- Email: [email protected]
Feeding effects of hypothalamic injection of melanocortin 4 receptor ligands
Brain Research 809 Ž1998. 302–306
Short communication
Feeding effects of hypothalamic injection of melanocortin 4 receptor ligands Silvia Q. Giraudo
a,b,)
, Charles J. Billington
a,b,c
, Allen S. Levine
a,b,c
a
b
Department of Medicine, UniÕersity of Minnesota, Minneapolis, MN USA Department of Food Science and Nutrition, UniÕersity of Minnesota, St. Paul, MN USA c Minnesota Obesity Center, VA Medical Center, Minneapolis, MN 55417, USA Accepted 11 August 1998
Abstract It has been reported that intraventricular administration of the melanocortin 4 receptor ŽMC4-R. agonist MT II and antagonist SHU9119 alter food intake. We found that MT II and SHU9119 have extremely potent effects on feeding when injected in the paraventricular nucleus ŽPVN., a site where MC4-R gene expression is very high. Our finding provides direct evidence that MC4-R signaling is important is mediating food intake and that melanocortin neurons in the PVN exert a tonic inhibition of feeding behavior. Chronic disruption of this inhibitory signal is a possible explanation of the agouti-obesity syndrome. q 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Paraventricular nucleus; MT II; SHU9119; MC4-receptor; Food intake
The control of body weight is a complex process that likely involves the interplay of numerous molecular mechanisms and neural circuits, many of them yet undefined. Recently, a link between melanocortin receptors and obesity has been shown based on studies of the action of the agouti pigmentation factor w2–4x. In the mouse the agouti protein normally is expressed in the skin where it regulates the synthesis of pigment by antagonism of the melanocortin-1 receptor ŽMC1-R., also known as the melanocyte-stimulating hormone receptor on the melanocytes w5x. Dominant alleles of the agouti locus resulting in widespread ectopic expression of agouti give rise to a pleiotropic obesity syndrome referred to as the obese yellow or agouti syndrome w1,12,13x. The obesity of the yellow mouse Žmaturity-onset. results from over expression of the agouti protein which competes with amelanocyte-stimulating hormone Ž a-MSH. for binding to melanocortin-4-receptor ŽMC4-R.. Similarly, genetic disruption of the MC4-R also causes obesity w4x. Thus, a-MSH derived from the arcuate nucleus pro-opioimelanocortin ŽPOMC. neurons, the primary source of ligand for MC4-R,
) Corresponding author. VA Medical Center, Research Service Ž151., One Veterans Drive, Minneapolis, MN 55417, USA. Fax: q1-612-7252093
appears to play a tonic inhibitory role in feeding and energy storage w9x. Fan et al. identified cyclic melanocortin analogues that are potent agonists or antagonists to the MC4-R w3x. Among them, the agonist MT II was found to produce a potent inhibition of feeding when given intracerebroventricularly and the antagonist SHU9119 Žagoutimimetic protein. was found to block the anorectic effect of MT II w3,11x. Consistent with a role in body weight regulation, the MC4-R is expressed in a number of hypothalamic sites, including the ventromedial, lateral, dorsomedial and paraventricular ŽPVN. nuclei, which play important roles in regulating feeding behavior w6x. Questions about which brain sites are critical for the MC4-R action remain, since specific site injections have not yet been reported. The current studies were aimed at confirming the participation of PVN MC4 receptors in the regulation of food intake by injecting an MC4-R agonist ŽMT II. and antagonist ŽSHU9119. into the PVN. Male Sprague–Dawley rats ŽHarlan, Madison, WI., weighing 225–250 g, were individually housed in conventional hanging cages with a 12 h lightr12 h dark photoperiod Žlights on at 0700. in a temperature controlled room Ž21–228C.. Rats were anesthetized with Nembutal Ž40 mgrkg. and fitted with 26 gauge stainless steel guide cannulas ŽPlastics One, Austin, TX. in the PVN. Stereo-
0006-8993r98r$ - see front matter q 1998 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 8 3 7 - 3
S.Q. Giraudo et al.r Brain Research 809 (1998) 302–306
taxic coordinates were determined from the rat brain atlas by Paxinos and Watson w8x and for PVN cannulations were: 0.5 mm lateral, 1.9 mm posterior to bregma, and 7.3
303
mm below the skull surface. The injector extended 1 mm beyond the tip of the guide cannula. For all cannulations, the incisor bar was set at 3.3 mm below the ear bars. At
Fig. 1. Cumulative food intake after 10 and 50 pmol MT II at 0–2 h ŽA., 2–4 h ŽB., and 4–24 h ŽC. PVN injection. Values are means" S.E.M. Values with different superscripts are significantly different Ž P - 0.0001..
