Dietary supplementation of alpha-linolenic acid in an enriched rapeseed oil diet protects from stroke.

Populations of Western countries are severely deficient in omega-3 intake, both in the form of alpha-linolenic acid (ALA) and the Long Chain derivatives (LC-n-3), Eicosa-Pentaenoic-Acid and Docosa-Hexaenoic-Acid. Omega-3 insufficiency is a risk factor for cardiovascular and cerebral diseases such as coronary heart disease and stroke. Stroke is a major cause of mortality and morbidity, and induces a significant socioeconomic cost and a marked increase in patient/family burden. To date, preventive treatments and neuroprotective drugs identified in preclinical studies failed in clinical trials, in part because of an inability to tolerate drugs at neuroprotective concentrations. Therefore testing alternative protective strategies, such as functional foods/nutraceuticals, are of considerable interest. We have previously demonstrated that a single injection of ALA reduced ischemic damage by limiting glutamate-mediated neuronal death, whereas repeated injections displayed additive protective benefits as a result of increased neurogenesis, synaptogenesis and neurotrophin expression. Because intravenous injections are not a suitable long-term strategy in humans, the present study investigated the effect of ALA supplementation by an experimental diet containing rapeseed oil (RSO, a rich source of ALA) as the only source of lipids for stroke prevention. We tested several experimental diets which included 5, 10, and 20% RSO-enriched diet and feeding paradigms (fresh diet was provided once or twice a week for 4 or 6 weeks). Our results showed that ALA supplemented diets are more sensitive to lipid peroxidation than a regular chow diet. Because the diet affected feeding behavior and animal growth, we defined concrete guidelines to investigate the effect of omega-3 supplementation on neuropathology. Among the different sets of experiments, animals fed with 10% and 20% RSO-enriched diet displayed a reduced mortality rate, infarct size and increased probability of spontaneous reperfusion in the post-ischemic period. In addition, a drastic reduction of lipid peroxidation levels was observed in the ischemic brain of RSO-fed animals. Overall, our findings provide new insights into the potential of employing rapeseed oil as a functional food/nutraceutical aiding in stroke prevention and protection.

Birth of two chimeric genes in the Hominidae lineage.

How genes with newly characterized functions originate remains a fundamental question. PMCHL1 and PMCHL2, two chimeric genes derived from the melanin-concentrating hormone (MCH) gene, offer an opportunity to examine such an issue in the human lineage. Detailed structural, expression, and phylogenetic analysis showed that the PMCHL1 gene was created near 25 million years ago (Ma) by a complex mechanism of exon shuffling through retrotransposition of an antisense MCH messenger RNA coupled to de novo creation of splice sites. PMCHL2 arose 5 to 10 Ma by an event of duplication involving a large chromosomal region encompassing the PMCHL1 locus. The RNA expression patterns of those chimeric genes suggest that they have been submitted to strong regulatory constraints during primate evolution.

Stromal-cell-derived factor 1alpha /CXCL12 modulates high-threshold calcium currents in rat substantia nigra.

Dopaminergic neurons of the substantia nigra constitutively express the CXCR4 receptor for the chemokine stromal-cell-derived factor 1alpha (CXCL12) but, to date, no direct effect of CXCR4 activation by CXCL12 on membrane conductance of dopaminergic neurons has been demonstrated. We tested the effects of CXCL12 on whole-cell currents of dopaminergic neurons recorded in patch clamp in substantia nigra slices and showed that CXCL12 (0.01-10 nm) increased the amplitude of total high-voltage-activated (HVA) Ca currents through CXCR4 activation. This effect was reversibly reduced by varpi-conotoxin-GVIA, suggesting that CXCL12 acted on N-type Ca currents, known to be involved in dopamine (DA) release. We therefore investigated the effects of CXCL12 on DA release from cultured dopaminergic neurons from the rat mesencephalon. In basal conditions, CXCL12 alone had no effect on DA release. When neurons were depolarized with KCl (20 mm), and thus when HVA Ca currents were activated, low CXCL12 concentrations (1-50 nm) increased DA release via CXCR4 stimulation. These data strongly suggest that the chemokine CXCL12 can act directly as a neuromodulator of dopaminergic neuronal electrical activity through the modulation of HVA currents.

Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5' region.

We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression. Another such site, located about 4,000 base pairs upstream from the AFP gene, seems to be correlated with the tissue specificity of the cells. DNase I-hypersensitive sites were mapped by using the indirect end-labeling technique with cloned genomic DNA probes. Three tissue-specific DNase I-hypersensitive sites were mapped in the 5' flanking region of the AFP gene when this gene was transcribed. Similarly, three tissue-specific DNase I-hypersensitive sites were detected upstream from the albumin gene in producing cell lines. In both cases, the most distal sites were maintained after cessation of gene activity and appear to be correlated with the potential expression of the gene. Interestingly, specific methylation sites are localized in the same DNA region as DNase I hypersensitive sites. This suggests that specific alterations of chromatin structure and changes in methylation pattern occur in specific critical regulatory regions upstream from the albumin and AFP genes in rat hepatoma cell lines.

Structure of the human melanin concentrating hormone mRNA.

The melanin-concentrating hormone (MCH) is a cyclic neuropeptide which induces skin paling and may be involved in the control of the pituitary adrenal axis in teleost fishes. We have recently cloned and characterized the salmon and rat MCH mRNAs and we report in the present paper the cloning and sequencing of the human counterpart. The deduced human MCH (hMCH) precursor is 165 amino acids long and as for rat and salmon, encodes the MCH peptide at the C-terminus. The human and rat MCH precursors are very similar to one another but differ extensively from the salmon counterpart. Strong sequence conservation was found in the regions of mammalian prohormones encoding the novel putative neuropeptides named NGE and NEI which we had originally identified in the rat MCH precursor. Furthermore, sequence identities, with perhaps functional implications, were found among the MCH, human ANF, and aplysia peptide A hormone precursors.

The rat melanin-concentrating hormone gene encodes an additional putative protein in a different reading frame.

The sequence of a novel melanin-concentrating hormone (MCH) transcript, composed of precisely joined exon I and exon III of the MCH gene, has been elucidated by RT-PCR analysis of hypothalamic RNA isolated from different rat strains. The deduced amino acid sequence predicted that it encodes in the exon I the same N-terminal moiety as MCH precursor part but diverges downstream, exon III being translated in a different reading frame than the MCH mRNA. This putative chimeric protein has been named MCH(M)-gene(G)-overprinted (O)-polypeptide(P). A peptide of 14 amino acids could be generated after cleavage at a basic site. Immunocytochemistry studies, using MGOP- and MCH-specific antisera, revealed overlapping expression in the dorsolateral hypothalamus. This suggests that MGOP or derived peptide(s) would participate in modulating the effect of pro-MCH generated peptides in the rat brain.

Impaired processing of brain proneurotensin and promelanin-concentrating hormone in obese fat/fat mice.

Mice homozygous for the fat mutation exhibit marked hyperpro-insulinemia and develop late onset obesity. The fat mutation was recently mapped to the gene encoding carboxypeptidase E (CpE), a processing enzyme involved in trimming C-terminal paired basic residues from prohormone-derived peptides. The mutation resulted in a loss of CpE activity that correlated with aberrant proinsulin processing. Neurotensin (NT) and melanin-concentrating hormone (MCH) are two neuropeptides that, among other central effects, inhibit food intake. Here, using RIA techniques coupled to reverse phase HPLC, we analyzed the processing products derived from the NT and MCH precursors in the brain of +/fat and fat/fat mice. Compared to control hypothalamic and brain extracts, fat/fat extracts had markedly reduced levels (>80%) of NT and neuromedin N (NN), another active pro-NT-derived peptide. In contrast, they exhibited high concentrations of biologically inactive NT-KR and NN-KR (NT and NN with a C-terminal Lys-Arg extension), two peptides that were undetectable in control extracts. MCH, which is located at the C-terminus of its precursor, was present in 2- to 3-fold higher amounts in fat/fat than in +/fat hypothalamus. Neuropeptide-Glu-Ile, another pro-MCH-derived neuropeptide separated from MCH by an Arg-Arg sequence, was present in amounts similar to those of MCH in control extracts. In contrast, neuropeptide-Glu-Ile was more than 10 times less abundant than MCH in extracts from obese mice. Our data are consistent with a deficit in CpE activity affecting the maturation of both pro-NT and pro-MCH. This suggests that abnormal neuropeptide and hormone precursor processing is a general phenomenon in fat/fat mice and supports the idea that defects in the production of neuropeptide involved in the control of feeding might lead to the development of obesity in these animals.

