Melanin-concentrating hormone: a functional melanocortin antagonist in the hypothalamus

David S. Ludwig1,2, Kathleen G. Mountjoy5, Jeffrey B. Tatro4, Jennifer A. Gillette3, Robert C. Frederich1, Jeffrey S. Flier1, and Eleftheria Maratos-Flier3

1 Division of Endocrinology and Metabolism, Department of Medicine, Beth Israel-Deaconess Medical Center, 2 Division of Endocrinology, Department of Medicine, Children's Hospital, and 3 Elliott P. Joslin Research Laboratory, Joslin Diabetes Center, Boston 02115; 4 Division of Endocrinology, Metabolism and Molecular Medicine, Department of Medicine, Tupper Research Institute, Tufts University School of Medicine, New England Medical Center, Boston, Massachussetts 02111; and 5 Research Center for Developmental Medicine and Biology, University of Auckland, Auckland, New Zealand

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Melanin-concentrating hormone (MCH) and alpha -melanocyte-stimulating hormone (alpha -MSH) demonstrate opposite actions on skin coloration in teleost fish. Both peptides are present in the mammalian brain, although their specific physiological roles remain largely unknown. In this study, we examined the interactions between MCH and alpha -MSH after intracerebroventricular administration in rats. MCH increased food intake in a dose-dependent manner and lowered plasma glucocorticoid levels through a mechanism involving ACTH. In contrast, alpha -MSH decreased food intake and increased glucocorticoid levels. MCH, at a twofold molar excess, antagonized both actions of alpha -MSH. alpha -MSH, at a threefold molar excess, blocked the orexigenic properties of MCH. MCH did not block alpha -MSH binding or the ability of alpha -MSH to induce cAMP in cells expressing either the MC3 or MC4 receptor, the principal brain alpha -MSH receptor subtypes. These data suggest that MCH and alpha -MSH exert opposing and antagonistic influences on feeding behavior and the stress response and may function in a coordinate manner to regulate metabolism through a novel mechanism mediated in part by an MCH receptor.

obesity; eating

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE CONTROL OF BODY WEIGHT is a complex process that involves integration of peripheral signals and central neural mechanisms (18). Recently, much attention has focused on the ob gene product leptin (44), levels of which communicate the status of peripheral energy stores to the brain via activation of hypothalamic leptin receptors, themselves a product of the ob gene (9, 10). The neural mechanisms through which leptin acts to influence energy balance are the subject of intense study. Neuropeptide Y (NPY) is the best characterized target of leptin action in the brain and is likely to participate in mediating the neural consequences of leptin deficiency (35, 38). However, the absence of a phenotype in NPY knockout mice (16) suggests the existence of other critical appetite-regulating pathways.

Several lines of evidence support a role for the central melanocortin system in the regulation of energy balance. First, targeted disruption of the melanocortin-4 receptor (MC4R), a G protein-coupled receptor expressed in the brain, results in obesity in mice (22). Second, melanocortin analogs with agonist or antagonist properties on MC3R and MC4R regulate feeding after intracerebroventricular (icv) administration to mice (17). Finally, obesity due to the dominant agouti alleles is the result of ectopic expression of the 131-amino acid agouti protein (15), a secreted protein that acts as an antagonist at MC1R and MC4R (8, 26).

The existence of a melanocortin system for the central regulation of energy balance raises questions about the identity of the neuropeptides involved in this system. Agonists for melanocortin receptors are presumably one or more central products of the POMC gene, such as alpha -melanocyte-stimulating hormone (alpha -MSH) (1). Although the agouti protein may function as a direct antagonist at MC4R, agouti is not normally expressed within the brain. Two observations suggest that melanin-concentrating hormone (MCH) may act as an endogenous functional antagonist of the central melanocortin system. MCH antagonizes the alpha -MSH- induced enhancement of the hippocampal response to auditory stimuli (27). In female rats, alpha -MSH increases aggressive and exploratory behaviors, and this effect is antagonized by MCH (19). In addition, MCH blocks the alpha -MSH-induced grooming behavior (34).

