Neuromedin S Is a Novel Anorexigenic Hormone
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《内分泌学杂志》
Department of Veterinary Physiology (T.I., Y.E., S.A., K.N., N.M.), Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2155
Department of Biochemistry (K.M., M.M., K.K.), National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka 565-8565
Laboratory of Veterinary Physiology (M.N.), Veterinary Medical Science, The University of Tokyo, Tokyo 113-8657, Japan
Abstract
A novel 36-amino acid neuropeptide, neuromedin S (NMS), has recently been identified in rat brain and has been shown to be an endogenous ligand for two orphan G protein-coupled receptors, FM-3/GPR66 and FM-4/TGR-1. These receptors have been identified as neuromedin U (NMU) receptor type 1 and type 2, respectively. In this study, the physiological role of the novel peptide, NMS, on feeding regulation was investigated. Intracerebroventricular (icv) injection of NMS decreased 12-h food intake during the dark period in rats. This anorexigenic effect was more potent and persistent than that observed with the same dose of NMU. Neuropeptide Y, ghrelin, and agouti-related protein-induced food intake was counteracted by coadministration of NMS. Icv administration of NMS increased proopiomelanocortin mRNA expression in the arcuate nucleus (Arc) and CRH mRNA in the paraventricular nucleus (PVN). Pretreatment with SHU9119 (antagonist for -MSH) and -helical corticotropin-releasing factor-(9–41) (antagonist for CRH) attenuated NMS-induced suppression of 24-h food intake. After icv injection of NMS, Fos-immunoreactive cells were detected in both the PVN and Arc. When neuronal multiple unit activity was recorded in the PVN before and after icv injection of NMS, a significant increase in firing rate was observed 5 min after administration, and this increase continued for 100 min. These results suggest that the novel peptide, NMS, may be a potent anorexigenic hormone in the hypothalamus, and that expression of proopiomelanocortin mRNA in the Arc and CRH mRNA in the PVN may be involved in NMS action on feeding.
Introduction
NEUROMEDIN U (NMU), originally isolated from porcine spinal cord, is a brain-gut peptide that has potent contractile activity on uterine smooth muscle (1). In previous studies, two orphan G protein-coupled receptors, FM-3/GPR66 and FM-4/TGR-1, were identified as NMU receptor type 1 (NMU1R) and type 2 (NMU2R), respectively (2, 3, 4, 5). Recently, a novel 36-amino acid neuropeptide was identified in rat brain as another endogenous ligand for FM-3/GPR66 and FM-4/TGR-1 using a reverse-pharmacological technique (6). This neuropeptide was designated neuromedin S (NMS) because it is specifically expressed in the suprachiasmatic nucleus (SCN). Although the NMS shares a C-terminal core structure (seven-amino acid residues) with NMU and activates both recombinant NMU1R and NMU2R expressed in Chinese hamster ovary cells, NMS is not a splice variant of NMU because both NMS and NMU genes were mapped to discrete chromosomes. In addition, although NMU mRNA was detected in peripheral and central organs (7), the distribution of NMS was limited to the testis, spleen and SCN (6). NMS was recently suggested to be involved in circadian oscillation systems because intracerebroventricular (icv) administration of NMS induces phase-dependent phase shifts in the circadian rhythm of locomotor activity in rats kept under constant darkness (6).
NMU1R is located in a wide range of peripheral tissues such as intestine, testis, pancreas, uterus, lung, and kidney. On the other hand, expression of NMU2R is limited to areas of the brain such as the paraventricular nucleus (PVN), along the wall of the third ventricle in the hypothalamus and the CA1 region of the hippocampus (2, 5, 8, 9). Immunohistochemical and in situ analysis has revealed NMU-immunoreactive neurons or NMU mRNA expression in the ventromedial hypothalamic region including the arcuate nucleus (Arc), pituitary, caudal brainstem region including the nucleus of the solitary tract, area postrema, dorsal motor nucleus of the vagus nerve and inferior olive, and spinal cord (2, 10, 11). NMU-immunoreactive fibers project prominently into the PVN, ventromedial nucleus, dorsomedial nucleus, and Arc. It has been well documented that the PVN and Arc of the hypothalamus play pivotal roles in the regulation of feeding behavior through a complex neuronal network composed of several orexigenic neuropeptides such as neuropeptide Y (NPY), agouti-related protein (AGRP) and ghrelin, and anorexigenic neuropeptides such as -MSH, cocaine- and amphetamine-regulated transcript, CRH, and leptin (12, 13). Icv administration of NMU suppresses both dark-phase food intake and fasting-induced feeding, suggesting that NMU acts as anorexigenic hormone (2, 3). Conversely, disruption of the NMU gene in mice [NMU knockout (KO) mice] resulted in severe obesity (14). Although ob/ob mice (mutant leptin-deficient mice) are known to be obese through a decrease in proopiomelanocortin (POMC) mRNA and an increase of NPY and AGRP mRNA in the Arc (15, 16, 17), obesity in NMU KO mice results specifically from a decrease of CRH mRNA in the PVN. Therefore, NMU and leptin share the mechanism of feeding suppression (14).
The fact that receptors for NMU have a high affinity for NMS suggests that NMS may also act on feeding. The NMS gene was mapped to chromosome 2q11.2 in humans, and this locus is consistent with one potential location of the quantitative trait loci implicated in obesity (18). These data also lead to speculation that NMS may play an important role in central regulation of feeding.
