PEGylation of Neuromedin U yields a promising candidate for the treatment of obesity and diabetes

Neuromedin U (NMU) is an endogenous peptide, whose role in the regulation of feeding and energy homeostasis is well documented. Two NMU receptors have been identified: NMUR1, expressed primarily in the periphery, and NMUR2, expressed predominantly in the brain. We recently demonstrated that acute peripheral administration of NMU exerts potent but acute anorectic activity and can improve glucose homeostasis, with both actions mediated by NMUR1. Here, we describe the development of a metabolically stable analog of NMU, based on derivatization of the native peptide with high molecular weight poly(ethylene) glycol (PEG) ('PEGylation'). PEG size, site of attachment, and conjugation chemistry were optimized, to yield an analog which displays robust and long-lasting anorectic activity and significant glucose-lowering activity in vivo. Studies in NMU receptor-deficient mice showed that PEG-NMU displays an expanded pharmacological profile, with the ability to engage NMUR2 in addition to NMUR1. In light of these data, PEGylated derivatives of NMU represent promising candidates for the treatment of obesity and diabetes.     

A potent neuromedin U receptor 2-selective alkylated peptide

Neuromedin U (NMU) mediates various physiological functions via NMUR1 and NMUR2 receptors. NMUR2 has been considered a promising treatment option for diabetes and obesity. Although NMU-8, a shorter peptide, has potent agonist activity for both receptors, it is metabolically unstable. Therefore, NMU-8 analogs modified with long-chain alkyl moieties via a linker were synthesized. An octadecanoyl analog (17) with amino acid substitutions [αMePhe¹⁹, Nle²¹, and Arg(Me)²⁴] and a linker [Tra-γGlu-PEG(2)] dramatically increased NMUR2 selectivity, with retention of high agonist activity. Subcutaneous administration of 17 induced anorectic activity in C57BL/6J mice. Owing to its high metabolic stability, 17 would be useful in clarifying the physiological role and therapeutic application of NMU.     

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.     

Syntheses of two neuromedin U (NMU) analogues and their comparative reducing food intake effect in rats

Summary.  To examine the roles of aromatic rings Tyr residues at positions 1 and 6 and Phe residues at positions 16, 17 and 19 of rat neuromedin U-23 (NMU-23) (Tyr-Lys-Val-Asn-Glu-Tyr-Gln-Gly-Pro-Val-Ala-Pro-Ser-Gly-Phe-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH₂) for reducing food intake activity in male Wistar rats, two NMU-23 analogues, [Phe(4F)^16,17,19]NMU-23 and [Tyr(Me)^1,6]NMU-23, were synthesized by Fmoc strategy of manual solid-phase method. The synthetic NMU-23 showed reducing effect on food intake in rats. [Phe(4F)^16,17,19]NMU-23 exhibited higher reducing food intake effect than that of NMU-23. On the contrary, [Tyr(Me)^1,6]NMU-23 showed no reducing effect on food intake in rats than that of NMU-23. Received July 6, 2002 Accepted October 30, 2002 Published online January 20, 2003 Acknowledgements We are grateful to Dr. Hiroshi Sekino of Kojinkai Central Hospital for giving us opportunities to do some experiments at Kidney Research Laboratory in Kojinkai Central Hospital in Sendai. Authors' address: Takashi Abiko, Ph. D., Research Laboratory, Global Shinwa Pharmaceutical Company, 4-17-29, Komatsushima, Aoba-ku, Sendai, 981-0905 Japan, E-mail: abiko@ma.mni.ne.jp Abbreviations: NMU, neuromedin U; TLC, thin layer chromatography; 4F, 4-fluoro; TFA, trifluoroacetic acid; Fmoc, 9-fluorenylmethoxycarbonyl; NPY, neuropeptide Y; Obu^t, tert-butoxy; Trt, trityl; HPLC, high performance liquid chromatography; FAB-MS, fast atom bombardment mass spectrometry; AcOH, acetic acid; DCC, N,N′-dicyclohexylcarbodiimide; HOBT, 1-hydroxybenzotriazole; Mts, mesitylenesulfonyl; Boc, tert-butoxycarbonyl; DMF, dimethyformamide; TMSOTf, trimethylsilyltrifrate; EtOH, ethanol.     

