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© 2004 Nature Publishing Group http://www.nature.com/natureneuroscience
Cholecystokinin-mediated suppression of feeding involves the brainstem melanocortin system Wei Fan, Kate L J Ellacott, Ilia G Halatchev, Kanji Takahashi, Pinxuan Yu & Roger D Cone Hypothalamic pro-opiomelanocortin (POMC) neurons help regulate long-term energy stores. POMC neurons are also found in the nucleus tractus solitarius (NTS), a region regulating satiety. We show here that mouse brainstem NTS POMC neurons are activated by cholecystokinin (CCK) and feedinginduced satiety and that activation of the neuronal melanocortin-4 receptor (MC4-R) is required for CCK-induced suppression of feeding; the melanocortin system thus provides a potential substrate for integration of long-term adipostatic and short-term satiety signals. Hypothalamic POMC neurons tonically inhibit food intake1 and are regulated by the long-term adipostatic factor leptin2–4. However, the central melanocortin system is also important in the acute regulation of satiety; in particular, central administration of melanocortins reduces food intake by decreasing meal size, a hallmark of satiety5,6. These hypothalamic neurons send fibers to MC4-R target sites in both the hypothalamus and brainstem, and melanocortin agonists administered to either region inhibit feeding7. Notably, in addition to expression in the arcuate nucleus of the hypothalamus (ARC), POMC is also expressed in the caudal aspect of the NTS8, the primary site of synapse of vagal afferent fibers transmitting satiety information from the gastrointestinal system. NTS neurons are activated by either electrical or CCK-induced stimulation of vagal afferent fibers. Furthermore, leptin and CCK act synergistically to inhibit feeding and activate NTS neurons9. Yet regulation of POMC cells in the NTS by metabolic state has not been reported. Here, we test the hypotheses that (i) brainstem POMC neurons are activated by satiety signals and (ii) central melanocortin signaling is required for the action of specific signals that acutely inhibit feeding. Intraperitoneal (i.p.) injection of CCK-8s (the sulfated 8-aminoacid form of cholecystokinin) significantly increased c-Fos immunoreactivity 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 ± 11 cells per section, n = 4; P < 0.001) (Fig. 1; compare Fig. 1a,d), as shown previously9. Immunohistochemical experiments using a previously characterized transgenic mouse that expresses enhanced green fluorescent protein (EGFP) under the control of the POMC promoter4 showed no significant difference in the number of POMC-EGFP–immunoreactive (IR) neurons in the NTS between saline- (Fig. 1b) and CCK-8streated 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 between groups treated with i.p. saline or CCK-8s (data not shown). We also examined a model of feeding-induced satiety. We gave POMC-EGFP mice a 5-d training in which they were allowed access to food for two
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 EGFPPOMC 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 neurons are 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 by POMC neurons in ARC and NTS, respectively. Blue, nuclei containing POMC neurons; yellow, nuclei containing MC4-R neurons that may serve to integrate 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, area postrema; DMV, dorsal motor nucleus of vagus. All studies followed the NIH Guide For the Care and Use of Laboratory Animals and were approved by the Oregon Health and Sciences University Animal Care and Use Committee. See Supplementary Methods online for details.
periods totaling 5 h (9:00–10:00 h, 14:00–18:00 h) and examined c-Fos immunoreactivity in the ARC and NTS at 11:00 h (see Supplementary Fig. 1 online; fed n = 8; fasted, n = 3, *** P < 0.001). Feeding activated c-Fos expression in ∼21% of ARC POMC neurons but also in 13% of NTS POMC neurons (Supplementary Fig. 1). Although a majority of c-Fos-IR cells in the ARC were POMC positive, only a few percent of those in the NTS were, showing the complexity of cells in the NTS
Vollum Institute, Oregon Health and Science University, Portland, Oregon 97239-3098, USA. Correspondence should be addressed to R.D.C. (
[email protected]) or W.F. (
[email protected]). Published online 14 March 2004; doi:10.1038/nn1214
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Figure 2 Brainstem MC4-R signaling is required for CCK-8s-induced feeding inhibition. (a) Mc3r–/– mice are fully responsive to CCK-induced inhibition 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 CCK8s (3 nmol/kg); the strains showed a comparable anorexigenic response to CCK-8s 30–180 min after treatment. (b) MC4-R is required for CCKinduced inhibition of feeding. After a 16-h fast, 9-week wild-type and Mc4r–/– 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 not Mc4r–/– mice. (c) Pharmacological blockade of central melanocortin receptors in rats partially blocks CCK-induced inhibition of feeding. Rats received 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 rats fully blocks CCK-induced inhibition of feeding. Rats received 4th-ventricle injections of a subthreshold dose of SHU9119 (0.2 nmol/4 µl) just before i.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 ttest (c,d). Data presented as mean ± s.e.m. *, P < 0.05; **, P < 0.01; ***, P < 0.001. See Supplementary Methods online for details.
involved in satiety. Previous work has shown that both catecholaminergic and glucagon-like peptide-1 (GLP-1)-positive cells in the NTS are involved in satiety10,11. Dual immunohistochemical analysis showed that although POMC-EGFP–IR cells and tyrosine hydroxylase–IR cells are found in the same region of the NTS, they are not coexpressed 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-IR neurons (Supplementary Fig. 2). To test whether feeding suppression by CCK-8s was dependent on central melanocortin signaling, we examined the ability of CCK-8s to inhibit food intake after a fast in three different mouse lines, two of which carry deletions of the genes encoding melanocortin receptors 3 and 4, respectively: C57BL/6J, C57BL/6J Mc3r–/–12 and C57BL/6J Mc4r–/–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-type and Mc3r–/– mice (Fig. 2a) and continued to inhibit food intake for up to 180 min in each strain. We then administered CCK-8s to female Mc4r–/– mice and age-matched female wild-type mice. CCK-8s significantly reduced food intake in wild-type mice, but not in Mc4r–/– mice
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(Fig. 2b), at time points from 30 to 180 min. Next we examined the site of action of endogenous melanocortins. We administered the MC3-R and MC4-R antagonist SHU9119 (ref. 14) to rats via either the 3rd or 4th ventricle to assess the relative contributions of forebrain and brainstem MC4-R target sites in CCK-mediated inhibition of feeding. We used subthreshold doses of SHU9119 previously determined not to stimulate food intake by these routes. Third-ventricular injection of SHU9119, expected to access both forebrain and brainstem MC4-R sites, partially attenuated the inhibition of food intake induced by i.p. injection of CCK-8s (Fig. 2c). Fourth-ventricle injection, which dyeinjection tests had shown primarily accesses brainstem sites, completely attenuated the CCK-8s-induced inhibition of food intake (Fig. 2d). Both CCK-8s and normal food-induced satiety activated a small group of NTS POMC neurons. These brainstem POMC cells are distinct from previously characterized GLP-1-positive and catecholaminergic NTS neurons. CCK-8s-induced inhibition of feeding also seems to depend on MC4-R signaling. These findings support a model in which brainstem MC4-R neurons, and possibly NTS POMC neurons, contribute to the satiety effects of CCK and other mealrelated satiety signals. Recently, electrical activation of cranial visceral afferents in the solitary tract was reported to activate POMC NTS neurons (Appleyard, S.M. et al., Soc. Neurosci. Abstr. 29, 231.11, 2003); however, the role of NTS POMC neurons in the perception of mealrelated satiety has not been established. The distribution of POMC neurons in the ARC, where they are sensitive to the adipostatic hormone leptin, and the NTS, where they are responsive to vagally mediated satiety signals, makes the central melanocortin system ideally suited for the integration of acute regulation of feeding behavior with the long-term control of energy stores (Fig. 1h) Resistance to factors such as CCK may explain, in part, the profound hyperphagia and increased meal size seen in obese subjects with mutations in Mc4r15. Note: Supplementary information is available on the Nature Neuroscience website. ACKNOWLEDGMENTS Supported by US National Institutes of Health grants DK55819 (R.D.C.) and DK62179 (W.F.), and a grant from the Wellcome Trust (K.L.J.E.). POMC-EGFP mice were a kind gift of M. Low (Oregon Health and Science University). COMPETING INTERESTS STATEMENT The authors declare competing financial interests; see Nature Neuroscience website for details. Received 15 September 2003; accepted 12 February 2004 Published online at http://www.nature.com/natureneuroscience/ 1. Fan, W., Boston, B.A., Kesterson, R.A., Hruby, V.J. & Cone, R.D. Nature 385, 165–168 (1997). 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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