the rin 30,31. The trkb hypomorph eomes severely oese (Fig. 3). The fbz llele ws krosse to the C57BL/6 geneti kgroun for t lest four genertions efore it ws use to generte fbz homozygous mie for hrteriztion of the oesity synrome. At 5 weeks of ge, the trkb mutnt strte to show higher oy weight ompre to its wil-type littermte (Fig. 3,). By 12 weeks, oth femle n mle trkb mutnts were mrkely hevier thn their wil-type littermtes. The oy weight of fbz heterozygous mie ws similr to tht of the wil-type littermtes, initing tht fbz is reessive muttion. To etermine whether weight gin in the trkb mutnt is ue to inrese iposity, we issete n weighe four ifferent ft ps from three femle fbz/fbz mie n three femle wil-type littermtes t 12 weeks of ge. The mss of eh ws gretly inrese in the trkb hypomorph (rown ft, 0.92 ± 0.10 g in the mutnt vs ± 0.06 g in the WT ontrol, P < 0.01; suutneous ft, 5.08 ± 1.43 g vs ± 0.16 g, P < 0.01; uterine ft, 5.38 ± 2.63 g vs ± 0.12 g, P < 0.05; perirenl ft, 1.14 ± 0.11 g vs ± 0.02 g, P < 0.01). The trkb mutnt showe ltere liner growth when oy length (nso nl) ws mesure t pproximtely 3 months of ge. The men lengths of mle n femle fbz/fbz mie were inrese pproximtely 8% n 13%, respetively, ompre to ontrols (Fig. 3). Reution in TrkB expression les to oesity We previously esrie the genertion y insertion of trkb DNA into the first oing exon of the trkb lous of trkb hypomorph, fbz/fbz, tht only expresses the full-length TrkB reeptor tyrosine Figure 2 Coexpression of TrkB n neuropepties in the hypothlmus. Expression of TrkB ws exmine y using n ult trkb lz /+ mouse. Most TrkB-expressing neurons o not express MCH () or orexins () in the lterl hypothlmus n CART () or NPY () in the ARC. White rrows inite representtive neurons expressing MCH, orexin, CART or NPY. White rrowhes inite representtive TrkB-expressing ells. A yellow rrow enotes ell expressing oth TrkB n neuropepties. Sle r, 20 µm. NATURE NEUROSCIENCE VOLUME 6 NUMBER 7 JULY

3 Figure 3 The trkb hypomorphi mutnt shows mturity-onset oesity n inrese liner growth. () The fbz/fbz mutnt is oese. The two mie re femle littermtes t 3 months of ge. () Both femle n mle trkb mutnt mie show inrese liner growth. Mesurements were from four pirs of femle n three pirs of mle mie t out 3 months of ge. Error rs re stnr error of the men (s.e.m.). **P < 0.01 y Stuent s t-test. () Weight gin of femle homozygous fbz/fbz mutnt mie (n = 7), heterozygous fbz/+ mie (n = 7) n wil-type (+/+) ontrols (n = 5). When the femle mutnt mie were 5 weeks or oler, their oy weights were signifintly higher thn those of their femle +/+ or fbz/+ littermtes (P < 0.05 y two-tile Stuent s t-test). () Weight gin of mle homozygous fbz/fbz mutnt mie (n = 6), heterozygous fbz/+ mie (n = 4) n wil-type (+/+) ontrols (n = 5). When the mle mutnt mie were 7 weeks or oler, their oy weights were signifintly higher thn those of their mle +/+ or fbz/+ littermtes (P < 0.05 y two-tile Stuent s t-test). Feeing ehvior of the trkb hypomorph The evelopment of oesity in the trkb mutnts inites tht these nimls hve elevte energy intke reltive to energy expeniture. Phenotypi effets on energy onsumption were first exmine y etermining the ily foo intke of the trkb mutnts t 5 n 9 weeks of ge. The mutnts showe mrke hyperphgi t oth ges, onsuming 75% more foo thn ontrols (Fig. 4). This likely ontriutes sustntilly to their evelopment of