Mice lacking melaninconcentrating

Transcription

1 . Reurn, S.., Ilton, E. S. & Velen, D. R. Quntittive etermintion of the oxition stte of iron in iotite using X-ry photeletron spetrosopy: II. In situ nlyses. Geohim. Cosmohim. At, 3±37 (997).. Hrris, J., Huthison, M. T., Hursthouse, M., Light, M. & Hrte,. A new tetrgonl silite minerl ourring s inlusions in lower-mntle imons. Nture 37, ± (997).. Mo, H.-K., Shen, G. & Hemley, R. J. Multivrile epenene of Fe-Mg prtitioning in the lower mntle. Siene 7, 9± (997). 7. Woo,. J. & Ruie, D. C. The effet of lumin on phse trnsformtions t the -kilometer isontinuity from Fe-Mg prtitioning experiments. Siene 73, ± (99).. Disko, M. M., Ahn, C. C. & Fultz,. Trnsmission Eletron Energy Loss Spetrometry in Mterils Siene (Minerls, Metls & Mterils So., Wrrenle, IL, 99). 9. Grvie, L. A. J. & Crven, A. J. High-resolution prllel eletron energy-loss spetrosopy of Mn L,3- eges in inorgni mngnese ompouns. hys. Chem. Minerls, 9± (99).. Grvie, L. A. J. & usek,. R. in oron Minerlogy, etrology n Geohemistry (es Grew, E. S. & Anovitz, L. M.) ±3 (Reviews in Minerlogy vol. 33, Minerlogil So. Am., Wshington DC, 99).. Grvie, L. A. J., Crven, A. J. & ryson, R. Use of eletron-energy loss ner-ege ne struture in the stuy of minerls. Am. Minerl. 79, ± (99).. Egerton, R. F. Eletron Energy-Loss Spetrosopy in the Eletron Mirosope n en (lenum, New York, 99). 3. vn er Ln, G. & Kirkmn, I. W. The p sorption spetr of 3 trnsition metl ompouns in tetrherl n otherl symmetry. J. hys.: Conens. Mtter., 9± (99).. rowning, N. D., Chisholm, M. F. & ennyook, S. J. Atomi-resolution hemil nlysis using snning trnsmission eletron mirosope. Nture 3, 3± (993).. rley, J.. Nnometer-sle minerlogy n petrogrphy of ne-grine ggregtes in nhyrous interplnetry ust prtiles. Geohim. Cosmohim. At, 3±3 (99).. Colliex, C. New trens in STEM-se nno-eels nlysis. J. Eletron Miros., ± (99). 7. Cznk, M., Myer, J. & Klein, U. Eletron spetrosopi imging (ESI): A new metho to revel the existene of nm-sle exsolution lmelle. Eur. J. Minerl. 9, 99± (997).. Righter, K. & Crmihel, I. S. E. Meg-xenorysts in lkli olivine slts: Frgments of isrupte ssemlges. Am. Minerl. 7, 3± (993). 9. MGuire, A. V., Frnis, C. A. & Dyr, M. D. Minerls stnrs for eletron miroproe nlysis of oxygen. Am. Minerl. 77, 7±9 (99). 3. Gumunsson, G. & Hollowy, J. R. Ativity±omposition reltionships in the system Fe-t t 3 n C n t tm n kr. Am. Minerl. 7, 7± (993). 3. Vn Aken,. A., Liesher,. & Styrs, V. S. Quntittive etermintion of iron oxition sttes in minerls using Fe L,3 -ege eletron energy-loss ner-ege struture spetrosopy. hys. Chem. Miner., 33±37 (99). regultor of feeing n energy lne whih ts ownstrem of leptin n the melnoortin system, n tht eletion of gene enoing single orexigeni peptie n result in lenness. The MCH gene ws isrupte in emryoni stem ells y homologous reomintion using trgeting vetor in whih exons ±3 of the MCH gene were reple y onstrut onsisting of phosphoglyerte kinse (GK) promoter n the neomyinresistne (neo r ) gene (GK-neo r ) (Fig. ). Two emryoni stem ell lines with orretly trgete reomintion were etete y Southern lot nlysis, n line of mie with isrupte MCH gene (MCH -/- ) ws estlishe (Fig. ). Reverse trnsription with polymerse hin retions (RT-CR; Fig. ) n in situ hyriiztion (Fig. ) on rme isruption of the MCH gene in MCH -/- mie. In ition, immunoytohemistry showe tht MCH ws not immunostine in the rin of MCH -/- mie (t not shown). MCH -/- mie were orn t the expete menelin frequeny, vile into ulthoo, n fertile, n they ppere phenotypilly Wil type roe N Trgete vetor Mutnt llele N N Exon I II III X A X (C) GK-neo X (C) Aknowlegements. We thnk. Rez n J. rley for isussions, n K. Righter, D. Cnil n G. Gumunsson for proviing us with smples. This work ws supporte y the Erth Sienes Division of the US NSF. WT K HR 7. K GK-neo Corresponene n requests for mterils shoul e resse to L.A.J.G. (e-mil: Igrvie@su.eu). -/- +/+ +/- -/- -/- +/- +/- +/+ +/- -/- M r (K) M NC. Mie lking melninonentrting