- Email: [email protected]
Effects of intracerebroventricular administration of neuromedin U or neuromedin S in steers
General and Comparative Endocrinology 163 (2009) 324–328
Contents lists available at ScienceDirect
General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen
Effects of intracerebroventricular administration of neuromedin U or neuromedin S in steers K. Yayou a,*, S. Kitagawa b, S. Ito c, E. Kasuya a, M. Sutoh d a
Laboratory of Neurobiology, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan Department of Animal Science, Utsunomiya University, Utsunomiya 321-8505, Japan c Laboratory of Animal Behavior, Tokai University, Aso-gun 869-1404, Japan d Endocrinology and Metabolism Research Team, National Institute of Livestock and Grassland Science, Tsukuba 305-0901, Japan b
a r t i c l e
i n f o
Article history: Received 19 March 2009 Revised 24 April 2009 Accepted 29 April 2009 Available online 12 May 2009 Keywords: Body temperature Hypothalamo–pituitary–adrenal axis Intracerebroventricular administration Neuromedin S Neuromedin U Steer
a b s t r a c t Although neuromedin U (NMU) and neuromedin S (NMS) are reported to modulate stress responses mainly through corticotropin-releasing hormone system in rodents, the in vivo effects of centrally administered NMU or NMS on stress regulation have not been fully elucidated in cattle. We examined adrenocorticotropic hormone levels, body temperature, and behavioral responses to intracerebroventricularly (ICV) administered rat NMU or rat NMS in steers. ICV NMU and NMS (0.2, 2, and 20 nmol/200 ll) evoked a dose-related increase in plasma cortisol concentrations (CORT). There was a significant time–treatment interaction for the time course of CORT (p < 0.001). ICV NMU evoked a dose-related increase in rectal temperature (RT). There was a significant time–treatment interaction for the change in RT from pre-injection value (p < 0.05). There was a significant difference among treatments in the percentage of time spent lying (Friedman’s test, v2 = 15.6, p < 0.01) and in the total number of head shaking (Friedman’s test, v2 = 14.49, p < 0.01). A high dose of NMS tended to shorten the duration of lying and increase the number of head shaking. These findings indicate that both central NMU and NMS might participate in controlling the hypothalamo–pituitary–adrenal axis, that central NMU might participate in controlling body temperature, and that central NMS is likely to be involved in behavioral activation in cattle. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction The neuropeptides neuromedin U (NMU) and neuromedin S (NMS) were identified as endogenous ligands for two orphan G protein-coupled receptors, FM-3/GPR66 and FM-4/TGR-1 (Minamino et al., 1985; Mori et al., 2005), currently identified as NMU type1 (NMUR1) and type-2 (NMUR2) receptors, respectively (Guan et al., 2001, Howard et al., 2000; Raddatz et al., 2000). Although NMU and NMS share a C-terminal core structure and activate recombinant NMUR1 and NMUR2 expressed in Chinese hamster ovary cells, they have been mapped to discrete chromosomes (Mori et al., 2005). Since the NMUR2 is highly expressed in the central nervous system (Guan et al., 2001; Howard et al., 2000; Raddatz et al., 2000), the physiological roles of these neuropeptides in the central nervous system have been investigated mainly in rodents primarily by intracerebroventricular (ICV) administration of the peptides. NMU and NMS function in the regulation of feeding behaviors
* Corresponding author. Address: Laboratory of Neurobiology, National Institute of Agrobiological Sciences, 2, Ikenodai, Tsukuba, Ibaraki 305-8602, Japan. Fax: +81 29 838 8610. E-mail address: [email protected] (K. Yayou). 0016-6480/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2009.04.033
(Howard et al., 2000; Ida et al., 2005; Peier et al., 2009), energy homeostasis (Hanada et al., 2003; Nakazato et al., 2000; Peier et al., 2009), circadian rhythms (Mori et al., 2005; Nakahara et al., 2004a), and stress responses (Hanada et al., 2001; Jaszberenyi et al., 2007). Although NMU and NMS bind to the same receptor, there are several discrepancies in the effects ICV administered neuropeptides. A nociceptive response was observed only after ICV NMU (Nakahara et al., 2004b; Yu et al., 2003). Increased release of luteinizing hormone was observed only after ICV NMS (Vigo et al., 2007). The anorexigenic effect through the corticotropinreleasing hormone (CRH) and a-melanocyte-stimulating hormone systems was greater and longer lasting after ICV NMS than that after ICV NMU (Ida et al., 2005; Peier et al., 2009). The antidiuretic action by arginine vasopressin (AVP) was stronger after ICV NMS than that after ICV NMU (Sakamoto et al., 2007). As to the function in stress response, both neuropeptides activated the HPA axis and induced grooming through the CRH system (Hanada et al., 2001; Jaszberenyi et al., 2007), but only NMU increased locomotor activity (Hanada et al., 2001; Wren et al., 2002). Recently we have shown that ICV administration of bovine-CRH (bCRH) and AVP activated HPA axis and that bCRH was more potent to stimulate HPA axis than AVP. We have also shown that ICV bCRH and AVP induced stereotyped behaviors in steers
K. Yayou et al. / General and Comparative Endocrinology 163 (2009) 324–328
and that the types of stereotyped behaviors induced were different between bCRH and AVP (Yayou et al., 2008). Although the result that CRH was more potent stimulator of HPA axis than AVP was consistent with the results in rodents, the behavioral data suggest the presence of species differences in the central roles of CRH and AVP to regulate behavioral responses under stressful conditions. Until now, no data have been published regarding the central effects of NMU and NMS in steers, though these peptides might have important roles to modulate stress responses through CRH and/or AVP. The biologically active region, the C-terminal region of the peptide, was conserved among various animal species including cattle (GenBank Accession Nos.: XP_001250700, region 193. . .217 for NMU; Q0VBW8, region 104. . .136 for NMS) (Mori et al., 2005, 2008). Moreover, the NMUR2 gene of cattle (GenBank Accession No. AAX46680) had high homology (76% identities, 86% positive) compared to that of rats (Hosoya et al., 2000). As part of the study of central modulation of stress response in cattle, we elucidated the central roles of NMU and NMS in cattle by examining changes in adrenocorticotropic hormone levels, body temperature, and behavioral responses after ICV administrations of rat NMU or rat NMS into the third ventricle of steers.
2. Materials and methods Experimental procedures and animal care were approved by the Institute Committee for Animal Use and Care at the National Institute of Agrobiological Sciences, Tsukuba, Japan. 2.1. Animals and surgery Six Holstein steers (7–9 months old, weighing 165–246 kg at the start of the experiment) were used in the experiment. They were castrated at least 2 months before the experiment. After stereotaxic surgery, animals were individually reared in a stanchion stall in an experimental room. They were fed 2 kg of concentrate feed and chopped timothy hay twice a day at 09.00 and 16.00 h. The amount of hay was changed according to body weight to maintain a daily body weight gain of 0.9 kg. Water was provided ad libitum. More than 2 weeks before the experiment, an 18 G stainless cannula (Eicom, Kyoto, Japan) was stereotaxically implanted into the third cerebral ventricle of each steer under isoflurane anesthesia according to a procedure reported earlier (Kasuya et al., 2006). The intracerebroventricular cannulation was ensured by the efflux of cerebrospinal fluid from the cannula. During the pre-experimental period, steers were continually tamed and allowed to adapt to their environment in order to minimize the impact of the stress of handling associated with ICV injections and blood sampling. On the day before the first experiment, each animal was fitted with an indwelling jugular catheter (Terufusion IVH catheter kit; Terumo, Tokyo, Japan) to collect blood samples. 2.2. Treatment solutions The injection vehicle was an artificial cerebrospinal fluid (aCSF: NaCl, 125 mM; KCl, 2.5 mM; NaH2PO4, 0.5 mM; Na2HPO4, 1.2 mM; CaCl2, 1.2 mM; MgCl2, 1.0 mM; NaHCO3, 27 mM) (Hashizume et al., 1994). The pH of the medium was adjusted to 7.4. Synthetic NMU and NMS (Peptide Institute, Inc., Osaka, Japan) were dissolved in aCSF solution at doses of 1, 10, and 100 nmol/ml and divided into 400 ll aliquots, which were stored at 20 °C before use.