Similarities in cellular expression and functions of melanin-concentrating hormone and atrial natriuretic factor in the rat digestive tract.

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.

Rat melanin-concentrating hormone messenger ribonucleic acid expression: marked changes during development and after stress and glucocorticoid stimuli.

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.

The rat melanin-concentrating hormone messenger ribonucleic acid encodes multiple putative neuropeptides coexpressed in the dorsolateral hypothalamus.

The melanin-concentrating hormone (MCH) is a cyclic neuropeptide, first isolated from salmon pituitary glands, which regulates melanin dispersion in the skin and perhaps the activity of the pituitary-adrenal axis in teleost fish. We have recently purified and characterized rat MCH (rMCH) and report here the cloning and sequencing of specific MCH cDNA isolated from a rat hypothalamic library. The sequence of rMCH found by DNA sequencing confirms the sequence deduced from the purified peptide. rMCH is located at the C-terminus of a protein precursor of 165 amino acid residues. Comparison of the amino acid sequence of prepro-MCH and that of the Aplysia peptide-A prohormone suggests that these proteins as well as other precursors may be evolutionarily related. Besides rMCH, two putative neuropeptides, termed NGE and NEI, might be generated from the same precursor. The rMCH precursor shared sequence identities with human GH-releasing factor and mammalian CRF in the regions encoding NGE and NEI. By immunohistochemical studies we have established that the amidated C-terminus of NEI is recognized by some alpha MSH and rat CRF antisera and that the C-terminal portion of NGE is responsible for the cross-reactivity revealed with one hGRF-(1-37) antiserum. Our results explain the staining of a discrete population of dorso-lateral hypothalamic neurons by heretofore seemingly unrelated antisera and provide evidence for the production of multiple novel neuropeptides from a common precursor.

The melanin-concentrating hormone system of the rat brain: an immuno- and hybridization histochemical characterization.

In addition to a nonadecapeptide homologous to the teleost melanin-concentrating hormone (MCH), the amino acid sequence predicted from a rat prepro-MCH (ppMCH) cDNA suggested that at least one (neuropeptide EI, or NEI), and possibly a second (NGE), additional neuropeptide may be encoded by this precursor. Cross-reactivity with epitopes of NEI or NGE can account for reported localization of alpha-MSH, rat CRF, and human GRF in rat dorsolateral hypothalamic neurons. We have used antisera raised against rat MCH and NEI in immunohistochemical studies at the light and electron microscopic levels, along with hybridization histochemical localization of ppMCH mRNA, to define the organization of this system. As expected, ppMCH mRNA is prominently expressed in cells in the lateral hypothalamic area and zona incerta. The MCH and NEI peptides were extensively colocalized in neurons in both of these areas. In addition, smaller cell groups in the olfactory tubercle and pontine tegmentum were also positively hybridized for ppMCH mRNA and immunostained for MCH and NEI. Fibers stained for MCH and NEI were similarly, and very broadly, distributed throughout the central nervous system in patterns that generally conformed with known projection fields of the lateral hypothalamic area and zona incerta. A differential distribution was seen in at least one region, the interanterodorsal nucleus of the thalamus, which contained a prominent terminal field stained for MCH but not NEI. At the electron microscopic level, MCH-stained perikarya displayed a prominent staining associated with the Golgi apparatus; this was not encountered in NEI-stained cells. Both peptides were distributed similarly in terminals in the lateral hypothalamic area and median eminence, with staining associated principally with dense-cored vesicles. The results suggest that ppMCH-derived peptides may serve as neurotransmitters or modulators of prominence in a surprisingly expansive projection field of incerto-hypothalamic neurons. The terminal distributions of this system seem most compatible with functional roles in generalized arousal and sensorimotor integration, processes previously implicated as being subject to modulation by the lateral hypothalamic area.