MCH, first isolated from salmon in 1983, is a cyclic peptide present in the brains of all vertebrate species examined (24). The name of this compound derives from its ability to cause melanosome aggregation in fish skin, an action that antagonizes the melanosome-dispersing effects of alpha -MSH (4). In mammals, high concentrations of MCH are found in neurons of the dorsolateral hypothalamus and the zona incerta, with extensive projections throughout the brain (6, 36). Recently, a role for MCH in feeding behavior was proposed based on several observations (31): ob/ob mice overexpress MCH mRNA compared with lean littermates, and fasting increases hypothalamic MCH mRNA expression in both ob/ob and wild-type mice. In addition, intracerebroventricular MCH administration stimulates food intake in the rat (31, 33). Previous studies have suggested an interaction between MCH and the hypothalamic-pituitary-adrenal (HPA) axis (5, 7). Given the above observations, we have examined the ability of MCH to function as a physiological antagonist of central melanocortin action with respect to feeding and the HPA axis.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals. The feeding studies employed male Long-Evans rats weighing 300-400 g. Animals were obtained from Zivic-Miller Laboratories (Zeleinople, PA) with 22-gauge cannulas placed in the lateral ventricle according to the following coordinates: AP -0.9 mm from bregma, DV -4.8 mm from dura, and ML +1.5 mm. Cannula placement was confirmed by demonstration of increased thirst (water intake >5 ml in 1 h) after administration of ANG II (50 ng icv) and by histology. The animals were housed singly, fed standard laboratory diet, and maintained on 12:12-h reverse day-night cycles at 20°C. The dark cycle began at 9:30 AM. Before the start of the studies, animals were handled daily until tame to minimize the effects of stress on food intake. Body weight was measured twice weekly. Any animal showing signs of illness, such as weight loss, poor grooming, or decreased activity, was removed from the study. Number of animals (n) for these experiments ranged from 8 to 18.

Studies of the HPA axis were generally conducted with doubly cannulated male Sprague-Dawley rats weighing 250-300 g. These animals were also obtained from Zivic-Miller Laboratories with intrajugular venous catheters (tip at right atrium) and intracerebroventricular cannulas at the coordinates described above. Central cannula placement was similarly confirmed by ANG II administration and histology. Animals were maintained on standard, not reverse, 12:12-h day-night cycles; otherwise, they were handled and fed as above. The study depicted in Fig. 5 employed Long-Evans rats, which had been rehabituated to a standard light cycle for 2 wk.

Feeding studies. To reduce variation in feeding response to neuropeptide administration, we employed a modification of the methods of Stanley and Leibowitz (37). This approach uses a highly palatable food to induce a reproducible hyperphagia, followed by a consistent period of sustained, although slower, feeding. The animals were given the palatable food (consisting of evaporated milk, dextrose, and powdered standard rodent food in a ratio of 1.0:1.0:1.8) on the evening before each study. For experiments depicted in Figs. 1 and 3, a fresh aliquot of palatable food was provided 1 h before administration of the appetite-modulating substance, and food intake was monitored at 2, 4, 6, and 24 h after intracerebroventricular injection by weighing the remaining food. For the experiment depicted in Fig. 2, animals were fasted overnight, and the fresh aliquot of food was given at the time of intracerebroventricular injection. This modification served to increase baseline food intake during the initial few hours, thereby providing a greater range in which the effects of the anorectic agent alpha -MSH could be demonstrated. Injections (icv) were conducted within 1 h of the beginning of the dark period under calm conditions in a procedure lasting ~1 min. Control animals, receiving 5 µl of artificial cerebrospinal fluid (CSF) instead of neuropeptide, were handled in an identical manner to experimental animals and were included in all studies. After injection, the animal was returned to its cage and left undisturbed for the remainder of the study.

HPA axis studies. Handling stress was produced as previously described (13). The animal was gently removed from its cage, and baseline blood samples were obtained through the indwelling venous catheter. Subsequently, the animal was stroked for 30 s, given the intracerebroventricular injection, and returned to its cage. The total procedure lasted ~4 min. Control animals, receiving 5 µl of artificial CSF, were simultaneously studied in an identical manner. Additional blood samples were obtained at 45 and 90 min. For the experiments depicted in Fig. 5, the animals were treated similarly, except that blood samples were taken by tail bleed at 1 h after intracerebroventricular injection (no baseline obtained) in a procedure lasting <60 s.

Hormone assays. Blood samples were immediately placed into heparinized tubes on ice, plasma was obtained by centrifugation within 2 h, and specimens were stored at -20°C until assayed. Corticosterone and ACTH were determined by 125I radioimmunoassays using kits from ICN Biomedicals (Costa Mesa, CA) and Incstar (Stillwater, MN), respectively.

