Introduction

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The rat/human melanin-concentrating hormone (MCH) consists of a 19 amino acid sequence presenting a high homology with the fish peptide originally described (Kawauchi et al., 1983; Vaughan et al., 1989; Nahon, 1994). Immunolocalization and/or in situ hybridization studies in rat have revealed the existence of MCH in the perikarya of lateral hypothalamus and the zona incerta, and fibers innervating the entire brain (Bittencourt et al., 1992). MCH mRNA and hormone precursor expression were also found in peripheral tissues (Hervieu and Nahon, 1995; Hervieu et al., 1996a,b), suggesting implication of this peptide in various physiological functions (Nahon, 1994). In the rat brain, MCH is expressed in the lateral hypothalamus, an area playing a central role in the control of energy homeostasis, feeding behavior, and body weight. Indeed, hypothalamic MCH messenger expression is increased by fasting in wild-type as well as in ob/ob mice, and intracerebroventricular injection of MCH stimulates food intake in rats (Qu et al., 1996; Rossi et al., 1997, 1999; Sahu, 1998; Ludwig et al., 1998) and mice (O. Della Zuana, unpublished data). In addition, transgenic mice that overexpress the MCH peptide are obese and developed particularly marked hyperphagia in response to high fat diet (Ludwig et al., 2001). Importantly, and, contrasting with other orexigenic factors such as neuropeptide Y or orexin (Erickson et al., 1996; Chemelli et al., 1999), MCH is the only peptide for which gene disruption results in hypophagic and lean mice (Shimada et al., 1998). There is thus a large body of evidence suggesting a critical role for MCH in food intake behavior and metabolism control in rodents (Tritos et al., 1998; Tritos and Maratos-Flier, 1999).

Most of the MCH functions have been investigated in the absence of information concerning its receptor (for review, see Nahon, 1994; Baker, 1994). Indeed, the MCH receptor has only been recently identified (Chambers et al., 1999; Lembo et al., 1999; Saito et al., 1999; for review, see Saito et al., 2000) through reverse pharmacology as the SLC-1 orphan G protein-coupled receptor (Kolakowski et al., 1996; Lakaye et al., 1998). This receptor is strongly and widely expressed in the rat brain and its distribution is in good agreement with that of the MCH peptide (Bittencourt et al., 1992; Hervieu et al., 2000). To characterize the MCH activity at its receptor, several structure-activity relationship studies based on salmon MCH analogs had been performed in the past, using fish, reptilian, or batracian bioassays (Kawazoe et al., 1987; Wilkes et al., 1984; Matsunaga et al., 1989). Recently, the availability of, on one hand, the human cloned receptor, and, on the other, in vitro functional assays have allowed the description of a wide diversity of MCH analogs of various potencies (Audinot et al., 2001a). In this study, the MCH fragment MCH_6-17 (Table 1) was shown to be the minimal sequence keeping potent biological activity. Designed from this sequence, S36057 (Table 1) has been found to be the most powerful MCH agonist, which, as a radioligand, represents a more potent, less hydrophobic, and more stable tool than [¹²⁵I][3-iodo Tyr¹³]-MCH itself (Audinot et al., 2001b).

The aim of the present study was to investigate the implication of the SLC-1 receptor in mediating the effects of MCH upon food intake in rat. We thus selected MCH analogs and tested them with rat recombinant SLC-1 receptor both for their functional activity (inhibition of intracellular cAMP) and their binding affinity using [¹²⁵I]S36057 as a radioligand. Furthermore, these analogs, injected i.c.v. in rats, were evaluated upon food intake. Indeed, there was a good correlation between their effects on feeding behavior and results obtained at the rat recombinant receptor, strongly suggesting that SLC-1 receptor mediates the central effects of MCH on food intake.

	Experimental Procedures

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Materials

Most of the natural and modified peptides used in these studies were custom-made by Neosystem SA (Strasbourg, France). They were all at least 95% pure as assessed by high-performance liquid chromatography and mass spectrometry (Audinot et al., 2001a). The amino acid structures of human, rat, mouse MCH, salmon MCH and MCH analogs used in these studies are shown in Table 1. Total RNA of heart, brain, lung, liver, and kidney obtained from a pool of Sprague-Dawley rats, as well as rat genomic DNA were obtained from CLONTECH (Palo Alto, CA). Total RNA of thymus, testis, and ovary were obtained from of a pool of Sprague-Dawley rats, purchased from Ambion (Austin, TX).