oesity. To etermine the impt of the trkb muttion on ptterns of ingestive ehvior, mie were hitute to single housing for 6 in ges equippe to ontinuously monitor feeing n rinking tivity. Mie were then monitore for n itionl 8, n t from these ys were verge for exmintion of intke ptterns. The intke ptterns re shown for mutnt n wiltype littermte pir (Fig. 4,). Similr to the ptterns oserve for this pir, mutnts frequently engge in prolonge perios of feeing n rinking in omprison to wil-type ontrols. These episoes were most frequent uring the rk yle ut lso ourre uring the light yle. In ition, prolonge perios of quiesene were ommonly oserve uring the light yle. Aompnying ltertions in the irin istriutions of feeing n rinking were lso seen, suh tht the perentges of totl foo n wter intke uring the rk yle were erese in the mutnt mie (how: WT 78 ± 2%, fbz/fbz 64 ± 3%, P = 0.001; wter: WT 84 ± 2%, fbz/fbz 66 ± 3%, P < 0.001). Overll, the trkb mutnts were foun to e signifintly hyperphgi n hyperipsi uring oth the light n rk yles (Fig. 4,e). MC4R regultes BDNF expression in the VMH These phenotypi nlyses inite tht the trkb hypomorph is remrkly similr to the MC4R / null mutnt in evelopment of oesity. The trkb hypomorph n MC4R / mutnt show mturity-onset oesity n inrese liner growth. In ition, they hve similr sex ifferenes in the oesity synrome. For exmple, femle n mle fbz/fbz mie, on verge, re 107% n 50% hevier thn their sex-mthe wil-type littermtes t 12 weeks of ge (Fig. 3,). Similrly, femle n mle MC4R / null mie, on verge, re 100% n 50% hevier thn their sex-mthe wil-type littermtes t 15 weeks of ge 24.Moreover, liner growth in these two mutnts is inrese over wil-type mie to similr extent (13% in fbz/fbz vs. 11% in MC4R / in femles; 8% in fbz/fbz vs. 7% in MC4R / in mles). Figure 4 Hyperphgi n hyperipsi of the trkb hypomorphi mutnt mie. () The foo intke of iniviully house mie ws mesure every y over one-week perio. The rs represent men of five mesurements for eh of five fbz/fbz mie (2 mle, 3 femle) n five sex-mthe littermte ontrols (+/+ or fbz/+). We use ifferent mie for foo intke ssys t the 5- week n 9-week stges. Error rs re s.e.m. Two-tile Stuent s t-test, *P < 0.05, **P < (,) Dily pttern of how intke n wter intke for wil-type () n mutnt littermte () in 6-min ins. Eh in represents the verge of 8 of intke t tht irin time. These verges were then smoothe y entere five-in moving verge. () Averge rk n light yle how intke uring 8 of home ge ehviorl monitoring. For ll home ge ehviorl monitoring, error rs inite s.e.m. for mutnt (n = 9) n wil-type (n = 10) mie, n omprisons were me y 2 2 repete-mesures ANOVA (genotype, yle). e There ws signifint effet of yle (F 1,17 = 109.4, P < 0.001) n genotype (F 1,17 = 60.8, P < 0.001), ut no intertion etween yle n genotype. (e) Averge rk n light yle wter intke uring 8 of home ge ehviorl monitoring. There ws signifint effet of yle (F 1,17 = 181.9, P < 0.001) n of genotype (F 1,17 = 98.0, P < 0.001) ut no intertion etween yle n genotype. 738 VOLUME 6 NUMBER 7 JULY 2003 NATURE NEUROSCIENCE

