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Melanin-concentrating hormone: from fish skin to skinny mammals
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Melanin-concentrating hormone: from fish skin to skinny mammals Pavlos Pissios and Eleftheria Maratos-Flier Obesity Section, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215, USA
In recent years, the key role of melanin-concentrating hormone (MCH) in regulating mammalian energy balance has been confirmed through several lines of evidence. When administered exogenously, MCH leads to a rapid and robust feeding response and chronic infusions result in the development of mild obesity. At the physiological level, it is known that MCH expression changes in states of altered energy balance, such as fasting and obesity. Genetic studies with mice have shown that ablation of either the gene for prepro-MCH or the gene encoding the MCH receptor leads to a lean phenotype. Finally, the administration of MCH antagonists appears to inhibit both feeding and the development of diet-induced obesity. The aim of this article is to review the recent data on MCH and MCH receptors in light of their emerging roles in energy homeostasis. Melanin-concentrating hormone was initially characterized as a circulating factor mediating color change in teleost fish 20 years ago [1], although speculation regarding the existence of such a factor dates back to studies of pigmentation changes in amphibians performed in the 1930s [2]. MCH was first referenced as ‘melanophoreconcentrating hormone’ in 1955 in studies examining the possible origin of a factor leading to lightening of fish color [3]. In fish, MCH is a cyclic 17-amino acid polypeptide with a cysteine – cysteine disulfide bond (Fig. 1). It is synthesized as a preprohormone in the pituitary and is secreted into the circulation. It acts on cells derived from neuroectoderm, designated melanophores, which contain large numbers of pigment-containing vesicles called melanosomes. In these target cells, MCH changes the distribution of the melanosomes by stimulating their migration to the perinuclear area, an effect accomplished by the association of the melanosomes with microtubulebased motor proteins. Agents causing aggregation, such as MCH, cause transport of melanosomes by dynein motors, whereas agents causing dispersion lead to transport along kinesin motors [4]. The dispersion or aggregation changes the refractive index of the fish scales; as melanosomes are dispersed, scales become darker and, as they aggregate, scales become lighter. MCH is not the only factor to mediate color change. Migration into the perinuclear area is also stimulated by melatonin [5]. Other agents, such as b-adrenergic agonists and melanocyte-stimulating hormone (a-MSH), mediate the dispersion of melanophores Corresponding author: E. Maratos-Flier ([email protected]).
away from the nucleus into the cytoplasm, which results in skin darkening. In fish scales, MCH and a-MSH act as functional antagonists mediating color change. MCH synthesis in mammals MCH was identified in mammalian brain several years after its discovery in fish. Several studies reported the presence of neurons, which coincidentally crossreacted with antibodies to a-MSH but did not stain with antibodies to other products of the preproopiomelanocortin gene (POMC) [6]. These neurons were later found to correspond to neurons that stained with antibodies directed against salmon MCH. The isolation of mammalian MCH was accomplished as part of a peptide isolation program that involved extraction from 60 000 rat hypothalamic fragments [7]. Mammalian MCH is a 19-amino acid peptide that has high homology to salmon MCH (Fig. 1). The N-terminus is extended by two additional amino acids; however, the loop structure is highly conserved. At the amino acid level, MCH is identical in all mammals analyzed so far, including mouse, rat, rabbit and human. In contrast to fish, MCH synthesis in the central nervous system (CNS) is limited to the magnocellular neurons in the lateral hypothalamus and the zona incerta (Fig. 2). These hypothalamic neurons are atypical in that they make monosynaptic connections throughout the brain, (a)
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Fig. 1. Schematic representation of mammalian and salmon melanin-concentrating hormone (MCH). The different residues between (a) mammalian and (b) salmon MCH are colored red. In addition to the substitutions, salmon MCH lacks two amino acids at the N-terminus compared with mammalian MCH.
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Fig. 2. MCH is synthesized in the lateral hypothalamus and the zona incerta. Immunohistochemical localization of MCH-synthesizing neurons in the rat hypothalamus. Abbreviations: 3V, third ventricle; Arc, arcuate nucleus; DMH, dorsomedial nucleus; F, fornix; LHA, lateral hypothalamus; MCH, melanin-concentrating hormone; ME, median eminence; VMH, ventromedial nucleus; ZI, zona incerta. Scale bar ¼ 1 mm.
projecting to the cortex, amygdala, nucleus acccumbens, olfactory tubercle and to various nuclei in the brainstem. No association between MCH and mammalian pigmentation has been found. Identification of rodent cDNA for MCH (the official gene symbol is Pmch) was reported in 1989 and analysis of the mRNA indicated that the Pmch transcript encodes a preprohormone with two other potential neuropeptide products [8]. Rat, mouse and human mRNA sequences have a high degree of homology, with 90% overall nucleotide identity. The preprohormone encoded comprises 165 residues. MCH is generated via a proteolytic cleavage at arginines 145 and 146. The preprohormone can also be processed to two additional products, designated neuropeptide E-I (NEI) and neuropeptide G-E (NGE). Immunocytochemical studies indicate that NEI is cleaved and present in the same perikarya as MCH. However, it is unclear whether NGE exists as a functional peptide product. In addition to the peptides NEI, MCH and possibly NGE that are cleaved from the MCH prohormone, two other products of the Pmch gene have been detected. One includes an alternatively spliced mRNA that produces a peptide in a different reading frame from that for MCH. The peptide named MGOP (MCH-gene-overprinted-polypeptide) contains 14 amino acids after processing of a putative upstream dibasic cleavage site, although only unprocessed peptide was found in the rat hypothalamus [9,10]. Antisense transcription of the DNA partially overlapping with the Pmch gene has also been detected. The gene was named AROM (antisense RNA overlapping MCH gene), and it appears to generate multiple transcripts by alternative splicing. However, the coding sequences of these peptides do not overlap with the coding sequences of the MCH prohormone. The functional importance of these novel transcripts is currently unknown [11]. The distribution of MCH in mammalian brains suggested that it might be involved in mediating http://tem.trends.com
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‘motivated behaviors’. However, the role of the peptide remained obscure until 1996 when, with the use of RT– PCR differential display, the expression of Pmch mRNA was found to be increased in leptin-deficient ob/ob mice [12]. The increase was similar to that seen with the known orexigenic peptide neuropeptide P-Y (NPY), suggesting that MCH might play a role in appetite. This was confirmed when it was found that intracerebroventricular (ICV) administration of 5 mg of MCH to rats led to a rapid increase in chow consumption. Compared with vehicle, MCH-treated animals eat two- to threefold more over a six-hour period. Feeding can also be induced by injection of MCH directly into the paraventricular nucleus [13]. Although repetitive injections of MCH to rats over a one-week period did not lead to the development of obesity [14], chronic infusions into the lateral ventricle led to both hyperphagia and weight gain [15]. In addition to the overexpression of Pmch seen in the ob/ob model, Pmch expression increases two- to threefold with fasting, an effect that is seen in both normal and ob/ob animals. Treatment with leptin blunts the fasting-induced increase of Pmch mRNA in both wild-type (wt) and ob/ob mice. The increase in Pmch expression in ob/ob animals indicates that other factors, in addition to leptin, also regulate Pmch expression [16]. Furthermore, Pmch expression changes in hyperleptinemic models of obesity. For example, in the obese uncoupling protein 1-ditheria toxin mouse model, where leptin levels are extremely high, Pmch expression is suppressed to a similar extent as that seen for mRNA encoding NPY. Treatment of animals with b3-adrenergic receptor agonists, which typically leads to anorexia, is associated with a rapid fall in leptin levels. These agents lead to an increase in Pmch mRNA expression, which is significantly greater than that seen with fasting [17]. As an orexigenic peptide, MCH interacts with other neuropeptides that influence feeding behavior. For example, MCH is a mutual functional antagonist of the anorectic peptide a-MSH, which inhibits appetite. Depending on the dose administered, MCH can antagonize the anorectic effect of a-MSH, which in turn can antagonize the orexigenic effect of MCH [18]. Further evidence regarding the role of MCH in energy homeostasis comes from mice in which the Pmch gene is either ablated or overexpressed. Mice lacking MCH are , 25% leaner than their wt littermates [19], as a result of a reduction in feeding and a slight increase in energy expenditure. Otherwise, the mice appear to be normal, with normal levels of activity as assessed by an open field test, and normal fertility when bred with wt C57BL/6 mice. Leptin levels are low, as would be expected in a lean mouse model. Low leptin levels would be expected to lead to an increase in NPY and agouti-related peptide (AgRP) and a concomitant decrease in Pomc expression, which encodes the a-MSH precursor in the arcuate nucleus. However, analysis at the mRNA level revealed that only Pomc was decreased. Expression of mRNA encoding NPY and AgRP was at a similar level to that seen in normal mice. This suggests that, in the absence of MCH, low levels of a-MSH might contribute to the lean phenotype or, alternatively, Pomc is undergoing a compensatory downregulation.
