Neuromedin U regulates the anti-tumor activity of CD8+ T cells and glycolysis of tumor cells in the tumor microenvironment of pancreatic ductal adenocarcinoma in an NMUR1-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a poor prognosis, which is lethal in approximately 90% of cases despite advanced standard therapies. A typical feature of PDAC is the immunosuppressive tumor microenvironment with multiple immunosuppressive factors including neurotransmitters. Recently, neuromedin U (NMU), a highly conserved neuropeptide with many physiological functions, has attracted attention for its roles in tumorigenesis and metastasis in several types of cancers. However, whether NMU affects PDAC progression remains unclear. In this study, using an orthotopic mouse model of PDAC in combination with bioinformatics analysis, we found that NMU was upregulated in tumor tissues from the patients with PDAC and positively correlated with a poor prognosis of the disease. Interestingly, knockout of the Nmu gene in mice enhanced the anti-tumor functions of tumor-infiltrating CD8+ T cells in an NMU receptor 1-dependent manner. Additionally, NMU promoted the glycolytic metabolism of mouse PDAC tumors. The activities of pyruvate kinase (PK) and lactate dehydrogenase (LDH), pivotal enzymes involved in the regulation of lactate production, were markedly reduced in tumor tissues from NMU-knockout mice. In vitro the presence of LDHA inhibitor can reduce the production of lactic acid stimulated by NMU, which can increase the anti-tumor activity of CD8+ T cells. Moreover, treatment of the pancreatic cancer cells with a phosphoinositide 3-kinase (PI3K) inhibitor diminished NMU-induced lactate production and the activities of PK and LDH, suggesting that NMU might regulate glycolysis via the PI3K/AKT pathway.

Neuromedin U Neurons in the Edinger-Westphal Nucleus Respond to Alcohol Without Interfering with the Urocortin 1 Response.

The Edinger-Westphal nucleus (EW) is a midbrain nucleus composed of a preganglionic, cholinergic subpopulation and a densely clustered peptidergic subpopulation (EWcp). The EWcp is one of the few brain regions that show consistent induction of FOS following voluntary alcohol intake. Previous results in rodents point to urocortin 1 (UCN1) as one of the peptides most involved in the control of ethanol intake and preference. Notably, the functions described for UCN1, such as reward processing, stress coping or the regulation of feeding behavior are similar to those described for the neuropeptide neuromedin U (NMU). Interestingly, NMU has been recently associated with the modulation of alcohol-related behaviors. However, little is known about the expression and functionality of NMU neurons in alcohol-responsive areas. In this study, we used the recently developed Nmu-Cre knock-in mouse model to examine the expression of NMU in the subaqueductal paramedian zone comprising the EWcp. We delved into the characterization and co-expression of NMU with other markers already described in the EWcp. Moreover, using FOS as a marker of neuronal activity, we tested whether NMU neurons were sensitive to acute alcohol administration. Overall, we provided novel insights on NMU expression and functionality in the EW region. We showed the presence of NMU within a subpopulation of UCN1 neurons in the EWcp and demonstrated that this partial co-expression does not interfere with the responsivity of UCN1-containing cells to alcohol. Moreover, we proposed that the UCN1 content in these neurons may be influenced by sex.

Pharmacomodulation of brain neuromedin U signaling as a potential therapeutic strategy.

Neuromedin U (NMU) belongs to a family of multifunctional neuropeptides that modulate the activity of several neural networks of the brain. Acting via metabotropic receptor NMUR2, NMU plays a role in the regulation of multiple systems, including energy homeostasis, stress responses, circadian rhythms, and endocrine signaling. The involvement of NMU signaling in the central regulation of important neurophysiological processes and its disturbances is a potential target for pharmacological modulation. Number of preclinical studies have proven that both modified NMU analogues such as PASR8-NMU or F4R8-NMU and designed NMUR2 agonists, for example, CPN-116, CPN-124 exhibit a distinct pharmacological activity especially when delivered transnasally. Their application can potentially be useful in the more convenient and safe treatment of obesity, eating disorders, Alzheimer's disease-related memory impairment, alcohol addiction, and sleep disturbances. Accumulating findings suggest that pharmacomodulation of the central NMU signaling may be a promising strategy in the treatment of several neuropsychiatric disorders.

