Introduction

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MCH is a cyclic 17-amino acid peptide, which was originally isolated from the salmon pituitary gland. This peptide regulates changes in skin color of teleosts (Kawauchi et al., 1983). A 19-amino acid-long MCH-like peptide was later identified in rat and human hypothalami (Vaughan et al., 1989; Mouri et al., 1993). Mammalian MCH is encoded by a precursor of 165 amino acids, which also gives rise to one well characterized peptide, neuropeptide-glutamic acid-isoleucine (NEI), and another putative peptide, neuropeptide-glycine-glutamic acid (NGE) (Nahon et al., 1989; Presse et al., 1990; Parkes and Vale, 1992). In rodents, MCH is prominently expressed in the perikarya of the lateral hypothalamus and the sub zona incerta and projects broadly throughout the central nervous system (Skofitsch et al., 1985; Bittencourt et al., 1992). The same locations for MCH perikarya were found in human (Bresson et al., 1989; Elias et al., 1998). This widespread projection distribution suggested early that MCH might be used as neurotransmitters/neuromodulators in a number of neural functions (Baker, 1994; Nahon, 1994). Among these functions, the regulatory role of MCH in feeding behavior has been the most intensely studied. Evidence of a major role in food intake behavior include the observation of increased feeding behavior in rats after i.c.v. administration of MCH (Qu et al., 1996; Rossi et al., 1997) and the up-regulation of MCH mRNA during fasting in the rat (Presse et al., 1996; Hervé and Fellmann, 1997) and in obese ob/ob mice (Qu et al., 1996; Tritos et al., 2001). Mice lacking the MCH gene display reduced body weight due to hypophagia and an increase in metabolic rate (Shimada et al., 1998), whereas transgenic mice overexpressing MCH are obese and develop hyperphagia (Ludwig et al., 2001). All these data suggested an important role for MCH as an orexigenic peptide and supported a considerable interest in the potential for the development of therapeutic drugs.

Efforts to identify the MCH receptor led initially to the discovery of a spliced variant of the seven transmembrane G-coupled protein named SLC-1 (Kolakowski et al., 1996) as a cognate MCH receptor and thereafter referred to as MCH1 (Bächner et al., 1999; Chambers et al., 1999; Lembo et al., 1999; Saito et al., 1999; Shimomura et al., 1999). SLC-1 is localized in specific brain regions involved in the control of feeding behavior (Hervieu et al., 2000). Interestingly, MCH1-deficient mice are lean due to hyperactivity and altered metabolism (Marsh et al., 2002). Very recently, a second MCH receptor, named here MCH2 (or MCH₂, MCHR2, MCH-2R, or SLT) was identified and characterized in mammalian tissues (An et al., 2001; Hill et al., 2001; Mori et al., 2001; Rodriguez et al., 2001; Sailer et al., 2001; Wang et al., 2001). This MCH receptor displayed a brain distribution that overlapped partially with that of SLC1. Furthermore, in contrast to MCH1, which signals to either Gi or Gq, depending on the transfected cell systems, MCH2 signaling operates apparently exclusively through Gq protein (for review, see Boutin et al., 2002; Hervieu et al., 2002).

In the rat and human brain, exclusively mature peptides, i.e., cyclic MCH and amidated NEI, were found (Takahashi et al., 1995; Hervieu et al., 1996). In contrast, mature MCH and NEI were not found in peripheral organs but a large MCH-immunoreactive (IR) form was identified in human and mouse (Viale et al., 1997, 1999) (Fig. 1A). This large MCH-IR peptide encompassed the NEI and MCH sequences in its C terminus (Fig. 1B). The differences in pro-MCH-derived peptides between brain and peripheral organs resulted from differential processing of pro-MCH by pro-convertases (Viale et al., 1999). It was therefore tempting to speculate that these different pro-MCH-derived peptides may have distinct physiological functions and/or various pharmacological profiles at the MCH receptors.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Mammalian pro-MCH and its peptide products. A, schematic representation of the mammalian pro-MCH and of its peptide detected either in the brain or in other organs. Hatched lines indicate that the N-terminal part of the large MCH-containing form identified at the periphery was not yet established. B, sequence of the C-terminal part of the mammalian pro-MCH. The positions of the cleavage sites at pairs of basic residues (in boldface type) are noted by arrowheads. NEI and MCH are shown.