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A strain of mice that overexpresses Pmch has also been generated from a P1 clone containing a large genomic fragment of the mouse Pmch gene [20]. Overexpression of Pmch was only seen in the neurons of the lateral hypothalamus and the levels of Pmch mRNA expression were twofold higher on average in the transgenic animals than in their wt littermates. On an FVB background, the mice had normal weight and normal feeding. When the transgene was bred to homozygosity and the homozygous animals were placed on a high-fat diet, the animals developed excessive obesity compared with normal control littermates. This appeared to be secondary to a trend towards hyperphagia seen in the Pmch-overexpressing animals. Another interesting feature of these animals was the significant hyperinsulinemia and islet cell hyperplasia, suggesting that MCH might play a role in the regulation of islets. However, it is unclear whether such a role would be mediated directly by local synthesis of MCH in islet ganglia or synapses from the CNS through paracrine regulation, or possibly via a secondary effect of MCH on an intermediate target tissue. Recently, chronic infusion in mice has reproduced the hyperphagic obese phenotype seen with overexpression of Pmch. Interestingly, these mice also showed a decrease in body temperature and a decrease in the oxidation of fatty acids in brown fat [21]. Additional effects of MCH in vivo in mice include an increase in water consumption, which is independent of feeding [22]. In conscious sheep, MCH has a diuretic effect but does not increase water consumption [23]. MCH signals through two G protein-coupled receptors Before the genetic identification of the rodent MCH receptor a significant body of literature reported on receptor expression with the use of radioreceptor assays utilizing either the iodinated native mammalian MCH or the biologically active Phe13Tyr19 analog [24]. Receptors were found in a variety of cells, including SVK14 keratinocytes [25] and mouse melanoma cells [24]. However, subsequent studies suggested that this binding was artifactitious in nature, resulting from physical properties of MCH and its analog and making the interpretation of binding assays problematic [26]. Indeed, subsequent studies of the SVK14 cell line indicate that these cells do not express mRNAs for either MCH receptor 1 (MCHR1) or MCH receptor 2 (MCHR2) [27]. The first high-affinity receptor for MCH was identified simultaneously by several groups using different pharmacological approaches [28 –31]. MCHR1 was found to be identical to the previously cloned orphan receptor SLC-1 and belongs to the superfamily of G protein-coupled receptors [32]. MCHR1 is 353 amino acids long, with all the hallmark features of the superfamily, including seven transmembrane helices, a DRY motif at the end the third intracellular loop and three potential glycosylation sites at the N-terminus. It shows the highest degree of homology to the somatostatin receptor family (, 35%), and the gene has been localized to chromosome 22q13.3 [32]. Comparison of the human and the rodent receptors shows a high degree of conservation between species (human – mouse, 95% identity; human –rat, 96% identity). Based on the homology to http://tem.trends.com
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the somatostatin receptors, which are coupled to Gi, it was quickly determined that MCHR1 could also suppress forskolin-stimulated cAMP and increase intracellular Ca2þ ([Ca2þ]i). Later work confirmed the ability of the receptor to couple to multiple G proteins, including Gi, Go and Gq [33]. However, the EC50 responses for cAMP inhibition and [Ca2þ]i increase suggest that the coupling is stronger to Gi than to Gq. Activation of MCHR1 also leads to the stimulation of protein kinase C, phospholipase C and extracellular-signal-regulated kinase pathways, at least in heterologous cell lines [33]. In the CNS, activation of the above signaling pathways has diverse effects, ranging from changes in gene expression to modulation of ion channel activity [34]. In addition, several ion channels are direct effectors of activated G proteins. For example, Gi-coupled receptors are known to inhibit voltage-gated Ca2þ channels (VGCCs) and to activate Kþ inward rectifying channels (Fig. 3) [35,36]. Both actions result in the inhibition of synaptic transmission. Consistent with these reports, MCH was found to inhibit Ca2þ currents flowing through the N-, P- and, to a lesser degree, the L-types of VGCCs in the neurons of the lateral hypothalamus [37], although activation of G protein-coupled inward rectifying channels by MCH has not been demonstrated. MCHR1 is most highly expressed in the brain, with moderate to weak expression in other tissues, including muscle, eye, tongue and adipose tissue [31]. The distribution of MCHR1 in the brain has been investigated in detail in the rat [38,39], and the expression pattern was found to be very broad throughout the CNS. Expression is seen in the cortex, basal ganglia, hypothalamus and
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Fig. 3. Hypothetical activation of signaling pathways by MCH receptors. Through coupling to multiple G (Gi/o/q) proteins, melanin-concentrating hormone (MCH) receptors activate phospholipase C (PLC), which catalyzes the production [from phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P3)] of diacylglycerol (DAG) and inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]. Ins(1,4,5)P3, in turn, causes an increase in intracellular calcium ([Ca2þ]i). Coupling to Gi (MCH receptor 1) causes inhibition of adenylate cyclase (AC) and therefore decreased production of cAMP. Extracellular-signal-regulated kinase (ERK) is also activated. Important effectors in the central nervous system include voltage-gated Ca2þ channels (VGCC) and G protein-coupled inward rectifying channels (GIRK), which are inhibited and activated by Gi proteins, respectively.
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brainstem. The presence of MCHR1 in the arcuate and ventromedial nuclei of the hypothalamus is consistent with MCH regulating food intake and energy balance. However, the highest level of expression is seen in extrahypothalamic areas that process olfactory information, namely the anterior olfactory nucleus, the piriform cortex and the olfactory tubercle. Very high levels of MCHR1 are also found in the shell of the nucleus accumbens, an area involved in rewarding aspects of motivated behaviors, such as food intake. Significant amounts are also present in the amygdala and hippocampus, potentially implicating MCH in regulating emotions, such as fear and anxiety, and also as a potential regulator of memory. Therefore, MCH is probably involved in many other behaviors, some of which might be important in food intake, such as olfaction and reward, in addition to regulating general mood and arousal levels. Similar patterns of expression of MCHR1 are seen in the mouse brain. Furthermore, MCHR1 expression in mice appears to be regulated by altered energy balance. Expression rises with fasting and this increase is inhibited by leptin administration [40]. Recently, MCHR1 has been inactivated in mice by homologous recombination [41,42]. Mice lacking functional MCHR1 maintained on regular chow diet exhibited decreased fat mass, increased activity and, surprisingly, were found to be hyperphagic compared with their wt littermates. When placed on a high-fat diet, MCHR1deficient mice gained significantly less weight, suggesting that inactivation of MCHR1 protected them from dietinduced obesity. Consistent with the above reports, pharmacological blockade of MCHR1 using specific antagonists produced similar effects [43]. Chronic administration of the MCHR1 antagonist SNAP-7941 to diet-induced obese rats caused a decrease in food intake and body weight. In addition to the beneficial effect on energy homeostasis, SNAP-7941 also acted as an anxiolytic. These results indicate that the MCH system is effective in reducing body weight under conditions in which leptin is ineffective and make the MCH receptor a viable target for the treatment of obesity. Two years after the cloning of MCHR1, database scanning revealed a second high-affinity receptor for MCH (MCHR2), with significant homology to the core region of MCHR1. The human receptor is 340 amino acids long and its gene resides on the long arm of chromosome 6 (6q16.2 – 6q21) [44– 49]. Analysis of the gene encoding MCHR2 revealed one non-coding and five coding exons that span .35 kb of chromosome 6 [44]. The overall homology between the two MCH receptors is quite low. They share only 38% identical amino acids, with the highest homology in the transmembrane domains that form part of the putative MCH-binding pocket. Figure 4 shows a space-filled model of the MCHR1. The colored residues are identical between the two MCH receptors. By looking directly into the cleft formed by the transmembrane helices the considerable sequence identity that underlies the high-affinity binding of these receptors to MCH is readily apparent. Overall, the expression profile of MCHR2 is similar to MCHR1, with the highest expression seen in the brain, notably in the frontal cortex, amygdala http://tem.trends.com
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Fig. 4. Melanin-concentrating hormone (MCH) receptor 1 and MCH receptor 2 exhibit significant sequence homology in the putative MCH-binding pocket. Spacefill model of the human MCH receptor 1 based on the crystal structure of bovine rhodopsin [56]. The colored residues correspond to identical amino acids between the two receptors. The view is from outside looking into the cleft formed by the seven transmembrane helices. The model was generated by the Swiss-model/Swiss PDB viewer and enhanced by POV-Ray [57].