Receptor binding and activation studies. To test the possibility that MCH acts by inhibition of alpha -MSH binding to melanocortin receptors, a variant of B16 melanoma cells, B16-4GF, which do not express alpha -MSH receptors, was transfected with expression plasmids containing cDNA encoding either rat MC3R or rat MC4R. 125I-[Nle4,D-Phe7]-alpha -MSH (125I-NDP-MSH) was prepared, and binding assays were performed using monolayers on 24-well plates as described (21). Specific binding was >95% of total binding as determined by its inhibition in the presence of 1 µM alpha -MSH. Effects of hormones on melanogenesis in B16-F1C29 mouse melanoma cells were assayed as described (39).

The potential ability of MCH to antagonize melanocortin peptide (alpha -MSH, gamma 1-MSH, gamma 2-MSH, or ACTH) action at MC3R or MC4R was studied using transfected human embryonic kidney 293 cells expressing rat, mouse, or human receptors. Adenylate cyclase activity was measured as previously described (29). Kaleidagraph software program (Synergy Software, Reading, PA) was used for fitting curves to the data and calculating EC50 and maximal response (Rmax) values by the method of least squares.

Peptides. MCH and alpha -MSH were obtained from Bachem Biosciences (King of Prussia, PA). The agents were dissolved in artificial CSF (in mM: 147 NaCl, 1.3 CaCl2, 0.9 MgCl2, and 4.0 KCl) and injected in a total volume of 5 µl over 1 min. Artificial CSF was sterilized by autoclave, and neuropeptide solutions were kept frozen until use. Recombinant mouse agouti protein, prepared as described (28), was obtained from Dr. Greg Barsh (Stanford, CA).

Statistics. Data were analyzed by ANOVA, using Statview 4.0 software from Abacus Concepts (Berkeley, CA). Error bars in figures represent SE.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effects of MCH and alpha -MSH on food intake. To quantitate changes in appetite after central neuropeptide treatment, we used a modification of the methods of Stanley and Leibowitz (37). This approach employs a highly palatable food to elicit a reproducible, transient hyperphagia, followed by a sustained period of feeding at a slower rate (see METHODS). Injection (icv) of MCH, in the range of 0.1-25 µg, stimulated feeding behavior in a dose-dependent manner (Fig. 1). alpha -MSH, as previously reported (40), reduced food intake by about half over a 4- to 6-h period (Fig. 2). The anorectic action of intracerebroventricular alpha -MSH was significantly attenuated by coadministration of MCH at a twofold molar excess (Fig. 2). Conversely, the appetite-stimulating effect of 5 µg of intracerebroventricular MCH was completely blocked by alpha -MSH (Fig. 3A). However, when MCH was administered at a three molar excess (25 µg), alpha -MSH had no effect on stimulation of feeding (Fig. 3B). Thus, in parallel with their behavior in the skin of teleost fish, MCH and alpha -MSH behave as functional antagonists in the regulation of food intake in the mammalian central nervous system.


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Fig. 1.   Melanin-concentrating hormone (MCH) stimulates feeding behavior. Various doses of MCH in artificial cerebrospinal fluid (CSF) were administered by intracerebroventricular (icv) injection to male Long-Evans rats at beginning of dark cycle, and food intake was subsequently monitored. For control vs. 5 and 25 µg, P < 0.001 at all time points; for control vs. 0.1 µg, P = NS.


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Fig. 2.   MCH antagonizes anorectic actions of alpha -melanocyte-stimulating hormone (alpha -MSH). Animals were fasted overnight to increase baseline food consumption as described in METHODS. Doses: alpha -MSH 10 µg; MCH 25 µg. For control vs. alpha -MSH, P < 0.001 at 2 h; for alpha -MSH vs. alpha -MSH + MCH, P < 0.05 at all time points.


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Fig. 3.   alpha -MSH blocks effects of low-dose but not high-dose MCH. alpha -MSH was given at 10 µg. A: for MCH 5 µg vs. control or MCH 5 µg + alpha -MSH, P < 0.05 at all time points. B: for control vs. MCH 25 µg or MCH 25 µg + alpha -MSH, P< 0.05 at all time points.