304
S.Q. Giraudo et al.r Brain Research 809 (1998) 302–306
least 7 days elapsed following surgery before experimental trials were conducted. PVN injections were administered in a 0.5 ml volume over 30 s.
Food was allowed ad libitum until the start of each experimental trial. Immediately before drug injection food was removed from the cages. Immediately following injec-
Fig. 2. Cumulative food intake after 10 and 50 pmol SHU9119 at 0–2 h ŽA., 2–4 h ŽB., and 4–24 h ŽC. PVN injection. Values are means" S.E.M. Values with different superscripts are significantly different Ž P - 0.0001..
S.Q. Giraudo et al.r Brain Research 809 (1998) 302–306
tions, pre-weighed chow hoppers were placed back in the rat cage. At 2 and 4 h, the pellets and collected spillage were weighed and subtracted from the initial weight to quantify the amount of food eaten. Following the experiments, brains were dissected out and stored in a 10% formaldehyde solution for later placement verification by histologic examination. Coronal sections Ž40 mm thick. through the brain were cut on a cryostat and mounted on glass microscope slides. The sections were stained with 0.1% Thionin ŽLauths Violet; Sigma, St. Louis, MO. solution, dried and coverslipped. Data from animals with incorrectly placed cannulas Ž n s 2. were discarded from the experiments. Ac-Nle-Asp-His-D-Phe-Arg-D-Trp-Lys-NH 2 ŽMT II. and Ac-Nle-Asp-His- D -NalŽ 2 X . -Arg- D -Trp-Lys-NH 2 ŽSHU9119. were purchased from Phoenix Pharmaceuticals, ŽMountain View, CA.. All drugs were dissolved in 0.9% saline just prior to use. In the first experiment PVN cannulated animals were stimulated to eat by overnight deprivation Ž18 h.. Immediately before having access to food, MT II was injected into the PVN at doses of 10 and 50 pmol. We injected the rats over a series of days, with 3 days between each trial, each animal receiving both doses Ž n s 14.. On each experimental day, a control group Žsaline. was included. Food intake was measured 2, 4 and 24 h post injection. In the second experiment cannulated rats were randomly assigned to treatment groups and received either saline, or PVN SHU9119 at doses of 10 pmol and 50 pmol. At least 3-day elapsed between injections and each rat received all treatments Ž n s 12.. Food intake was measured at 2, 4 and 24 h post injection. Data were analyzed by a one factor repeated measures ANOVA and means were compared using Scheffe’s F-test. All data are expressed as mean " S.E.M. In the first experiment we found a main effect of drug administration on deprivation induced feeding. Doses of 10 and 50 pmol of MT II strongly suppressed deprivation-induced feeding at 0–2 h Ž F2,41 s 12.993, p s 0.0001. and 0–4 h Ž F2,41 s 11.887, p s 0.0002. after PVN injection ŽFig. 1.. Four hours after injection, 10 and 50 pmol doses of MT II significantly reduced food intake relative to the control treatment by 23 and 48%, respectively. However, when measures were collected at 24 h after MT II administration, only 50 pmol was found to significantly reduce food intake Ž F2,41 s 18.876, p s 0.0001.. In experiment 2, the agouti-like protein SHU9119 significantly stimulated feeding significantly 0–2 h Ž F2,35 s 11.601, p s 0.0004., 0–4 h Ž F2,35 s 23.393, p s 0.0001. and 4–24 h Ž F2,35 s 35.128, p s 0.0001. ŽFig. 2. after 10 and 50 pmol PVN injection. Stimulation of feeding was evident at 2 h post treatment, and continued during 4 to 24 h to produce a mean increase in food intake of 27 and 52%, relative to vehicle injected rats. Consistent with previous findings in mice w3x, PVN administration of the MC4-receptor agonist MT II at doses