The neuromedin U system: Pharmacological implications for the treatment of obesity and binge eating behavior.

Neuromedin U (NMU) is a bioactive peptide produced in the gut and in the brain, with a role in multiple physiological processes. NMU acts by binding and activating two G protein coupled receptors (GPCR), the NMU receptor 1 (NMU-R1), which is predominantly expressed in the periphery, and the NMU receptor 2 (NMU-R2), mainly expressed in the central nervous system (CNS). In the brain, NMU and NMU-R2 are consistently present in the hypothalamus, commonly recognized as the main "feeding center". Considering its distribution pattern, NMU revealed to be an important neuropeptide involved in the regulation of food intake, with a powerful anorexigenic ability. This has been observed through direct administration of NMU and by studies using genetically modified animals, which revealed an obesity phenotype when the NMU gene is deleted. Thus, the development of NMU analogs or NMU-R2 agonists might represent a promising pharmacological strategy to treat obese individuals. Furthermore, NMU has been demonstrated to influence the non-homeostatic aspect of food intake, playing a potential role in binge eating behavior. This review aims to discuss and summarize the current literature linking the NMU system with obesity and binge eating behavior, focusing on the influence of NMU on food intake and the neuronal mechanisms underlying its anti-obesity properties. Pharmacological strategies to improve the pharmacokinetic profile of NMU will also be reported.

Neuromedin U regulates food intake of Siberian sturgeon through the modulation of central and peripheral appetite factors.

Neuromedin U (NMU) has a critical function on the regulation of food intake in mammals, while the information is little in teleost. To investigate the function of NMU on appetite regulation of Siberian sturgeon (Acipenser baerii), this study first cloned nmu cDNA sequence that encoded 154 amino acids including NMU-25 peptide. Besides, the results showed that nmu mRNA was widely distributed in various tissues especially in the hypothalamus and telencephalon. The results of nutritional status (pre-feeding and post-feeding, fasting and re-feeding) experiments showed that nmu mRNA expression was significantly decreased at 1 and 3 h after feeding in different brain regions. Similarly, after feeding, the expression of nmu significantly decreased in peripheral tissues. Moreover, nmu expression in the hypothalamus was significantly increased after fasting 1 d, but decreased after fasting 17 d, which was significantly reversed after re-feeding. However, other brain regions like telencephalon and peripheral tissues like oesophagus, intestinum valvula and liver have different change patterns. Further study showed that acute i.c.v. and i.p. injection of NMU and chronic i.p. injection of NMU significantly reduced the food intake in a dose-dependent mode. In addition, the expressions of several critical appetite factors (nmu, aplein, cart, cck, ghrelin, npy, nucb2, pyy and ucn3) were significantly affected by acute NMU-25 administration in the hypothalamus, intestinum valvula and liver. These results indicate that NMU-25 has the anorexigenic function on food intake by affecting different appetite factors in Siberian sturgeon, which provides a foundation for further exploring the appetite regulation networks in fish.

Neuromedin U secreted by colorectal cancer cells promotes a tumour-supporting microenvironment.

BACKGROUND: Neuromedin U (NMU) was identified as one of the hub genes closely related to colorectal cancer (CRC) progression and was recently shown to be a motility inducer in CRC cells. Its autocrine signalling through specific receptors increases cancer cell migration and invasiveness. Because of insufficient knowledge concerning NMU accessibility and action in the tumour microenvironment, its role in CRC remains poorly understood and its potential as a therapeutic target is still difficult to define. METHODS: NMU expression in CRC tissue was detected by IHC. Data from The Cancer Genome Atlas were used to analyse gene expression in CRC. mRNA and protein expression was detected by real-time PCR, immunoblotting or immunofluorescence staining and analysed using confocal microscopy or flow cytometry. Proteome Profiler was used to detect changes in the profiles of cytokines released by cells constituting tumour microenvironment after NMU treatment. NMU receptor activity was monitored by detecting ERK1/2 activation. Transwell cell migration, wound healing assay and microtube formation assay were used to evaluate the effects of NMU on the migration of cancer cells, human macrophages and endothelial cells. RESULTS: Our current study showed increased NMU levels in human CRC when compared to normal adjacent tissue. We detected a correlation between high NMUR1 expression and shorter overall survival of patients with CRC. We identified NMUR1 expression on macrophages, endothelial cells, platelets, and NMUR1 presence in platelet microparticles. We confirmed ERK1/2 activation by treatment of macrophages and endothelial cells with NMU, which induced pro-metastatic phenotypes of analysed cells and changed their secretome. Finally, we showed that NMU-stimulated macrophages increased the migratory potential of CRC cells. CONCLUSIONS: We propose that NMU is involved in the modulation and promotion of the pro-metastatic tumour microenvironment in CRC through the activation of cancer cells and other tumour niche cells, macrophages and endothelial cells. Video abstract.