In this study, we have first compared the effect of pro-MCH_131-165 (referred to as NEI-MCH) versus mature MCH upon food intake behavior. We found that i.c.v. administration of NEI-MCH induced a sustained feeding effect by comparison with MCH. We explored thereafter several hypotheses, which were not mutually exclusive. NEI-MCH could be more potent than MCH and/or display differential activity at MCH1 or MCH2 receptors. Alternatively, enhanced food intake behavior might result from a better resistance to proteases of NEI-MCH than MCH. Therefore, the pharmacological profiles of MCH and NEI-MCH were compared at the MCH1 and MCH2, and the kinetics of peptide degradation by proteases and its sensitivity to inhibitors were studied in detail.

	Materials and Methods

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Peptides, Enzymes, and Peptidase Inhibitors

Rat/human MCH and NEI-MCH were custom-synthesized by Bachem Biochemistry (Voisins-le-Bretonneux, France). Aminopeptidase M (microsomal, type IV) and peptidase inhibitors were obtained from Sigma-Aldrich Chimie (Saint Quentin Fallavier, France). Dr. Auberger (Université de Médecine, Nice, France) kindly provided purified endopeptidase 24.11.

Central Peptide Administration and Effect on Food Intake

Animals. These studies were conducted with male Wistar rats weighing between 310 and 370 g (Charles River, St. Aubin-les-Elbeuf, France). All animals were maintained on a 12-h light/dark cycle (lights on 7:30 AM) at 22 ± 3°C and 55% relative humidity. The animals were supplied with food in the form of 6-mm pellets (A03, UAR laboratory chow; Epinay, Villemuisson, France) and tap water ad libitum. The food had the following composition: 67.5% food flour, 26.5% saccharose, 5% gum tragacanth, and 1.25% magnesium sterate. All animal procedures complied with French laws regarding animal experimentation (Decree no. 87-848, October 19, 1987, and the Ministerial Decree of April 19, 1988) and were pre-approved by the ethical committee of the Servier Research Institute.

Intracerebroventicular Cannula Implantation. Rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.), and their heads were placed into a stereotaxic device (Apparatus MM, Unimechanique, France). Stainless steel guide cannula (Plastics One Inc. Phymep, Châtillon, France) were then implanted into the right lateral ventricle according to the following coordinates: (AP = 0.8 mm, L = 1.2 mm, and V = 3.5 mm to the bregma). After implantation, each cannula was closed with a stainless steel obturator and kept in place with dental cement (Kulzer/Heraeus, Dormagen, Germany). During the following 7-day recovery period, the animals were housed individually and handled daily to minimize nonspecific stress.

Central Peptide Administration. On the day of study the animals were lightly anesthetized with Forene (Abbott Laboratories, Pomezia, Italy). Thereafter, peptide solutions or artificial CSF were infused slowly into the right lateral ventricle over a 30-s period (final volume administered, 2.5 µl).

Feeding Studies. The animals were distributed into matched experimental and control groups and then injected i.c.v. with either artificial CSF or peptides in artificial CSF. The injections were performed at 9:00 AM, shortly after the beginning of the light phase. After injection, the animals were returned to their home cage, which contained a known weight of food pellets contained in a spill-free cup. At different times during the test period, the food container was briefly removed and weighed to determine quantity of food consumed. Each food intake measurement was corrected for spillage.

Binding Assay

Human embryonic kidney or CHO cell lines stably expressing the rat MCH1 or the human MCH2 have been described elsewhere (Rodriguez et al., 2001; Suply et al., 2001). For binding studies at the MCH1 receptor, proteins (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, and 0.5% BSA) in a final volume of 250 µl containing 0.04 nM [¹²⁵I]S36057 and test peptides (Audinot et al., 2001b). 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. For displacement experiments, inhibition constants were calculated according to the Cheng-Prusoff equation: K_i = IC₅₀/[1 = (L/K_d)], where IC₅₀ is the inhibitory concentration, L is the concentration, and K_d is the dissociation constant of the radioligand.

