Suppressive Effects of Neuromedin U Receptor 2-Selective Peptide Agonists on Appetite and Prolactin Secretion in Mice

Abstract

Neuromedin U receptor 2 (NMUR2), which is expressed in the central nervous system (CNS) including the hypothalamus, has been noted as a therapeutic target against obesity. We previously reported that intranasal administration of CPN-219, a NMUR2-selective hexapeptide agonist, suppresses body weight gain in mice; however, there is no detailed information regarding its CNS effects. Recently, in addition to appetite suppression, stress responses and regulation of prolactin (PRL) secretion have also attracted attention. NMUR2 expressed in the hypothalamic tuberoinfundibular dopaminergic neurons has emerged as an alternative target for treating hyperprolactinemia. Here, CPN-219 decreased food intake up to 24 h after administration at a dose of 200 nmol, resulting in body weight gain suppression, although grooming and anxiety-like behaviors were transiently induced. Interestingly, the restraint stress-induced increase in plasma PRL levels was significantly suppressed at a lower dose of 20 nmol, indicating the potential for drug development as an anti-PRL agent of NMUR2-selective agonists.

Neuromedin U (NMU), originally isolated from the porcine spinal cord,¹ is a neuropeptide that activates two types of receptors (NMUR1 and NMUR2). NMUR1 mRNA is highly expressed in peripheral tissues such as the small intestine and lungs, whereas NMUR2 mRNA is mainly detected in the central nervous system (CNS), including the hypothalamus.²⁻⁴ Intracerebroventricular NMU administration in rats decreases food intake and body weight via secretion of corticotropin-releasing hormone (CRH) from the hypothalamic paraventricular nucleus (PVN).^5,6 Therefore, NMU has attracted attention as an anorectic peptide for more than 2 decades. Obesity and overweight are global health issues that are risk factors for various disorders, including diabetes and cardiovascular disease.^7,8 In the drug discovery approach targeting NMU receptors, fatty acid, protein, and polymer modifications have been applied to NMU for improving in vivo retention, and those conjugates showed appetite suppression and body-weight-lowering effects.⁹⁻¹⁶ Among them, Nagai et al. reported that the NMUR2-selective agonist bearing 20 kDa poly(ethylene glycol) (PEG) subcutaneously injected into diet-induced obese mice had higher efficacy against obesity and lower induction of diarrhea as an adverse effect compared to the NMUR1-selective one.¹⁴ It also indicated that the PEGylated NMUR2 agonists activate the hypothalamic arcuate nucleus (ARC). In 2014, Benzon et al. suggested that NMUR2 signaling in the rat PVN was important for the anorectic response to high-fat diet intake.¹⁷ Based on these reports, an approach effectively acting on NMUR2 in the CNS, particularly in the hypothalamus, is noteworthy.

Intranasal (i.n.) administration is a promising strategy for drug delivery to the brain.¹⁸ The olfactory and trigeminal nerve pathways have been suggested as delivery routes for drugs injected into the nasal cavity.¹⁸⁻²¹ High delivery efficiency to the CNS and avoidance of peripheral adverse effects are expected in nose-to-brain delivery. We previously reported that CPN-219, a hexapeptidic NMUR2-selective agonist (Figure 1), dose-dependently suppressed body weight gain in mice by i.n. administration but not by intraperitoneal injection.^22,23 However, detailed evaluations of the CNS effects of CPN-219, including appetite suppression, have not yet been carried out.

Figure 1.

Sequence of human NMU and CPN-219 structure. CPN-219 was previously obtained through a structure–activity relationship (SAR) study based on the C-terminal amidated heptapeptide (position 19–25 on human NMU) completely shared among mammals, which is a core for activation of both NMURs.^22,23

Besides its antiobesity effects, several studies have suggested that NMU is related to the stress response. In 2001, Hanada et al. first demonstrated that intracerebroventricularly (icv) administered NMU induces grooming behavior via CRH in rats.²⁴ Similarly, De Prins et al. reported that icv injection of NMU-8, a short version of endogenous NMU, in mice affected the induction of grooming and the increase of c-Fos immunoreactivity in PVN and ARC.²⁵ Moreover, Nakahara et al. indicated that NMU suppresses prolactin (PRL) release from the pituitary gland via dopaminergic neurons of ARC, which is stimulated by restraint stress, lactation, and pseudopregnancy.²⁶ Although it is attracting attention like this, there are no reports to investigate stress response in i.n. administration of NMUR2 agonists, including CPN-219, so far.

In this study, we evaluated feeding- and stress-related behaviors and the effects on PRL secretion after i.n. administration of CPN-219 to mice.

