Neuropeptide Y (NPY), one of the most abundant peptides in rat and human brains, appears to act in the hypothalamus to stimulate feeding. It was first suggested that the NPY Y1 receptor (Y1R) was involved in feeding stimulated by NPY. More recently a novel NPY receptor subtype (Y5R) was identified in rat and human as the NPY feeding receptor subtype. There is, however, no absolute consensus since selective Y1R antagonists also antagonize NPY-induced hyperphagia. Nevertheless, new anti-obesity drugs may emerge from further pharmacological characterization of the NPY receptors and their antagonists. A large panel of Y1R and Y5R antagonists (such as CGP71683A, BIBO3304, BIBP3226, 1229U91, and SYNAPTIC and BANYU derivatives but also patentable in-house-synthesized compounds) have been evaluated through in vitro and in vivo tests in an attempt to establish a predictive relationship between the binding selectivity for human receptors, the potency in isolated organs assays, and the inhibitory effect on food intake in both normal and obese hyperphagic rodents. Although these results do not allow one to conclude on the implication of a single receptor subtype at the molecular level, this approach is crucial for the design of novel NPY receptor antagonists with potential use as anti-obesity drugs and for evaluation of their possible adverse peripheral side effects, such as hypotension.

INTRODUCTION:The purpose of this study was to test the continuing validity of the hypothesis that neuropeptide Y (NPY) produced in the brain controls food intake through an interaction with the NPY Y 5 receptor subtype.METHODS: The hypothesis was tested using CGP 71683A a potent and highly selective non-peptide antagonist of the NPY Y 5 receptor which was administered into the right lateral ventricle of obese Zucker faafa rats. RESULTS: Intraventricular injection of 3.4 nmolakg NPY increased food intake during a 2 h test period. Doses of CGP 71683A in excess of 15 nmolakg (i.cv.) resulted in blockade of the increase in food intake produced by NPY. Repeated daily injection of CGP 71683A (30 ± 300 nmolakg, i.cv.) immediately before the dark phase produced a dose-dependent and slowly developing decrease in food intake. CGP 71683A has a low af®nity for NPY Y 1 , Y 2 and Y 4 receptors but a very high af®nity for the NPY Y 5 receptor (Ki, 1.4 nM). Surprisingly, CGP 71683A had similarly high af®nity for muscarinic receptors (Ki, 2.7 nM) and for the serotonin uptake recognition site (Ki, 6.2 nM) in rat brain. Anatomic analysis of the brain after treatment with CGP 71683A demonstrated an in¯ammatory response associated with the fall in food intake. CONCLUSIONS: While the fall in food intake in response to CGP 71683A may have a Y 5 component, interactions with other receptors or in¯ammatory mediators may also play a role. It is concluded that CGP 71683A is an imprecise tool for investigating the role of the NPY Y 5 receptor in the control of physiological processes including food intake. International Journal of Obesity (2001) 25, 84 ± 94

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide, with a major role in stimulation of feeding behavior in mammals. MCH signals in the brain occur via two seventransmembrane G protein-coupled receptors, namely MCH1 (SLC-1, MCH 1 , MCH-R1, or MCH-1R) and MCH2 (SLT, MCH 2 , MCH-R2, or MCH-2R). In this study, we demonstrate that the pro-MCH 131-165 peptide neuropeptide-glutamic acid-isoleucine (NEI)-MCH is more potent than MCH in stimulating feeding in the rat. Using rat MCH1-expressed human embryonic kidney 293 cells, we show that NEI-MCH exhibits 5-fold less affinity in a binding assay and 2-fold less potency in a cAMP assay than MCH. A similar 7-to 8-fold shift in potency was observed in a Ca 2ϩ i assay using rat MCH1 or human MCH2-transfected Chinese hamster ovary cell models. This demonstrates that NEI-MCH is not a better agonist than MCH at either of the MCH receptors. Then, we compared the proteolysis resistance of MCH and NEI-MCH to rat brain membrane homogenates and purified proteases. Kinetics of peptide degradation using brain extracts indicated a t 1/2 of 34.8 min for MCH and 78.5 min for NEI-MCH with a specific pattern of cleavage of MCH but not NEI-MCH by exo-and endo-proteases. Furthermore, MCH was found highly susceptible to degradation by aminopeptidase M and endopeptidase 24.11, whereas NEI-MCH was fully resistant to proteolysis by these enzymes. Therefore, our results strongly suggest that reduced susceptibility to proteases of NEI-MCH compared with MCH account for its enhanced activity in feeding behavior. NEI-MCH represents therefore the first MCH natural functional "superagonist" so far described.

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