325
2.3. Experimental procedure Experiments were performed between 13.30 and 15.30 h. At 14.00 h (time 0 in the experiment), the animal was lightly restrained with a rope and 0.2, 2, or 20 nmol of NMU or NMS, each dissolved in 200 ll aCSF, was injected into the third ventricle via the implanted cannula at a rate of 200 ll/20 s. Serial blood samples for measurements of plasma cortisol concentrations (CORT) were collected via an indwelling jugular catheter at 30, 0, 10, 20, 30, 40, 50, 60, and 90 min. Concurrently, rectal temperature (RT) was checked with a mercury thermometer. A network camera and recording program (BB-HCM581 and BB-HNP11; Panasonic, Osaka, Japan) were used to record the animal behavior continuously. To minimize the carry-over effect of each treatment, each steer was randomly assigned to each of the seven treatments and an interval of at least 2 days was kept between successive treatments, depending on the recovery of the animal’s condition. 2.4. Data analysis Blood samples were taken into prechilled tubes containing EDTA and stored on ice until the end of the experiment. After the experiment, the tubes were centrifuged and plasma samples were stored at 20 °C until assay. CORT were obtained by enzyme immunoassay (Sakumoto et al., 2003) using peroxidase-labeled cortisol (FKA403, final dilution 1:40,000; Cosmo Bio Co., Ltd., Tokyo, Japan) and anti-cortisol serum (FKA404E, final dilution 1:70,000; Cosmo Bio Co.). The standard curve ranged from 0.16 to 160 ng/ml, and the ED50 of the assay was 3.2 ng/ml. The intra-assay coefficient of variation (CV) was 3.5% at 24 ng/ml. The inter-assay CV was 8.6% at 24 ng/ml. The area under the cortisol concentration curve between 0 and 90 min after the injection was calculated. Raw data for RT were converted to changes in RT from the pre-injection value (mean of values at 30 and 0 min). Continuous behavior sampling was performed for 90 min after the injection. The percentage of time spent lying or ruminating for 90 min after the injection was calculated. Occurrences of selfgrooming, head shaking, head rubbing, and water access were also analyzed. 2.5. Statistical analysis The effects of time and treatments on CORT and the changes in RT were analyzed using the repeated statement of the SAS GLM procedure with animals and treatments as the sole source of variation in the whole plot, and time as the source of variation in the subplot. The differences in variation with time depended on whether the time–treatment interaction was significant. Moreover, after transposing the dataset with respect to each animal, the variations with time within each treatment were analyzed as a randomized blocks design of the SAS GLM procedure with animals and time as the main effects in the model. If there was a significant main effect of time, the statistical differences from the pre-injection values ( 30 and 0 min) within each treatment were analyzed using the contrast statement of the SAS GLM procedure followed by calculation of the Scheffe F-value. In addition, the areas under the curve (AUC) for CORT were calculated from 0 to 90 min with basal concentrations subtracted during each treatment. The AUCs were analyzed as a randomized blocks design using the GLM procedure of SAS with animals as the block and treatments as the main effect in the model. If there was a significant main effect of treatments, the statistical differences among the treatments were analyzed using the contrast statement of the SAS GLM procedure followed by calculation of the Scheffe F-value. For RT, the maximum changes in RT from the pre-injection value during 90 min after the injection (0–90 min) were statistically assessed in the
K. Yayou et al. / General and Comparative Endocrinology 163 (2009) 324–328
same way as AUCs for CORT. Behavioral data were statistically assessed by Friedman’s test followed by Nemenyi multiple comparison if there was a significant effect of treatments.
A
3. Results
A Plasma Cortisol Concentration (ng/ml)
20
aCSF NMU2 NMS0.2 NMS20
NMU0.2 NMU20 NMS2
15
*
*
† 10
5
0
30
60
AUC from 0-90 min (ng·min/ml)
*
*
0.4
†
0.3 0.2 0.1
0
30
60
90
Time after the Injection (min)
B
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 aCSF
0.2
2.0
NMU
20.0
0.2
2
20
NMS
90
Time after the Injection (min) 500 450 400 350 300 250 200 150 100 50 0 -50 -100 -150
NMU0.2 NMU20 NMS2
Fig. 2. Effects of intracerebroventricular injections of 200 ll aCSF and 0.2, 2, and 20 nmol neuromedin U (NMU) or neuromedin S (NMS) on temporal changes in mean (+SEM) change in rectal temperature (RT) from the pre-injection value (mean of values at 30 and 0 min) in steers (A) and the mean (±SEM) of the maximum change in RT from the pre-injection value during 90 min after the injection (0– 90 min) (B). *Significant difference from the pre-injection value (p < 0.05). Tendency to differ from the pre-injection value (p < 0.1). There was a significant main effects of treatment (p < 0.05) on the maximum changes in RT.
0 -30
B
0.5
0 -30 -0.1
Maximum Changes in RT during 90 min after the injection (°C)
NMU and NMS evoked a dose-related increase in CORT after ICV administration (Fig. 1). There was a significant time–treatment interaction for CORT (p < 0.001). Time effects were seen with 0.2 (p < 0.1) and 20 (p < 0.001) nmol NMU treatments, and with 0.2 (p < 0.1), 2 (p < 0.005), and 20 (p < 0.001) nmol NMS treatments. Compared with the levels in the control period ( 30 and 0 min), the CORT was significantly higher at 60 min (p < 0.005) with 20 nmol NMU. With 20 nmol NMS treatment, CORT tended to be higher at 50 min (p < 0.1) and was significantly higher at 90 min (p < 0.05) (Fig. 1A). There was a significant main effect of treatment (p < 0.005) on the CORT AUC. The AUC response to 20 nmol NMS tended to be higher than that of aCSF (p < 0.1) (Fig. 1B). ICV NMU evoked a dose-related increase in RT (Fig. 2). There was a significant time–treatment interaction for the changes in RT (p < 0.001). Time effects were seen with 2 (p < 0.001) and 20 (p < 0.001) nmol NMU treatments and with 2 (p < 0.05) and 20 (p < 0.01) nmol NMS treatments. Compared with the pre-injection value (mean of values at 30 and 0 min), the changes in RT was
aCSF NMU2 NMS0.2 NMS20
0.6
Mean Changes in RT (°C)
326
b
a
aCSF
0.2
2
NMU (nmol)
20
0.2
2
20
NMS (nmol)
Fig. 1. Effects of intracerebroventricular injections of 200 ll aCSF and 0.2, 2, and 20 nmol neuromedin U (NMU) or neuromedin S (NMS) on temporal changes in mean (+SEM) plasma cortisol concentrations (CORT) in steers (A) and the mean (±SEM) area under the CORT curve (AUC) from 0 to 90 min after the injection (B). * Significant difference from the pre-injection value ( 30 and 0 min) (p < 0.05) Tendency to differ from the pre-injection value (p < 0.1). Different superscript letters indicate statistical differences (p < 0.1 between a and b).