4 To exmine whether MC4R signling regultes BDNF expression in the VMH, we first etermine whether POMC- or AgRP-expressing neurons projet to the VMH in mie. We use ntioies to gmmmelnoyte-stimulting hormone (γmsh), POMC-erive peptie, to revel POMC fiers (Supplementry Fig. 1 online). Consistent with wht hs een reporte in rts 32, the numer of POMC-immunoretive fiers in the VMH is fewer thn the numer in the DMH, ut is omprle to the numer oserve in the LH.As the POMC fiers in the VMH ontin outons, these fiers must innervte VMH neurons. Similrly, VMH neurons lso reeive innervtion from AgRP-immunoretive fiers (Supplementry Fig. 1 online). In ition, there re sustntil numers of POMC n AgRP fiers surrouning the VMH, whih my innervte enrites extening from neurons within the VMH. Next we quntifie expression of BDNF y using in situ hyriiztion in the A y mutnt in whih etopilly expresse gouti loks the funtion of the MC4R 22,23,26.Compre to sex-mthe wil-type littermtes, BDNF expression in the VMH ws reue y 30 40% in the A y mutnt (Fig. 5). The t suggest tht reution in MC4R signling results in ownregultion of BDNF expression in the VMH. We lso use in situ hyriiztion to quntify BDNF expression in the VMH of MC4R / mie, emonstrting tht it is lso signifintly reue in the VMH (Fig. 5). To etermine whether MC4R signling utely regultes the level of BDNF mrna in the VMH, we use wil-type mie to exmine the possiility tht ministrtion of melnoortin reeptor gonist reverses the inhiitory effet of foo eprivtion on BDNF expression in the VMH. Similr to the results shown in Fig. 1, 47 h of fsting resulte in n pproximtely 65% reution in the level of BDNF mrna in the VMH tht ws not prevente y intrereroventriulr injetion of rtifiil ererospinl flui (ACSF; Fig. 5). Compre to ACSF ministrtion lone, however, ministrtion of n MC4R gonist, MTII, signifintly inrese (y 70%) the level of BDNF mrna in the VMH of foo-eprive mie within 3 h of tretment (Fig. 5). Although the level of BDNF mrna in the VMH of MTII-trete fooeprive mie ws still signifintly lower thn tht in the VMH of vehile-trete fe mie, these results inite tht the melnoortin pthwy is t lest prtilly responsile for ownregultion of VMH BDNF uring foo eprivtion. Figure 5 Melnoortins regulte the levels of BDNF mrnas in the VMH. () Expression of BDNF in the VMH is signifintly reue in the A y mutnt. Expression levels of BDNF in the VMH were etermine with in situ hyriiztion on C57BL/6J A y / mie n C57BL/6J wil-type littermtes t 7 9 weeks of ge. In the first experiment, we use four mle wil-type mie (25.9 ± 1.7 g) n three mle A y / mie (28.5 ± 1.3 g). In experiment 2, we use two mles n one femle for eh genotype. () Defiieny in MC4R signling reues expression of BDNF in the VMH. Expression levels of BDNF in the VMH were etermine with in situ hyriiztion on mle MC4R / null mutnts (n = 6) n C57BL/6J wil-type mie (n = 6) t 10 weeks of ge. () Intrereroventriulr injetion of MTII inrese the levels of BDNF mrnas in the VMH of foo-eprive mie. ACSF (n = 5) or MTII (n = 4) ws injete into the thir ventrile of foo-eprive femle C57BL/6J wil-type mie t 10 weeks of ge. As ontrol, ACSF ws lso injete into the thir ventrile of normlly fe femle C57BL/6J mie (n = 5). The levels of BDNF in the VMH were etermine with in situ hyriiztion on ryostt rin setions s esrie in Methos. Two-tile Stuent s t-test, *P < 0.05, **P < MC4R regultes BDNF expression in seletive VMH neurons Neurons in hypothlmi nulei re extremely heterogeneous. To etermine whether etopi expression of gouti reues expression of BDNF in ll BDNF-expressing VMH neurons or seletively olishes BDNF expression in only some, we rosse the BDNF lz /+ mouse to the A y mutnt n use β-gltosise s mrker to exmine