hormone re hypophgi n len Msko Shim, Nihols A. Tritos², rfor. Lowell, Jeffrey S. Flier & Eleftheri Mrtos-Flier³ Division of Enorinology, eth Isrel Deoness Meil Center, 33 rookline Avenue, oston, Msshusetts, USA ² Reserh Division, Joslin Dietes Center, Joslin le, oston, Msshusetts, USA Feeing is in uene y hypothlmi neuropepties tht promote (orexigeni pepties) or inhiit feeing. Of these, neuropeptie Y (NY) in the rute nuleus n melnin-onentrting hormone (MCH) 3 n orexins/hyporetins, in the lterl hypothlmus hve reeive ttention euse their expression is inrese uring fsting n euse they promote feeing when ministere entrlly. Surprisingly, sene of the orexigeni neuropeptie NY fils to lter feeing or oy weight in norml mie.as e ieny of single omponent of the pthwy tht limits foo intke (suh s leptin or reeptors for melnoortin-) 7, uses oesity, it hs een suggeste tht orexigeni signls re more reunnt thn those limiting foo intke 7,. To e ne further the physiologil role of MCH n to test the reunny of orexigeni signls, we generte mie rrying trgete eletion of the MCH gene. MCH-e ient mie hve reue oy weight n lenness ue to hypophgi (reue feeing) n n inppropritely inrese metoli rte, espite their reue mounts of oth leptin n rute nuleus pro-opiomelnoortin messenger RNA. Our results show tht MCH is ritil... MCH Leptin reeptor (long form) +/+ +/- ND -/- NDNC Figure Trgete isruption of the MCH gene in mie., Top, the trgete region of the MCH gene lous. Mile, the GK±neo r ssette ws use to isrupt the MCH gene oing region. ottom, the expete trgete llele. Restrition enzyme sites:, glii; N, NoI; X, XI; A, A ;,sti; (C), ClI in polylinker. The thik lk r inites the genomi 3-p glii±noi proe. WT, wil-type; HR, the homologous reominnt llele., Southern lot nlysis of mouse til genomi DNA. Genotypes re shown t the top., Top, etetion of MCH mrna in the hypothlmus using RT-CR. The mouse genotype n totl mount of RNA (in ng) use for RT-CR in eh group is shown t the top. ottom, RT-CR nlysis, using primers for the long form of the leptin reeptor n the sme RNA s in the top pnel. M, mrker; NC, negtive ontrol; ND, not one., In situ hyriiztion with igoxigenin-lelle rioproe ws use to nlyse MCH expression in the hypothlmus of wil-type (left) n MCH -/- (right) mie. 3V, thir ventrile. Nture Mmilln ulishers Lt 99 7 NATURE VOL 39 7 DECEMER 99

2 norml y gross inspetion exept for their reue size. The men oy weight of oth mle n femle MCH -/- mie ws reue, n this ws rst pprent etween n weeks of ge. At 7 weeks of ge, mle MCH -/- mie weighe % less (Fig. ) n femle MCH -/- mie weighe % less (Fig. ) thn ontrol mie; weights of heterozygous MCH +/- mie tene to e slightly ut not signi ntly lower thn weights of ontrol mie (Fig., ). Crss nlysis showe tht MCH -/- mie h reue triglyerie ontent; they were lener thn ontrol littermtes (Fig. ). We mesure nose±nus length of mle mie t weeks of ge; this mesurement i not vry etween groups (MCH +/+, 9: :; MCH -/-,9::m; n ˆ in eh group). The reue oy weight n len phenotype were ssoite with hypophgi, s -week-ol MCH -/- mie ingeste % fewer lories thn ontrol mie over -hour perio (Fig. ). The entire reution in foo intke ourre uring the rk yle, n the mount of foo onsume uring the light perio, lthough smll frtion of totl ily intke, ws signi ntly inrese ompre with ontrol nimls, initing tht MCH my exert its primry effet on feeing uring the rk perio (Fig. ). However, s MCH -/- mie onsume most of their lories uring the rk yle, like norml mie, it seems tht MCH is not neessry for the norml iurnl feeing pttern (Fig. ). Consistent with their len phenotype, the MCH -/- mie showe erese leptin levels, eing %, 9% n % of ontrol levels t :, : n oy weight (g) oy weight (g) Mle Femle Time (week) Foo intke (g) Ft in -week-ol mie (%) oy weight (g) 3 Dy Night Whole y Figure oy weight, ft omposition n foo intke in MCH -/- n ontrol mie fe on how iet.,, Growth urves. Three to four mie were house per ge. Mle: MCH +/+, n ˆ ; MCH +/-, n ˆ 9; MCH -/-, n ˆ 3. Femle: MCH +/+, n ˆ ; MCH +/-, n ˆ ; MCH -/-, n ˆ., erentge of triglyeries reltive to oy weight in MCH -/- n MCH +/+ mie. Open irles, wil-type mie; open tringles, MCH +/- mie; she irles, MCH -/- mie., Foo intke y -week-ol wiltype (n ˆ ; white rs) n MCH -/- (n ˆ 9; she rs) mle mie uring the light n rk yles. Mie were house iniviully ys efore the experiment n the vlues were etermine over -y perio. Dt re men intke per y s.e.m. Asterisk inites, :; oule sterisk inites, :; gger inites, :; oule gger inites, :. Sttistil signi ne ws etermine y Fisher's LSD (post-ho lest signi nt ifferene). :, respetively (, : versus ontrol) (Fig. 3). As MCH hs een propose to ply role in motivte ehviours, we stuie the ehviour of MCH -/- mie using n open- el loomotion test 9, in whih MCH -/- mie were s tive s wil-type ontrols. Control mle mie (n ˆ ) rosse squres over minutes wheres MCH -/- mle mie (n ˆ ) rosse 3 squres over the sme intervl. We stuie potentil effets of MCH e ieny on neuroenorine funtion. Thyroxine levels (MCH +/+ mie, :7 : mgl ; MCH -/- mie, : : mgl ; men s:e:m:) n ortiosterone levels (MCH +/+ mie, :9 :ngml ; MCH -/- mie, :7 :3ngml ) mesure t : were not signi ntly ifferent in -week-ol mle MCH -/- mie n ontrols. Levels of loo gluose n insulin mesure in -week-ol mles were lso not signi ntly ifferent (gluose: MCH +/+ mie, 7:7 :3mgl ; MCH -/- mie, : :9mgl ; insulin; MCH +/+ mie, :99 : ng ml ; MCH -/- mie, :7 :7 ng ml - ; n ˆ 7 eh group). The reution in oy weight in MCH -/- mie exeee the egree to whih foo intke ws reue, initing tht inrese metoli rte might ontriute to the lower weight. Retl temperture, n imperfet initor of metoli rte, ws unhnge, eing 37:9 :3 C in ontrol nimls (n ˆ ) n 37: : C in MCH -/- nimls (n ˆ ). We therefore mesure the metoli rte of these mie in the fe stte. MCH -/- mie show rtes of oxygen onsumption tht re slightly ut not signi ntly higher thn ontrol rtes when expresse on per mouse sis (MCH +/+ mie, : : ml O per min per mouse; MCH -/- mie, : : ml O per min per mouse). However, the rte of oxygen onsumption is inrese y % in MCH -/- mie when normlize to oy mss (MCH -/- mie, 3: :ml O per min per kg oy weight; MCH +/+ mie, 7: :ml O per min per kg oy weight). Whether expresse per mouse or per kg oy weight, this rte of lsm leptin level (ng ml ) Foo intke fter h strvtion (g) 7 3 : : : : Time (h) Hours fter refeeing oy weight reution fter h strvtion (%) oy weight uring foo shift (g) Wil type MCH (-/-) Dys fter strting foo shift Figure 3 Leptin levels n feeing response in MCH -/- mie., Leptin levels in - week-ol mle MCH -/- (n ˆ ; lle irles) n MCH +/+ (n ˆ ; open irles) mie. Dt re mens s.e.m., Cumultive foo intke in response to strvtion in MCH -/- ( lle irles) n MCH +/+ (open irles) mie., oy-weight reution fter -h strvtion., Response of wil-type (open irles) n MCH -/- ( lle irles) mie to foo shift. Asterisk inites, :; oule sterisk inites, :. Sttistil signi ne ws etermine y Fisher's LSD. Nture Mmilln ulishers Lt 99 NATURE VOL 39 7 DECEMER

3 oxygen onsumption my re et n impire ility of MCH -/- mie to ppropritely regulte their metoli rte in response to their reue feeing n lower mounts of leptin (see elow). Thus, MCH e ieny promotes weight loss y reuing feeing n my lso limit the norml suppression of metoli rte uring foo restrition. This omintion of results ws preite for NY -/- mie on the sis of the results of intrererl injetions of this neuropeptie, ut mie e ient in NY showe no suh efets. We stuie the response of MCH -/- mie to foo eprivtion. MCH -/- mie respone to hours of strvtion with ompenstory hyperphgi equl to tht of wil-type mie (Fig. 3). Thus, like NY, MCH is not essentil for the hyperphgi response to strvtion. However, two lines of eviene inite tht MCH e ieny my rete suseptiility to the verse effets of strvtion. First, when ge-mthe mie were strve for hours, weight loss ws greter in MCH -/- mie thn in ontrols (7: :% vs : :9% weight loss, respetively; n ˆ,, :) (Fig. 3). Seon, when ge-mthe mie were strve for hours, 3 of MCH -/- ut of MCH +/+ mie ie uring the lst hours of the fst. The preise sis for these verse effets of strvtion is not yet known, ut inpproprite levels of thermogenesis, ouple with reue initil ft stores, woul e expete to use greter weight loss, n erlier eth, in the sene of foo intke. To further explore the response to ltere foo vilility, we use restrite-feeing prigm. Foo ws offere only uring the light yle, etween : n :, to 7-week-ol mie. Over 9-y perio, wil-type ontrols respone to this stress with % reution in oy weight, wheres MCH -/- mie showe 3% weight loss (, :) (Fig. 3). In ontrst, foo intke fell to the sme egree in wil-type n MCH -/- mie (t not shown), onsistent with the possiility tht MCH e ieny uses inrese thermogenesis uner these irumstnes. The hypophgi n lenness of MCH -/- mie le to the proution of lower leptin levels, whih shoul use hyperphgi. However, in the ontext of MCH e ieny, low leptin levels resulte in erese rther thn inrese foo intke, initing tht MCH my e essentil for the hyperphgi response to leptin e ieny uner these onitions. We lso exmine the effet of exogenous Chnge in oy weight fter leptin injetion (%) efore injetion Dy Dy Chnge in foo intke fter leptin injetion (%) efore injetion Dy Dy Figure Response of six MCH +/+ (open irles) n six MCH -/- mie (she irles) to reominnt mouse leptin, ministere t ose of mg per g oy weight twie ily (9: n :), vs sline ontrol (n ˆ 3; tringles). Vlues for, the hnge in oy weight n, the hnge in foo intke re the per ent hnge from 7-y seline perio. Asterisk inites, :. Sttistil signi ne ws etermine y Fisher's LSD. leptin on MCH -/- mie. Leptin ws injete t ose of mg per g oy weight twie y for ys. Leptin ws le to reue oth foo intke n oy weight within the initil -hour perio (Fig. ). The initil response of MCH -/- mie to leptin ws twie tht of wil-type mie (, :). This exggerte response to exogenous leptin, whih ws lso seen in NY -/- mie, ws ttenute y y. As leptin inhiits foo intke in MCH -/- mie, suppression of MCH is unlikely to e the mehnism y whih leptin inhiits foo intke in norml mie. Furthermore, MCH my, either iretly or iniretly, oppose the effet of leptin in iminishing foo intke. Finlly, to ssess the stte of hypothlmi ompenstion in response to oth MCH e ieny n lower leptin levels, we stuie hypothlmi expression of mrnas enoing four other ppetiteregulting neuropepties in MCH -/- mie, nmely NY, orexin, Agouti-relte protein (AGR),3 n pro-opiomelnoortin (OMC),. Expression of NY n AGR in the rute nuleus n of orexin in the lterl hypothlmus remine unhnge in MCH -/- mie in the fe stte. However, expression of OMC in the rute nuleus ws mrkely suppresse (erese y 3% ompre with ontrols;, :) (Fig. ). As leptin is positive regultor of rute OMC expression,7 the reue OMC expression oul result from lower leptin levels, whih, in turn, result from len oy omposition. Lower leptin mounts i not inrese NY expression in MCH -/- mie ut i in wil-type mie. These results might inite tht OMC expression is more sensitive to the reution in leptin levels thn is NY expression. Alterntively, MCH e ieny oul reue rute OMC expression iretly, through the loss of iret innervtion of rute OMC-expressing neurons y MCH-expressing neurons; MCH immunoretivity is seen in the rute nuleus of wil-type mie 9, onsistent with iiretionl ommunition etween OMC- n MCH-expressing systems. There re iiretionl onnetions etween rute neurons expressing NY, AGR n OMC n lterl hypothlmi neurons expressing MCH n orexins. The lower expression of rute OMC, whih in other ontexts promotes hyperphgi n oesity, might e expete to limit the onsequenes of MCH e ieny. However, our results show tht the ility of low rute OMC expression (n/or low OMC:AGR rtio) to use hyperphgi n oesity is limite in the sene of n intt MCH gene. Our MCH -/- mie hve eletion of the entire oing region of the MCH gene, whih lso enoes the neuropepties EI n GE. There is little informtion ville on the iologil role of these two pepties; iret ministrtion of either peptie lone or in omintion in rts hs no effet on eting ehviour (E.M.-F., unpulishe oservtions). In theory, the phenotypes of MCH -/- mie oul result from e ieny of ny or ll of these pepties. Expression of hypothlmi mrna (%) NY Orexin AGR OMC Figure Expression of neuropeptie mrnas in hypothlmi of -week-ol wiltype (n ˆ ; white rs) n MCH -/- (n ˆ ; she rs) mie. Asterisk inites, :. Sttistil signi ne ws etermine with the Mnn±Whitney test. Nture Mmilln ulishers Lt 99 7 NATURE VOL 39 7 DECEMER 99