and nucleus accumbens [44,45,49]. Expression is also detected, at least by quantitative RT–PCR, in several tissues, including intestine, adipose tissue and prostate [49]. Curiously, MCHR2 does not appear to be present in many species. Tan and co-workers performed a detailed investigation of several species for the presence of MCH receptors [50]. They found that rodents do not have MCHR2. Guinea pigs and rabbits carry non-functional alleles, whereas carnivores, such as ferrets and dogs, have both MCH receptors. Monkeys and humans also have both MCH receptors. Expression of recombinant MCHR2 in heterologous cell lines demonstrated the ability of the receptor to increase [Ca2þ]i levels. However, MCHR2 was unable to decrease cAMP levels and was insensitive to pertussis toxin, suggesting that it couples exclusively to Gq [44,45,49]. This is in contrast to MCHR1, which appears to couple to multiple G proteins, at least in heterologous cell lines. The overall distribution of MCHR2 in the CNS suggests that it might be involved in aspects of physiology similar to those of MCHR1. However, the functional importance of MCHR2 is currently difficult to assess without an appropriate animal model. MCH outside the CNS In mammals, the potential significance of MCH action outside the CNS remains controversial. MCH expression has been reported in the testis, stomach and intestine [51], and one recent report suggested that prepro-MCH might also be expressed in immune cells [52] and in endothelial cells derived from human skin [53]. Furthermore, MCH receptor expression in the periphery has also been reported. Both primary rat adipocytes and 3T3-L1cells are known to express the receptor and to respond to MCH [54], and direct effects of MCH on the leptin promoter have been demonstrated in 3T3-L1 cells [54]. Receptor expression has also been shown in ciliary epithelium
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[55]. Other reports have demonstrated the ability of MCHR1 to activate intracellular signaling pathways in monocytes, melanocytes and islet cells [39,52]. Although these findings suggest that MCH signaling in the periphery might be important, the physiological role outside of pigmentation in fish remains unclear. Conclusions A role for MCH in energy homeostasis is now clear. Genetic ablation of either the MCH prohormone or MCHR1 results in leanness. MCH, injected ICV, stimulates feeding acutely and chronic infusions lead to the development of obesity. However, several important questions remain. The mechanisms underlying the effects of MCH on energy homeostasis are not clear, although food intake, locomotor activity and resting energy expenditure probably play a major role. Differences exist between mice lacking the MCH prohormone and mice lacking MCHR1. Those lacking MCHR1 are hyperphagic, hypermetabolic and have higher locomotor activity, whereas those lacking the MCH prohormone have slightly decreased feeding and slightly increased energy expenditure. These differences could potentially be explained by ablation of the NEI and NGE peptides in addition to MCH in the prohormonedeficient mice. The second MCH receptor, MCHR2, has relatively low homology to MCHR1 and does not exist in rodent species. Hence, its potential role in feeding and energy balance remains uncertain. Furthermore, the widespread reach of MCH neurons and the broad expression of MCH receptors, especially in regions concerned with olfactory processing and motivated behaviors, make MCH an ideal candidate for the integration of various homeostatic and sensory stimuli necessary for the proper coordination of energy intake and expenditure. Future studies will address these important questions. Acknowledgements We thank Richard Bradley and Dan Trombly for careful reading of the article and Elena Nikiforova for technical assistance. EMF is a recipient of NIH grants DK53978 and DK56113. EMF also participates in program project DKPP56116.
References 1 Kawauchi, H. et al. (1983) Characterization of melanin-concentrating hormone in chum salmon pituitaries. Nature 305, 321– 323 2 Hogben, L.T. and Slome, D. (1931) The pigmentary effector system VI. The dual character of endocrine coordination in amphibian colour change. Proc. R. Soc. London B. Biol. Sci. 108, 10 – 53 3 Enami, M. (1955) Melanophore-concentrating hormone (MCH) of possible hypothalamic origin in the catfish, parasilurus. Science 121, 36 – 37 4 Rogers, S.L. et al. (1997) Regulated bidirectional motility of melanophore pigment granules along microtubules in vitro. Proc. Natl. Acad. Sci. U. S. A. 94, 3720– 3725 5 Filadelfi, A.M. and Castrucci, A.M. (1994) Melatonin desensitizing effects on the in vitro responses to MCH, a-MSH, isoproterenol and melatonin in pigment cells of a fish (S. marmoratus), a toad (B. ictericus), a frog (R. pipiens), and a lizard (A. carolinensis), exposed to varying photoperiodic regimens. Comp. Biochem. Physiol. A. Physiol. 109, 1027 – 1037 6 Kohler, C. et al. (1984) A diffuse a MSH-immunoreactive projection to the hippocampus and spinal cord from individual neurons in the lateral hypothalamic area and zona incerta. J. Comp. Neurol. 223, 501 – 514 http://tem.trends.com
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7 Vaughan, J.M. et al. (1989) Characterization of melanin-concentrating hormone from rat hypothalamus. Endocrinology 125, 1660 – 1665 8 Nahon, J.L. et al. (1989) The rat melanin-concentrating hormone messenger ribonucleic acid encodes multiple putative neuropeptides coexpressed in the dorsolateral hypothalamus. Endocrinology 125, 2056– 2065 9 Toumaniantz, G. et al. (1996) The rat melanin-concentrating hormone gene encodes an additional putative protein in a different reading frame. Endocrinology 137, 4518 – 4521 10 Toumaniantz, G. et al. (2000) Differential neuronal expression and projections of melanin-concentrating hormone (MCH) and MCH-geneoverprinted-polypeptide (MGOP) in the rat brain. Eur. J. Neurosci. 12, 4367– 4380 11 Borsu, L. et al. (2000) The AROM gene, spliced mRNAs encoding new DNA/RNA-binding proteins are transcribed from the opposite strand of the melanin-concentrating hormone gene in mammals. J. Biol. Chem. 275, 40576 – 40587 12 Qu, D. et al. (1996) A role for melanin-concentrating hormone in the central regulation of feeding behaviour. Nature 380, 243 – 247 13 Rossi, M. et al. (1999) Investigation of the feeding effects of melanin concentrating hormone on food intake – action independent of galanin and the melanocortin receptors. Brain Res. 846, 164 – 170 14 Rossi, M. et al. (1997) Melanin-concentrating hormone acutely stimulates feeding, but chronic administration has no effect on body weight. Endocrinology 138, 351 – 355 15 Della-Zuana, O. et al. (2002) Acute and chronic administration of melanin-concentrating hormone enhances food intake and body weight in Wistar and Sprague-Dawley rats. Int. J. Obes. Relat. Metab. Disord. 26, 1289– 1295 16 Tritos, N.A. et al. (2001) Characterization of melanin concentrating hormone and preproorexin expression in the murine hypothalamus. Brain Res. 895, 160 – 166 17 Mantzoros, C.S. et al. (1996) Activation of b3 adrenergic receptors suppresses leptin expression and mediates a leptin-independent inhibition of food intake in mice. Diabetes 45, 909 – 914 18 Ludwig, D.S. et al. (1998) Melanin-concentrating hormone: a functional melanocortin antagonist in the hypothalamus. Am. J. Physiol. 274, E627– E633 19 Shimada, M. et al. (1998) Mice lacking melanin-concentrating hormone are hypophagic and lean. Nature 396, 670– 674 20 Ludwig, D.S. et al. (2001) Melanin-concentrating hormone overexpression in transgenic mice leads to obesity and insulin resistance. J. Clin. Invest. 107, 379 – 386 21 Ito, M. et al. (2003) Characterization of MCH-mediated obesity in mice. Am. J. Physiol. Endocrinol. Metab. 284, E940– E945 22 Clegg, D.J. et al. (2003) Intraventricular melanin concentrating hormone stimulates water intake independent of food intake. Am. J. Physiol. Regul. Integr. Comp. Physiol. 284, R494– R499 23 Parkes, D.G. (1996) Diuretic and natriuretic actions of melanin concentrating hormone in conscious sheep. J. Neuroendocrinol. 8, 57 – 63 24 Drozdz, R. and Eberle, A.N. (1995) Synthesis and iodination of human (phenylalanine 13, tyrosine 19) melanin-concentrating hormone for radioreceptor assay. J. Pept. Sci. 1, 58 – 65 25 Burgaud, J.L. et al. (1997) Melanin-concentrating hormone binding sites in human SVK14 keratinocytes. Biochem. Biophys. Res. Commun. 241, 622 – 629 26 Kokkotou, E. et al. (2000) Characterization of [Phe(13), Tyr(19)]-MCH analog binding activity to the MCH receptor. Neuropeptides 34, 240– 247 27 Audinot, V. et al. (2002) SVK14 cells express an MCH binding site different from the MCH1 or MCH2 receptor. Biochem. Biophys. Res. Commun. 295, 841 – 848 28 Bachner, D. et al. (1999) Identification of melanin concentrating hormone (MCH) as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1). FEBS Lett. 457, 522 – 524 29 Chambers, J. et al. (1999) Melanin-concentrating hormone is the cognate ligand for the orphan G protein-coupled receptor SLC-1. Nature 400, 261 – 265 30 Lembo, P.M. et al. (1999) The receptor for the orexigenic peptide melanin-concentrating hormone is a G protein-coupled receptor. Nat. Cell Biol. 1, 267 – 271