Effects of MCH and alpha -MSH on HPA axis. To further assess this functional antagonism, we determined the effects of these two peptides on the activation of the HPA axis. HPA axis activity was assessed by determination of plasma corticosterone, the predominant circulating glucocorticoid in rodents, after a mild handling stress, as described in METHODS. Consistent with prior reports (13), handling produced a moderate rise in corticosterone, from 3.1 ± 0.1 to 10.2 ± 2.8 µg/dl at 45 min (Fig. 4A). Central administration of MCH not only blocked this stress-induced rise but also reduced 45-min corticosterone levels 60% below baseline values, from 5.8 ± 1.1 to 2.4 ± 0.5 µg/dl (Fig. 4A). The change in corticosterone level was associated with decreased ACTH levels (Fig. 4B). In contrast, alpha -MSH increased both corticosterone and ACTH levels (Fig. 4, A and B). Furthermore, MCH at a twofold molar excess prevented the alpha -MSH-induced rise in corticosterone (Fig. 5) and ACTH (data not shown).


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Fig. 4.   MCH decreases and alpha -MSH increases plasma corticosterone levels through an ACTH-dependent mechanism. Male Sprague-Dawley rats were subjected to a mild handling stress and then given 5 µg of MCH or 5 µg of alpha -MSH by icv injection. Blood was obtained for hormone determination via indwelling intrajugular catheters at 0, 45, and 90 min. A: corticosterone. For control vs. alpha -MSH, P < 0.0001 at 45 min; for control vs. MCH, P < 0.02 at 45 min. B: change in ACTH from baseline. For control vs. alpha -MSH, P < 0.0001; for control vs. MCH, P = 0.02 at 90 min.


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Fig. 5.   Antagonistic action of MCH and alpha -MSH on hypothalamic-pituitary-adrenal axis activity. Male Long-Evans rats were given icv injections, and blood was obtained at 1 h. Control, artificial CSF only. Doses: alpha -MSH 5 µg; alpha -MSH 5 µg + MCH 18.5 µg. For alpha -MSH vs. alpha -MSH + MCH, P = 0.02.

MCH does not antagonize alpha -MSH via known melanocortin receptors. Five subtypes of the melanocortin receptor have been identified (12). Among these, the brain and especially the hypothalamus contain mRNA encoding only two principal subtypes, MC3R and MC4R (29, 32), whereas mRNA coding for other known melanocortin receptors is either absent or present in exceedingly low amounts (3, 20). To determine whether MCH may antagonize alpha -MSH action by inhibiting alpha -MSH activation of the MC3R or MC4R, we tested the ability of MCH to inhibit agonist binding to, and activation of, these receptors in vitro. At concentrations of 0.1 nM-1 µM, MCH had no effect on the binding of the superpotent alpha -MSH agonist, 125I-NDP-MSH, to heterologous cells stably expressing the rat MC3R (B16-G4F-rMC3) or the rat MC4R (B16-G4F-rMC4). In contrast, alpha -MSH and the MCH receptor antagonist, mouse agouti protein, produced the expected dose-dependent inhibition of tracer binding (Fig. 6, A and B). Similarly, concentrations of MCH from 4 to 400 nM did not affect the ability of alpha -MSH, gamma 1-MSH, gamma 2-MSH, or ACTH to activate adenylate cyclase in cell lines bearing MC3R or MC4R (neither EC50 nor Rmax was altered). For example, the EC50 for activation of MC3R with ACTH is 3.85 × 10-9 M in the absence of MCH and 2.99 × 10-9 M in the presence of 400 µM of MCH. Rmax is 0.95 without MCH and 0.93 in the presence of 400 µM MCH.


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Fig. 6.   Lack of MCH effects on 125I-NDP-MSH binding to melanocortin receptor subtypes. Iodinated 125I-NDP-MSH was added to cells grown on 24-well plates for 30 min at 20°C in presence or absence of indicated peptide. Binding was specific and inhibited by both alpha -MSH and recombinant agouti protein. Addition of MCH at concentrations up to 10-6 M had no effect on binding of ligand. Similar results were seen with B16-G4F-rMC3 cells (A) and B16-G4F-rMC4 cells (B). MC3R binding was examined in 3 separate experiments; MC4R binding studies were performed twice. SEs are not shown but ranged between 1 and 12% of mean control binding. MCH at concentrations of up to 10-6 M had no effect on binding, whereas alpha -MSH and mouse agouti protein showed expected dose-dependent inhibition of binding.

