Chapter II The melanin-concentrating hormone

CHAPTER II

The melanin-concentrating hormone GUILLAUME J. HERVIEU, LAURENCE MAULON-FERAILLE, JONATHAN K. CHAMBERS, JANE E. CLUDERAY, SHELAGH WILSON, FRANCOISE PRESSE AND JEAN-LOUIS NAHON

The purpose of this review is to present the most recent findings on structural analysis and neurobiology of melanin-concentrating hormone (MCH), with special emphasis on the cloning, functional characterisation and neuronal distribution of at least two MCH receptors. It was a long-standing and important achievement in the field. First, the reader is proposed a survey of the milestones that marked the discovery of the peptide MCH in 1983 until the characterisation of two MCH receptors, named MCH-R1 and MCH-R2, in the late 1990s and the development of MCH-R1 antagonists that show potential anxiolytic, antidepressant and/or anorectic actions in 2002. Previous reviews have been already published on the functions and brain localisation of MCH in fishes and mammals (Eberle, 1988; Baker, 1991, 1994; Nahon et al., 1993; Nahon, 1994; Knigge et al., 1996; Griffond and Baker, 2002) and on the pairing of the MCH ligand with two subtype receptors so far (Saito et al., 2000; Boutin et al., 2002; Griffond and Baker, 2002). Recent neuroanatomical comments related to the feeding properties of MCH have been written by Sawchenko (1998); Tritos and Maratos-Flier (1999); Kilduff and de Lecea (2001). The effect of a pharmaceutically drug discovery molecule acting as an antagonist at one of the MCH receptor, MCH-R1, and affecting both affective and energy balances, is discussed by Schwartz and Gelling (2002).

1. A SURVEY OF THE MELANIN-CONCENTRATING SYSTEM: SEMINAL BACKGROUND STUDIES AND PHARMACEUTICAL INTEREST

1.1. MCH HAS A CONCERTED SET OF ACTIONS IN THE FISH MCH was first isolated from fish teleost pituitaries in 1983 by its role in melanin-concentration action (Kawauchi et al., 1983). The 'MCH enigma' was solved. For 50 years, ichthyologists and life scientists interested in pigmentary control had wondered if two substances with opposite actions existed to regulate the fish colour tegument for the component dark/light. It was already known that s-melanin-stimulating hormone (MSH), one of the very first neuropeptides ever biochemically isolated, had darkening properties by allowing melanin to spread with melasome suborganelles. Viewed from a pharmacological aspect, one was asking if the inhibition of the MSH darkening properties just happening by biological

Handbook of Chemical Neuroanatomy, Vol. 20: Peptide Receptors, Part H R. Quirion, A. Bjrrklund and T. Hrkfelt, editors 9 2003 Elsevier Science B.V. All rights reserved.

31

Ch. H

G.J. Hervieu et al.

desensitisation/degradation or by active pharmacological/functional antagonism of another substance or both. MCH is classified as a melanotropin, i.e. a factor implicated in regulating colour tegument (with a potency ECs0 as low as pM range to evoke melanin concentration in certain teleost fishes). However, unlike the MSH and adrenocorticotropic hormone (ACTH) peptide family, all derived from the prepro-opio-melanocortin (POMC) precursor, MCH is not classified as a melanocortin because it lacks the consensus melanocortin sequence - H i s - P h e - A r g - T r p - . Consistent with that, MCH does not signal through the specific set of not less than six G-protein-coupled receptor (GPCR) proteins binding MSHs and ACTH, and termed from MC-1 to MC-6 binding (see Adan and Gipsen, 1997). Intriguingly, it may be possible that MCH and MSH are evolutionary related as suggested by Matsunaga et al. (1986). In the fish, few other functions than melanophore concentration were noted for MCH, i.e. an action of the peptide regulating the stress axis (see Baker, 1994) and more recently, stimulating the immune system (Harris and Bird, 2000). It may well illustrate a coordinated, goal-orientated function of the peptide in the vital neural activation related to a potentially hazardous situation perceived by the fish. This could be particularly pertinent in predatory situations, when camouflage can be achieved by matching tegument colour to background environment colour through a simple monocolour balance from dark to light, and when neurobehavioural mechanisms are triggered that should normally optimise the animal's response in dealing with dangerous and potentially fatal threatening stimuli. 1.2. MCH ALSO EXISTS IN MAMMALS Immunochemical studies indicated the very probable existence of an MCH-like factor in the rat, as an antiserum raised against salmon MCH resulted in a very strong and localised immunosignal within the hypothalamus in rats (Zamir et al., 1986a,b) (see Fig. 1). The rat orthologue peptide was purified in the laboratory of Wilie Vale at the Clayton Foundation for Peptide Studies, The Salk Institute in California in 1989 from 60,000 hypothalami using the same extracts that served to purify the growth hormone-releasing hormone (Vaughan et al., 1989). Rat and salmon MCH displayed strong homology mainly within the loop structure (Fig. 2A). Molecular studies allowed the identification of the genetic rat sequence encoding the prepro-MCH precursor and establish the possibility that two other

......... ~.

'~

~ "~,.

tubercle

~I

thalamus

~. . . . . . .

;i

',~.

.................................... :................. ,~::~:.~,~, ..........................................

.........

o

ongata

Fig. 1. MCH-containing neurons and projections in the rat. MCH-producing cells and projections are noted, respectively, by dots and lines.

32

The melanin-concentrating hormone

Ch. H

A

Rat MCH

H-~pmel~---]z~tz_eulz~~.c~ ~t]z~=~lGzy~ v~ ~ ~ ~-oc~ ~IG~F~-]-C~

Salmon MCH

B

mRNA

Precursor

wz

-65 +1 = ', 5' UT +1

Translated region

Pro

+495 ,I

+687 I(A) n 3' UT

v

I II ......III I I I III

v v +165

I INGEINE=IMCHI

signal

Peptides

~

MCH

O

H

H

NEI

H E I G D E E N S A K F P I

NGE

H

NH2 OH

Fig. 2. (A) Comparison of the rat and salmon MCH. Identical amino acids are boxed. (B) Schematic representation of the MCH mRNA precursor and peptide sequences. Basic amino acid residues are noted in black in the precursor. Arrowheads indicate putative or overt cleavage sites.

peptides, namely neuropeptide-GE (NGE) and neuropeptide-EI (NEI), could be released upon proteolytic maturation of the prepro-MCH precursor (Breton et al., 1989; Nahon et al., 1989) (Fig. 2B). 1.3. MCH AS A 'GUT-BRAIN' PEPTIDE A new mammalian peptidergic system had thus emerged. It would fit within the general rules that govern neuropeptidergic expression: notably, the neuropeptide is in fact both expressed in the central nervous system (see Fig. 1; Bittencourt et al., 1992) and in the periphery, notably and not surprisingly in the enteric nervous system. By different techniques either related to mRNA studies (Northern blot, mRNA in situ hybridisation, RT-PCR) or peptide studies (immunohistochemistry, radio-immunoassays), MCH mRNA and peptides have been characterised in the peripheral tissues of the mouse (Breton et al., 1993b), rat (Hervieu and Nahon, 1995; Takahashi et al., 1995) and human (Takahashi et al., 1995; Viale et al., 1997). Expression level is much lower in the periphery than in the brain. There is a widespread expression of MCH in the gastrointestinal organs (stomach, duodenum, jejunum, ileum, colon), in the immune system (thymus, spleen), in the cardiopulmonary tissues (heart, lung) and in the reproductive organs (testis, ovary). Histological studies showed that in the testis, the MCH peptide is present in the Sertoli cells and is also strikingly localised to the germ cell nuclei (Hervieu and Nahon, 1995; Hervieu et al., 1996b) while in the gastrointestinal tract, the peptide is produced by white cells and is shown to regulate the hydro-ionic balance of the gastrointestinal tract in an in vivo model (Hervieu et al., 1996a). As alluded before, immunomodulatory effect of MCH have been reported on rainbow trout phagocytes and leucocytes (Harris and Bird, 2000). These characteristics are not at odds with general features of neuropeptides and the established concept of a dual and reciprocal communication between the nervous, humoral and immune system (Blalock, 1989). 33

Ch. H

G.J. Hervieu et al.

1.4. "A [MAMMALIAN] PEPTIDE STILL IN SEARCH OF FUNCTIONS" (Bittencourt et al., 1992) For 20 years, it is fair to say that it proved difficult to assign to the mammalian MCH a clear set of biological actions. First of all, there is no evidence for a role of MCH in the control of mammalian melanogenesis (reviewed by Baker, 1994; Nahon, 1994), nor melanoma incidence (see gene array study by Bittner et al., 2000). That MCH does not act directly on pigmentary control may be partially explained by a strikingly different neuroanatomical location of the fish and rodent brain MCH system. While in the fish MCH is a hypophysiotropic factor released from the pituitary with quasi-instant control over the pigmentory system, there is, on the contrary, a marked low peptide content at both the median eminence and pituitary levels. Rat MCH is neither synthesised in the neurohypophysis, nor in the hypothalamic nuclei with strong neural connections to the median eminence, part of the hypothalamo-anterior pituitary portal system that links hypothalamus to the adenohypophysis gland in the rat. Also, of particular importance, numerous, but unsuccessful, attempts by laboratories have been made to identify MCH receptor(s) for more than a decade. Undoubtedly, the absence of a simple MCH bioactivity assay (i.e. melanin concentration in fish skin bioassay) and its non-reliable characteristics, have certainly refrained attempts to characterise the receptor. Significant chemistry work was performed in Switzerland by Alex Eberle, a leader in the melanocortins field. Photoactivable and radio-active MCH peptide mimetics are used to demonstrate evidence of specific MCH receptor-like entities in a number of mouse melanomas (Drozdz et al., 1995). It is well known that peptides are difficult to experiment with, mostly because of their usually high molecular weight and other unfavourable generally hydrophobic physicochemical properties. They render peptides 'sticky' to the tube in vitro and when injected in vivo, peptides bind mostly to albumins in the peripheral blood and to brain ventricular component (see study by Bittencourt and Sawchenko, 2000, discussing if centrally administered neuropeptides access cognate receptors, using the CRF system as a case study). Peptides are also readily degradable by enzymes. Their usual short shelf-life in biological fluids and the sheer multiplicity of product degradation by-passes that may still retain pharmacological activity, add another degree of complexity for studying neuropeptidergic systems. Amongst other endocrine and neural systems, the brain contains probably the richest source of peptides, both in terms of diversity and abundance. Brain central effects of a peptide can very often be only evidenced through surgical intervention and intracerebral administration (intrathecal/intracerebroventricular). To demonstrate an in vivo central effect of any compound, it is most preferred to experiment with a compound that is not a peptide, is orally available and at least reasonably penetrant into the brain. It is perhaps why few out of the sheer plethora of naturally purified and in silico potential identified peptides, fulfil the criteria as neurotransmitter. Meanwhile, sporadic evidence attributed the peptide a role in sensory gating (Miller et al., 1993), epileptogenesis (Knigge et al., 1997), and, in the regulation of the hypothalamopituitary adrenal axis, though reported with somehow opposite actions (Jezova et al., 1992; Bluet-Pajot et al., 1995; Ludwig et al., 1998). It was possibly a lead in confirming an evolutionary conservation of the peptide function to intervene in the stress axis, both in the fish and in the rat.

34

The melanin-concentrating hormone

Ch. H

1.5. MCH REGULATES FOOD INTAKE IN RATS Strong and sudden impetus to research in the MCH field probably all came from a study published in Nature in 1996 showing that the peptide induces feeding behaviour (Qu et al., 1996). Numerous review works have been published looking at the possibility of developing drugs that would regulate the energy balance, notably to fight obesity, a leading cause of mortality in the 'Western world' (see Campfield, 1998; Bray and Tartaglia, 2000; Kopelman, 2000; Clapham et al., 2001; see also reviews on the neurocircuitries implicated in the regulation of feeding behaviour, e.g. Morley, 1987; Flier and Maratos-Flier, 1998; Elmquist et al., 1999; Schwartz et al., 2000). From that point, interest in MCH was no more purely academic. Major pharmaceuticals and biotechs entered a race to characterise the receptor (SmithKline Beecham, Glaxo-Wellcome, Schering-Plough, Synaptic, Merck, Sharp and Dohm, Takeda), and to develop peptide and non-peptide mimetics ligand of MCH (legacy SmithKline Beecham, GlaxoSmithKline, Schering-Plough, Servier, Synaptic, Takeda). Major academic laboratories studying the feeding behaviour entered the competition too. 1.6. SLC-1 AND ANOTHER ORPHAN GPCR ARE PARALOGUE RECEPTORS FOR MCH An orphan GPCR called SLC-1 (GPR24) was identified in 1996 by Kolakowski et al., 1996. SLC-1 has most homology with the somatostatin receptor family, but does not bind somatostatin-14, nor somatostatin-28, nor corticostatin. Kolakowski then commented that 'the abundance of SLC-1 mRNA expression in brain with regional localisation to discrete areas involved in functions such as emotion, memory and sensory perception, make the isolation of the endogenous ligand of this receptor an important priority. This heralds the exciting potential of identifying a novel peptidergic neurotransmitter signalling system in the brain' (Kolakowski et al., 1996). Darlinson and Richter (1999), while reviewing evolutionary aspects of paired peptidergic ligand/receptor systems as well as orphan ligands or receptors, also highlighted the importance in identifying the natural ligand of SLC-1. SLC-1 was later characterised as being a receptor for MCH (Chambers et al., 1999; Saito et al., 1999; Bachner et al., 1999; Lembo et al., 1999; Shimomura et al., 1999; reviewed in Saito et al., 2000). Some of the approaches used a method best described as 'reverse pharmacology approach' (RPMA) (e.g. Chambers et al., 1999; for review, Stadel et al., 1997) while others approaches relied on systematic agonist compound bank screening or biochemical purification of ligands that can activate the transfected receptor in heterologous cell lines. No more than a year later, a second receptor with low sequence identity to that of MCH-R1 was identified as a biologically relevant second MCH receptor subtype or a paralogue to MCH-R1 (reviewed in Boutin et al., 2002). 1.7. THE MCH SYSTEM APPEARS AS A COMPLEX EVOLUTIONARY MODEL The MCH and its receptor system, apart from its biological significance, seems to have had a very intriguing purely genetic evolution history. There are two very distinct loci for human MCH gene chromosomal localisation. A truncated version of the MCH gene was identified with a different chromosomal localisation to that of the 'authentic' MCH gene (Breton et al., 1993a,b; Pedeutour et al., 1994). It is only found in the Hominidae (Viale et al., 1998, 2000). That very example was published in the Science issue reporting the data on human genome sequencing by Craig Venter and colleagues as part of phylogenetic case study (Courseaux 35

Ch. H

G.J. Hervieu et al.

and Nahon, 2001). Interestingly, this gene may code for a putative nuclear protein, the signal peptide of the pro-MCH being replaced by a nuclear-localisation signal (NLS) in the new gene (Viale et al., 2000). Coincidentally perhaps, Hervieu et al. (1996a,b) localised MCHlike immunoreactivity in the nuclei of germ cells. Prepro-MCH antisense transcripts were characterised in the rat (Hervieu and Nahon, 1995), the human (Miller et al., 1998) and cell lines (Presse et al., 1997; Borsu et al., 2000). Toumaniantz et al. (1996) characterised a peptide called MGOP that could be derived from the alternative splicing of the prepro-MCH mRNA. Regarding MCH receptors, data seem firmly to establish that non-human species (rat, mouse, hamster, guinea, pig, and rabbit) do not have functional MCH-R2 receptors, or encode a non-functional MCH-R2 pseudogene while retaining MCH-R1 functional expression (see Tan et al., 2002). Such a late evolutionary process for both a variant putative peptide as well as a receptor subtype must be seen as a rare occurrence.

2. THE PRO-MCH GENE, REGULATION OF EXPRESSION AND PRECURSOR PROCESSING 2.1. STRUCTURE, CHROMOSOMAL MAPPING AND EVOLUTION OF THE PRO-MCH GENE AND LINKED GENES The organisation of the gene encoding the MCH precursor in the rat, mouse and human (reviewed in Nahon et al., 1993; Nahon, 1994) is clearly established. This gene comprises three exons and two introns coveting about 1.4 kb of genomic DNA (Fig. 3A). The first exon encodes the 5'-untranslated region of the mRNA and the N-terminal part of pro-MCH including the signal peptide that allows targeting to the secretory pathway. The second exon comprises the sequence corresponding to NGE, NEI and the first three amino acids of MCH. The last 15 amino acids of MCH and the T-untranslated amino acids are localised on exon III. Intron B splits, methionine codon with the nucleotide A on exon II and the bases TG on exon III. This intronic organisation is identical in the rat mouse and human MCH gene. This unusual intron position for a neuropeptide encoding gene is of primary importance to generate by alternative splicing a new protein named MCH-gene-overprinted-polypeptide (MGOP) (Toumaniantz et al., 1996) (Fig. 3B). In addition, dense MGOP projections were encountered in the suprachiasmatic, ventromedial and arcuate nuclei, as well as median eminence, suggesting neuroendocrine functions for MGOE Recently, comparison of MCH and MGOP distribution in the rat brain revealed a striking colocalisation in neurones of the zona incerta/lateral hypothalamus and unique expression of MGOP in neurones of the hypothalamic periventricular nucleus and of many brain areas (cortex, amygdala, caudate putamen, lateral septal nucleus) (Toumaniantz et al., 2000). Characterisation of high molecular weight MCH gene transcripts in PC12 cells and rat tissues revealed the existence of antisense RNAs complementary to the MCH gene (Presse et al., 1997; Borsu et al., 2000). This followed the discovery of shorter natural antisense transcript in the rat gut (Hervieu and Nahon, 1995). Human antisense prepro-MCH mRNAs were also reported by Miller et al. (1998) and Viale et al. (2000). In the pheochromocytoma cell line PC12, two classes of antisense RNAs were found: (1) non-coding unspliced RNAs overlapping exon II/exon III and flanking intronic sequences of the MCH gene; and (2) alternative spliced mRNAs coding for new RNA/DNA binding proteins. We named this gene AROM for Antisense-RNA-overlapping-MCH. This gene maps at the same locus than 36

The melanin-concentrating hormone

Ch. H

Fig. 3. The MCH gene. (A) Comparison of the human, mouse and rat MCH genes. Exons and introns are noted by boxes and lines, respectively. Percent identities are indicated. (B) Structure of the MCH and MGOP mRNA and deduced proteins. Alternative splicing is noted by dotted line.

MCH gene and appears highly conserved among rat, mouse and human (Borsu et al., in preparation). Because of the reciprocal regulation of MCH and AROM gene expression in PC12 cells (Borsu et al., 2000) and in vivo (Presse and Nahon, unpublished data) it is feasible that AROM mRNA and/or proteins may control MCH gene expression but this remains to be directly tested. The pro-MCH (PMCH) locus was localised to chromosome 12q23 in human (Pedeutour et al., 1994; Viale et al., 1997). We previously hypothesised that the MCH gene could be a candidate gene for the Darier's disease and spinocerebellar ataxia type 2, but further studies excluded MCH and identified the actual genes involved in these diseases (Sanpei et al., 1996; Sakuntabhai et al., 1999). A second MCH-like gene system was identified in the human genome. Indeed a truncated version (named variant MCH gene) of the authentic MCH was found duplicated on the long an short arms of the human chromosome 5 (reviewed in Nahon, 1994). Interestingly, this gene may code for a putative nuclear protein, the signal peptide of the pro-MCH being replaced by a nuclear-localisation signal (NLS) in the new gene (Viale et al., 2000). Phylogenetic analysis revealed that the variant MCH gene arose by very complex mechanisms only in the Hominidae lineage (Viale et al., 1998; Courseaux and Nahon, 2001). Thus, the MCH gene family provides a unique model to investigate the structural and functional switches of genes that diverged during late primate evolution. 2.2. REGULATION OF PREPRO-MCH GENE EXPRESSION The regulation of MCH gene expression has been most extensively investigated with in vivo models. In particular, MCH mRNA increases during postnatal development at the 37

Ch. H

G.J. Hervieu et al.

suckling-transition time in rodents (Presse et al., 1992; Breton et al., 1993b; Brischoux et al., 2001) and exhibits a marked circadian variation (Presse and Nahon, 1993; Bluet-Pajot et al., 1995), consistent with the important role of MCH in feeding behaviour, locomotor activity or higher integrative behaviours. Furthermore, low level of MCH mRNA expression was found associated with short-term footshock stress (Presse et al., 1992) and dehydration or hypertonic saline regimen (Presse and Nahon, 1993), suggesting an inhibitory function for the MCH system in the stress response and fluid homeostasis (see below). Furthermore, an increase in MCH mRNA was recently reported (Herv6 et al., 1998) following acute polyethylene glycol (PEG) injection that induced a reduction of the extracellular fluid volume. The apparent discrepancy between previous and recent data on the regulation of MCH mRNA following osmotic challenges most likely resulted from a differential effect of the experimental models on volaemia or osmolability as discussed by Herv6 et al. (1998). During recent years, a number of studies have accumulated with respect to variations of MCH mRNA expression, feeding behaviour and possibly obesity. First, activation of MCH neurones was observed following insulin and 2-deoxyglycose injections in the rat (BahjaouiBouhaddi et al., 1994; Presse et al., 1996) or lesions of the neuromedial hypothalamic nuclei (VMN) (Griffond et al., 1995). Then, direct involvement of the MCH neuronal system in the control of feeding behaviour was supported by the increase of MCH mRNA levels observed following food-deprivation in the rats (Presse et al., 1996; Herv6 and Fellmann, 1997) and mice (Qu et al., 1996). Finally, overexpression of MCH mRNA and/or pro-MCH derived peptides was found in various obese rodents, including ob/ob mice (Qu et al., 1996; Mondal et al., 2002); Huang et al., 1999), db/db mice (Mizuno et al., 1998; Mondal et al., 2002), fat~fat mice (Rovbre et al., 1996) and Ay/a (agouti) mice (Hanada et al., 2000). Alteration in adiposity may also have some influences on the MCH expression. This was exemplified by the decrease of MCH mRNA observed in brown adipose tissue-deficient mice which developed both obesity and hyperleptinaemia (Tritos et al., 1998a) and the higher levels of MCH mRNA found in thin ewes by comparison with the fat animals (Henry et al., 2000). Leptin either decreased MCH gene expression following acute injection in rats (Sahu, 1998) and ob/ob mice (Tritos et al., 2001) or, conversely, stimulated MCH mRNA peptide expression under chronic treatment in lean and ob/ob mice (Huang et al., 1999). Stricker-Krongrad et al. (2001) recently reported that in obese hyperphagic Zucker, the absence of leptin signaling in rats is associated with an increased hypothalamic expression and circulating release of MCH and that it probably contributed to their obesity syndrome. Using canine distemper virus (CDV) which can target hypothalamic nuclei, and lead to obesity syndrome in the late stages of infection, Verlaeten et al. (2001) showed a specific down-regulation of melanin-concentrating hormone precursor mRNA (ppMCH) in infected obese mice. The MCH gene expression is decreased in adult male rats treated with lipopolysaccharide (LPS), an inflammatory agent. Anorexia is often a consequence of inflammatory processes and the down-regulation of MCH gene expression may contribute to hypophagic behaviours (Sergeyev et al., 2001). Apart from regulation by stress, food or water deprivation, the MCH neuronal system is also affected by the steroid hormones status of the animals. Early studies (Parkes and Vale, 1992b; Presse et al., 1992) have suggested that endogenous glucocorticoids in adrenalectomised rats or addition of dexamethasone in vivo or in hypothalamic cells in culture, stimulated the synthesis of MCH mRNA and pro-MCH derived peptides (MCH and NEI). The effects of gonadal steroids on MCH mRNA or peptide production were recently examined in models of ovariectomised (OVX) female rats (Murray et al., 2000) or OVX female macaques (Viale et al., 1999a). The animals were exposed to oestradiol benzoate to suppress secretion of luteinising hormone (LH) in the first step, then a 'mid-cycle-like' LH-surge operated 38

The melanin-concentrating hormone

Ch. H

naturally in the primates or after administration of progesterone in the rat model. In the rats, MCH mRNA content decreased in a subpopulation of cell bodies of the medial zona incerta following the oestradiol benzoate-treatment. Negative regulation by oestrogen on MCH expression was further confirmed in an oestrogen-cachexia-induced model. Oestrogeninduced anorexia in rodents is associated with decreased MCH gene expression (Mystkowski et al., 2000). Surprisingly, in view of the effect of MCH on the LH release (see below), the addition of progesterone provoked a LH surge but did not affect the reduction of MCH mRNA levels observed in the zona incerta. In the macaques, oestradiol treatment induced basic variations in MCH and NEI contents that paralleled those of gonadotrophin-releasing hormone (GnRH). This suggested that pro-MCH-derived peptides may indeed participate in the regulation of the LH secretion through interaction with the GnRH neural system localised in the medial preoptic area. Furthermore, in addition to mature MCH or NEI, other MCH-ir and NEI-ir products were identified in oestradiol-treated OVX monkeys, suggesting that post-translational regulation at the level of processing and/or degradation of pro-MCH may operate (Viale et al., 1999a). 2.3. PEPTIDE CHARACTERISATION AND PRECURSOR PROCESSING Regarding the structure of the mammalian pro-MCH derived peptides, it is worth noting that exclusively mature peptides, i.e. cyclic MCH and amidated NEI, were found in the rat and human brains (Hervieu et al., 1996a; Viale et al., 1997) (Fig. 4A). Conversely, neither MCH nor NEI were found in human peripheral organs and a larger peptidic form was identified in the colon, thymus or adipose tissues (Viale et al., 1997) (Fig. 4A). This large MCH-ir peptide contained, at least, a NEI sequence in its N-terminus and ends with a C-terminal MCH sequence (Viale et al., 1997). Similarly, pro-MCH processing intermediates are likely to exist in the rat gut (Hervieu et al., 1996a), testis (Hervieu et al., 1996b) and spleen (Hervieu and Nahon, unpublished data) but their structures remained elusive. More recently, a pro-MCH derivative similar to this detected in the human peripheral organs was characterised in the mouse spleen (Viale et al., 1999b) and fat tissues (Viale and Nahon, unpublished data). In this context, the processing of MCH precursor was examined using various cellular systems and an in vivo PC2 KO-mouse model (Viale et al., 1999b) (Fig. 4B). The main finding was that active pro-convertase (PC2) is necessary and sufficient to generate NEI in the brain and in PC12 cells. In contrast, several PC, including PC1/3, PC2 and PC5/6A may cleave at the appropriate site to produce MCH either in vaccinia expression systems or in the mouse brain. Incidently, co-localisation studies demonstrated simultaneous expression of MCH mRNA and PC2 in all MCH-expressing cell bodies of the lateral hypothalamus, whereas only 15-20% of these cells contained PC1. Therefore, PC2 is likely to be the key enzyme that cleaves MCH-precursor to generate MCH and NEI in the brain. The counterpart in peripheral organs remained unknown.

