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Cholecystokinin-mediatedsuppression of feeding involvesthe brainstem melanocortinsystemWei Fan, Kate L J Ellacott, Ilia G Halatchev, Kanji Takahashi,Pinxuan Yu & Roger D Cone

Hypothalamic pro-opiomelanocortin (POMC) neurons helpregulate long-term energy stores. POMC neurons are also foundin the nucleus tractus solitarius (NTS), a region regulatingsatiety. We show here that mouse brainstem NTS POMCneurons are activated by cholecystokinin (CCK) and feeding-induced satiety and that activation of the neuronalmelanocortin-4 receptor (MC4-R) is required for CCK-inducedsuppression of feeding; the melanocortin system thus providesa potential substrate for integration of long-term adipostaticand short-term satiety signals.

Hypothalamic POMC neurons tonically inhibit food intake1 and areregulated by the long-term adipostatic factor leptin2–4. However, thecentral melanocortin system is also important in the acute regulationof satiety; in particular, central administration of melanocortinsreduces food intake by decreasing meal size, a hallmark of satiety5,6.These hypothalamic neurons send fibers to MC4-R target sites inboth the hypothalamus and brainstem, and melanocortin agonistsadministered to either region inhibit feeding7. Notably, in addition toexpression in the arcuate nucleus of the hypothalamus (ARC), POMCis also expressed in the caudal aspect of the NTS8, the primary site ofsynapse of vagal afferent fibers transmitting satiety information fromthe gastrointestinal system. NTS neurons are activated by either elec-trical or CCK-induced stimulation of vagal afferent fibers.Furthermore, leptin and CCK act synergistically to inhibit feedingand activate NTS neurons9. Yet regulation of POMC cells in the NTSby metabolic state has not been reported. Here, we test the hypothesesthat (i) brainstem POMC neurons are activated by satiety signals and(ii) central melanocortin signaling is required for the action of spe-cific signals that acutely inhibit feeding.

Intraperitoneal (i.p.) injection of CCK-8s (the sulfated 8-amino-acid form of cholecystokinin) significantly increased c-Fosimmunoreactivity in the NTS (saline 3 ± 1 cells per section, n = 6;CCK-8s 3.5 µg/kg, 54 ± 11 cells per section, n = 4; 10 µg/kg, 80 ± 11cells per section, n = 4; P < 0.001) (Fig. 1; compare Fig. 1a,d), as shownpreviously9. Immunohistochemical experiments using a previouslycharacterized transgenic mouse that expresses enhanced green fluores-cent protein (EGFP) under the control of the POMC promoter4 showedno significant difference in the number of POMC-EGFP–immunoreac-tive (IR) neurons in the NTS between saline- (Fig. 1b) and CCK-8s-treated mice (Fig. 1e). Of the NTS POMC-EGFP neurons, >30%coexpressed c-Fos immunoreactivity after CCK-8s treatment (Fig.1f,g). c-Fos expression in the ARC did not differ significantly betweengroups treated with i.p. saline or CCK-8s (data not shown). We alsoexamined a model of feeding-induced satiety. We gave POMC-EGFPmice a 5-d training in which they were allowed access to food for two

periods totaling 5 h (9:00–10:00 h, 14:00–18:00 h) and examined c-Fosimmunoreactivity in the ARC and NTS at 11:00 h (see SupplementaryFig. 1 online; fed n = 8; fasted, n = 3, *** P < 0.001). Feeding activatedc-Fos expression in ∼ 21% of ARC POMC neurons but also in 13% ofNTS POMC neurons (Supplementary Fig. 1). Although a majority ofc-Fos-IR cells in the ARC were POMC positive, only a few percent ofthose in the NTS were, showing the complexity of cells in the NTS

NATURE NEUROSCIENCE VOLUME 7 | NUMBER 4 | APRIL 2004 335

Vollum Institute, Oregon Health and Science University, Portland, Oregon 97239-3098, USA. Correspondence should be addressed to R.D.C. (cone@ohsu.edu) orW.F. (fanw@ohsu.edu).