305
of 10 and 50 pmol caused a reduction of food intake at 2 and 4 h. Also, the 50 pmol dose reduced food intake up to 24 h after administration into the PVN ŽFig. 1.. Thiele et al. reported similar results when doses of 100 and 1000 pmol MT II was given into the third ventricle of rats w11x. Their highest dose reduced feeding up to 48 h. In the case of the MC4-R antagonist SHU9119, doses of 10 and 50 pmol increased feeding significantly above the control group ŽFig. 2.. This potent stimulus lasted up to 24 h. Recently, Seeley et al. have shown that 500 pmol of SHU9119 injected intraventricularly blocked the ability of leptin to reduce long-term food intake and body weight; a dose SHU9119 that had no effect on food intake by itself w10x. In the Seeley study, only the 1000 pmol of SHU9119 increased food intake significantly at 4 and 24 h after ventricular administration. In mice, Fan et al. found no effect of SHU9119 during daytime feeding, however a dose of 3000 pmol induced a significant increase of feeding at night. A significant difference between previous studies and ours is the specific site of injection. Previous studies have used injection of agonistrantagonist of the MC4-R intracerebroventricularly or into lateral or third ventricles w3,10,11x. We found that both MT II and SHU9119 have extremely potent effects on feeding behavior when injected into the PVN, a site were MC4-R gene expression is very high. Mountjoy et al. reported that high concentrations of the MC4-R mRNA are found in both the parvicellular and magnocellular neurons of the paraventricular nucleus w6,7x. Our findings provide direct evidence that MC4-R signaling is important in mediating food intake. The data suggest that melanocortin neurons in the PVN exert a tonic inhibition of feeding behavior. Chronic disruption of this inhibitory signal is a likely explanation of the agouti-obesity syndrome. The current studies bring additional information on the MC4-R and its agonistrantagonist peptides on a specific site of action, the PVN. The small doses used and the long term effects on food intake indicate that the PVN is one primary site of action for MC4-R.
Acknowledgements This research was supported by the General Research Funds of the Veterans Administration Medical Center, the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK 50456 and by the National Institute of Drug Abuse Grant DA-03999.
References w1x G.A. Bray, D.A. York, Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis, Physiol. Rev. 59 Ž1979. 719–809. w2x R.D. Cone, D. Lu, S. Koppula, D.I. Vage, H. Klungland, B. Boston,
306
w3x
w4x
w5x
w6x
w7x
S.Q. Giraudo et al.r Brain Research 809 (1998) 302–306 W. Chen, D.N. Orth, C. Pouton, R.A. Kesterson, The melanocortin receptors: agonists, antagonists, and the hormonal control of pigmentation, Recent Prog. Horm. Res., 51 Ž1996. 287–317; discussion, 318 Ž1996.. W. Fan, B.A. Boston, R.A. Kesterson, V.J. Hruby, R.D. Cone, Role of melanocortinergic neurons in feeding and the agouti obesity syndrome, Nature 385 Ž1997. 165–168, see comments. D. Huszar, C.A. Lynch, V. Fairchild-Huntress, J.H. Dunmore, Q. Fang, L.R. Berkemeier, W. Gu, R.A. Kesterson, B.A. Boston, R.D. Cone, F.J. Smith, L.A. Campfield, P. Burn, F. Lee, Targeted disruption of the melanocortin-4 receptor results in obesity in mice, Cell 88 Ž1997. 