Identification and functional analysis of a novel ligand for G protein-coupled receptor, Neuromedin S

We identified a novel 36-amino acid neuropeptide in rat brain as an endogenous ligand for the G protein-coupled receptors FM-3/GPR66 and FM-4/TGR-1, which were identified to date as the neuromedin U (NMU) receptors, and designated this peptide neuromedin S (NMS) because it was specifically expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus. NMS shared a C-terminal core structure with NMU. NMS mRNA was highly expressed in the central nervous system, spleen and testis. In rat brain, NMS expression was restricted to the ventrolateral portion of the SCN and has a diurnal peak under light/dark cycling, but remains stable under constant darkness. Intracerebroventricular (ICV) administration of NMS in rats induced nonphotic type phase shifts in the circadian rhythm of locomotor activity. ICV injection of NMS also decreased 12-h food intake during the dark period in rats. This anorexigenic effect was more potent than that observed with the same dose of NMU. ICV administration of NMS increased proopiomelanocortin (POMC) mRNA expression in the arcuate nucleus (Arc) and corticotropin-releasing hormone mRNA in the paraventricular nucleus, and induced c-Fos expression in the POMC neurons in the Arc. These findings suggest that NMS is implicated in the regulation of circadian rhythm and feeding behavior.     

Central effects of neuromedin U in the regulation of energy homeostasis

Neuromedin U (NMU) is a brain-gut peptide whose peripheral activities are well-understood but whose central actions have yet to be clarified. The recent identification of two NMU receptors in rat brain has provided a springboard for further investigation into its role in the central nervous system. Intracerebroventricular administration of NMU to free-feeding rats decreased food intake and body weight. Conversely, NMU increased gross locomotor activity, body temperature, and heat production. NMU, a potent endogenous anorectic peptide, serves as a catabolic signaling molecule in the brain. Further investigation of the biochemical and physiological functions of NMU will help our better understanding of the mechanisms of energy homeostasis.     

Synthesis and evaluation of novel lipidated neuromedin U analogs with increased stability and effects on food intake

Neuromedin U (NMU) is a 25 amino acid peptide expressed and secreted in the brain and gastrointestinal tract. Data have shown that peripheral administration of human NMU decreases food intake and body weight and improves glucose tolerance in mice, suggesting that NMU receptors constitute a possible anti‐diabetic and anti‐obesity drug target. However, the clinical use of native NMU is hampered by a poor pharmacokinetic profile. In the current study, we report in vitro and in vivo data from a series of novel lipidated NMU analogs. In vitro plasma stability studies of native NMU were performed to investigate the proteolytic stability and cleavage sites using LC–MS. Native NMU was found to be rapidly cleaved at the C‐terminus between Arg²⁴ and Asn²⁵, followed by cleavage between Arg¹⁶ and Gly¹⁷. Lipidated NMU analogs were generated using solid‐phase peptide synthesis, and in vitro potency was investigated using a human embryonic kidney 293‐based inositol phosphate accumulation assay. All lipidated analogs had preserved in vitro activity on both NMU receptors with potency improving as the lipidation site was moved away from the receptor‐interacting C‐terminal octapeptide segment. In vivo efficacy was assessed in lean mice as reduction in food intake after acute subcutaneous administration of 1, 0.3, 0.1, and 0.03 µmol/kg. These lipidated NMU analogs prolonged the anorectic effect of NMU in a dose‐dependent manner. This was likely an effect of improved pharmacokinetic properties because of improved vitro plasma stability. Accordingly, the data demonstrate that lipidated NMU analogs may represent drug candidates for the treatment of obesity. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Development of a neuromedin U–human serum albumin conjugate as a long‐acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide

Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half‐life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half‐life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA–NMU displayed long‐lasting, potent anorectic, and glucose‐normalizing activity. When compared side by side with a previously described PEG conjugate, HSA–NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU‐based therapeutics are promising candidates for the treatment of obesity and diabetes. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Distribution and characterisation of neuromedin U-like immunoreactivity in rat brain and intestine and in guinea pig intestine