significantly higher at 60 (p < 0.01) and 90 min (p < 0.005) with 20 nmol NMU and tended to be higher at 90 min (p < 0.1) with 2 nmol NMU (Fig. 2A). There was a significant main effect of treatment (p < 0.05) on the maximum changes in RT from the pre-injection value during 90 min after the injection (Fig. 2B). Table 1 summarizes the data for behaviors exhibited during 90 min after ICV injection of aCSF, NMU, or NMS. There was a significant difference among the seven treatments in percentage of time spent lying (Friedman’s test, v2 = 15.6, p < 0.01) and in the total number of head shaking (Friedman’s test, v2 = 14.49, p < 0.01). The percentage of time spent lying after the injection of 20 nmol NMS was significantly less than that after 2 nmol NMS (Nemenyi’s test, p < 0.05), and tended to be less than that after 0.2 nmol of NMS (Nemenyi’s test, p < 0.1). 4. Discussion In the present study, we confirmed that ICV administration of either rat NMU or rat NMS activated the HPA axis in steers, evoking marked cortisol responses, suggesting that NMU and NMS are involved in the control of the HPA axis in this species. We also demonstrated that ICV injection of rat NMU induced increase in body temperature in steers, suggesting that NMU might be involved in
327
K. Yayou et al. / General and Comparative Endocrinology 163 (2009) 324–328
Table 1 Effects of intracerebroventricular injections of 200 ll aCSF and 0.2, 2 and 20 nmol of neuromedin U (NMU) or neuromedin S (NMS) on behavior during 90 min after the injection in steers. aCSF
NMU
NMS
0.2 nmol
2 nmol
20 nmol
0.2 nmol
2 nmol
20 nmol
77c 9
75.1a 20.7
39.4b,d 30.4
v2
p
15.6
0.004
Lying (%) Mean SD
57.4 28.7
78.5 16.1
64.2 11.5
64.3 18.4
Ruminating (%) Mean SD
30.6 8
41.4 11.6
32.6 19.6
45.1 13.6
40.3 17.9
40 17.4
43.5 21.2
4.71
0.619
Self-grooming (No.) Mean 11 SD 10.4
4.4 4.2
5.8 7.6
8.3 5.1
6 5.4
8.3 10.1
12 10.2
5.01
0.577
Head shaking (No.) Mean 0 SD 0
0 0
0.7 1.6
0.2 0.4
0 0
0.2 0.4
1.7 2
14.49
0.009
Head rubbing (No.) Mean 3.3 SD 3.5
0.8 0.8
2.7 2.7
4.3 1.8
3 3
2.3 2.3
4.2 2.2
7.27
0.304
Water access (No.) Mean 6.2 SD 4.4
3 3.1
3.2 3.9
4 2.4
2.2 2.2
4 3.4
6.8 7.8
6.29
0.412
p: p-values obtained by Friedman’s test. Different superscript letters indicate statistical differences (Nemenyi multiple comparison: p < 0.05: between a and b, and p < 0.1: between c and d).
thermoregulation in this species. A high dose of NMS tended to shorten the duration of lying and increase the number of head shaking in steers, suggesting that NMS might induce restlessness in this species. Although both NMU and NMS reduced food intake when administered centrally via NMUR2 in rodents (Ida et al., 2005; Peier et al., 2009), the steers fed normally at evening feeding time, 2 h after the injection of NMU or NMS (our personal observation). Mean of average daily weight gain of the steers over the total time of experiment was 0.75 kg/day. Therefore the chronic effects of NMU or NMS injection on energy expenditure were less in the present experimental condition. The present finding that ICV administration of NMU and NMS activated the HPA axis in cattle is similar to results obtained in rats (Brighton et al., 2004; Ida et al., 2005; Ozaki et al., 2002). The effect was more persistent after a high dose of NMS than after a high dose of NMU, since a significantly higher CORT than the pre-injection value was still present 90 min after the injection of NMS 20 nmol but not after NMU 20 nmol treatment. In the central nervous system, NMU and NMS act on the same receptor, NMUR2, and stimulate the HPA axis via the CRH-mediated pathway modulated by the paraventricular nucleus of the hypothalamus (PVN) (Brighton et al., 2004; Ida et al., 2005; Jaszberenyi et al., 2007; Ozaki et al., 2002; Wren et al., 2002). The neuronal multiple unit activity in the PVN induced by ICV NMS lasted longer than that induced by ICV NMU (Ida et al., 2005). ICV NMU and NMS also induced a dose-dependent increase in the plasma concentration of arginine vasopressin (AVP) and oxytocin, with activation of the PVN and the supraoptic nucleus of the hypothalamus (SON) (Ozaki et al., 2002; Sakamoto et al., 2007, 2008). In cattle, we previously demonstrated that both ICV CRH and AVP could activate the HPA axis in steers and that CRH was more potent than AVP in stimulating the HPA axis (Yayou et al., 2008). Although further studies to examine the downstream neuroendocrine targets of the NMUR2 using specific antagonist for CRH, AVP, or oxytocin are required to determine exact roles of central NMU and NMS in regulating the HPA axis in cattle, we speculate at this point that central NMU and NMS might regulate the HPA axis by modulating the activity of CRH, AVP, and oxytocin neurons in the PVN and SON like rodents.
In the present study, ICV administration of NMU increased body temperature, whereas NMS did not increase body temperature remarkably. This result is consistent with the results in rats (Jaszberenyi et al., 2007; Howard et al., 2000; Nakazato et al., 2000). NMU also increased non-exercise activity thermogenesis after bilateral application directly to the PVN and arcuate nucleus of the hypothalamus (Novak et al., 2006). Since increase in body temperature induced by ICV NMU was not observed in CRH-deficient mice (Hanada et al., 2003), increase in body temperature observed after ICV NMU in the present study might have been induced through the CRH pathway. In our previous study, however, neither ICV CRH nor AVP induced changes in body temperature in steers, and we considered that the lack of effect of ICV CRH and AVP on thermoregulation in cattle was due to species differences in autonomic nervous regulation of cardiac function, which is parasympathetic-predominant in cattle but sympathetic-predominant in rodents (Matsui and Sugano, 1989; Yayou et al., 1993), or due to insufficient behavioral activation to increase sympathetic nervous activity on behavioral restriction by a stanchion (Yayou et al., 2008). Another candidate downstream target which would modulate increase in body temperature in the present study is oxytocin. A hyperthermic action of ICV oxytocin was reported in mice (Mason et al., 1986) and rabbits (Lipton and Glyn, 1980) and ICV NMU stimulated oxytocin neurons within the PVN and SON in rats (Ozaki et al., 2002) Although NMS also stimulates CRH and oxytocin neurons in rats (Sakamoto et al., 2007, 2008), ICV NMS showed a tendency to lower core temperature in rats, partly because of activation of central dopaminergic neurons (Jaszberenyi et al., 2007). Although further studies to examine the downstream neuroendocrine targets of ICV NMU using specific antagonist for oxytocin are required in cattle, we speculate at this point that central NMU might regulate increase in body temperature by modulating the activity of oxytocin neurons unlike rodents. A high dose of NMS had tendencies to shorten the duration of lying and to increase the number of head shaking, which suggest that NMS might cause restlessness in cattle. In rats, NMS activated grooming (Jaszberenyi et al., 2007) and NMU activated grooming and increased overall locomotor activity (Hanada et al., 2001; Wren et al., 2002) which are reported to be CRH-mediated (Hanada et al., 2001; Jaszberenyi et al., 2007) and might be mediated by
328
K. Yayou et al. / General and Comparative Endocrinology 163 (2009) 324–328
stimulation of dopamine secretion via the CRHR1 pathway (Jaszberenyi et al., 2007). In our previous study in cattle, however, ICV CRH did not induce either stereotypical grooming or behavioral activation; rather ICV AVP induced restlessness including head shaking (Yayou et al., 2008). There might be species differences in the downstream regulation of behavioral responses by central NMS. Although further studies are required to determine exact roles of central NMS in regulating restlessness in cattle, we speculate at this point that central NMS might regulate restlessness by modulating the activity of AVP neurons unlike rodents. In the present study, NMU and NMS increased the CORT after ICV administration, suggesting that both central NMU and NMS might have roles in the control of the HPA axis in cattle. We also showed that ICV NMU increased body temperature, suggesting that central NMU might participate in the control of body temperature in cattle. Behavioral activation induced by ICV NMS suggests that NMS are likely to be involved in the control of behavior in this species. Although further research are necessary using specific antagonist for CRH, AVP, or oxytocin to confirm downstream neuroendocrine targets of NMUR2, these result together with our previous findings on the central roles of CRH and AVP in cattle suggest that central NMU and NMS might take part in the regulation of HPA axis, body temperature, and behavioral activation via different pathways from those of rodents at least partly. Acknowledgments The authors thank the members of Livestock Research Support Center of the National Institute of Livestock and Grassland Science to support accurate research works, feeding and management of experimental animals. This work was partially supported by a Grant-in-Aid for Scientific Research (B) (No. 20380151) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References Brighton, P.J., Szekeres, P.G., Willars, G.B., 2004. Neuromedin U and its receptors: structure, function, and physiological roles. Pharmacol. Rev. 56, 231–248. Guan, X.M., Yu, H., Jiang, Q., Van Der Ploeg, L.H., Liu, Q., 2001. Distribution of neuromedin U receptor subtype 2 mRNA in the rat brain. Brain Res. Gene Expr. Patterns 1, 1–4. Hanada, R., Nakazato, M., Murakami, N., Sakihara, S., Yoshimatsu, H., Toshinai, K., Hanada, T., Suda, T., Kangawa, K., Matsukura, S., Sakata, T., 2001. A role for neuromedin U in stress response. Biochem. Biophys. Res. Commun. 289, 225– 228. Hanada, T., Date, Y., Shimbara, T., Sakihara, S., Murakami, N., Hayashi, Y., Kanai, Y., Suda, T., Kangawa, K., Nakazato, M., 2003. Central actions of neuromedin U via corticotropin-releasing hormone. Biochem. Biophys. Res. Commun. 311, 954– 958. Hashizume, T., Haglof, S.A., Malven, P.V., 1994. Intracerebral methionineenkephalin, serum cortisol, and serum beta-endorphin during acute exposure of sheep to physical or isolation stress. J. Anim. Sci. 72, 700–708. Hosoya, M., Moriya, T., Kawamata, Y., Ohkubo, S., Fujii, R., Matsui, H., Shintani, Y., Fukusumi, S., Habata, Y., Hinuma, S., Onda, H., Nishimura, O., Fujino, M., 2000. Identification and functional characterization of a novel subtype of neuromedin U receptor. J. Biol. Chem. 275, 29528–29532. Howard, A.D., Wang, R., Pong, S.S., Mellin, T.N., Strack, A., Guan, X.M., Zeng, Z., Williams Jr., D.L., Feighner, S.D., Nunes, C.N., Murphy, B., Stair, J.N., Yu, H., Jiang, Q., Clements, M.K., Tan, C.P., McKee, K.K., Hreniuk, D.L., McDonald, T.P., Lynch, K.R., Evans, J.F., Austin, C.P., Caskey, C.T., Van der Ploeg, L.H., Liu, Q., 2000. Identification of receptors for neuromedin U and its role in feeding. Nature 406, 70–74. Ida, T., Mori, K., Miyazato, M., Egi, Y., Abe, S., Nakahara, K., Nishihara, M., Kangawa, K., Murakami, N., 2005. Neuromedin S is a novel anorexigenic hormone. Endocrinology 146, 4217–4223.