Cloning of Rat SLC-1 Receptor

Poly(A⁺) RNA from a pool of rat hypothalami was reverse transcribed with oligo(dT)_12-18 using reverse Transcriptase Superscript II (Invitrogen, Cergy Pontoise, France). First-strand cDNA (corresponding to 1 µg of total RNA) was subjected to 35 cycles of amplification using primers based on GenBank entry (accession no. AF008650) describing the rat SLC-1 receptor (rSLC-1; forward primer 23-43 and the reverse primer 1065-1084). After an initial cycle of denaturation at 94°C for 1 min, PCR was carried out for 35 cycles with the following cycle conditions: 94°C, 1 min; 55°C, 1 min; and 72°C, 3 min with a postincubation of 72°C for 7 min. The expected 1061-base pair fragment was isolated and inserted into pcDNA3.1(+) (Invitrogen, Carlsbad, CA). The recombinant plasmid pcDNA3.1-rSLC-11 was sequenced on both strands by automated sequencing (ABI 377; Applied Biosystems, Foster City, CA).

Tissue Expression of Rat SLC-1 Receptor Messenger RNA

First-strand cDNA from different rat tissues (corresponding to 1 µg of total RNA) was subjected to 30 cycles of amplification using the primers based on GenBank entries (accession no. AF008650) for the rat SLC-1 receptor (forward primer 23-43 and the reverse primer 384-403). PCR products were separated by agarose (1%) gel electrophoresis and transferred onto Hybond N+ membranes (Amersham Pharmacia Biotech, Orsay, France). Hybridization was performed at 42°C using a ³²P-labeled oligoprobe (214-236).

Establishment of Stable Cell Line Expressing Rat SLC-1 Receptor

HEK293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. They were seeded at 5 × 10⁶ cells in a 75-cm² culture flask. Twenty-four hours later, the cells were transfected with 10 µg of the recombinant plasmid pcDNA3.1-rSLC-1, using LipofectAMINE as described by the manufacturer (Invitrogen). The day after transfection, cells were trypsinized, resuspended in complete Dulbecco's modified Eagle's medium containing 800 µg/ml of geneticin, and seeded at different dilutions in 96-well plates, which were then kept for 2 to 3 weeks in a humidified CO₂ incubator. At the end of this selection period, isolated cellular clones were selected, amplified, and further characterized by cAMP experiments.

Intracellular cAMP Assay

Intracellular cAMP was measured using flashplates (SMP004; PerkinElmer Life Science Products, Boston, MA). In brief, forskolin (15 µM) and test peptides diluted in 0.1% bovine serum albumin were added to 96-well flashplates. Incubation was started with the addition of HEK293 cells stably expressing the rat SLC-1 receptor (35,000 cells/well). After 15 min at 37°C, the incubation was stopped by the addition of the revelation mix. Two hours later, plates were counted on a TopCount (Packard, Rungis, France).

Membrane Preparation

Cell lines stably expressing the rat SLC-1 receptor were grown to confluence, harvested in phosphate-buffered saline containing 2 mM EDTA, and centrifuged at 1000g for 5 min (4°C). The resulting pellet was suspended in 20 mM HEPES, pH 7.5, containing 5 mM EGTA and homogenized using a Kinematica Polytron. The homogenate was then centrifuged (95,000g, 30 min, 4°C) and the resulting pellet suspended in 50 mM HEPES, pH 7.5, 10 mM MgCl₂, and 2 mM EGTA. Determination of protein content was performed according to the method of Lowry et al. (1951). Aliquots of membrane preparations were stored at 80°C until use.

[¹²⁵I]S36057 Binding

Membranes (10-25 µg/ml) were incubated for 90 min at room temperature in binding buffer (25 mM HEPES, pH 7.4, 1 mM CaCl₂, 5 mM MgCl₂, 0.5% bovine serum albumin) in a final volume of 250 µl containing, or where otherwise stated, 0.04 nM [¹²⁵I]S36057 (Amersham Pharmacia Biotech) and test peptides (Neosystem SA). Nonspecific binding was defined with 1 µM MCH. The reaction was stopped by rapid filtration through GF/B unifilters presoaked with 0.5% polyethyleneimine, followed by three successive washes with ice-cold buffer.