To examine whether NMS is involved in feeding regulation, the effects of central administration of NMS and NMU on food intake were investigated in rats, and the cellular mechanisms involved were analyzed.
Materials and Methods
Animals
Male Wistar rats (Charles River Japan, Inc., Yokohama, Japan), weighing 300–350 g, were housed in individual Plexiglas cages in an animal room maintained under a constant light-dark cycle (light on from 0700–1900 h) and temperature (22 ± 1 C) for at least 1 wk. Food and water were provided ad libitum except during the fasting experiments. All procedures were done in accordance with the Japanese Physiological Society’s guidelines for animal care.
Feeding experiments
Cannulation for icv injection was performed described previously (19). After surgery, all rats were housed individually in Plexiglas cages. During a 6-d postoperative recovery, the rats became accustomed to the handling procedure. In the first experiment, various doses of rat NMS and NMU were dissolved in saline, and 10 μl of solution was injected through a 27-gauge injection cannula connected to a 50-μl Hamilton syringe into each free-moving rat at 1845 h; 12-h food intake was then examined. We also examined the diurnal effect of NMS on food intake by icv injection of NMS at 0900 h. Rat NMS and NMU were synthesized by an Fmoc solid-phase method on a peptide synthesizer (433A; Applied Biosystems, Foster City, CA). In the second experiment, rats were fasted for 8 h from 0100 h at night, and then centrally injected with NMS or NMU (0.5 or 1 nmol) at 0845 h. In the third experiment, single NPY (0.5 nmol), ghrelin (0.5 nmol) or AGRP (1 nmol), and mixed NPY, ghrelin or AGRP + NMS (0.5 or 1 nmol) or NMU (0.5 or 1 nmol) (each peptide was mixed in 10 μl of saline solution) was administered to free-feeding rats at 0845 h and 2-h food intake was measured. NPY, ghrelin and AGRP were purchased from the Peptide Institute, Inc. (Osaka, Japan). In the fourth experiment, 1 nmol NMS was injected 1 h after pretreatment with 1, 5, or 10 μg -helical corticotropin-releasing factor-(9–41) (-hCRF) (Sigma, St. Louis, MO) or 0.1, 0.5, or 1 nmol SHU9119 (Bachem, Budendorfm, Switzerland) at 0745 h to 8-h fasted rats or intact rats, and 2-h and 24-h food intake was examined, respectively.
c-Fos immunohistochemistry
Ninety minutes before perfusion, rats were injected with NMS, NMU (1 nmol per rat) or saline (n = 3 per group) in the lateral ventricle to study the immunostaining of c-Fos-expressing neurons. After the rats had been perfused with fixative [4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4)], the brain was removed immediately, fixed in fixative and embedded in O.C.T. compound (Tissue-Tek, Tokyo, Japan) at –20 C. Frozen serial brain sections (40 μm thick) were incubated for 1 d with goat anti-c-Fos antiserum (Santa Cruz Biotechnology, Santa Cruz, CA; final dilution 1:1500) and visualized by the avidin-biotin complex method (Vectastain Elite ABC kit; Vector Laboratories, Inc., Burlingame, CA) using 0.02% 3,3'-diaminobenzidine tetrahydrochloride (Sigma) and 0.005% hydrogen peroxide in 50 mM Tris-HCl (pH 7.6).
Quantitative RT-PCR
To quantify POMC and CRH mRNA in the Arc and PVN after icv injection of NMS, 1 nmol NMS was injected into rats at 1845 h, 4 h before collection of Arc and PVN tissue for mRNA extraction. After the brain tissues had been frozen, the Arc and PVN were dissected out. Total RNA was extracted from the Arc and PVN using an RNeasy Mini kit (QIAGEN, Hilden, Germany) and then synthesized into first-strand cDNA. Quantitative RT-PCR was conducted with a LightCycler system (Roche, Basel, Switzerland) using a LightCycler-FastStart DNA Master SYBR Green I kit (Roche). The primer set used for rat POMC was 5'-GACCTCACCACGGAAAGCAACCTG-3' and 5'-ACTTCCGGGGATTTTCAGTCAAGGG-3', and for rat CRH was 5'-ATCTCACCTTCCACCTTCTG-3' and 5'-GTGTGCTAAATGCAGAATCG-3'. Known amounts of rat POMC and CRH cDNA were used to obtain a standard curve. Rat glyceraldehyde-3-phosphate dehydrogenase mRNA was also measured as an internal control. The primer set used for rat glyceraldehyde-3-phosphate dehydrogenase was 5'-CGGCAAGTTCAACGGCACA-3' and 5'-AGACGCCAGTAGACTCCACGACA-3'.