Modulatory effect of long-term treatment with escitalopram and clonazepam on the expression of anxiety-related neuropeptides: neuromedin U, neuropeptide S and their receptors in the rat brain.

BACKGROUND: Newly identified multifunctional peptidergic modulators of stress responses: neuromedin U (NMU) and neuropeptide S (NPS) are involved in the wide spectrum of brain functions. However, there are no reports dealing with potential molecular relationships between the action of diverse anxiolytic or antidepressant drugs and NMU and NPS signaling in the brain. The present work was therefore focused on local expression of the aforementioned stress-related neuropeptides in the rat brain after long-term treatment with escitalopram and clonazepam. METHODS: Studies were carried out on adult, male Sprague-Dawley rats that were divided into 3 groups: animals injected with saline (control) and experimental individuals treated with escitalopram (at single dose 5 mg/kg daily), and clonazepam (at single dose 0.5 mg/kg). All individuals were sacrificed under anaesthesia and the whole brain excised. Total mRNA was isolated from homogenized samples of amygdala, hippocampus, hypothalamus, thalamus, cerebellum and brainstem. Real time-PCR method was used for estimation of related NPS, NPS receptor (NPSR), NMU, NMU and receptor 2 (NMUR2) mRNA expression. The whole brains were also sliced for general immunohistochemical assessment of the neuropeptides expression. RESULTS: Chronic administration of clonazepam resulted in an increase of NMU mRNA expression and formation of NMU-expressing fibers in the amygdala, while escitalopram produced a significant decrease in NPSR mRNA level in hypothalamus. Long-term escitalopram administration affects the local expression of examined neuropeptides mRNA in a varied manner depending on the brain structure. CONCLUSIONS: Pharmacological effects of escitalopram may be connected with local at least partially NPSR-related alterations in the NPS/NMU/NMUR2 gene expression at the level selected rat brain regions. A novel alternative mode of SSRI action can be therefore cautiously proposed.

Nmu/Nms/Gpr176 Triple-Deficient Mice Show Enhanced Light-Resetting of Circadian Locomotor Activity.

The suprachiasmatic nucleus (SCN) is the master circadian clock in mammals and is properly entrained by environmental light cycle. However, the molecular mechanism(s) determining the magnitude of phase shift by light is still not fully understood. The orphan G-protein-coupled receptor Gpr176 is enriched in the SCN, controls the pace (period) of the circadian rhythm in behavior but is not apparently involved in the light entrainment; Gpr176-/- animals display a shortened circadian period in constant darkness but their phase-resetting responses to light are normal. Here, we performed microarray analysis and identified enhanced mRNA expression of neuromedin U (Nmu) and neuromedin S (Nms) in the SCN of Gpr176-/- mice. By generating C57BL/6J-backcrossed Nmu/Nms/Gpr176 triple knockout mice, we noted that the mutant mice had a greater magnitude of phase shift in response to early subjective night light than wildtype mice, while Nmu/Nms double knockout mice as well as Gpr176 knockout mice are normal in the phase shifts induced by light. At the molecular level, Nmu-/-Nms-/-Gpr176-/- mice had a reduced induction of Per1 and cFos mRNA expression in the SCN by light and mildly upregulated circadian expression of Per2, Prok2, Rgs16, and Rasl11b. These expressional changes may underlie the phenotype of the Nmu/Nms/Gpr176 knockout mice. Our data argue that there is a mechanism requiring Nmu, Nms, and Gpr176 for the proper modulation of light-induced phase shift in mice. Simultaneous modulation of Nmu/Nms/Gpr176 may provide a potential target option for modulating the circadian clock.