3. FEATURES OF THE MCH SYSTEM IN THE RAT CNS

Long before MCH was known in the rat brain, immunoreactive signals related to the POMCderived 0~-MSH were observed where immunosignals for the other POMC-derived peptides could not be seen. This suggested that the antiserum recognised an as yet unknown factor, bearing immunological resemblance with the c~-MSH. By retrograde tracing studies combined to immunohistochemistry, a system with lateral hypothalamic origin was identified and which 39

Ch. II

G.J. Hervieu et al.

A

Hypothalamus 15 600

NEI

400

200

200

100

15

5

200]

25 (rain)

25

Periphery 400

15

300

32

300

MCH

45 (min)

35

32

'Or0

200 100

15

B eRA,N

I

25 (min)

NEI

35

|S

~NH2

I'

45 (min)

MCH

'

I

or.', 129-130

I

QEKRE

PERIPHERY

N =l

14]~4~

MC.

I

MCH

I

IGRRD

NEI

II

Fig. 4. MCH precursor processing and peptide production. (A) Schematic representation of RP-HPLC analysis of protein extracts from hypothalamus or peripheral organs. Mature NEI and MCH migrate at 15 and 32 min, respectively. At the periphery a larger product migrates at 45 min (Viale et al., 1997). (B) Role of pro-convertases in pro-MCH processing. Sequences at the cleavage sites are noted. The question marks indicate that PC involved at the putative cleavage site is unknown.

innervated the entire neuraxis. It was termed the 'et-2' system (for second ot-MSH system). That system was to be later identified as the MCH system. The antiserum was targeting the amidated motif of NEI (Nahon et al., 1989). The ~-MSH is amidated, as could also be the NEI, a peptide of the pro-MCH precursor protein, because of a consensus motive for amidation. At the same time, it brings support for the predicted amidated NEI to exist in the rat brain (see Eberle, 1988; Baker, 1994; Nahon, 1994; Sawchenko, 1998). The seminal paper by Bittencourt et al. (1992) is a very thorough description of the MCH system in the rat, and pertinently proposed some directions in which to investigate the role of the peptide. Other neuroanatomical studies have been done in the guinea pig (Knigge et al., 1996), in the monkey (Bittencourt et al., 1998) and the human (Elias et al., 1998, 2001).

40

The melanin-concentrating hormone

Ch. II

3.1. A STRIKING HYPOTHALAMIC LOCALISATION OF THE MCH IMMUNOREACTIVE CELL BODIES The hypothalamic location of MCH neurones is striking: more than 95% of the MCH gene neurones in the CNS are hypothalamic with more than 90,000 cells there expressing the MCH gene (see Knigge et al., 1996). The MCH gene expression level here is just below those of oxytocin and vasopressin (Bittencourt et al., 1992). In fact, hypothalamic MCH peptide levels are amongst the highest in the brain (Nahon, 1994) and animals do not need to receive colchicine treatment (which inhibits axonal transport of prepro-MCH out of the cell body) in order to allow visualisation of MCH cell body immunosignals (Bittencourt et al., 1992). Interestingly, the high level of MCH gene expression was confirmed in substractive molecular biology experiments at the Salk Institute: two clones identified as both the MCH and the hypocretins/orexins were read-out as being particularly strongly enriched in rat hypothalamic cDNA libraries (Gautwik et al., 1996). MCH is an excellent peptidergic marker of the lateral hypothalamus and would stand as the only specific peptidergic marker of the lateral hypothalamus until the discovery of the orexins/hypocretins (Sakurai et al., 1998). Bittencourt, Presse and colleagues identified secondary sites of brain MCH expression in the olfactory tubercle and in a previously uncharacterised part of the paramedian pontine reticular formation in the rat (Bittencourt et al., 1992; Presse et al., 1992) but not in the monkey (Bittencourt et al., 1998). Of interest, lactation induces novel hypothalamic expression site of the female rat MCH system in the medial preoptic nucleus, preoptic periventricular nucleus and rostral area of the paraventricular hypothalamus (Knollema et al., 1992). Some differences are noted in the way MCH neurones are organised in the hypothalamus of different animals: the MCH cell group is always positioned in the lateral hypothalamus and zona incerta but with differences in the subdivisions of these regions: for instance, a tail of immunopositive cells extend in the guinea pig until reaching the posterior hypothalamus at the supramammillary commissure level (see Knigge et al., 1996). A reliable feature for all species studied so far (mouse, rat, hamster, guinea pig, rabbit, dog, monkey, human) is that the MCH cell group do not populate any of the traditional proper hypothalamic nuclei (see Knigge et al., 1996). In the human, no differences was reported regarding the number and position of MCH cell bodies in males versus females. The most prominent cluster of MCH cells ran along the entire rostrocaudal extension of the fornix. The MCH perikarya were observed extending back into the posterior hypothalamic area, just above the mammillary body and close to the third ventricle (Elias et al., 1998). MCH gene expression is noted at early stages of development: in the rat, MCH mRNA and peptide are detected at day 13 of gestation while in the human, MCH immunoreactivity is observed at the 7th week of foetal life (Bresson et al., 1987; Brischoux et al., 2001). The ontogeny of rat hypothalamic MCH neurones was carefully studied using the bromodeoxyuridine method, combined to immunochemical techniques, in order to determine the period of birth of these neurones. The maturation of the total MCH neuronal population occurred throughout prenatal stages and the early postnatal period. No subpopulations of MCH neurones could be identified based on ontogenic criteria, but it was found that the spatiotemporal pattern of MCH cell genesis had striking similarities with the one described for the hypothalamic parvicellular neuroendocrine neurones (Brischoux et al., 2001). Cytological features of the non-colchicinised MCH neuropeptidergic adult rat hypothalamic cell include: a medium-size, a shape ranging from multipolar to fusiform, giving rise to 2-5 41

Ch. II

G.J. Hervieu et al.

primary dendrites and common secondary dendritic branching. Varicose axons (0.1-0.2 ~m in diameter) arose from labelled perikarya or the proximal portion of primary dendrites. Large and bipolar neurones (12 x 40 ~m) are observed at the level of the internal capsula (Bittencourt et al., 1992; Knigge et al., 1996). Electron microscopy using an antiserum directed towards the rat MCH revealed the ultrastructural details of MCH neurones: densecored granules (75-200 nm in diameter) in a moderate number, distributed throughout the cytoplasm, with immunosignals located in a subset of Golgi saccules. Labelled cell bodies are often apposed to vascular elements such as venules, separated by fine interposed astrocytic processes. Axons are most commonly unmyelinated and terminals are characteristic of a neuropeptidergic neurone with small electron-lucent vesicles and large dense-cored vesicles. These vesicles were detected at the level of the external lamina of the median eminence (Bittencourt et al., 1992). 3.2. FEATURES OF THE MCH INNERVATION WITHIN THE MAMMALIAN BRAIN MCH nerve immunoreactivity is broadly distributed within the rat brain (neocortex, allocortex and hippocampal formation, basal ganglia, diencephalon, brainstem/pons/reticular formation, myelencephalon) (Figs. 1 and 9G) with very similar projections to these of orexins (Peyron et al., 1998; see also Chapter 5). The peptide was not detected in the anterior pituitary. Extremely dense terminal fields of MCH nerves are observed in parts of the extrapyramidal system, the reticular formation throughout the brainstem, the precerebellar nuclei and the spinal cord. In the neocortex, the MCH immunosignals distributes within laminae, a characteristic of a non-specific cortical afferent innervation, which appears to be a basis for generalised cortical arousal. In the diencephalon, MCH is not predicted to have major neuroendocrine effects because of a relatively low peptide content in key areas like the paraventricular and supraoptic nuclei and also, a relatively sparse innervation of median eminence. Other MCH-immunoreactive territories enriched with MCH are the autonomic-related structures of the brainstem, though the levels of peptide are much lower than in the forebrain. Signals are observed in all sorts of nuclei (motor nucleus of the V, parabrachial nuclei, locus coeruleus) and reach the spinal cord, particularly enriched around the central canal (Bittencourt et al., 1992; Bittencourt and Elias, 1998; Sawchenko, 1998). Some investigations have more especially focused on the MCH immunoreactive projections to the parabrachial nucleus and pontine gustatory area (Touzani et al., 1993), within the hypothalamus (Broberger et al., 1998; Elias et al., 1998; Broberger, 1999; Abrahamson et al., 2001; Abrahamson and Moore, 2001), to the peri-aqueductal grey matter (PAG; Elias and Bittencourt, 1997) and to the medial septum and thoracic spinal cord (Bittencourt and Elias, 1998). By combining retrograde tracing studies and immunochemistry, it was shown that projections to the periaqueduct area originated from two major sources: the zona incerta supplied afferents via a medial pathway that entered the PAG dorsally at rostral levels, and a pathway originating in the lateral hypothalamus that entered the PAG ventrally at more caudal levels. The medial subdivision of the PAG, which encompassed the Gerrits column I contained the greatest level of MCH-ir fibres (Elias and Bittencourt, 1997). In the spinal cord, MCH-ir fibres were concentrated primarily in lamina X (surrounding the central canal) and secondarily in layers I, III and IV. In the septal complex, MCH-ir fibres were enriched in the medial aspects and in the vertical and horizontal limbs of the medial septum and the nucleus of the diagonal band (Bittencourt and Elias, 1998). The medial area of the parabrachial nucleus, also termed the pontine taste area, receives many ascending gustatory afferents and is implicated 42

The melanin-concentrating hormone

Ch. H

in the palatability of nutrients. Fifty to 60% of the MCH-ir neurones from the juxtacapsular region and 20-30% of neurones in the perifornical area project to the taste area (Touzani et al., 1993). A recent study using neurotropic virus pseudorabies in conjunction with the immunocytochemical localisation of MCH to better define the pathways in the rat hypothalamus related to the reflex control of thermogenesis has shown that MCH neurones from the lateral hypothalamus project to brown adipose tissue (Oldfield et al., 2002). These data set the scene for the peptide to play a role in an extremely vast array of brain activities, ranging from neocortical to autonomic modulations. MCH appears as to be one out of the many other factors which contribute to the bridging of diverse biological systems, such as mood regulation, reward system, feeding and water intake, sexual behaviour, motor and somatosensory controls. It is presently uncertain to what extent the peptide is significantly implicated within each of them but evidences are accumulating that MCH is a key regulator in body homeostasis by its role on those diverse systems. 3.3. COLOCALISATION DATA It is a rule of thumb that neuropeptides usually colocalise with other neuromodulators within the cell that produces them. To date, investigations have mainly focused on feeding regulators. 3.3.1. Neurochemical colocalisation

As expected, MCH coexists with the NEI peptide (released from the prepro-MCH precursor protein) with a 96% overlap except in the interanterodorsal nucleus of the thalamus (Bittencourt et al., 1992). It has still not yet been proven that the NGE, the other peptide potentially released from the prepro-MCH precursor protein, also co-exists in MCH neurones. Also in the lateral hypothalamus, the MGOP-ir cells have a very similar location to that of MCH cells (Toumaniantz et al., 1996). Overt co-localisation of the MGOP-ir and MCH-ir was demonstrated in the LHA whereas only MGOP-ir was found in neurons of the periventricular area of the hypothalamus (Toumaniantz et al., 2000). MCH and orexin form distinct cell population groups in the lateral hypothalamus of the rodent (less than 1% overlap; Broberger et al., 1998; Elias et al., 1998) and the human (Elias et al., 1998). MCH co-exist with the anorectic neuropeptide cocaine- and amphetamine-regulated transcript (CART) (65% of the MCH population; Broberger, 1999; Elias et al., 2001; Brischoux et al., 2001) and glutamate (Abrahamson and Moore, 2001; Abrahamson et al., 2001). Comforting the role of MCH in energy balance, the MCH neurones receive a dense innervation by NPY, AgRP and ~-MSH neurones of the basal hypothalamus (Elias et al., 1998) and harbour on the cell membrane leptin receptor (Hakansson et al., 1998a) as well as the NK3 receptor (55% of the MCH population, Griffond et al., 1997) and contain within their nuclei STAT-3-1ike immunosignal (Hakansson et al., 1998). In a more recent study, Iqbal et al. (2001) showed that all MCH neurones harbour the long form of the leptin receptor, OB-Rb. MCH neurones innervating VIP and AVP fibres coming from the hypothalamic suprachiasmatic nucleus (Abrahamson et al., 2001) could implicate the MCH neurones as a component of the arousal system. Lastly, strong acetylcholine-esterase activity and immunoreactivity is present in the MCH neurones at reticular and nuclear envelope level, suggesting a possible cholinergic receptivity (see Griffond et al., 1998).

43

Ch. H

G.J. Hervieu et al.

3.3.2. Functional colocalisation

Energy metabolism is also thought to be critically controlled by the LHA, as it contains a population of neurones which is sensitive to glucose levels and is activated by hypoglycaemia. The identity of these glucostat cells has remained elusive, probably because the lateral hypothalamus is not as topologically organised as other major nuclei of the hypothalamus. Cells are scattered within the medial forebrain bundle, the most prominent brain fibre network bundle. This has added a great deal of complexity to the design of experiments to study the LHA that would not damage the medial forebrain bundle itself (see Bernardis and Bellinger, 1996). The principal path of MCH projections though the basal forebrain, hypothalamus and rostral midbrain is virtually superimposable upon a map of sites showing increased metabolic activity in response to rewarding electrical stimulations of the LHA (see Bittencourt et al., 1992; Sawchenko, 1998). This data would imply the MCH neuronal population be the brain glucose-sensor neuronal group. However, experimental data do not favour that hypothesis as shown by injection of non-metabolisable glucose analog such as goldthioglucose, which does not affect MCH gene expression (Grillon et al., 1997) and 2-deoxyglucose used on slice cultures of rat hypothalamus (Bayer et al., 1999a). Rather, it would seem that the orexin neurones are the anatomical substrate of the hypothalamic glucostat, as reported by an activation of the orexin neurons lateral hypothalamic for instance by insulin-induced acute hypoglycaemia (Moriguchi et al., 1999). 3.4. NEUROCHEMICAL ENVIRONMENT AND SURVIVAL OF MCH NEURONES 3.4.1. Neurochemical environment

The MCH neurones are strongly intermingled with orexin, GABA, dopamine, neurotensin and galanin neurones. MCH neurones are surrounded by a rich neuropil containing dopamine, serotonin, neurotensin, galanin, neurophysin, GABA, NPY, ~-MSH, AgRR somatostatin, VIR AVP and neurokinin B. Nodular figures and pericellular baskets suggest the occurrence of synapses between the different systems (see Griffond et al., 1998). 3.4.2. Survival of M C H neurones in culture

Compagnone et al. (1991) devised a serum-free medium culture system for growing hypothalamic neurones containing MCH immunoreactive cells. Using a co-culture model, it was shown that diffusible factors from the arcuate nucleus and the diagonal band increased the number and the size of the MCH neurones. The glia in the arcuate nucleus produced factors important for MCH neurite outgrowth and expressed inhibitory factors, preventing the adhesion of MCH cells on arcuate glial cells. Contacts between MCH and dopaminergic cells were increased (Compagnone et al., 1993). Hypothalamic slices prepared from 6- to 8-day-old rats and containing the MCH neurones, when treated with carbachol, a cholinergic agonist, resulted in an increase of MCH gene expression, whereas that regulation was abolished using atropine and hexamethonium, respectively, a muscarinic and nicotinic antagonist, respectively (Bayer et al., 1999b). That mice deleted for the muscarinic M3 receptor have reduced level of MCH (Yamada et al., 2001) comes as supportive evidence for a close interaction between the MCH and cholinergic systems. Also hypothalamic slices containing the MCH neurones prepared from 6- to 8-day-old rats, could be maintained for 1 month in culture with MCH 44

The melanin-concentrating hormone

Ch. H

neurones responsive to leptin at day 10 of growth (Bayer et al., 1999c). Organotypic cultures might thus represent an adequate model in which to investigate further the pharmacology and physiology of the MCH system. A study using synaptically coupled neurones from the rat embryonic hypothalamus in tissue culture has shown that MCH exerted profound inhibition of synaptic activity on those cells. Both glutamate and GABA synaptic transmission were inhibited by MCH (Gao and van den Pol, 2001). In a study on the hippocampus, Varas et al. (2002) observed a long-lasting potentiation on the hippocampal evoked response on dentate gyms induced by MCH. 3.5. PHYSIOLOGICAL SECRETION OF MCH To date, dexamethasone, cAMP analogues, phorbol esters, noradrenaline and glutamate/ NMDA have been shown to induce the release of MCH from neurones in culture while CRF inhibits MCH release (see Nahon, 1994). 3.6. PERIPHERAL PLASMATIC AND CENTRAL MCH While Takahashi et al. (1995) were unable to detect rat peripheral blood circulating levels of MCH, a recent study using a commercial radio-immunoassay has shown that MCH-like immunoreactive material was detectable with levels ranging from 54 to 400 pg/ml of plasma (Bradley et al., 2000). A recent topic in neuropeptidergy has been to propose transcytosis of peptides between the brain and the periphery (in both ways) (refer to: Strand et al., 1994; Crawley and McLean, 1996). Sensor-systems at the periphery-brain barrier level exist in order to transduce an humoral messenger into a brain signal, like for leptin. It implies a cooperation at the blood-brain barrier (BBB) level mostly while circumventricular organs (e.g. median eminence, choroid plexus, area postrema... ) vasculature, though devoid of BBB, are not a first-place of entry because part of the ependyme is joined by tight junctions. Peptides can cross the BBB due to their lipophilicity, but the existence of saturable transporters for peptides across the blood-brain barrier has been reported (refer to: Strand et al., 1994; Crawley and McLean, 1996; Gao et al., 2000). Such a system could exist for MCH. While almost all of the peptides and polypeptides tested so far cross the BBB at a faster rate than the vascular marker albumin, the MCH analog, [125I][Phe13,Tyr19]MCH, did not cross faster than Tc-99m-albumin. This is probably because of its binding to serum proteins (Kastin et al., 1999; see also Kastin et al., 2000). 3.7. DEGRADATION OF MCH BY PEPTIDASES The endopeptidase EC 3.4.24.11 (neutral endopeptidase, enkephalinase) in vitro attacked MCH at three sites of the molecule with an apparent affinity of about 12 txM and a kcat of 4 rain -1 . The first site of cleavage was at CysV-Met 8, i.e. within the peptide loop formed by the internal disulphide bridge, and necessary to bioactivity. NEP could therefore be considered as one of the main MCH-inactivating peptidases since the degradation products generated are probably devoid of biological activity (Checler et al., 1992; Maulon-Feraille et al., 2002). NEP is particularly abundant in choroid tissues and meninges which are the first sites sensing the i.c.v. MCH injection. Hence it is probable that MCH may have a short half-life in the blood and cerebrospinal fluid (CSF). However, it has to be noticed that the dipeptide H-NEIMCH-OH is fully resistant to degradation by both aminopeptidase M and endopeptidase 24.11 as well as by exo- and endo-proteases using brain extracts and purified proteases. It retains 45

Ch. H

G.J. Hervieu et al.

biological activity on MCH-R1 and has physiologically a more potent orexigenic effect than MCH itself (Maulon-Feraille et al., 2002). It may well be that the dipeptide is a physiological agonist at MCH-R1.

4. C E N T R A L E F F E C T S OF MCH We shall focus here on three cerebral functions supported by substantial, although sometimes controversial, experimental evidence. 4.1. MCH AND THE REGULATION OF THE HPA (Fig. 5) Historically, the first study dealing with the MCH role in mammals was to test the effect of salmon MCH on ACTH release from isolated rat pituitary cells (Baker et al., 1985). This initiated the long series of conflicting results since the direct inhibitory action of salmon MCH (Baker et al., 1985) could not be reproduced using the synthetic rat MCH (Navarra et al., 1990). In the same line, Jezova et al. (1992) reported a central stimulatory effect of rat MCH on basal ACTH release after intracerebroventricular (i.c.v.) administration in conscious rats. Another study, however, found that i.c.v, injection of rat MCH (and NEI) did not modify the basal secretion of ACTH at day or night time (Bluet-Pajot et al., 1995). On the contrary, MCH appears to inhibit ACTH secretion after an ether stress (Bluet-Pajot et al., 1995) or after a mild handling stress (Ludwig et al., 1998). Interestingly, NEI (Bluet-Pajot et al., 1995) or MSH (Ludwig et al., 1998) can prevent the inhibitory action of MCH on ACTH release. Since none of these peptides may antagonise the binding of MCH ligand or activation of the MCH receptor in transfected cells (see below), it is likely that they act through their own

Fig. 5. The hypothalamic-pituitary-adrenal axis and central neurotransmitter circuitry. During stress, stimuli are

integrated at the levels of the brainstem and hypothalamus. +, activator of the stress response; -, inhibitor of the stress response. Stress leads to secretion of glucocorticoids which in turn inhibit the activators at multiple levels in the brain and possibly induces putative activator (like MCH) in the hypothalamus. 46

The melanin-concentrating hormone

Ch. H

receptor either on the same cellular target or downstream of MCH in the stress-responseregulating pathway. Lastly, another work has reported that MCH administered intravenously (i.v.) induced a strong and significant dose-dependent increase in plasmatic corticosterone level (Ashmeade et al., 2000). It is thus presently a confusing situation where both activatory and inhibitory actions of MCH on the stress axis are reported and it is hoped that some light can be shed on these discrepancies in the near future. The anxiolytic and antidepressant properties of SNAP-7941, an MCH-R1 antagonist (Borowsky et al., 2002), may, however, favour the hypothesis that MCH is activatory within the stress axis. 4.2. MCH AND REPRODUCTIVE FUNCTIONS Based on neuroanatomical considerations, Gonzalez, Wilson and colleagues made a comprehensive study of MCH and sexual or reproductive behaviours in female rats. Infusion of rat MCH in the medial preoptic area (MPOA) or the ventromedial nucleus (VMN) of the hypothalamus stimulated lordosis of sexually non-receptive rats (Gonzalez et al., 1996). MCH administered into the MPOA or the median eminence has also a stimulatory effect on the LH release (Gonzalez et al., 1997a). Interestingly, this action seems of true physiological significance since MCH antiserum injected in the MPOA fully prevented the progesteroneinduced rise in LH release. Furthermore, MCH and MSH had opposite effects on LH release after injection in MPOA or ME (Gonzalez et al., 1997a). Later, the same group (Murray et al., 2000) confirmed the stimulatory action of MCH on LH secretion when injected into the MPOA and suggested that this peptide could be a weak agonist at the MC5 receptor. However, another laboratory (Tsukamura et al., 2000) found very recently that central injection of rat MCH decreased both plasma LH concentrations and LH pulse frequencies. There is no obvious explanation for these opposite results other than the fact that the stimulatory effect was noted when MCH was injected at low concentrations (100 ng/side) into discrete hypothalamic areas (MPOA, VMN) whereas the inhibitory effect was observed when highest doses (1-10 ~g/animal) were infused into the third cerebroventricle. Another group recently observed an effect of MCH on the stimulation of both LH and FSH gonadotropins from proestrous pituitaries similar to the effect produced by luteinising hormone-releasing hormone (LHRH). Simultaneous incubation of pituitaries with MCH and LHRH did not modify LH, but increased the FSH release induced by LHRH. This suggest that MCH could be involved in the regulation of preovulatory gonadotropin secretion (Chiocchio et al., 2001). 4.3. A ROLE FOR MCH IN REGULATING WATER BALANCE Given the particularly robustly strong (and quasi-uniquely localised) MCH gene expression in the lateral hypothalamus and zona incerta in the all mammals studied so far (rat, guinea pig, mouse, monkey, human; see Section 3.1), a role for MCH in the regulation of the hydric and energy balance was suggested on the basis of functional neuroanatomical considerations. The evidence for a role of MCH in water balance was known prior to the biochemical purification of the rat MCH: a 5-day salt loading triggered an elevation of MCH immunoreactivity in the hypothalamus and pituitary (Zamir et al., 1986a). Gene-expression studies showed that rat hypothalamic MCH gene expression was down-regulated by a 6-day salt-loading experiment. Females and males show differences and clusters of MCH cells within subdivisions of the lateral hypothalamus do not behave evenly (Presse and Nahon, 1993). Also, the MCH is a secretagogue of arginine-vasopressin (AVP) as demonstrated in vivo (Forsling and Zhou, 1997). 47

Ch. II

G.J. Hervieu et al.