Unlike the mutant MCH receptor-deficient B16-G4F mouse melanoma subline, other B16 cells express the native MC1R, a melanocortin receptor subtype suggested by one report to be expressed in a small population of midbrain neurons in the rat (42). MCH (0.1 nM-1.0 µM) had no effect on 125I-NDP-MSH binding in B16-F1-C23 cells (data not shown) or on alpha -MSH-induced melanogenesis in B16-F1-C29 cells (Fig. 7). A slight trend toward stimulation of melanogenesis at the highest concentrations tested was noted (0.1 and 1 µM), consistent with its reported alpha -MSH-like activity in melanophores at high concentrations (Fig. 7). In contrast, mouse agouti protein, at a concentration of 10-7 M, inhibited melanogenesis (26) and stimulation of cAMP accumulation induced by alpha -MSH and NDP-MSH in the B16-F1-C29 cells (data not shown). Taken together, these results suggest that MCH inhibits alpha -MSH action by a mechanism other than interference with agonist activation of known melanocortin receptor subtypes.


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Fig. 7.   Lack of MCH effect on melanogenesis in B16-F1C29 cells. alpha -MSH stimulated melanogenesis in a dose-dependent manner. MCH showed a minimal stimulation at very high doses and did not inhibit alpha -MSH effect. Each point represents a mean of 4 wells.

    DISCUSSION
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Introduction
Methods
Results
Discussion
References

This study demonstrates that MCH and alpha -MSH produce functionally opposing actions within the mammalian central nervous system with regard to feeding and HPA axis. Interestingly, this mutual antagonism extends from effects on fish melanophores to complex behavior in the mammalian central nervous system. These antagonistic actions involve several pathways, two of which are described in this paper, regulation of food intake and the HPA axis, as well as actions on complex behaviors related to grooming, auditory gating, and aggression, which have previously been described (19, 27, 34). These actions are mediated by at least two receptors, MC4R (responding to alpha -MSH) and an as yet unidentified receptor for MCH. MCH is well placed both anatomically (lateral hypothalamus) and functionally to be a key participant alongside products of the POMC gene in the melanocortin system for regulation of energy balance.

Central MCH administration stimulates food consumption through a dose range of 0.1-25 µg in accordance with, and extending, a recent observation by us (31). This result is at variance with that of Presse et al. (30), who described decreased food intake after administration of MCH into the lateral ventricle at extremely low doses (1 ng-0.1 mg). An orexigenic effect of MCH was recently reported by Rossi et al. (33) using a dose similar to ours. This group examined doses as low as 10 ng in an attempt to duplicate the data of Presse et al., which demonstrated inhibition in low doses. Like us, Rossi et al. failed to see inhibition of eating at any dose. We believe that these data clearly demonstrate an orexigenic effect of MCH. At present, both the precise anatomic targets of MCH and the physiological concentrations of MCH at these targets are unknown. Hence, it is difficult to distinguish between a physiological and a pharmacological effect. This is a problem in assessing action of many centrally administered compounds, such as NPY (11). Further studies involving direct injection of MCH into potential target areas as well as measurement of ambient MCH levels will be required to address this question.

We have also confirmed that alpha -MSH can inhibit food intake after intracerebroventricular injection in rats. The fact that higher doses of one neuropeptide (MCH) can antagonize the actions of the other (alpha -MSH), and vice versa, is of interest and can be viewed as a mutually functional antagonism. The simplest hypothesis to explain the observed functional antagonism of alpha -MSH by MCH is a model in which MCH binds competitively to central MCH receptors and prevents their activation by alpha -MSH (26). However, the fact that MCH does not affect alpha -MSH binding or alpha -MSH action on cells expressing either cloned MC3R or MC4R excludes this possibility, at least with regard to these previously identified receptors.

The receptor through which MCH exerts its actions, whether in teleost fish or mammalian brain, has yet to be identified, although specific MCH binding sites have been demonstrated in B16F10 melanoma cells (14). It remains to be determined whether this receptor or some other species mediates the central actions of MCH. It should be noted that the physiological antagonism of two molecules is most often exerted through binding to unique receptors (i.e., insulin vs. glucagon, parathyroid hormone vs. calcitonin) that converge on downstream signaling pathways, rather than through competitive interactions at a common receptor. Thus identification of the central MCH receptor is an important goal.