Multiple unit activity (MUA) recording
Rats were fitted with chronically implanted electrode arrays as described previously (20). Briefly, the electrode assembly consisted of four 75-mm Teflon-insulated platinum (90%)-iridium (10%) wires (A-M Systems, Inc., Sequim, WA) encased in a stainless steel guide tube (650 mm diameter; Inter Medical, Fukuoka, Japan). The stainless steel tube served as a ground. The impedance of each platinum-iridium electrode measured at 1 kHz was 50–100 k. According to the stereotaxic atlas of the rat brain (Paxinos and Watson, Ref.27) described by Albe-Fessard et al., the electrodes were implanted unilaterally into the left side of the PVN and fixed to the skull with anchor screws and dental cement. At the same time, an icv cannula was implanted slantingly into the right lateral cerebral ventricle. After a recovery period of 5 d, MUA was recorded as follows: signals were passed through a buffer amplifier, amplified by a biophysical amplifier (MEG-2100; Nihon Kohden, Tokyo, Japan) with low and high cutoff frequencies of 500 Hz and 10 kHz, respectively, and displayed on an oscilloscope (DS-8812; Iwatsu, Tokyo, Japan). Neural spikes were discriminated by their amplitude, and the number of spikes was counted with a pulse counter (ET-612J; Nihon Kohden) and integrated for 1 sec. Outputs were recorded as a histogram on a thermal recorder (WR8500; Graphtec, Tokyo, Japan) and with a powerLab (AD Instrument, Castle Hill, Australia), respectively. On the day of the experiment, the MUA electrode was attached to the buffer amplifier under isoflurane inhalation anesthesia (Univentor 400; Univentor, Zejtun, Malta). Rats were maintained under anesthesia with 1.5% isoflurane (Abbott Laboratories, Abbott Park, IL). At 15 min after the beginning of stable MUA volley, rats received icv administration of 1 nmol NMS, NMU, or saline. At 120 min after administration, electrical stimulation was applied for 1 sec through the MUA electrode with pulses (1 mA) from an electric stimulator (RGF-4A; Radionics, Burlington, MA) to check the site of the electrode.
Statistical analysis
The data (mean ± SEM) were analyzed statistically by ANOVA with the post hoc Fisher’s test. P < 0.05 was considered statistically significant.
Results
Intracerebroventricular injection of NMS reduced 12-h food intake during the dark period in a dose-dependent manner (Fig. 1A). This effect of NMS was more potent than that of NMU because a smaller dose of NMS was effective at suppressing feeding (Fig. 1A). We also measured 12-h water intake after NMS or saline injection before the onset of dark period. A quantity of 1 nmol of NMS, but not 0.5 nmol, significantly decreased water intake during dark phase [NMS 1 nmol, 34.75 ± 3.26 ml (P < 0.05 vs. saline); 0.5 nmol, 44.74 ± 4.89 ml; saline, 47.17 ± 4.54 ml]. Although feeding suppression by 1 nmol NMU recovered completely within 2 d, suppression by the same dose of NMS continued at least for 3 d starting from 1845 h (Fig. 1B). Icv injection of 1 nmol NMS and NMU into 8-h fasted rats also resulted in a decrease in food intake for 2 h. On the other hand, at a dose of 0.5 nmol, only NMS injection suppressed food intake (Fig. 1C).
Although icv injection of NPY, ghrelin, and AGRP significantly increased food intake, this peptide-induced food intake was reduced by coadministration of NMS or NMU (Fig. 1D). In these cases, the suppressive effect with NMS was more potent than that with NMU. We also examined the diurnal effect of NMS on food intake by icv injection of NMS at 0900 h. There was no significant difference in food intake during the 12-h light period on the first, second, and third day between the NMS- and saline-treated groups (first 12-h light period 1.9 ± 0.62 vs. 2.4 ± 0.64 g; second 12-h light period 2.4 ± 0.52 vs. 2.5 ± 0.44 g; third 12-h light period 2.5 ± 0.72 vs. 2.4 ± 0.48 g; NMS vs. saline). However, NMS suppressed significantly 12 h dark food intake for 3 d starting from 0900 h.
To understand the cellular mechanisms involved in NMS-induced suppression of feeding, POMC and CRH mRNA expression and the expression of c-Fos protein were investigated. Icv administration of NMS augmented the levels of Arc POMC and PVN CRH mRNA (Fig. 2, A and B). The involvement of POMC and CRH in NMS-induced suppression of feeding was therefore investigated using an antagonist for these peptides. Pretreatment with both SHU9119 (an antagonist for -MSH) and -hCRF (an antagonist for CRH) attenuated NMS-induced suppression of food intake in a dose-dependent manner in fasted rats. Whereas only SHU9119 significantly blocked the effect of NMS on 2-h food intake (Fig. 2C), both -hCRF and SHU9119 blocked the effect of NMS on 24-h food intake (Fig. 2D). The central distributions of c-Fos immunoreactive cell were similar in NMS- and NMU-injected rats. The hypothalamic PVN (Fig. 3, A and D), Arc (Fig. 3, B and E), supraoptic nucleus (Fig. 3, C and F) and SCN (data not shown) expressed the c-Fos protein strongly. In saline-treated rats, no c-Fos immunoreactivity was observed in any of these regions (data not shown).
Neuronal electrical activity in the PVN was then measured before and after icv administration of 1 nmol NMS and NMU using a MUA recording system. This method has practical advantages, in that continuous and real-time analysis of hypothalamic neural activity can performed in vivo. In the frequency-time histograms, MUA could be influenced within 5 min by NMS and NMU (Fig. 4, A and B). The most active MUA induced by NMS was observed between 20 min and 100 min and decreased gradually thereafter. Although NMU also increased MUA immediately after injection, the effect was weaker than that of NMS. We analyzed the total spike count at 30-min intervals for 120 min (Fig. 4C). Although a significant increase in the spike count was observed only between 30 and 60 min after icv injection of NMU, the increase continued for at least 120 min in NMS-treated rats. As shown in Fig. 4D, the recording sites of these MUA volleys were located adjacent to the PVN.