Lactation induces novel hypothalamic expression of MCH in the preoptic area and paraventricular hypothalamus (Knollema et al., 1992). Also MCH evokes the release of oxytocin in a rat hypothalamo-pituitary explants (Parkes and Vale, 1992a). Lactation, which can be considered as an energy-deficient component, is an obvious link between feeding behaviour and internal fluid regulation. 4.4. MCH AND THE CONTROL OF FEEDING BEHAVIOUR It is now clearly established that MCH is a critical factor involved in feeding behaviour and energy regulation (Presse et al., 1996; Qu et al., 1996; Rossi and Bloom, 1997; Ludwig et al., 1998; Rossi et al., 1999; Edwards et al., 1999) (Fig. 6). The determining role of the LHA in controlling feeding behaviour made the MCH an early putative candidate for that role. One can grossly define the lateral hypothalamus as a 'feeding centre'. A lesioned or chemically damaged LHA makes the animals die by voluntary starvation (the so-called syndrome of aphagia). There was a long-term controversy dating back from the 1940s and 1950s, which led to the proposal of a dual centre hypothesis for feeding intake control where the LHA would be the feeding centre, while the ventromedial hypothalamic nucleus would be the satiety centre (Hetherington and Ranson, 1940; Anand and Brobeck, 1951). The mechanistic as well as the detailed anatomical observations would finally support that hypothesis and equally revealed a great hormonal/neural neurochemical diversity in natural agents regulating feeding behaviour (Morley, 1987; Elmquist et al., 1999). However, while clearly demonstrated for NPY, the implication of MCH within energy balance was a more frustrating area of investigation, as the lateral hypothalamus is extremely difficult to experiment with. Also feeding intake is an extremely complex behaviour to analyse. Leptin is produced by adipocytes in the periphery upon satiety and transmits to brain to induce food intake cessation. It acts within the hypothalamus where it regulates an increasingly reported intermingled circuitry of neuromodulators (mainly neuropeptides) and neurotransmitters (noradrenaline, glutamate, GABA, serotonin) (for reviews see Morley, 1987; Schwartz et al., 2000). A first report established that MCH was anorectic at specific times of the circadian rhythm (Presse et al., 1996). An orexigenic action of MCH was subsequently identified (Qu et al.,

Fig. 6. Effects of MCH on feeding behavior. Intracerebroventricular injection of MCH may inhibit or activate food-intake according to the studies of Presse et al. (1996) or Qu et al. (1996).

48

The melanin-concentrating hormone

Ch. H

1996) and confirmed by others (Rossi and Bloom, 1997; Ludwig et al., 1998; Rossi et al., 1999; Edwards et al., 1999). The discrepancy may be due to the use of a low dose of peptide in the first study and the existence of a biphasic effect of MCH on appetite and food consumption. Indeed, the anorectic effect of MCH was characterised by i.c.v, injection of MCH in very low doses of peptides (1-100 ng/animal) in male Wistar rats with prior fasting for 2-24 h. This reduction in food intake was found also when 1 ng rat MCH was bilaterally infused into the central borders of the ZI-LHA in the morning, but not at the beginning of the feeding active period (Presse et al., 1996). Orexigenic effects of MCH was found with i.c.v. injection of 5 [xg MCH at different times of the dark phase consistently increased feeding for 2-4 h in Long Evans rats (Qu et al., 1996). The appetite-stimulating effect of MCH occurred both in the light and dark phases of the day (Rossi and Bloom, 1997) and is comparable to that of orexins and galanin (Edwards et al., 1999). Rat strain differences in terms of MCH sensitivity were identified (Della-Zuana et al., 2002). Confirmation of an orexigenic role for MCH came from studies showing that mice deleted for the prepro-MCH gene had a hypophagic and lean phenotype with virtually no fat deposit and an altered metabolism (Shimada et al., 1998). It was the first example that deletion of a gene encoding a single orexigenic peptide can result in leanness. Possible pathophysiological implication of MCH in feeding disorders was indicated by a three-fold increase of MCH mRNA and peptide in human obese as compared to lean subjects (Zhang et al., 1998). However, one would expect MCH to have a chronic orexigenic action that leads to weight gain to cause obesity, as for NPY. Twice daily administration of MCH only caused an transient increase in food intake for 5 consecutive days, after which time the effect was lost. Daily food intake and weight were not altered too (Rossi and Bloom, 1997). These data are in contrast to those obtained by Della-Zuana et al. (2002) who made a continuous infusion of MCH in Wistar or Sprague-Dawley rats (8 txg/animal/day) and +/+, +/ob or ob/ob mice (4 Ixg/animal/day) and reported both stimulation of feeding and enhanced body weight after 5 days in rat and 4 days in mice. Long-term infusion of the peptide still resulted in a feeding-promoting effect of MCH after 12 days of administration in both Wistar and Sprague-Dawley rats. Also in satiated C57B1/6J mice (both ob/ob and ob/+), feeding was still stimulated after more than a week. Also, MCH overexpression in transgenic mice leads to obesity as well as insulin resistance (Ludwig et al., 2001). So, while the role of MCH on short-term feeding behaviour seemed clearly established, its effect on long-term weight remained controversial. As alluded in Section 3.7, the dipeptide H-NEI-MCH-OH is resistant to degradation by exo- and endo-proteases to which MCH is highly sensitive and has been shown to have a more potent feeding-inducing effect than MCH itself (Maulon-Feraille et al., 2002). A cell-specific precursor processing may well naturally produce the dipeptide and one would expect a longer-lasting effect than MCH on activating food intake (see Section 2.3). Functional interactions of MCH with others peptidic systems involved in feeding control and energy balance homeostasis are now well documented. Indeed, MCH may act as a functional antagonist of MSH in food intake (Ludwig et al., 1998), consistent with the evolutionary conserved blocking action of either of these peptides under several paradigms (reviewed in Tritos and Maratos-Flier, 1999). Manipulation of these peptidergic systems with MCH agonists and ~-MSH antagonists quite reproducibly triggers a feeding intake effect. Both MCH and MSH peptides are part of a new feeding regulatory circuitry with the Agouti protein and its related protein, AgRP, and the newly discovered Mahogany protein (see Schwartz et al., 2000). Other anorectic peptides, such as glucagon-like peptide (GLP)-I or neurotensin, prevent also the appetite stimulating effect of MCH with differential action on 49

Ch. H

G.J. Hervieu et al.

the NPY neuronal system (Tritos et al., 1998b). The hypothesis that MCH and NPY act on feeding behaviour through independent pathways was recently sustained by Della-Zuana et al. (2002), using selective NPY receptor antagonists. However, it has also been found that two structurally different NPY Y-1-receptor antagonists, namely BIBO 3304 and GR231118, could inhibit MCH-induced feeding which may mean that the orexigenic action of MCH involves the Y-1-receptor (Chaffer and Morris, 2002). Galanin and MCH would control food-intake behaviour by using separate, parallel neuronal circuits (Rossi et al., 1999). The orexin-induced feeding effect is independent from that of MCH (Lopez et al., 2002). In summary, the MCH action on food consumption is independent from the galanin and melanocortin receptors (Rossi et al., 1999), possibly from the NPY pathway (Della-Zuana et al., 2002) but is abolished with ct-MSH (Ludwig et a1.,'1998) as well as neurotensin and GLP- 1 (Tritos et al., 1998b). Further evidence of a paramount role for MCH in regulating energy balance come from neuroendocrine studies on the influence of MCH on the hypothalamo-pituitary-thyroid (HPT) axis. The thyroid axis is important in energy homeostasis and starvation leads to profound suppression of the HPT axis. MCH suppresses TRH release from hypothalamic explants as well as decreasing plasmatic levels of TSH (Kennedy et al., 2001). In conclusion, there is now strong evidence supporting a crucial role for MCH in the hypothalamic control of feeding and long-term body weight maintenance. The additional effects on the HPA axis, reproduction and other higher integrative functions (reviewed in Knigge et al., 1996) could represent integrative mechanisms aimed at producing the most appropriate and coordinated response to food stimulation.

5. THE MCH RECEPTORS

For more than 15 years, advances in understanding of MCH biology have been hampered by the lack of information about the MCH receptor(s). This was mostly due to technical difficulties inherent in the cyclical nature of the MCH molecule and the absence of a simple assay to monitor MCH bioactivity. Using a reverse-pharmacology approach (see Stadel et al., 1997), Chambers et al. (1999) identified an MCH receptor. SLC-1, previously an orphan receptor (Kolakowski et al., 1996), was shown to be activated by MCH with high specificity and affinity. Another research group also identified the same MCH receptor by using a brain purification extract approach (Saito et al., 1999). Several other reports describing the identification of SLC-1 as being an MCH receptor followed (Bachner et al., 1999; Lembo et al., 1999; Shimomura et al., 1999; reviewed by Saito et al., 2000). A second human MCH receptor, named, MCH-2, MCH2, MCH-2R or MCH-R2 or SLT was subsequently characterised with little sequence identity to SLC-1 (An et al., 2001; Hill et al., 2001; Moil et al., 2001; Rodriguez et al., 2001; Sailer et al., 2001; Wang et al., 2001). Consequently to the discovery of a second MCH receptor subtype, SLC-1 is also called MCH-R1 or MCH-1 or MCH1 (reviewed by Saito et al., 2000; Boutin et al., 2002). 5.1. BIOASSAYS AVAILABLE FOR MELANOTROPINS Bioassays are based on the ability of MCH to induce pigment aggregation in melanophores of lower vertebrates (amphibians and teleost fish species; mostly Hyla, Anolis, Xenopus, Ctenopharyngodon, Synbranchus). The hormonal response is quantified by determination 50

The melanin-concentrating hormone

Ch. H

of the degree of skin darkening, either by naked eye estimation of colour change, by microscopic observation of melanophores, or by photoelectric measurements of reflection or absorption of light. Sensitivity of these assays are high (subpicomolar and subfemtomolar ranges, respectively, in in vivo and in vitro melanophore assay). The response of isolated melanophores to hormonal aggregation is complete within 20-40 min. However, the routine use of isolated melanophores is cumbersome given the need for large quantities of organelles. Consequently, isolated skin is a more practical tool for repetitive serial tests (see Eberle, 1988). The use of these assays has defined gross, but key, pharmacophores for MCH bioactivity (see Section 5.6). But a functional MCH receptor has not been characterised by cloning from the teleost melanosomes to date. 5.2. MCH-BINDING SITES A small number of studies have used radiolabelled forms of MCH to characterise high affinity binding sites in tissue and cell preparations. Qualitative studies with tritiated MCH provided the first evidence of MCH binding sites in mammalian tissues (Drozdz and Eberle, 1995a) but quantitative studies were greatly facilitated by the development of a biologically active, high specific activity radioiodinated ligand, [125I][Phe13,Tyr19]MCH (Drozdz and Eberle, 1995b), which has been used to characterise specific high affinity binding sites on SVK14 keratinocytes (Burgaud et al., 1997) and mouse melanoma cells (Drozdz et al., 1995). The use of MCH radioiodinated on Tyr 13 ([125I]MCH) was initially avoided as it was shown to lack biological activity (Eberle, 1988). However, studies with a cloned MCH receptor, SLC-1 (see below) have demonstrated the ability of this ligand to radiolabel MCH receptors with high affinity (Chambers et al., 1999). This radioligand was subsequently used to characterise MCH binding sites in membrane preparations from several areas of the human brain, with similar (subnanomolar) affinities for MCH to those reported at the cloned receptor (Sone et al., 2000). No autoradiographic studies of binding site distribution in tissue sections have been reported, most likely due to the technical problems of lipophilicity and sensitivity to oxidation associated with MCH. More recently, oxidatively stable (Hintermann et al., 1999) and photoreactive (Drozdz et al., 1999) analogues of radioiodinated MCH have been developed. Hintermann et al. (2001a) reported further evidence of MCH binding sites on the mouse B 16 melanoma cell line. In addition, Audinot et al. (2001b) reported the synthesis of [125I]$36057, a shortened, more stable and weakly hydrophobic peptide analogue of MCH, which proved to be a more potent and more stable radioligand than [125I][3-iodo-Tyr13]MCH and may represent a reliable tool for binding assays in the search of novel MCH ligand receptors as well as providing great help for autoradiographic studies of the MCH receptors. These new ligands may circumvent some of the technical difficulties of the past, and may provide the means to identify and clone novel subtypes of MCH receptor. The Synaptic compound SNAP-7941, an MCH-R1 antagonist, appears that it will be a valuable tool to that end (Borowsky et al., 2002). 5.3. MOLECULAR CLONING, CHROMOSOMAL LOCALISATION, AND PHYLOGENY 5.3.1. SLC-1 Diverse approaches were used by a number of groups to identify firstly the human (Chambers et al., 1999; Saito et al., 1999), and later the rat (Bachner et al., 1999; Lembo et al., 1999; Shimomura et al., 1999) and mouse (Kokkotou et al., 2001) orphan G-protein-coupled receptor designated SLC-1 as a receptor for MCH. Three forms of the SLC-1 protein have been char51

Ch. H

G.J. Hervieu et al.

acterised from human cDNA libraries: depending on the initiation codon used for translation, three proteins of 353, 417 and 422 amino acids could be potentially produced (Shimomura et al., 1999). Also, two splice variant forms of human SLC-1, differing in length and sequence at the N-terminus, have been cloned from cDNA libraries. The shorter version, a 353 amino acid sequence which arises from the excision of a single exon during processing (Lakaye et al., 1998), has high homology to equivalent expressed sequences in rat and mouse, and has been characterised as a functional MCH receptor. However, the putative protein product of the longer splice variant (Kolakowski et al., 1996), which arises from the failure to splice out the single intron, lacks glycosylation sites in the extracellular N-terminal tail region (Lakaye et al., 1998) and does not appear to be expressed efficiently on cell membranes in the heterologous systems used so far (Chambers et al., 1999). Consequently, there is no evidence yet that this longer form is a functional receptor. Also, rat and mouse equivalents of the longer form have not been successfully cloned from expression libraries, and the predicted protein products deduced from equivalent genomic sequences in rat, mouse and man would be predicted to exhibit low homology in the N-terminal region. For these reasons, it is likely that the longer form, although expressed, may represent an aberrant processing of SLC-1 primary transcripts, which appeared after the evolutionary divergence of primates and rodents (Fig. 7A). The resulting mRNA produced from the slc-1 gene locus is reported with a size of 2.4 kb (Kolakowski et al., 1996) in the rat and human or 2.0 kb (Saito et al., 1999) in the rat by Northern blot. The rat and human orthologue SLC-1 protein sequences are greatly conserved (Fig. 7B; accession number: U71092 for human sequence; U77953 for the rat sequence). In the human, the SLC- 1 gene is located on the chromosome 22ql 3.3 (Kolakowski et al., 1996). Highest sequence identity with other receptors than SLC-1 is observed with the somatostatin receptor (SSTR) gene family (Fig. 7C). SLC-1 encodes 40% protein sequence identity with the SSTRs in the transmembrane domains and an overall 29-32% with the SSTRs. Compared with other SSTRs, the SLC-1 protein has a large amino acid terminus and a short cytoplasmic tail. SLC-1 has also 26-30% protein sequence identity with the opioid receptors (see Darlinson and Richter, 1999). 5.3.2. MCH2 MCH2 was initially identified in a genomic survey sequence as being homologous to SLC-1 receptors (An et al., 2001; Hill et al., 2001; Mori et al., 2001; Sailer et al., 2001; Wang et al., m~

Fig. 7. SLC-1 dendrogram and schematic representation comparing human and rat SLC-1 sequences. (A) Schematic representation comparing the human 'long' and 'short' SLC-1 sequences. In the rat, normal expression of SLC-1 involves processing to excise a single intron, producing a 353-amino acid form of SLC-1. However, in the human, abberant processing results in the production of a longer form of SLC-1 protein (402 aa), in addition to the 353-amino acid receptor. The longer form, although found in cDNA libraries, lacks glycosylation signals in the N-terminal region, and has not been demonstrated to be a functional MCH receptor. Genomic sequences are indicated by thick bars, and protein products by thin bars. Dark arrows indicate areas of homology, and arrows with crosses indicate sequences lacking in homology. Large open arrows depict translation, splicing and transcription. Small triangles indicate consensus sites for asparagine-linked glycosylation. (B) Schematic representation comparing human and rat orthologue SLC-1 sequences. An alignment between the protein sequences of the human (HSllCBY) and the rat (RNllCBY) orthologue SLC-1 sequences. White-boxed residues indicate species amino-acid mismatch. (C) Dendrogram. A dendrogram indicating the sequence identity of SLC-1 with opioid/nociceptin (in grey), somatostatin and other GPCR receptors: SLC-1 as well as GPR7 and GPR8 sequences are classified as somatostatin-receptor-like/opioid-receptor-like sequences.

52

The melanin-concentrating hormone

Ch. H

2001; Rodriguez et al., 2002). Using this sequence, a full-length cDNA was generated from human foetal brain tissue with an open reading frame of 1023 bp, encoding a polypeptide of 340 amino acids, with 38% identity to SLC-1 and with many of the structural features conserved in G-protein-coupled receptors (Hill et al., 2001). Indeed the receptor contains a short N-terminus, seven distinct hydrophobic membrane-spanning domains and the highly conserved DRY motif located at the interface between the third transmembrane helix and the

53

Ch. H

G.J. Hervieu et al.

cytoplasm. The receptor has only one initiator methionine in the open reading frame, which contrasts with three such putative initiator methionines in SLC-1. There are two putative N-linked glycosylation sites and there is no characteristic signal peptide. BLAST analysis of public databases revealed SLC-1 to be its most homologous relative. The gene locus is located to the human chromosome 6q14,3-q15 and Northern-blot data showed a 4-kb transcript predominantly expressed in the brain (Rodriguez et al., 2002). The two GPCRs are 57% identical at the nucleotide level, 59% similar and 38% identical at the amino acid level. A short splice-variant of MCH2 (with a predicted protein sequence lacking the two last transmembrane domain) was identified by RACE-PCR but not Northern blot (An et al., 2001; Rodriguez et al., 2002). 5.4. SIGNALLING Intracellular signalling resulting from the activation of native MCH receptors has been reported in melanophores of the tilapia, Oreochromis niloticus (Oshima and Wannitikul, 1996). In these fish cells, MCH produced a clear reduction of cAMP levels and forskolin inhibited the aggregation response to MCH. In rat synaptically coupled lateral hypothalamic neurones in tissue culture, blockade of the Gi/o protein with pertussis toxin eliminated the actions of MCH (Gao and van den Pol, 2001). 5.4.1. SLC-1 The intracellular signalling events following SLC-1 MCH receptor activation have so far been largely explored in mammalian cell lines heterologously expressing recombinant SLC-1 (Chambers et al., 1999; Lembo et al., 1999; Saito et al., 1999; Shimomura et al., 1999) (Table 1). In these cells, MCH induced a pertussis toxin sensitive, dose-dependent inhibition of forskolin-elevated levels of intracellular cAMP, demonstrating that SLC-1 couples to Gproteins of the Gi/o class. At higher doses, MCH caused a SLC-1 mediated transient elevation of intracellular Ca 2+ concentrations in these cells, suggesting coupling to Gaq proteins. However, this response was inhibited by pertussis toxin (Lembo et al., 1999), suggesting that G~u subunits released from G~i/o proteins may be responsible via a direct activation of PLC~. Further evidence for coupling to G-proteins of the Gi/o class and activation of phospholipase C has been observed after heterologous expression of SLC-1 in Xenopus oocytes, where MCH

T A B L E 1. Reported potencies in functional assays of mammalian MCH at SLC-1 expressed stably in CHO and HEK-293 cells, and transiently in COS-7 cells and Xenopus oocytes Literature reference

Species h o m o l o g u e of SLC-1

Parental host cell

C h a m b e r s et al. (1999)

human

HEK-293

Saito et al. (1999)

human

CHO

L e m b o et al. (1999) S h i m o m u r a et al. (1999) B a c h n e r et al. (1999) M a c d o n a l d et al. (2000)

rat rat rat human

HEK-293 CHO oocyte COS-7

[Ca 2+]int

7.91 18.2 119 . -

.

Inositol phosphate

[cAMP]int

GTP-y-S binding

GIRK current

-

0.28

-

-

-

4.1

-

-

-

3.2 0.2

8.4 -

2.3

-

-

. 18.5

.

Values are given in nM. G I R K , G-protein gated i n w a r d l y rectifying p o t a s s i u m currents ( c o m p i l e d f r o m the literature).

54

The melanin-concentrating hormone

Ch. H

induced activation of both GIRK mediated currents and Ca2+-dependent chloride currents (Bachner et al., 1999).

5.4.2. MCH2 HEK293 cells transfected with MCH2 receptors responded to nanomolar concentrations of MCH with an increase in concentrations of intracellular Ca 2+ (An et al., 2001; Hill et al., 2001). Similar data were obtained in CHO cells transfected with MCH2 receptors (An et al., 2001; Mori et al., 2001; Sailer et al., 2001). Microphysiometry (Hill et al., 2001), binding studies (An et al., 2001; Hill et al., 2001; Sailer et al., 2001), inositol phosphate turnover assay (An et al., 2001; Sailer et al., 2001) and assay for inhibition of forskolin-induced intracellular accumulation of cAMP (Mori et al., 2001) were also used to show the specific response of MCH2 to MCH. Pretreatment with pertussis toxin had no effect on calcium mobilisation (An et al., 2001; Hill et al., 2001; Sailer et al., 2001) and inositol production (An et al., 2001) by MCH in these cells. MCH2 cannot reduce forskolin-stimulated cAMP production (Sailer et al., 2001). Those evidence suggest that the MCH2 receptor is coupled to G-proteins of the Gq/11 subfamily. 5.5. PHARMACOLOGY 5.5.1. SLC-1 SLC-1 was originally identified as a somatostatin receptor-like sequence, although somatostatin does not interact with SLC-1. Indeed, in the original identification of SLC-1 as an MCH receptor, over 500 known and putative mammalian neuropeptides were observed to lack detectable agonist activity (Chambers et al., 1999). SLC-1 is activated by [Phe13,Tyr19]MCH and salmon MCH with approximately three-fold less potency than mammalian MCH. A putative variant form of MCH (Breton et al., 1993a), differing from authentic MCH by four amino acids, and likely not naturally expressed (Miller et al., 1998; Viale et al., 2000), was 100-1000 times less active than MCH. This suggests that variant MCH is not a natural ligand for SLC- 1. Early work on the pharmacology of MCH has been limited to the fish heptadecapeptide homolog of MCH, using a melanophore pigment assay. These studies have demonstrated that reduction of the intramolecular disulphide bond of MCH to produce a linear molecule results in loss of activity (Kawazoe et al., 1987). Also, ring contraction analogues, demonstrated that the size of the cyclic peptide is important (Lebl et al., 1988), and the minimally active sequence of fish MCH, with activity equal to native peptide, is MCH(5-15) (Matsunaga et al., 1989). In addition, chemical modification of Tyr or Arg residues in fish MCH results in a significant loss of activity (Kawazoe et al., 1987). This led to the idea that radio-iodination of MCH on its natural tyrosine residue was resulting in a near complete loss of bioactivity in all possible situation, and that this ligand could not be used as a tracer to unravel the MCH receptor binding signature. It proved to be very wrong as [125I]MCH binds the SLC-1 protein quite as potently as [125I][Phe13,Tyr19]MCH (see Chambers et al., 1999). Interestingly, a number of pharmacological differences have been reported between the cloned MCH receptor and high affinity MCH binding sites on SVK14 keratinocytes (Burgaud et al., 1997) and mouse melanoma cells (Drozdz et al., 1995; Hintermann et al., 2001a). Binding on these sites was weakly displaced by a number of atrial, brain, and C-type natriuretic peptides (Ki values between 116 and 365 nM). However, such peptides had no 55

Ch. H

G.J. Hervieu et al.

detectable affinity for, or functional activity at SLC-1. In addition, the affinity of MCH for sites on keratinocytes and melanoma cells is over 275-fold lower than reported at SLC-1 (Ki values of 65-93 and 12-120 nM vs. 0.043 nM). These pharmacological differences may arise from the expression of SLC-1 in different hosts, or may be due to novel subtypes of MCH receptors in these cells. It will therefore be of great interest to investigate the expression of SLC-l-like sequences in these cells. Of interest, the recent characterisation of [lZSI]MCH binding in the human brain indicates a similar pharmacology to that reported for SLC-1, except that CNP-22 (but not ANP or BNP-32) was observed to partially inhibit binding at high concentrations (Sone et al., 2000). The low affinity of CNP-22 suggests it is unlikely to act as a natural agonist at MCH binding sites, but may prove a useful tool to discriminate between receptor subtypes.