Central Peptide Administration

Animals. The feeding experiments in these studies were conducted using male Wistar rats weighing 325 to 350 g (Iffa Credo, L'arbresle, France). The animals were housed individually in wire-bottomed cages in a room with a 12-h light/dark cycle (lights on 7:30 AM) at 22 ± 30°C and 55% relative humidity. Six-millimeter-diameter food pellets of the following composition (67.5% food flour, 26.5% saccharose, 5% gum tragacanth, 1.25% magnesium stearate; A03 UAR, Orge, France) were available ad libitum. The pellets were available to the animals from a food hopper attached to the side of the cage. All animal procedures described in this study complied with French laws regulating animal experimentation (Decree no. 87-848 19 October 1987 and the ministerial Decree of 10 April 1988) and were also approved by the ethics committee of the Servier Research Institute.

Implantation of Intraventricular Cannulae. Rats were anesthetized with sodium pentobarbital (60 mg/kg i.p.; Sanofi, Libourne, France) and a stainless steel guide cannulae (Plastic Products Co., Roanoke, VA) stereotaxically implanted into the right lateral ventricle at the following coordinates relative to the bregma: AP, 0.8 mm; L, 1.2 mm; and V, 3.5 mm.

Peptide Injection. After a 7-day recovery period, during which the animals were handled every day to minimize nonspecific stress, they were lightly anesthetized with Forène (Abbott Laboratories, Queenborough, UK). MCH (4.4 nmol/kg; Bachem Biochimie SARL, Voisins-le-Bretonneux, France) was then slowly injected through the intraventricular cannulae over a 30-s period in a final volume of 5 µl. Immediately after injection, the animals quickly recovered and were returned to their home cages. Animals that exhibited a robust increase in food intake, i.e., indicating correct cannulae placement, were randomly assigned to different groups and studied in one of the following protocols.

Effect of Different Peptides on Spontaneous Food Intake. The injections were performed at 9:00 AM shortly after the beginning of the light phase. Each Wistar rat was injected i.c.v. either with artificial cerebrospinal fluid (aCSF, 5 µl) or with a single dose (1-10 nmol/kg in 5 µl) of peptide dissolved in aCSF. The animals were then returned to their home cage, which contained a known weight of food pellets in a spill-free cup. Measurements of food intake were made for 2 h after injection. For each animal, food spillage was quantified during each time period and food intake was corrected for this loss. At the end of the experiments, the animals were euthanized and the position of the cannulae assessed by the injection of 100 µl of Evans blue (2 mg/kg) followed by visual examination of the brain slices. Only data obtained from animals with correctly positioned cannulae were included in the final data analysis.

Data and Statistical Analysis

All statistical analyses were done with a 5% significance threshold. For in vitro data, IC₅₀ and K_I were estimated on the overall values by nonlinear regression using the program PRISM (GraphPad Software Inc., San Diego, CA). For displacement experiments, inhibition constants (K_i) were calculated according to the Cheng-Prusoff equation: K_i = IC₅₀/[1 + (L/K_D)], where IC₅₀ is the inhibitory concentration₅₀, L is the concentration, and K_D the dissociation constant of the radioligand. Difference of potency between IC₅₀ values or K_i values of two peptides can be statistically performed by comparing their confidence limits. For in vivo data, significance of effects was determined by a one way analysis of variance followed by a Newman-Keuls test for pairwise comparison.

