NMU Central Signaling And Characterization Of The Small-molecule NMU2R Agonists

Neuromedin U(NMU)plays important roles in CNS in regulation of food intake and energy homeostasis.Therefore,investigation into the NMU central signaling is of great importance in terms of its potency to be a drug target of metabolic diseases such as type 2 diabetes and obesity.However,the crosstalk between NMU signal transduction pathway and other energy homeostasis-related pathways in CNS has not yet been elucidated.The present study was conducted to investigate the possible interaction in controlling feeding behavior between the NMU pathway and melanocortins(MC) system in CNS in rats treated intracerebraventricularly(ICV)with NMU,the endogenous ligand of NMU2 receptor(NMU2R),and/or SHU9119,a specific antagonist of melanocortin receptors 3 and 4.Meanwhile,after screening of the activities of synthetic NMU2R agonists in human NMU2R cell line,one agonist candidate,EUK2085,was selected for characterization of its actions in vivo in regulating food intake and body weight in rats.The effects of EUK2085 on central expressions of NMU as well as MC3/4R were also examined.1.Effects of intracerebroventricular administration of NMU on food intake, water drink and body weight in the ratsTo identify the role of NMU in the regulation of feeding behavior,the NMU were ICV administrated into rats,and the changes in food intake,water drink and the body weight were measured.The results revealed that the food intake was significantly reduced in the rats with ICV administration of NMU,and simultaneously,the water drink and the body weight gain in these rats were also decreased as compared with those of the control rats,confirming the role of NMU2R in controlling food intake and body weight in CNS. 2.The inhibitory role of central NMU in feeding behavior is partially mediated by melanocortin pathway in ratsTo explore the underlying mechanism of central NMU2R signaling in keeping energy homeostasis,a possible relationship between NMU2R and MC3/4R pathways in regulation of food intake and body weight in rats was preliminarily investigated. NMU and SHU9119 were ICV administrated to the fasting and non-fasting rats.The effects of NMU2R on feeding behavior and body weights and their relationships with the role of MC3/4R signaling were evaluated in the absence or presence of SHU9119 in the rat.The results showed that the anorexigenic effect of NMU could be partially inhibited by ICV co-administration of NMU and SHU9119.Furthermore,ICV injection of NMU showed a similar pattern of reduced anorexigenic effect of NMU in SHU9119-pretreated rats.These results suggest that the central modulation of NMU2R on feeding behavior is associated,at least in part,with melanocortin receptor pathway.3.Activity analysis of NMU2R agonistsWe have identified small molecules with NMU2R agonist properties from our traditional Chinese medicine(TCM)compound library.More than 100 synthetic analogs derived from the modifications of these natural structures have also been available for further structure and activity relationship(SAR)studies.In this study, the in vitro activities of these analogs were measured using an optimized NMU2R cell-based assay.The results showed that,for example,the EC50 values of the analogs EUK2040,EUK2047 and EUK2085 were 16.666μM,33.723μM and 47.044μM respectively,suggesting that these compounds could be good candidates for the further evaluation of their NMU2R agonist roles in vivo.4.Effects of ICV administration of EUK2085 on the food and water intake and body weight changingTo further assess the actions of the NMU2R agonist compounds in vivo in the brain,EUK2085 was selected in the study to demonstrate the potential NMU2R agonist efficacy of the compound in regulating feeding behavior in the rats using the ICV administration procedure.Meanwhile,the changes in several homeostasis-related genes(such as NMU,POMC and MC4)in the hypothalamus were also analyzed in the rats treated with EUK2085.The results showed that the food intake was significantly increased,while the expressions of NMU,POMC and MC4R mRNA in the hypothalamus were not changed,in the rats with ICV administration of EUK2085 compared to the control rats.