5.5.2. MCH2 Rat atrial natriuretic peptide (ANP)(1-28), rat ANP(3-28), human C-type natriuretic peptide22, human brain natriuretic peptide-32, y-endorphin, ~-MSH, somatostatin-14, somatostatin28, cortistatin-14,; neuropeptide-EI (NEI), neuropeptide-GE (NGE), MCH-gene-overprinted peptide-14 (MGOP-14), and variant neuropeptide-EI (vNEI) were inactive as agonists or antagonists at concentrations up to 10 I~M when tested in an intracellular Ca 2+ assay. 5.6. LIGAND-RECEPTOR STRUCTURE-ACTIVITY RELATIONSHIPS The first mechanistic understanding of the interaction of mammalian MCH with SLC-1 was recently explored in a study by Schering-Plough which combined site-directed mutagenesis of SLC-1 and Ala-scanned mammalian MCH (Macdonald et al., 2000). This study confirmed some earlier observations: the entire MCH cyclic ring Cys7-Cys 16 is necessary for biological activity whereas the N- or C- linear portions are not critical. It also revealed that Arg 11 of MCH and Asp 123 in the third transmembrane domain of the SLC-1 protein are both required for the formation of peptide/receptor complex and that charge inversion does not reverse bioactivity (Asp 11 in MCH with D123R or D123K). Asp 123 is found in bioamine receptors as well as somatostatin and opioid receptors. The work also demonstrated that [Lys11]MCH is a partial agonist on SLC-l-transfected cells (67% of maximum response in calcium response as read out by FLIPR) whereas D-Arg 11 is a weak competitive functional antagonist (potency in the ~M range). All other substitutions at that location 11 in the MCH primary sequence abolished peptide bioactivity. Bednarek et al. (2001) reported that the sequence Arg-cyclo(S-S)(Cys-Met-Leu-Gly-Arg-Val-TyrArg-Pro-Cys) appears to constitute the 'active core' that is necessary for agonist potency at both human MCH receptor paralogues. Audinot et al. (2001a) who did the alanine scan of the dodecapeptide MCH(6-17) (MCH ring between Cys 7 and Cys 16, with a single extra amino acid at the N terminus (Arg 6) and at the C-terminus (Trp17), found it to be the minimal sequence required for a full and potent agonistic response on cAMP formation and [35S]GTP u binding. They showed that only 3 of 8 amino acids of the ring, namely Met 8, Arg 11, and Tyr 13, were essential to elicit full and potent responses in both tests. More recent peptide analog studies have shown that compounds with Ava in positions 9, 10 and/or 14, 15 revealed that the Leu9-Gly 1~ and Argl4-Prol5 segments of the disulphide ring are the principal structural elements determining hMCH-1R selectivity and ability to act as a hMCH-1R antagonist (Bednarek et al., 2002a). In addition, structural changes in positions 6 and 10 results in peptide conformations that allow for efficient interactions with hMCH-1R are 56

The melanin-concentrating hormone

Ch. H

unfavourable for molecular recognition at hMCH-2R (Bednarek et al., 2002b). The dipeptide H-NEI-MCH-OH is an agonist on MCH-R1 (Maulon-Feraille et al., 2002). Non-peptide antagonists at MCH-R1 such as the biphenyl carboxamide GSK compound (Witty et al., 2002), the Takeda compound T-226296 (Takekawa et al., 2002) or the synaptic compound SNAP-7941 (Borowsky et al., 2002) should allow to shed further light on the key interaction between the ligand and the receptor. 5.7. CENTRAL AND PERIPHERAL DISTRIBUTION OF THE MCH RECEPTOR SLC-1 IN THE MAMMALS

5.7.1. Overall distribution of SLC-1 mRNA and protein in the rodents The distribution of the SLC-1 mRNA has been reported by several teams (Kolakowski et al., 1996; Chambers et al., 1999; Lembo et al., 1999; Saito et al., 1999, 2001a; Hervieu et al., 2000; Tan et al., 2002), either by Northern blot and/or mRNA in situ hybridisation (ISH) as well as RT-PCR. In a study using in situ hybridisation with oligoprobes and immunohistochemistry, we reported the distribution of SLC-1 mRNA and its protein product in the rat brain and spinal cord (Hervieu et al., 2000). SLC-1 mRNA and protein were found to be widely and strongly expressed throughout the brain. Immunoreactivity was observed in areas that largely overlapped with regions mapping positive for mRNA. SLC-1 signals were observed in the cerebral cortex, caudate-putamen, hippocampal formation, amygdala, hypothalamus and thalamus, as well as in various nuclei of the mesencephalon and rhombencephalon. The distribution of the receptor mRNA and immunolabelling was in good general agreement with the previously reported distribution of MCH. In a study using riboprobe in situ hybridisation, Saito et al. reported similar results, except that SLC-1 gene expression could not be detected in the olfactory bulb, some diencephalic nuclei and the cerebellum (Saito et al., 2001a). Interestingly Saito et al. (1999) reported SLC-1 gene expression in the eye. Hintermann et al. (2001b) extended that observation and showed MCH-R expression was observed at both the mRNA and protein levels in primary porcine ciliary pigmented epithelial cells and on a human non-pigmented ciliary epithelial cell line. A crosslinking study resulted in a labelled 44-kDa protein, consistent with the molecular weight of the receptor. The retina is phylogenetically related to the hypothalamus and direct monosynaptic contact links both regions. Saito et al. (2001b) reported SLC-1 expression in the human melanoma cell line SK-MEL-37. Northern-blot experiments broadly confirm these data: the SLC-1 gene is strongly detected in the rat frontal cortex, striatum, thalamus and pons (but not in cerebellum) (Kolakowski et al., 1996) and in the whole brain as well as in the eye (Saito et al., 1999). These data are consistent with the known biological effects of MCH in the brain, such as modulation of the energy balance, stress response, sexual behaviour, anxiety, learning, seizure production, memory retention, grooming and sensory gating and with a role for SLC-1 in mediating these physiological actions.

5.7.2. Quantitative RT-PCR (Taqman analysis) of SLC-1 gene expression in rat CNS and PNS Our results show that SLC-1 is widely expressed in the rat nervous system, with mRNA detected in all tissues that were tested (Fig. 8A). However, some variation in expression is observed, with higher expression in amygdala, cerebral cortex (all divisions), hippocampus, 57

Ch. H

G.J. Hervieu et al.

hypothalamus and substantia nigra, and lower levels of expression in striatum, thalamus, cerebellum, rhombencephalon, spinal cord and dorsal root ganglia (DRG).

58

The melanin-concentrating hormone

Ch. H

5.7.3. lmmunochemical studies

In immunochemical experiments (Figs. 8-16), adult male Wistar rats (200-250 g, Charles River, UK) were used, kept in a fixed 12-h light-dark cycle with food and water provided ad libitum. All probe sequence (DNA and peptide) were checked for uniqueness to SLC-1 protein using BLAST. 5.7.3.1. mRNA in situ hybridisation (ISH)

The mRNA localisation experiments were performed with oligonucleotides designed from the rat SLC-1 orthologue sequence (Lakaye et al., 1998). A substantial amount of specific autoradiographic signal was obtained, with discrete anatomical localisation, using the radiolabelled antisense oligoprobes used in the study (Fig. 8B). In control experiments, sense oligoprobes produced no substantial tissue labelling (Fig. 8C). Furthermore, preincubation of radiolabelled antisense oligoprobes with an excess of cold antisense probes (Fig. 8D), and the use of RNase A pretreated sections (Fig. 8E) similarly resulted in a diminution of the signal intensity as compared to that obtained by using antisense oligoprobes (Fig. 8B). Autoradiographic signals were detected throughout the rat nervous system (brain and spinal cord). Thus specific signals corresponding to the SLC-1 mRNA were observed in the cortex (CTX), the olfactory regions (anterior olfactory nucleus and olfactory tubercle; respectively AON and OT), the basal ganglia (caudate-putamen and amygdala; respectively, CP and Amygd.), the hippocampal formation (hi), the diencephalon (thalamus, habenula and hypothalamus; respectively Th, H, Hyp) and various midbrain areas (superior and inferior colliculi, pons, reticular formation, nucleus of the solitary tract and cerebellum; respectively SC, IC, Pn, RF, NTS, Cb). The pattern of distribution was in agreement with the one reported by Taqman experiments. Also that mRNA distribution pattern was quite conserved in other small rodents such as the mouse (Fig. 8F,G) and the guinea pig (Fig. 8H), when compared to the rat.

+....._

Fig. 8. slc-1 gene expression in rodents. (A) SLC-1 mRNA expression in rat CNS and PNS by quantitative RT-PCR analysis. SLC-1 mRNA expression in rat CNS and PNS. SLC-1 expression has been normalised to the expression of the housekeeping gene GAPDH. Bars indicate the mean values derived from three independent RT-PCRs; error bars indicate standard error of the mean. SLC-1 mRNA is detectable in all areas tested with highest levels of expression observed in amygdala, cerebral cortex (all divisions), hippocampus, hypothalamus and substantia nigra. (B-E) mRNA in situ hybridisation in the rat brain for SLC-1. Consecutive rat brain sagittal sections were hybridised with a mix of SLC-1 radiolabelled antisense oligonucleotides (B), a mix of SLC-1 radiolabelled sense oligonucleotides (C), a mix of SLC-1 radiolabelled antisense oligonucleotides and a 100• excess of unlabelled radiolabelled antisense oligonucleotides (D). In E, rat brain sagittal sections were pretreated with the RNase A and were hybridised with a mix of SLC-1 radiolabelled antisense oligonucleotides. The use of sense oligoprobe competed radiolabelled antisense oligoprobes with an excess of cold antisense probes and the use of Rnase A pretreated sections resulted in an absence or diminution (D,E) of the signal intensity as compared to the one obtained by using antisense oligoprobes (B). Specific signals corresponding to the SLC-1 mRNA were observed in the cortex (CTX), the olfactory regions (anterior olfactory nucleus and olfactory tubercle; respectively, AON and OT), the basal ganglia (caudate-putamen and amygdala; respectively, CP and Amyg.), the hippocampal formation (hi), the diencephalon (thalamus and hypothalamus; respectively, Th, Hyp) and various midbrain areas (superior and inferior colliculi, pons, reticular formation, nucleus of the solitary tract and cerebellum; respectively, SC, IC, Pn, RF, NTS, Cbgr) (Hervieu et al., 2000). (F-H) mRNA in situ hybridisation in the mouse and guinea pig. Similar mRNA distribution patterns to the rat were observed in the mouse (F: antisense probe; G: antisense probe with an excess of cold probe) and guinea pig (H: rat antisense probe) brains. Abbreviations: same as in B-E; MRN: mesencephalic reticular nucleus. Scale bars: B-E, 0.35 cm; F-G, 0.3 cm; H, 0.55 cm.

59

Ch. H

G.J. Hervieu et al.

5. 7.3.2. Antiserum immunochemistry A rabbit polyclonal antiserum was raised against the extreme C-terminal hexadecapeptide H-SNAQTADEERTESKGT-OH (amino acids 338-353) derived from the human SLC-1 sequence (Kolakowski et al., 1996) with total sequence identity with the rat orthologue protein sequence (Lakaye et al., 1998). Immunohistochemistry and immunocytochemistry were carried out with an avidin-biotin complex system with either a peroxidase or a fluorescence reporter as previously described (Hervieu and Emson, 1998; Hervieu et al., 2000). Control experiments included the omission of the primary antiserum, the use of rabbit preimmune serum and preabsorbing the antiserum with the immunogenic peptide. Preabsorption controls were done with 10 gM of the immunogenic peptide (incubated overnight with the antiserum prior to the incubation on sections).

Specificity profile of the antiserum: Immunocytochemistry and Western blot using human SLC-1 transfected cells. Specificity of the antisera was investigated on SLC-1- HEK 293 transfected cells versus wild-type cells, with or without preabsorption, with or without primary antisera in a fluorescence procedure. For Western blot analysis, cell extracts were resolved by SDS-PAGE (4-20%), transferred onto nitrocellulose and revealed using an immunoglobulin fraction of the crude anti-SLC-1 antiserum at 1:2000 and a chemiluminescence detection. Specificity of the antibody was confirmed by preabsorption of antibody with the immunogenic peptide (2 gM) and by omitting the primary antibody. Specific immunocytochemical signals (Fig. 9A) largely confined to the plasma membrane, as opposed to the control condition using no primary serum (Fig. 9B) or the antiserum preabsorbed with the synthetic peptide (Fig. 9C), were generated with SLC-l-transfected HEK-293 cells incubated with the antiserum.

Fig. 9. Immunochemical specificity of the anti-SLC-1 antiserum. (A-C) Specificity of the SLC-1 antiserum: immunocytochemistry. HEK 293-SLC-l-transfected cells grown on chamber microscopic slides were incubated with affinity-purified MCH receptor antiserum (A), with no primary serum (B) and with affinity-purified MCH receptor antiserum preadsorbed with the synthetic peptide (C). Specific membrane-associated immunostaining can be in condition A (Hervieu et al., 2000). (D) Specificity of the SLC-1 antiserum: Western-blot. Immunoglobulin fraction of the crude MCH receptor antiserum detected several bands in SLC-1 transfected cells (lane 1) in comparison to untransfected (lane 3) HEK293 cells. Bands were identified at 60 kDa and may represent the glycosylated form of the receptor. High molecular weight forms may represent receptor aggregates. Pre-absorbed antibody failed to detect these bands in transfected (lane 2) or untransfected (lane 4) cells (Hervieu et al., 2000). (E,E') The MCH system in the hypothalamus. Strong MCH gene expression was detected in the lateral hypothalamic area and dopaminergic zone of the zona incerta (LHA and ZIda) by in situ hybridisation with rat MCH antisense oligoprobe (E). Strong gene expression was corroborated by strong immunohistochemical signals obtained with a rabbit polyclonal antiserum raised against the rat MCH. Immunostained cells (arrow) were intermingled within a rich network of labelled varicosities (arrowhead). (Bittencourt et al., 1992). (F-/) Specificity of the SLC-1 antiserum: Immunohistochemistry. The rat sagittal sections were incubated with the affinity-purified followed by immunohistochemistry reported with peroxidase (F,H,/). Immunosignals were observed in the cortex (CTX), the olfactory regions (AON, OB and OT), the basal ganglia (CP, ACB, HDB), the hippocampal formation (hi and SUB), the diencephalon (LHA, VP) and various midbrain and hindbrain areas (SC, IC, LL, NTS, PSV, SPV, Cb, MV). When the section was incubated with the antiserum preabsorbed with the synthetic peptide, no signals were observed (/). There was a good overlapping between the distribution of the SLC-1 protein-like immunoreactivity and the MCH peptide (G; adapted from Bittencourt et al., 1992; a schematic representation of the MCH projections through the rat brain). Calibration bars: A-C, 35 ~m; E, 0.3 cm; E', 70 txm; F, H, I, 0.30 cm. See Section 8 for abbreviations (Hervieu et al., 2000).

60

The melanin-concentrating hormone

Ch. H

61

Ch. H

G.J. Hervieu et al.

Western blot analysis using the crude anti-SLC-1 antiserum revealed several specific bands in SLC-1 0verexpressing cells (Fig. 9D). Two prominent molecular forms were present at 60 and 120 kDa. Immunosignals were absent with wild-type HEK cells (lane 3) or when the antiserum had been preabsorbed with the synthetic peptide (2 ~M) and tested either with SLC-1 transfected (lane 2) or wild-type HEK 293 cell extracts (lane 4). Since the predicted molecular weight of SLC-1 is 38.9 kDa, it was likely that the 60-kDa band could represent a glycosylated form of the receptor. Indeed, amongst putative posttranslational modifications are three N-glycosylation sites in the human N-terminal presumed extracellular tail (namely NAS; NTS, NLT; respective position of the arginine residue: 13, 16 and 23). By treatment with N-glycosidase F, the apparent MW of the immunosignal was shifted from 60 kDa to 40 kDa (not shown). Also apparent mobility of the migrating protein could be affected by phosphorylation as several potential phosphorylation sites are present in the C-terminal presumed intracellular tail as follows: two protein kinase C phosphorylation sites: 317-319 TFR; 325-327 SVK and one casein kinase II phosphorylation site (342-345 TADE). It is likely that the 120-kDa band may represent receptor aggregates. Immunohistochemistry using rat brain tissue sections. A distinctive immunostaining pattern was obtained after incubation of brain sections in the affinity-purified anti-SLC-1 antiserum (Fig. 9F,H). SLC-1 immunosignals were observed in the cerebral cortex, caudate-putamen, hippocampal formation, amygdala, hypothalamus and thalamus, as well as in various nuclei of the mesencephalon and rhombencephalon. In control experiments using the anti-SLC-1 antiserum preabsorbed with the immunogenic peptide, a great reduction in immunosignal intensity could be observed (Fig. 9I). At the same anatomical level, the SLC-1 immunostained regions in the rat brain (Fig. 9F) were largely identical with MCH immunostained regions in the rat brain (Fig. 9I). Cellular morphological features of the SLC-1 immunostained rat brain cells SLC-l-like immunoreactivity was found in cells with the morphology of projection neurones (e.g. Fig. 12F) and interneurones (e.g. Fig. 12D). Glial cell immunostaining was observed, but labelling could not be fully competed with the preadsorbed antiserum. Furthermore, signals were mainly confined to plasma membranes of cells, as would be expected for a G-protein-coupled receptor. Confocal fluorescence analysis of tissue sections revealed mainly discrete punctate staining of plasma membranes (Fig. 12D) which was in contrast to the more continuous pattern of staining of entire cell membranes seen in SLC-1 transfected cells (Fig. 9A). This may well be explained by the different densities of receptors in these cells, with a much higher abundance of receptors in the transfected cells. The antiserum revealed some nerve fibre staining. Axons with collaterals and dendrites from the many interneurones were labelled in the cerebral cortex (e.g. Fig. 12A).

Fig. 10. The distribution of the SLC-1 mRNA and protein in the rat nervous system. Several plans of sections illustrating the rat distribution of the SLC-1 mRNA (A, C, E, G, L K) and protein (B, D, F, H, J, L) from the forebrain to the spinal cord. A good overlap between the mRNA and protein signals was observed. Key hypothalamic regions involved in feeding control such as the DMH and VMH are shown in G and H. Calibration bars: A, B, 0.2 cm; C, D, 0.33 cm; E, F, 0.2 cm; G, 0.3 cm; H, 280 Ixm; L J, 0.2 cm; K, L, 0.07 cm. See Section 8 for abbreviations (Hervieu et al., 2000).

62

The melanin-concentrating hormone

Ch. H

63

Ch. H

G.J. Hervieu et al.

5. 7.3.3. A comparison between the mRNA and protein distribution pattern of SLC-1 products in the rat nervous system With the 'caveat' that neuronally expressed proteins are expected to be identified far away from the cell body they originate, receptor immunoreactivity was observed in areas that largely overlapped with regions mapping positive for mRNA. This is illustrated in Fig. 10 with a set of rat nervous system sections representative of the forebrain, brainstem and spinal cord, presented in parasagittal, sagittal and coronal views.

5.7.3.4. Neuroanatomical localisation studies: rat brain and spinal cord localisation of the MCH receptor SLC-1 Please also refer to Table 2 and Figs. 8B, 9F, H, 10-13, 15 and 16.

Cell groups Forebrain. A strong labelling was noticed at many subregional levels in the isocortex amongst them the primary and secondary motor area (MOp, MOs), primary and secondary somatosensory areas (SSp, SSs), gustatory area (GU), the infralimbic and prelimbic areas (ILA, PL), the orbital area (ORB), anterior cingulate area in both dorsal and ventral parts (ACAd, v), visceral area (VISC), agranular insular area in both dorsal and ventral parts (Ald and Alv), retrosplenial cortex (RSP) in both dorsal, ventral and lateral agranular parts (RSPd, v, agl), posterior-parietal region association area (PTLp), primary, ventral and dorsal auditory areas (AUDp, v, d), ventral temporal association area (TEv), the ectorhinal area (ECT), a number of visual areas (primary, anterolateral, rostrolateral and anteromedial visual areas (respectively, VISp, a, rl and am) and the claustrum (CLA). The olfactory cortex was one of the richest in terms of both SLC-1 mRNA and protein labelling. Heavy labelling of the olfactory region was seen in the main olfactory bulb (MOB), the anterior olfactory nucleus (AON) and olfactory tubercle (OT) where immunostaining of the layer 3 (polymorph layer) was more pronounced than in the molecular and pyramidal layers. Dense signals were observed in the taenia tecta (TT) both in the ventral part (TTv) and the dorsal part (TTd), mainly in layer 3, and in the major islands of Calleja (islm). Strong signals were observed in the piriform cortex (PIR) in the layer II (pyramidal) and the dorsal endopiriform nucleus (EPd) while much weaker immunosignals were seen in the post piriform transition area (TR).

Fig. 11. A rostrocaudal distribution of the SLC-1 protein in the rat brain. The SLC-1 mRNA was localised in some key-forebrain regions such as the isocortex, olfactory system/hippocampal formation, nucleus accumbens, caudateputamen and other basal ganglia (amygdala), medial septum, and nucleus of the diagonal band. A particularly dense staining was obtained in various hypothalamic (SO, AHN, PVH, MEPO, MPO, MPN, VMH, LHA, PM, PV, PH, LM, MM) and thalamic (RT, AV, AD, IAD, MH, ZI, CL, PVT, OP, STN, PF, VPL) nuclei. Note also the metathalamic signals obtained in the geniculate nuclei (lateral and medial) and in their functionally associated colliculus nuclei (SC, IC). Note also the signals obtained in the oculomotor nerve (III), the caudal part of the substantia nigra (SNpr), the specific staining of the caudal part of the interpeduncular nucleus (IPN) and the staining in other various midbrain nuclei (PAG, VTA, APN, RN, NB, DR, VTN, TRN, NTB, POR, PB-KF, DTN, VCO). Lastly, note the signals obtained in various nuclei of the reticular formation (PRN, MARN, GRN), in the cochlear nuclei (DCO, VCO), in the vestibular nuclei (MV), in the olivary nuclei (SOC1 and IO), in the trigeminal system (PSV, SPV), in the facial nerve (VII), in the locus coeruleus (LC) and the grey matter of the spinal cord. Calibration bars: A-U, 0.2 cm; V-W, 0.15 cm; X-Z', 0.1 cm. See Section 8 for abbreviations (Hervieu et al., 2000). 64

The melanin-concentrating hormone

Ch. II

65

Ch. H

66

G.J. Hervieu et al.

The melanin-concentrating hormone

Ch. H

67

Ch. H

68

G.J. Hervieu et al.