the effet of gouti on expression of BDNF. Compre with BDNF lz /+ mie, fewer neurons expresse BDNF in the VMH of A y /;BDNF lz /+ mie (Fig. 6 ). Furthermore, it ppere tht the numer of neurons expressing BDNF in the ul prt of the VMH ws more severely reue in A y mie (Supplementry Fig. 2 online). The effet of gouti on BDNF expression seems speifi to the VMH. BDNF expression in the A y mutnt remins norml in the DMH, LH (Fig. 6,), PVN Figure 6 Melnoortins regulte expression of BDNF in seletive popultions of neurons in the VMH. (,) Expression of BDNF in the hypothlmus of BDNF lz /+ n A y /;BDNF lz /+ mie. Expression of BDNF is revele with nti-β-gltosise immunohistohemistry in BDNF lz /+ mie. Sle r, 200 µm. (,) Expression of BDNF is reue in seletive popultions of neurons in the VMH of the A y mutnt. Sme mgnifition in f, h n i; sle r in is 100 µm. (e,f) Expression of BDNF in the PVN is not ffete in the A y mutnt. (g) MTII inue expression of BDNF in the VMH of A y /;BDNF lz /+ mie. BDNF-expressing neurons in the VMH were ounte on setions (pproximtely regm 1.8 mm) from sline-injete (n = 5) n MTII-injete (n = 5) mie. Two-tile Stuent s t-test, *P < (h,i) Representtive setions of sline- n MTII-injete A y /;BDNF lz /+ mie. There re more intensely stine neurons in the outline re of the VMH from the MTII-injete mouse. PVN, prventriulr hypothlmus. e f g h i NATURE NEUROSCIENCE VOLUME 6 NUMBER 7 JULY

5 Figure 7 TrkB ts ownstrem of MC4R to regulte feeing response to ietry ft. (,) Boy weight n energy intke of femle fbz/fbz (n = 4) n ontrol littermtes (+/+ or fbz/+, n = 4). The verge weights of the fbz/fbz n ontrol mie t the strt of the experiment were 21.7 ± 1.1 n 19.0 ± 0.6 g, respetively. () Averge ily energy intke of fbz/fbz (n =8) n ontrol littermtes (n = 9) on the low-ft n moerte-ft iets. The ily energy intke on two iets is n verge from 7 on the low-ft iet n 7 on the moerte-ft iet, respetively. () BDNF infusion i not ffet energy intke of the A y mie on the low-ft iet, ut i suppress their energy intke on the moerte-ft iet. The ily energy intke on the low-ft iet n moerte-ft iet is n verge from the first 3 n the lst 8 of the stuy, respetively. (e) Weight gin of wil-type mie trete with PBS (n = 5) or BDNF (n = 5) n A y mie trete with PBS (n = 7) or BDNF (n = 5). Infusion of BDNF suppresse weight gin of A y mie on the moerte-ft iet. (f) Boy weight hnges of wiltype n A y mie trete with either PBS or BDNF over 9- perio on the moerte-ft iet. Vlues represent men ± s.e.m. Stuent s t-test, *P < 0.05, **P < (Fig. 6e,f), hippompus n ererl ortex (t not shown). Reution in the numer of BDNF expressing neurons in the VMH is unlikely to e ue to ell eth euse resyl violet stining shows norml VMH in the A y /;BDNF lz /+ mutnt (t not shown). To further onfirm tht melnoortins utely regulte BDNF expression in the VMH, we injete MTII into the orsl thir ventrile of A y /;BDNF lz /+ mie. MTII injetion inrese the numer of intensely stine neurons in the re of the VMH where expression of BDNF is reue in the A y mie (Fig. 6h,i). Compre to sline-injete rins, the numer of neurons expressing β-gltosise ws inrese y 39% in the ul VMH of MTII-trete rins (P = 0.017) (Fig. 6g). Thus, results inite tht n MC4R gonist utely inreses expression of BDNF in set of VMH neurons, even in the presene of the MC4R ntgonist gouti. This is onsistent with the previous oservtion tht MTII