131–141. D. Lu, D. Willard, I.R. Patel, S. Kadwell, L. Overton, T. Kost, M. Luther, W. Chen, R.P. Woychik, W.O. Wilkison et al., Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor, Nature 371 Ž1994. 799–802. K.G. Mountjoy, M.T. Mortrud, M.J. Low, R.B. Simerly, R.D. Cone, Localization of the melanocortin-4 receptor ŽMC4-R. in neuroendocrine and autonomic control circuits in the brain, Mol. Endocrinol. 8 Ž1994. 1298–1308. K.G. Mountjoy, J. Wong, Obesity, diabetes and functions for proopi-
w8x w9x
w10x
w11x
w12x
w13x
omelanocortin-derived peptides, Mol. Cell. Endocrinol. 128 Ž1997. 171–177. G. Paxinos, C. Watson, The rat brain in stereotaxic coordinates, San Diego, 1986. R. Poggioli, A.V. Vergoni, A. Bertolini, ACTH-Ž1-24. and a-MSH antagonize feeding behavior stimulated by kappa opiate agonists, Peptides 7 Ž1986. 843–848. R.J. Seeley, K.A. Yagaloff, S.L. Fisher, P. Burn, T.E. Thiele, G. van Dijk, D.G. Baskin, M.W. Schwartz, Melanocortin Receptors in Leptin Effects 390 Ž1997. 349. T.E. Thiele, G. van Dijk, K.A. Yagaloff, S.L. Fisher, M. Schwartz, P. Burn, R.J. Seeley, Central infusion of melanocortin agonist MTII in rats: assessment of c-Fos expression and taste aversion, Am. J. Physiol. 274 Ž1998. R248–54. G.L. Wolff, D.B. Galbraith, O.E. Domon, J.M. Row, Phaeomelanin synthesis and obesity in mice. Interaction of the viable yellow ŽAvy. and sombre Žeso. mutations, J. Hered. 69 Ž1978. 295–298. G.L. Wolff, D.W. Roberts, D.B. Galbraith, Prenatal determination of obesity, tumor susceptibility, and coat color pattern in viable yellow ŽAvyra. mice. The yellow mouse syndrome, J. Hered. 77 Ž1986. 151–158.
Short communication
Feeding effects of hypothalamic injection of melanocortin 4 receptor ligands Silvia Q. Giraudo
a,b,)
, Charles J. Billington
a,b,c
, Allen S. Levine
a,b,c
a
b
Department of Medicine, UniÕersity of Minnesota, Minneapolis, MN USA Department of Food Science and Nutrition, UniÕersity of Minnesota, St. Paul, MN USA c Minnesota Obesity Center, VA Medical Center, Minneapolis, MN 55417, USA Accepted 11 August 1998
Abstract It has been reported that intraventricular administration of the melanocortin 4 receptor ŽMC4-R. agonist MT II and antagonist SHU9119 alter food intake. We found that MT II and SHU9119 have extremely potent effects on feeding when injected in the paraventricular nucleus ŽPVN., a site where MC4-R gene expression is very high. Our finding provides direct evidence that MC4-R signaling is important is mediating food intake and that melanocortin neurons in the PVN exert a tonic inhibition of feeding behavior. Chronic disruption of this inhibitory signal is a possible explanation of the agouti-obesity syndrome. q 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Paraventricular nucleus; MT II; SHU9119; MC4-receptor; Food intake
The control of body weight is a complex process that likely involves the interplay of numerous molecular mechanisms and neural circuits, many of them yet undefined. Recently, a link between melanocortin receptors and obesity has been shown based on studies of the action of the agouti pigmentation factor w2–4x. In the mouse the agouti protein normally is expressed in the skin where it regulates the synthesis of pigment by antagonism of the melanocortin-1 receptor ŽMC1-R., also known as the melanocyte-stimulating hormone receptor on