Intracellular cAMP Assay

Intracellular cAMP assay was determined using the flashplate technology (SMP004; PerkinElmer Life Sciences, Boston, MA). In brief, 15 µM forskolin and test peptides diluted in 0.1% BSA were added into 96-well flashplates, and incubation was started with addition of cells (35,000 cells per well). After 15 min at 37°C, incubation was stopped by the addition of the revelation mix, and 2 h later, plates were counted on a TopCount (Packard Instrument Company, Inc., Downers Grove, IL).

Calcium Flux Measurements

Stable CHO cells expressing either the rat MCH1 or the human MCH2 receptors were seeded (40,000 cells) into 96-well black-walled culture plates coated with poly-D-lysine 24 h before assay. Cells were loaded with a calcium kit assay buffer (Molecular Devices Corp., Sunnyvale, CA) containing 2.5 mM probenecid at 37°C for 1 h in 6% CO₂ atm. After 10 s, the peptide was added. Increases of intracellular calcium in the presence of peptides were monitored using the fluorimetric imaging plate reader detection system (Molecular Devices Corp.) at 488 nm for 120 s.

Preparation of Brain Extracts

The brains from adult male Wistar rats were dissected, washed with 0.1 M sodium-phosphate buffer (Na₂HPO₄/NaH₂PO₄), pH 7.4, and homogenized by polytron in a 20 mM Tris/HCl pH 7.5 buffer. All tissue homogenates were diluted before use in the studies outlined below. The protein content of the extract preparation was determined by Bradford's protein assay.

Peptide Incubation and Reverse Phase (RP)-HPLC Analysis

Peptide incubation with several peptidases was previously described (Checler et al., 1992). In brief, synthesized peptides (2 nmol) were incubated with peptidases or brain extracts for various times at 37°C in a final volume of 100 µl of 20 mM Tris/HCl, pH 7.5 buffer, containing 0.02% BSA. Incubations were terminated by acidification (10 µl of 1 M HCl), and the incubation mixtures were centrifuged (10,000g, 10 min). Supernatants were submitted to HPLC in an RP column (C-18) and a gradient elution of 20 to 60% acetonitrile in 0.1% trifluoroacetic acid for 50 min at a flow rate of 1 ml/min. Degradation fragments were assessed by UV absorbance (230 nm).

Inhibitor Studies

Enzyme inhibitors were added 10 min before peptide incubation. Final concentrations of inhibitors in the incubate were 10 µM bestatin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µM leupeptin, 1 µM phosphoramidon, 1 µM pepstatin, and 10 µM E64.

Structural Analysis

Peptides and fragments obtained from incubation were assessed by mass spectrophotometry. Mass measurements were made on a Voyager-DE PRO MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a 1.3-m ion flight tube, delayed extraction technology, and a reflector system. Lyophilized samples were dissolved in water/acetonitrile (1:1, 0.1% trifluoroacetic acid) and mixed (1:1) with a-cyano-4-hydroxycinnamic acid matrix (20 mg/ml) on the sample plate. Spectra were recovered in positive ion linear (for NEI-MCH) or reflector mode (for MCH) with a 20-kV acceleration voltage. Mass spectra were obtained by accumulating 100 shots for each sample and by averaging five measurements per sample. Calibration of the system was done for each sample by close external calibration using a mixture of calibration peptides (mass range 905.05-5734.59 Da). Processing mass spectra was done in the Dta Explorer 3.2 software, and theoretical masses were calculated with GPMAW 4.02. In some instances, sequencing of the peptide fragments was performed using a Procise sequencer (Applied Biosystems).

Data and Statistical Analysis

For in vitro data (binding assay, cAMP, and calcium content determination) significance of difference between K_i and IC₅₀ was determined by an unpaired two-tailed t test. The same test was used to compare t_1/2 difference in in vitro peptide degradation studies. For in vivo feeding behavior data, significance of effects was determined by a three-way analysis of variance followed by a Newman-Keuls test for pair-wise comparison.