	Results

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Expression of SLC-1 Receptor mRNA in Rat. We investigated the distribution of the rat SLC-1 receptor mRNA in several rat tissues using RT-PCR and Southern blotting (Fig. 1). By selecting the primers on either side of the splice junction, and performing a negative reaction consisting of RT-PCR without the addition of reverse transcriptase, it was possible to check that the samples were free of contamination with genomic DNA. A ³²P-labeled oligoprobe for the rat SLC-1 receptor transcript showed massive expression in brain, skeletal muscle, and ovary, and less expression in testis and adipose tissue (Fig. 1A). There was no detectable expression of the SLC-1 receptor mRNA in heart, liver, lung, kidney, and thymus. The hypothalamus also expressed mRNA for the SLC-1 receptor. This was not linked to genomic contamination, as attested by the higher molecular weight of the PCR product obtained with the rat genomic DNA (Fig. 1B).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Tissues distribution of SLC-1 receptor mRNA in the Sprague-Dawley rat. Analysis of total RNA from indicated rat tissues (A) and rat hypothalamus (B). Southern blot of RT-PCR amplicons from rat cDNA amplified with the 23 to 43 and 384 to 403 primers and revealed by the 32P-labeled oligoprobe 214 to 236 (+). Negative reactions of RT-PCR in the absence of reverse transcriptase addition were also performed to test genomic DNA contamination (). PCR product of genomic DNA obtained in the same conditions is larger than cDNA RT-PCR product.

Characterization of HEK293 Cell Line Stably Expressing Rat SLC-1 Receptor. To examine ligand binding properties and signal transduction through the rat SLC-1 receptor, the coding sequence was subcloned in the mammalian expression vector pcDNA3.1(+) and transfected into HEK293 cells. In HEK293 cells stably expressing the rat SLC-1 receptor coding sequence, MCH induced a dose-dependent inhibition of intracellular cAMP (Fig. 2), while no effect was observed in native cells (data not shown). A robust but transient elevation of intracellular calcium in the presence of MCH was also observed in transfected cells (data not shown). [¹²⁵I]S36057-specific binding to rat SLC-1 receptors was saturable (Fig. 3) and best fitted by a single-site analysis with a K_D of 0.029 ± 0.002 nM and a B_max of 1608 ± 122 fmol/mg of protein (n = 4), while, in contrast, no specific binding was detected in native cells (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Dose-dependent inhibition of forskolin-induced cAMP in HEK293 cells stably expressing the rat SLC-1 receptor. Effect of S36057, MCH, salmon MCH, linear MCH (C1), and minimal MCH agonist MCH6-17 at rat recombinant SLC-1 receptor. Peptides were tested for their potency to inhibit the forskolin-elevated levels of cAMP in rSLC-1/HEK cells. Data were pooled from all the experiments and were expressed as means ± S.E.M.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Saturation binding experiments and Scatchard plot of the specific binding of [125I]S36057 at membranes from HEK293 cells stably expressing the rat SLC-1 receptor. Saturation binding isotherm was obtained using [125I]S36057 in the concentration range of 5 to 280 pM. A, total binding (), nonspecific binding determined with 1 µM MCH (), and specific binding () are represented. B, Scatchard plot of the specific binding isotherm. Points shown are from a representative experiment performed in triplicate and repeated at least three times.

Characterization of MCH Analogs with Rat Recombinant SLC-1 Receptor: Effect on cAMP Levels. Peptides including the MCH analogs S36057, MCH, [F¹³;Y¹⁹]-MCH, salmon MCH, and the dodecapeptide MCH_6-17 dose dependently inhibited the formation of intracellular cAMP (Fig. 2, A and B; Table 2) with potencies in the nanomolar range. A second group of five peptides also showed some activity in this test, but it was approximately 10- to 1000-fold less potent compared with the first set of active peptides (Fig. 2; Table 2). Indeed, substitution of Val¹² or Arg¹⁴ by Ala (C3 and C5) appeared to be less deleterious than Met⁸ substitution (C2) or Cys substitutions by Ser residues leading to linear peptides (C1 and S36077). Finally, other modifications such as Tyr¹³ by Ala (C4) or Arg¹¹ by His (C6) substitution or amino acid deletion inside the cystine loop (C7 and C8) led to inactive compounds (Fig. 2C; Table 2). Results obtained in the cAMP assay at the rat receptor were highly correlated (r = 0.98, P < 0.0001, n = 10) with those previously reported with its human counterpart (Fig. 4).

View this table: [in this window] [in a new window]   TABLE 2 Characterization of MCH analogs in rat: in vitro potency and affinity at recombinant SLC-1 receptor and in vivo effects upon food intake In vitro results were obtained from HEK293 cells stably expressing the rat SLC-1 receptor. IC50 (inhibitory concentration50) represents the concentration of peptide inducing 50% of inhibition of forskolin-induced intracellular cAMP level. Ki (inhibition constant) values from [125I]S36057 binding experiments determined on membrane preparation. Results are expressed as the mean value of at least three separate determinations each performed in duplicate or triplicate and 95% confidence limits (CL). Effect upon food intake in rat, according to the statistical significance shown in Figs. 7 to 9, are represented in arbitrary units: very strong effect (++), strong effect (+), limited effect (+/), or inactivity ().