The melanin-concentrating hormone

Ch. H

Immunostaining was more concentrated in layers III (deep supragranular pyramidal) and V (infragranular pyramidal) with stained cells resembling multipolar interneurones and principal neurones (see Fig. 12). In the hippocampal formation, immunolabelled cells were observed in the dorsal parts (SUBd) and ventral parts (SUBv) of the subiculum (SUB). The post-subiculum (POST) and presubiculum (PRE) were also immunostained unlike the parasubiculum (PAR). The entorhinal area (ENT) was moderately immunostained in its lateral (ENT1) and medial (ENTm) parts. In the hippocampus (hi), immunosignals were mainly located in the stratum pyramidale (sp) in Ammon's horn and the granule cell layer of the dentate gyrus (DGsg). Also the crest of the dentate gyrus was immunostained both in the molecular (DGcr-mo) and granule cell layer (DGcr-gr) levels as seen at a more ventrocaudal level. Lastly, a pronounced immunostaining was present within the indusium griseum (IG). In the amygdala, immunostaining for the MCH receptor SLC-1 was observed in the nucleus of the olfactory tract (NLOT), basolateral, basomedial, intercalated nuclei (respectively BLA, BMA, IA) and anterior area (AAA). In the septal regions, the septum was particularly enriched in SLC-1 signals with labelling in the medial septum nucleus (MS), the nucleus of the diagonal band of Broca (NDB) and the lateral septum (LS). On a sagittal section, immunostaining could be seen in the horizontal limb of the diagonal band (HDB) and the vertical limb of the diagonal band (VDB). At the level of the corpus striatum, a very dense immunolabelling was seen in the striatum (caudate-putamen, fundus of the striatum and nucleus accumbens; CP, FS and NA) as well as in the pallidum where particularly strong signals were expressed within the substantia innominata (SI) including the magnocellular preoptic nucleus (MA) and the globus pallidus (GP) in contiguity with the labelling found in the SI. In the thalamus, at the epithalamic level, signals were more prominent in the medial habenula (MH) than in the lateral habenula (LH). The thalamus was reasonably enriched with SLC-1 signals. Progressing rostrally to caudally, in the dorsal thalamus, on a ventral-caudal axis, SLC-1 receptor signals were detected in the paraventricular nucleus thalamus (PVT) at low levels while being much more abundant in the anterodorsal (AD), anteroventral (AV) and lateroposterior (LP) thalamus nuclei. Immunosignals were also dense in the ventral thalamic complex including the ventral anterior-lateral complex thalamus (VAL), ventral posterior medial nucleus thalamus (VPM) and ventral posterolateral nucleus thalamus (VPL). At that level, immunosignals were also detected in the posterior complex thalamus (PO), and the parafascicular nucleus thalamus (PF). In the ventral part of the thalamus, the reticular nucleus thalamus (RT), the subthalamic nucleus (STN) and its overlying zona incerta (ZI) were also immunopositive for SLC-1 protein. More rostrally, the subdopaminergic cell group of the zona incerta (ZIda) also expressed the SLC-1 gene. In the metathalamus, the lateral geniculate complex (LG) including its dorsal, ventral, ventrolateral and ventromedial parts (LGd, v, vl, vm) as well as the intergeniculate leaflet (IGL) contained SLC-1 protein. Also, immunosignals were observed in the ventral part of the medial geniculate nuclei (MGv). Dense immunosignals were observed in the hypothalamus. In the periventricular zone, the median preoptic nucleus (MEPO) was the most rostral hypothalamic region to be labelled. Staining was also observed in the suprachiasmatic (SCN), supraoptic (SO), arcuate (AN), and paraventricular (PVH) as well as periventricular (PV) nuclei. In the medial hypothalamus, strong signals were obtained at the level of the anterior nucleus (AHN) in the anterior (AHNa) and central (AHNc) parts. In the tuberal area, staining was seen in the ventromedial (VMH) and the dorsomedial nuclei (DMH) while in the lateral zone, staining was noticed in the lateral area (LHA). Lastly, the posterior nucleus (PH) and 69

Ch. H

G.J. Hervieu et al.

TABLE 2. Distribution of slc-1 mRNA and immunoreactivity in the rat brain Region

mRNA

Immunoreactivity

Olfactory bulbs

+++

+++

+ +++ +++ +++ +++

+ +++ +++ +++ +++

+ ++ + ++

+ ++ + ++

++

++

+++ ++

++++ +++

+++ +++ +++ +++ +++

+++ +++ +++ +++ +++

+++ ++

+++ ++

++ ++ ++ ++ ++ +++

+++ ++ ++ + +++ +++

+++ + + + ++ + + +++ ++ + +++ +++ ++ +++ +++ +++

+++ ++ + + ++ + + ++ ++ + +++ +++ + +++ +++ +++

Telencephalon Olfactory system Dorsal endopiriform nucleus Islands of Calleja Olfactory nuclei Piriform cortex Tenia tecta Neocortex Agranular insular cortex Frontal cortex Granular insular cortex Parietal cortex Metacortex Cingulate/retrosplenial cortex Basal Ganglia Caudate putamen Globus pallidus Hippocampal formation CA1 region CA2 region CA3 region Dentate gyrus Subiculum Amygdala Amygdaloid nuclei Substantia innominata Septal and basal magnocellular nuclei Accumbens nucleus Bed nucleus of the stria terminalis Lateral septal nucleus, dorsal part Lateral septal nucleus, ventral part Medial septal nucleus Nucleus of the horizontal limb of the diagonal band

Diencephalon Thalamus Anterodorsal thalamic nucleus Anteroventral thalamic nucleus Centrolateral thalamic nucleus Centromedial thalamic nucleus Geniculate nuclei Interanterodorsal thalamic nucleus Intermediodorsal thalamic nucleus Lateral habenular nucleus Medial habenular nucleus Parafascicular thalamic nucleus Paratenial thalamic nucleus Paraventricular thalamic nucleus Reticular thalamic nucleus Reuniens thalamic nucleus Submedius thalamic nucleus Ventral posterolateral thalamic nucleus Ventral posteromedial thalamic nucleus

70

The melanin-concentrating hormone

Ch. H

TABLE 2 (continued) Region Thalamus (continued) Ventrolateral thalamic nucleus Ventromedial thalamic nucleus Zona incerta Hypothalamus Anterior hypothalamic area Arcuate hypothalamic nucleus Dorsomedial hypothalamic nucleus Lateral hypothalamic area (LHA) Lateral mammillary nucleus Medial mammillary nucleus Medial preoptic area Medial preoptic nucleus Paraventricular hypothalamic nucleus Periventricular hypothalamic nucleus Posterior hypothalamic area Supraoptic nucleus Ventromedial hypothalamic nucleus

mRNA

Immunoreactivity

++ § §247247

++ §247 ++§

§247 +§ §247247 §247 §247247 +§247 §247 §247 §247247 §247 § §247247 §247247

§247 + §247247 §247 §247247 §247247 § + §247247 ++§ + §247247 §247247

§247 §247247 §247247 +§ +++ +§ + ++ ++ +§247 ++ ++

§247 §247247 §247247 +++ +++ +++ § +§ ++ §247 ++ §

++ n.d. ++ §247 + §247247 §247 n.d. n.d.

++ ++§ ++ ++§ + §247247 §247 §247247 §247247

+++ ++

+++ ++

Mesencephalon Anterior pretectal nucleus Dorsal tegmental nucleus Inferior colliculus Interpeduncular nuclei Oculomotor nucleus Periaqueductal grey Principal sensory trigeminal nucleus Raphe nuclei Red nucleus Substantia nigra Superior colliculus Ventral tegmental area

Rhombencephalon Cochlear nucleus complex Facial nucleus Parabrachial nuclei Locus coeruleus Nucleus of the solitary tract Olivary complex Pontine reticular nucleus Spinal trigeminal nucleus Vestibular nucleus

Cerebellum Cerebellar cortex Deep cerebellar nuclei

The relative density of labelling is classified as: absent ( - ) , sparse (+), moderate ( + + ) , extensive ( + + + ) , not determined (n.d.) (partially adapted from Hervieu et al., 2000).

mammillary body including the dorsal pre- (PM), medial (MM), lateral (LM), lateral supra(SUM1) and ventral tuberomammillary nucleus (TMv) displayed clear SLC-1 immunoreactive signals. 71

Ch. H

G.J. Hervieu et al.

Brainstem. In the sensory systems, many immunosignals were present in the visual areas

such as the superior colliculus (SC) (with a discernible enrichment of signals in the zonal, optic, intermediate (SCig-b/c) and deep (SCdg) grey layers). In the pretectal regions, immunostaining was present in the anterior pretectal nucleus (APN), the olivary pretectal nucleus (OP) and the nucleus of the optic tract (NOT). In the somatosensory areas, the principal sensory nucleus of the trigeminal (PSV) was stained as well as the spinal nucleus of the trigeminal (SPV), the cuneate (CU) and the external cuneate (ECU) nuclei. The oral (rostrodorsomedial and ventrolateral; SPVO rdm and vl), interpolar (SPVI) and caudal (SPVC) segments of the spinal nucleus were immunolabelled. In the auditory areas, both the dorsal (DCO) and ventral (VCO) cochlear nuclei were immunolabelled. Strong immunostaining was found in the nucleus of the trapezoid body (NTB) and the leminisci (LL) such as the nucleus of the lateral lemniscus (NLL). Also SLC-1 immunosignals were encountered in the superior olivary complex (SOC) including the complex SOC itself and the periolivary region (POR). The inferior colliculus (IC) in all its subdivisions (external, dorsal, central; e, d, c) was particularly rich in SLC-1 signals as was also the nucleus brachium (NB). The vestibular areas displayed high levels of SLC-l-like immunoreactivity. All vestibular nuclei including the medial (MV), superior (SUV), lateral (LV) and spinal vestibular nuclei (SPIV) were immunostained. Also, the nucleus prepositus (PRP) from the perihypoglossal nuclei was labelled. The gustatory areas exhibited immunolabelling in the medial zone of the nucleus of the solitary tract (NTSm). In the visceral areas, the nucleus of the solitary tract (NTS) contained protein in its intermediate (NTSi) and ventrolateral (NTSvl) parts. Similarly the lateral division of the parabrachial nucleus (PB1) contained immunostaining as well as its contiguous Kolliker-Fuse nucleus (KF). In the motor systems, the nucleus of oculomotor nerve III was immunostained as well as the facial nucleus (cranial nerve VII), the nucleus ambiguus (AMB), the superior salivatory nucleus (SSN) and the Edinger-Westphal nucleus (EW). A robust immunostaining was seen in the extrapyramidal system, particularly in the substantia nigra (SN) with signals in the pars reticulata and to a lesser extent in the pars compacta (SNpc). The ventral tegmental area (VTA) was immunostained. In the pre- and post-cerebellar nuclei, the red nucleus (RN) contained obvious immunolabelling as well as the pontine central grey (PG) and the tegmental reticular nucleus (TRN). More caudally, the inferior olivary complex (IO) was immunopositive in all its subdivisions. In the cerebellum, there was mRNA labelling in the granular cell layer of the cerebellar cortex (Cbgr) as well as immunolabelling. In addition, the interpositus cerebellar nucleus was immunostained in both its anterior and posterior subdivisions (IntA; IntP). In the reticular core, staining was strong in the central grey of the brain: in particular, SLC1 protein was detected in the many divisions of the peri-aqueductal grey (PAG); i.e. dorsal, ventrolateral and dorsolateral; d, vl and dl. The ventral (VTN) and dorsal (DTN) tegmental nuclei were also immunostained. The locus coeruleus (LC) was quite strongly positive for

Fig. 12. SLC-1 immunoreactivity in the neocortical areas of the rat brain. Numerous SLC-1 immunostained

cells were, respectively, detected throughout the isocortex such as in the ventral (A) and dorsal (B,F) parts of the retrosplenial cortex (respectively, RSPv and RSPd), the primary motor cortical area (MOp; C and D; D by confocal microscopy), the secondary motor cortical area, (MOs; B), the primary somatosensory cortex (SSs; E), the posterior-parietal region association area (PTLp; G) and the primary auditory cortex (AUDp; H). In all cases, cells were labelled on the membrane. Calibration bars (in Ixm):A, C, H, 70; B, 140; D, 20; E, F, G, 35 (Hervieu et al., 2OOO). 72

The melanin-concentrating hormone

Ch. H

73

Ch. H

G.J. Hervieu et al.

SLC-1 protein staining. The LC staining was encompassed within the immunostained pontine central grey (PCG) In the raphe, immunostaining was observed in the dorsal nucleus raphe (DR), the central linear nucleus raphe (CL1), the nucleus raphe magnus (RM) and the medial and lateral part of the superior central nucleus raphe (CSm and CS1). More caudally, immunostaining was present in the nucleus raphe pallidus (RPA). There was a pronounced labelling of the interpeduncular nucleus (IPN), particularly in the central subnucleus (IPNc). In the reticular formation, staining was quite widespread. It was indeed observed in the mesencephalic reticular nucleus (MRN) including its retrotuberal area (RR), in the pedunculopontine nucleus (PPN), in the pontine reticular nucleus (PRN), in the gigantocellular reticular nucleus (GRN), the parvicellular reticular nucleus (PARN) the magnocellular reticular nucleus (MARN) and also found in the medullary reticular nucleus (MDRN). Spinal cord (lumbar segment). There was strong mRNA labelling and immunolabelling in the lumbar part of the spinal cord. All subdivisions of the grey matter (dorsal and ventral horns) were labelled (ventromedial, dorsomedial, intermediolateral, central, ventrolateral, dorsolateral and retrodorsolateral). The immunolabelling was particularly pronounced in the dorsal horn. Fibre tracts Most fibres were devoid of mRNA or immunolabelling such as the cranial nerve (olfactory limb of the anterior commissure, aco; spinal tract of the trigeminal nerve, sptV; the facial nerve, VIIn; the trapezoid body of the cochlear nerve, tb), the spinal nerves such as the medial lemniscus (ml) of the dorsal column, the cerebellum such as the middle cerebellar peduncle (mcp), the lateral forebrain bundle system such as the corpus callosum (cc) including the anterior forceps (fa), genu (ccg) and external capsule (ec), as well as the corticospinal tract (cst) including the pyramidal tract (py), the extrapyramidal fibre system such as the rubrospinal tract (rust), the medial forebrain bundle system (MFBS) such as the temporal limb of the anterior commissure (act), dorsal hippocampal commissure (dhc). However, the MFBS-habenula related fasciculus retroflexus (fr) was immunostained and the internal capsula (ic) was densely labelled. There was also dense immunoreactivity in some of the white matter tracts of the spinal cord such as the laterocorticospinal tract (lct), the laterospinocortical tract (lsc), the lateral spinothalamic tract (lst) and the anterior corticothalamic tract (act).

5.7.4. Peripheral and central distribution studies of SLC-1 regional gene expression sites in the human (Fig. 17) Northern blot experiments reported expression of the slc-1 gene in the frontal cortex, hypothalamus, basal forebrain, midbrain, amygdala, hippocampus, subthalamus, substantia

Fig. 13. SLC-1 immunoreactivityin the hippocampal formation. Hippocampal SLC-1 labelling was observed in the pyramidal layers of the Ammon's horns (CA1-3 spd) as well as in the dentate gyrus (DG). Immunostaining was seen in the CA1 near the fasciola cinerea (B) and in a more lateral position (C). A stronger immunostaining was seen in the CA2 (D, F) and CA3 (E). The immunolabelling was present on the membrane of the pyramidal cells (F). The dentate gyrus was stained in the granular (DGsg) and polymorph (DGpo) cell layers (G). Calibration bars (in Ixm):A, 600; B-E, G, 70; F, 35 (Hervieu et al., 2000).

74

The melanin-concentrating hormone

Ch. H

75

Ch. H

G.J. Hervieu et al.

nigra, thalamus, corpus callosum, liver and heart while no signals were observed in the caudate-putamen, pancreas, kidney, muscle, lung and placenta (Kolakowski et al., 1996). The slc-1 gene is also expressed in adrenal glands as well as a variety of adrenal tumours, ganglioneuroblastomas and neuroblastoma (Takahashi et al., 2001). By Taqman, the SLC-1 receptor gene level expression was dramatically higher in the brain and the pituitary than in other tissues. The receptor was expressed at quite low levels in peripheral tissues regulating energy balance (stomach, intestine, adipose tissue, pancreas, skeletal muscle) and immune/haematopoietic system (spleen, lymphocytes, bone marrow, but not in macrophages) (Fig. 17A). In the human brain, the gene expression distribution pattern was similar to that found in the rat. Expression was high in the cerebral cortex (cingulate, medial frontal and superior frontal gyri), quite high in the hippocampus and hypothalamus, moderate to low in the basal ganglia (amygdala >> caudate nucleus, putamen, striatum, substantia nigra, globus pallidus) and low in the thalamus, spinal cord, cerebellum and medulla oblongata. (Fig. 17B). mRNA in situ experiments confirmed slc-1 gene expression in the neocortex (Fig. 17C), the hippocampus (Fig. 17D) and the cerebellum (Fig. 17F). Immunoreactive cells were also detected in the dentate gyms (Fig. 17E).

5.7.5. Autoradiographic ligand studies The synaptic compound SNAP-7941, an MCH-R1 non-peptidic antagonist, was used tritiated and applied to rat brain sections (Borowsky et al., 2002). Specific labelling was detected in the cerebral cortex, claustrum, several limbic structures (hippocampus, septum and nucleus of the diagonal band, bed nucleus of the stria terminalis, amygdala). Dense signals were recorded in dopaminergic regions such as the neostriatum and the nucleus accumbens. In the hypothalamus, discrete binding sites were revealed in the ventromedial and medial mammillary nuclei. The serotoninergic dorsal raphe and noradrenergic locus coerulus were also radiolabelled. This parallels the mRNA (Lembo et al., 1999; Saito et al., 1999, 2001a; Hervieu et al., 2000) and protein (Hervieu et al., 2000) distribution of the MCH-R1 gene product. 5.8. CENTRAL AND PERIPHERAL DISTRIBUTION OF THE MCH RECEPTOR MCH2 IN THE MAMMALS Several reports have provided data regarding the MCH-R2 gene expression sites in the human (An et al., 2001; Hill et al., 2001; Moil et al., 2001; Sailer et al., 2001) and the rhesus monkey (Sailer et al., 2001). Strikingly, several non-human species (rat, mouse, hamster, guinea, pig, and rabbit) do not have functional MCH-R2 receptors, or encode a non-functional MCH-R2 pseudogene while retaining MCH-R1 expression (Tan et al., 2002). MCH2 gene expression is

Fig. 14. SLC-1 immunoreactivity in the basal ganglia. A strong immunostaining was found in the basal ganglia as

illustrated in A with a sagittal section: dense immunosignals were seen in the substantia nigra, subthalamic nucleus (STN), lateral segment of the globus pallidus (GP1). Immunosignals were also observed in the caudate-putamen, shell of the nucleus accumbens (NacS) and ventral pallidum (VPal). Note also the labelling within the internal capsula (ic). Higher magnifications are presented for the medial and lateral segments of the globus pallidus (B), caudate-putamen (B,C), subthalamic nucleus (D) and substantia nigra pars reticulata (E). In the caudate-putamen, immunosignals are located on the cell membrane of aspiny neurones. Calibration bars (in Ixm): A, 800; B, 140; C, 35; D, E, 280 (Hervieu et al., 2000). 76

The melanin-concentrating hormone

Ch. H

77

Ch. H

G.J. H e r v i e u et al.

much higher in the brain than in the periphery. The tissue distributions of MCH1 and MCH2 receptors using quantitative TaqMan RT-PCR (Hill et al., 2001; Moil et al., 2001) and Northern blot (An et al., 2001) were compared. The mRNA profiles of the two receptors were relatively similar, showing predominant expression in the brain. The distribution of the two receptors in the individual regions of the brain is similar but there are subtle differences. For example, the contribution of the hypothalamus, locus coeruleus, medulla oblongata and cerebellum to the MCH1 profile is greater than the contribution of these regions to the MCH2 profile (Hill et al., 2001). One of the more prominent differences was the increase in pituitary contribution to the MCH1 profile compared with the MCH2 profile (Hill et al., 2001). In the rhesus monkey, by in situ hybridisation, MCH2 gene expression was detected in the cerebral cortex, hippocampus, and hypothalamus, with lower levels in the caudate nucleus, putamen and thalamus. In the hypothalamus, MCH2 gene is strongly expressed in the anterior and lateral areas (unlike MCH1) but not in the dorsomedial area (unlike MCH1) (Sailer et al., 2001). 5.9. NEUROFUNCTIONAL ANALYSIS Functional implications based on SLC-1 localisation within the rat nervous system has been proposed in several reports (Hervieu et al., 2000; Kilduff and de Lecea, 2001; Saito et al., 2001a). The widespread and generally dense cortical distribution of the MCH receptor is consistent with the suggestion that the peptide is involved in generalised cortical arousal and sensorimotor integration (Bittencourt et al., 1992; Nahon, 1994). The substantia innominata and parabrachial nuclei receive dense MCH innervation and are both SLC-1 immunopositive. A particularity of these cell groups is that their cortical projections conform to the description of non-specific cortical afferents. Neurones in the lateral hypothalamus and zona incerta, where the MCH gene is strongly expressed, and where the mRNA and protein SLC-1 are strongly expressed, are also associated with diffused arousal functions and sensorimotor integration (see Bittencourt et al., 1992). Together with the presence of SLC-1 labelling in many parts of the limbic system and the medial septum, these findings may explain the role of MCH in sensory conditioning, since the peptide diminishes the ability of rats to appropriately filter sensory clues as found in a CNS auditory gating paradigm (Miller et al., 1993). The prediction is that MCH thus lessens the chances of a behavioural change in response to a new sound. Impairment in sensory processing (such as loss of activation in auditory association areas in response to external speech) is often encountered in people with schizophrenia who have a pronounced tendency to misinterpret significant sounds in a noisy environment. The subiculum, Ammon's horn (strata oriens and pyramidale) and dentate gyrus regions were strongly immunoreactive to the SLC-1 antisera in both the rat and human. Together with the immunostaining observed in other forebrain regions such as the amygdaloid regions and the

.......+

Fig. 15. SLC-1 immunoreactivity in the forebrain/diencephalon. A dense population of MCH receptor SLC-1

immunostained cells was detected in the supraoptic nucleus (SON; A), the suprachiasmatic nucleus (SCN; B), the posterior region of the anterior nucleus (AHNp; C). At the level of the paraventricular nucleus (PVH), there was a more pronounced staining of the lateral zone of the posterior magnocellular region (PVHpml) as opposed to a lighter immunostaining observed in the dorsal parvicellular, medio-parvicellular and the dorsal zone of the medial parvicellular region (resp. PVHdp, PVHmpv and PVHmpd; D). Immunostained cells were detected as well in the arcuate nucleus (AN; E), the posterior region of the dorsomedial nucleus (DMHp; F) and the lateral hypothalamic area (LHA; G). In the posterior hypothalamus, MCH receptor immunostained cells were seen in the tuberomammillary nucleus (TMv; H). Calibration bars: A-H, 140 txm (Hervieu et al., 2000). 78

The melanin-concentrating hormone

Ch. H

79

Ch. H

G.J. H e r v i e u et al.

bed nucleus of the stria terminalis, these results may be functionally related to the action of MCH on passive avoidance, a behavioural paradigm associated with cognition and learning. In that work, MCH hastened the extinction of the passive avoidance response (commonly used to measure cognitive alterations). The ~-MSH in that paradigm showed the opposite action (McBride et al., 1994). MCH was also shown to affect memory retention when infused into the hippocampus and amygdala (Monzon et al., 1999). In keeping with this, our results appeared to indicate a particular representation of SLC-1 labelling in anatomical regions implicated in the control of vigilance. The SLC-1 protein is present in the locus coerulus, identified as a major nucleus to set levels of arousal and behavioural vigilance (Foote et al., 1983). MCH hypothalamic neurones project to the locus coerulus (Bittencourt et al., 1992). Moreover, immunostaining was also observed in many other regions that control the arousal state. It includes the metathalamic geniculate bodies, the thalamic paraventricular and reticular nuclei, the hypothalamic suprachiasmatic and tuberomammillary nuclei, the preoptic area, the mesencephalic reticular formation, and the pontine, raphe and full medullar nuclei. In all of these regions, SLC-1 gene was expressed as a protein with MCH-like associated immunoreactivity signatures (Bittencourt et al., 1992). Also the MCH receptor is present the diagonal band of Broca and medial septum in the forebrain, the laterotegmental nucleus and the pedunculopontine nucleus in the reticular formation in cholinergic regions. These nuclei are known to be involved in electroencephalogram (EEG) desynchronisation (as one of the indicators for wake/sleep cycles). Interestingly, a strong immunostaining was observed in the fasciculus retroflexus, a cholinergic fibre bundle originating in the habenula and projecting to the interpeduncular nucleus and various paramedian midbrain nuclei. MCH neurones have been shown to be responsive to acetylcholine stimulation, and carbachol has been shown to induce a rapid increase in hypothalamic MCH mRNA expression (Bayer et al., 1999a,b). MCH neurones are likely to be cholinoreceptive (see Section 3.4.2). There might be a more specific aspect of MCH control on sleep mechanisms as SLC-1 immunostaining was observed in the oculomotor nerve (III) and the red nucleus (RN). The third cranial nerve, passing through the red nucleus, controls the extrinsic ocular muscles as well as the pupillary sphincter and the ciliary muscles. Also immunostaining was found in the anterior pretectal area. This midbrain area receives afferents from the retina and the visual association cortex and controls pupillary reflexes. There may be grounds here for a role in sensorimotor integration played by SLC-1 at the level of the sensory systems. The dense labelling seen in the piriform cortex could be related to the anti-seizure activity of the MCH reported by Knigge and Wagner (1997). Indeed, i.c.v, injections of MCH given 15 min before pentylenetetrazole (PTZ) intraperitoneal injection prevents seizure activity triggered by PTZ in rat and guinea pig (Knigge and Wagner, 1997). SLC-1 labelling was also represented in the extrapyramidal motor system and throughout many areas of the mesencephalic, pontine and medullary reticular formations associated with locomotor activity. Much of this labelling may also be