	Results

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Changes in Food Intake after Acute Intracerebroventricular Injection of MCH and NEI-MCH. Compared with control values, injection of MCH dose dependently stimulated food intake (Fig. 2A). The maximum increase in food intake was obtained with doses of 5 to 15 nmol/kg. NEI-MCH also dose dependently stimulated food intake above control values with a maximum response again achieved at doses of 5 to 15 nmol/kg (Fig. 2B). Statistical comparison of the results revealed that each injected dose of NEI-MCH produced a significantly greater effect on food intake than did MCH (n = 7). Consequently, the maximum increase in food intake obtained with NEI-MCH was also significantly greater than with MCH. One day after treatment, there was no significant difference in food intake between control and peptide-injected rats except with NEI-MCH-treated rats at the highest dose. NEI treatment with the same doses did not increase food intake (results not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Changes in food intake after acute i.c.v. injection of MCH and NEI-MCH. Either MCH or NEI-MCH was injected i.c.v. at time 0, and cumulative food intake was measured over the following 6-h period in satiated rats. Because of the large number of rats involved (n = 7), each dose of MCH, NEI-MCH, and a control group was run on a separate day. The control groups for each dose of peptide are shown as filled symbols and correspond to the same convention as each dose of the peptides. Food intake for all groups was analyzed by three-way analysis of variance (treatment × dose × time). Post hoc test was by complementary analysis based on the Newman-Keuls test after pooling all levels of time. , significantly different from the appropriate control group; +, MCH and NEI-MCH are significantly different from each other.

Binding Affinity and Functional Activity of MCH and NEI-MCH at the Rat MCH1 and Human MCH2 Receptors. First, binding affinities of MCH and NEI-MCH were evaluated at membranes from the CHO cell line stably expressing the rat MCH1 receptor, using [¹²⁵I]S36057 as a radioligand. Both NEI-MCH and MCH dose dependently displaced [¹²⁵I]S36057 binding at the rat MCH₁ receptor (Fig. 3A). However NEI-MCH was 5-fold less potent than MCH (K_i values of 0.73 ± 0.11 nM and 0.16 ± 0.04 nM, respectively, p = 0.0082, n = 3). Second, potency and efficacy of these two peptides were evaluated upon forskolin-induced intracellular cAMP measurement. Both peptides dose dependently inhibited intracellular cAMP level with a similar efficacy (Fig. 3B), NEI-MCH was apparently slightly less potent than MCH, but differences did not reach significance (EC₅₀ values of 1.05 ± 0.28 nM and 0.46 ± 0.08 nM, respectively, n = 5). Third, potency of these two peptides was evaluated in another functional test, the induction of intracellular calcium rise at the rat MCH1 receptor but also at the human MCH2 receptor. In agreement with the binding and intracellular cAMP results observed at the MCH1 receptor, NEI-MCH was as efficient as but less potent (7-fold) than MCH (Fig. 4A; EC₅₀ values of 13 ± 0.5 and 1.79 ± 0.55 nM, respectively, p < 0.001, n = 4). Interestingly, at the human MCH2 receptor, a similar 8-fold lower potency was found for NEI-MCH compared with MCH (Fig. 4B; 95 ± 2 nM and 12 ± 3 nM, respectively; p < 0.001, n = 4), whereas both peptides were as efficient.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Binding affinity and functional activity of MCH and NEI-MCH at the rat MCH1 receptor. Concentration-response effect of MCH and NEI-MCH upon [125I]S36057-specific binding to membranes expressing the rat MCH1 receptor (A) and upon the inhibition of forskolin-induced intracellular cAMP level in cells stably expressing the rat MCH1 receptor (B). Points shown are from a representative experiment performed in triplicate and repeated independently at least three times.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effect of MCH and NEI-MCH upon intracellular calcium level in cells expressing the rat MCH1 or the human MCH2 receptors. Concentration-response effect of MCH and NEI-MCH upon intracellular calcium in cells expressing the MCH1 (A) or the MCH2 (B) receptors. Results are expressed as the mean percentage of the calcium peak height with the peak height of 1 µM MCH taken as 100%. Points shown are from a representative experiment performed in triplicate and repeated independently four times.