The regulation of albumin and alpha-fetoprotein gene expression in mammals.

Albumin and alpha-fetoprotein (AFP) are two plasma proteins synthesized by the liver and the yolk sac. The production of these major proteins is subject to considerable and characteristic variations during both the course of development and hepatic carcinogenesis. It is therefore a system of choice for the analysis of genetic expression during normal differentiation and the cancerous state of eukaryotic cells. The knowledge of regulatory mechanisms at the cellular and molecular levels of the albumin and AFP genes has recently made great progress: 1) the cells which are responsible for the synthesis of albumin and AFP in the liver and other organs have been defined by conjointly using in vitro and in vivo molecular hybridization techniques; 2) the organization of these genes and their adjoining regions has been established in the rat, the mouse and man; 3) the level at which the synthesis of these two proteins is regulated has been determined; it is the transcriptional level. The transcriptional regulation of the albumin and AFP genes could be the result of genome and/or chromatin conformation level modifications. Different groups have shown that: 1) the global structure of the albumin and AFP genes does not change during the course of development and hepatic carcinogenesis; 2) modifications at the level of the methylation of certain specific cytosines could be associated with the variations in the transcription of these genes; 3) global or local (hypersensitive sites with DNase I) changes of chromatin conformation could be correlated to the potential or the overt activity of the transcription of these genes. Very recently certain 'regulatory' regions having cis 'enhancer' or 'silencer' properties have been detected upstream from the albumin and AFP genes. These regions are hypothesized to be DNA 'target' sequences on which trans-acting regulatory factors are fixed and which control the transcription of these genes. Starting from the framework of this recent work, a model of albumin and AFP gene regulation is proposed.

Differential regulation of melanin-concentrating hormone gene expression in distinct hypothalamic areas under osmotic stimulation in rat.

Melanin-concentrating hormone and associated peptides represent a novel peptide neuronal system that may be involved in the control of water homeostasis in mammals. We have examined the effect of 24 h dehydration or salt-loading over a period of six days, on melanin-concentrating hormone messenger RNA levels in rat brains by using complementary methods of Northern blotting and in situ hybridization histochemistry. In response to one to six day salt-loading regimen, hypothalamic melanin-concentrating hormone messenger RNA content in male or female rats decreased by two to three-fold. Levels of melanin-concentrating hormone messenger RNA in the hypothalamus were also dramatically decreased following dehydration in female rats whereas contrasting responses were noted in male rats. In addition, no significant variation in the low levels of melanin-concentrating hormone gene transcripts in medulla pons and cortex was found after osmotic stimulus. In agreement with Northern blot data, in situ hybridization studies revealed that the majority of the melanin-concentrating hormone-expressing neurons in the anterior part of the lateral hypothalamus of dehydrated or salt-loaded rats expressed lower amounts of melanin-concentrating hormone messenger RNAs than those found in control rats. Interestingly, less variation was found in the posterior part of the lateral hypothalamus. Furthermore few clusters of cells, located in zona incerta and near the internal capsula and fornix, increased their contents in melanin-concentrating hormone messenger RNA in salt-loaded but not in dehydrated rats suggesting that melanin-concentrating hormone gene expression may be regulated differently by various osmotic stimuli. Finally, diurnal variations in melanin-concentrating hormone messenger RNA contents were observed in normal and dehydrated rats with highest levels around 22.00 h and lowest levels during daylight hours. However, the up-regulation of melanin-concentrating hormone gene activity at night was found lower in dehydrated rats than in control animals suggesting that osmotic stress may interfere with the generation of the diurnal pattern of melanin-concentrating hormone messenger RNA expression. Altogether, our results indicate that osmotic stimulations lead to a selective and conspicuous inhibition of melanin-concentrating hormone gene activity in the whole hypothalamus of rat. We suggest that the melanin-concentrating hormone neuronal system plays an important role in integration processes relative to nocturnal regulation of water homeostasis and drinking behavior.

Hypothalamic melanin-concentrating hormone and alpha-neoendorphin-immunoreactive neurons project to the medial part of the rat parabrachial area.