injetion signifintly inhiits foo intke in A y mie 33. TrkB regultes feeing response to ietry ft On the sis of these finings, we resone tht BDNF my e ownstrem omponent in the MC4R-meite ontrol of energy lne. MC4R is require for the feeing response to ietry ft 27.Trnsition from low-ft iet (Purin iet 5001, 12.8% ft lories) to moerteft iet (Purin iet 5015, 25.1% ft lories) les to rmti inrese in energy intke n rte of weight gin in the MC4R / mie, ut oes not inrese the rte of weight gin in wil-type mie. In ontrst, leptin-efiient o/o mie re similrly hyperphgi on oth the lowft n moerte-ft iets n o not exhiit n inrese in the rte of weight gin on the moerte-ft iet 27.To exmine whether mie with efiient BDNF-meite signling exhiit n norml response to the moerte-ft iet similr to tht of MC4R / mie, the weight n foo intke of the nimls were monitore for 3 on the low-ft iet n then for 7 on the moerte-ft iet, followe y nother 4 on the low-ft iet. Femle fbz/fbz mie gine weight to similr extent s ontrol mie (WT or fbz/+ mie) on the low-ft iet, ut gine weight t mrkely inrese rte on the moerte-ft iet (Fig. 7). Mle fbz/fbz mie showe similr norml response in weight gin to the iet trnsition (t not shown). e f The weight gin in the fbz/fbz mie ws ssoite with shrp inrese in energy intke on the moerte-ft iet (Fig. 7). The trkb mutnt mie n their littermte ontrols onsume similr mount of the low-ft iet (Fig. 7; 4.9 ± 0.2 g for ontrols vs. 5.4 ± 0.4 g for fbz/fbz, P = 0.307). Trnsition from the low-ft to the moerte-ft iet le ontrol mie to signifintly reue foo intke (4.9 ± 0.2 g on the low-ft iet vs. 3.9 ± 0.2 g on the moerte-ft iet, P = 0.003), therey mintining similr energy intke level (Fig. 7,). In ontrst, the fbz/fbz mie gretly inrese foo intke fter the iet ws swithe (Fig. 7). On verge, the fbz/fbz mie ingeste 71% more lories on the moerte-ft thn on the low-ft iet (Fig. 7). These results inite tht tivtion of the TrkB reeptor is require for the norml feeing response to elevte ietry ft. We then etermine whether BDNF is ownstrem of MC4R y ttempting to suppress with infusion of BDNF into the rin hyperphgi n the inrese rte of weight gin oserve in A y mie on the moerte-ft iet. For two weeks, PBS or BDNF (50 ng/h) were infuse into the lterl ventriles of femle A y n WT C57BL/6 littermtes using osmoti pumps. Trnsition from the low-ft to the moerte-ft iet le to n inrese in energy intke in the PBS-trete A y mie (Fig. 7; 11.2 ± 0.4 kl on the low-ft iet vs ± 0.5 kl on the moerte-ft iet, P < 0.001). BDNF infusion signifintly suppresse hyperphgi in the A y mie on the moerte-ft iet without ltering energy intke on the low-ft iet (Fig. 7). Trnsition from the low-ft to the moerte-ft iet le to fster weight gin in the PBStrete A y mie thn in the PBS-trete, wil-type mie. BDNF infusion reue the rte of weight gin in these A y mie on the moerte-ft iet to level similr tht oserve in wil-type mie (Fig. 7e). Over the 9- perio on the moerte-ft iet, BDNF infusion i not signifintly reue weight gin in wil-type mie (P = 0.244), ut gretly reue it in the A y mie from 15.3% to 7.6% (P = ) (Fig. 7f). The t support moel in whih BDNF t lest prtilly meites the effet of MC4R on the feeing response to ietry ft. DISCUSSION Although ilterl lesions in the VMH proue hyperphgi n oesity, the mehnism through whih VMH neurons ontrol energy lne is not known. Our results show tht BDNF is highly expresse in 740 VOLUME 6 NUMBER 7 JULY 2003 NATURE NEUROSCIENCE