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31 Saito, Y. et al. (1999) Molecular characterization of the melaninconcentrating-hormone receptor. Nature 400, 265 – 269 32 Kolakowski, L.F. Jr et al. (1996) Characterization of a human gene related to genes encoding somatostatin receptors. FEBS Lett. 398, 253 – 258 33 Hawes, B.E. et al. (2000) The melanin-concentrating hormone receptor couples to multiple G proteins to activate diverse intracellular signaling pathways. Endocrinology 141, 4524– 4532 34 Curtis, J. and Finkbeiner, S. (1999) Sending signals from the synapse to the nucleus: possible roles for CaMK, Ras/ERK, and SAPK pathways in the regulation of synaptic plasticity and neuronal growth. J. Neurosci. Res. 58, 88 – 95 35 Dolphin, A.C. (1998) Mechanisms of modulation of voltage-dependent calcium channels by G proteins. J. Physiol. 506, 3 – 11 36 Yamada, M. et al. (1998) G protein regulation of potassium ion channels. Pharmacol. Rev. 50, 723 – 760 37 Gao, X.B. and Van Den Pol, A.N. (2002) Melanin-concentrating hormone depresses L-, N-, and P/Q-type voltage-dependent calcium channels in rat lateral hypothalamic neurons. J. Physiol. 542, 273 – 286 38 Hervieu, G.J. et al. (2000) The distribution of the mRNA and protein products of the melanin-concentrating hormone (MCH) receptor gene, slc-1, in the central nervous system of the rat. Eur. J. Neurosci. 12, 1194 – 1216 39 Saito, Y. et al. (2001) Expression of the melanin-concentrating hormone (MCH) receptor mRNA in the rat brain. J. Comp. Neurol. 435, 26 – 40 40 Kokkotou, E.G. et al. (2001) Melanin-concentrating hormone receptor is a target of leptin action in the mouse brain. Endocrinology 142, 680 – 686 41 Marsh, D.J. et al. (2002) Melanin-concentrating hormone 1 receptordeficient mice are lean, hyperactive, and hyperphagic and have altered metabolism. Proc. Natl. Acad. Sci. U. S. A. 99, 3240– 3245 42 Chen, Y. et al. (2002) Targeted disruption of the melanin-concentrating hormone receptor-1 results in hyperphagia and resistance to dietinduced obesity. Endocrinology 143, 2469 – 2477 43 Borowsky, B. et al. (2002) Antidepressant, anxiolytic and anorectic effects of a melanin-concentrating hormone-1 receptor antagonist. Nat. Med. 8, 825 – 830 44 An, S. et al. (2001) Identification and characterization of a
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melanin-concentrating hormone receptor. Proc. Natl. Acad. Sci. U. S. A. 98, 7576 – 7581 Sailer, A.W. et al. (2001) Identification and characterization of a second melanin-concentrating hormone receptor, MCH-2R. Proc. Natl. Acad. Sci. U. S. A. 98, 7564 – 7569 Rodriguez, M. et al. (2001) Cloning and molecular characterization of the novel human melanin-concentrating hormone receptor MCH2. Mol. Pharmacol. 60, 632 – 639 Mori, M. et al. (2001) Cloning of a novel G protein-coupled receptor, SLT, a subtype of the melanin-concentrating hormone receptor. Biochem. Biophys. Res. Commun. 283, 1013 – 1018 Wang, S. et al. (2001) Identification and pharmacological characterization of a novel human melanin-concentrating hormone receptor, mch-r2. J. Biol. Chem. 276, 34664 – 34670 Hill, J. et al. (2001) Molecular cloning and functional characterization of MCH2, a novel human MCH receptor. J. Biol. Chem. 276, 20125 – 20129 Tan, C.P. et al. (2002) Melanin-concentrating hormone receptor subtypes 1 and 2: species-specific gene expression. Genomics 79, 785– 792 Hervieu, G. and Nahon, J.L. (1995) Pro-melanin concentrating hormone messenger ribonucleic acid and peptides expression in peripheral tissues of the rat. Neuroendocrinology 61, 348 – 364 Verlaet, M. et al. (2002) Human immune cells express ppMCH mRNA and functional MCHR1 receptor. FEBS Lett. 527, 205– 210 Hoogduijn, M.J. et al. (2002) Melanin-concentrating hormone and its receptor are expressed and functional in human skin. Biochem. Biophys. Res. Commun. 296, 698– 701 Bradley, R.L. et al. (2002) Melanin-concentrating hormone activates signaling pathways in 3T3-L1 adipocytes. Am. J. Physiol. Endocrinol. Metab. 283, E584– E592 Hintermann, E. et al. (2001) Expression of the melanin-concentrating hormone receptor in porcine and human ciliary epithelial cells. Invest. Ophthalmol. Vis. Sci. 42, 206 – 209 Palczewski, K. et al. (2000) Crystal structure of rhodopsin: a G proteincoupled receptor. Science 289, 739 – 745 Guex, N. and Peitsch, M.C. (1997) SWISS-MODEL and the SwissPdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714 – 2723
In the June issue of Endeavour: Parents and children: ideas of heredity in the 19th century by John C. Waller Fertility or sterility? Darwin, Naudin and the problem of experimental hybridity by Joy Harvey Mendel and modern genetics: the legacy for today by Garland E. Allen C.D. Darlington and the ’invention’ of the chromosome by Oren Harman Relics, replicas and commemorations by Soraya de Chadarevian Why celebrate the golden jubilee of the double helix? by Robert Olby Portraits of Dorothy Hodgkin by Patricia Fara Sequencing the genome from nematode to human: changing methods, changing science by Rachel Ankeny God’s signature: DNA profiling, the new gold standard in forensic science by Michael Lynch
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Melanin-concentrating hormone: from fish skin to skinny mammals Pavlos Pissios and Eleftheria Maratos-Flier Obesity Section, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215, USA
In recent years, the key role of melanin-concentrating hormone (MCH) in regulating mammalian energy balance has been confirmed through several lines of evidence. When administered exogenously, MCH leads to a rapid and robust feeding response and chronic infusions result in the development of mild obesity. At the physiological level, it is known that MCH expression changes in states of altered energy balance, such as fasting and obesity. Genetic studies with mice have shown that ablation of either the gene for prepro-MCH or the gene encoding the MCH receptor leads to a lean phenotype. Finally, the administration of MCH antagonists appears to inhibit both feeding and the development of diet-induced obesity. The aim of this article is to review the recent data on MCH and MCH receptors in light of their emerging roles in energy homeostasis. Melanin-concentrating hormone was initially characterized as a circulating factor mediating color change in teleost fish 20 years ago [1], although speculation regarding the existence of such a factor dates back to studies of pigmentation changes in amphibians performed in the 1930s [2]. MCH was first referenced as ‘melanophoreconcentrating hormone’ in 1955 in studies examining the possible origin of a factor leading to lightening of fish color [3]. In fish, MCH is a cyclic 17-amino acid polypeptide with a cysteine – cysteine disulfide bond (Fig. 1). It is synthesized as a preprohormone in the pituitary and is secreted into the circulation. It acts on cells derived from neuroectoderm, designated melanophores, which contain large numbers of pigment-containing vesicles called melanosomes. In these target cells, MCH changes the distribution of the melanosomes by stimulating their migration to the perinuclear area, an effect accomplished by the association of the melanosomes with microtubulebased motor proteins. Agents causing aggregation, such as MCH, cause transport of melanosomes by dynein motors, whereas agents causing dispersion lead to transport along kinesin motors [4]. The dispersion or aggregation changes the refractive index of the fish scales; as melanosomes are dispersed, scales become darker and, as they aggregate, scales become lighter. MCH is not the only factor to mediate color change. Migration into the perinuclear area is also stimulated by melatonin [5]. Other agents, such as b-adrenergic agonists and melanocyte-stimulating hormone (a-MSH), mediate the dispersion of melanophores Corresponding author: E. Maratos-Flier ([email protected]).