Degradation Studies with Brain Extracts. Synthetic MCH or NEI-MCH were incubated with rat brain homogenates (0.5 mg/ml protein, final concentration) for up to 2 h. The remaining peptides were separated by RP-HPLC and analyzed by UV. Kinetics of MCH and NEI-MCH are illustrated in Fig. 5. After 2 h of incubation, 41 ± 2.5% of NEI-MCH was recovered whereas only 19 ± 0.5% of MCH remained in the incubation. MCH declined quickly (t_1/2 = 32.80 min), whereas the decrease of NEI-MCH was slower (t_1/2 = 78.5 min). We analyzed further the pattern of degradation and identified the metabolic products resulting from MCH-NEI and MCH cleavages. Four major peaks were observed after MCH degradation in brain extracts (Fig. 6, left panel). The primary metabolites of MCH eluted with a retention time (Rt) of 28 min (peak I), 27 min (peak II), 26 min (peak III), and 23 min (peak IV). Formation of peak II decreased dramatically after 15 min of incubation. After 1 h of incubation, a group of minor peaks (V and VI) was observed with a Rt between 45 and 50 min (not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Kinetics of MCH and NEI-MCH degradation in rat brain extracts. Peptides were incubated for up to 2 h at 37°C with brain extracts (0.5 mg/ml protein concentration). Separation and quantification was performed on RP-HPLC/UV as indicated under Materials and Methods. Exponential equation was fitted to determine half-time and the initial velocity of the peptide hydrolysis. Each point corresponds to the mean of three independent experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Time courses of MCH (left panel) and NEI-MCH (right panel) hydrolysis by rat brain membranes. MCH or NEI-MCH (2 nmol) was incubated for the indicated times at 37°C with 50 µg of brain extracts in a final volume of 100 µl of Tris/HCl, pH 7.4. Incubation mixtures were analyzed by RP-HPLC as described under Materials and Methods. Arrowheads indicate the retention times of MCH or NEI-MCH synthetic peptides. Roman numbers indicate the peptide-containing peaks that were analyzed by MALDI-TOF mass spectrometry.

View larger version (36K): [in this window] [in a new window]   Fig. 7.   Sequence of MCH and metabolites identified by mass assignments. Letters in bold indicate oxidized residues. Proposed sites of cleavage by endopeptides are indicated by arrowheads. The degradation products found in every HPLC peaks (numbered as in Fig. 6) were recovered and analyzed by mass spectrometry as described under Materials and Methods.

Inhibition of Degradation in Brain Extracts. Various exo- and endopeptidase activities are present in the rat brain extracts. Peptidase inhibitors were tested as protecting agents of MCH or NEI-MCH degradation at different times (10 and 30 min in Table 1). Aminopeptidase inhibitor, bestatin, and the selective inhibitor of endopeptidase 24.11, phosphoramidon, reduced markedly MCH degradation even after 90 min (55% intact MCH; data not shown). Cysteine protease inhibitor E64 protected also MCH from degradation, albeit less efficiently than bestatin or phosphoramidon. Other peptidase inhibitors proved to be ineffective such as the inhibitor of aspartic protease, pepstatin, or partially active close to t_1/2 such as the inhibitor of serine protease, PMSF. Surprisingly, leupeptin, an inhibitor of trypsin-like activities, enhanced MCH degradation at the earliest time. Preincubation of brain extracts with PMSF, leupeptin, or phosphoramidon but not bestatin, pepstatin, or E64 led to strong protection of NEI-MCH, therefore indicating that proteolysis of MCH and NEI-MCH was due to different peptidases.

Degradation in Vitro by Endopeptidase 24.11 or Aminopeptidase M. Because endopeptidase 24.11 and aminopeptidase M represent the strongest candidates as MCH degrading enzymes, we examined in vitro the relative susceptibility of MCH and NEI-MCH to hydrolysis by these enzymes. As shown in Fig. 8, after 2 h of incubation, the MCH was readily degraded by purified endopeptidase 24.11 or aminopeptidase M to two major fragments and four peptides, respectively. In contrast, NEI-MCH was fully resistant to both enzymes.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   MCH and NEI-MCH hydrolysis by aminopeptidase M and endopeptidase 24.11. MCH (top) or NEI-MCH (bottom) was incubated for 60 min at 37°C in a final volume of 100 µl of Tris/HCl, pH 7.4, without protease (control), or in the presence of 100 nM endopeptidase 24.11 or 200 nM aminopeptidase M. Incubations were analyzed by RP-HPLC as described under Materials and Methods.

	Discussion

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Recent structure-activity studies have suggested that the feeding action of MCH in the rat brain appeared to be mediated by MCH1 (Suply et al., 2001). However, a second MCH receptor has been recently characterized that could also participate in this function (for review, see Boutin et al., 2002; Hervieu et al., 2002).