Discussion
In the present study, the novel peptide, NMS, was demonstrated to be a potent anorexigenic hormone in rats. Central administration of NMS reduced the daily dark period food intake and 8-h fasting-induced food intake. This suppression of feeding is unlikely to be due to any side effects of NMS because NMS-injected rats did not show any abnormal behavior (such as glooming behavior, searching behavior, attaching behavior, and barrel rolling). Although NMU has been well documented to reduce food intake in rats (2, 3), the relative potency of NMS on suppression of food intake was stronger than that of NMU because a smaller dose of NMS significantly suppressed food intake. Considering that NMS contains the core active C terminus of NMU and binds to the same receptors (NMU1R and NMU2R) as NMU (6), NMS-induced suppression of food intake can be assumed. In a previous study, the distribution of NMS mRNA was investigated in various rat tissues by quantitative RT-PCR (6). NMS mRNA was expressed mainly in the hypothalamus, spleen, and testis. In the hypothalamus, however, NMS mRNA was expressed predominantly in the SCN, with only very slight expression in other brain regions including the PVN and Arc. In situ hybridization histochemistry also showed that NMS mRNA expression was restricted to the SCN. No hybridization signal was observed in any other brain region. The fact that the relative potency of NMS in suppressing food intake was stronger than that of NMU despite the lower expression of NMS mRNA than NMU mRNA in the PVN and Arc suggests that the feeding regulation effect may differ between NMS and NMU. Especially, in the case of NMS, its action on the PVN and Arc through the NMS projection from the SCN may be important.
When the NMS was injected at 0900 h, there was no significant difference in food intake during the 12-h light period, suggesting the diurnal variation in the anorexigenic effect of NMS. Although the interpretation of these data is difficult because of the very low feeding activity in the beginning of the light period, this diurnal difference may be due to diurnal variation of NMU receptors in SCN (21) or diurnal variation of NMS secretion (6) in autocrine regulation.
It is not known why NMS-induced suppression of food intake is more potent and continues for a longer time than with NMU. There was no difference in the distribution of c-Fos expression between NMS- and NMU-injected rats. However, neural MUA records showed a clear difference between the rats. There was a greater increase in firing rate of PVN neurons in NMS-treated rats than in NMU-treated rats, and this increased effect continued for a long period of time after NMS injection. This potent and long-term increase of firing rate by NMS may cause the powerful and long-term suppression of food intake. Alternatively, the possibility that NMS may act on another unknown receptor cannot be excluded.
NPY, ghrelin, and AGRP-induced food intake was counteracted by coadministration of NMS, suggesting that the NPY, ghrelin, and AGRP are independently antagonistic with NMS for feeding regulation.
Hanada et al. (14) reported that icv injection of NMU in rats did not affect POMC mRNA expression in the Arc but augmented CRH mRNA expression in the PVN. In addition, CRH KO mice did not show any reduction in food intake after NMU injection (22). Therefore, it has been speculated that an increase in CRH, but not -MSH, is the primary cause of NMU-induced suppression of food intake. In the present study, NMS increased both POMC and CRH mRNA expression. These results indicate that the cellular mechanism of suppression of food intake by NMS may be different from that by NMU, and both CRH and -MSH may be involved in NMS-induced suppression of food intake. This hypothesis is supported by the following results: pretreatment with antagonists for -MSH and CRH blocked NMS-induced suppression of food intake.
It is questionable why receptors for NMS and NMU are the same; nevertheless, the downstream mechanism of feeding regulation by NMS and NMU is different. Recent studies demonstrate that NMU, NMS, NMU1R, and NMU2R mRNA each have an intrinsic rhythmic expression in the SCN with a different circadian pattern (6, 21). Because the SCN sends neural projections into the PVN and Arc (23, 24), these different rhythmic expressions may relate to the different effects of NMS and NMU. Of course, as mentioned above, NMS may act on a receptor other than NMU1R and NMU2R. Either way, it is unknown why NMS, but not NMU, stimulates the POMC system in the Arc, but a different downstream mechanism may explain the difference in effectiveness and duration of action between NMU and NMS.
Wren et al. (25) reported that leptin was able to stimulate NMU release in hypothalamic explants in vitro. In contrast, Hanada et al. (14). showed that the anorexigenic effect of NMU is independent of leptin in NMU KO mice because NMU and leptin reduced food intake in ob/ob mice and NMU KO mice, respectively. Wren et al. measured NMU content using an antibody raised in a rabbit immunized with synthetic NMU-8. Because NMU-8 is the core active C terminus of NMS and NMU, the antibody must recognize both NMS and NMU. We had also raised antiserum against synthetic NMU-8 and established a RIA for NMU (26). Rat NMS and NMU were equally recognized with the serum on a molar basis (data not shown) and could not separate NMS and NMU in this RIA system. Therefore, NMU release stimulated by leptin in hypothalamic explants presented by Wren et al. might be NMS. If this is the case, NMS is the downstream signal pathway for leptin. NMS is a novel anorexigenic hormone, and further investigation of the function of NMS will help in our understanding of weight control mechanisms and should facilitate the study of eating disorders.
Footnotes
This study was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture, Japan (to N.M., K.N., K.K., and M.M.), Nissan Science Foundation (K.N.), the Program for Promotion of Fundamental Studies in Health Sciences of Pharmaceuticals and Medical Devices Agency (to K.K.), and the Program for promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN) (to N.M.).
Abbreviations: AGRP, Agouti-related protein; Arc, arcuate nucleus; -hCRF, -helical corticotropin-releasing factor-(9–41); icv, intracerebroventricular; KO, knockout; MUA, multiple-unit activity; NMS, neuromedin S; NMU, neuromedin U; NPY, neuropeptide Y; POMC, proopiomelanocortin; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus.