Jaszberenyi, M., Bagosi, Z., Thurzo, B., Foldesi, I., Telegdy, G., 2007. Endocrine and behavioral effects of neuromedin S. Horm. Behav. 52, 631–639. Kasuya, E., Kushibiki, S., Sutoh, M., Saito, T., Ito, S., Yayou, K., Sakumoto, R., Hodate, K., 2006. Effect of melatonin injected into the third ventricle on growth hormone secretion in Holstein steers. J. Vet. Med. Sci. 68, 1075–1080. Lipton, J.M., Glyn, J.R., 1980. Central administration of peptides alters thermoregulation in the rabbit. Peptides 1, 15–18. Mason, G.A., Caldwell, J.D., Stanley, D.A., Hatley, O.L., Prange Jr., A.J., Pedersen, C.A., 1986. Interactive effects of intracisternal oxytocin and other centrally active substances on colonic temperatures of mice. Regul. Pept. 14, 253– 260. Matsui, K., Sugano, S., 1989. Relation of intrinsic heart rate and autonomic nervous tone to resting heart rate in the young and the adult of various domestic animals. Nippon Juigaku Zasshi 51, 29–34. Minamino, N., Kangawa, K., Matsuo, H., 1985. Neuromedin U-8 and U-25: novel uterus stimulating and hypertensive peptides identified in porcine spinal cord. Biochem. Biophys. Res. Commun. 130, 1078–1085. Mori, K., Miyazato, M., Ida, T., Murakami, N., Serino, R., Ueta, Y., Kojima, M., Kangawa, K., 2005. Identification of neuromedin S and its possible role in the mammalian circadian oscillator system. EMBO J. 24, 325– 335. Mori, K., Miyazato, M., Kangawa, K., 2008. Neuromedin S: discovery and functions. Results Probl. Cell Differ. 46, 201–212. Nakahara, K., Hanada, R., Murakami, N., Teranishi, H., Ohgusu, H., Fukushima, N., Moriyama, M., Ida, T., Kangawa, K., Kojima, M., 2004a. The gut-brain peptide neuromedin U is involved in the mammalian circadian oscillator system. Biochem. Biophys. Res. Commun. 318, 156–161. Nakahara, K., Kojima, M., Hanada, R., Egi, Y., Ida, T., Miyazato, M., Kangawa, K., Murakami, N., 2004b. Neuromedin U is involved in nociceptive reflexes and adaptation to environmental stimuli in mice. Biochem. Biophys. Res. Commun. 323, 615–620. Nakazato, M., Hanada, R., Murakami, N., Date, Y., Mondal, M.S., Kojima, M., Yoshimatsu, H., Kangawa, K., Matsukura, S., 2000. Central effects of neuromedin U in the regulation of energy homeostasis. Biochem. Biophys. Res. Commun. 277, 191–194. Novak, C.M., Zhang, M., Levine, J.A., 2006. Neuromedin U in the paraventricular and arcuate hypothalamic nuclei increases non-exercise activity thermogenesis. J. Neuroendocrinol. 18, 594–601. Ozaki, Y., Onaka, T., Nakazato, M., Saito, J., Kanemoto, K., Matsumoto, T., Ueta, Y., 2002. Centrally administered neuromedin U activates neurosecretion and induction of c-fos messenger ribonucleic acid in the paraventricular and supraoptic nuclei of rat. Endocrinology 143, 4320–4329. Peier, A., Kosinski, J., Cox-York, K., Qian, Y., Desai, K., Feng, Y., Trivedi, P., Hastings, N., Marsh, DJ., 2009. The anti-obesity effects of centrally administered neuromedin U and neuromedin S are mediated predominantly by the neuromedin U receptor 2 (NMUR2). Endocrinology. doi:10.1210/en.2008-1772. Raddatz, R., Wilson, A.E., Artymyshyn, R., Bonini, J.A., Borowsky, B., Boteju, L.W., Zhou, S., Kouranova, E.V., Nagorny, R., Guevarra, M.S., Dai, M., Lerman, G.S., Vaysse, P.J., Branchek, T.A., Gerald, C., Forray, C., Adham, N., 2000. Identification and characterization of two neuromedin U receptors differentially expressed in peripheral tissues and the central nervous system. J. Biol. Chem. 275, 32452– 32459. Sakamoto, T., Mori, K., Nakahara, K., Miyazato, M., Kangawa, K., Sameshima, H., Murakami, N., 2007. Neuromedin S exerts an antidiuretic action in rats. Biochem. Biophys. Res. Commun. 361, 457–461. Sakamoto, T., Mori, K., Miyazato, M., Kangawa, K., Sameshima, H., Nakahara, K., Murakami, N., 2008. Involvement of neuromedin S in the oxytocin release response to suckling stimulus. Biochem. Biophys. Res. Commun. 375, 49–53. Sakumoto, R., Kasuya, E., Komatsu, T., Akita, T., 2003. Central and peripheral concentrations of tumor necrosis factor-alpha in Chinese Meishan pigs stimulated with lipopolysaccharide. J. Anim. Sci. 81, 1274–1280. Vigo, E., Roa, J., López, M., Castellano, JM., Fernandez-Fernandez, R., Navarro, VM., Pineda, R., Aguilar, E., Diéguez, C., Pinilla, L., Tena-Sempere, M., 2007. Neuromedin S as novel putative regulator of luteinizing hormone secretion. Endocrinology 148, 813–823. Wren, A.M., Small, C.J., Abbott, C.R., Jethwa, P.H., Kennedy, A.R., Murphy, K.G., Stanley, S.A., Zollner, A.N., Ghatei, M.A., Bloom, S.R., 2002. Hypothalamic actions of neuromedin U. Endocrinology 143, 4227–4234. Yayou, K., Annen, Y., Kuwahara, M., Tsubone, H., Sugano, S., 1993. Studies on the autonomic nervous function in Tsukuba emotional rats – with special reference to their cardiac function. J. Vet. Med. Sci. 55, 1017–1023. Yayou, K., Sato, Y., Ito, S., Nakamura, M., 2008. Comparison between the central effects of CRH and AVP in steers. Physiol. Behav. 93, 537–545. Yu, X.H., Cao, C.Q., Mennicken, F., Puma, C., Dray, A., O’Donnell, D., Ahmad, S., Perkins, M., 2003. Pro-nociceptive effects of neuromedin U in rat. Neuroscience 120 (2), 467–474.