Neurons in the middle and posterior parts of the lateral hypothalamus project to the parabrachial area, and in particular to the gustatory relay-station located in the medial part of this area. In the present study we have examined some of the neuropeptide immunoreactivities of the lateral hypothalamus neurons that project to the gustatory region of the parabrachial area. By coupling retrograde transport and immunohistochemistry, we found that 50-60% of medial parabrachial area-projecting cells located in the juxta-capsular region of the posterior lateral hypothalamus are labeled by rat melanin-concentrating hormone antiserum, while 28% of the retrogradely labeled neurons located in the perifornical lateral hypothalamus are visualized with alpha-neoendorphin antiserum. Moreover, a large number of terminals distributed throughout the parabrachial nucleus are immunoreactive to melanin-concentrating hormone or alpha-neoendorphin antisera. These immunoreactivities are not co-localized within the same lateral hypothalamic neurons. The potential role of these peptidergic projections in the reward mechanisms elicited in the medial parabrachial area and in the control of palatability is discussed.

Melanin-concentrating hormone is a potent anorectic peptide regulated by food-deprivation and glucopenia in the rat.

Melanin-concentrating hormone is a cyclic nonadecapeptide that is produced almost exclusively in neurons of the lateral hypothalamus and sub zona incerta areas while fibers are widespread in the rat brain. Such a localization strongly suggests that this peptide might participate as neurotransmitter/neuromodulator in the control of feeding behavior. In this study we examined first the influence of rat melanin-concentrating hormone on feeding behavior at different times either in the light or in the dark period (light off at 18.00 h) of the day in fed Wistar rats. Intracerebroventricular injection of rat melanin-concentrating hormone (1-100 ng per rat) at 18.00 h reduced food consumption as early as 2 h after injection and for the next 24 h. In addition, similar anorectic effect was noted after bilateral administration of 1 ng melanin-concentrating hormone into the lateral hypothalamic area at 11.30 h but not at 16.30 h. These findings strongly suggest that rat melanin-concentrating hormone may exert inhibitory control over food intake behavior depending on the circadian rhythm. Second, we investigated the modifications induced by food deprivation/refeeding on melanin-concentrating hormone messenger RNA levels in Wistar rats. Total RNA was isolated from whole hypothalamic dissections and the contents of melanin-concentrating hormone messenger RNA, beta-actin messenger RNA (taken as sample control) and neuropeptide Y messenger RNA (taken as control of food-deprivation paradigms) were assessed by using northern blotting. The time-course of messenger RNA expression was determined in groups of rats deprived for 24, 48 and 72 h and revealed a three-fold induction of melanin-concentrating hormone messenger RNA by 24 and 48 h, with reduced increase at 72 h. As expected, the same treatment led to a three-fold increase in neuropeptide Y messenger RNA content by 48 and 72 h. Refeeding groups of animals for up to 72 h after 24 h of food deprivation resulted in full restoration of melanin-concentrating hormone messenger RNA levels by 24 h. Strikingly, a large range of variations in melanin-concentrating hormone messenger RNA content between individuals was observed in food-deprived versus controls or refed rats suggesting that genetic or environmental factors may alter response in melanin-concentrating hormone gene activity after food deprivation. Finally, we investigated the effects of short-term glucoprivation induced by intraperitoneal administration of either 2-deoxy-D-glucose or insulin on melanin-concentrating hormone messenger RNA expression. A transitory increase in melanin-concentrating hormone messenger RNA content was noted 1 h after 2-deoxy-D-glucose injection while melanin-concentrating hormone messenger RNA levels rose two-fold only 5 h after insulin treatment. These results indicate that 2-deoxy-D-glucose and insulin activate melanin-concentrating hormone gene expression through likely distinct regulatory pathways.

The melanin-concentrating hormone: from the peptide to the gene.