Peptide Tool-Driven Functional Elucidation of Biomolecules Related to Endocrine System and Metabolism.

The enhancement of basic research based on biomolecule-derived peptides has the potential to elucidate their biological function and lead to the development of new drugs. In this review, two biomolecules, namely "neuromedin U (NMU)" and "myostatin," are discussed. NMU, a neuropeptide first isolated from the porcine spinal cord, non-selectively activates two types of receptors (NMUR1 and NMUR2) and displays a variety of physiological actions, including appetite suppression. The development of receptor-selective regulators helps elucidate each receptor's detailed biological roles. A structure-activity relationship (SAR) study was conducted to achieve this purpose using the amidated C-terminal core structure of NMU for receptor activation. Through obtaining receptor-selective hexapeptide agonists, molecular functions of the core structure were clarified. Myostatin is a negative regulator of skeletal muscle growth and has attracted attention as a target for treating atrophic muscle disorders. Although the protein inhibitors, such as antibodies and receptor-decoys have been developed, the inhibition by smaller molecules, including peptides, is less advanced. Focusing on the inactivation mechanism by prodomain proteins derived from myostatin-precursor, a first mid-sized α-helical myostatin-inhibitory peptide (23-mer) was identified from the mouse sequence. The detailed SAR study based on this peptide afforded the structural requirements for effective inhibition. The subsequent computer simulation proposed the docking mode at the activin type I receptor binding site of myostatin. The resulting development of potent inhibitors suggested the existence of a more appropriate binding mode linked to their ß-sheet forming properties, suggesting that further investigations might be needed.

Central dopaminergic, serotoninergic, as well as GABAergic systems mediate NMU-induced hypophagia in newborn chicken.

AIM: Dopaminergic, serotoninergic, and GABAergic systems influence feeding; however, it is unknown how these chemicals interact with neuromedin U (NMU)-induced feeding in birds. In the current study, ten trials were conducted to determine the links between the above-mentioned systems and NMU. MATERIALS AND METHODS: In the foremost experimentation, chickens were given intracerebroventricularly injections of NMU (0.1, 1, and 10 µg). NMU (10 µg), SCH23390 (5 nmol), a D1 receptor antagonist, and NMU + SCH23390 were administered in the second experiment. In subsequent experiments, instead of SCH23390, were applied AMI-193 (5 nmol D2 receptor antagonist), NGB2904 (6.4 nmol D3 receptor antagonist), L-741,742 (6 nmol D4 receptor antagonist), 6-OHDA (2.5 nmol dopamine inhibitor), SB242084 (5-HT2c receptor antagonist, 1.5 µg), 8-OH-DPAT (5-HT1A receptor agonist, 15.25 nmol), picrotoxin (GABAA receptor antagonist, 0.5 µg), and CGP54626 (GABAB receptor antagonist, 20 ng). Then, cumulative intake of food was recorded for 2 h. RESULTS: According to the results, NMU reduced feeding when compared to the control group (p < 0.05). The NMU-induced hypophagia was reduced with co-injection of NMU and SCH23390 (p < 0.05). Hypophagia was diminished with NMU and AMI-193 (p < 0.05). NMU + NGB2904 and NMU + L-741,742 co-injections had no influence (p > 0.05). 6-OHDA reduced the hypophagia (p < 0.05). NMU and SB242084 decreased the hypophagia (p < 0.05), whereas NMU and 8-OH-DPAT had no effect (p > 0.05). The effects were amplified with picrotoxin (p < 0.05). NMU with CGP54626 had no influence on the hypophagia (p > 0.05). CONCLUSION: Thus, NMU-induced hypophagia is probably mediated by D1/D2, 5-HT2c, and GABAA receptors in neonatal chicks.

Neuromedin U: potential roles in immunity and inflammation.

