Melanin-concentrating hormone (MCH) is a cyclic neuropeptide, which centrally regulates food intake and stress. MCH induces food intake in rodents and, more generally, acts as an anabolic signal in energy regulation. In addition, MCH seems to be activatory on the stress axis. Two receptors for MCH in humans have very recently been characterised, namely, MCH-R1 and MCH-R2. MCH-R1 has received considerable attention, as potent and selective antagonists acting at that receptor display anxiolytic, antidepressant and/or anorectic properties. Feeding and affective disorders are both debilitating conditions that have become serious worldwide health threats. There are as yet no efficient and/or safe cures that could contain the near-pandemia phenomen of both diseases. Thus, the discovery of MCH-R1 antagonists may lead to the development of valuable drugs to treat obesity, anxiety and depressive syndromes. In addition, it opens wide avenues to probe additional functions of the peptide, both in the brain and in the peripheral nervous system.

Several psychiatric diseases, including schizophrenia, are thought to have a developmental aetiology, but to date no clear link has been made between psychiatric disease and a specific developmental process. LPA(1) is a G(i)-coupled seven transmembrane receptor with high affinity for lysophosphatidic acid. Although LPA(1) is expressed in several peripheral tissues, in the nervous system it shows relatively restricted temporal expression to neuroepithelia during CNS development and to myelinating glia in the adult. We report the detailed neurological and behavioural analysis of mice homozygous for a targeted deletion at the lpa(1) locus. Our observations reveal a marked deficit in prepulse inhibition, widespread changes in the levels and turnover of the neurotransmitter 5-HT, a brain region-specific alteration in levels of amino acids, and a craniofacial dysmorphism in these mice. We suggest that the loss of LPA(1) receptor generates defects resembling those found in psychiatric disease.

Melanin-concentrating hormone (MCH) is implicated in the control of a number of hormonal axes including the hypothalamic-pituitary adrenal (HPA) axis. Previous studies have shown that there is evidence for both a stimulatory and an inhibitory action on the HPA axis; therefore, we attempted to further characterize the effects of MCH on this axis. Intracerebroventricular injection of MCH increased circulating adrenocorticotropic hormone (ACTH) at 10 min post injection. Injection of MCH directly into the paraventricular nucleus (PVN) was found to increase both circulating ACTH and corticosterone 10 min after injection. Additionally, MCH was found to increase corticotropin-releasing factor (CRF) release from hypothalamic explants, and this effect was abolished by the specific SLC-1 antagonist SB-568849. Neuropeptide EI, a peptide from the same precursor as MCH was also found to increase CRF release from explants. These results suggest that MCH has a stimulatory role in the HPA axis via SLC-1, and that MCH exerts its effects predominantly through the PVN CRF neuronal populations

Apelin peptides have recently been identified to be the endogenous ligands for the G protein-coupled receptor APJ. However, little is known about the physiological roles of this ligand-receptor pairing. In the present study we investigated the pharmacology of several apelin analogues at the human recombinant APJ receptor using radioligand binding and functional assays. This has led to the identification of key residues in the apelin peptide required for functional potency and binding affinity through structure-activity studies. In particular, we have identified that replacement of leucine in position 5, or arginine in position 2 and 4 of the C -terminal apelin peptide, apelin-13, resulted in significant changes in pharmacology. We also investigated the detailed localization of pre-proapelin and APJ receptor mRNA in a wide range of human, rat and mouse tissues using quantitative RT-PCR, and carried out a detailed immunohistochemical study of the distribution of the APJ receptor in rat brain and spinal cord. Interestingly, the APJ receptor was not only co-localized in white matter with GFAP in the spinal cord, but was also clearly localized on neurones in the brain, suggesting that this receptor and its peptide may be involved in a wide range of biological process yet to be determined.