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Department of Biochemistry (K.M., M.M., K.K.), National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka 565-8565
Laboratory of Veterinary Physiology (M.N.), Veterinary Medical Science, The University of Tokyo, Tokyo 113-8657, Japan
Abstract
A novel 36-amino acid neuropeptide, neuromedin S (NMS), has recently been identified in rat brain and has been shown to be an endogenous ligand for two orphan G protein-coupled receptors, FM-3/GPR66 and FM-4/TGR-1. These receptors have been identified as neuromedin U (NMU) receptor type 1 and type 2, respectively. In this study, the physiological role of the novel peptide, NMS, on feeding regulation was investigated. Intracerebroventricular (icv) injection of NMS decreased 12-h food intake during the dark period in rats. This anorexigenic effect was more potent and persistent than that observed with the same dose of NMU. Neuropeptide Y, ghrelin, and agouti-related protein-induced food intake was counteracted by coadministration of NMS. Icv administration of NMS increased proopiomelanocortin mRNA expression in the arcuate nucleus (Arc) and CRH mRNA in the paraventricular nucleus (PVN). Pretreatment with SHU9119 (antagonist for -MSH) and -helical corticotropin-releasing factor-(9–41) (antagonist for CRH) attenuated NMS-induced suppression of 24-h food intake. After icv injection of NMS, Fos-immunoreactive cells were detected in both the PVN and Arc. When neuronal multiple unit activity was recorded in the PVN before and after icv injection of NMS, a significant increase in firing rate was observed 5 min after administration, and this increase continued for 100 min. These results suggest that the novel peptide, NMS, may be a potent anorexigenic hormone in the hypothalamus, and that expression of proopiomelanocortin mRNA in the Arc and CRH mRNA in the PVN may be involved in NMS action on feeding.
Introduction
NEUROMEDIN U (NMU), originally isolated from porcine spinal cord, is a brain-gut peptide that has potent contractile activity on uterine smooth muscle (1). In previous studies, two orphan G protein-coupled receptors, FM-3/GPR66 and FM-4/TGR-1, were identified as NMU receptor type 1 (NMU1R) and type 2 (NMU2R), respectively (2, 3, 4, 5). Recently, a novel 36-amino acid neuropeptide was identified in rat brain as another endogenous ligand for FM-3/GPR66 and FM-4/TGR-1 using a reverse-pharmacological technique (6). This neuropeptide was designated neuromedin S (NMS) because it is specifically expressed in the suprachiasmatic nucleus (SCN). Although the NMS shares a C-terminal core structure (seven-amino acid residues) with NMU and activates both recombinant NMU1R and NMU2R expressed in Chinese hamster ovary cells, NMS is not a splice variant of NMU because both NMS and NMU genes were mapped to discrete chromosomes. In addition, although NMU mRNA was detected in peripheral and central organs (7), the distribution of NMS was limited to the testis, spleen and SCN (6). NMS was recently suggested to be involved in circadian oscillation systems because intracerebroventricular (icv) administration of NMS induces phase-dependent phase shifts in the circadian rhythm of locomotor activity in rats kept under constant darkness (6).
NMU1R is located in a wide range of peripheral tissues such as intestine, testis, pancreas, uterus, lung, and kidney. On the other hand, expression of NMU2R is limited to areas of the brain such as the paraventricular nucleus (PVN), along the wall of the third ventricle in the hypothalamus and the CA1 region of the hippocampus (2, 5, 8, 9). Immunohistochemical and in situ analysis has revealed NMU-immunoreactive neurons or NMU mRNA expression in the ventromedial hypothalamic region including the arcuate nucleus (Arc), pituitary, caudal brainstem region including the nucleus of the solitary tract, area postrema, dorsal motor nucleus of the vagus nerve and inferior olive, and spinal cord (2, 10, 11). NMU-immunoreactive fibers project prominently into the PVN, ventromedial nucleus, dorsomedial nucleus, and Arc. It has been well documented that the PVN and Arc of the hypothalamus play pivotal roles in the regulation of feeding behavior through a complex neuronal network composed of several orexigenic neuropeptides such as neuropeptide Y (NPY), agouti-related protein (AGRP) and ghrelin, and anorexigenic neuropeptides such as -MSH, cocaine- and amphetamine-regulated transcript, CRH, and leptin (12, 13). Icv administration of NMU suppresses both dark-phase food intake and fasting-induced feeding, suggesting that NMU acts as anorexigenic hormone (2, 3). Conversely, disruption of the NMU gene in mice [NMU knockout (KO) mice] resulted in severe obesity (14). Although ob/ob mice (mutant leptin-deficient mice) are known to be obese through a decrease in proopiomelanocortin (POMC) mRNA and an increase of NPY and AGRP mRNA in the Arc (15, 16, 17), obesity in NMU KO mice results specifically from a decrease of CRH mRNA in the PVN. Therefore, NMU and leptin share the mechanism of feeding suppression (14).
The fact that receptors for NMU have a high affinity for NMS suggests that NMS may also act on feeding. The NMS gene was mapped to chromosome 2q11.2 in humans, and this locus is consistent with one potential location of the quantitative trait loci implicated in obesity (18). These data also lead to speculation that NMS may play an important role in central regulation of feeding.
To examine whether NMS is involved in feeding regulation, the effects of central administration of NMS and NMU on food intake were investigated in rats, and the cellular mechanisms involved were analyzed.