Since the discovery of neuromedin U (NmU) from porcine spinal cord in 1985, this neuropeptide has been subsequently identified in many other species with multiple physiological and pathophysiological roles detected, ranging from smooth muscle contraction, feeding, energy balance to tumorigenesis. Intriguingly, NmU is also emerging to play pro-inflammatory roles involving immune cell activation and cytokine release in a neuron-dependent or neuron-independent manner. The NmU-mediated inflammatory responses have already been observed in worm infection, sepsis, autoimmune arthritis and allergic animal models. In this review, we focus on the roles of NmU in immunity and inflammation by highlighting the interactions between NmU and immune cells, summarizing the signalling mechanism involved in their reactions and discussing its potential contributions to inflammatory diseases.

Comparison of physiological functions between neuromedin U-related peptide and neuromedin S-related peptide in the rat central nervous system.

Two novel peptides, neuromedin U precursor-related peptide (NURP) and neuromedin S precursor-related peptide (NSRP), are produced from neuromedin U (NMU) and neuromedin S (NMS) precursors, respectively, as these precursors have multiple consensus sequences for proteolytic processing. Our group has shown previously that one of these two novel peptides, NURP, stimulates body temperature and locomotor activity, but not food intake. However, the physiological function of the other peptide, NSRP, has remained unclear. Therefore, the aim of this study was to characterize differences in the regions of the rat brain targeted by the NMU/NMS peptide family, including NURP and NSRP, and their physiological functions. First, we explored the regions of c-Fos expression after intracerebroventricular (i.c.v.) injection of NURP and NSRP and found that these were fewer than after i.c.v. injection of NMU and NMS in the hypothalamus, possibly because NURP and NSRP cannot activate NMU/NMS receptors. In the ventral subiculum, which is one region of the hippocampus, c-Fos expression was evident only after i.c.v. injection of NURP. We also examined the effects of NSRP on food intake, body temperature and locomotor activity. Like NURP, NSRP increased both body temperature and locomotor activity, but not food intake, indicating that NSRP is also a functional peptide. However, these effects of NSRP were distinctly weaker than those of NURP. These findings suggest differences in the affinity of NURP and/or NSRP for specific receptors, or in their respective biological activities.

Neuromedin U suppresses prolactin secretion via dopamine neurons of the arcuate nucleus.

Neuromedin U (NMU) has a precursor that contains one additional peptide consisting of 33 or 36 amino acid residues. Recently, we identified this second peptide from rat brain and designated it neuromedin U precursor-related peptide (NURP), showing it to stimulate prolactin release from the pituitary when injected via the intracerebroventricular (icv) route. Here, we examined whether NMU, like NURP, also stimulates prolactin release. Unlike NURP, icv injection of NMU significantly decreased the secretion of prolactin from the pituitary. This suppression of prolactin release by NMU was observed in hyper-prolactin states such as lactation, stress, pseudopregnancy, domperidone (dopamine antagonist) administration, and icv injection of NURP. Immunohistochemical analysis revealed that icv injection of NMU induced cFos expression in dopaminergic neurons of the arcuate nucleus, but not the substantia nigra. Mice with double knockout of NMU and neuromedin S (NMS), the latter also binding to NMU receptors, showed a significant increase of the plasma prolactin level after domperidone treatment relative to wild-type mice. These results suggest that NMU and NURP may play important reciprocal roles in physiological prolactin secretion.

Neuromedin U (NMU) regulates osteoblast differentiation and activity.

Osteoporosis is a disease of low bone mass that places individuals at enhanced risk for fracture, disability, and death. Osteoporosis rates are expected to rise significantly in the coming decades yet there are limited pharmacological treatment options, particularly for long-term management of this chronic condition. The drug development pipeline is relatively bereft of new strategies, causing an urgent and unmet need for developing new strategies and targets for treating osteoporosis. Here, we examine a lesser-studied bone remodeling pathway, Neuromedin U (NMU), which is expressed in the bone microenvironment along with its cognate receptors NMU receptor 1 (NMUR1) and 2 (NMUR2). We independently corroborate a prior report that global loss of NMU expression leads to high bone mass and test the hypothesis that NMU negatively regulates osteoblast differentiation. Consistent with this, in vitro studies reveal NMU represses osteoblastic differentiation of osteogenic precursors but, in contrast, promotes osteoblastic marker expression, proliferation and activity of osteoblast-like cells. Phospho-profiling arrays were used to detail differential signaling outcomes that may underlie the opposite responses of these cell types. Collectively, our findings indicate that NMU exerts cell-type-specific responses to regulate osteoblast differentiation and activity.