Contents lists available at ScienceDirect
General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen
Effects of intracerebroventricular administration of neuromedin U or neuromedin S in steers K. Yayou a,*, S. Kitagawa b, S. Ito c, E. Kasuya a, M. Sutoh d a
Laboratory of Neurobiology, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan Department of Animal Science, Utsunomiya University, Utsunomiya 321-8505, Japan c Laboratory of Animal Behavior, Tokai University, Aso-gun 869-1404, Japan d Endocrinology and Metabolism Research Team, National Institute of Livestock and Grassland Science, Tsukuba 305-0901, Japan b
a r t i c l e
i n f o
Article history: Received 19 March 2009 Revised 24 April 2009 Accepted 29 April 2009 Available online 12 May 2009 Keywords: Body temperature Hypothalamo–pituitary–adrenal axis Intracerebroventricular administration Neuromedin S Neuromedin U Steer
a b s t r a c t Although neuromedin U (NMU) and neuromedin S (NMS) are reported to modulate stress responses mainly through corticotropin-releasing hormone system in rodents, the in vivo effects of centrally administered NMU or NMS on stress regulation have not been fully elucidated in cattle. We examined adrenocorticotropic hormone levels, body temperature, and behavioral responses to intracerebroventricularly (ICV) administered rat NMU or rat NMS in steers. ICV NMU and NMS (0.2, 2, and 20 nmol/200 ll) evoked a dose-related increase in plasma cortisol concentrations (CORT). There was a significant time–treatment interaction for the time course of CORT (p < 0.001). ICV NMU evoked a dose-related increase in rectal temperature (RT). There was a significant time–treatment interaction for the change in RT from pre-injection value (p < 0.05). There was a significant difference among treatments in the percentage of time spent lying (Friedman’s test, v2 = 15.6, p < 0.01) and in the total number of head shaking (Friedman’s test, v2 = 14.49, p < 0.01). A high dose of NMS tended to shorten the duration of lying and increase the number of head shaking. These findings indicate that both central NMU and NMS might participate in controlling the hypothalamo–pituitary–adrenal axis, that central NMU might participate in controlling body temperature, and that central NMS is likely to be involved in behavioral activation in cattle. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction The neuropeptides neuromedin U (NMU) and neuromedin S (NMS) were identified as endogenous ligands for two orphan G protein-coupled receptors, FM-3/GPR66 and FM-4/TGR-1 (Minamino et al., 1985; Mori et al., 2005), currently identified as NMU type1 (NMUR1) and type-2 (NMUR2) receptors, respectively (Guan et al., 2001, Howard et al., 2000; Raddatz et al., 2000). Although NMU and NMS share a C-terminal core structure and activate recombinant NMUR1 and NMUR2 expressed in Chinese hamster ovary cells, they have been mapped to discrete chromosomes (Mori et al., 2005). Since the NMUR2 is highly expressed in the central nervous system (Guan et al., 2001; Howard et al., 2000; Raddatz et al., 2000), the physiological roles of these neuropeptides in the central nervous system have been investigated mainly in rodents primarily by intracerebroventricular (ICV) administration of the peptides. NMU and NMS function in the regulation of feeding behaviors
* Corresponding author. Address: Laboratory of Neurobiology, National Institute of Agrobiological Sciences, 2, Ikenodai, Tsukuba, Ibaraki 305-8602, Japan. Fax: +81 29 838 8610. E-mail address: [email protected] (K. Yayou). 0016-6480/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2009.04.033
(Howard et al., 2000; Ida et al., 2005; Peier et al., 2009), energy homeostasis (Hanada et al., 2003; Nakazato et al., 2000; Peier et al., 2009), circadian rhythms (Mori et al., 2005; Nakahara et al., 2004a), and stress responses (Hanada et al., 2001; Jaszberenyi et al., 2007). Although NMU and NMS bind to the same receptor, there are several discrepancies in the effects ICV administered neuropeptides. A nociceptive response was observed only after ICV NMU (Nakahara et al., 2004b; Yu et al., 2003). Increased release of luteinizing hormone was observed only after ICV NMS (Vigo et al., 2007). The anorexigenic effect through the corticotropinreleasing hormone (CRH) and a-melanocyte-stimulating hormone systems was greater and longer lasting after ICV NMS than that after ICV NMU (Ida et al., 2005; Peier et al., 2009). The antidiuretic action by arginine vasopressin (AVP) was stronger after ICV NMS than that after ICV NMU (Sakamoto et al., 2007). As to the function in stress response, both neuropeptides activated the HPA axis and induced grooming through the CRH system (Hanada et al., 2001; Jaszberenyi et al., 2007), but only NMU increased locomotor activity (Hanada et al., 2001; Wren et al., 2002). Recently we have shown that ICV administration of bovine-CRH (bCRH) and AVP activated HPA axis and that bCRH was more potent to stimulate HPA axis than AVP. We have also shown that ICV bCRH and AVP induced stereotyped behaviors in steers
K. Yayou et al. / General and Comparative Endocrinology 163 (2009) 324–328
and that the types of stereotyped behaviors induced were different between bCRH and AVP (Yayou et al., 2008). Although the result that CRH was more potent stimulator of HPA axis than AVP was consistent with the results in rodents, the behavioral data suggest the presence of species differences in the central roles of CRH and AVP to regulate behavioral responses under stressful conditions. Until now, no data have been published regarding the central effects of NMU and NMS in steers, though these peptides might have important roles to modulate stress responses through CRH and/or AVP. The biologically active region, the C-terminal region of the peptide, was conserved among various animal species including cattle (GenBank Accession Nos.: XP_001250700, region 193. . .217 for NMU; Q0VBW8, region 104. . .136 for NMS) (Mori et al., 2005, 2008). Moreover, the NMUR2 gene of cattle (GenBank Accession No. AAX46680) had high homology (76% identities, 86% positive) compared to that of rats (Hosoya et al., 2000). As part of the study of central modulation of stress response in cattle, we elucidated the central roles of NMU and NMS in cattle by examining changes in adrenocorticotropic hormone levels, body temperature, and behavioral responses after ICV administrations of rat NMU or rat NMS into the third ventricle of steers.
2. Materials and methods Experimental procedures and animal care were approved by the Institute Committee for Animal Use and Care at the National Institute of Agrobiological Sciences, Tsukuba, Japan. 2.1. Animals and surgery Six Holstein steers (7–9 months old, weighing 165–246 kg at the start of the experiment) were used in the experiment. They were castrated at least 2 months before the experiment. After stereotaxic surgery, animals were individually reared in a stanchion stall in an experimental room. They were fed 2 kg of concentrate feed and chopped timothy hay twice a day at 09.00 and 16.00 h. The amount of hay was changed according to body weight to maintain a daily body weight gain of 0.9 kg. Water was provided ad libitum. More than 2 weeks before the experiment, an 18 G stainless cannula (Eicom, Kyoto, Japan) was stereotaxically implanted into the third cerebral ventricle of each steer under isoflurane anesthesia according to a procedure reported earlier (Kasuya et al., 2006). The intracerebroventricular cannulation was ensured by the efflux of cerebrospinal fluid from the cannula. During the pre-experimental period, steers were continually tamed and allowed to adapt to their environment in order to minimize the impact of the stress of handling associated with ICV injections and blood sampling. On the day before the first experiment, each animal was fitted with an indwelling jugular catheter (Terufusion IVH catheter kit; Terumo, Tokyo, Japan) to collect blood samples. 2.2. Treatment solutions The injection vehicle was an artificial cerebrospinal fluid (aCSF: NaCl, 125 mM; KCl, 2.5 mM; NaH2PO4, 0.5 mM; Na2HPO4, 1.2 mM; CaCl2, 1.2 mM; MgCl2, 1.0 mM; NaHCO3, 27 mM) (Hashizume et al., 1994). The pH of the medium was adjusted to 7.4. Synthetic NMU and NMS (Peptide Institute, Inc., Osaka, Japan) were dissolved in aCSF solution at doses of 1, 10, and 100 nmol/ml and divided into 400 ll aliquots, which were stored at 20 °C before use.