Materials and Methods
Animals
Male Wistar rats (Charles River Japan, Inc., Yokohama, Japan), weighing 300–350 g, were housed in individual Plexiglas cages in an animal room maintained under a constant light-dark cycle (light on from 0700–1900 h) and temperature (22 ± 1 C) for at least 1 wk. Food and water were provided ad libitum except during the fasting experiments. All procedures were done in accordance with the Japanese Physiological Society’s guidelines for animal care.
Feeding experiments
Cannulation for icv injection was performed described previously (19). After surgery, all rats were housed individually in Plexiglas cages. During a 6-d postoperative recovery, the rats became accustomed to the handling procedure. In the first experiment, various doses of rat NMS and NMU were dissolved in saline, and 10 μl of solution was injected through a 27-gauge injection cannula connected to a 50-μl Hamilton syringe into each free-moving rat at 1845 h; 12-h food intake was then examined. We also examined the diurnal effect of NMS on food intake by icv injection of NMS at 0900 h. Rat NMS and NMU were synthesized by an Fmoc solid-phase method on a peptide synthesizer (433A; Applied Biosystems, Foster City, CA). In the second experiment, rats were fasted for 8 h from 0100 h at night, and then centrally injected with NMS or NMU (0.5 or 1 nmol) at 0845 h. In the third experiment, single NPY (0.5 nmol), ghrelin (0.5 nmol) or AGRP (1 nmol), and mixed NPY, ghrelin or AGRP + NMS (0.5 or 1 nmol) or NMU (0.5 or 1 nmol) (each peptide was mixed in 10 μl of saline solution) was administered to free-feeding rats at 0845 h and 2-h food intake was measured. NPY, ghrelin and AGRP were purchased from the Peptide Institute, Inc. (Osaka, Japan). In the fourth experiment, 1 nmol NMS was injected 1 h after pretreatment with 1, 5, or 10 μg -helical corticotropin-releasing factor-(9–41) (-hCRF) (Sigma, St. Louis, MO) or 0.1, 0.5, or 1 nmol SHU9119 (Bachem, Budendorfm, Switzerland) at 0745 h to 8-h fasted rats or intact rats, and 2-h and 24-h food intake was examined, respectively.
c-Fos immunohistochemistry
Ninety minutes before perfusion, rats were injected with NMS, NMU (1 nmol per rat) or saline (n = 3 per group) in the lateral ventricle to study the immunostaining of c-Fos-expressing neurons. After the rats had been perfused with fixative [4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4)], the brain was removed immediately, fixed in fixative and embedded in O.C.T. compound (Tissue-Tek, Tokyo, Japan) at –20 C. Frozen serial brain sections (40 μm thick) were incubated for 1 d with goat anti-c-Fos antiserum (Santa Cruz Biotechnology, Santa Cruz, CA; final dilution 1:1500) and visualized by the avidin-biotin complex method (Vectastain Elite ABC kit; Vector Laboratories, Inc., Burlingame, CA) using 0.02% 3,3'-diaminobenzidine tetrahydrochloride (Sigma) and 0.005% hydrogen peroxide in 50 mM Tris-HCl (pH 7.6).
Quantitative RT-PCR
To quantify POMC and CRH mRNA in the Arc and PVN after icv injection of NMS, 1 nmol NMS was injected into rats at 1845 h, 4 h before collection of Arc and PVN tissue for mRNA extraction. After the brain tissues had been frozen, the Arc and PVN were dissected out. Total RNA was extracted from the Arc and PVN using an RNeasy Mini kit (QIAGEN, Hilden, Germany) and then synthesized into first-strand cDNA. Quantitative RT-PCR was conducted with a LightCycler system (Roche, Basel, Switzerland) using a LightCycler-FastStart DNA Master SYBR Green I kit (Roche). The primer set used for rat POMC was 5'-GACCTCACCACGGAAAGCAACCTG-3' and 5'-ACTTCCGGGGATTTTCAGTCAAGGG-3', and for rat CRH was 5'-ATCTCACCTTCCACCTTCTG-3' and 5'-GTGTGCTAAATGCAGAATCG-3'. Known amounts of rat POMC and CRH cDNA were used to obtain a standard curve. Rat glyceraldehyde-3-phosphate dehydrogenase mRNA was also measured as an internal control. The primer set used for rat glyceraldehyde-3-phosphate dehydrogenase was 5'-CGGCAAGTTCAACGGCACA-3' and 5'-AGACGCCAGTAGACTCCACGACA-3'.