The prognostic value of neuromedin U in patients with hepatocellular carcinoma.

BACKGROUND: Neuromedin U (NMU) is a neuropeptide belonging to the neuromedin family. Recently, significant associations between NMU and several cancers have been reported. However, no studies have examined the association between NMU and hepatocellular carcinoma (HCC). The purpose of this study was to examine the role of NMU in HCC. METHODS: An enzyme-linked immunosorbent assay was used to measure the level of NMU protein in the sera of patients with hepatic hemangioma and HCC. NMU and cytokine mRNA expression was assessed in HCC samples via RT-qPCR. A tissue microarray consisting of 228 HCC peri- and intra-tumor tissues was used to detect NMU expression via immunohistochemical analysis. The association between NMU expression and overall survival (OS) and disease-free survival (DFS) was analyzed by Kaplan-Meier curves, the log-rank test, and Cox proportional hazard model. RESULTS: The level of NMU protein was increased in the sera of HCC patients (p = 0.006). NMU was expressed in intercellular space, rather than in hepatocytes or HCC cells. The prognosis of HCC patients with high NMU expression in peri-tumor tissue was significantly poorer than that of patients with low NMU expression (OS: p = 0.002, DFS: p = 0.033). Peri-tumor NMU expression was also a significant independent prognostic factor for OS (hazard ratio: 1.541, 95% confidence interval: 1.092-2.175, p = 0.014). The level of NMU expression was positively associated with M2 macrophage percentage and the levels of type-2 inflammatory cytokines in HCC tissue. CONCLUSIONS: NMU may serve as a novel prognostic biomarker for HCC patients, although further validation is needed in the future. The activation of M2 macrophages and a type-2 inflammatory response may involve in the role of NMU in patients with HCC.

Effects of neuromedin U-8 on stress responsiveness and hypothalamus-pituitary-adrenal axis activity in male C57BL/6J mice.

Neuromedin U (NMU) is a highly conserved neuropeptide that has been implicated in the stress response. To better understand how it influences various aspects of the stress response, we studied the effects of intracerebroventricular NMU-8 administration on stress-related behavior and activity of the hypothalamus-pituitary-adrenal (HPA) axis in male C57BL/6J mice. We investigated these NMU-8 effects when mice remained in their home cage and when they were challenged by exposure to forced swim stress. NMU-8 administration resulted in increased grooming behavior in mice that remained in their home cage and in a significant increase in c-Fos immunoreactivity in the paraventricular hypothalamus (PVH) and arcuate nucleus (ARC). Surprisingly, NMU-8 administration significantly decreased plasma corticosterone concentrations. Furthermore, NMU-8 administration increased immobility in the forced swim test in both naïve mice and mice that were previously exposed to swim stress. The effect of NMU-8 on c-Fos immunoreactivity in the PVH was dependent on previous exposure to swim stress given that we observed no significant changes in mice exposed for the first time to swim stress. In contrast, in the ARC we observed a significant increase in c-Fos immunoreactivity regardless of previous stress exposure. Interestingly, NMU-8 administration also significantly decreased plasma corticosterone concentrations in mice that were exposed to single forced swim stress, while this effect was no longer observed when mice were exposed to forced swim stress for a second time. Taken together, our data indicate that NMU-8 regulates stress responsiveness and suggests that its effects depend on previous stress exposure.

Transnasal Delivery of the Peptide Agonist Specific to Neuromedin-U Receptor 2 to the Brain for the Treatment of Obesity.