Published online 14 March 2004; doi:10.1038/nn1214

Figure 1 CCK-8s activates POMC neurons in the NTS. (a) Saline (i.p.)activates c-Fos (red) in only a few NTS neurons (arrows). Scale bar = 70 µm.(b) Anti-GFP antibodies detect POMC neurons (green) in NTS of the EGFP-POMC mouse. (c) POMC neurons are not activated by saline treatment. (d) CCK-8s (10 µg/kg, i.p.) activates c-Fos (red) in NTS neurons. (e) CCK-8s(10 µg/kg, i.p.) does not alter expression of POMC in NTS (compare b,e). (f) CCK-8s (10 µg/kg, i.p.) activates c-Fos in NTS POMC neurons (red, c-Fos;green, GFP; yellow-orange, c-Fos + GFP). (g) ∼ 30% of NTS POMC neuronsare activated by i.p. CCK-8s (3.5 or 10 µg/kg; ***, P < 0.001 vs. saline,statistical test done by one-way ANOVA with Dunnett’s post hoc test). (h) Receipt of long-term adipostatic signals and acute satiety signals byPOMC neurons in ARC and NTS, respectively. Blue, nuclei containing POMCneurons; yellow, nuclei containing MC4-R neurons that may serve tointegrate adipostatic and satiety signals. Red arrows, adipostatic signaling;green arrows, satiety signaling. BST, bed nucleus of stria terminalus; CEA,central nucleus of amygdala; PVN, paraventricular nucleus of hypothalamus;LH, lateral hypothalamic area; LPB, lateral parabrachial nucleus; AP, areapostrema; DMV, dorsal motor nucleus of vagus. All studies followed the NIHGuide For the Care and Use of Laboratory Animals and were approved by theOregon Health and Sciences University Animal Care and Use Committee.See Supplementary Methods online for details.

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involved in satiety. Previous work has shown that both catecholamin-ergic and glucagon-like peptide-1 (GLP-1)-positive cells in the NTSare involved in satiety10,11. Dual immunohistochemical analysisshowed that although POMC-EGFP–IR cells and tyrosine hydroxy-lase–IR cells are found in the same region of the NTS, they are notcoexpressed in the same neurons (see Supplementary Fig. 2 online).Likewise, POMC and GLP-1 do not colocalize in NTS neurons:POMC-EGFP–IR neurons are focused more medially than GLP-1-IRneurons (Supplementary Fig. 2).

To test whether feeding suppression by CCK-8s was dependent oncentral melanocortin signaling, we examined the ability of CCK-8s toinhibit food intake after a fast in three different mouse lines, two ofwhich carry deletions of the genes encoding melanocortin receptors 3and 4, respectively: C57BL/6J, C57BL/6J Mc3r–/–12 and C57BL/6JMc4r–/–13. Administration of CCK-8s i.p. after a 16-h fast produced a≥50% inhibition of food intake in the first 30 min in both wild-typeand Mc3r–/– mice (Fig. 2a) and continued to inhibit food intake for upto 180 min in each strain. We then administered CCK-8s to femaleMc4r–/– mice and age-matched female wild-type mice. CCK-8s signifi-cantly reduced food intake in wild-type mice, but not in Mc4r–/– mice

(Fig. 2b), at time points from 30 to 180 min. Next we examined the siteof action of endogenous melanocortins. We administered the MC3-Rand MC4-R antagonist SHU9119 (ref. 14) to rats via either the 3rd or4th ventricle to assess the relative contributions of forebrain and brain-stem MC4-R target sites in CCK-mediated inhibition of feeding. Weused subthreshold doses of SHU9119 previously determined not tostimulate food intake by these routes. Third-ventricular injection ofSHU9119, expected to access both forebrain and brainstem MC4-Rsites, partially attenuated the inhibition of food intake induced by i.p.injection of CCK-8s (Fig. 2c). Fourth-ventricle injection, which dye-injection tests had shown primarily accesses brainstem sites, completelyattenuated the CCK-8s-induced inhibition of food intake (Fig. 2d).

Both CCK-8s and normal food-induced satiety activated a smallgroup of NTS POMC neurons. These brainstem POMC cells are dis-tinct from previously characterized GLP-1-positive and cate-cholaminergic NTS neurons. CCK-8s-induced inhibition of feedingalso seems to depend on MC4-R signaling. These findings support amodel in which brainstem MC4-R neurons, and possibly NTS POMCneurons, contribute to the satiety effects of CCK and other meal-related satiety signals. Recently, electrical activation of cranial visceralafferents in the solitary tract was reported to activate POMC NTSneurons (Appleyard, S.M. et al., Soc. Neurosci. Abstr. 29, 231.11, 2003);however, the role of NTS POMC neurons in the perception of meal-related satiety has not been established. The distribution of POMCneurons in the ARC, where they are sensitive to the adipostatic hor-mone leptin, and the NTS, where they are responsive to vagally medi-ated satiety signals, makes the central melanocortin system ideallysuited for the integration of acute regulation of feeding behavior withthe long-term control of energy stores (Fig. 1h) Resistance to factorssuch as CCK may explain, in part, the profound hyperphagia andincreased meal size seen in obese subjects with mutations in Mc4r15.