To investigate the effect of CPN-219 on feeding behavior, mice were individually housed, and the amount of food and water consumed was measured daily. Intranasally administered CPN-219 (200 nmol) at day 0 significantly suppressed body weight gain, as previously reported (Figure 2A).²² Daily food intake of mice administered CPN-219 was significantly lower on day 1 than that of vehicle-treated mice (Figure 2B), whereas daily water intake was similar between the two groups (Figure 2C). These results suggested that appetite suppression for 24 h after CPN-219 administration at least partly contributed to body weight gain suppression.

Figure 2.

Time courses of body weight (A), food consumption (B), and water intake (C) after a single i.n. administration of CPN-219 (200 nmol) or vehicle (saline) at day 0. Each point represents the mean ± SD (N = 8). *p < 0.05 vs vehicle by two-way repeated measures ANOVA followed by a Tukey–Kramer test.

As an undesirable effect, rapid tachyphylaxis development upon repeated dosing is of concern for some NMU analogs;²⁷ we previously reported the possibility of developing tachyphylaxis with a hexapeptidic NMUR1-selective agonist subcutaneously injected for 3 days.²⁸ In this study, daily i.n. administration of CPN-219 for 3 days was similarly performed to evaluate whether tachyphylaxis was caused based on the time course of body weight and food intake. Following CPN-219 administration on days 0–2, the body weight relative to day 0 was maintained below 100% until day 3 (Figure 3A), and daily food intake decreased on days 1–3 (Figure 3B). The daily water intake was not significantly different between the two groups, although the water intake on days 4 and 5 was higher than that on day 3 in the CPN-219 group (Figure 3C). These results suggest that repeated i.n. administration of CPN-219 to mice continuously suppressed appetite without the rapid development of tachyphylaxis.

Figure 3.

Time courses of body weight (A), food consumption (B), and water intake (C) with repeated i.n. administration of CPN-219 (200 nmol) or vehicle (saline) at days 0–2. Each point represents the mean ± SD (N = 8). **p < 0.01 vs vehicle, and ^##p < 0.01 vs day 3 of CPN-219 by two-way repeated measures ANOVA followed by a Tukey–Kramer test.

Next, stress-related behaviors in the mice were analyzed by using a video tracking system (Figure 4). The total distance traveled, cumulative grooming time, and inner ratio were measured as indicators of activity, stress response behavior, and anxiety-like behavior using a square open field of 40 × 40 × 50 cm³ at 45 min and 24 h after i.n. administration. The inner ratio is the percentage of time spent in the inner zone, which is defined as a 20 × 20 cm² square in the center of the field.²⁹ Mice placed in a novel environment explore it entirely, including the inner zone, while mice prefer the area near the wall as an innate habit, called thigmotaxis.³⁰ Since the behavior based on this habit is increased by strengthening anxiety, the inner ratios in the open field test are decreased. At 45 min after i.n. CPN-219 administration, the cumulative grooming times and inner ratios significantly increased and decreased, respectively, although the total distance traveled was not altered (Figure 4). The levels were the same between the two groups after 24 h. It is considered that the reduced inner ratio at 24 h compared to that at 45 min in the vehicle group was due to acclimatization in the second trial. Stress-related behaviors 3 h after CPN-219 administration were similarly evaluated (Figure S1). CPN-219 reproducibly suppressed body weight gain and appetite in mice, similar to the results shown in Figure 2. However, cumulative grooming times, inner ratios, and total distance traveled were not altered. These results indicate that transient stress responses and anxiety-like behaviors were induced by i.n. CPN-219 administration.

Figure 4.

At 45 min and 24 h after CPN-219 administration, activity (A), stress response behavior (B), and anxiety-like behavior (C) of mice were evaluated in the open field. Each column represents the mean ± SD (N = 8). *p < 0.05 and **p < 0.01 vs 45 min of vehicle, and ^##p < 0.01 vs 45 min of CPN-219 by two-way repeated measures ANOVA followed by a Tukey–Kramer test.