The neuropeptides orexin‐A and orexin‐B are produced in neurons of the lateral hypothalamic area and have been implicated to be involved in the regulation of food/water intake and sleep–wake control. The orexins act at two different G‐protein‐coupled orexin receptors (OX‐R1 and OX‐R2) that are derived from separate genes and expressed differentially throughout the central nervous system. In the present study, we have used a polyclonal antipeptide antiserum to analyse in detail the distribution of OX‐R1‐immunoreactive neurons in the rat hypothalamus. In order to identify the chemical mediators of orexin action in the hypothalamus, the OX‐R1‐containing neurons were characterized with regard to the content of peptides shown previously to affect ingestive and drinking behaviour. Neurons containing OX‐R1 immunoreactivity were widely distributed in the hypothalamus with cell bodies located in the suprachiasmatic, periventricular, paraventricular (both magno‐ and parvocellular division), supraoptic, arcuate, ventromedial, dorsomedial and tuberomammillary nuclei and the lateral hypothalamic area. In magnocellular neurons of the paraventricular and supraoptic nuclei, OX‐R1 immunoreactivity was seen in both vasopressin‐ and oxytocin‐containing neurons. OX‐R1 immunoreactivity was demonstrated in vasopressin and vasoactive intestinal polypeptide (VIP) neurons of the suprachiasmatic nucleus, in somatostatin neurons of the periventricular nucleus and in corticotropin‐releasing hormone (CRH) neurons of the parvocellular paraventricular nucleus. In the arcuate nucleus, OX‐R1 immunoreactivity was present in neuropeptide Y (NPY) and agouti‐related peptide (AGRP) neurons of the ventromedial part as well as in proopiomelanocortin (POMC) and cocaine‐ and amphetamine‐regulated transcript (CART) neurons of the ventrolateral division. In the lateral hypothalamic area, OX‐R1 immunoreactivity was demonstrated in melanin‐concentrating hormone (MCH)‐ and orexin‐containing neurons. In the hypothalamic tuberomammillary nucleus, OX‐R1‐immunoreactivity was shown in many histamine‐containing neurons. The results support the idea that orexins have important actions on hypothalamic neurons that control food intake and fluid balance, but also that orexins may regulate other neuroendocrine systems.

Melanin-concentrating hormone (MCH) is a hypothalamic orexigenic peptide. Recently, an orphan G-protein-coupled receptor (SLC-1) was identified that binds MCH with high affinity. Here, we demonstrate the mRNA expression of this receptor in insulin-producing cells including CRI-G1 and RINm5F cells, and in rat islets of Langerhans. Immunofluorescence studies in CRI-G1 and RINm5F cell-lines demonstrated cell-surface expression of the receptor. Rat MCH significantly stimulated insulin secretion in both cell-lines. The potency and the efficacy of MCH were significantly increased in the simultaneous presence of forskolin, suggesting that MCH may amplify the insulinotropic effect of cyclic AMP elevating stimuli. Salmon MCH, which differs from rat/human MCH by six amino acids, was less efficacious than rat/human MCH in stimulating insulin release. The data provide evidence for the expression of MCH receptors in insulin producing cells. The insulinotropic effect of MCH may contribute to the regulation of metabolism and energy balance by this peptide.

Uridine 5'-diphosphoglucose (UDP-glucose) has a well established biochemical role as a glycosyl donor in the enzymatic biosynthesis of carbohydrates. It is less well known that UDP-glucose may possess pharmacological activity, suggesting that a receptor for this molecule may exist, Here, we show that UDP-glucose, and some closely related molecules, potently activate the orphan G protein-coupled receptor KIAA0001 heterologously expressed in yeast or mammalian cells. Nucleotides known to activate P2Y receptors were inactive, indicating the distinctly novel pharmacology of this receptor. The receptor is expressed in a wide variety of human tissues, including many regions of the brain. These data suggest that some sugar-nucleotides may serve important physiological roles as extracellular signaling molecules in addition to their familiar role in intermediary metabolism.

Melanin concentrating hormone (MCH) and neuropeptide EI (NEI) are two peptides produced from the same precursor in mammals, by cleavage at the Arg145-Arg146 site and the Lys129-Arg130 site, respectively. We performed co-localization studies to reveal simultaneously the expression of MCH mRNA and proconvertases (PCs) such as PC1/3 or PC2. In the rat hypothalamus, PC2 was present in all MCH neurons, and PC1/3 was present in about 15-20% of these cells. PC1/3 or PC2 was not found in MCH-positive cells in the spleen. In GH4C1 cells co-infected with vaccinia virus (VV):pro-MCH along with VV:furin, PACE4, PC1/3, PC2, PC5/6A, PC5/6B, or PC7, we observed only efficient cleavage at the Arg145-Arg146 site to generate mature MCH. Co-expression of pro-MCH together with PC2 and 7B2 resulted in very weak processing to NEI. Comparison of pro-MCH processing patterns in PC1/3- or PC2-transfected PC12 cells showed that PC2 but not PC1/3 generated NEI. Finally, we analyzed the pattern of pro-MCH processing in PC2 null mice. In the brain of homozygotic mutants, the production of mature NEI was dramatically reduced. In contrast, MCH content was increased in the hypothalamus of PC2 null mice. In the spleen, a single large MCH-containing peptide was identified in both wild type and PC2 null mice. Together, our data suggest that pro-MCH is processed differently in the brain and in peripheral organs of mammals. PC2 is the key enzyme that produces NEI, whereas several PCs may cleave at the Arg145-Arg146 site to generate MCH in neuronal cell types.