The functional antagonism of MCH and alpha -MSH also extends to regulation of the HPA axis. A strong relationship between the regulation of feeding and the HPA axis has previously been noted. Actions of peripheral signals, such as leptin (2), and central neuropeptides, such as NPY and corticotropin-releasing hormone, on both of these axes have been amply documented. Here we show that MCH inhibits glucocorticoid secretion by an ACTH-dependent mechanism. This finding builds on previous reports that salmon MCH (bearing a 70% homology in amino acid sequence to mammalian peptide) inhibited ACTH release from isolated rat pituitary in vitro (5) and that central MCH injection in rats reduced peak circadian ACTH levels and inhibited stress-induced ACTH secretion (7). Another study, however, found increased ACTH levels after intracerebroventricular injection (23). Although the reason for this discrepancy remains unexplained, our data support the role of MCH as an inhibitor of ACTH secretion. In contrast, alpha -MSH was shown in the present study to increase glucocorticoid levels by stimulating ACTH secretion. This in vivo demonstration of alpha -MSH stimulation of ACTH release is consistent with previous studies showing that alpha -MSH stimulates release of ACTH from primary cultures of rat anterior pituitary cells (25). Finally, we show that MCH can fully attenuate the effects of alpha -MSH on corticosterone levels. Future studies will address the possibility that this antagonism is involved in the integration of feeding and the HPA axis under one or more physiological conditions.

An interesting parallel can be drawn to mice with mutations at the agouti locus (28). The agouti gene product, normally expressed in the skin of various mammals, competitively inhibits the binding of alpha -MSH to the MC1R (26). As a result of this antagonism, the black hair produced under the influence of alpha -MSH develops a band of yellow, providing a dusky appearance (called agouti) to the coat. The viable yellow and lethal yellow mouse mutations cause ubiquitous overexpression of agouti protein and result, as expected, in a completely yellow coat color (26). In addition to pigmentary changes, however, the agouti mutants manifest hyperphagia and obesity (43). Moreover, they also have lower corticosterone levels than other obese mouse strains (41). These phenotypic changes, analogous to the acute effects of MCH described here, may demonstrate the consequences of chronic melanocortin antagonism by ectopically expressed agouti in the hypothalamus.

The mechanisms underlying the pleiotropic metabolic effects of agouti overexpression are not known; the most likely candidates for central molecular targets mediating agouti action are MC4R, for which agouti is a potent and specific antagonist (26), and perhaps MC3R. Despite the qualitatively similar effects of agouti overexpression and acute MCH administration on feeding behavior and the HPA axis, the present results indicate that MCH does not antagonize these central melanocortin receptors. Thus MCH may comprise a functional hypothalamic analog of agouti protein, operating through a unique, as yet unidentified receptor. It is also possible that MCH antagonizes other appetite suppressing peptides (GLP-1 and urocortin) and that alpha -MSH can antagonize orexigenic peptides, such as NPY. Further studies are required to address this issue.

    ACKNOWLEDGEMENTS

We thank Drs. Joe Majzoub and Lou Muglia for thoughtful suggestions, Jean Flanagan and Margaret Entwistle for expert technical assistance, and Drs. Greg Barsh and Mike Ollman for kindly providing the mouse agouti protein to the laboratory of J. B. Tatro. We also thank Jennifer Marron for assistance in preparing the manuscript.

    FOOTNOTES

The animals used were housed at the Diabetes and Endocrinology Research Center Animal Core Facility at the Joslin Diabetes Center or the Beth Israel Deaconess Hospital.

D. S. Ludwig was supported by grants from the Charles H. Hood Foundation and the Lawson Wilkins Pediatric Endocrine Society. J. S. Flier was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R08-DK-02440, and J. B. Tatro was supported by National Institute of Mental Health Grant MH-44694. This work was supported by grants from the American Diabetes Association, DERC Pilot and Feasibility Study (to E. Maratos-Flier), the Health Research Council of New Zealand, and a Wellcome Trust Senior Research Fellowship (to K. G. Mountjoy).

Present address of R. C. Frederich: Div. of Endocrinology, Department of Medicine, Chandler Medical Center, University of Kentucky, Lexington, KY 40536.

Address for reprint requests: E. Maratos-Flier, Joslin Diabetes Center, One Joslin Place, Rm. 620, Boston, MA 02215.

Received 20 June 1997; accepted in final form 16 December 1997.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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