Multiple unit activity (MUA) recording
Rats were fitted with chronically implanted electrode arrays as described previously (20). Briefly, the electrode assembly consisted of four 75-mm Teflon-insulated platinum (90%)-iridium (10%) wires (A-M Systems, Inc., Sequim, WA) encased in a stainless steel guide tube (650 mm diameter; Inter Medical, Fukuoka, Japan). The stainless steel tube served as a ground. The impedance of each platinum-iridium electrode measured at 1 kHz was 50–100 k. According to the stereotaxic atlas of the rat brain (Paxinos and Watson, Ref.27) described by Albe-Fessard et al., the electrodes were implanted unilaterally into the left side of the PVN and fixed to the skull with anchor screws and dental cement. At the same time, an icv cannula was implanted slantingly into the right lateral cerebral ventricle. After a recovery period of 5 d, MUA was recorded as follows: signals were passed through a buffer amplifier, amplified by a biophysical amplifier (MEG-2100; Nihon Kohden, Tokyo, Japan) with low and high cutoff frequencies of 500 Hz and 10 kHz, respectively, and displayed on an oscilloscope (DS-8812; Iwatsu, Tokyo, Japan). Neural spikes were discriminated by their amplitude, and the number of spikes was counted with a pulse counter (ET-612J; Nihon Kohden) and integrated for 1 sec. Outputs were recorded as a histogram on a thermal recorder (WR8500; Graphtec, Tokyo, Japan) and with a powerLab (AD Instrument, Castle Hill, Australia), respectively. On the day of the experiment, the MUA electrode was attached to the buffer amplifier under isoflurane inhalation anesthesia (Univentor 400; Univentor, Zejtun, Malta). Rats were maintained under anesthesia with 1.5% isoflurane (Abbott Laboratories, Abbott Park, IL). At 15 min after the beginning of stable MUA volley, rats received icv administration of 1 nmol NMS, NMU, or saline. At 120 min after administration, electrical stimulation was applied for 1 sec through the MUA electrode with pulses (1 mA) from an electric stimulator (RGF-4A; Radionics, Burlington, MA) to check the site of the electrode.
Statistical analysis
The data (mean ± SEM) were analyzed statistically by ANOVA with the post hoc Fisher’s test. P < 0.05 was considered statistically significant.
Results
Intracerebroventricular injection of NMS reduced 12-h food intake during the dark period in a dose-dependent manner (Fig. 1A). This effect of NMS was more potent than that of NMU because a smaller dose of NMS was effective at suppressing feeding (Fig. 1A). We also measured 12-h water intake after NMS or saline injection before the onset of dark period. A quantity of 1 nmol of NMS, but not 0.5 nmol, significantly decreased water intake during dark phase [NMS 1 nmol, 34.75 ± 3.26 ml (P < 0.05 vs. saline); 0.5 nmol, 44.74 ± 4.89 ml; saline, 47.17 ± 4.54 ml]. Although feeding suppression by 1 nmol NMU recovered completely within 2 d, suppression by the same dose of NMS continued at least for 3 d starting from 1845 h (Fig. 1B). Icv injection of 1 nmol NMS and NMU into 8-h fasted rats also resulted in a decrease in food intake for 2 h. On the other hand, at a dose of 0.5 nmol, only NMS injection suppressed food intake (Fig. 1C).
Although icv injection of NPY, ghrelin, and AGRP significantly increased food intake, this peptide-induced food intake was reduced by coadministration of NMS or NMU (Fig. 1D). In these cases, the suppressive effect with NMS was more potent than that with NMU. We also examined the diurnal effect of NMS on food intake by icv injection of NMS at 0900 h. There was no significant difference in food intake during the 12-h light period on the first, second, and third day between the NMS- and saline-treated groups (first 12-h light period 1.9 ± 0.62 vs. 2.4 ± 0.64 g; second 12-h light period 2.4 ± 0.52 vs. 2.5 ± 0.44 g; third 12-h light period 2.5 ± 0.72 vs. 2.4 ± 0.48 g; NMS vs. saline). However, NMS suppressed significantly 12 h dark food intake for 3 d starting from 0900 h.
To understand the cellular mechanisms involved in NMS-induced suppression of feeding, POMC and CRH mRNA expression and the expression of c-Fos protein were investigated. Icv administration of NMS augmented the levels of Arc POMC and PVN CRH mRNA (Fig. 2, A and B). The involvement of POMC and CRH in NMS-induced suppression of feeding was therefore investigated using an antagonist for these peptides. Pretreatment with both SHU9119 (an antagonist for -MSH) and -hCRF (an antagonist for CRH) attenuated NMS-induced suppression of food intake in a dose-dependent manner in fasted rats. Whereas only SHU9119 significantly blocked the effect of NMS on 2-h food intake (Fig. 2C), both -hCRF and SHU9119 blocked the effect of NMS on 24-h food intake (Fig. 2D). The central distributions of c-Fos immunoreactive cell were similar in NMS- and NMU-injected rats. The hypothalamic PVN (Fig. 3, A and D), Arc (Fig. 3, B and E), supraoptic nucleus (Fig. 3, C and F) and SCN (data not shown) expressed the c-Fos protein strongly. In saline-treated rats, no c-Fos immunoreactivity was observed in any of these regions (data not shown).
Neuronal electrical activity in the PVN was then measured before and after icv administration of 1 nmol NMS and NMU using a MUA recording system. This method has practical advantages, in that continuous and real-time analysis of hypothalamic neural activity can performed in vivo. In the frequency-time histograms, MUA could be influenced within 5 min by NMS and NMU (Fig. 4, A and B). The most active MUA induced by NMS was observed between 20 min and 100 min and decreased gradually thereafter. Although NMU also increased MUA immediately after injection, the effect was weaker than that of NMS. We analyzed the total spike count at 30-min intervals for 120 min (Fig. 4C). Although a significant increase in the spike count was observed only between 30 and 60 min after icv injection of NMU, the increase continued for at least 120 min in NMS-treated rats. As shown in Fig. 4D, the recording sites of these MUA volleys were located adjacent to the PVN.