6 the VMH where its expression is regulte y nutritionl stte. We re not wre of nother moleule in the VMH tht is regulte y foo eprivtion. Furthermore, our results show tht MC4R-meite signling stimultes expression of BDNF in the VMH n tht infusion of BDNF into the rin resues the norml feeing response of A y mie to ietry ft. These results suggest tht BDNF is one of the moleules tht meite the funtion of the VMH in the ontrol of energy lne. As BDNF is key regultor of neuronl evelopment, neuroevelopmentl nomlies my ontriute to the oesity phenotype in BDNF n trkb mutnt mie. Sustntil eviene inites, however, tht the efet of energy homeostsis in these mutnts oes reflet n ult funtion of BDNF. First, infusion into the ult rin of BDNF, ut not NGF or NT-3, reverses hyperphgi n oesity of BDNF +/ mie 3.Seon, eletion of the BDNF gene in the rin, using re trnsgene uner the ontrol of the C 2+ /lmoulinepenent kinse II (CMKII) promoter les to oesity 4.Most evelopmentl proesses hve een omplete efore this promoter is first tivte uring the thir week of postntl evelopment. Thir, ministrtion of BDNF inreses energy expeniture in / mie 6.Fourth, foo eprivtion speifilly reues the level of BDNF mrna in the VMH (Figs. 1 n 5). Finlly, MC4R signling regultes BDNF expression in the VMH (Figs. 5 n 6). Therefore, the oesity phenotypes oserve in BDNF n trkb mutnts nnot ompletely result from evelopmentl nomlies. There re severl possile mehnisms through whih MC4R signling oul influene BDNF expression in the VMH. One possiility is iret signling through MC4R-expressing neurons within the VMH. MC4R is expresse within the VMH n the VMH reeives innervtions from rute POMC- or AgRP-expressing neurons 32,34.The level of POMC innervtion within the VMH is low, ut is omprle to tht in the LH (Supplementry Fig. 1 online). In ition, there re sustntil numer of POMC n AgRP fiers surrouning the VMH. These fiers my innervte the enrites extening from neurons within the VMH 35, therey regulting BDNF expression within the VMH. Finlly, it is lso possile tht regultion involves one or more itionl neurons tht my resie outsie of the VMH n whose projetions ontrol BDNF expression within the VMH. Foo eprivtion for 2 results in 2/3 reution in the levels of BDNF mrnas in the VMH, whih is sustntilly ut inompletely reverse y ministrtion of n MC4R gonist. Among potentil resons for the inompleteness of reversl re suoptiml tivtion of MC4R reeptors ue to poor penetrtion or rpi turnover of MTII, ssy of BDNF mrna efore elevte synthesis hs estlishe new equilirium level, n possile involvement of itionl signling pthwys whose tivities re regulte y feeing sttus, ut o not involve the MC4R. Where re the TrkB-expressing neurons through whih BDNF regultes feeing ehvior n energy lne? Trgete eletion of floxe BDNF llele with CMKII-riven re results in oesity 4,suggesting tht the BDNF-expressing neurons in the VMH re proly popultion of exittory neurons tht express enogenous CMKII. BDNF is nterogrely trnsporte to postsynpti neurons in n tivityepenent mnner 36, so the TrkB-expressing neurons oul e lote in ny of the rin regions tht reeive projetions from the VMH. Neurons in the VMH projet to mny trgets within n outsie the hypothlmus, inluing the DMH, nterior hypothlmus, e nulei of the stri terminlis, myglr nulei, nuleus of the solitry trt n periqueutl