away from the nucleus into the cytoplasm, which results in skin darkening. In fish scales, MCH and a-MSH act as functional antagonists mediating color change. MCH synthesis in mammals MCH was identified in mammalian brain several years after its discovery in fish. Several studies reported the presence of neurons, which coincidentally crossreacted with antibodies to a-MSH but did not stain with antibodies to other products of the preproopiomelanocortin gene (POMC) [6]. These neurons were later found to correspond to neurons that stained with antibodies directed against salmon MCH. The isolation of mammalian MCH was accomplished as part of a peptide isolation program that involved extraction from 60 000 rat hypothalamic fragments [7]. Mammalian MCH is a 19-amino acid peptide that has high homology to salmon MCH (Fig. 1). The N-terminus is extended by two additional amino acids; however, the loop structure is highly conserved. At the amino acid level, MCH is identical in all mammals analyzed so far, including mouse, rat, rabbit and human. In contrast to fish, MCH synthesis in the central nervous system (CNS) is limited to the magnocellular neurons in the lateral hypothalamus and the zona incerta (Fig. 2). These hypothalamic neurons are atypical in that they make monosynaptic connections throughout the brain, (a)
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Fig. 1. Schematic representation of mammalian and salmon melanin-concentrating hormone (MCH). The different residues between (a) mammalian and (b) salmon MCH are colored red. In addition to the substitutions, salmon MCH lacks two amino acids at the N-terminus compared with mammalian MCH.
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Fig. 2. MCH is synthesized in the lateral hypothalamus and the zona incerta. Immunohistochemical localization of MCH-synthesizing neurons in the rat hypothalamus. Abbreviations: 3V, third ventricle; Arc, arcuate nucleus; DMH, dorsomedial nucleus; F, fornix; LHA, lateral hypothalamus; MCH, melanin-concentrating hormone; ME, median eminence; VMH, ventromedial nucleus; ZI, zona incerta. Scale bar ¼ 1 mm.
projecting to the cortex, amygdala, nucleus acccumbens, olfactory tubercle and to various nuclei in the brainstem. No association between MCH and mammalian pigmentation has been found. Identification of rodent cDNA for MCH (the official gene symbol is Pmch) was reported in 1989 and analysis of the mRNA indicated that the Pmch transcript encodes a preprohormone with two other potential neuropeptide products [8]. Rat, mouse and human mRNA sequences have a high degree of homology, with 90% overall nucleotide identity. The preprohormone encoded comprises 165 residues. MCH is generated via a proteolytic cleavage at arginines 145 and 146. The preprohormone can also be processed to two additional products, designated neuropeptide E-I (NEI) and neuropeptide G-E (NGE). Immunocytochemical studies indicate that NEI is cleaved and present in the same perikarya as MCH. However, it is unclear whether NGE exists as a functional peptide product. In addition to the peptides NEI, MCH and possibly NGE that are cleaved from the MCH prohormone, two other products of the Pmch gene have been detected. One includes an alternatively spliced mRNA that produces a peptide in a different reading frame from that for MCH. The peptide named MGOP (MCH-gene-overprinted-polypeptide) contains 14 amino acids after processing of a putative upstream dibasic cleavage site, although only unprocessed peptide was found in the rat hypothalamus [9,10]. Antisense transcription of the DNA partially overlapping with the Pmch gene has also been detected. The gene was named AROM (antisense RNA overlapping MCH gene), and it appears to generate multiple transcripts by alternative splicing. However, the coding sequences of these peptides do not overlap with the coding sequences of the MCH prohormone. The functional importance of these novel transcripts is currently unknown [11]. The distribution of MCH in mammalian brains suggested that it might be involved in mediating http://tem.trends.com
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‘motivated behaviors’. However, the role of the peptide remained obscure until 1996 when, with the use of RT– PCR differential display, the expression of Pmch mRNA was found to be increased in leptin-deficient ob/ob mice [12]. The increase was similar to that seen with the known orexigenic peptide neuropeptide P-Y (NPY), suggesting that MCH might play a role in appetite. This was confirmed when it was found that intracerebroventricular (ICV) administration of 5 mg of MCH to rats led to a rapid increase in chow consumption. Compared with vehicle, MCH-treated animals eat two- to threefold more over a six-hour period. Feeding can also be induced by injection of MCH directly into the paraventricular nucleus [13]. Although repetitive injections of MCH to rats over a one-week period did not lead to the development of obesity [14], chronic infusions into the lateral ventricle led to both hyperphagia and weight gain [15]. In addition to the overexpression of Pmch seen in the ob/ob model, Pmch expression increases two- to threefold with fasting, an effect that is seen in both normal and ob/ob animals. Treatment with leptin blunts the fasting-induced increase of Pmch mRNA in both wild-type (wt) and ob/ob mice. The increase in Pmch expression in ob/ob animals indicates that other factors, in addition to leptin, also regulate Pmch expression [16]. Furthermore, Pmch expression changes in hyperleptinemic models of obesity. For example, in the obese uncoupling protein 1-ditheria toxin mouse model, where leptin levels are extremely high, Pmch expression is suppressed to a similar extent as that seen for mRNA encoding NPY. Treatment of animals with b3-adrenergic receptor agonists, which typically leads to anorexia, is associated with a rapid fall in leptin levels. These agents lead to an increase in Pmch mRNA expression, which is significantly greater than that seen with fasting [17]. As an orexigenic peptide, MCH interacts with other neuropeptides that influence feeding behavior. For example, MCH is a mutual functional antagonist of the anorectic peptide a-MSH, which inhibits appetite. Depending on the dose administered, MCH can antagonize the anorectic effect of a-MSH, which in turn can antagonize the orexigenic effect of MCH [18]. Further evidence regarding the role of MCH in energy homeostasis comes from mice in which the Pmch gene is either ablated or overexpressed. Mice lacking MCH are , 25% leaner than their wt littermates [19], as a result of a reduction in feeding and a slight increase in energy expenditure. Otherwise, the mice appear to be normal, with normal levels of activity as assessed by an open field test, and normal fertility when bred with wt C57BL/6 mice. Leptin levels are low, as would be expected in a lean mouse model. Low leptin levels would be expected to lead to an increase in NPY and agouti-related peptide (AgRP) and a concomitant decrease in Pomc expression, which encodes the a-MSH precursor in the arcuate nucleus. However, analysis at the mRNA level revealed that only Pomc was decreased. Expression of mRNA encoding NPY and AgRP was at a similar level to that seen in normal mice. This suggests that, in the absence of MCH, low levels of a-MSH might contribute to the lean phenotype or, alternatively, Pomc is undergoing a compensatory downregulation.