Melanin-concentrating hormone (MCH) is a cyclic peptide originally isolated from chum salmon pituitaries, then structurally characterized from rat hypothalami. In the fish, MCH induces melanin concentration within the melanophores and may inhibit ACTH secretion in vitro and under stressful conditions in vivo. In the rat, MCH modulates ACTH release in vivo and oxytocin secretion in vitro. However, on the basis of neuroanatomical features, that is, cell bodies almost exclusively confined to the lateral area of the hypothalamus and the zona incerta, while fibers are observed throughout whole rat or human brains, this peptide appears to participate as a neurotransmitter/neuromodulator in the control of goal-oriented behaviors and/or general arousal in mammals. The knowledge of structural and regulatory features of the MCH precursor, mRNA, and genes at the cellular and molecular levels has recently made great progress. (1) The cells expressing MCH and associated peptides have been defined conjointly using radioimmunoassay, immunocytochemistry, and in vitro and in vivo molecular hybridization techniques. (2) The organization of the precursor deduced from cDNA cloning has been established and led to the discovery of two novel putative peptides named NEI and NGE. (3) The regulation of MCH mRNA and peptide production has been explored during the course of development in rodent and human and under a variety of paradigms (neurogenic or osmotic stress, hormonal stimuli, etc.). (4) The structure of the MCH genes has been determined in salmon, rat, mouse, and human and revealed striking exon-intron organization differences between fish and mammals. Strong homology, with a likely functional implication, was found between salmon MCH mRNA and 7SL RNA, a structural RNA involved in protein translocation. Furthermore, a variant gene that may encode slightly different MCH was found exclusively in primates. (5) Chromosomal assignment of the authentic and variant MCH genes in rat and human indicates that these genes may be good candidates involved in neurodegenerative or psychiatric disorders. Based on the framework of these studies, a working model of MCH regulation/function in mammalian brain is finally proposed.

Isolation and characterization of the human melanin-concentrating hormone gene and a variant gene.

Melanin-concentrating hormone (MCH) is a cyclic peptide found expressed almost exclusively in the hypothalamus while MCH-containing fibers project throughout the brain of many vertebrates including man. In fishes, MCH induces melanin concentration within the melanophores and may inhibit ACTH secretion. In mammals, MCH modulates ACTH release in vivo and participates as a neuromediator in the control of complex behaviors such as water and food intake. Salmon, rat and human MCH cDNAs have been cloned and structures of deduced mRNAs and precursors have been elucidated. In this report we determine the nucleotide sequence of two human MCH (hMCH) genes and demonstrate that both genes are expressed in human brain. Cloning from three genomic libraries and sequencing of one class of hMCH genomic DNA reveal high similarity between coding regions and the C-terminal part of the hMCH prohormone. However no sequence identity was found in the N-terminal and 5' end non-coding regions of the gene between them even within 6.5 kilobases (kb) upstream from the truncation point. Using polymerase chain reaction (PCR) analysis we have identified RNA populations that are derived from this gene in human brain. For that reason, this gene is a variant rather than a pseudogene. The authentic hMCH gene could only be cloned by using the PCR technique. With primers specific to 5'-end and 3'-end regions of the MCH mRNA we amplified a 1400 bp fragment as well as other shorter PCR products from human genomic DNA. The longest PCR fragment contains 3 exons encompassing most of the 5' untranslated and all of the coding and 3' untranslated sequences of the hMCH mRNA, that are separated by two introns of 350 and 271 bp, respectively. Interestingly the second intron dissects the hMCH peptide sequence in both the authentic and the variant gene. A strikingly high degree of homology was found between the variant and authentic hMCH genes, including intronic sequences, suggesting that these two genomic sequences diverged very recently during evolution. A strong homology was also noted between the exons and intervening sequences of the human and rat MCH genes. Altogether, our results provide the first strong evidence for the existence of two distinct MCH genes expressing prohormones with different MCH and neuropeptide EI (NEI) sequences in human and along with in vivo and in vitro findings, suggest that these neuropeptides may influence the activity of numerous mammalian neuronal systems.

Lithium increases melanin-concentrating hormone mRNA stability and inhibits tyrosine hydroxylase gene expression in PC12 cells.