325
2.3. Experimental procedure Experiments were performed between 13.30 and 15.30 h. At 14.00 h (time 0 in the experiment), the animal was lightly restrained with a rope and 0.2, 2, or 20 nmol of NMU or NMS, each dissolved in 200 ll aCSF, was injected into the third ventricle via the implanted cannula at a rate of 200 ll/20 s. Serial blood samples for measurements of plasma cortisol concentrations (CORT) were collected via an indwelling jugular catheter at 30, 0, 10, 20, 30, 40, 50, 60, and 90 min. Concurrently, rectal temperature (RT) was checked with a mercury thermometer. A network camera and recording program (BB-HCM581 and BB-HNP11; Panasonic, Osaka, Japan) were used to record the animal behavior continuously. To minimize the carry-over effect of each treatment, each steer was randomly assigned to each of the seven treatments and an interval of at least 2 days was kept between successive treatments, depending on the recovery of the animal’s condition. 2.4. Data analysis Blood samples were taken into prechilled tubes containing EDTA and stored on ice until the end of the experiment. After the experiment, the tubes were centrifuged and plasma samples were stored at 20 °C until assay. CORT were obtained by enzyme immunoassay (Sakumoto et al., 2003) using peroxidase-labeled cortisol (FKA403, final dilution 1:40,000; Cosmo Bio Co., Ltd., Tokyo, Japan) and anti-cortisol serum (FKA404E, final dilution 1:70,000; Cosmo Bio Co.). The standard curve ranged from 0.16 to 160 ng/ml, and the ED50 of the assay was 3.2 ng/ml. The intra-assay coefficient of variation (CV) was 3.5% at 24 ng/ml. The inter-assay CV was 8.6% at 24 ng/ml. The area under the cortisol concentration curve between 0 and 90 min after the injection was calculated. Raw data for RT were converted to changes in RT from the pre-injection value (mean of values at 30 and 0 min). Continuous behavior sampling was performed for 90 min after the injection. The percentage of time spent lying or ruminating for 90 min after the injection was calculated. Occurrences of selfgrooming, head shaking, head rubbing, and water access were also analyzed. 2.5. Statistical analysis The effects of time and treatments on CORT and the changes in RT were analyzed using the repeated statement of the SAS GLM procedure with animals and treatments as the sole source of variation in the whole plot, and time as the source of variation in the subplot. The differences in variation with time depended on whether the time–treatment interaction was significant. Moreover, after transposing the dataset with respect to each animal, the variations with time within each treatment were analyzed as a randomized blocks design of the SAS GLM procedure with animals and time as the main effects in the model. If there was a significant main effect of time, the statistical differences from the pre-injection values ( 30 and 0 min) within each treatment were analyzed using the contrast statement of the SAS GLM procedure followed by calculation of the Scheffe F-value. In addition, the areas under the curve (AUC) for CORT were calculated from 0 to 90 min with basal concentrations subtracted during each treatment. The AUCs were analyzed as a randomized blocks design using the GLM procedure of SAS with animals as the block and treatments as the main effect in the model. If there was a significant main effect of treatments, the statistical differences among the treatments were analyzed using the contrast statement of the SAS GLM procedure followed by calculation of the Scheffe F-value. For RT, the maximum changes in RT from the pre-injection value during 90 min after the injection (0–90 min) were statistically assessed in the
K. Yayou et al. / General and Comparative Endocrinology 163 (2009) 324–328
same way as AUCs for CORT. Behavioral data were statistically assessed by Friedman’s test followed by Nemenyi multiple comparison if there was a significant effect of treatments.
A
3. Results
A Plasma Cortisol Concentration (ng/ml)
20
aCSF NMU2 NMS0.2 NMS20
NMU0.2 NMU20 NMS2
15
*
*
† 10
5
0
30
60
AUC from 0-90 min (ng·min/ml)
*
*
0.4
†
0.3 0.2 0.1
0
30
60
90
Time after the Injection (min)
B
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 aCSF
0.2
2.0
NMU
20.0
0.2
2
20
NMS
90
Time after the Injection (min) 500 450 400 350 300 250 200 150 100 50 0 -50 -100 -150
NMU0.2 NMU20 NMS2
Fig. 2. Effects of intracerebroventricular injections of 200 ll aCSF and 0.2, 2, and 20 nmol neuromedin U (NMU) or neuromedin S (NMS) on temporal changes in mean (+SEM) change in rectal temperature (RT) from the pre-injection value (mean of values at 30 and 0 min) in steers (A) and the mean (±SEM) of the maximum change in RT from the pre-injection value during 90 min after the injection (0– 90 min) (B). *Significant difference from the pre-injection value (p < 0.05). Tendency to differ from the pre-injection value (p < 0.1). There was a significant main effects of treatment (p < 0.05) on the maximum changes in RT.
0 -30
B
0.5
0 -30 -0.1
Maximum Changes in RT during 90 min after the injection (°C)
NMU and NMS evoked a dose-related increase in CORT after ICV administration (Fig. 1). There was a significant time–treatment interaction for CORT (p < 0.001). Time effects were seen with 0.2 (p < 0.1) and 20 (p < 0.001) nmol NMU treatments, and with 0.2 (p < 0.1), 2 (p < 0.005), and 20 (p < 0.001) nmol NMS treatments. Compared with the levels in the control period ( 30 and 0 min), the CORT was significantly higher at 60 min (p < 0.005) with 20 nmol NMU. With 20 nmol NMS treatment, CORT tended to be higher at 50 min (p < 0.1) and was significantly higher at 90 min (p < 0.05) (Fig. 1A). There was a significant main effect of treatment (p < 0.005) on the CORT AUC. The AUC response to 20 nmol NMS tended to be higher than that of aCSF (p < 0.1) (Fig. 1B). ICV NMU evoked a dose-related increase in RT (Fig. 2). There was a significant time–treatment interaction for the changes in RT (p < 0.001). Time effects were seen with 2 (p < 0.001) and 20 (p < 0.001) nmol NMU treatments and with 2 (p < 0.05) and 20 (p < 0.01) nmol NMS treatments. Compared with the pre-injection value (mean of values at 30 and 0 min), the changes in RT was
aCSF NMU2 NMS0.2 NMS20
0.6
Mean Changes in RT (°C)
326
b
a
aCSF
0.2
2
NMU (nmol)
20
0.2
2
20
NMS (nmol)
Fig. 1. Effects of intracerebroventricular injections of 200 ll aCSF and 0.2, 2, and 20 nmol neuromedin U (NMU) or neuromedin S (NMS) on temporal changes in mean (+SEM) plasma cortisol concentrations (CORT) in steers (A) and the mean (±SEM) area under the CORT curve (AUC) from 0 to 90 min after the injection (B). * Significant difference from the pre-injection value ( 30 and 0 min) (p < 0.05) Tendency to differ from the pre-injection value (p < 0.1). Different superscript letters indicate statistical differences (p < 0.1 between a and b).