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A strain of mice that overexpresses Pmch has also been generated from a P1 clone containing a large genomic fragment of the mouse Pmch gene [20]. Overexpression of Pmch was only seen in the neurons of the lateral hypothalamus and the levels of Pmch mRNA expression were twofold higher on average in the transgenic animals than in their wt littermates. On an FVB background, the mice had normal weight and normal feeding. When the transgene was bred to homozygosity and the homozygous animals were placed on a high-fat diet, the animals developed excessive obesity compared with normal control littermates. This appeared to be secondary to a trend towards hyperphagia seen in the Pmch-overexpressing animals. Another interesting feature of these animals was the significant hyperinsulinemia and islet cell hyperplasia, suggesting that MCH might play a role in the regulation of islets. However, it is unclear whether such a role would be mediated directly by local synthesis of MCH in islet ganglia or synapses from the CNS through paracrine regulation, or possibly via a secondary effect of MCH on an intermediate target tissue. Recently, chronic infusion in mice has reproduced the hyperphagic obese phenotype seen with overexpression of Pmch. Interestingly, these mice also showed a decrease in body temperature and a decrease in the oxidation of fatty acids in brown fat [21]. Additional effects of MCH in vivo in mice include an increase in water consumption, which is independent of feeding [22]. In conscious sheep, MCH has a diuretic effect but does not increase water consumption [23]. MCH signals through two G protein-coupled receptors Before the genetic identification of the rodent MCH receptor a significant body of literature reported on receptor expression with the use of radioreceptor assays utilizing either the iodinated native mammalian MCH or the biologically active Phe13Tyr19 analog [24]. Receptors were found in a variety of cells, including SVK14 keratinocytes [25] and mouse melanoma cells [24]. However, subsequent studies suggested that this binding was artifactitious in nature, resulting from physical properties of MCH and its analog and making the interpretation of binding assays problematic [26]. Indeed, subsequent studies of the SVK14 cell line indicate that these cells do not express mRNAs for either MCH receptor 1 (MCHR1) or MCH receptor 2 (MCHR2) [27]. The first high-affinity receptor for MCH was identified simultaneously by several groups using different pharmacological approaches [28 –31]. MCHR1 was found to be identical to the previously cloned orphan receptor SLC-1 and belongs to the superfamily of G protein-coupled receptors [32]. MCHR1 is 353 amino acids long, with all the hallmark features of the superfamily, including seven transmembrane helices, a DRY motif at the end the third intracellular loop and three potential glycosylation sites at the N-terminus. It shows the highest degree of homology to the somatostatin receptor family (, 35%), and the gene has been localized to chromosome 22q13.3 [32]. Comparison of the human and the rodent receptors shows a high degree of conservation between species (human – mouse, 95% identity; human –rat, 96% identity). Based on the homology to http://tem.trends.com
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the somatostatin receptors, which are coupled to Gi, it was quickly determined that MCHR1 could also suppress forskolin-stimulated cAMP and increase intracellular Ca2þ ([Ca2þ]i). Later work confirmed the ability of the receptor to couple to multiple G proteins, including Gi, Go and Gq [33]. However, the EC50 responses for cAMP inhibition and [Ca2þ]i increase suggest that the coupling is stronger to Gi than to Gq. Activation of MCHR1 also leads to the stimulation of protein kinase C, phospholipase C and extracellular-signal-regulated kinase pathways, at least in heterologous cell lines [33]. In the CNS, activation of the above signaling pathways has diverse effects, ranging from changes in gene expression to modulation of ion channel activity [34]. In addition, several ion channels are direct effectors of activated G proteins. For example, Gi-coupled receptors are known to inhibit voltage-gated Ca2þ channels (VGCCs) and to activate Kþ inward rectifying channels (Fig. 3) [35,36]. Both actions result in the inhibition of synaptic transmission. Consistent with these reports, MCH was found to inhibit Ca2þ currents flowing through the N-, P- and, to a lesser degree, the L-types of VGCCs in the neurons of the lateral hypothalamus [37], although activation of G protein-coupled inward rectifying channels by MCH has not been demonstrated. MCHR1 is most highly expressed in the brain, with moderate to weak expression in other tissues, including muscle, eye, tongue and adipose tissue [31]. The distribution of MCHR1 in the brain has been investigated in detail in the rat [38,39], and the expression pattern was found to be very broad throughout the CNS. Expression is seen in the cortex, basal ganglia, hypothalamus and
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Fig. 3. Hypothetical activation of signaling pathways by MCH receptors. Through coupling to multiple G (Gi/o/q) proteins, melanin-concentrating hormone (MCH) receptors activate phospholipase C (PLC), which catalyzes the production [from phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P3)] of diacylglycerol (DAG) and inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]. Ins(1,4,5)P3, in turn, causes an increase in intracellular calcium ([Ca2þ]i). Coupling to Gi (MCH receptor 1) causes inhibition of adenylate cyclase (AC) and therefore decreased production of cAMP. Extracellular-signal-regulated kinase (ERK) is also activated. Important effectors in the central nervous system include voltage-gated Ca2þ channels (VGCC) and G protein-coupled inward rectifying channels (GIRK), which are inhibited and activated by Gi proteins, respectively.
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brainstem. The presence of MCHR1 in the arcuate and ventromedial nuclei of the hypothalamus is consistent with MCH regulating food intake and energy balance. However, the highest level of expression is seen in extrahypothalamic areas that process olfactory information, namely the anterior olfactory nucleus, the piriform cortex and the olfactory tubercle. Very high levels of MCHR1 are also found in the shell of the nucleus accumbens, an area involved in rewarding aspects of motivated behaviors, such as food intake. Significant amounts are also present in the amygdala and hippocampus, potentially implicating MCH in regulating emotions, such as fear and anxiety, and also as a potential regulator of memory. Therefore, MCH is probably involved in many other behaviors, some of which might be important in food intake, such as olfaction and reward, in addition to regulating general mood and arousal levels. Similar patterns of expression of MCHR1 are seen in the mouse brain. Furthermore, MCHR1 expression in mice appears to be regulated by altered energy balance. Expression rises with fasting and this increase is inhibited by leptin administration [40]. Recently, MCHR1 has been inactivated in mice by homologous recombination [41,42]. Mice lacking functional MCHR1 maintained on regular chow diet exhibited decreased fat mass, increased activity and, surprisingly, were found to be hyperphagic compared with their wt littermates. When placed on a high-fat diet, MCHR1deficient mice gained significantly less weight, suggesting that inactivation of MCHR1 protected them from dietinduced obesity. Consistent with the above reports, pharmacological blockade of MCHR1 using specific antagonists produced similar effects [43]. Chronic administration of the MCHR1 antagonist SNAP-7941 to diet-induced obese rats caused a decrease in food intake and body weight. In addition to the beneficial effect on energy homeostasis, SNAP-7941 also acted as an anxiolytic. These results indicate that the MCH system is effective in reducing body weight under conditions in which leptin is ineffective and make the MCH receptor a viable target for the treatment of obesity. Two years after the cloning of MCHR1, database scanning revealed a second high-affinity receptor for MCH (MCHR2), with significant homology to the core region of MCHR1. The human receptor is 340 amino acids long and its gene resides on the long arm of chromosome 6 (6q16.2 – 6q21) [44– 49]. Analysis of the gene encoding MCHR2 revealed one non-coding and five coding exons that span .35 kb of chromosome 6 [44]. The overall homology between the two MCH receptors is quite low. They share only 38% identical amino acids, with the highest homology in the transmembrane domains that form part of the putative MCH-binding pocket. Figure 4 shows a space-filled model of the MCHR1. The colored residues are identical between the two MCH receptors. By looking directly into the cleft formed by the transmembrane helices the considerable sequence identity that underlies the high-affinity binding of these receptors to MCH is readily apparent. Overall, the expression profile of MCHR2 is similar to MCHR1, with the highest expression seen in the brain, notably in the frontal cortex, amygdala http://tem.trends.com
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Fig. 4. Melanin-concentrating hormone (MCH) receptor 1 and MCH receptor 2 exhibit significant sequence homology in the putative MCH-binding pocket. Spacefill model of the human MCH receptor 1 based on the crystal structure of bovine rhodopsin [56]. The colored residues correspond to identical amino acids between the two receptors. The view is from outside looking into the cleft formed by the seven transmembrane helices. The model was generated by the Swiss-model/Swiss PDB viewer and enhanced by POV-Ray [57].