the melanocytes w5x. Dominant alleles of the agouti locus resulting in widespread ectopic expression of agouti give rise to a pleiotropic obesity syndrome referred to as the obese yellow or agouti syndrome w1,12,13x. The obesity of the yellow mouse Žmaturity-onset. results from over expression of the agouti protein which competes with amelanocyte-stimulating hormone Ž a-MSH. for binding to melanocortin-4-receptor ŽMC4-R.. Similarly, genetic disruption of the MC4-R also causes obesity w4x. Thus, a-MSH derived from the arcuate nucleus pro-opioimelanocortin ŽPOMC. neurons, the primary source of ligand for MC4-R,
) Corresponding author. VA Medical Center, Research Service Ž151., One Veterans Drive, Minneapolis, MN 55417, USA. Fax: q1-612-7252093
appears to play a tonic inhibitory role in feeding and energy storage w9x. Fan et al. identified cyclic melanocortin analogues that are potent agonists or antagonists to the MC4-R w3x. Among them, the agonist MT II was found to produce a potent inhibition of feeding when given intracerebroventricularly and the antagonist SHU9119 Žagoutimimetic protein. was found to block the anorectic effect of MT II w3,11x. Consistent with a role in body weight regulation, the MC4-R is expressed in a number of hypothalamic sites, including the ventromedial, lateral, dorsomedial and paraventricular ŽPVN. nuclei, which play important roles in regulating feeding behavior w6x. Questions about which brain sites are critical for the MC4-R action remain, since specific site injections have not yet been reported. The current studies were aimed at confirming the participation of PVN MC4 receptors in the regulation of food intake by injecting an MC4-R agonist ŽMT II. and antagonist ŽSHU9119. into the PVN. Male Sprague–Dawley rats ŽHarlan, Madison, WI., weighing 225–250 g, were individually housed in conventional hanging cages with a 12 h lightr12 h dark photoperiod Žlights on at 0700. in a temperature controlled room Ž21–228C.. Rats were anesthetized with Nembutal Ž40 mgrkg. and fitted with 26 gauge stainless steel guide cannulas ŽPlastics One, Austin, TX. in the PVN. Stereo-
0006-8993r98r$ - see front matter q 1998 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 8 3 7 - 3
S.Q. Giraudo et al.r Brain Research 809 (1998) 302–306
taxic coordinates were determined from the rat brain atlas by Paxinos and Watson w8x and for PVN cannulations were: 0.5 mm lateral, 1.9 mm posterior to bregma, and 7.3
303
mm below the skull surface. The injector extended 1 mm beyond the tip of the guide cannula. For all cannulations, the incisor bar was set at 3.3 mm below the ear bars. At
Fig. 1. Cumulative food intake after 10 and 50 pmol MT II at 0–2 h ŽA., 2–4 h ŽB., and 4–24 h ŽC. PVN injection. Values are means" S.E.M. Values with different superscripts are significantly different Ž P - 0.0001..
304
S.Q. Giraudo et al.r Brain Research 809 (1998) 302–306
least 7 days elapsed following surgery before experimental trials were conducted. PVN injections were administered in a 0.5 ml volume over 30 s.
Food was allowed ad libitum until the start of each experimental trial. Immediately before drug injection food was removed from the cages. Immediately following injec-
Fig. 2. Cumulative food intake after 10 and 50 pmol SHU9119 at 0–2 h ŽA., 2–4 h ŽB., and 4–24 h ŽC. PVN injection. Values are means" S.E.M. Values with different superscripts are significantly different Ž P - 0.0001..