Neuromedin U-8 (NMU-8) is a peptide isolated from porcine spinal cord which contracts blood vessels and the uterus. Antisera were raised against NMU-8 and used in a radioimmunoassay (RIA) together with HPLC to characterize NMU-like immunoreactivity (NMU-LI) in tissues extracts of rat brain and gut and guinea pig gut. Samples of duodenum, ileum and distal colon were taken from both species, and processed for detection of NMU-LI by fluorescence immunohistochemistry. In RIA the antiserum had no cross-reactivity with neuropeptide Y, vasoactive intestinal peptide or the C-terminal hexapeptide of pancreatic polypeptide. Preincubation of antiserum with any of these peptides had no effect on the NMU-LI staining. In rats the highest content of NMU-LI was found in the ileum and the lowest in the cerebral cortex and striatum. HPLC studies showed that at least two molecular forms of NMU-LI were present in both species. In rat small intestine, subpopulations of submucous and myenteric neurones were stained; nerve fibres and terminals within these ganglia and in the mucosa were also seen. NMU-LI was sparse in the muscle. In guinea pig ileum small populations of nerve terminals were seen in both myenteric and submucous ganglionated plexuses. No endocrine cells were stained in either species.     

Identification of neuromedin S and its possible role in the mammalian circadian oscillator system

The discovery of neuropeptides has resulted in an increased understanding of novel regulatory mechanisms of certain physiological phenomena. Here we identify a novel neuropeptide of 36 amino-acid residues in rat brain as an endogenous ligand for the orphan G protein-coupled receptor FM-4/TGR-1, which was identified to date as the neuromedin U (NMU) receptor, and designate this peptide 'neuromedin S (NMS)' because it is specifically expressed in the suprachiasmatic nuclei (SCN) of the hypothalamus. NMS shares a C-terminal core structure with NMU. The NMS precursor contains another novel peptide. NMS mRNA is highly expressed in the central nervous system, spleen and testis. In rat brain, NMS expression is restricted to the core of the SCN and has a diurnal peak under light/dark cycling, but remains stable under constant darkness. Intracerebroventricular administration of NMS in rats activates SCN neurons and induces nonphotic type phase shifts in the circadian rhythm of locomotor activity. These findings suggest that NMS in the SCN is implicated in the regulation of circadian rhythms through autocrine and/or paracrine actions.     

Synthesis of two neuromedin U (NMU) analogues and their comparative effect of reducing food intake in rats

To examine the roles of aromatic rings, Tyr residues at positions 1 and 5 and Phe residues at positions 16, 17, and 19 of rat neuromedin U-23 (NMU-23) (Tyr-Lys-Val-Asn-Glu-Tyr-Gln-Gly-Pro-Val-Ala-Pro-Ser-Gly-Gly-Phe-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2) for reducing food intake activity in male Wistar rats, two NMU-23 analogues, [Phe(4F)16,17,19] NMU-23 and [Tyr(Me)1,6]NMU-23, were synthesized by Fmoc strategy of manual solid-phase method. The synthetic NMU-23 showed reducing effect on food intake in rats. [Phe(4F)16,17,19]NMU-23 exhibited higher reducing food in take effect than that of NMU-23. On the contrary, [Tyr(- Me)1,6]NMU-23 showed no reducing effect on food intake in rats than that of NMU-23.     

Comparison of the anorexigenic activity of CRF family peptides

Corticotropin releasing factor (CRF) family peptides have an important role in the control of food intake. We investigated the effects of CRF family peptides on food intake and body weight gain in mice. Of the CRF family peptides, including CRF, urocortin1 (Ucn1), urocortin2 (Ucn2) and urocortin3 (Ucn3), peripherally administered Ucn1 was shown to have the most potent inhibitory effect on the food intake and body weight gain of both lean and high fat fed obese mice. In addition, repeated administration of Ucn1 lowered blood glucose and acylated ghrelin, and decreased the visceral fat weight of high fat fed obese mice.     

Neuromedin U has a novel anorexigenic effect independent of the leptin signaling pathway

Neuromedin U (NMU) is a hypothalamic neuropeptide that regulates body weight and composition. Here we show that mice lacking the gene encoding NMU (Nmu(-/-) mice) develop obesity. Nmu(-/-) mice showed increased body weight and adiposity, hyperphagia, and decreased locomotor activity and energy expenditure. Obese Nmu(-/-) mice developed hyperleptinemia, hyperinsulinemia, late-onset hyperglycemia and hyperlipidemia. Notably, however, treatment with exogenous leptin was effective in reducing body weight in obese Nmu(-/-) mice. In addition, central leptin administration did not affect NMU gene expression in the hypothalamus of rats. These results indicate that NMU plays an important role in the regulation of feeding behavior and energy metabolism independent of the leptin signaling pathway. These characteristic functions of NMU may provide new insight for understanding the pathophysiological basis of obesity.     