and nucleus accumbens [44,45,49]. Expression is also detected, at least by quantitative RT–PCR, in several tissues, including intestine, adipose tissue and prostate [49]. Curiously, MCHR2 does not appear to be present in many species. Tan and co-workers performed a detailed investigation of several species for the presence of MCH receptors [50]. They found that rodents do not have MCHR2. Guinea pigs and rabbits carry non-functional alleles, whereas carnivores, such as ferrets and dogs, have both MCH receptors. Monkeys and humans also have both MCH receptors. Expression of recombinant MCHR2 in heterologous cell lines demonstrated the ability of the receptor to increase [Ca2þ]i levels. However, MCHR2 was unable to decrease cAMP levels and was insensitive to pertussis toxin, suggesting that it couples exclusively to Gq [44,45,49]. This is in contrast to MCHR1, which appears to couple to multiple G proteins, at least in heterologous cell lines. The overall distribution of MCHR2 in the CNS suggests that it might be involved in aspects of physiology similar to those of MCHR1. However, the functional importance of MCHR2 is currently difficult to assess without an appropriate animal model. MCH outside the CNS In mammals, the potential significance of MCH action outside the CNS remains controversial. MCH expression has been reported in the testis, stomach and intestine [51], and one recent report suggested that prepro-MCH might also be expressed in immune cells [52] and in endothelial cells derived from human skin [53]. Furthermore, MCH receptor expression in the periphery has also been reported. Both primary rat adipocytes and 3T3-L1cells are known to express the receptor and to respond to MCH [54], and direct effects of MCH on the leptin promoter have been demonstrated in 3T3-L1 cells [54]. Receptor expression has also been shown in ciliary epithelium
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[55]. Other reports have demonstrated the ability of MCHR1 to activate intracellular signaling pathways in monocytes, melanocytes and islet cells [39,52]. Although these findings suggest that MCH signaling in the periphery might be important, the physiological role outside of pigmentation in fish remains unclear. Conclusions A role for MCH in energy homeostasis is now clear. Genetic ablation of either the MCH prohormone or MCHR1 results in leanness. MCH, injected ICV, stimulates feeding acutely and chronic infusions lead to the development of obesity. However, several important questions remain. The mechanisms underlying the effects of MCH on energy homeostasis are not clear, although food intake, locomotor activity and resting energy expenditure probably play a major role. Differences exist between mice lacking the MCH prohormone and mice lacking MCHR1. Those lacking MCHR1 are hyperphagic, hypermetabolic and have higher locomotor activity, whereas those lacking the MCH prohormone have slightly decreased feeding and slightly increased energy expenditure. These differences could potentially be explained by ablation of the NEI and NGE peptides in addition to MCH in the prohormonedeficient mice. The second MCH receptor, MCHR2, has relatively low homology to MCHR1 and does not exist in rodent species. Hence, its potential role in feeding and energy balance remains uncertain. Furthermore, the widespread reach of MCH neurons and the broad expression of MCH receptors, especially in regions concerned with olfactory processing and motivated behaviors, make MCH an ideal candidate for the integration of various homeostatic and sensory stimuli necessary for the proper coordination of energy intake and expenditure. Future studies will address these important questions. Acknowledgements We thank Richard Bradley and Dan Trombly for careful reading of the article and Elena Nikiforova for technical assistance. EMF is a recipient of NIH grants DK53978 and DK56113. EMF also participates in program project DKPP56116.
References 1 Kawauchi, H. et al. (1983) Characterization of melanin-concentrating hormone in chum salmon pituitaries. Nature 305, 321– 323 2 Hogben, L.T. and Slome, D. (1931) The pigmentary effector system VI. The dual character of endocrine coordination in amphibian colour change. Proc. R. Soc. London B. Biol. Sci. 108, 10 – 53 3 Enami, M. (1955) Melanophore-concentrating hormone (MCH) of possible hypothalamic origin in the catfish, parasilurus. Science 121, 36 – 37 4 Rogers, S.L. et al. (1997) Regulated bidirectional motility of melanophore pigment granules along microtubules in vitro. Proc. Natl. Acad. Sci. U. S. A. 94, 3720– 3725 5 Filadelfi, A.M. and Castrucci, A.M. (1994) Melatonin desensitizing effects on the in vitro responses to MCH, a-MSH, isoproterenol and melatonin in pigment cells of a fish (S. marmoratus), a toad (B. ictericus), a frog (R. pipiens), and a lizard (A. carolinensis), exposed to varying photoperiodic regimens. Comp. Biochem. Physiol. A. Physiol. 109, 1027 – 1037 6 Kohler, C. et al. (1984) A diffuse a MSH-immunoreactive projection to the hippocampus and spinal cord from individual neurons in the lateral hypothalamic area and zona incerta. J. Comp. Neurol. 223, 501 – 514 http://tem.trends.com
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7 Vaughan, J.M. et al. (1989) Characterization of melanin-concentrating hormone from rat hypothalamus. Endocrinology 125, 1660 – 1665 8 Nahon, J.L. et al. (1989) The rat melanin-concentrating hormone messenger ribonucleic acid encodes multiple putative neuropeptides coexpressed in the dorsolateral hypothalamus. Endocrinology 125, 2056– 2065 9 Toumaniantz, G. et al. (1996) The rat melanin-concentrating hormone gene encodes an additional putative protein in a different reading frame. Endocrinology 137, 4518 – 4521 10 Toumaniantz, G. et al. (2000) Differential neuronal expression and projections of melanin-concentrating hormone (MCH) and MCH-geneoverprinted-polypeptide (MGOP) in the rat brain. Eur. J. Neurosci. 12, 4367– 4380 11 Borsu, L. et al. (2000) The AROM gene, spliced mRNAs encoding new DNA/RNA-binding proteins are transcribed from the opposite strand of the melanin-concentrating hormone gene in mammals. J. Biol. Chem. 275, 40576 – 40587 12 Qu, D. et al. (1996) A role for melanin-concentrating hormone in the central regulation of feeding behaviour. Nature 380, 243 – 247 13 Rossi, M. et al. (1999) Investigation of the feeding effects of melanin concentrating hormone on food intake – action independent of galanin and the melanocortin receptors. Brain Res. 846, 164 – 170 14 Rossi, M. et al. (1997) Melanin-concentrating hormone acutely stimulates feeding, but chronic administration has no effect on body weight. Endocrinology 138, 351 – 355 15 Della-Zuana, O. et al. (2002) Acute and chronic administration of melanin-concentrating hormone enhances food intake and body weight in Wistar and Sprague-Dawley rats. Int. J. Obes. Relat. Metab. Disord. 26, 1289– 1295 16 Tritos, N.A. et al. (2001) Characterization of melanin concentrating hormone and preproorexin expression in the murine hypothalamus. Brain Res. 895, 160 – 166 17 Mantzoros, C.S. et al. (1996) Activation of b3 adrenergic receptors suppresses leptin expression and mediates a leptin-independent inhibition of food intake in mice. Diabetes 45, 909 – 914 18 Ludwig, D.S. et al. (1998) Melanin-concentrating hormone: a functional melanocortin antagonist in the hypothalamus. Am. J. Physiol. 