4 However, given the ility of MCH to promote feeing 3,3 n the sene of eviene tht NEI n NGE promote suh n effet, it is resonle to ssume tht MCH e ieny is the primry, or even sole, use of the phenotype reporte here. MCH is yli 9-mino-i polypeptie whose expression is limite to the lterl hypothlmus n zon inert,. It hs een implite s n importnt regultor of eting ehviour, euse entrl ministrtion of MCH promotes feeing, n MCH mrna mounts rise s result of strvtion n leptin e ieny 3. MCH-expressing neurons re well ple ntomilly to prtiipte in feeing ehviour, euse they mke monosynpti onnetions with severl res in the rin involve in integrting inputs relte to tste, olftion n viserl senstions, inluing the nuleus of the solitry trt, the prrhil nuleus n the insulr n meil prefrontl ortex. MCH-expressing neurons hve therefore een suspete to prtiipte in omplex integrtive ehviours, n the ute feeing response to MCH 3,3 inite tht feeing ws mong these ehviours. The phenotype of the MCH -/- mouse reporte here strongly supports this hypothesis. As the lenness of MCH -/- mie ours espite reue expression of leptin n rute OMC, whih proue oesity when MCH is present 7,,, MCH ppers to e ritil effetor of energy lne ownstrem of leptin n the OMC/melnoortin system. Further geneti rosses etween MCH -/- mie n mie with ifferent uses of oesity will e require to test this hypothesis iretly. Finlly, s MCH -/- mie re len, ntgonists of MCH tion my e effetive tretments for oesity. Methos Cretion of MCH -/- mie. We sreene mouse C9 SvJ genomi lirry (Genome Systems) using -se-pir (p) CR frgment, generte on the sis of the pulishe sequene of the murine MCH gene, s proe. One of three lones ws mppe; it onsiste of the -kilose (k) vetor n 7-k genomi insert ontining the MCH oing region etween, k of 9 n k of 39 nking sequene. We reple,. k-frgment extening from 3 p upstrem of the trnsltion initition oon (XI site) n p ownstrem of the stop oon (A III site)) of this lone with GK±neo r ssette. This trgeting vetor in pgem (romeg) ws linerize t the SlI site n eletroporte into the J emryoni stem line 7 (ells provie y E. Li n A. Shrp); seletion of G-resistnt lones ws one s esrie 7.We ienti e trgete lones y Southern lot nlysis, using 3-p gii±noi frgment lote 9 of the trgeting vetor s proe; of lones were positive. These positive lones were injete into C7/ emryos t the lstoyst stge. Chimeri offspring were mte with C7/ mie. Germline trnsmission of the mutnt llele ws etermine y Southern nlysis of mouse til genomi DNA. One line of mie rrying the isrupte MCH gene ws generte, n F 3 hyris were use in ll experiments. We reversetrnsrie mg of totl RNA from the hypothlmus of mouse from eh group n mpli e liquots equivlent to, n. ng totl RNA y CR, using the following primers: sense, 9-ATGGCAAAGATGACTCTCTCT- 39; n ntisense, 9-GACTTGCCAACATGGTCGGTA-39, s esrie. In situ hyriiztion ws one using igoxigenin-lelle MHC omplementry RNA proe. Stuies of MCH -/- mie. Mie were mintine in ily yle of h light (:±:) n h rkness (:±:) n were llowe free ess to how (urin Formul ) n wter. Totl oy lipi ontent ws ssesse using loholi potssium hyroxie igestion with sponi tion of ll fts, neutrliztion n then enzymti etermintion of glyerol (Sigm) s esrie 9. Leptin n insulin onentrtions were mesure y RIA (Lino Reserh), s were ortiosterone n thyroxine levels (ICN). Gluose ws mesure using One Touh meter (Lifesn). Retl temperture ws mesure with retl proe (Yellow Springs Instrument). Oxygen onsumption ws mesure in -week-ol mle wil-type (n ˆ ) n MCH (n ˆ ) mie using n Oxymx.93 (Columus Instruments). Response to strvtion in MCH -/- n MCH +/+ mie. We eprive -weekol mie (n ˆ ) of foo for h, eginning t 9:; the mie were then llowe free ess to how. To mesure umultive foo intke fter refeeing, we mesure the weight of onsume foo t,,, n h fter refeeing. oy weights were mesure fter the -h strvtion perio n fter refeeing. Foo-shift experiment. To etermine the response of wil-type n MCH -/- mie to foo shift, oy-weight-mthe nimls (wil-type mle, n ˆ 7; MCH -/- mle, n ˆ ) were iniviully house n llowe free ess to how for only h uring the light yle (:±:) for ys. oy weights were mesure t :. Animls were llowe free ess to wter uring the perio. Response to leptin. To etermine the response to leptin, mie were injete intrperitonelly with reominnt mouse leptin (Eli Lilly) t ose of mg per g oy weight twie ily (9: n :). Three ontrol mie were injete with sline. Expression of hypothlmi mrnas. Mie were kille t 9: y ministrtion of pentoritl n were perfuse trnsrilly rst with sline n then with % uffere formlin. In situ hyriiztion ws one with proes for NY, OMC, AGR n orexin mrnas. The imges pture on lm were qunti e with omputing ensitometer (Moleulr Dynmis) n ImgeQunt Softwre (Moleulr Dynmis). We integrte the sorne in retngulr res enompssing eh nuleus of interest (rute nuleus for NY, OMC n AGR, n lterl hypothlmus for orexin) over eh set of rin setions, n sutrte kgroun ensity from n jent re without spei hyriiztion signl. We etermine sttistil signi ne with the Mnn±Whitney test. Reeive July; epte Otoer 99.. Flier, J. S. & Mrtos-Flier, E. Oesity n the hypothlmus: novel pepties for new pthwys. Cell 9, 37± (99).. Dryen, S., Frnkish, H., Wng, Q. & Willims, G. Neuropeptie Yn energy lne: one wy he for the tretment of oesity? Eur. J. Clin. Invest., 93±3 (99). 3. Qu, D. et l. A role for melnin-onentrting hormone in the entrl regultion of feeing ehviour. Nture 3, 3±7 (99).. Skuri, T. et l. Orexinsn orexin reeptors: fmily of hypothlmi neuropepties n G proteinouple reeptors tht regulte feeing ehvior. Cell 9, 73± (99).. e Lee, L. et l. The hyporetins: hypothlmus-spei pepties with neuroexittory tivity. ro. Ntl A. Si. USA 9, 3±37 (99).. Erikson, J., Clegg, K. & lmiter, R. Sensitivity to leptin n suseptiility to seizures of mie lking neuropeptie Y. Nture 3, ± (99). 7. Zhng, Y. et l. ositionl loning of the mouse o gene n its humn homologue. Nture 37, ± 3 (99).. Huszr, D. et l. Trgete isruption of the melnoortin- reeptor results in oesity in mie. Cell, 3± (997). 9. Krezel, W. et l. Impire loomotion n opmine signling in retinoi reeptor mutnt mie. Siene 79, 3±7 (99).. Ahim, R. S., rkrn, D. & Flier, J. S. ostntl leptin surge n regultion of irin rhythm of leptin y feeing. Implitions for energy homeostsis n neuroenorine funtion. J. Clin. Invest., ±7 (99).. Hls, J. et l. Weight reuing effet of the plsm protein enoe y the oese gene. Siene 9, 3± (99).. Ollmnn, M. M. et l. Antgonism of entrl melnoortin reeptors in vitro n in vivo y Agoutirelte protein. Siene 7, 3±3 (997). 3. Shutter, J. R. et l. Hypothlmi expression of ART, novel gene relte to gouti, is up-regulte in oese n ieti mutnt mie. Genes Dev., 93± (997).. Tsujii, S. & ry, G. A. Aetyltion lters the feeing response to MSH n et-enorphin. rin Res. ull. 3, ±9 (99).. Fn, W., oston,. A., Kesterson, R. A., Hruy, V. J. & Cone, R. D. Role of melnoortinergi neurons in feeing n the gouti oesity synrome. Nture 3, ± (997).. Thornton, J. E., Cheung, C. C., Clifton, D. K. & Steiner, R. A. Regultion of hypothlmi proopiomelnoortin mrna y leptin in o/o mie. Enorinology 3, 3± (997). 7. Mizuno, T. M. et l. Hypothlmi pro-opiomelnoortin mrna is reue y fsting in o/o n / mie, ut is stimulte y leptin. Dietes 7, 9±97 (99).. Ahim, R. S. et l. Role of leptin in the neuroenorine response to fsting. Nture 3, ± (99). 