Regulation of gonadotropin secretion and puberty onset by neuromedin U

Neuromedin U (NMU), an anorexigenic peptide, was originally isolated from porcine spinal cord in 1985. As NMU is abundant in the anterior pituitary gland, we investigated the effects of NMU on gonadotropin secretion. Both NMU and its receptors, NMUR1 and NMUR2, were expressed in the pituitary gland. NMU suppressed LH and FSH releases from rat anterior pituitary cells. Moreover, NMU-deficient mice exhibit an early onset of vaginal opening. The LHbeta/FSHbeta ratio, which is an index of puberty onset, is high in young NMU-deficient mice. These results indicate that NMU suppresses gonadotropin secretion and regulates the onset of puberty.     

Amylin receptors mediate the anorectic action of salmon calcitonin (sCT)

The teleost salmon calcitonin (sCT), but not mammalian CT, shows similar biologic actions in the skeletal muscle as amylin and calcitonin gene-related peptide (CGRP). The peptides have also been shown to reduce food intake in rams. Because sCT, but not amylin, binds irreversibly to amylin binding sites, the aim of the present study was to compare the anorectic potency of both peptides. To determine whether sCT reduces food intake through interaction with amylin binding sites, we also tested whether appropriate antagonists (CORP 8-37, AC 187) attenuate the anorectic effect of sCT. Finally, we wanted to know whether rat calcitonin (rCT) and sCT reduce food intake to the same extent. Peptides were injected intraperitoneally at dark onset in 24 h food-deprived rats. At doses of 5 or 0.5 microg/kg, the anorectic effect of sCT was more potent and lasted much longer (e.g. 5 microg/kg: sCT > 10 h; amylin approx. 2 h) than that of amylin. Both CORP 8-37 and AC 187 (10 microg/kg) markedly reduced the anorectic action of sCT (0.5 microg/kg). In contrast to sCT, rCT (0.5 microg/kg) had no effect on food intake. It is concluded that sCT s anorectic effect is partly mediated by amylin receptors. Irreversible binding of sCT to amylin receptors may lead to a stronger and prolonged effect in comparison to amylin due to a sustained activation of the binding sites. Similar to other actions of CTs, the anorectic potency of sCT in rats was higher than that of mammalian (rat) CT. This agrees with binding profiles of amylin, sCT, and rCT at amylin binding sites as observed in in vitro studies.     

Neuromedin U-immunoreactivity in the nervous system of the small intestine of the pig and its coexistence with substance P and CGRP

Summary In the small intestine of the pig, neuromedin U (NMU)-immunoreactivity was mainly confined to the nerve plexus of the inner submucosal and mucosal regions. After colchicine treatment, a high number of immunoreactive nerve cell bodies was observed in the plexus submucosus internus (Meissner), whereas only a low number was found in the plexus submucosus externus (Schabadasch). The plexus myentericus as well as the aganglionic nerve meshworks in the circular and longitudinal smooth muscle layers almost completely lacked NMU-immunoreactivity. Double-labeling experiments demonstrated the occurrence of distinct NMU-containing neuron populations in the plexus submucosus internus: (1) relatively large type-II neurons revealing immunoreactivity for NMU and calcitonin gene-related peptide (CGRP) and/or substance P (SP); (2) a group of small NMU- and SP-immunoreactive neurons; (3) a relatively low number of small neurons displaying immunoreactivity for NMU but not for SP. Based on its distributional pattern, it is concluded that NMU plays an important role in the regulation and control of mucosal functions.     