S.Q. Giraudo et al.r Brain Research 809 (1998) 302–306
tions, pre-weighed chow hoppers were placed back in the rat cage. At 2 and 4 h, the pellets and collected spillage were weighed and subtracted from the initial weight to quantify the amount of food eaten. Following the experiments, brains were dissected out and stored in a 10% formaldehyde solution for later placement verification by histologic examination. Coronal sections Ž40 mm thick. through the brain were cut on a cryostat and mounted on glass microscope slides. The sections were stained with 0.1% Thionin ŽLauths Violet; Sigma, St. Louis, MO. solution, dried and coverslipped. Data from animals with incorrectly placed cannulas Ž n s 2. were discarded from the experiments. Ac-Nle-Asp-His-D-Phe-Arg-D-Trp-Lys-NH 2 ŽMT II. and Ac-Nle-Asp-His- D -NalŽ 2 X . -Arg- D -Trp-Lys-NH 2 ŽSHU9119. were purchased from Phoenix Pharmaceuticals, ŽMountain View, CA.. All drugs were dissolved in 0.9% saline just prior to use. In the first experiment PVN cannulated animals were stimulated to eat by overnight deprivation Ž18 h.. Immediately before having access to food, MT II was injected into the PVN at doses of 10 and 50 pmol. We injected the rats over a series of days, with 3 days between each trial, each animal receiving both doses Ž n s 14.. On each experimental day, a control group Žsaline. was included. Food intake was measured 2, 4 and 24 h post injection. In the second experiment cannulated rats were randomly assigned to treatment groups and received either saline, or PVN SHU9119 at doses of 10 pmol and 50 pmol. At least 3-day elapsed between injections and each rat received all treatments Ž n s 12.. Food intake was measured at 2, 4 and 24 h post injection. Data were analyzed by a one factor repeated measures ANOVA and means were compared using Scheffe’s F-test. All data are expressed as mean " S.E.M. In the first experiment we found a main effect of drug administration on deprivation induced feeding. Doses of 10 and 50 pmol of MT II strongly suppressed deprivation-induced feeding at 0–2 h Ž F2,41 s 12.993, p s 0.0001. and 0–4 h Ž F2,41 s 11.887, p s 0.0002. after PVN injection ŽFig. 1.. Four hours after injection, 10 and 50 pmol doses of MT II significantly reduced food intake relative to the control treatment by 23 and 48%, respectively. However, when measures were collected at 24 h after MT II administration, only 50 pmol was found to significantly reduce food intake Ž F2,41 s 18.876, p s 0.0001.. In experiment 2, the agouti-like protein SHU9119 significantly stimulated feeding significantly 0–2 h Ž F2,35 s 11.601, p s 0.0004., 0–4 h Ž F2,35 s 23.393, p s 0.0001. and 4–24 h Ž F2,35 s 35.128, p s 0.0001. ŽFig. 2. after 10 and 50 pmol PVN injection. Stimulation of feeding was evident at 2 h post treatment, and continued during 4 to 24 h to produce a mean increase in food intake of 27 and 52%, relative to vehicle injected rats. Consistent with previous findings in mice w3x, PVN administration of the MC4-receptor agonist MT II at doses
305
of 10 and 50 pmol caused a reduction of food intake at 2 and 4 h. Also, the 50 pmol dose reduced food intake up to 24 h after administration into the PVN ŽFig. 1.. Thiele et al. reported similar results when doses of 100 and 1000 pmol MT II was given into the third ventricle of rats w11x. Their highest dose reduced feeding up to 48 h. In the case of the MC4-R antagonist SHU9119, doses of 10 and 50 pmol increased feeding significantly above the control group ŽFig. 2.. This potent stimulus lasted up to 24 h. Recently, Seeley et al. have shown that 500 pmol of SHU9119 injected intraventricularly blocked the ability of leptin to reduce long-term food intake and body weight; a dose SHU9119 that had no effect on food intake by itself w10x. In the Seeley study, only the 1000 pmol of SHU9119 increased food intake significantly at 4 and 24 h after ventricular administration. In mice, Fan et al. found no effect of SHU9119 during daytime feeding, however a dose of 3000 pmol induced a significant increase of feeding at night. A significant difference between previous studies and ours is the specific site of injection. Previous studies have used injection of agonistrantagonist of the MC4-R intracerebroventricularly or into lateral or third ventricles w3,10,11x. We found that both MT II and SHU9119 have extremely potent effects on feeding behavior when injected into the PVN, a site were MC4-R gene expression is very high. Mountjoy et al. reported that high concentrations of the MC4-R mRNA are found in both the parvicellular and magnocellular neurons of the paraventricular nucleus w6,7x. Our findings provide direct evidence that MC4-R signaling is important in mediating food intake. The data suggest that melanocortin neurons in the PVN exert a tonic inhibition of feeding behavior. Chronic disruption of this inhibitory signal is a likely explanation of the agouti-obesity syndrome. The current studies bring additional information on the MC4-R and its agonistrantagonist peptides on a specific site of action, the PVN. The small doses used and the long term effects on food intake indicate that the PVN is one primary site of action for MC4-R.