We have shown here that pro-MCH_131-165 (NEI-MCH) stimulates food intake in the rats and that this action is more sustained than MCH. Because NEI-MCH acts as a superagonist, we hypothesized that it may be a better ligand or activator of signaling pathways than MCH at either of the MCH receptors. Previous studies have identified the structural domains of MCH responsible for binding and affinity to MCH1 (MacDonald et al., 2000; Audinot et al., 2001a) and MCH2 (Bednarek et al., 2001; Rodriguez et al., 2001). These studies have defined the region between Arg¹¹ and Arg¹⁴ as a potential site of interaction with MCH1 and MCH_6-17 (ring between Cys⁷ and Cys¹⁶) as a minimal sequence required for an agonistic response. NEI-MCH possess the same C terminus as MCH but 16 additional amino acids at its N terminus, which may have a notable effect on the MCH receptor binding/signal transduction activities. Our results obtained with rat MCH1 stably transfected human embryonic kidney 293 cells were comparable for the [¹³⁵I]S36057 binding assay, the cAMP assay, and the Ca²⁺_i assay. NEI-MCH was a weaker agonist than MCH in all three tests. Furthermore, NEI-MCH did not display a greater efficacy/potency than MCH at the second MCH receptor. On the contrary, we observed a 7- to 8-fold lower potency than MCH to either of the human MCH receptors. These data indicate that the superagonist effect of NEI-MCH observed on feeding behavior does not result from a better affinity and/or efficacy of the peptide at MCH1 or MCH2, at least in transfected cell models. Obviously, we cannot formally exclude that NEI-MCH recognizes in the rat brain a receptor that does not correspond to the two MCH receptors so far described. However, it is unlikely that the heretofore uncharacterized NEI receptor mediates the orexigenic effect of NEI-MCH in the rat brain. Previous studies (Rossi et al., 1997) and our data deny any role for NEI in the control of feeding behavior.

The relative sensitivity of NEI-MCH and MCH to proteolytic activities was then assessed by incubating the synthetic peptides with crude brain extracts. Strikingly, MCH was degraded at a much faster rate than NEI-MCH (see Fig. 5). Less than 20% of intact MCH was still present in brain extracts after 2 h of incubation, whereas more than 40% of NEI-MCH remained intact under the same conditions. Furthermore, the patterns of NEI-MCH and MCH degradation were clearly different. MCH appeared rapidly cleaved by endopeptidase 24.11 since phosphoramidon protected MCH from enzyme degradation during the first 10 min of incubation with brain extracts (see Table 1). The Asp¹-Phe², Glu¹⁸-Val¹⁹, and Cys⁷-Met⁸ bonds are the initial sites of hydrolysis of MCH by brain extracts (Fig. 6, peaks I, II, and III). Using pure preparation of endopeptidase 24.11, Checler et al. (1992) have monitored the stepwise degradation of MCH and revealed that Cys⁷-Met⁸ bond was the first site of attack by this enzyme in vitro. Here, we confirmed this result. In addition, MCH was also highly susceptible to aminopeptidases and carboxypeptidases as expected from its free N and C termini. Peaks II to IV of the RP-HPLC analysis contained essentially sequential degradation products of MCH at both termini. Consistent with these data, MCH was readily degraded by aminopeptidase M in vitro (Checler et al., 1992) and likely in vivo, since preincubation of brain extracts with bestatin reduced the MCH degradation (Table 1). The subsequent steps of MCH catabolism involve the shortening of the C terminus and the opening of the ring, likely by an endopeptidase 24.11-like activity. Final products of in vitro MCH degradation by purified endopeptidase 24.11 were generated by cleavage within the loop and at the Cys¹⁶-Trp¹⁷ bond (Checler et al., 1992).