Neuromedin U is involved in nociceptive reflexes and adaptation to environmental stimuli in mice

Following our recent observations of inactivity and slowed movement in neuromedin U knockout (NMU KO) mice, we compared nociceptive reflexes and environmental adaptation in NMU KO and wild-type mice. Hot plate and formalin tests revealed that reflexes to heat and pain were significantly decreased in NMU KO mice. Conversely, intracerebroventricular injection of NMU into wild-type mice stimulated nociceptive reflexes in a dose-dependent manner. After NMU injection, increased c-Fos expression was observed in a wide range of locations in hypothalamus, brainstem, and spinal cord. NMU mRNA expression increased in the spinal cord, but not in the hypothalamus, 2 and 4 h after formalin injection. When their light-dark cycle was advanced or retarded by 5 h, NMU KO mice required a longer time to re-entrain into the new light-dark cycle than did wild-type mice. Wild-type mice displayed increased blood pressure after their environmental temperature was changed from 23 to 37 degrees C, whereas no such increase was observed in NMU KO mice. Blood corticosterone levels were significantly increased after 10 min of immobilization stress in wild-type mice, but not in NMU KO mice. These results suggest that endogenous NMU may be involved in reflexes and adaptation to environmental stimuli.     

Potent antiobesity effect of a short-length peptide YY-analogue continuously administered in mice

The gastrointestinal peptide, peptide YY_3–36 (PYY_3–36) and its shorter peptide analogues have been reported to reduce appetite by activating the neuropeptide Y2 receptor (Y2R), which is associated with obesity and other metabolic diseases. A 14-amino acid PYY analogue, Ac-[d-Pro²⁴,Cha^27,28,36,Aib³¹]PYY(23–36) (3), showed high binding affinity and agonist activity for the Y2R, similar to that of PYY_3–36, but had weak anorectic activity upon continuous administration in lean mice. Three amino acid substitutions [Pya(4)²⁶, Aib²⁸, Lys³⁰], which contributed to the decreased hydrophobicity of 3, efficiently increased its anorectic activity. The compound containing these three amino acids, Ac-[d-Pro²⁴,Pya(4)²⁶,Cha^27,36,Aib^28,31,Lys³⁰]PYY(23–36) (22), exerted more potent and durable food intake suppression than that by PYY_3–36 in lean mice, as well as excellent Y2R agonist activity (EC₅₀: 0.20 nM) and good subcutaneous bioavailability (66.6%). The 11-day continuous administration of 22 at 1 mg/kg/day successfully produced antiobese and antidiabetic effects, with more than 20% body weight loss in obese and Type 2 diabetes ob/ob model mice.     

PEGylation of cholecystokinin prolongs its anorectic effect in rats

The anorectic compound CCK-9 was coupled to polyethylene glycol 5 kDa, 10 kDa, 20 kDa and 30 kDa, under different reaction conditions. Conjugates were purified by HPLC and characterized by MALDI-TOF MS. A 96% PEGylation yield was obtained in buffer pH 7.5 after 6h reaction at 20 degrees C. The anorectic activity was tested in vivo in rats. A single bolus intra-peritoneal injection of non-modified CCK-9 resulted in a significant initial food intake reduction 30 min after food presentation (87% compared to paired control group). When PEG-CCK-9 conjugates modified with polymers of molecular weight up to 20 kDa were injected, lower but statistically significant initial food intake reductions were obtained (76% for PEG 10 kDa-CCK-9 conjugate compared to control group). The cumulative food intake reduction of non-modified CCK-9 is normalized within 1-2h, whereas the PEG-CCK-9 molecules showed a prolonged anorectic activity lasting for 6h for PEG 5 kDa-CCK-9; 23 h for PEG 10 kDa-CCK-9 and between 8h and 23 h for PEG 20 kDa-CCK-9. For PEG 30 kDa-CCK-9 conjugate, neither an initial nor a cumulative FI reduction was observed. PEG-CCK-9 conjugates show a significantly prolonged anorectic activity in comparison to the non-modified peptide. This effect is most evident for the PEG 10 kDa-CCK-9 conjugate.     

Isolation and microsequence analysis of a novel form of neuromedin U from guinea pig small intestine

A multidimensional chromatographic regimen has been used to isolate and purify a peptide showing immunoreactivity for neuromedin U from guinea pig small intestine. Microsequence Edman N-terminal analysis and C-terminal analysis by enzymatic digestion showed this peptide to be a nonapeptide with the following sequence: H-Gly-Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2. The C-terminal octapeptide of this sequence is the same as porcine NMU-8, and the C-terminal heptapeptide is identical to rat NMU(17-23).