9. ittenourt, J. C. & Elis, C. F. Dienephli origins of melnin-onentrting hormone immunoretive projetions to meil septum/igonl n omplex n spinl or using two retrogre uoresent trers. Ann. NY A. Si., ± (993).. Elis, C. F. et l. Chemilly e ne projetions linking the meiosl hypothlmus n the lterl hypothlmi re. J. Comp. Neurol. (in the press).. Krue, H. et l. Severe erly-onset oesity, renl insuf ieny n re hir pigmenttion use y OMC muttions in humns. Nture Genet. 9, ±7 (99).. Nhon, J. L. The melnin-onentrting hormone: from the peptie to the gene. Crit. Rev. Neuroiol., ± (99). 3. Rossi, M. et l. Melnin-onentrting hormone utely stimultes feeing, ut hroni ministrtion hs no effet on oy weight. Enorinology 3, 3±3 (997).. ittenourt, J. C. et l. The melnin-onentrting hormone system of the rt rin: n immuno- n hyriiztion histohemil hrteriztion. J. Comp. Neurol. 39, ± (99).. Montgue, C. T. et l. Congenitl leptin e ieny is ssoite with severe erly-onset oesity in humns. Nture 37, 93±9 (997).. reton, C., resse, F., Hervieu, G. & Nhon, J. L. Struture n regultion of the mouse melnin onentrting hormone mrna n gene. Mol. Cell. Neurosi., 7± (993). 7. Li, E., Suov, H. M., Lee, K. F., Evns, R. M. & Jenish, R. Norml evelopment n growth of mie rrying trgete isruption of the lph retinoi i reeptor gene. ro. Ntl A. Si. USA 9, 9±9 (993).. Mrks, D. L. et l. Simultneous visuliztion of two ellulr mrna speies in iniviul neurons y use of new oule in situ hyriiztion metho. Mol. Cell. Neurosi. 3, 39± (993). Nture Mmilln ulishers Lt 99 NATURE VOL 39 7 DECEMER

5 9. Lowell,.. et l. Development of oesity in trnsgeni mie fter geneti ltion of rown ipose tissue. Nture 3, 7±7 (993). Aknowlegements. We thnk J. Mstitis, C. ehn n C. Lee for tehnil ssistne, J. Elmquist for help in nlysing n interpreting rin ntomy, n D. S. Luwig for the lone use in mking the onstrut for the knokout mie. This work ws supporte in prt y grnts from NIH to J.S.F. n E.M.-F., from the Amerin Dietes Assoition to E.M.-F., n from Eli Lilly to J.S.F. n E.M.-F., n y the Trnsgeni Core of the oston Oesity Nutrition Reserh Center. M.S. ws supporte y the nyu Fellowship in Lipi Metolism n Atheroslerosis whih is sponsore y nyu hrmeutil Co Lt n the Merk Fountion. Corresponene n requests for mterils shoul e resse to E.M.-F. (e-mil: emrt@josl. hrvr.eu). GAA reeptors funtion s heteromeri ssemly of the suunits GAA R n GAA R Kenneth A. Jones, eth orowsky, Joe A. Tmm, Dougls A. Crig, Mrgret M. Durkin, Meng Di, Wen-Jeng Yo, Mry Johnson, Cryn Gunwlsen, Ling-Yn Hung, Cheng Tng, Qunrong Shen, John A. Slon, Kelley Morse, Thoms Lz, Kelli E. Smith, Dhnpln Ngrthnm, Stewrt A. Nole, Theres A. rnhek & Christophe Gerl Synpti hrmeutil Corportion, College Ro, rmus, New Jersey 7, USA The prinipl inhiitory neurotrnsmitter GAA (g-minoutyri i) exerts its effets through two lign-gte hnnels, GAA A n GAA C reeptors, n thir reeptor, GAA (ref. ), whih ts through G proteins to regulte potssium n lium hnnels. Cells heterologously expressing the lone DNA enoing the GAA R protein exhiit high-f nity ntgonist-ining sites, ut they proue little of the funtionl tivity expete from stuies of enogenous GAA reeptors in the rin. Here we esrie new memer of the GAA polypeptie fmily, GAA R, tht shows sequene homology to GAA R. Neither GAA R nor GAA R, when expresse iniviully, tivtes GIRK-type potssium hnnels; however, the omintion of GAA R n GAA R onfers roust stimultion of hnnel tivity. oth genes re o-expresse in iniviul neurons, n oth proteins o-lolize in trnsfete ells. Moreover, immunopreipittion experiments inite tht the two polypepties ssoite with eh other, proly s heteroimers. Severl G-protein-ouple reeptors (GCRs) exist s high-moleulr-weight speies, onsistent with the formtion of imers y these reeptors 3±7, ut the relevne of these speies for the funtioning of GCRs hs not een estlishe. We hve now shown tht o-expression of two GCR strutures, GAA R n GAA R, elonging to the sme sufmily is essentil for signl trnsution y GAA reeptors. To n other genes relte to the GAA R gene, we serhe the expresse sequene tgs (ESTs) of Gennk using the GAA R sequene. (Throughout the text, `GAA R' refers to the GAA R splie vrint.) Two entries h sores tht suggeste signi nt homology to GAA R. We use oligonuleotie proes from these sequenes to isolte full-length lone from rt hypothlmi omplementry DNA lirry. Sequene nlysis of this new lone showe tht it hs.