Melanin-concentrating hormone (MCH) is a cyclic peptide involved in the regulation of food-intake behaviour and stress response in mammals. Expression of the MCH gene predominates in hypothalamic neurons. Mechanisms governing the regulation of expression of MCH gene in established cell lines were not explored yet. Here, we analysed the actions of nerve growth factor (NGF), dexamethasone, forskolin and lithium on MCH mRNA levels in the PC12 pheochromocytoma cell line. We compared them with those observed on tyrosine hydroxylase (TH) mRNA, constitutively expressed in PC12 cells, and neurotensin (NT) mRNA, taken as a control. In untreated cells, MCH RNA species of high molecular weight were found. Exposure of cells at a combination of NGF and lithium resulted in decreased expression of these MCH RNAs and in the transient production of mature MCH mRNA. Strikingly, after short exposure of PC12 cells to NGF, lithium per se elicited a marked increase in MCH mRNA levels whilst it exerted a potent inhibitory action on TH mRNA expression. Detailed investigations revealed that lithium enhanced MCH mRNA expression through post-transcriptional mechanisms whereas it regulated TH gene expression mainly at the level of transcription. These results demonstrate that lithium, an agent widely used for treatment of manic depressive illness, can exert an opposite effect on MCH and TH mRNA production in PC12 cells. The MCH gene system in NGF-treated PC12 cells provides a good opportunity for studying the effect of lithium on gene expression at post-transcriptional levels in a neuron-like cellular model.

The melanin-concentrating hormone gene in human: flanking region analysis, fine chromosome mapping, and tissue-specific expression.

Genomic sequences encoding the human melanin-concentrating hormone (MCH) were isolated from a YAC library and subcloned in pUC vector using a novel E. coli transformation method. A 4.1-kb fragment encompassing approximately 1.0 kb of the 5'-end-flanking region, the three exons-two introns of the coding region and approximately 1.7 kb of the 3'-end-flanking region, was sequenced. Comparison with the rat MCH gene indicated strong conservation in the 5'-flanking region, in particular over the putative TATA box, CAAT box, GRE and AP-1 elements that could potentially regulate MCH gene expression. FISH with a fluorescent MCH genomic probe on human chromosomes and PCR analysis of a YAC panel mapped MCH to chromosome 12q23.1 in a region flanked by D12S1074 and D12S1030 markers. Expression of the MCH RNA species and pro-MCH-derived peptides (MCH and NEI) was investigated in human tissues by combining Northern blotting, RT-PCR, in situ hybridization, immunohistochemistry and RIA. In the human brain, MCH mRNA and MCH/NEI peptides were predominantely expressed in the lateral hypothalamus in agreement with the known distribution of MCH expression in rat. In addition, MCH gene products were detected in extra-hypothalamic sites, such as the pallidum, neocortex and cerebellum. In peripheral tissues, MCH mRNA was identified in several organs, including the thymus, brown adipose tissue, duodenum and testis. An additional shorter MCH gene transcript, likely the result of alternate splicing, was revealed in several brain areas and peripheral tissues. While only fully processed MCH and NEI were found in hypothalamus, a different peptide form, bearing MCH and NEI epitopes, was detected in peripheral organs. This represents the first evidence for differential processing of pro-MCH in mammals.

Identification of a Single Melanin-Concentrating Hormone Messenger Ribonucleic Acid in Coho Salmon: Structural Relatedness with 7SL Ribonucleic Acid.

Abstract Melanin-concentrating hormone (MCH) is a cyclic neuropeptide possessing antagonistic function to alpha-melanocyte-stimulating hormone and corticotropin-releasing factor in the control of melanosome dispersion within melanophores and adrenocorticotropin release in fish. We have isolated and characterized MCH cDNAs from coho salmon (Oncorhyncus kisutch). The precursor protein predicted by the longest cDNA consists of 132 amino-acids with a characteristic signal peptide at the N-terminus and the biologically active salmon MCH (sMCH) peptide at the C-terminus. The coho sMCH mRNA and protein sequences are very similar but not identical to the previously reported chum or chinook salmon counterparts, suggesting the existence of species polymorphism. Sequence similarities were revealed between alpha-melanocyte-stimulating hormone and part of the C-terminal domain of sMCH precursor. Two sMCH genes were found in coho salmon. By contrast to other salmon species, only one major sMCH mRNA was detected in coho species suggesting that differential MCH gene expression might occur in salmon. In addition, under low stringency oligoprobes complementary to the sMCH RNA recognize a 0.3 kb RNA which was identified as the 7SL RNA. The regions conserved between those RNAs fold in a similar secondary structure. These similarities might reflect common ancestry which may have functional significance.