significantly higher at 60 (p < 0.01) and 90 min (p < 0.005) with 20 nmol NMU and tended to be higher at 90 min (p < 0.1) with 2 nmol NMU (Fig. 2A). There was a significant main effect of treatment (p < 0.05) on the maximum changes in RT from the pre-injection value during 90 min after the injection (Fig. 2B). Table 1 summarizes the data for behaviors exhibited during 90 min after ICV injection of aCSF, NMU, or NMS. There was a significant difference among the seven treatments in percentage of time spent lying (Friedman’s test, v2 = 15.6, p < 0.01) and in the total number of head shaking (Friedman’s test, v2 = 14.49, p < 0.01). The percentage of time spent lying after the injection of 20 nmol NMS was significantly less than that after 2 nmol NMS (Nemenyi’s test, p < 0.05), and tended to be less than that after 0.2 nmol of NMS (Nemenyi’s test, p < 0.1). 4. Discussion In the present study, we confirmed that ICV administration of either rat NMU or rat NMS activated the HPA axis in steers, evoking marked cortisol responses, suggesting that NMU and NMS are involved in the control of the HPA axis in this species. We also demonstrated that ICV injection of rat NMU induced increase in body temperature in steers, suggesting that NMU might be involved in
327
K. Yayou et al. / General and Comparative Endocrinology 163 (2009) 324–328
Table 1 Effects of intracerebroventricular injections of 200 ll aCSF and 0.2, 2 and 20 nmol of neuromedin U (NMU) or neuromedin S (NMS) on behavior during 90 min after the injection in steers. aCSF
NMU
NMS
0.2 nmol
2 nmol
20 nmol
0.2 nmol
2 nmol
20 nmol
77c 9
75.1a 20.7
39.4b,d 30.4
v2
p
15.6
0.004
Lying (%) Mean SD
57.4 28.7
78.5 16.1
64.2 11.5
64.3 18.4
Ruminating (%) Mean SD
30.6 8
41.4 11.6
32.6 19.6
45.1 13.6
40.3 17.9
40 17.4
43.5 21.2
4.71
0.619
Self-grooming (No.) Mean 11 SD 10.4
4.4 4.2
5.8 7.6
8.3 5.1
6 5.4
8.3 10.1
12 10.2
5.01
0.577
Head shaking (No.) Mean 0 SD 0
0 0
0.7 1.6
0.2 0.4
0 0
0.2 0.4
1.7 2
14.49
0.009
Head rubbing (No.) Mean 3.3 SD 3.5
0.8 0.8
2.7 2.7
4.3 1.8
3 3
2.3 2.3
4.2 2.2
7.27
0.304
Water access (No.) Mean 6.2 SD 4.4
3 3.1
3.2 3.9
4 2.4
2.2 2.2
4 3.4
6.8 7.8
6.29
0.412
p: p-values obtained by Friedman’s test. Different superscript letters indicate statistical differences (Nemenyi multiple comparison: p < 0.05: between a and b, and p < 0.1: between c and d).
thermoregulation in this species. A high dose of NMS tended to shorten the duration of lying and increase the number of head shaking in steers, suggesting that NMS might induce restlessness in this species. Although both NMU and NMS reduced food intake when administered centrally via NMUR2 in rodents (Ida et al., 2005; Peier et al., 2009), the steers fed normally at evening feeding time, 2 h after the injection of NMU or NMS (our personal observation). Mean of average daily weight gain of the steers over the total time of experiment was 0.75 kg/day. Therefore the chronic effects of NMU or NMS injection on energy expenditure were less in the present experimental condition. The present finding that ICV administration of NMU and NMS activated the HPA axis in cattle is similar to results obtained in rats (Brighton et al., 2004; Ida et al., 2005; Ozaki et al., 2002). The effect was more persistent after a high dose of NMS than after a high dose of NMU, since a significantly higher CORT than the pre-injection value was still present 90 min after the injection of NMS 20 nmol but not after NMU 20 nmol treatment. In the central nervous system, NMU and NMS act on the same receptor, NMUR2, and stimulate the HPA axis via the CRH-mediated pathway modulated by the paraventricular nucleus of the hypothalamus (PVN) (Brighton et al., 2004; Ida et al., 2005; Jaszberenyi et al., 2007; Ozaki et al., 2002; Wren et al., 2002). The neuronal multiple unit activity in the PVN induced by ICV NMS lasted longer than that induced by ICV NMU (Ida et al., 2005). ICV NMU and NMS also induced a dose-dependent increase in the plasma concentration of arginine vasopressin (AVP) and oxytocin, with activation of the PVN and the supraoptic nucleus of the hypothalamus (SON) (Ozaki et al., 2002; Sakamoto et al., 2007, 2008). In cattle, we previously demonstrated that both ICV CRH and AVP could activate the HPA axis in steers and that CRH was more potent than AVP in stimulating the HPA axis (Yayou et al., 2008). Although further studies to examine the downstream neuroendocrine targets of the NMUR2 using specific antagonist for CRH, AVP, or oxytocin are required to determine exact roles of central NMU and NMS in regulating the HPA axis in cattle, we speculate at this point that central NMU and NMS might regulate the HPA axis by modulating the activity of CRH, AVP, and oxytocin neurons in the PVN and SON like rodents.
In the present study, ICV administration of NMU increased body temperature, whereas NMS did not increase body temperature remarkably. This result is consistent with the results in rats (Jaszberenyi et al., 2007; Howard et al., 2000; Nakazato et al., 2000). NMU also increased non-exercise activity thermogenesis after bilateral application directly to the PVN and arcuate nucleus of the hypothalamus (Novak et al., 2006). Since increase in body temperature induced by ICV NMU was not observed in CRH-deficient mice (Hanada et al., 2003), increase in body temperature observed after ICV NMU in the present study might have been induced through the CRH pathway. In our previous study, however, neither ICV CRH nor AVP induced changes in body temperature in steers, and we considered that the lack of effect of ICV CRH and AVP on thermoregulation in cattle was due to species differences in autonomic nervous regulation of cardiac function, which is parasympathetic-predominant in cattle but sympathetic-predominant in rodents (Matsui and Sugano, 1989; Yayou et al., 1993), or due to insufficient behavioral activation to increase sympathetic nervous activity on behavioral restriction by a stanchion (Yayou et al., 2008). Another candidate downstream target which would modulate increase in body temperature in the present study is oxytocin. A hyperthermic action of ICV oxytocin was reported in mice (Mason et al., 1986) and rabbits (Lipton and Glyn, 1980) and ICV NMU stimulated oxytocin neurons within the PVN and SON in rats (Ozaki et al., 2002) Although NMS also stimulates CRH and oxytocin neurons in rats (Sakamoto et al., 2007, 2008), ICV NMS showed a tendency to lower core temperature in rats, partly because of activation of central dopaminergic neurons (Jaszberenyi et al., 2007). Although further studies to examine the downstream neuroendocrine targets of ICV NMU using specific antagonist for oxytocin are required in cattle, we speculate at this point that central NMU might regulate increase in body temperature by modulating the activity of oxytocin neurons unlike rodents. A high dose of NMS had tendencies to shorten the duration of lying and to increase the number of head shaking, which suggest that NMS might cause restlessness in cattle. In rats, NMS activated grooming (Jaszberenyi et al., 2007) and NMU activated grooming and increased overall locomotor activity (Hanada et al., 2001; Wren et al., 2002) which are reported to be CRH-mediated (Hanada et al., 2001; Jaszberenyi et al., 2007) and might be mediated by
328
K. Yayou et al. / General and Comparative Endocrinology 163 (2009) 324–328
stimulation of dopamine secretion via the CRHR1 pathway (Jaszberenyi et al., 2007). In our previous study in cattle, however, ICV CRH did not induce either stereotypical grooming or behavioral activation; rather ICV AVP induced restlessness including head shaking (Yayou et al., 2008). There might be species differences in the downstream regulation of behavioral responses by central NMS. Although further studies are required to determine exact roles of central NMS in regulating restlessness in cattle, we speculate at this point that central NMS might regulate restlessness by modulating the activity of AVP neurons unlike rodents. In the present study, NMU and NMS increased the CORT after ICV administration, suggesting that both central NMU and NMS might have roles in the control of the HPA axis in cattle. We also showed that ICV NMU increased body temperature, suggesting that central NMU might participate in the control of body temperature in cattle. Behavioral activation induced by ICV NMS suggests that NMS are likely to be involved in the control of behavior in this species. Although further research are necessary using specific antagonist for CRH, AVP, or oxytocin to confirm downstream neuroendocrine targets of NMUR2, these result together with our previous findings on the central roles of CRH and AVP in cattle suggest that central NMU and NMS might take part in the regulation of HPA axis, body temperature, and behavioral activation via different pathways from those of rodents at least partly. Acknowledgments The authors thank the members of Livestock Research Support Center of the National Institute of Livestock and Grassland Science to support accurate research works, feeding and management of experimental animals. This work was partially supported by a Grant-in-Aid for Scientific Research (B) (No. 20380151) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References Brighton, P.J., Szekeres, P.G., Willars, G.B., 2004. Neuromedin U and its receptors: structure, function, and physiological roles. Pharmacol. Rev. 56, 231–248. Guan, X.M., Yu, H., Jiang, Q., Van Der Ploeg, L.H., Liu, Q., 2001. Distribution of neuromedin U receptor subtype 2 mRNA in the rat brain. Brain Res. Gene Expr. Patterns 1, 1–4. Hanada, R., Nakazato, M., Murakami, N., Sakihara, S., Yoshimatsu, H., Toshinai, K., Hanada, T., Suda, T., Kangawa, K., Matsukura, S., Sakata, T., 2001. A role for neuromedin U in stress response. Biochem. Biophys. Res. Commun. 289, 225– 228. Hanada, T., Date, Y., Shimbara, T., Sakihara, S., Murakami, N., Hayashi, Y., Kanai, Y., Suda, T., Kangawa, K., Nakazato, M., 2003. Central actions of neuromedin U via corticotropin-releasing hormone. Biochem. Biophys. Res. Commun. 311, 954– 958. Hashizume, T., Haglof, S.A., Malven, P.V., 1994. Intracerebral methionineenkephalin, serum cortisol, and serum beta-endorphin during acute exposure of sheep to physical or isolation stress. J. Anim. Sci. 72, 700–708. Hosoya, M., Moriya, T., Kawamata, Y., Ohkubo, S., Fujii, R., Matsui, H., Shintani, Y., Fukusumi, S., Habata, Y., Hinuma, S., Onda, H., Nishimura, O., Fujino, M., 2000. Identification and functional characterization of a novel subtype of neuromedin U receptor. J. Biol. Chem. 275, 29528–29532. Howard, A.D., Wang, R., Pong, S.S., Mellin, T.N., Strack, A., Guan, X.M., Zeng, Z., Williams Jr., D.L., Feighner, S.D., Nunes, C.N., Murphy, B., Stair, J.N., Yu, H., Jiang, Q., Clements, M.K., Tan, C.P., McKee, K.K., Hreniuk, D.L., McDonald, T.P., Lynch, K.R., Evans, J.F., Austin, C.P., Caskey, C.T., Van der Ploeg, L.H., Liu, Q., 2000. Identification of receptors for neuromedin U and its role in feeding. Nature 406, 70–74. Ida, T., Mori, K., Miyazato, M., Egi, Y., Abe, S., Nakahara, K., Nishihara, M., Kangawa, K., Murakami, N., 2005. Neuromedin S is a novel anorexigenic hormone. Endocrinology 146, 4217–4223.