Finally, we examined the suppressive effect of CPN-219 on restraint stress-induced PRL secretion. Plasma PRL levels after subjecting vehicle-administered mice (stressed vehicle) to restraint stress for 40 min were elevated compared with those without stress subjection (unstressed vehicle) (Table 1). This restraint stress-induced increase in PRL concentration was significantly suppressed by i.n. CPN-219 administration at a dose of 20 nmol (stressed CPN-219), and there was no significant difference in the levels (0.49 ± 0.92 ng/mL) in comparison with the unstressed vehicle (0.45 ± 0.14 ng/mL). Plasma PRL concentrations in CPN-219-administered mice without stress were below the detection limit (unstressed CPN-219). In the alternative experiment, intranasally administered CPN-219 (20 nmol) reproducibly suppressed the elevation of plasma PRL levels induced by restraint stress, along with the case at a dose of 200 nmol (Table S1). Previously, we reported that the body weight gain is not affected at a dose of 25 μg of CPN-219 (corresponding to 26 nmol as acetate salts).²² Therefore, we believe that the action of CPN-219 on dopaminergic neurons is greater than that on CRH neurons.

Table 1. Restraint Stress-Induced Increase of Plasma PRL Levels Suppressed by CPN-219 (20 nmol) Administration^a.

administration (i.n.)	stress	PRL concn (ng/mL plasma)
vehicle	–	0.45 ± 0.14**
	+	2.04 ± 0.95
CPN-219	–	<0.15##
	+	0.49 ± 0.92##

^aEach value represents the mean ± SD (unstressed (−): N = 8; stressed (+): N = 7). **p < 0.01 vs unstressed vehicle, and ^##p < 0.01 vs stressed vehicle by one-way repeated measures ANOVA followed by a Tukey–Kramer test.

Additionally, to investigate the sustainability of the anti-PRL effect, plasma PRL levels at 90 min after i.n. CPN-219 administration were measured. Elevation of plasma PRL levels by stress subjection for 40 min just before blood collection (time range: 50–90 min) was not significantly suppressed by CPN-219 (Table S2), suggesting that the duration of suppressive effect toward PRL secretion was limited.

In summary, CPN-219 administered to the nasal cavity suppressed appetite for up to 24 h, although stress-related grooming and anxiety-like behaviors were induced within a limited time window, suggesting that it at least partly contributes to body weight gain suppression. Interestingly, restraint stress-induced PRL secretion was suppressed by i.n. CPN-219 administration at a lower dose than that inducing appetite suppression.

In clinical practice, prolactinoma, a secretory pituitary tumor, presents with hyperprolactinemia; it leads to several psychologically burdensome symptoms, such as oligomenorrhea, infertility, and galactorrhea in women and hypogonadism, decreased libido, erectile dysfunction, infertility, and gynecomastia in men.³³ Cabergoline, a dopamine agonist, is usually used as first-line therapy due to its fewer side effects compared to bromocriptine. However, since dopamine agonists act on the brain nonspecifically, various side effects, including nausea, constipation, and psychiatric symptoms, are known to be induced.³³ There are some case reports that cabergoline induces psychosis and mania;³⁴⁻³⁶ those episodes can lead to interruption in therapy, which is generally taken for longer than a year. However, the confined action of NMU on hypothalamic tuberoinfundibular dopaminergic neurons, especially ARC, is expected to be an alternative therapeutic strategy against hyperprolactinemia since the substantia nigra, one of the main regions of dopamine release, was not activated by its icv injection.²⁶ Therefore, the present findings that plasma PRL levels were modulated by the less invasive i.n. administration of NMUR2 agonists contribute to the advancement of anti-PRL therapy.

In the future, a pharmaceutical approach using some kind of additive^31,32 to improve bioavailability and development of drug seeds possessing prolonged residence time on NMUR2 may be necessary for the enhancement of the anti-PRL effect, including activity duration. Meanwhile, the study based on the NMUR2 agonist treatment in a longer time scale would provide valuable information directing therapeutic application, including antigenicity.

Glossary

Abbreviations

ARC

arcuate nucleus

CNS

central nervous system

CRH

corticotropin-releasing hormone

Fmoc

9-fluorenylmethyloxycarbonyl

icv

intracerebroventricular

i.n.

intranasal

NMU

neuromedin U

NMUR1/2

neuromedin U receptor 1/2

PEG

poly(ethylene glycol)

PRL

prolactin

PVN

paraventricular nucleus

RP-HPLC

reverse-phase high-performance liquid chromatography

SAR

structure–activity relationship

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.3c00541.

• Materials and experimental procedures, Tables S1–S3, and Figure S1 (PDF)

Author Contributions

All authors were involved in the preparation of this manuscript, including the conception and design of the study (A.N. and K.T.), peptide synthesis (K.T.), animal studies (A.N., Y.S., and K.T.), PRL measurement (A.N.), data analysis (A.N. and Y.S.), and writing and editing of the manuscript (A.N., K.M., K.N., and K.T.). All the authors approved the final version of the manuscript.

Takeda Science Foundation (K.T.).

The authors declare no competing financial interest.