._____.+

Fig. 16. SLC-1 immunoreactivity in the brainstem and lumbar spinal cord. Immunostained MCH receptor staining

was detected in the periaqueductal grey matter (PAG; A), the pontine grey (PG; B), the granular layer of the cerebellum (Gr; C) and in the locus coeruleus (LC; D). In the spinal cord, SLC-l-like immunoreactivity was present within the grey matter of the spinal cord with a more pronounced staining of the dorsal horn as opposed to the ventral one (E,F). In both horns, immunostained cells were detected (G,H). The spinal tracts such as the laterocorticospinal tract (lct), the laterospinocortical tract (lsc), the lateral spinothalamic tract (lst) and the anterior corticothalamic tract (act) were moderately labelled (E). Calibration bars (in Ixm): A, D, 70; B, C, 140; E, F, 280: G, H, 35 (Hervieu et al., 2000). 80

The melanin-concentrating hormone

Ch. H

81

Ch. H

G.J. Hervieu et al.

concerned with a possible involvement of MCH in central pattern generator circuitry as well as brainstem-controlled motor behaviour. It should be noted, however, that targeted deletion of the MCH gene does not appear to affect locomotor behaviour in the mutant mice (Shimada et al., 1998) and MCH itself has no action on locomotor activity but antagonises MSH-induced hyperlocomotor behaviour (Sanchez et al., 1997). This sets a possible scene for MCH neurones being involved in the circuitry and activity of extrapyramidal motor pathways and it points out a possible role of MCH for the prevention of generalised seizure and basal ganglia disorders. Strong SLC-1 signals were reported in the basal ganglia for the messenger (Lembo et al., 1999; Saito et al., 1999; Hervieu et al., 2000; Saito et al., 2001a), the protein (Hervieu et al., 2000) and protein binding sites (Borowsky et al., 2002). Dense immunostaining was found in the anterior hypothalamic area and the zona incerta both involved in drinking behaviour. This could be linked with the observed effects of dehydration and salt-loading on MCH gene expression activity (see Section 3.2 and Section 5.3) and the effect of intestinal MCH on regulating the hydro-mineral balance in the gastrointestinal tract (Hervieu and Nahon, 1995). SLC-1 immunoreactivity also appears to be related to processing systems for visual and auditory stimuli. The visual pathway conveys regulatory information through the lateral geniculate nucleus, a thalamic relay receiving afferents from the retina, the parabrachial region, the hypothalamic tuberomammillary region and the superior colliculus, and sending projections to the visual cortex and the reticular thalamic nucleus. Information is then relayed back to the dorsal thalamus. All of these regions exhibit substantial SLC-1 immunoreactivity. Interestingly, Saito et al. (1999) have also reported slc-1 gene expression in the eye. SLC1 receptors are also probably involved in auditory processing since immunostaining was observed in the auditory cortex, medial geniculate nucleus, inferior colliculus, the lateral lemniscus, the ventral and dorsal cochlear nuclei and the olivary complex. SLC-1 is clearly involved in many diverse motor and sensory systems. As discussed earlier, staining observed in specific mid- and hindbrain nuclei (oculomotor nucleus, red nucleus and the anterior pretectal area) may further indicate that MCH, acting through SLC-1 receptors, plays a role in general sensorimotor integration in these systems. Attention has recently been very much focused on the involvement of MCH in feeding and energy balance. The presence of SLC-1 in the arcuate, ventromedial, dorsomedial and paraventricular nuclei of the hypothalamus, as well as many gustatory regions, indicates that the receptor could mediate the reported orexigenic effects of MCH (Qu et al., 1996; Rossi and Bloom, 1997; Ludwig et al., 1998) summarised earlier in Section 5.4. Interestingly,

Fig. 17. The SLC-1 gene expression pattern in the human. (A) By Taqman, the SLC-1 receptor gene level

expression was dramatically higher in the brain and the pituitary than in other tissues. The receptor was expressed at quite low levels in peripheral tissues regulating energy balance (stomach, intestine, adipose, pancreas, skeletal muscle) and immune/haematopoietic system (spleen, lymphocytes, bone marrow but not in macrophages). (B) In the human brain, the gene expression distribution pattern was similar to that found in the rat. Expression was high in the cerebral cortex (cingulate, medial frontal and superior frontal gyri), quite high in the hippocampus and hypothalamus, middle to low in the basal ganglia (amygdala >> caudate nucleus, putamen, striatum, substantia nigra, globus pallidus) and low in the thalamus, spinal cord, cerebellum and medulla oblongata. (C-F) mRNA in situ experiments confirmed human SLC-1 gene expression in the cortex (C), the hippocampus (D) and the cerebellum (F). Immunoreactive cells were also detected in the dentate gyms (E). Scale bars: C, F, 0.6 cm; D, 0.5 cm; E, 35 Ixm. Abbreviations: (C) Caud., caudate; Put, putamen; MOrG, medial orbital gyms; POrG, posterior orbital gyms; GR, gyms rectus; (D) ARG, Andreas Retzius gyms; sms, superficial medullary stratum; ICG, isthmus of the cingulate gyms; PHG, parahippocampal gyms; DG, dentate gyms. 82

The melanin-concentrating hormone

Ch. H

83

Ch. H

G.J. Hervieu et al.

SLC-1 immunoreactivity was present within the medial hypothalamus, a region where there is prepro-MCH mRNA and peptide novel expression in lactating rats (Knollema et al., 1992). Transgenic mice lacking the MCH gene have a lean and hypophagic phenotype and this is described as the first example that 'deletion of a gene encoding a single orexigenic peptide can result in leanness' (Shimada et al., 1998). Also targeted disruption of the melaninconcentrating hormone receptor-1 results in resistance to diet-induced obesity (Chen et al., 2002), hyperphagia (Chen et al., 2002; Marsh et al., 2002), leanness, hyperactivity, and hyperphagic and altered metabolism (Marsh et al., 2002). Physiological structure-activity studies with a variety of MCH peptide analogues indicated a strong correlation between their effects upon food intake and their potency obtained at the rat SLC-1 receptor. This would indicate the relevance of the SLC-1 receptor in feeding behaviour (Haynes et al., 2001; Suply et al., 2001). Finally, the anorectic property of SNAP-7941, a specific MCH-R1 antagonist (Borowsky et al., 2002), are all proof that MCH, at least through its signalling to MCH-R1, is essential to energy balance homeostasis. A parallel line of evidence reinforces the important role of MCH in the feeding response. MCH is a regulator of glucocorticoid secretion (see Section 5.1). It is well established that the nutritional status of mammals and activity of the HPA axis are inter-related. In pathological situations, the overactivity of the HPA axis (elevated circulating ACTH and glucocorticoid blood levels) is a hallmark of comorbidity with obesity (see Peeke and Chrousos, 1995). Glucocorticoid excess induces abdominal obesity, insulin resistance, diabetes and hypertension. All of this experimental evidence may suggest a pathophysiological role for centrally acting MCH being involved in the development of obesity. The SLC-1 immunostaining observed in the hypothalamic paraventricular nucleus could be associated with the neurodocrine effects of MCH on the stress response. MCH has indeed been found either to stimulate (Jezova et al., 1992; Ashmeade et al., 2000) or inhibit (Ludwig et al., 1998; Bluet-Pajot et al., 1995) the HPA axis through an action on pituitary ACTH and/or hypothalamic CRH neurones. Both i.c.v, and i.v. injection of MCH evoked changes in HPA activity and suggest an action at both hypothalamic and peripheral levels, i.c.v. administration of MCH in conscious rats potently activated the CRH-like immunoreactive neuronal population of the parvicellular paraventricular hypothalamic nucleus (Parkes et al., 1992) and both rat and human pituitary glands expressed quite strongly the SLC-1 receptor. Intriguingly, the pairing of MCH and ~-MSH again appears as an evolutionary-acquired functional antagonism feature: MCH antagonises the effects of the melanocortin on grooming and locomotor activities in the rat (Sanchez et al., 1997; see Baker, 1994; Tritos and MaratosFlier, 1999). Excessive grooming behaviour is induced by melanocortins and is observed during mild stress situations and following exposure to novel stimuli, translating very often as a whole into anxious behaviours. MCH is reported to be anxiogenic when injected into the hypothalamic preoptic area (Gonzalez et al., 1996) or anxiolytic following i.c.v, administration (Monzon et al., 1999, 2001). There might be an overall outcome of the implication of MCH through SLC-1 in the stress neuroendocrine pathway. The stress axis is a key component of body homeostasis, itself dependent on fuel/nutrients available to appropriately maintain vital vegetative functions as others. Dysregulation of the stress axis may lead humans and other animal models to become statistically highly sensitive to affective disorders, of which abnormal feeding behaviour (anorexia and bulimia) is a frequent comorbidity component. Human clinical data have long been accumulated about the effect of glucocorticoid treatment on inducing obesity and depression in patients as well as immune disorders, amongst many other dysregulations. Fuel-deficient animals are immunodepressed, as depressed animals including major depressive humans are. That MCH 84

The melanin-concentrating hormone

Ch. H

by itself already has wide physiological actions in the fish as a stress, immune and pigmentary modulator, may mean that dysregulation in one of the system could potentially translate in clinically relevant human disorders, and worsen because of cascading to other interelated systems. Depressive states are often associated with anxiety behaviours. As a cardinal link, CRH appears to be a key messenger in mood disorders and anxiety. CRH mediates very wide and profound stress-induced changes in the autonomic nervous system, neuroendocrine function and behaviour (see Koob, 1999). There is substantial evidence for the hyperactivity of the HPA axis in the aetiology of affective illnesses as shown by up to two-thirds of drug-free depressed patients (depression, post-traumatic stress disorder, anxiety and anorexia nervosa) having hypercortisolaemia, enlarged adrenal and pituitary glands, elevated cerebrospinal fluid levels of CRH, blunted neuroendocrine response to synthetic GC (dexamethasone) challenge, cognitive impairments which may be consistent with a toxic activity of the chronically high levels of brain cortisol and the down-regulation of its receptors in the hippocampal formation (see Koob, 1999). This endocrinopathy is largely related to the hypersecretion of CRH as also suggested by the down-regulation of receptor level. Also, key neuromodulators implicated in affective disorders are regulated by MCH: MCH affects amine release thereby reducing serotonergic activity and inhibiting dopamine release (Gonzalez et al., 1997b). Thus dysregulations in the MCH system could potentially impact on affective behaviours. The very recent report by Synaptic Inc. showing the antidepressant and anxiolytic actions of SNAP-7941, an MCH-R1 antagonist (Borowsky et al., 2002), gives a strong credential to that hypothesis. The presence of SLC-1 protein in other major neuroendocrine regions such as the hypothalamic supraoptic, arcuate and the medial preoptic nuclei is consistent with MCH regulating oxytocin (Parkes and Vale, 1992a) and luteinising hormone release (Gonzalez et al., 1997a). This may possibly translate into sexual behaviour regulation (see Sections 4.2 and 5.2). Both the nucleus accumbens and ventral tegmental area were SLC-l-immunoreactive. The former nucleus is a major recipient of the mesolimbic dopaminergic projection from the ventral tegmental area and plays a key role in reward mechanisms. This brain region may mediate the positive reinforcing effects of food and thus provide an additional control by MCH on feeding behaviour. Borowsky et al. (2002) have shown that the SNAP-7941 compound does not inhibit food intake because of a taste-aversion effect. It should be borne that leptin is part of the reward system by regulating the incentive value of food (Fulton, 2000; see also Filglewicz and Wood, 2000). Melanotropins are known to be implicated in drug-seeking behaviours (see Eberle, 1988; Adan and Gipsen, 1997) and the primary location of action seems to be in the peri-aqueductal grey matter, which receives an important but separate hypothalamic innervation of both MCH and MSH. Bittencourt noted that the presence of MCH fibres with varicosities indicate that the PAG is a site of MCH fibre ending and presumably peptide release and not just a location of fibres of passage. Also it is well-known that the CRH pathway is potently implicated in the physiology of addiction and withdrawal behaviour (e.g. CRH increases the predisposition to self-administer drugs as observed in a stressful context). It is conceivable that MCH may be a component within the numerous neuromediators regulating reward mechanisms.

6. CONCLUSION The phylogenetic distribution of MCH has provided an interesting problem for biologists who wish to reconcile the melanophore modulation of this peptide in lower vertebrates 85

Ch. II

G.J. Hervieu et al.

with heretofore unknown functions in mammalian brain. The pairing of MCH to its SLC-1 receptor intervened some 15 years after the isolation of the MCH peptide. While data had been gathered on the biology of MCH for that period (with a landmark study demonstrating that central MCH induces feeding intake), no information was available on the receptor. As alluded in the introduction, peptides are difficult molecular entities to work with. However, it is strongly suggested and suggested again 20 years after that peptides may only show significant up to dramatic biological relevance in a pathophysiological context (see Hockfelt, 1991; Hockfelt et al., 2000). This should provide impetus to unravel the functions of many other peptides. In particular, one eagerly awaits to know about the functions of the NGE, NEI, MGOP and potential human variant MCH. The main effect of MCH in teleost concerns pigmentary control. That is also a functional boundary between fish and mammals. MCH is not involved in pigmentary control in mammals. But is that as clear-cut? A human melanoma cell line SL-MEL-37 harboured MCH receptors (MCH-R1) (Saito et al., 2001b). Pigmentory cell of the eye express the SLC-1 receptor (Hintermann et al., 2001b). In fact, there is recent evidence for a role in pigmentogenesis as a very recent study has reported that the first discovered paralogue of both MCH receptors so far characterised, MCH-R1, is an auto-antigen associated with vitiligo, a common depigmenting disorder resulting from the loss of melanocytes in the skin. The study also reported that anti-MCH-R1 IgG were naturally inhibiting MCH binding to its receptor MCH-R1 (Kemp et al., 2002). This may be coincidental however as may peptidergic systems are present in melanocytes for no direct functions per se on pigmentory control. Lastly, does MCH act as a feeding factor in fish? A recent transgenic fish medaka strain overexpressing the MCH gene was established and its phenotypic features were examined (Kinoshita et al., 2001). Development, growth, feeding behaviour, and reproduction of transgenics did not differ significantly among transgenic and non-transgenic siblings. The result whereby enhanced MCH expression induced a change in body colour, but no remarkable abnormalities. The review has presented the characterisation of SLC-1 and MCH2 as being two MCH receptors and should open wide avenues for probing additional functions of the peptide, both in the brain and in the periphery.

7. ABBREVIATIONS

Anatomical (adapted from Paxinos and Watson, 1998 and Swanson, 1998) AAA

ac(o, t) ACA(v,d) ACB ACT AD AH AHN(a, p) Al(d) (v) AM AMB Amygd. 86

anterior amygdaloid nucleus anterior commissure (olfactory, temporal) limb anterior cingulate area nucleus accumbens anterior corticothalamic tract anterodorsal nucleus thalamus anterior hypothalamus anterior hypothalamic nucleus (anterior, posterior part) agranular insular area (dorsal)(ventral) part anteromedial nucleus thalamus nucleus ambiguus amygdala

The melanin-concentrating hormone

AN AOL AON APN AQ ARG AUDp AUDv AV BLA BMA BST C(M) (L) CA(l) (2) (3) CA(so) (sp) Caud. Put. CB(gr) cc(g) CEA1 CG CL CLA CLi CM COA cpd CS(m) cst

CTX DCO DG(s g, cr) dhc DLL DMH(p) DR DRG DTN ec

ECT ECU EP EPN(d) EW fa fi fr Fr FRP(am)

Ch. H

arcuate nucleus anterior olfactory nucleus, lateral part anterior olfactory nucleus anterior pretectal nucleus cerebral aqueduct Andreas Retzius gyrus primary auditory area ventral auditory area anteroventral nucleus thalamus basolateral nucleus amygdala basomedial basolateral nucleus amygdala bed nuclei of the stria terminalis (mediocentral) (laterocentral) nucleus thalamus Ammon's horn field (1) (2) (3) field CA stratum (oriens) (pyramidal) caudate putamen cerebellum (granular cell layer of the cerebellar cortex) corpus callosum (genu) central nucleus amygdala central grey central lateral nucleus claustrum caudal linear nucleus of raphe central medial nucleus thalamus cortical nucleus amygdala cerebral peduncle superior central nucleus raphe, medial part corticospinal tract neocortex dorsal cochlear nucleus dentate gyrus (granule cell, corona radiata) layer dorsal hippocampal commissure dorsal nucleus lateral lemniscus dorsomedial hypothalamus (posterior part) dorsal nucleus raphe dorsal root ganglion dorsal tegmental nuclei extermal commissure ectorhinal area external cuneate nucleus endopiriform nucleus entopeduncular nucleus (dorsal part) Edinger-Westphal nucleus corpus callosum, anterior forceps fimbria fasciculus retroflexus frontal cortex frontoparietal/frontal pole cortex (motor area) 87

Ch. II

FS GP(1) GRN GU Hab HDB HF hi HYP IA IA(D) (M) IC(c, d, e) ICG IG III ILL int Int(A) (P) IO IP, IPN islm KF LA Lat LAV LC lct LD LG(d, v, m, 1) LH LHA LL LM LPO LS(d) (v) lsc 1st LV MA Sam MARN mcp MD MDRNv ME(ex) MEA 88

G.J. Hervieu et al.

fundus of the striatum globus pallidus (lateral segment) gigantocellular reticular nucleus gustatory area habenula nucleus horizontal limb diagonal band hippocampal formation hippocampus hypothalamus intercalated nuclei amygdala interantero(dorsal) (medial) nucleus thalamus inferior colliculus (central, dorsal, external) nucleus isthmus of the cingulate gyrus indusium griseum principal oculomotor nucleus intermediate nucleus of the lateral lemniscus internal capsule interpositus cerebellar nucleus (Int) (anterior) (posterior) subdivisions inferior olivary complex interpeduncular nucleus central subnucleus major islands of Calleja Kolliker-Fuse subnucleus lateral nucleus amygdala lateral cerebellar nucleus lateral vestibular nucleus locus coeruleus laterocorticospinal tract laterodorsal thalamus lateral geniculate complex (dorsal, ventral, medial, lateral) part lateral habenula lateral hypothalamic area lateral leminisci lateral mammillary nucleus lateral preoptic area lateral septum (dorsal) (ventral) segments laterospinocortical tract lateral spinothalamic tract lateral vestibular nucleus magnocellular preoptic nucleus mammillary nuclei magnocellular reticular nucleus middle cerebellar peduncle mediodorsal nucleus thalamus medullary reticular nucleus, ventral part median eminence (external lamina) median nucleus amygdala

The melanin-concentrating hormone

MEPO MG(d, v) MH MidThal ml MM NO(p) (s) MOB(opl) moV MPN MPO MRN MS MV(m) (v) NA NB NDB NLL NLOT NTS(i) (m) (rm) (vl) OCP OP OT (3) PA PAG(d, vl, dl, m) PAR PARN PB(1) (mm) PCG PCN PF PG PGRN(1) PH PHG PIR(2) PMd PO POLF POR PorG POST PP PPN PRE

Ch. H

median preoptic nucleus medial geniculate complex (dorsal, ventral element) medial habenula median part of the thalamus medial lemniscus medial mammillary nucleus (primary) (secondary) motor area main olfactory bulb (outer plexiform layer) motor root of the trigeminal nerve medial preoptic nucleus (MPN) medial preoptic area mesencephalic reticular nucleus septal nucleus (MS) medial vestibular nucleus (magnocellular)(parvicellular) parts nucleus accumbens nucleus brachium inferior colliculus nucleus of the diagonal band nucleus of lateral lemniscus nucleus of the lateral olfactory tract nucleus of the solitary tract (intermediate) (medial) (rostral zone of the rostral part) (ventrolateral) parts occipital lobe (neocortex) olivary pretectal nucleus olfactory tubercle (polymorph layer) posterior nucleus amygdala periaqueductal grey matter (dorsal, ventrolateral, dorsolateral, medial) parasubiculum parvicellular reticular nucleus parabrachial nucleus, (lateral) (mediomedial) part pontine central grey paracentral nucleus thalamus parafascicular nucleus thalamus pontine grey paragigantocellular reticular nucleus (lateral part) posterior hypothalamus nucleus parahippocampal gyrus piriform cortex (pyramidal layer) dorsal premammillary nucleus posterior complex thalamus primary olfactory cortex periolivary nuclei posterior orbital gyrus postsubiculum posterior pituitary (neurohypophysis) pedunculopontine nucleus presubiculum 89

Ch. H

PreCBL Pretect PRN(c) (r) PrS PSV PT PTLp PV(i) (p) PVH(dp, mpv, pml, pmd, pv)

PVT PY RE RH RN RPA RR RSP(d) (v) RT rust

SC (zo) (op) (sg) (ig) (dg) SEP

SEZ/RC SG SI sms

SN p(c) (r) so

SO SOC(1) Sp Cd sp, SP SPIV sptV SPV(o, i, 1, c) SPVO(rdm, vl) SS(p) (s) STN SUB(d) (v) SUMI SUV tb 90

G.J. Hervieu et al.

precerebellar nuclei pretectal areas pontine reticular nucleus (caudal) (rostral) part presubiculum principle sensory trigeminal nerve parathenial thalamus nucleus posterior-parietal region association area periventricular nucleus hypothalamus (intermediate) (posterior) part paraventricular nucleus hypothalamus (dorsal parvicellular) (medio-parvicellular) (posterior magnocellular- lateral zone) (medial parvicellular-dorsal zone) (periventricular zone) periventricular nucleus thalamus pyramidal tract nucleus reuniens rhomboid nucleus red nucleus nucleus raphe pallidus mesencephalic reticular nucleus, retrotuberal area retrosplenial cortex, (dorsal) (ventral) part reticular nucleus thalamus rubrospinal tract superior colliculus (zonal) (optic) (intermediate grey) (superficial grey) (deep grey) layer septum subependymal zone/rhinocele supragenual nucleus substantia innominata superficial medullary stratum substantia nigra pars (compacta) (reticulata) stratum oriens supraoptic nucleus superior olivary complex (lateral part) spinal cord pyramidal layer spinal vestibular nucleus spinal tract of the trigeminal nerve nucleus spinal tract trigeminal nerve (oral) (interpositus) (lateral) (caudal) part nucleus spinal tract trigeminal nerve, oral part (rostrodorsomedial, ventrolateral) (primary) (secondary) somatosensory area subthalamic nucleus subiculum (dorsal) (Bd) (ventral) parts supramammillary nucleus (lateral) superior vestibular nucleus trapezoid body

The melanin-concentrating hormone

TEv TMv TR TRN TT(d)(v3) V (pc) (mo) v3 VAL VCO VDB Vest VII VIIn VIS(al) (am) (li) (11) (p) (pl) (pro)

VISC VL VLL VM VMH VP VP(L) (M) VTA VTN ZI(da)

Ch. H

ventral temporal association area tuberomammillary nucleus post piriform transition area tegmental reticular nucleus taenia tecta (dorsal)(ventral-layer 3) trigeminal nerve (parvicellular part of the motor nucleus) (motor root)/motor nucleus of the trigeminal nerve third ventricle ventral anterior-lateral complex thalamus ventral cochlear nucleus vertical limb of the diagonal band vestibular nuclei facial nerve facial nucleus visual area (anterolateral) (anteromedial) (intermediolateral) (laterolateral) primary) (posterolateral) (posteriomedial) visceral area ventrolateral nucleus thalamus ventral nucleus lateral lemniscus ventral medial nucleus thalamus ventromedial area hypothalamus ventroposterior thalamic complex ventral postero (lateral) (medial) nucleus thalamus ventral tegmental area ventral tegmental nucleus zona incerta (dopaminergic cell group of the)

Miscellaneous

BSA cDNA DAB EDTA GPCR HEK i.c.v. -ir i.v. KLH -li mRNA NGS PAGE PBS PMSF RPMA RT-PCR

bovine serum albumin complementary deoxyribonucleic acid 3,3'-diaminobenzidine ethylene diamine tetraacetate G-protein-coupled receptor human embryonic kidney intracerebroventicular -immunoreactive intravenous keyhole limpet haemocyanin -like messenger ribonucleic acid normal goat serum polyacrylamide gel electrophoresis phosphate-buffered saline phenylmethylsulfonylfluoride reverse pharmacology approach reverse transcription followed by polymerase chain reaction 91

Ch. H

SDS

(T)TBS v/v w/v

G.J. Hervieu et al.

sodium dodecyl sulphate (Tween 20) Tris-buffered saline volume per volume weight per volume

8. A C K N O W L E D G E M E N T S

This study was supported by SmithKline Beecham Pharmaceuticals, Research and Development (G.J.H., J.K.C., J.E.C., S.W.) and the Centre National de la Recherche Scientifique (CNRS), the Institut de Recherche Servier, Nestle, and Danone Institute (J.-L.N., EE, L.M.-E). We wish to thank SmithKline Beecham Bioinformatics (Simon Topp), SmithKline Beecham Biopharmaceuticals (Paul Murdoch), SmithKline Beecham Neuroscience Research (David Harrison, Peter Maycox) for their contributions.