Note: Supplementary information is available on the Nature Neuroscience website.

ACKNOWLEDGMENTSSupported by US National Institutes of Health grants DK55819 (R.D.C.) andDK62179 (W.F.), and a grant from the Wellcome Trust (K.L.J.E.). POMC-EGFPmice were a kind gift of M. Low (Oregon Health and Science University).

COMPETING INTERESTS STATEMENTThe authors declare competing financial interests; see Nature Neuroscience websitefor details.

Received 15 September 2003; accepted 12 February 2004Published online at http://www.nature.com/natureneuroscience/

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2. Cheung, C.C., Clifton, D.K. & Steiner, R.A. Endocrinol. 138, 4489–4492 (1997).3. Elias, C.F. et al. Neuron 23, 775–786 (1999).4. Cowley, M.A. et al. Nature 411, 480-484 (2001).5. Williams, D.L., Grill, H.J., Weiss, S.M., Baird, J.P. & Kaplan, J.M.

Psychopharmacology 161, 47–53 (2002).6. Azzara, A.V., Sokolnicki, J.P. & Schwartz, G.J. Physiol. Behav. 77, 411–416

(2002).7. Grill, H.J., Ginsberg, A.B., Seeley, R.J. & Kaplan, J.M. J. Neurosci. 18,

10128–10135 (1998).8. Joseph, S.A., Pilcher, W.H. & Bennet-Clarke, C. Neurosci. Lett. 38, 221–225

(1983).9. Wang, L., Martinez, V., Barrachina, M.D. & Tache, Y. Brain Res. 791, 157–166

(1998).10. Rinaman, L. Am. J. Physiol. 277, R582–R590 (1997).11. Luckman, S. J. Neuroendocrinol. 4, 149–152 (1992).12. Butler, A.A. et al. Endocrinol. 141, 3518–3521 (2000).13. Huszar, D. et al. Cell 88, 131–141 (1997).14. Hruby, V.J. et al. J. Med. Chem. 38, 3454–3461 (1995).15. Farooqi, I.S. et al. N. Engl. J. Med. 348, 1160–1163 (2003).

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Figure 2 Brainstem MC4-R signaling is required for CCK-8s-inducedfeeding inhibition. (a) Mc3r–/– mice are fully responsive to CCK-inducedinhibition of feeding. After a 16-h fast, 5–10-month wild-type C57BL/6J(C57) and Mc3r–/– mice (MC3-RKO) were injected i.p. with saline or CCK-8s (3 nmol/kg); the strains showed a comparable anorexigenic response toCCK-8s 30–180 min after treatment. (b) MC4-R is required for CCK-induced inhibition of feeding. After a 16-h fast, 9-week wild-type andMc4r–/– mice (MC4-RKO) were injected i.p. with saline or CCK-8s (3 nmol/kg). CCK-8s significantly reduced food intake in wild-type but notMc4r–/– mice. (c) Pharmacological blockade of central melanocortinreceptors in rats partially blocks CCK-induced inhibition of feeding. Ratsreceived 3rd-ventricle injections of a subthreshold dose of SHU9119(0.375 nmol/4 µl) 10–15 min before i.p. injection of CCK-8s (3 nmol/kg).(d) Pharmacological blockade of brainstem melanocortin receptors in ratsfully blocks CCK-induced inhibition of feeding. Rats received 4th-ventricleinjections of a subthreshold dose of SHU9119 (0.2 nmol/4 µl) just beforei.p. injection of CCK-8s (3 nmol/kg). Data given as mean ± s.e.m.Statistical analyses were done using one-way ANOVA (a,b) or unpaired t-test (c,d). Data presented as mean ± s.e.m. *, P < 0.05; **, P < 0.01; ***, P < 0.001. See Supplementary Methods online for details.

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