-kilose open reing frme tht is preite to enoe protein of 9 mino is. A LAST serh of the GenEML tse inite tht this mino-i sequene ws most losely relte to tht of GAA R, exhiiting 3% n % ientity overll n within the preite trnsmemrne omins, respetively (Fig. ). The struturl similrity to GAA R inite tht this sequene might enoe new GAA polypeptie, whih we refer to s GAA R. The next most relte sequenes were other memers of the metotropi glutmte reeptor (mglur) fmily, with ±% overll mino-i ientity. Like GAA R n other memers of the mglur fmily, GAA R ontins lrge mino-terminl extrellulr omin with regions of homology to teril mino-i-ining proteins. We stuie the istriution of GAA R messenger RNA within the entrl nervous system y in situ hyriiztion. Strong hyriiztion signls were oserve in regions of the rt rin tht hve high ensities of GAA -reeptor-ining sites 9, suh s the hippompus, meil henul, thlmus n ereellum (Fig. ±e). There ws high egree of similrity in the istriution n intensity of GAA R n GAA R hyriiztion signls. ostsynpti inhiition of neurons y GAA -reeptor tivtion is use y the opening of inwrly retifying K + hnnels (GIRKs) ±3. We ssesse the ility of either GAA R or GAA R to regulte K + urrents in ells trnsfete with GIRK n GIRK suunits ±. Xenopus ooytes injete with either GAA R or GAA R mrnas file to generte GAA-evoke GIRK urrents (Fig. ). The longer splie vrint of GAA R, GAA R, i not stimulte GIRK tivity either, s reporte elsewhere,7. In mmmlin host, HEK93 ells, smll gonistevoke urrents (± pa) were oserve in of ells expressing GAA R; similr wek urrents were evoke in of 3 ells expressing GAA R. In ontrst, lrge urrents were proue in % of ells expressing the glnin reeptor GlR (ref. ) (Fig. i). The overlpping expression ptterns of GAA R n GAA R trnsripts in the rin inite tht the orresponing proteins might e o-expresse in iniviul neurons n tht oth might e require for funtionl tivity. When GAA R n GAA R were o-injete into ooytes together with GIRK suunits, the pplition of GAA proue roust K + urrents (Fig., ). Responses to GAA were olishe in ooytes pretrete with pertussis toxin, showing tht reeptor stimultion les to tivtion of heterotrimeri G protein of the G o /G i lss. The GAAinue urrents showe the following properties, whih suggest tht the urrents were meite y GIRK hnnels: rst, epeneny on rise externl K + onentrtion; seon, strong inwr reti tion; thir, reversl potentil (-3 mv) ner the preite equilirium potentil for K + ; n fourth, sensitivity to lok y mm + (Fig. ). The phrmology of gonists t the GAA R/GAA R omintion ws omprle to tht reporte for ntive reeptors 9,. GAA, lofen n 3-minopropylmethyl phosphini i (3- AMA) exhiite hlf-mximlly effetive onentrtions (EC vlues) of :3 :(n ˆ ), 3:3 :(n ˆ ), n : :3 (n ˆ ) mm, respetively (Fig., g). Conentrtion±effet urves for GAA were shifte to the right, in n pprently ompetitive mnner, y well-hrterize GAA -seletive ntgonists (Fig. h). Estimtes of f nity of the ntgonists CG (33: :3 nm; n ˆ ) n CG (: : nm; n ˆ ) for GAA R/GAA R were similr to vlues reporte in previous eletrophysiologil stuies using rin tissue 9,, s well s to those otine y mesuring isplement of riolign from ells expressing GAA R lone. The pperne of roust funtionl responses fter GAA R/ GAA R o-expression ws lso oserve in HEK93 ells. Upon oexpression of GAA R n GAA R together with GIRKs, GAA evoke urrents in 7 of reore ells (Fig. e, i). These urrents were loke y low onentrtions of the ompetitive GAA - reeptor ntgonist CG (ref. ) n y the GIRK-hnnel loker + (Fig. f), initing tht they resulte from the stimultion of GAA reeptors n the susequent tivtion of GIRKs. Lrge-mplitue urrents were lso oserve when GAA R ws pire with the GAA R splie vrint (; 7 pa; n ˆ 9). To Nture Mmilln ulishers Lt 99 7 NATURE VOL 39 7 DECEMER 99