gry Some of these trgets suh s the DMH, nuleus of the solitry trt n periqueutl gry hve een implite in the regultion of energy intke To ientify the ells through whih TrkB regultes feeing ehvior will require extensive nlyses using region-speifi n ell type speifi elimintion n overexpression of the TrkB reeptor. It hs een shown tht BDNF is require for gusttory evelopment 43,44 n tht TrkB is expresse in the olftory ul 45.Sine the moerte-ft iet is more pltle, these oservtions rise the possiility tht evelopmentl efets n/or mlfuntion of the gusttory or olftory system my ffet feeing ehvior of the trkb mutnt on the moerte-ft iet. A efet in the gusttory or olftory system most likely results in loss of the ility to istinguish the low-ft iet n moerte-ft iet in the trkb mutnt. If so, gusttory or olftory efet my reue ingestion of the moerte-ft iet. Therefore, efet in the gusttory or olftory system is unlikely to signifintly ontriute to hyperphgi on higher ft iets in the trkb mutnt. In onlusion, our results inite tht BDNF-to-TrkB signling is n importnt ownstrem trget of MC4R-meite signling tht prtiiptes in the regultion of energy lne n feeing ehvior. The oesity phenotypes oserve in nimls with ompromise MC4R n TrkB signling thus seem to e use y efiits in ifferent steps of ommon or overlpping signling pthwy. In the future, it will e importnt to ientify the neurons n ellulr mehnisms tht ontrol eh step of this pthwy. METHODS Mouse strins. Mie were mintine on norml 12 h/12 h light/rk yle with regulr mouse how n wter liitum. A y mie n wil-type littermtes were purhse from the Jkson Lortories. Genertion of fbz/+, trkb lz /+ n BDNF LZ /+ mie were s esrie previously 28,30,31.The MC4R / null mie were offspring from rosses etween MC4R / mie tht h een krosse to C57BL/6J for five genertions. The ontrol C57BL/6J wil-type mie were purhse from Jkson Lortories. Animl proeures were pprove y the University of Cliforni Sn Frniso Committee on Animl Reserh. Feeing ehvior. Mie were iniviully house for t lest 3 efore mesurement. Over 5, regulr mouse how ws weighe n provie to 5- or 9-week ol mie liitum with mesurement t noon eh y n reful monitoring for spillge. Eh group onsiste of three femles n two mles. The 5-week ontrol group ontine one fbz heterozygous n four wil-type mie, wheres the 9-week ontrol group onsiste of three fbz heterozygous n two wil-type mie. Low-ft Purin iet 5001 (23.0% protein, 4.5% ft, 5.3% rue fier, 49% rohyrte, totl igestile nutrient 76%, 3.04 kl/g metolizle energy) n moerte-ft Purin iet 5015 (17.0% protein, 11.0% ft, 3.0% rue fier, 53.5% rohyrte, totl igestile nutrient 88%, 3.73 kl/g metolizle energy) were use to monitor the feeing response of mie to ietry ft. Mie were isolte n fe with Purin iet 5001 one week efore ny mesurement of foo intke n oy weight. Home ge ehviorl monitoring. Mle mie were limte to iniviul housing in feeing n liking monitoring ges (plexiglss, m with feeers n wter ottles mounte t one en; AFL monitors, DiLog Instruments) for 6 n then monitore for n itionl 8 of t olletion. Aitionl etils re provie in Supplementry Methos online. Histology. X-gl stining n immunohistohemistry were performe s esrie previously 31. Anti-β-gltosise (1:4,000) ws from Cppel Reserh Regents. Anti-γ-MSH n nti-agrp were provie y S. Wtson (University of Mihign, Ann Aror, Mihign) n G. Brsh (Stnfor University, Stnfor, Cliforni), respetively. For in situ hyriiztion, mie were nesthetize n fixe with sequentil trnsril perfusion of PBS n 4% prformlehye. The rins were issete, immerse in 30% surose, n then frozen. Alterntively, mie were kille y nek islotion n the rins were issete n frozen immeitely. BDNF in situ hyriiztion ws performe on ryostt oronl setions t 30 µm using 35 S-lele ntisense RNA proes omplementry to the oing region of the mouse BDNF DNA. After hyriiztion n wshes, se- NATURE NEUROSCIENCE VOLUME 6 NUMBER 7 JULY