9. REFERENCES Abrahamson EE, Moore RY (2001): The posterior hypothalamic area: chemoarchitecture and afferent connections. Brain Res 889:1-22. Abrahamson EE, Leak RK, Moore RY (2001): The suprachiasmatic nucleus projects to posterior hypothalamic arousal systems. NeuroReport 12 (2):435-440. Adan RAH, Gipsen WH (1997): Brain melanocortin receptors: from cloning to function. Peptides 18 (8):12791287. An S, Cutler G, Zhao JJ, Huang SG, Tian H, Li W, Liang L, Rich M, Bakleh A, Juan Du J, Chen JL, Dai K (2001): Identification and characterization of a melanin-concentrating hormone receptor. Proc Natl Acad Sci USA 98:7576-7581. Anand BK, Brobeck JR (1951): Localisation of a 'feeding center' in the hypothalamus of the rat. Proc Soc Exp Biol Med 77:323-324. Ashmeade TE, Jones DNC, Munton RP, Shilliam C, Gartlon JE, Parker F, Hervieu G, Heidbreder CA (2000): An investigation into the effect of MCH on neuroendocrine markers in the rat. Eur J Neurosci 12 (Suppl 11):217.2, p. 476. Audinot V, Lahaye C, Suply T, Rovere-Johene C, Rodriguez M, Nicolas JP, Beauverger P, Cardinaud B, Galizzi JP, Fauchere JL, Nahon JL, Boutin JA (2001a): Structure-activity relationship studies of melanin concentrating hormone (MCH)-related peptide ligands at SLC-1, the human MCH receptor. J Biol Chem 276 (17):1355413562. Audinot V, Lahaye C, Suply T, Beauverger P, Rodriguez M, Galizzi JP, Fauchere JL, Boutin JA (2001b): [I-125]$36057: a new and highly potent radioligand for the melanin-concentrating hormone receptor. Br J Pharmacol 133 (3):371-378. Bachner D, Kreienkamp H, Weise C, Buck F, Richter D (1999): Identification of melanin concentrating hormone (MCH): as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1):. FEBS Lett 457 (3):522-524. Bahjaoui-Bouhaddi M, Fellmann D, Griffond B, Bugnon C (1994): Insulin treatment stimulates the rat melaninconcentrating hormone-producing neurons. Neuropeptides 24:251-258. Baker BI (1991): Melanin-concentrating hormone: a general vertebrate neuropeptide. Int Rev Cytol 126:1-47. Baker BI (1994): Melanin-concentrating hormone updated: functional considerations. Trends Endocrinol Metab 5 (3):120-126. Baker BI, Bird DJ (2002): Neuronal organization of the melanin-concentrating hormone system in primitive actinopterygians: evolutionary changes leading to teleosts. J Comp Neurol 442 (2):99-114. Baker BI, Bird DJ, Buckingham JC (1985): Salmonid melanin-concentrating hormone inhibits corticotropin release. J Endocrinol 106:R5-R8. Bayer L, Poncet F, Fellmann D, Griffond B (1999a): MCH expression in slice cultures of rat hypothalamus is not affected by 2-deoxyglucose. Neurosci Lett 267:77-80. Bayer L, Risold PY, Griffond B, Fellmann D (1999b): Rat diencephalic neurons producing melanin-concentrating hormone are influenced by ascending cholinergic projections. Neuroscience 91 (3): 1087-1101.

92

The melanin-concentrating hormone

Ch. H

Bayer L, Jacquemard C, Fellmann D, Griffond B (1999c): Survival of rat MCH neurons in hypothalamus slice culture: histological, pharmacological and molecular studies. Cell Tissue Res 297:23-33. Bednarek MA, Feighner SD, Hreniuk DL, Palyha OC, Morin NR, Sadowski SJ, MacNeil DJ, Howard AD, Van der Ploeg LHY (2001): Short segment of human melanin-concentrating hormone that is sufficient for full activation of human melanin-concentrating hormone receptors 1 and 2. Biochemistry 40 (31):9379-9386. Bednarek MA, Hreniuk DL, Tan C, Palyha OC, MacNeil DJ, Van der Ploeg LHY, Howard AD, Feighner SD (2002a): Synthesis and biological evaluation in vitro of selective, high affinity peptide antagonists of human melanin-concentrating hormone action at human melanin-concentrating hormone receptor 1. Biochemistry 41 (20):6383-6390. Bednarek MA, Tan C, Hreniuk DL, Palyha OC, MacNeill DJ, Van der Ploeg LHY, Howard AD, Feighner SD (2002b): Synthesis and biological evaluation in vitro of a selective, high potency peptide agonist of human melanin-concentrating hormone action at human melanin-concentrating hormone receptor 1. J Biol Chem 277 (16):13821-13826. Bernardis LL, Bellinger LL (1996): The lateral hypothalamic area revisited: ingesting behavior. Neurosci Behav Rev 20 (2): 189-287. Bittencourt JC, Elias CF (1998): MCH and NEI projections from the LHA and ZI to the medial septal nucleus and spinal cord: a study using multiple neuronal tracers. Brain Res 805:1-19. Bittencourt JC, Sawchenko PE (2000): Do centrally administered neuropeptides access cognate receptors?: an analysis in the central CRF system. J Neurosci 20 (3):1142-1156. Bittencourt JC, Presse F, Arias C, Peto C, Vaughan J, Nahon JL, Vale W, Sawchenko PE (1992): The melaninconcentrating hormone system of the rat brain: an immuno- and hybridization histochemical characterisation. J Comp Neurol 319:218-245. Bittencourt JC, Frigo R, Rissman RA, Casatti CA, Nahon JL, Bauer JA (1998): The distribution of MCH in the monkey brain. Brain Res 804:140-143. Bittner M, Meltzer P, Chen Y, Jiang MM, Seftor E, Hendrix M, Radmacher M, Trent J (2000): Molecular classification of cutaneous malignant melanoma by gene expression profiling. Nature 406:536-540. Blalock JE (1989): The bidirectional communication between the neural and immune systems. Physiol Rev 69 (1):1-32. Bluet-Pajot MT, Presse F, Voko Z, Hoeger C, Mounier J, Epelbaum J, Nahon JL (1995): Neuropeptide E-I antagonises the action of melanin-concentrating hormone on stress-induced release of adrenocorticotropin in the rat. J Neuroendocrinol 7:297-303. Borowsky B, Durkin M, Ogozaler K, Marzabadi MR, DeLeon J, Heurich R, Lightblau H, Shaposhnik Z, Daniewska I, Blackburn TP, Blanchek TA, Gerald C, Vaysse PJ, Forray C (2002): Antidepressant, anxiolytic and anorectic properties of MCH-R1 antagonist. Nat Med 8 (8):825-830. Borsu L, Presse F, Nahon JL (2000): The AROM gene, spliced mRNAs encoding new DNA/RNA-binding proteins are transcribed from the opposite strand of the MCH gene in mammals. J Biol Chem 275 (51):40576-40587. Boutin JA, Suply T, Audinot V, Rodriguez M, Beauverger P, Nicolas JP, Galizzi JP, Fauchfre JL (2002): Melaninconcentrating hormone and its receptors: state of the art. Can J Physiol Pharmacol 80 (5):388-395. Bray GA, Tartaglia LA (2000): Medicinal strategies in the treatment of obesity. Nature 404:672-677. Bradley RL, Kokkotou EG, Maratos-Flier E, Cheatham B (2000): MCH regulates leptin synthesis and secretion in rat adipocytes. Diabetes 49:1073-1077. Bresson JL, Clavequin MC, Fellman D, Bugnon C (1987): Donn6es ontog6n6tiques sur la population d'interneurones peptidergiques ~ immunor6activit6 de type GRF37 de l'hypothalamus postero-lat6ral humain. Etudes immunocytochimiques ~ l'aide d'un IS anti-GRF37 et d'un IS anti-MCH. CR Soc Biol 181:376-382. Bresson JL, Clavequin MC, Fellman D, Bugnon C (1989): Human hypothalamic neuronal system revealed with a salmon MCH antiserum. Neurosci Lett 102:39-43. Breton C, Fellman D, Bugnon C (1989): Clonage et sequencage d'ADNc du precurseur commun de trois neuropeptides hypothalamiques immunologiquement apparentes a la somatocrinine humaine 1-37, a l'alpha melanotropine e t a 1' hormone de melanoconcentration du saumon. CR Acad Sci Paris 309:749-754. Breton C, Schorpp M, Nahon JL (1993a): Isolation and characterization of the human melanin-concentrating hormone gene and a variant gene. Mol Brain Res 18:297-310. Breton C, Presse F, Hervieu G, Nahon JL (1993b): Structure and regulation of the mouse melanin-concentrating hormone mRNA and gene. Mol Cell Neurosci 4:271-284. Brischoux F, Fellmann D, Risold PY (2001): Ontogenetic development of the diencephalic MCH neurons: a hypothalamic 'MCH area' hypothesis. Eur J Neurosci 13:1733-1744. Broberger C (1999): Hypothalamic CART' neurons: histochemical relationship to TRH MCH, orexin/hypocretrin and NPY. Brain Res 848:101-113.

93

Ch. H

G.J. Hervieu et al.

Broberger C, de Lecea L, Sutcliffe JG, Hokfelt T (1998): Hypocretin/orexin- and melanin-concentrating hormoneexpressing cells form distinct populations in the rodent lateral hypothalamus: relationship to the neuropeptide Y and agouti gene-related protein systems. J Comp Neurol 402:460-474. Burgaud JL, Poosti R, Fehrentz JA, Martinez J, Nahon JL (1997): Melanin-concentrating hormone binding sites in human svkl4 keratinocytes. Biochem Biophys Res Commun 241:622-629. Campfield A (1998): Strategies and potential molecular targets for obesity treatment. Science 280:1383-1387. Chambers J, Ames RS, Bergsma D, Muir A, Fitzgerald LR, Hervieu G, Dytko GM, Foley JJ, Martin J, Liu WS, Park J, Ellis C, Ganguly S, Konchar S, Cluderay J, Leslie R, Wilson S, Sarau HM (1999): Melanin-concentrating hormone is the cognate ligand for the orphan G-protein-coupled receptor SLC-1. Nature 400:261-265. Chaffer CL, Morris MJ (2002): The feeding response to melanin-concentrating hormone is attenuated by antagonism of the NPYYl-receptor in the rat. Endocrinology 143 (1):191-197. Clapham JC, Arch JRS, Taddayyon M (2001): Anti-obesity drugs: a critical review of current therapies and future opportunities. Pharmacol Ther 89:81-121. Checler F, Dauch P, Barelli H, Nahon JL, Vincent JP (1992): Hydrolysis of rat melanin-concentrating hormone by endopeptidase 24.11 (neutral endopeptidase). J Biochem 286 (1):217-221. Chen YY, Hu CZ, Hsu CK, Zhang Q, Bi C, Asnicar M, Hsiung HM, Fox N, Slieker LJ, Yang DD, Heiman ML, Shi YG (2002): Targeted disruption of the melanin-concentrating hormone receptor-1 results in hyperphagia and resistance to diet-induced obesity. Endocrinology 143 (7):2469-2477. Chiocchio SR, Gallardo MGP, Louzan P, Gutnisky V, Tramezzani JH (2001): Melanin-concentrating hormone stimulates the release of luteinizing hormone-releasing hormone and gonadotropins in the female rat acting at both median eminence and pituitary levels. Biol Reprod 64 (5):1466-1472. Compagnone N, Fellman D, Bugnon C (1991): Expression of peptides derived from the MCH precursor in serum-free culture of rat fetal hypothalamic neurons: role of attachment factors. Dev Neurosci 13:417-423. Compagnone N, Fellman D, Bugnon C, Jacquemard C (1993): Diffusible factors from rat arcuate nucleus and Broca's diagonal band nucleus increase size and neurite outgrowth, respectively, of cultured MCH neurones. Brain Res 628:137-144. Courseaux A, Nahon JL (2001): Birth of two chimeric genes in the Hominidae lineage. Science 291 (5507):12931295. Crawley JN, McLean S (1996): Neuropeptides: basis and clinical advances. Ann New York Acad Sci 780:000-000. Dallvecchia-Adams S, Kuhar MJ, Smith Y (2002): CART peptide projections in the ventral midbrain: colocalisation with GABA, MCH, dynorphin, and synaptic interactions with dopamine neurons. J Comp Neurol 448:360-372. Darlinson MG, Richter D (1999): Multiple genes for neuropeptides and their receptors: co-evolution and physiology. Trends Neurosci 22 (2):81-88. Delecea L, Kilduff TS, Peyrin C, Foye PE, Danielson PE, Fukuhara C, Battenberg ELF, Gautvik VT, Bartlett FS, Frankel WN, van den Pol AN, Bloom FE, Gautvik KM, Sutcliffe JG (1998): The hypocretins: hypothalamusspecific peptides with neuroexcitatory activity. Proc Natl Acad Sci USA 95:322-327. Della-Zuana O, Presse F, Ortola C, Duhault J, Nahon JL, Levens N (2002): Acute and chronic administration od MCH enhances food intake and body weight in Wistar and Sprague-Dawley rats. Int J Obesity 26:1-7. Drozdz R, Eberle AN (1995a): Binding sites for melanin-concentrating hormone (MCH): In brain synaptosomes and membranes from peripheral tissues identified with highly tritiated MCH. J Receptor Signal Transduct Res 15:487-502. Drozdz R, Eberle AN (1995b): Synthesis and iodination of human (phenylalanine 13, tyrosine 19): melaninconcentrating hormone for radioreceptor assay. J Pept Sci 1:58-65. Drozdz R, Siegrist W, Baker BI, Chlubadetapia J, Eberle AN (1995): Melanin-concentrating hormone binding to mouse melanoma cells in vitro. FEBS Lett 359:199-202. Drozdz R, Hintermann E, Tanner H, Zumsteg U, Eberle AN (1999): (D-(p-benzoylphenylalanine):(13):, tyrosine(19):):-melanin-concentrating hormone, a potent analogue for MCH receptor crosslinking. J Pept Sci 5:234-242. Eberle AN (1988): The Melanotropins-Chemistry, Physiology and Mechanisms of Action. Karger, Basle. Edwards CMB, Abusnana S, Sunter D, Murphy KG, Ghatei MA, Bloom SR (1999): The effect of the orexins on food intake: comparison with NPY, MCH and galanin. J Endocrinol 160:R7-R12. Elias CF, Bittencourt JC (1997): Study of the origins of MCH and NEI immunoreactive projections to the periaqueductal gray matter. Brain Res 755:255-271. Elias CF, Saper CB, Eleftheria MF, Tritos NA, Lee C, Kelly J, Tatro JB, Hoffman GE, Ollmann MM, Barsh GS, Sakurai T, Yanagisawa M, Elmquist JK (1998): Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area. J Comp Neurol 402:442-459.

94

The melanin-concentrating hormone

Ch. H

Elias CF, Lee C, Kelly JF, Ahima RS, Kuhar M, Saper CB, Elmquist JK (2001): Characterisaton of CART neurons in the rat and human hypothalamus. J Comp Neurol 432:1-19. Elmquist JK, Elias CF, Saper CB (1999): From lesions to leptin: Hypothalamic control of food intake and body weight. Neuron 22:221-223. Filglewicz DE Wood SC (2000): Adiposity signals and brain reward mechanisms. Trends Pharmacol Sci 21:235236. Flier JS, Maratos-Flier E (1998): Obesity and the hypothalamus: novel peptides for new pathways. Cell 92:437440. Foote SL, Bloom FE, Aston-Jones G (1983): Nucleus locus coeruleus: new evidence of anatomical and physiological specificity. Physiol Rev 63:844-914. Forsling ML, Zhou Y (1997): MCH and vasopressin release in the rat. J Physio1504:223. Fulton S (2000): Modulation of brain reward circuitry by leptin. Science 287:125-128. Gao B, Hagenbuch B, Kullak-Ublick GA, Benke D, Aguzzi A, Meier P (2000): Organic anion-transporting polypeptides mediate transport of opioid peptides across the blood-brain barrier. J Pharmacol Exp Ther 294 (1):73-79. Gao X-B, van den Pol AN (2001): MCH depresses synaptic activity of glutamate and GABA neurons from rat lateral hypothalamus. J Physiol 533 (1):237-252. Gautwik KM, de Lecea L, Gautwik VT, Danielson PE, Tranque P, Dopazo A, Bloom FE, Sutcliffe JG (1996): Overview of the most prevalent hypothalamus-specific mRNAs, as identified by directional tag PCR substraction. Proc Natl Acad Sci USA 88:10208-10212. Gonzalez MI, Vaziri S, Wilson CA (1996): Behavioral effects of alpha-MSH and MCH after central administration in the female rat. Peptides 17 (1):171-17'7. Gonzalez MI, Baker BI, Wilson CA (1997a): Stimulatory effect of MCH on LH release. Neuroendocrinology 66:254-262. Gonzalez MI, Kalia V, Hole DR, Wilson CA (1997b): Alpha-MSH and MCH modify monoaminergic levels in the preoptic area of the rat. Peptides 18 (3):387-392. Griffond B, Baker BI (2002): Cell and molecular cell biology of melanin-concentrating hormone. Int Rev Cytol 213:233-277. Griffond B, Deray A, Nguyen NU, Colard C, Fellmann D (1995): The synthesis of melanin-concentrating hormone is stimulated by ventromedial hypothalamic lesions in the rat lateral hypothalamus: a time-course study. Neuropeptides 28:267-275. Griffond B, Ciofi P, Bayer L, Jacquemard C, Fellman D (1997): Immunocytochemical detection of the neurokinin B receptor (NK3): on MCH neurons in rat brain. J Chem Neuranat 12:183-189. Griffond B, Grillon S, Herve C, Deray A, Fellman D (1998): Morphofunctional changes in MCH-producing neurones in relation to the control of food and water intake. Ann New York Acad Sci 839:219-222. Grillon S, Herve C, Griffond B, Fellmann D (1997): Exploring the expression of the melanin-concentrating hormone messenger RNA in the rat lateral hypothalamus after goldthioglucose injection. Neuropeptides 31:131136. Hakansson ML, Brown H, Ghilardi N, Skoda RC, Meister B (1998): Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus. J Neurosci 18 (1):559-572. Hanada R, Nakazato M, Matsukara S, Murakami N, Yoshimatsu H, Sakata T (2000): Differential regulation of MCH and orexin genes in the agouti-related protein/MC-R4 system. Biochem Biophys Res Commun 268:88-91. Harris J, Bird DJ (2000): Supernatants from leucocytes treated with MCH and alpha-MSH have a stimulatory effect on rainbow trout phagocytes in vitro. Vet Immunol Immunopathol 76:117-124. Harrison DC, Medhurst AD, Bond BC, Campbell CA, Davis RE Philpott KL (2000): The use of quantitative RT-PCR to measure mRNA expression in a rat model of focal ischemia - - caspase-3 as a case study. Mol Brain Res 75:143-149. Hawes BE, Kil E, Green B, O'Neill K, Fried S, Graziano MP (2000): The melanin-concentrating hormone receptor couples to multiple G proteins to activate diverse intracellular signaling pathways. Endocrinology 141 (12):4524-4532. Haynes AC, Stevens AJ, Muir A, Avenell KY, Clapham JC, Tadayyon M, Darker JG, Wroblowski B, O'Toole CJ, Witty D, Arch JRS (2001): A MCH-R1 antagonist reduces food intake and body weight gain in rats and mice. North American Association for the Study of Obesity, Canada, Quebec. Published in Obesity Res 9 (Suppl. 9): O152. Henry BA, Tilbrook AJ, Dunshea FR, Rao A, Blache D, Martin GB, Clarke IJ (2000): Long-term alterations in adiposity affect the expression of melanin-concentrating hormone and enkephalin but not proopiomelanocortin in the hypothalamus of ovariectomized ewes. Endocrinology 141 (4): 1506-1514.

95

Ch. H

G.J. Hervieu et al.

Herv6 C, Fellmann D (1997): Changes in rat melanin-concentrating hormone and dynorphin messenger ribonucleic acids induced by food deprivation. Neuropeptides 31:237-242. Herv6 C, Colard C, Grillon S, Fellmann D, Griffond B (1998): Polyethylene glycol-induced hypovolemia affects the expression of MCH mRNA, but not dynorphin or secretogranin II mRNAs, in the rat lateral hypothalamus. Neurosci Lett 248:133-137. Hervieu G, Emson PC (1998): The localisation of somatostatin receptor 1 ssh immunoreactivity in the rat brain using an N-teminal specific antibody. Neuroscience 85 (4):1263-1284. Hervieu G, Nahon JL (1995): Pro-melanin-concentrating hormone messenger ribonucleic acid and peptides expression in peripheral tissues of the rat. Neuroendocrinology 61:348-364. Hervieu G, Volant K, Grishina O, Descroix-Vagne M, Nahon JL (1996a): Similarities in cellular expression and functions of melanin-concentrating hormone and atrial natriuretic factor in the rat digestive tract. Endocrinology 137 (2):561-571. Hervieu G, Segretain D, Nahon JL (1996b): Developmental and stage-dependent expression of melaninconcentrating hormone in the nuclei of rodent germ cell. Biol Reprod 54:1161-1171. Hervieu GJ, Cluderay JE, Harrison D, Meakin J, Maycox P, Nasir S, Leslie RA (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. Hetherington AW, Ranson SW (1940): Hypothalamic lesions and adiposity in the rat. Anat 78:149-172. Hill J, Duckworth M, Murdoch P, Rennie G, Sabido-David C, Ames RS, Szekeres P, Wilson S, Bergsma DJ, Gloger IS, Levy DS, Chambers JK, Muir AI (2001): Molecular cloning and functional characterisation of MCH2, a novel human MCH receptor. J Biol Chem 276 (23):20125-20129. Hintermann E, Drozdz R, Tanner H, Eberle AN (1999): Synthesis and characterization of new radioligands for the mammalian melanin-concentrating hormone (MCH): receptor. J Recept Signal Transd Res 19:411-422. Hintermann E, Tanner H, Talke-Messerer C, Schlumberger S, Zumsteg U, Eberle AN (2001a): Interaction of melanin-concentrating hormone (MCH):, neuropeptide E-I (NEI): 9 neuropeptide G-E (NGE), and alpha-MSH with melanocortin and MCH receptors on mouse B16 melanoma cells. J Recept Signal Transd Res 21 (1):93116. Hintermann E, Erb C, Talke-Messerer C, Liu R, Tanner H, Flammer J, Eberle AN (2001b): Expression of the melanin-concentrating hormone receptor in porcine and human ciliary epithelial cells. Invest Ophthal Vis Sci 42 (1):206-209. Hockfelt T (1991): Neuropeptides in perspective: the last ten years. Neuron 7:867-879. Hockfelt T, Broberger C, Xu DZ-Q, Sergeyev V, Ubink R, Diez M (2000): Neuropeptides, an overview. Neuropharmacology 39:1337-1356. Huang Q, Viale A, Picard F, Nahon JL, Richard D (1999): Effects of leptin on MCH expression in the brain of lean and obese mice. Neuroendocrinology 69:145-153. Iqbal J, Pompolo S, Murakami T, Grouzmann E, Sakurai T, Meister B, Clarke IJ (2001): Immunohistochemical characterization of localization of long-form leptin receptor (OB-Rb): in neurochemically defined cells in the ovine hypothalamus. Brain Res 920 (1-2):55-64. Jezova D, Bartanutsz V, Westergren I, Johansson B, Rivier J, Vale W, Rivier C (1992): Rat melanin-concentrating hormone stimulates adrenocorticotropin secretion: evidence for a site of action in brain regions protected by the blood brain barrier. Endocrinology 130:1021-1029. Kastin AJ, Pan WH, Maness LM, Banks WA (1999): Peptides crossing the blood-brain barrier: some unusual observations. Brain Res 848 (1-2):96-100. Kastin AJ, Akerstrom V, Hackler L, Zadina JE (2000): [Phe(13):,Tyr(19):]-melanin-concentrating hormone and the blood-brain barrier: role of protein binding. J Neurochem 74 (1):385-391. Kawauchi H, Kawazoe I, Tsubokawa M, Kishida M, Baker BI (1983): Characterisation of MCH in chum salmon pituitaries. Nature 305:321-323. Kawazoe I, Kawauchi H, Hirano T, Naito N (1987): Structure-activity relationships of melanin-concentrating hormone. Int J Pept Protein Res 29:714-721. Kemp EH, Waterman EA, Hawes BE, O'Neill K, Gottumukkala RVSRK, Gawkrodger DJ, Weetman AP, Watson PF (2002): The melanin-concentrating hormone receptor 1, a novel target of autoantibody responses in vitiligo. J Clin Invest 109 (7):923-930. Kennedy AR, Todd JF, Stanley SA, Abbott CR, Small CJ, Ghatei MA, Bloom SR (2001): Melanin-concentrating hormone (MCH): suppresses thyroid stimulating hormone (TSH): release, in vivo and in vitro, via the hypothalamus and the pituitary. Endocrinology 142 (7):3265-3268. Kilduff TS, de Lecea L (2001): Mapping of the mRNAs for the hypocretin/orexin and MCH receptors: network of overlapping peptide systems. J Comp Neurol 435:1-5.