Jaszberenyi, M., Bagosi, Z., Thurzo, B., Foldesi, I., Telegdy, G., 2007. Endocrine and behavioral effects of neuromedin S. Horm. Behav. 52, 631–639. Kasuya, E., Kushibiki, S., Sutoh, M., Saito, T., Ito, S., Yayou, K., Sakumoto, R., Hodate, K., 2006. Effect of melatonin injected into the third ventricle on growth hormone secretion in Holstein steers. J. Vet. Med. Sci. 68, 1075–1080. Lipton, J.M., Glyn, J.R., 1980. Central administration of peptides alters thermoregulation in the rabbit. Peptides 1, 15–18. Mason, G.A., Caldwell, J.D., Stanley, D.A., Hatley, O.L., Prange Jr., A.J., Pedersen, C.A., 1986. Interactive effects of intracisternal oxytocin and other centrally active substances on colonic temperatures of mice. Regul. Pept. 14, 253– 260. Matsui, K., Sugano, S., 1989. Relation of intrinsic heart rate and autonomic nervous tone to resting heart rate in the young and the adult of various domestic animals. Nippon Juigaku Zasshi 51, 29–34. Minamino, N., Kangawa, K., Matsuo, H., 1985. Neuromedin U-8 and U-25: novel uterus stimulating and hypertensive peptides identified in porcine spinal cord. Biochem. Biophys. Res. Commun. 130, 1078–1085. Mori, K., Miyazato, M., Ida, T., Murakami, N., Serino, R., Ueta, Y., Kojima, M., Kangawa, K., 2005. Identification of neuromedin S and its possible role in the mammalian circadian oscillator system. EMBO J. 24, 325– 335. Mori, K., Miyazato, M., Kangawa, K., 2008. Neuromedin S: discovery and functions. Results Probl. Cell Differ. 46, 201–212. Nakahara, K., Hanada, R., Murakami, N., Teranishi, H., Ohgusu, H., Fukushima, N., Moriyama, M., Ida, T., Kangawa, K., Kojima, M., 2004a. The gut-brain peptide neuromedin U is involved in the mammalian circadian oscillator system. Biochem. Biophys. Res. Commun. 318, 156–161. Nakahara, K., Kojima, M., Hanada, R., Egi, Y., Ida, T., Miyazato, M., Kangawa, K., Murakami, N., 2004b. Neuromedin U is involved in nociceptive reflexes and adaptation to environmental stimuli in mice. Biochem. Biophys. Res. Commun. 323, 615–620. Nakazato, M., Hanada, R., Murakami, N., Date, Y., Mondal, M.S., Kojima, M., Yoshimatsu, H., Kangawa, K., Matsukura, S., 2000. Central effects of neuromedin U in the regulation of energy homeostasis. Biochem. Biophys. Res. Commun. 277, 191–194. Novak, C.M., Zhang, M., Levine, J.A., 2006. Neuromedin U in the paraventricular and arcuate hypothalamic nuclei increases non-exercise activity thermogenesis. J. Neuroendocrinol. 18, 594–601. Ozaki, Y., Onaka, T., Nakazato, M., Saito, J., Kanemoto, K., Matsumoto, T., Ueta, Y., 2002. Centrally administered neuromedin U activates neurosecretion and induction of c-fos messenger ribonucleic acid in the paraventricular and supraoptic nuclei of rat. Endocrinology 143, 4320–4329. Peier, A., Kosinski, J., Cox-York, K., Qian, Y., Desai, K., Feng, Y., Trivedi, P., Hastings, N., Marsh, DJ., 2009. The anti-obesity effects of centrally administered neuromedin U and neuromedin S are mediated predominantly by the neuromedin U receptor 2 (NMUR2). Endocrinology. doi:10.1210/en.2008-1772. Raddatz, R., Wilson, A.E., Artymyshyn, R., Bonini, J.A., Borowsky, B., Boteju, L.W., Zhou, S., Kouranova, E.V., Nagorny, R., Guevarra, M.S., Dai, M., Lerman, G.S., Vaysse, P.J., Branchek, T.A., Gerald, C., Forray, C., Adham, N., 2000. Identification and characterization of two neuromedin U receptors differentially expressed in peripheral tissues and the central nervous system. J. Biol. Chem. 275, 32452– 32459. Sakamoto, T., Mori, K., Nakahara, K., Miyazato, M., Kangawa, K., Sameshima, H., Murakami, N., 2007. Neuromedin S exerts an antidiuretic action in rats. Biochem. Biophys. Res. Commun. 361, 457–461. Sakamoto, T., Mori, K., Miyazato, M., Kangawa, K., Sameshima, H., Nakahara, K., Murakami, N., 2008. Involvement of neuromedin S in the oxytocin release response to suckling stimulus. Biochem. Biophys. Res. Commun. 375, 49–53. Sakumoto, R., Kasuya, E., Komatsu, T., Akita, T., 2003. Central and peripheral concentrations of tumor necrosis factor-alpha in Chinese Meishan pigs stimulated with lipopolysaccharide. J. Anim. Sci. 81, 1274–1280. Vigo, E., Roa, J., López, M., Castellano, JM., Fernandez-Fernandez, R., Navarro, VM., Pineda, R., Aguilar, E., Diéguez, C., Pinilla, L., Tena-Sempere, M., 2007. Neuromedin S as novel putative regulator of luteinizing hormone secretion. Endocrinology 148, 813–823. Wren, A.M., Small, C.J., Abbott, C.R., Jethwa, P.H., Kennedy, A.R., Murphy, K.G., Stanley, S.A., Zollner, A.N., Ghatei, M.A., Bloom, S.R., 2002. Hypothalamic actions of neuromedin U. Endocrinology 143, 4227–4234. Yayou, K., Annen, Y., Kuwahara, M., Tsubone, H., Sugano, S., 1993. Studies on the autonomic nervous function in Tsukuba emotional rats – with special reference to their cardiac function. J. Vet. Med. Sci. 55, 1017–1023. Yayou, K., Sato, Y., Ito, S., Nakamura, M., 2008. Comparison between the central effects of CRH and AVP in steers. Physiol. Behav. 93, 537–545. Yu, X.H., Cao, C.Q., Mennicken, F., Puma, C., Dray, A., O’Donnell, D., Ahmad, S., Perkins, M., 2003. Pro-nociceptive effects of neuromedin U in rat. Neuroscience 120 (2), 467–474.