Melanin-concentrating hormone (NIGH) is a cyclic peptide predominantly expressed in the hypothalamus of mammals. This peptide modulates the stress response and regulates many goal-oriented behaviors in the rat brain. MCH mRNA and peptides generated from the precursor, namely MCH and neuropeptide (N) glutamic acid (E) isoleucine (I) amide (NEI), were also found in rodent peripheral tissues including those in adult testis. In the present study, we first examined the cellular distribution and content of MCH gene products and peptide in the testes of adult rats. Using reverse-transcription polymerase chain reaction with MCH gene primers and in situ hybridization with specific P-33-labeled oligoprobes, we characterized the MCH RNA species. Pro-MCH products were revealed through use of immunoperoxidase detection and an RIA with specific MCH and NEI antisera. Both MCH gene transcripts and MCH peptide were found within germ cells at the periphery of some of the seminiferous tubules of adult rats.We further investigated temporal expression of MCH in 7-mu m sections of testes from adult rats and mice. MCH was predominantly found in nuclei of spermatogonia at stages II-IV of spermatogenesis and in nuclei of leptotene and zygotene spermatocytes (at stages IX-XIII). MCH was markedly absent in preleptotene spermatocytes at the stage VII-VIII, and MCH immunoreactivity was no longer detectable as spermatocytes underwent the pachytene step. MCH immunoreactivity was also found in some peritubular cells, mainly at stages II-IV.Finally, we studied MCH expression during puberty in the rat, in sterile mutant mice, and in one adult man. We found predominant staining with MCH antiserum over nuclei of immature germ cells as early as 10 days postpartum in the rat and further confirmed the absence of staining except for that in spermatogonia and early spermatocytes during rat postnatal development. MCH distribution was found to be similar in normal and sterile mutant mice, suggesting that MCH expression is not dependent upon the early steps of spermiogenesis. MCH immunoreactivity was found to be confined to the nuclei of spermatogonia and early spermatocytes in the adult man. Our results indicate that MCH is present predominantly within nuclei of spermatogonia and primary spermatocytes in three mammalian species and that its expression is under strong stage-specific and developmental regulation. This peptide may play a role during stem cell renewal and/or differentiation of early spermatocytes.

Melanin-concentrating hormone (MCH) is a cyclic peptide isolated first from salmon brain, then from rat and human hypothalamus. We have recently found expression of MCH messenger RNA and encoded peptides, e.g. MCH and neuropeptide-glutamic acid-isoleucine, within the rat gastrointestinal (GI) tract, but their cellular origin was unclear. Furthermore, similarities in the localization of rat atrial natriuretic factor (ANF) and rat MCH immunoreactivities within intestine suggested functional convergence. In the present study we determined first the presence and distribution of MCH messenger RNA and encoded peptides in the GI tract by combining in situ hybridization and immunohistochemical analysis. Our data revealed numerous MCH-containing cells located in the lamina propria and submucosa at both duodenal and colonic levels. Second, the localisation of MCH- and arginine vasopressin- or ANF-containing cells appears similar at the duodenal and colonic levels, respectively. Colocalization of MCH/neuropeptide-glutamic acid-isoleucine immunoreactivity (-IR) and catecholamine indicated that MCH-expressing cells are probably antigen-presenting cells forming part of the enterochromaffin cell system. Third, we performed reverse phase HPLC coupled to RIA to characterize MCH-like materials in different portions of the rat gut. Crude acidic extracts of rat intestine contained about 2-3 pmol/g tissue of MCH-IR, close to the values found in brain extracts. Reverse phase HPLC of MCH-IR in the GI tract revealed that only 10-30% of the immunoreactivity corresponded to mature MCH, whereas the rat brain contained 94% mature peptide. Finally, we compared the effect of MCH and ANF on water and electrolyte secretions at different levels of the GI tract by using the in situ ligated loop technique. Similar effects were noted for ANF and MCH; both stimulated water, Na, and K fluxes at the proximal colon level and increased Na and K fluxes in the duodenum. However, only ANF increased water and Cl fluxes in the duodenum and decreased bicarbonate secretion in the ileum, whereas MCH increased bicarbonate absorption in the jejunum. The dose required was 10 nmol/100 g.h for MCH, i.e. 10 times more than for the ANF. These studies strongly suggest that MCH produced by antigen-presenting cells of the lamina propria may have an important role, similar to that of ANF at the colonic level, in the physiology of the GI tract.