View larger version (10K): [in this window] [in a new window]   Fig. 4.   Correlation plot of the MCH analogs potencies in the cAMP assay, at rat or human SLC-1 receptors stably expressed in HEK293 cells. Potencies expressed as pIC50 (= logIC50) were calculated from Table 2 for the rat receptor and from Audinot et al. (2001a) for the human receptor. The correlation coefficient was r = 0.95 (P < 0.0001, slope = 0.85, n = 10).

Characterization of MCH Analogs at Rat Recombinant SLC-1 Receptor: Binding Affinities. Peptide compounds could be classified according to their binding affinity by a similar order to their functional efficacy in the cAMP assay. The first group, including the most potent peptides, displaced [¹²⁵I]S36057 binding (Fig. 5A; Table 2) with K_i values in the subnanomolar range. The second group of peptides (Fig. 5B; Table 2) exhibited affinities in the nanomolar range for C3 and C5 (Fig. 5B), while the less active peptides (C1, C2, and S36077) in the cAMP assay were also the less potent peptides in the [¹²⁵I]S36057 binding assay (Fig. 5B; Table 2). Finally, the third group of compounds included peptides that were inactive in the cAMP assay (C4, C6, C7, and C8) and also unable to displace [¹²⁵I]S36057 binding (Fig. 5C; Table 2). Data obtained from the cAMP assay were highly correlated (r = 0.97, P < 0.0001, n = 9) with those obtained from the binding test (Fig. 6).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Concentration isotherms of [125I]S36057-specific binding in the presence of MCH analogs at rat SLC-1 receptors stably expressed in HEK293 cells. Compounds are gathered according to their affinity: potent peptides (A), intermediate-to-weak peptides (B), and inactive peptides (C). Data were pooled from all the experiments and were expressed as means ± S.E.M.

View larger version (8K): [in this window] [in a new window]   Fig. 6.   Correlation plot of binding affinity and functional potency of MCH analogs at rat SLC-1 receptor stably expressed in HEK293 cells. Affinities expressed as pKi (= logKi) were determined in [125I]S36057 displacement experiments and pIC50 (= logIC50) in cAMP assay. Data were calculated from Table 2. The correlation coefficient was r = 0.98 (P < 0.0001, slope = 1.30, n = 10).

Changes in Food Intake after Acute Central Administration of Different Peptides. All these MCH analogs (with the exception of S36077), were evaluated in rat, upon the cumulative food intake 2 h following i.c.v. injection. A significant effect of these peptides was searched in the dose range of 2 to 10 nmol/rat. MCH-induced stimulation of food intake was apparent 1 h after i.c.v. injection (data not shown) and was sustained for 2 h (Fig. 7A). Similarly to MCH, salmon MCH also increased cumulative food intake (Fig. 7B). These two natural peptides defined a group of particularly active compounds upon food intake, which also included the three MCH analogs [F¹³;Y¹⁹]-MCH (Fig. 7C), MCH_6-17 (Fig. 7D), and S36057 (Fig. 7E). Effect of MCH_6-17 appeared more pronounced at least at the lowest dose (Fig. 7B). Some peptides stimulated food intake, but with less effect than compounds of the first group (Fig. 8). In this second group of compounds, C2 and C5 presented limited but significant effects on food intake (Fig. 8, A and B), although compounds C1 and C3 nonsignificantly stimulated food intake (Fig. 8, C and D). Finally, analogs (C4, C6, C7, and C8) weakly potent or inactive in vitro were also inactive in vivo upon food intake (Fig. 9). At the highest doses (8-10 nmol/rat), some of the tested compounds (MCH_6-17, C2, C3, C4, C5, C7, and C8) induced apparent behavioral side effects like "barrel rolling", hyperventilation, sedation, or epilepsy.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Effect of "potent" MCH analogs upon cumulative food intake 2 h after acute i.c.v. administration. Vehicle (CSF-control), MCH (A), salmon MCH (B), [F13;Y19]-MCH (C), MCH6-17 (D), and S36057 (E) (2-10 nmol/rat) were injected at the beginning of the light period in satiated male Wistar rats. Cumulative food intake, represented in grams, was measured 2 h after injection. Results are expressed as mean ± S.E. from 5 to 21 animals in each group. Statistics by one-way ANOVA followed by Newman-Keuls test at each time point: *P < 0.05 versus control group.