7 tions were expose on Bet-Mx Hyperfilm (Amershm). Imges from two setions tht overe the ul VMH n were seprte y 150 µm in eh mouse were snne t 1,200.p.i. n the optil ensity of in situ signls ws etermine using NIH Imge. MTII injetion. Ten-week-ol femle wil-type C57BL/6J mie were fooeprive for 44 h n nesthetize with vertin. The orsl thir ventrile (2 mm ul from regm, 2.5 mm eep) ws injete with 4 nmol MTII (Bhem) or rtifiil ererospinl flui (ACSF) in volume of 3 µl with Hmilton syringe over 20-s perio. ACSF ws lso injete into group of normlly fe mie. The neele stye in the rin for nother 15 s efore it ws remove. Three hours lter, mie were kille y ervil islotion, n their rins were immeitely issete out n frozen. To etermine the effet of MTII injetion on expression of BDNF in A y mie, 3 nmol of MTII or sline ws intrereroventriulrly injete into A y /; BDNFlZ/+ mie. Six hours lter, the rins were fixe n proesse for ntiβ-gltosise immunohistohemistry. The VMH of eh rin t pproximtely regm 1.8 mm ws photogrphe, n β-gltosise-expressing neurons were ounte (y n experimenter lin to tretment group). BDNF infusion. Femle A y mie n wil-type littermtes were isolte n fe with Purin iet 5001 one week efore pump implnttion. Alzet osmoti pumps (moel 1002, 0.25 µl/h for 14 ) were fille with PBS or humn reominnt BDNF (Amgen) in PBS t onentrtion of 200 ng/µl. The osge of BDNF ws hosen oring to previous stuy 3.Pumps were then tthe to n Alzet rin infusion kit n the entire pprtus ws prime overnight in sterile sline t 37 C. The tip of n infusion theter ws positione in the lterl ventrile (0.5 mm ul n 1 mm lterl from regm, 2.5 mm eep) of mie nesthetize with vertin n seure with Lotisite gel. Strting y fter surgery, oy weight n foo intke were mesure ily. Upon ompletion of the 2-week stuy, the position of the nnul tip n the volume of remining solution in the pump were etermine. Animls were exlue from nlysis if they ie (n = 1), ppere sik (n = 1) or h misple or mlfuntioning pumps (n = 2). Note: Supplementry informtion is ville on the Nture Neurosiene wesite. ACKNOWLEDGMENTS We thnk M. Dlmn for onstrutive suggestions, J. Qiu for help in issetion of ft ps, J. MKen for tehnil ssistne n H. Chen for emonstrtion of implnttion of osmoti pumps. E.H.G. is Howr Hughes Meil Institute (HHMI) Physiin Postotorl Fellow. This work hs een supporte y the HHMI n the Ntionl Institute of Neurologil Disorers n Stroke (L.F.R.). COMPETING INTERESTS STATEMENT The uthors elre tht they hve no ompeting finnil interests. Reeive 24 April; epte 7 My 2003 Pulishe online 8 June 2003; oi: /nn Hung, E.J. & Reihrt, L.F. Neurotrophins: roles in neuronl evelopment n funtion. Annu. Rev. Neurosi. 24, (2001). 2. Lyons, W.E. et l. Brin-erive neurotrophi ftor efiient mie evelop ggressiveness n hyperphgi in onjuntion with rin serotonergi normlities. Pro. Ntl. A. Si. USA 96, (1999). 3. 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