Melanin-concentrating hormone (MCH) is a cyclic peptide which is predominantly synthetized in the hypothalamus of fish and mammalian brains. In the present paper we examined the expression of MCH mRNA and pro-MCH-derived peptides, i.e. MCH and neuropeptide-(N)-glutamic acid (E) isoleucine (I) amide (NEI), in peripheral tissues of adult rodents. By means of polymerase chain reaction (PCR) of reverse-transcribed RNA, low levels of MCH gene transcripts were detected reliably in testis, stomach, and intestine of Sprague-Dawley and Wistar rats, whereas strong expression was found in hypothalamus. Subsequent sequence analysis of the PCR products verified the authenticity of MCH mRNA found in hypothalamus and stomach. The length of MCH RNA species was measured by Northern blot and multiple MCH RNA species were detected in both rat species. Shortest polyadenylated tails were found in MCH RNAs isolated from the peripheral organs by comparison with hypothalamus MCH RNAs of Wistar rats. In order to localize MCH expression in gastrointestinal and genital tracts of Wistar rats we performed in situ hybridization with specific 33P-labeled oligoprobes joined to immunocytochemical studies with rat MCH or NEI antisera. In testis, the MCH transcripts and pro-MCH-derived peptide immunoreactivities were found at the periphery of the seminiferous tubules, suggesting expression in Sertoli cells. Studies with MCH oligoprobes and antisera directed towards MCH, NEI and alpha A-inhibin revealed similar pattern of expression in isolated Sertoli cells from Swiss mice, indicating that MCH RNA species were actually synthesized and translated in these cells. In the gastrointestinal (GI) tract, the cells expressing MCH RNA species and pro-MCH-derived peptides were predominantly expressed in the antral portion of the stomach and duodenum. Strikingly, distinct oligoprobes, recognizing antisense MCH transcript, revealed a pattern of hybridization in the GI tract similar to this observed with oligoprobes revealing the mature MCH mRNA. Furthermore, total RNA from the pyloric junction, duodenum, jejunum, ileum and hypothalamus as well appeared to contain RNA complementary to MCH mRNA suggesting therefore that antisense MCH RNA species may play a general role in regulation of MCH synthesis. Taken together, our present and previous data indicate that authentic MCH RNA species and translational products are expressed in various rodent tissues at the periphery. The cellular location suggests that MCH and associated peptides may play a role in spermatogenesis and in digestive processes.

Melanin-concentrating hormone (MCH) is a cyclic peptide (FIGURE 1) originally isolated from chum salmon pituitaries and characterized by Kawauchi et a/.' It is now well established that this heptadecapeptide is involved in regulating color change through stimulation of melanin granule aggregation within melanophores of melanocytes in teleost f i ~ h e s . ~ ~ Melanin dispersion in teleost melanophores is, indeed, under dual control by MCH and a-melanin stimulating hormone (a-MSH), an amidated peptide generated from proopiomelanocortin (POMC).S Paradoxally, high concentrations of salmon MCH (sMCH) cause pigment dispersion in amphibians and reptiles but also in fishes; this a-MSH-like activity may be attributable to sequences present outside the ring structure of s M C H . ~ . ~ Apart from its action on the melanophore, sMCH may inhibit corticotropin-releasing factor (CRF) induced ACTH secretion and a-MSH release from fish pituitary cells in ~ i t r o . * , ~ More recent studies have shown that MCH release is enhanced under stressful conditions,'' and that it may modulate the response of stress in fish." The discovery of sMCH and the availability of antiserum against it have made possible the characterization of the MCH counterpart in mammals. Using bioassay, radioimmunoassay, or immunohistochemical methods, an MCH-like peptide was seen throughout the brain but perikarya were found exclusively in hyp~tha lamus . '~'~ The locations of MCH-like neurons were mapped in the ventral aspect of zona incerta (ZI) and in the lateral hypothalamus (LH) in rat'**'-' and human.I6 These areas are directly concerned with many important aspects of drinking and ingestive behavior^'^-^^ and are thought to contribute to the control of behavioral arousal and emotion.21,22 In view of these anatomical features and possible involvement of MCH in the control of hypothalamopituitary axis (HPA) in rat,8x13 it was of interest to analyze in detail the MCH neuronal system in mammals.