274, E627– E633 19 Shimada, M. et al. (1998) Mice lacking melanin-concentrating hormone are hypophagic and lean. Nature 396, 670– 674 20 Ludwig, D.S. et al. (2001) Melanin-concentrating hormone overexpression in transgenic mice leads to obesity and insulin resistance. J. Clin. Invest. 107, 379 – 386 21 Ito, M. et al. (2003) Characterization of MCH-mediated obesity in mice. Am. J. Physiol. Endocrinol. Metab. 284, E940– E945 22 Clegg, D.J. et al. (2003) Intraventricular melanin concentrating hormone stimulates water intake independent of food intake. Am. J. Physiol. Regul. Integr. Comp. Physiol. 284, R494– R499 23 Parkes, D.G. (1996) Diuretic and natriuretic actions of melanin concentrating hormone in conscious sheep. J. Neuroendocrinol. 8, 57 – 63 24 Drozdz, R. and Eberle, A.N. (1995) Synthesis and iodination of human (phenylalanine 13, tyrosine 19) melanin-concentrating hormone for radioreceptor assay. J. Pept. Sci. 1, 58 – 65 25 Burgaud, J.L. et al. (1997) Melanin-concentrating hormone binding sites in human SVK14 keratinocytes. Biochem. Biophys. Res. Commun. 241, 622 – 629 26 Kokkotou, E. et al. (2000) Characterization of [Phe(13), Tyr(19)]-MCH analog binding activity to the MCH receptor. Neuropeptides 34, 240– 247 27 Audinot, V. et al. (2002) SVK14 cells express an MCH binding site different from the MCH1 or MCH2 receptor. Biochem. Biophys. Res. Commun. 295, 841 – 848 28 Bachner, D. et al. (1999) Identification of melanin concentrating hormone (MCH) as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1). FEBS Lett. 457, 522 – 524 29 Chambers, J. et al. (1999) Melanin-concentrating hormone is the cognate ligand for the orphan G protein-coupled receptor SLC-1. Nature 400, 261 – 265 30 Lembo, P.M. et al. (1999) The receptor for the orexigenic peptide melanin-concentrating hormone is a G protein-coupled receptor. Nat. Cell Biol. 1, 267 – 271
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31 Saito, Y. et al. (1999) Molecular characterization of the melaninconcentrating-hormone receptor. Nature 400, 265 – 269 32 Kolakowski, L.F. Jr et al. (1996) Characterization of a human gene related to genes encoding somatostatin receptors. FEBS Lett. 398, 253 – 258 33 Hawes, B.E. et al. (2000) The melanin-concentrating hormone receptor couples to multiple G proteins to activate diverse intracellular signaling pathways. Endocrinology 141, 4524– 4532 34 Curtis, J. and Finkbeiner, S. (1999) Sending signals from the synapse to the nucleus: possible roles for CaMK, Ras/ERK, and SAPK pathways in the regulation of synaptic plasticity and neuronal growth. J. Neurosci. Res. 58, 88 – 95 35 Dolphin, A.C. (1998) Mechanisms of modulation of voltage-dependent calcium channels by G proteins. J. Physiol. 506, 3 – 11 36 Yamada, M. et al. (1998) G protein regulation of potassium ion channels. Pharmacol. Rev. 50, 723 – 760 37 Gao, X.B. and Van Den Pol, A.N. (2002) Melanin-concentrating hormone depresses L-, N-, and P/Q-type voltage-dependent calcium channels in rat lateral hypothalamic neurons. J. Physiol. 542, 273 – 286 38 Hervieu, G.J. et al. (2000) The distribution of the mRNA and protein products of the melanin-concentrating hormone (MCH) receptor gene, slc-1, in the central nervous system of the rat. Eur. J. Neurosci. 12, 1194 – 1216 39 Saito, Y. et al. (2001) Expression of the melanin-concentrating hormone (MCH) receptor mRNA in the rat brain. J. Comp. Neurol. 435, 26 – 40 40 Kokkotou, E.G. et al. (2001) Melanin-concentrating hormone receptor is a target of leptin action in the mouse brain. Endocrinology 142, 680 – 686 41 Marsh, D.J. et al. (2002) Melanin-concentrating hormone 1 receptordeficient mice are lean, hyperactive, and hyperphagic and have altered metabolism. Proc. Natl. Acad. Sci. U. S. A. 99, 3240– 3245 42 Chen, Y. et al. (2002) Targeted disruption of the melanin-concentrating hormone receptor-1 results in hyperphagia and resistance to dietinduced obesity. Endocrinology 143, 2469 – 2477 43 Borowsky, B. et al. (2002) Antidepressant, anxiolytic and anorectic effects of a melanin-concentrating hormone-1 receptor antagonist. Nat. Med. 8, 825 – 830 44 An, S. et al. (2001) Identification and characterization of a
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melanin-concentrating hormone receptor. Proc. Natl. Acad. Sci. U. S. A. 98, 7576 – 7581 Sailer, A.W. et al. (2001) Identification and characterization of a second melanin-concentrating hormone receptor, MCH-2R. Proc. Natl. Acad. Sci. U. S. A. 98, 7564 – 7569 Rodriguez, M. et al. (2001) Cloning and molecular characterization of the novel human melanin-concentrating hormone receptor MCH2. Mol. Pharmacol. 60, 632 – 639 Mori, M. et al. (2001) Cloning of a novel G protein-coupled receptor, SLT, a subtype of the melanin-concentrating hormone receptor. Biochem. Biophys. Res. Commun. 283, 1013 – 1018 Wang, S. et al. (2001) Identification and pharmacological characterization of a novel human melanin-concentrating hormone receptor, mch-r2. J. Biol. Chem. 276, 34664 – 34670 Hill, J. et al. (2001) Molecular cloning and functional characterization of MCH2, a novel human MCH receptor. J. Biol. Chem. 276, 20125 – 20129 Tan, C.P. et al. (2002) Melanin-concentrating hormone receptor subtypes 1 and 2: species-specific gene expression. Genomics 79, 785– 792 Hervieu, G. and Nahon, J.L. (1995) Pro-melanin concentrating hormone messenger ribonucleic acid and peptides expression in peripheral tissues of the rat. Neuroendocrinology 61, 348 – 364 Verlaet, M. et al. (2002) Human immune cells express ppMCH mRNA and functional MCHR1 receptor. FEBS Lett. 527, 205– 210 Hoogduijn, M.J. et al. (2002) Melanin-concentrating hormone and its receptor are expressed and functional in human skin. Biochem. Biophys. Res. Commun. 296, 698– 701 Bradley, R.L. et al. (2002) Melanin-concentrating hormone activates signaling pathways in 3T3-L1 adipocytes. Am. J. Physiol. Endocrinol. Metab. 283, E584– E592 Hintermann, E. et al. (2001) Expression of the melanin-concentrating hormone receptor in porcine and human ciliary epithelial cells. Invest. Ophthalmol. Vis. Sci. 42, 206 – 209 Palczewski, K. et al. (2000) Crystal structure of rhodopsin: a G proteincoupled receptor. Science 289, 739 – 745 Guex, N. and Peitsch, M.C. (1997) SWISS-MODEL and the SwissPdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714 – 2723
In the June issue of Endeavour: Parents and children: ideas of heredity in the 19th century by John C. Waller Fertility or sterility? Darwin, Naudin and the problem of experimental hybridity by Joy Harvey Mendel and modern genetics: the legacy for today by Garland E. Allen C.D. Darlington and the ’invention’ of the chromosome by Oren Harman Relics, replicas and commemorations by Soraya de Chadarevian Why celebrate the golden jubilee of the double helix? by Robert Olby Portraits of Dorothy Hodgkin by Patricia Fara Sequencing the genome from nematode to human: changing methods, changing science by Rachel Ankeny God’s signature: DNA profiling, the new gold standard in forensic science by Michael Lynch
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