In contrast to MCH, NEI-MCH appears fully resistant to degradation by endopeptidase 24.11 and aminopeptidase M. The resistance to aminopeptidases may be explained by the absence of specific residues at its N terminus (see Fig. 1B). The lack of proteolysis by endopeptidase 24.11 is more surprising. NEI-MCH has identical bonds, Cys⁷-Met⁸ and Cys¹⁶-Trp¹⁷, that are the corresponding sites of endopeptidase 24.11 proteolysis of MCH in brain extracts (the present study) and in vitro (Checler et al., 1992). However, these bonds were not selectively cleaved by endopeptidase 24.11 within NEI-MCH. These findings suggest that NEI-MCH is not a natural substrate for neuropeptidase 24.11 and also emphasize the importance of secondary structure in the metabolism of neuropeptides. These data are consistent with studies that have suggested that the structural differences in the various natriuretic peptides play a role in the differential effect of endopeptidase 24.11 (Kenny et al., 1993; Watanabe et al., 1997). Results obtained from mass spectrometry did not allow us to identify the peptide fragments generated by NEI-MCH degradation, and the nature of the UV peaks in HPLC data remains unknown. However, pretreatment of brain membrane with phosphoramidon partly protects NEI-MCH from degradation. This suggests that NEI-MCH may be the substrate of other members of the neuropeptidase 24.11 family (for review, see Turner et al., 2001). Similarly, peptidases that belong to the serine protease and trypsin-like families may be involved in the degradation of NEI-MCH as suggested by the partial protection elicited by PMSF and leupeptin, respectively (Table 1).

Are endopeptidase 24.11 and aminopeptidases/carboxypeptidases truly physiological MCH-inactivating peptidases? Structure-activity studies performed with a number of cyclic fragments and MCH analogs demonstrated that the last two and the first five amino acids of the cyclic MCH_1-19 are not essential for full potency in rat/human MCH₁-transfected cellular models (MacDonald et al., 2000; Audinot et al., 2001a; Bednarek et al., 2001; Suply et al., 2001) and in vivo (Suply et al., 2001). This suggests that shortened cyclic peptides on the N- or C-terminal parts could be still active in the rat brain. In contrast, linear compound or deletion in the ring structure led to complete inactivity in binding/functional assays using MCH₁-transfected cells and in fish or rat bioassays (Audinot et al., 2001a; Bednarek et al., 2001; Suply et al., 2001). This allows us to consider endopeptidase 24.11 and related enzymes as overt MCH-inactivating peptidases.

Because NEI-MCH is not degraded by endopeptidase 24.11 and amino- or carboxypeptidases in vitro and in brain homogenates, it could be assumed that the observed potentiation of NEI-MCH effect on appetite reflects its protection from proteolytic degradation by these enzymes. However further studies are needed to evaluate the metabolism of pro-MCH-derived peptides in vivo.

A puzzling question concerns the occurrence of the NEI-MCH peptide in vivo. In the mammalian brain, 95% of the MCH immunoreactivity was found to correspond to mature cyclic peptide under resting conditions (Takahashi et al., 1995; Hervieu et al., 1996). In the peripheral tissues of human (Viale et al., 1997), mouse (Viale et al., 1999), and rat (Hervieu et al., 1996), most, if not all, of MCH-IR was found to be associated with a large MCH-IR containing peptide. This pro-MCH-derived peptide contained also a NEI sequence and may represent a processing intermediate of the MCH precursor (Viale et al., 1997). The pro-hormone convertase PC2 was found to be necessary and/or sufficient to generate mature NEI and MCH in neuronal cells, but the factors that control the processing of pro-MCH in the peripheral organs remain unknown (Viale et al., 1999). NEI-MCH (or larger) peptide could be produced in the brain under specific stimuli or associated with physiopathological states such as obesity. Alternatively, this pro-MCH-derived peptide could be synthesized at the periphery and cross the blood-brain barrier by diffusion once dissociated from putative serum proteins (Kastin et al., 1999, 2000) or via brain areas lacking the blood-brain barrier such as the circumventricular organs.

In conclusion, we demonstrate here that injection of pro-MCH_131-165 peptide (NEI-MCH) in the rat brain stimulates feeding more potently than MCH itself. This effect was not associated with a greater affinity to either MCH1 or MCH2 or activation of a secondary pathway but results likely from an increase of stability and resistance to endopeptidase 24.11 and aminopeptidase M. This report raises also the possibility of the existence of natural ligands generated by the processing of the precursor MCH that would have stronger effects than mature MCH in the brain. This point seems to be important in the context of putative therapeutic indications involving the MCH receptors.