Melanin-concentrating hormone (MCH) and associated peptides, designated NEI and NGE, are predominantly expressed in hypothalamic neurons which project widely throughout the mammalian brain. These peptides might be involved as neuromodulators in the control of goal-directed behaviors, the integration of the stress response, and/or the regulation of arousal in general. In vivo studies of this peptidic system using a transgenic mouse model call for information about the structure and regulation of the mouse MCH (mMCH) gene. One complementary DNA (cDNA) for mouse prepro-MCH was isolated from a brain library by using a rat MCH cDNA as probe. This cDNA contains an open reading frame coding for a 165-amino acid precursor that displays about 90% sequence identity with rat and human prepro-MCHs. Most of the structural portion of the mMCH gene was cloned and characterized using the polymerase chain reaction (PCR). Strong conservation in exon-intron organization and primary sequences was found among the mouse, rat, and human genes, suggesting that coding and noncoding regions have important biological functions. Developmentally regulated expression of mMCH gene in mouse hypothalamus was examined by Northern blot hybridization. Up to 10-fold changes in the relative mMCH mRNA contents were observed during postnatal development, characterized by a peak at the weaning period. Moreover, striking variations in mMCH mRNA length, due to a poly(A) tail, were revealed during postnatal life. Tissue distribution of mMCH gene transcripts was investigated by means of the PCR and Northern blot procedures. Expression of the mMCH gene was revealed in heart, intestine, spleen, and testis and was found to be regulated in a developmentally programmed manner. Strikingly, short as well as long mMCH RNA species were identified at the periphery. Taken together, our data indicate that both transcriptional and post-transcriptional mechanisms regulate the expression of the MCH gene in mouse brain and at the periphery.

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide first isolated from fish and rats. MCH may be involved in the control of the hypothalamic-pituitary-adrenocortical axis and, more generally, of specific goal-oriented behaviors and homeostatic functions in mammals. In this paper we examine 1) the cellular distribution of MCH gene transcripts in the rat central nervous system, 2) the changes in neuronal expression of MCH mRNA during rat development, and 3) the effects of stress and hormonal stimuli on rat MCH (rMCH) gene activity. Northern blot analysis and in situ hybridization histochemistry show that mature rMCH mRNA (1.0 kilobase) is very abundant in the zona incerta and the dorsolateral hypothalamus. While this is in agreement with previous peptide mapping by immunohistochemical techniques, a surprising new result is that a few clusters of rMCH mRNA-containing cells are found outside the hypothalamus, in the olfactory tubercle and the pontine tegmentum. Developmentally, rMCH mRNA is detected on embryonic day 18; its level increases gradually during early postnatal life and rises abruptly at weaning to reach a constant value in adult rats. In addition, striking variations in rMCH mRNA length occur during postnatal development and are found to be variations in the polyadenylate tail. Interestingly, this structural modification appears to be independent of the increase in rMCH mRNA levels. The regulation of rMCH mRNA expression by glucocorticoids and chronic stress is examined by Northern blot analysis. Chronic intermittent footshock stress causes a 58% or 29% decrease in rMCH mRNA content in the whole hypothalamus after a 1- or 3-day regimen, respectively. In contrast, the rMCH mRNA level returns to normal after a 7-day regimen. Two weeks after adrenalectomy (ADX) the whole hypothalamus rMCH mRNA content decreases 2.5-fold, but rises close to the control value 3 weeks after ADX. Dexamethasone administration 2 weeks after ADX not only reverses the fall in rMCH mRNA, it even provokes a slight increase (123% of control). No change in rMCH mRNA length is observed after chronic stress or ADX and dexamethasone injection. These results provide evidence for a negative regulation of rMCH gene expression by stress and suggest a major role for glucocorticoids in a positive feedback control of rMCH gene activity.