Discussion
In the present study, the novel peptide, NMS, was demonstrated to be a potent anorexigenic hormone in rats. Central administration of NMS reduced the daily dark period food intake and 8-h fasting-induced food intake. This suppression of feeding is unlikely to be due to any side effects of NMS because NMS-injected rats did not show any abnormal behavior (such as glooming behavior, searching behavior, attaching behavior, and barrel rolling). Although NMU has been well documented to reduce food intake in rats (2, 3), the relative potency of NMS on suppression of food intake was stronger than that of NMU because a smaller dose of NMS significantly suppressed food intake. Considering that NMS contains the core active C terminus of NMU and binds to the same receptors (NMU1R and NMU2R) as NMU (6), NMS-induced suppression of food intake can be assumed. In a previous study, the distribution of NMS mRNA was investigated in various rat tissues by quantitative RT-PCR (6). NMS mRNA was expressed mainly in the hypothalamus, spleen, and testis. In the hypothalamus, however, NMS mRNA was expressed predominantly in the SCN, with only very slight expression in other brain regions including the PVN and Arc. In situ hybridization histochemistry also showed that NMS mRNA expression was restricted to the SCN. No hybridization signal was observed in any other brain region. The fact that the relative potency of NMS in suppressing food intake was stronger than that of NMU despite the lower expression of NMS mRNA than NMU mRNA in the PVN and Arc suggests that the feeding regulation effect may differ between NMS and NMU. Especially, in the case of NMS, its action on the PVN and Arc through the NMS projection from the SCN may be important.
When the NMS was injected at 0900 h, there was no significant difference in food intake during the 12-h light period, suggesting the diurnal variation in the anorexigenic effect of NMS. Although the interpretation of these data is difficult because of the very low feeding activity in the beginning of the light period, this diurnal difference may be due to diurnal variation of NMU receptors in SCN (21) or diurnal variation of NMS secretion (6) in autocrine regulation.
It is not known why NMS-induced suppression of food intake is more potent and continues for a longer time than with NMU. There was no difference in the distribution of c-Fos expression between NMS- and NMU-injected rats. However, neural MUA records showed a clear difference between the rats. There was a greater increase in firing rate of PVN neurons in NMS-treated rats than in NMU-treated rats, and this increased effect continued for a long period of time after NMS injection. This potent and long-term increase of firing rate by NMS may cause the powerful and long-term suppression of food intake. Alternatively, the possibility that NMS may act on another unknown receptor cannot be excluded.
NPY, ghrelin, and AGRP-induced food intake was counteracted by coadministration of NMS, suggesting that the NPY, ghrelin, and AGRP are independently antagonistic with NMS for feeding regulation.
Hanada et al. (14) reported that icv injection of NMU in rats did not affect POMC mRNA expression in the Arc but augmented CRH mRNA expression in the PVN. In addition, CRH KO mice did not show any reduction in food intake after NMU injection (22). Therefore, it has been speculated that an increase in CRH, but not -MSH, is the primary cause of NMU-induced suppression of food intake. In the present study, NMS increased both POMC and CRH mRNA expression. These results indicate that the cellular mechanism of suppression of food intake by NMS may be different from that by NMU, and both CRH and -MSH may be involved in NMS-induced suppression of food intake. This hypothesis is supported by the following results: pretreatment with antagonists for -MSH and CRH blocked NMS-induced suppression of food intake.
It is questionable why receptors for NMS and NMU are the same; nevertheless, the downstream mechanism of feeding regulation by NMS and NMU is different. Recent studies demonstrate that NMU, NMS, NMU1R, and NMU2R mRNA each have an intrinsic rhythmic expression in the SCN with a different circadian pattern (6, 21). Because the SCN sends neural projections into the PVN and Arc (23, 24), these different rhythmic expressions may relate to the different effects of NMS and NMU. Of course, as mentioned above, NMS may act on a receptor other than NMU1R and NMU2R. Either way, it is unknown why NMS, but not NMU, stimulates the POMC system in the Arc, but a different downstream mechanism may explain the difference in effectiveness and duration of action between NMU and NMS.
Wren et al. (25) reported that leptin was able to stimulate NMU release in hypothalamic explants in vitro. In contrast, Hanada et al. (14). showed that the anorexigenic effect of NMU is independent of leptin in NMU KO mice because NMU and leptin reduced food intake in ob/ob mice and NMU KO mice, respectively. Wren et al. measured NMU content using an antibody raised in a rabbit immunized with synthetic NMU-8. Because NMU-8 is the core active C terminus of NMS and NMU, the antibody must recognize both NMS and NMU. We had also raised antiserum against synthetic NMU-8 and established a RIA for NMU (26). Rat NMS and NMU were equally recognized with the serum on a molar basis (data not shown) and could not separate NMS and NMU in this RIA system. Therefore, NMU release stimulated by leptin in hypothalamic explants presented by Wren et al. might be NMS. If this is the case, NMS is the downstream signal pathway for leptin. NMS is a novel anorexigenic hormone, and further investigation of the function of NMS will help in our understanding of weight control mechanisms and should facilitate the study of eating disorders.
Footnotes
This study was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture, Japan (to N.M., K.N., K.K., and M.M.), Nissan Science Foundation (K.N.), the Program for Promotion of Fundamental Studies in Health Sciences of Pharmaceuticals and Medical Devices Agency (to K.K.), and the Program for promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN) (to N.M.).
Abbreviations: AGRP, Agouti-related protein; Arc, arcuate nucleus; -hCRF, -helical corticotropin-releasing factor-(9–41); icv, intracerebroventricular; KO, knockout; MUA, multiple-unit activity; NMS, neuromedin S; NMU, neuromedin U; NPY, neuropeptide Y; POMC, proopiomelanocortin; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus.
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