96

The melanin-concentrating hormone

Ch. H

Kinoshita M, Morita T, Toyohara H, Hirata T, Sakaguchi M, Ono M, Inoue K, Wakamatsu Y, Ozato K (2001): Transgenic medaka overexpressing a melanin-concentrating hormone exhibit lightened body color but no remarkable abnormality. Mar Biotechnol 3 (6):536-543. Knigge KM, Wagner JE (1997): Melanin-concentrating hormone involvement in PTZ-induced seizure in rat and guinea pig. Peptides 18 (7):1095-1097. Knigge KM, Baxter-Grillo D, Speciale J, Wagner J (1996): Melanotropic peptides in the mammalian brain: the melanin-concentrating hormone. Peptides 17 (6): 1063-1073. Knollema S, Brown ER, Vale W, Sawchenko PE (1992): Novel hypothalamic and preoptic sites of pre-proMCH mRNA and peptide expression in lactating rats. J Neuroendocrinol 4:709-718. Kokkotou EG, Tritos NA, Mastaitis JW, Slieker L, Maratos-Flier E (2001): Melanin-concentrating hormone receptor is a target of leptin action in the mouse brain. Endocrinology 142 (2):680-686. Kolakowski LF, Jung BP, Nguyen T, Johnson MP, Lynch KR, Cheng R, Heng HHQ, George SR, O'Dowd BF (1996): Characterization of a human gene related to genes encoding somatostatin receptors. FEBS Lett 398:253258. Koob GF (1999): Corticotropin-releasing factor, norepinephrine, and stress. Biol Psychiatry 46:1167-1180. Kopelman PG (2000): Obesity as a medical problem. Nature 404:635-643. Lakaye B, Minet A, Zorzi W, Grisar T (1998): Cloning of the rat brain cdna encoding for the slc-1 g proteincoupled receptor reveals the presence of an intron in the gene. Biochim Biophys Acta Mol Cell Res 1401:216220. Lebl M, Hruby VJ, Castrucci AM, Visconti MA, Hadley ME (1988): Melanin concentrating hormone analogues: contraction of the cyclic structure. 1. Agonist activity. J Med Chem 31:949-954. Lembo PMC, Grazzini E, Cao J, Hubatsch DA, Pelletier M, Hoffert C, St Onge S, Pou C, Labrecque J, Groblewski T, O'Donnell D, Payza K, Ahmad S, Walker P (1999): The receptor for the orexigenic peptide melanin-concentrating hormone is a G-protein-coupled receptor. Nat Cell Biol 1:267-271. Lopez M, Seoane LM, Garcia MD, Dieguez C, Senaris R (2002): Neuropeptide Y, but not agouti-related peptide or melanin-concentrating hormone, is a target peptide for orexin-A feeding actions in the rat hypothalamus. Neuroendocrinology 75 (1):34-44. Ludwig DS, Mountjoy KG, Tato JB, Gillette JA, Frederich RC, Flier JS, Maratos-Flier E (1998): Melaninconcentrating hormone: a functional antagonist in the hypothalamus. Am J Physiol 274 [Endocrinol. Metab. 37]:E627-E633. Ludwig DS, Tritos NA, Mastaitis JW, Kulkarni R, Kokkotou E, Elmquist J, Lowell B, Flier JS, Maratos-Flier E (2001): MCH overexpression in transgenic mice leads to obesity and insulin resistance. J Clin Invest 107 (3):379-386. Macdonald D, Murgolo N, Zhang RM, Durkin JP, Yao XR, Strader CD, Graziano MP (2000): Molecular characterization of the melanin-concentrating hormone/receptor complex: identification of critical residues involved in binding and activation. Mol Pharmacol 58:217-225. Marsh DJ, Weingarth DT, Novi DE, Chen HY, Trumbauer ME, Chen Guan XM, Jiang MM, Feng Y, Camacho RE, Shen Z, Frazier EG, Yu H, Metzger JM, Kuca SJ, Shearman LP, Gopal-Truter S, MacNeil DJ, Strack AM, Maclntyre DE, Van der Ploeg LHT, Qian S (2002): Melanin-concentrating hormone 1 receptor-deficient mice are lean, hyperactive, and hyperphagic and have altered metabolism. Proc Natl Acad Sci USA 99 (5):3240-3245. Matsunaga TO, Castrucci AM, Hadley ME, Hruby VJ (1986): MCH: Structure-function aspects of its MSH-like activities. Peptides 10:773-778. Matsunaga TO, Castrucci AM, Hadley ME, Hruby VJ (1989): Melanin concentrating hormone (MCH): synthesis and bioactivity studies of MCH fragment analogues. Peptides 10:349-354. McBride RB, Becwith E, Swenson RR, Sawyer TK, Hadley ME, Matsungaga TO, Hruby VJ (1994): The actions of melanin-concentrating hormone on passive avoidance in rats: a preliminary study. Peptides 15 (4):757-759. Maulon-Feraille L, Della Zuana O, Suply T, Rovere-Jovene C, Audinot V, Leven N, Boutin JA, Duhault J, Nahon JL (2002): Appetite-boosting property of pro-melanin concentrating hormone(131-165): (neuropeptide-glutamic acid-isoleucine): is associated with proteolytic resistance. J Pharm Exp Ther 302 (2):766-773. Miller CL, Hruby VJ, Matsunaga TO, Bickford PC (1993): Alpha-MSH and MCH are functional antagonists in a CNS auditory gating paradigm. Peptides 14:431-440. Miller CL, Burmeister M, Thompson RC (1998): Antisense expression of the human preproMCH gene. Brain Res 803:86-94. Mizuno TM, Kleopoulos SP, Bergen HT, Roberts JL, Priest CA, Mobbs CV (1998): Hypothalamic POMC mRNA is reduced by fasting in ob/ob and db/db mice, but is stimulated by leptin. Diabetes 47:294-297. Mondal MS, Nakazato M, Matsukura S (2002): Characterization of orexins (hypocretins): and melaninconcentrating hormone in genetically obese mice. Reg Pept 104 (1-3 (Special Issue)):21-25.

97

Ch. H

G.J. Hervieu et al.

Monzon ME, de Souza MM, Izquierdo LA, Barros DM, de Bariglio SR (1999): MCH modifies memory retention in rats. Peptides 20:1517-1519. Monzon ME, Varas MM, De Barioglio SR (2001): Anxiogenesis induced by nitric oxide synthase inhibition and anxiolytic effect of melanin-concentrating hormone (MCH): in rat brain. Peptides 22 (7):1043-1047. Mori M, Harada M, Terao Y, Sugo T, Watanabe T, Shinomura Y, Abe M, Shintani Y, Onda H, Nishimura O, Fujino M (2001): Cloning of a novel GPCR SLT, a subtype of the MCH receptor. Biochem Biophys Res Commun 283:1013-1018. Moriguchi T, Sakurai T, Nambu T, Yanagisawa M, Goto K (1999): Neurons containing orexin in the lateral hypothalamic area of the adult rat brain are activated by insulin-induced acute hypoglycemia. Neurosci Lett 264:101-104. Morley JE (1987): Neuropeptide regulation of appetite and weight. Endocr Rev 8 (3):256-287. Murray JF, Mercer JG, Adan RAH, Datta J, Aldairy C, Moar KM, Baker BI, Stock MJ, Wilson CA (2000): The effect of leptin on LH release is exerted in the zona incerta and mediated by MCH. J Neuroendocrinol 12:1133-1139. Mystkowski P, Seeley RJ, Hahn TM, Baskin DG, Havel PJ, Matsumuto AM, Wilkinson CW, Peacock-Kinzig K, Blake KA, Schwatz M (2000): Hypothalamic MCH and estrogen-induced weight loss. J Neurosci 20 (22):86378642. Nahon JL (1994): The melanin-concentrating hormone: from the peptide to the gene. Crit Rev Neurobiol 8 (4):221262. Nahon JL, Presse F, Bittencourt JC, Sawchenko P, Vale W (1989): The rat melanin-concentrating hormone mRNA encodes multiple putative neuropeptides coexpressed in the dorsolateral hypothalamus. Endocrinology 125:20562065. Nahon JL, Presse F, Breton C, Hervieu G, Schorpp M (1993): Melanotropic peptides. In: Structure and regulation of the melanin-concentrating hormone gene. Ann New York Acad Sci 680:111-129. Navarra P, Tsagarakis S, Coy DH, Rees LH, Besser GP, Grossman AB (1990): Rat melanin concentrating hormone does not modify the release of CRH-41 from rat hypothalamus or ACTH from the anterior pituitary in vitro. J Endocrinol 127:R1-R4. Oldfield BJ, Giles ME, Watson A, Anderson C, Colvill LM, McKinley MJ (2002): The neurochemical characterisation of hypothalamic pathways projecting polysynaptically to brown adipose tissue in the rat. Neuroscience 110 (3):515-526. Oshima N, Wannitikul P (1996): Signal transduction of MCH in melanophores of the tilapia, Oreochromis Niloticus. Zool Sci 13:351-356. Parkes D, Vale W (1992a): Contrasting actions of MCH and NEI on posterior pituitary functions. In: Structure and regulation of the melanin-concentrating hormone gene. Ann New York Acad Sci 680:588-590. Parkes DG, Vale W (1992b): Secretion of melanin-concentrating hormone and neuropeptide-EI from cultured rat hypothalamic cells. Endocrinology 131:1826-1831. Parkes DG, Rivest S, Rivier C, Sawchenko PE, Vale W (1992): MCH and NEI activate hypothalamic CRF neurons in conscious rats. Soc Neurosci 18:120. Paxinos G, Watson C (1998): The Rat Brain in Stereotaxic Coordinates. 3rd edn. Academic Press, San Diego. Pedeutour F, Szpirer C, Nahon JL (1994): Assignment of the human proMCH gene (PMCH) to chromosome 12q23-q24 and two variant genes (PMCHL1 and PMCHL2) to chromosome 5p14 and 5q12-q13. Genomics 19(1):31-33. Peeke PM, Chrousos GP (1995): Hypercortisolism and obesity. Stress: basic mechanisms and clinical implications. Ann New York Acad Sci 711:665-676. Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, Kilduff TS (1998): Neurons containing hypocretin (orexin): project to multiple neuronal systems. J Neurosci 18:9996-10015. Presse F, Nahon JL (1993): Differential regulation of Melanin-Concentrating Hormone gene expression in distinct hypothalamic areas under osmotic stimulation in rat. Neuroscience 55:709-720. Presse F, Hervieu G, Imaki T, Sawchenko PE, Vale W, Nahon JL (1992): Rat melanin-concentrating hormone messenger ribonucleic acid expression: marked changes during development and after stress and glucocorticoid stimuli. Endocrinology 131:1241-1250. Presse F, Sorokovsky I, Max JP, Nicolaidis S, Nahon JL (1996): MCH is a potent anorectic peptide regulated by food-deprivation and glucopenia in the rat. Neuroscience 71 (3):735-745. Presse F, Cardona B, Borsu L, Nahon JL (1997): Lithium increases melanin-concentrating hormone mRNA stability and inhibits tyrosine hydroxylase gene expression in PC12 cells. Mol Brain Res 52(2):270-283. Qu D, Ludwig DS, Gammeltoft S, Piper M, Pelleymounter MA, Cullen MJ, Mathes WF, Przypek J, Kanarek R,

98

The melanin-concentrating hormone

Ch. H

Maratos-Flier E (1996): A role for melanin-concentrating hormone in the central regulation of feeding behaviour. Nature 380:243-244. Risold PY, Griffond B, Kilduff TS, Sutcliffe JG, Fellman D (1999): Preproorexin and PRL-like immunoreactivity are coexpressed by neurones of the rat lateral hypothalamus. Neurosci Lett 259:153-156. Rodriguez M, Beauverger P, Naime I, Rique H, Ouvry C, Souchaud S, Dromaint S, Nagel N, Suply T, Audinot V, Boutin JA, Galizzi JP (2002): Cloning and molecular characterization of the novel human melanin-concentrating hormone receptor MCH2. Mol Pharmacol 60 (4):632-639. Rossi M, Bloom SR (1997): MCH acutely stimulates feeding, but chronic administration has no effect on body weight. Endocrinology 138 (1):351-355. Rossi M, Beak SA, Choi S-J, Small CJ, Morgan DGA, Ghatei MA, Smith DM, Bloom SR (1999): Investigation of the feeding effects of MCH on food intake-action independent of galanin and the melanocortin receptors. Brain Res 846:164-170. Rov~re C, Viale A, Nahon JL, Kitabgi P (1996): Impaired processing of brain pro-neurotensin and pro-melaninconcentrating hormone in obese fat/fat mice. Endocrinology 137:2954-2958. Sahu A (1998): Evidence suggesting that galanin, MCH, neurotensin, POMC and NPY are targets of leptin signalliung in the hypothalamus. Endocrinology 139 (2):795-798. Sailer AW, Sano H, Zeng Z, McDonald TE Pan J, Pong SS, Feighner SD, Tan CP, Fukami T, Iwaasa H, Hreniuk DL, Morin NR, Sharon J, Sadowski SJ, Ito M, Ito M, Bansal A, Ky B, Figueroa DJ, Jiang Q, Austin CP, MacNeil DJ, Ishihara A, Ihara M, Kanatani A, Van der Ploeg LHT, Howard AD, Liu Q (2001): Identification and characterization of a second melanin-concentrating hormone receptor, MCH-2R. Proc Natl Acad Sci USA 98:7564-7569. Saito Y, Nothacker HP, Wang ZW, Lin SHS, Leslie F, Civelli O (1999): Molecular characterization of the melanin-concentrating-hormone receptor. Nature 400:265-269. Saito Y, Nothacker HP, Civelli O (2000): Melanin-concentrating-hormone receptor: an orphan receptor fits the key. Trends Endocr Metabol 11 (8):299-303. Saito Y, Cheng M, Leslie FM, Civelli O (2001a): Expression of the melanin-concentrating-hormone receptor mRNA in the rat brain. J Comp Neurol 435:26-40. Saito Y, Wang Z, Hagino-Yamagishi K, Civelli O, Kawashima S, Maruyama K (2001b): Endogenous MCH receptor SLC-1 in the human melanoma SK-MEL-37 cells. Biochem Biophys Res Commun 289:44-50. Sakuntabhai A, Ruiz-Perez V, Carter S, Jacobsen N, Burge S, Monk S, Smith M, Munro CS, O'Donovan M, Craddock N, Kucherlapati R, Rees JL, Owen M, Lathrop GM, Monaco AP, Strachan T, Hovnanian A (1999): Mutations in ATP2A2, encoding a Ca 2+ pump, cause Dafter disease. Nat Genet 21:271-277. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Anaka H, Williams SC, Richardson JA, Kozlowski GP, Wilson S, Arch JRS, Buckingham RE, Haynes AC, Carr SA, Annan RS, NcNulty DE, Liu W-S, Terret JA, Elshourbagy NA, Bergsma DJ, Yanagisawa M (1998): Orexins and orexin receptors: a family of hypothalamic neuropeptides and G-protein-coupled receptors that regulate feeding behavior. Cell 92:573-585. Sanchez M, Baker I, Celis M (1997): Melanin-concentrating hormone (MCH): antagonizes the effects of alphaMSH and neuropeptide E-I on grooming and locomotor activities in the rat. Peptides 18:393-396. Sanpei K, Takano H, Igarashi S, Sato T, Oyake M, Sasaki H, Wakisaka A, Tashiro K, Ishida Y, Ikeuchi T, Koide M, Saito M, Sato A, Tanaka T, Hanyu S, Takiyama Y, Nishizawa M, Shimizu N, Nomura Y, Segawa M, Iwabuchi K, Eguchi I, Tanaka H, Takahashi H, Tsuji S (1996): Identification of the spinocerebellar ataxia type 2 gene using a direct identification of repeat expansion and cloning technique DIRECT. Nat Genet 14:277-284. Sawchenko PE (1998): Toward a new neurobiology of energy balance, appetite and obesity. The anatomists weigh-in. J Comp Neurol 402:435-441. Schwartz MW, Gelling RW (2002): Rats lighten up with MCH antagonist. Nature Medicine 8(8): 779-781. Schwartz MW, Woods SC, Porte D, Seeley RJ, Baskin DG (2000): Central nervous system control of food intake. Nature 404:661-671. Sergeyev V, Broberger C, Hokfelt T (2001): Effect of LPS administration on the expression of POMC, NPY, galanin, CART and MCH mRNAs in the rat hypothalamus. Mol Brain Res 90:93-100. Shimada M, Tritos N, Bardford Lowell A, Flier J, Maratos-Flier E (1998): Mice lacking melanin-concentrating hormone are hypophagic and lean. Nature 396:670-674. Shimomura Y, Mori M, Sugo T, Ishibashi Y, Abe M, Kurokawa T, Onda H, Nishimura O, Sumino Y, Fujino M (1999): Isolation and identification of melanin-concentrating hormone as the endogenous ligand of the SLC-1 receptor. Biochem Biophys Res Commun 261:622-626. Sone M, Takahashi K, Murakami O, Totsune K, Arihara Z, Satoh F, Sasano H, Ito H, Mouri T (2000): Binding sites for melanin-concentrating hormone in the human brain. Peptides 21:245-250.

99

Ch. H

G.J. H e r v i e u et al.

Stadel JM, Wilson S, Bergsma DJ (1997): Orphan G protein-coupled receptors: a neglected opportunity for pioneer drug discovery. Trends Pharmacol Sci 18:430-437. Strand FL, Beckwith B, Chronwall B, Sandman CA (1994): Models of neuropeptide actions. Ann New York Acad Sci 739:000-000. Stricker-Krongrad A, Dimitrov T, Beck B (2001): Central and peripheral dysregulation of melanin-concentrating hormone in obese Zucker rats. Mol Brain Res 92 (1-2):43-48. Suply T, Della Zuana O, Audinot V, Rodriguez M, Beauverger P, Duhault J, Canet E, Galizzi JP, Nahon JL, Levens N, Boutin JA (2001): SLC-1 receptor mediates effect of melanin-concentrating hormone on feeding behavior in rat: a structure-activity study. J Pharmacol Exp Ther 299 (1):137-146. Swanson LW (1998): Brain Maps: Structure of the Rat Brain, 2nd edn., Elsevier, Amsterdam. Takahashi K, Suzuki H, Totsume K, Murakami O, Satoh F, Sone M, Sasano H, Mouri T, Shibahara S (1995): MCH in human and rat. Neuroendocrinology 61:493-498. Takahashi K, Totsume K, Murakami O, Sone M, Kitamuro T, Noshiro T, Hayashi Y, Sasano H, Shibahara S (2001): Expression of melanin-concentrating hormone receptor messenger ribonucleic acid in tumor tissues of pheochromocytoma, ganglioneuroblastoma, and neuroblastoma. J Clin Endocrinol Metab 86 (1):369-374. Takekawa S, Asami A, Ishihara Y, Terauchi J, Kato K, Shimomura Y, Mori M, Murakoshi H, Suzuki N, Nishimura O, Fujino M (2002): T-226296: a novel, orally active and selective melanin-concentrating hormone receptor antagonist. Eur J Pharmacol 438 (3):129-135. Tan CP, Sano H, Iwaasa H, Pan J, Sailer AW, Hreniuk DL, Feighner SD, Palyha OC, Pong SS, Figueroa DJ, Austin CP, Jiang MM, Yu H, Ito J, Ito M, Guan XM, MacNeil DJ, Kanatani A, Van der Ploeg LHT, Howard AD (2002): Melanin-concentrating hormone receptor subtypes 1 and 2: Species-specific gene expression. Genomics 79 (6):785-792. Toumaniantz G, Bittencourt JC, Nahon JL (1996): The rat MCH gene encodes an additional protein in a different reading frame. Endocrinology 137 (10):4518-4521. Toumaniantz G, Ferreira PC, Allaeys I, Bittencourt JC, Nahon JL (2000): Differential neuronal expression and projections of MCH and MGOP in the rat brain. Eur J Neurosci 12:4367-4380. Touzani K, Tramu G, Nahon JL, Velley L (1993): Hypothalamic MCH and alpha-neoendorphin-immunoreactive neurones project to the medial part of the rat parabranchial area. Neuroscience 53 (3):865-876. Tritos NA, Maratos-Flier E (1999): Two important systems in energy homeostasis: melanocortins and MCH. Neuropeptides 33:339-349. Tritos NA, Elmquist JK, Mastaitis JW, Flier JS, Maratos-Flier E (1998a): Characterization of expression of hypothalamic appetite-regulating peptides in obese hyperleptinemic brown adipose tissues-deficient (uncoupling protein-promoter-driven diphtheria toxin A) mice. Endocrinology 139:4634-4641. Tritos NA, Vicent D, Gillette J, Ludwig DS, Flier ES, Maratos-Flier E (1998b): Functional interactions between MCH, NPY and anorectic peptides in the rat hypothalamus. Diabetes 47:1687-1692. Tritos NA, Mastaitis JW, Kokkotou E, Maratos-Flier E (2001): Characterization of melanin concentrating hormone and preproorexin expression in the murine hypothalamus. Brain Res 895(1-2):160-166. Tsukamura H, Thompson RC, Tsukahara S, Ohkura S, Maekawa F, Moriyama R, Niwa Y, Foster DL, Maeda KI (2000): Intracerebroventricular administration of melanin-concentrating hormone suppresses pulsatile luteinizing hormone release in the female rat. J Neuroendocrinol 12 (6):529-534. Varas M, Perez M, Ramirez O, de Barioglio SR (2002): Melanin concentrating hormone increase hippocampal synaptic transmission in the rat. Peptides 23 (1):151-155. Vaughan JM, Fisher WH, Hoeger C, River J, Vale W (1989): Characterisation of melanin-concentrating hormone from rat hypothalamus. Endocrinology 125:1660-1665. Verlaeten O, Griffond B, Khuth ST, Giraudon P, Akaoka H, Belin MF, Fellmann D, Bernard A (2001): Down regulation of melanin concentrating hormone in virally-induced obesity. Mol Cell Endocrinol 181 (1-2):207-219. Viale A, Zhixing Y, Breton C, Pedeutour F, Coquerel A, Jordan D, Nahon JL (1997): The melanin-concentrating hormone gene in human: flanking region analysis, fine chromosome mapping, and tissue-specific expression. Mol Brain Res 46(1-2):243-255. Viale A, Ortola C, Richard F, Vernier P, Presse F, Schilling S, Dutrillaux B, Nahon JL (1998): Emergence of a brain-expressed variant melanin-concentrating hormone gene during higher primate evolution: a gene 'in search of a function'. Mol Biol Evol 15:196-214. Viale A, Kerdelhue B, Nahon JL (1999a): 17~-Estradiol regulation of melanin concentrating hormone and neuropeptide-E-I contents in cynomolgus monkeys: a preliminary study. Peptides 20:553-559. Viale A, Ortola C, Hervieu G, Furuta M, Barbero P, Steiner D, Seidah N, Nahon JL (1999b): Cellular localization and role of prohormone convertases in the processing of pro-melanin concentrating hormone in mammals. J Biol Chem 274:6536-6545. 100

The m e l a n i n - c o n c e n t r a t i n g h o r m o n e

Ch. H

Viale A, Courseaux A, Presse F, Ortola C, Breton C, Jordan D, Nahon JL (2000): Structure and expression of the variant melanin-concentrating hormone genes: only PMCHL1 is transcribed in the developing human brain and encodes a putative protein. Mol Biol Evol 17:1626-1640. Wang SK, Behan J, O'Neill K, Weig B, Fried S, Laz T, Bayne M, Gustafson E, Hawes BE (2001): Identification and pharmacological characterization of a novel human melanin-concentrating hormone receptor, MCH-R2. J Biol Chem 276 (37):34664-34670. Witty DR, Hadley MS, Hervieu GJ, Jeffrey R Johnson CN, Jones M, Muir A, O'Hanlon PJ, O'Toole CR Riley GJ, Stemp G, Stevens AJ, Thewlis KM, Wilson S, Winborn KY, Wroblowski B (2002): Biphenyl carboxamide antagonists of the human melanin-concentrating hormone receptor 11CBy (SLC-1); discovery and SAR, Medicinal Chemistry Symposium, Barcelona, Spain, September 2002. Yamada M, Mikayama T, Duttaroy A, Yamanaka A, Moriguchi T, Makita R, Ogawa M, Chou CJ, Xia B, Crawley JN, Felder CC, Deng C-X, Wess J (2001): Mice lacking the M3 muscarinin receptor are hypophagic and lean. Nature 410:207-212. Zamir N, Skotfish G, Banson ML, Jacobowitz DM (1986a): MCH: unique peptide neuronal system in the rat brain and pituitary gland. Proc Natl Acad Sci USA 83:1528-1531. Zamir N, Skotfish G, Jacobowitz DM (1986b): Distribution of immunoreactive MCH in the rat CNS. Brain Res 373:240-245. Zhang R Liang JD, Sandusky GE, Burguera B, Considine RV, Hyde TM, Caro JF (1998): Hypothalamic MCH mRNA protein are increased in human obesity. Satellite Symposium: Ninth International Congress of Obesity, France, Proceedings of the Symposium, Int J Obesity, p. 51.

101