Acknowledgements This research was supported by the General Research Funds of the Veterans Administration Medical Center, the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK 50456 and by the National Institute of Drug Abuse Grant DA-03999.
References w1x G.A. Bray, D.A. York, Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis, Physiol. Rev. 59 Ž1979. 719–809. w2x R.D. Cone, D. Lu, S. Koppula, D.I. Vage, H. Klungland, B. Boston,
306
w3x
w4x
w5x
w6x
w7x
S.Q. Giraudo et al.r Brain Research 809 (1998) 302–306 W. Chen, D.N. Orth, C. Pouton, R.A. Kesterson, The melanocortin receptors: agonists, antagonists, and the hormonal control of pigmentation, Recent Prog. Horm. Res., 51 Ž1996. 287–317; discussion, 318 Ž1996.. W. Fan, B.A. Boston, R.A. Kesterson, V.J. Hruby, R.D. Cone, Role of melanocortinergic neurons in feeding and the agouti obesity syndrome, Nature 385 Ž1997. 165–168, see comments. D. Huszar, C.A. Lynch, V. Fairchild-Huntress, J.H. Dunmore, Q. Fang, L.R. Berkemeier, W. Gu, R.A. Kesterson, B.A. Boston, R.D. Cone, F.J. Smith, L.A. Campfield, P. Burn, F. Lee, Targeted disruption of the melanocortin-4 receptor results in obesity in mice, Cell 88 Ž1997. 131–141. D. Lu, D. Willard, I.R. Patel, S. Kadwell, L. Overton, T. Kost, M. Luther, W. Chen, R.P. Woychik, W.O. Wilkison et al., Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor, Nature 371 Ž1994. 799–802. K.G. Mountjoy, M.T. Mortrud, M.J. Low, R.B. Simerly, R.D. Cone, Localization of the melanocortin-4 receptor ŽMC4-R. in neuroendocrine and autonomic control circuits in the brain, Mol. Endocrinol. 8 Ž1994. 1298–1308. K.G. Mountjoy, J. Wong, Obesity, diabetes and functions for proopi-
w8x w9x
w10x
w11x
w12x
w13x
omelanocortin-derived peptides, Mol. Cell. Endocrinol. 128 Ž1997. 171–177. G. Paxinos, C. Watson, The rat brain in stereotaxic coordinates, San Diego, 1986. R. Poggioli, A.V. Vergoni, A. Bertolini, ACTH-Ž1-24. and a-MSH antagonize feeding behavior stimulated by kappa opiate agonists, Peptides 7 Ž1986. 843–848. R.J. Seeley, K.A. Yagaloff, S.L. Fisher, P. Burn, T.E. Thiele, G. van Dijk, D.G. Baskin, M.W. Schwartz, Melanocortin Receptors in Leptin Effects 390 Ž1997. 349. T.E. Thiele, G. van Dijk, K.A. Yagaloff, S.L. Fisher, M. Schwartz, P. Burn, R.J. Seeley, Central infusion of melanocortin agonist MTII in rats: assessment of c-Fos expression and taste aversion, Am. J. Physiol. 274 Ž1998. R248–54. G.L. Wolff, D.B. Galbraith, O.E. Domon, J.M. Row, Phaeomelanin synthesis and obesity in mice. Interaction of the viable yellow ŽAvy. and sombre Žeso. mutations, J. Hered. 69 Ž1978. 295–298. G.L. Wolff, D.W. Roberts, D.B. Galbraith, Prenatal determination of obesity, tumor susceptibility, and coat color pattern in viable yellow ŽAvyra. mice. The yellow mouse syndrome, J. Hered. 77 Ž1986. 151–158.