View larger version (27K): [in this window] [in a new window]   Fig. 8.   Effect of "mild" MCH analogs upon cumulative food intake 2 h after acute i.c.v. administration. Vehicle (CSF-control) or weak agonist compounds C2 (A), C5 (B), C1 (C), and C3 (D) (2-10 nmol/rat) were injected at the beginning of the light period in satiated male Wistar rats. Cumulative food intake, represented in grams, was measured 2 h after injection. Results are expressed as mean ± S.E. from 5 to 11 animals in each group. Statistics by one-way ANOVA followed by Newman-Keuls test at each time point: *P < 0.05 versus control group.

View larger version (21K): [in this window] [in a new window]   Fig. 9.   Effect of "inactive" MCH analogs upon cumulative food intake 2 h after acute i.c.v. administration. Vehicle (CSF-control) or compounds (C4, C6, C7, and C8) (2-10 nmol/rat) were injected at the beginning of the light period in satiated male Wistar rats. Cumulative food intake, represented in grams, was measured 2 h after injection. Results are expressed as mean ± S.E. from 5 to 11 animals in each group. Statistics by one-way ANOVA followed by Newman-Keuls test at each time point.

	Discussion

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Part of the evidences supporting an orexigenic role for MCH in food intake behavior came from the acute i.c.v. injections of MCH, its analog [F¹³;Y¹⁹]-MCH (Drozdz et al., 1995), or salmon MCH in rats and mice (Qu at al., 1996; Kokkotou et al., 2000; O. Della Zuana, unpublished data). In the present study we tried to determine whether the effects of MCH on feeding behavior were mediated through the described SLC-1 receptor. We first determined the functional and binding profiles of a range of MCH analogs in an in vitro model consisting of the rat SLC-1 receptor stably expressed in HEK293 cells. Effects of these MCH analogs were then evaluated upon food intake in rats. Finally, we studied the repartition of messenger RNA coding for the SLC-1 receptor in various rat tissues, and discuss the potential role of this receptor in the energy balance homeostasis, at the periphery.

SAR at Rat SLC-1 Receptor. Recently, a large study including 57 MCH analogs was performed with the human SLC-1 receptor, giving a great deal of information concerning MCH structure-activity relationships (Audinot et al., 2001a). There, it was shown that the minimal MCH sequence to retain potent biological activity was limited to the dodecapeptide MCH_6-17. Starting from this sequence, a large number of substitutions and/or deletions have been done to further assess MCH structure-activity relationships at the human receptor. In the present report some of these MCH analogs, from highly potent agonists to completely inactive compounds, were evaluated both at the recombinant rat SLC-1 receptor and in vivo upon food intake.

Potential Roles of SLC-1 Receptor at Periphery. We studied the expression of SLC-1 receptor in peripheral organs. SLC-1 receptor mRNA expression could not be detected in heart, liver, lung, kidney, or thymus. Contradictory to Saito et al. (1999), SLC-1 receptor expression was found in testis, probably because the PCR assay is more sensitive than the Northern blot approach. Expression of SLC-1 receptor found in reproductive organs reinforces the possible implication of MCH in reproductive functions and sexual behaviors (Hervieu et al., 1996a; Murray et al., 2000). We detected the presence of the SLC-1 receptor mRNA in adipose tissue and skeletal muscle. Indeed, a recent report has shown the presence of these receptor mRNAs in isolated adipocytes and the involvement of MCH peptide in the expression and secretion of leptin (Bradley et al., 2000). In addition, expression of both the peptide and the receptor in pancreas, as well as MCH regulation of insulin release in pancreatic cell lines, has been reported (Tadayon et al., 2000). Taken together, these results reinforce the hypothesis of the general implication of MCH and its cognate receptor in the regulation of energy balance with peripheral actions, in addition to the described effect in the central regulation of feeding (Tritos and Maratos-Flier, 1999).