Obesity and metabolic syndrome are threats to the health of large population worldwide as they are associated with high mortality, mainly linked to cardiovascular diseases. Recently, CPN-116 (CPN), which is an agonist peptide specific to neuromedin-U receptor 2 (NMUR2) that is expressed predominantly in the brain, has been developed as a new therapeutic candidate for the treatment of obesity and metabolic syndrome. However, treatment with CPN poses a challenge due to the limited delivery of CPN to the brain. Recent studies have clarified that the direct anatomical connection of the nasal cavity with brain allows delivery of several drugs to the brain. In this study, we confirm the nasal cavity as a promising CPN delivery route to the brain for the treatment of obesity and metabolic syndrome. According to the pharmacokinetic study, the clearance of CPN from the blood was very rapid with a half-life of 3 min. In vitro study on its stability in the serum and cerebrospinal fluid (CSF) indicates that CPN was more stable in the CSF than in the blood. The concentration of CPN in the brain was higher after nasal administration, despite its lower concentrations in the plasma than that after intravenous administration. The study on its pharmacological potency suggests the effective suppression of increased body weight in mice in a dose-dependent manner due to the direct activation of NMUR2 by CPN. This results from the higher concentration of corticosterone in blood after nasal administration of CPN as compared to nasal application of saline. In conclusion, the above findings indicate that the nasal cavity is a promising CPN delivery route to the brain to treat obesity and metabolic syndrome.

Design and synthesis of peptidic partial agonists of human neuromedin U receptor 1 with enhanced serum stability.

Neuromedin U (NMU) activates two receptors (NMUR1 and NMUR2) and is a promising candidate for development of drugs to combat obesity. Previously, we obtained hexapeptides as selective full NMUR agonists. Development of a partial agonist which mildly activates receptors is an effective strategy which lead to an understanding of the functions of NMU receptors. In 2014, we reported hexapeptide 3 (CPN-124) as an NMUR1-selective partial agonist but its selectivity and serum stability were unsatisfactory. Herein, we report the development of a hexapeptide-type partial agonist (8, CPN-223) based on a peptide (3) but with higher NMUR1-selectivity and enhanced serum stability. A structure-activity relationship study of synthetic pentapeptide derivatives suggested that a hexapeptide is a minimum structure consistent with both good NMUR1-selective agonistic activity and serum stability.

A chemically stable peptide agonist to neuromedin U receptor type 2.

Neuromedin U (NMU) is a peptide with appetite suppressive activity and other physiological activities via activation of the NMU receptors NMUR1 and NMUR2. In 2014, we reported the first NMUR2 selective agonist, 3-cyclohexylpropionyl-Leu-Leu-Dap-Pro-Arg-Asn-NH2 (CPN-116). However, we found that CPN-116 in phosphate buffer is unstable because of Nα-to-Nß acyl migration at the Dap residue. In this study, the chemical stability of CPN-116 was evaluated under various conditions, and it was found to be relatively stable in buffers such as HEPES and MES. We also performed a structure-activity relationship study to obtain an NMUR2-selective agonist with improved chemical stability. Consequently, CPN-219 bearing a Dab residue in place of Dap emerged as a next-generation hexapeptidic NMUR2 agonist.

Purification and identification of native forms of goldfish neuromedin U from brain and gut.

Neuromedin U (NMU) plays important roles in energy homeostasis in rodents and birds. Previously, our group has isolated four cDNAs encoding precursor proteins of NMU from the goldfish brain and gut, and it was assumed that these transcripts are produced by alternative splicing. We have also demonstrated that intracerebroventricular (ICV) injection of putative goldfish NMU inhibits food intake. However, as native goldfish NMU has not yet been identified, we attempted to purify it from goldfish brain and gut extracts. To assess NMU activity in fractions at each purification step, we measured changes in the intracellular concentrations of Ca2+ using HEK293 cells expressing goldfish NMU-R1 or -R2. We isolated a 25-amino-acid peptide (NMU-25) from the brain and gut and found that its primary structure is similar to that of mammalian NMU. Another 21-amino-acid peptide (NMU-21) was purified from the brain, but not from the gut. Furthermore, a 9-amino-acid peptide (NMU-9) identical to the C-terminus of NMU-21 and -25 was also isolated from the brain and gut. Treatment with synthetic NMU-9, -21 and -25 dose-dependently increased the intracellular Ca2+ concentration in mammalian cells expressing goldfish NMU-R1 and -R2. We also examined the effect of ICV-administered synthetic goldfish NMUs on goldfish food intake. NMU-25 inhibited food intake to the same degree as NMU-21. However, the inhibitory effect of NMU-9 was slightly weaker than those of NMU-21 and -25. These results indicate that several molecular forms of NMU exist in the goldfish brain and gut, and that all of them play physiological roles via NMU-R1 and NMU-R2.