AJP - Endo Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 293: E1265-E1273, 2007. First published August 28, 2007; doi:10.1152/ajpendo.00425.2007
0193-1849/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/5/E1265    most recent
00425.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vigo, E.
Right arrow Articles by Tena-Sempere, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vigo, E.
Right arrow Articles by Tena-Sempere, M.

Novel role of the anorexigenic peptide neuromedin U in the control of LH secretion and its regulation by gonadal hormones and photoperiod

Eva Vigo, Juan Roa, Rafael Pineda, Juan M. Castellano, Victor M. Navarro, Enrique Aguilar, Leonor Pinilla, and Manuel Tena-Sempere

Department of Cell Biology, Physiology and Immunology, Faculty of Medicine, University of Córdoba, Cordoba; and CIBER (CB06/03) Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, Madrid, Spain

Submitted 4 July 2007 ; accepted in final form 23 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuromedin U (NMU) is a widely spread neuropeptide, with predominant expression at the gastrointestinal tract and brain, putatively involved in the regulation of a diversity of biological functions, including food intake, energy balance and circadian rhythms; all closely related to reproduction. Yet, the implication of NMU in the control of the gonadotropic axis remains scarcely studied. We report herein analyses on the hypothalamic expression and function of NMU in different physiological and experimental states of the rat reproductive system. Expression of NMU mRNA at the hypothalamus was persistently detected along female postnatal development, with maximum levels in adulthood that fluctuated across the cycle and were modulated by ovarian steroids. Acute central administration of NMU evoked increases of serum LH levels in pubertal female rats, while repeated injection of NMU tended to advance vaginal opening. Likewise, central injection of NMU increased serum LH concentrations in cycling female rats, with peak responses in estrus. In contrast, NMU significantly inhibited preelevated LH secretion in gonadectomized and kisspeptin-treated rats. Finally, in noncycling females due to photoperiodic manipulation (constant light), hypothalamic NMU mRNA levels were markedly depressed, but relative LH responses to exogenous NMU were significantly augmented. All together, our present data support a predominant stimulatory role of NMU in the control of the female gonadotropic axis, which appears under the influence of developmental, hormonal, and photoperiodic cues, and might contribute to the joint regulation of energy balance, biological rhythms, and reproduction.

neuromedin S; luteinizing hormone; estrous cycle; estrogen; progesterone; puberty; photoperiod; suprachiasmatic nucleus; rat

NEUROMEDIN U (NMU) was originally identified in the porcine spinal cord (26), and later demonstrated as a highly conserved neuropeptide found in a diversity of species, including mammals, amphibians and birds (3). Distribution of NMU has been reported as relatively ubiquitous, with prominent expression in the gastrointestinal tract, pituitary and hypothalamus; the latter including the dorsomedial, arcuate (ARC) and suprachiasmatic (SCN) nuclei (2, 3, 15). The biological actions of NMU are conducted through interaction with two different G protein-coupled receptors: FM-3/GPR66, also known as NMU receptor type-1 (NMU1R), and FM4/TGR-1 or NMU receptor type 2 (NMU2R) (12, 18, 19, 23, 39, 40). Interestingly, NMU1R and NMU2R show rather distinct patterns of distribution: NMU1R is expressed in a variety of peripheral tissues, such as intestine, testis, pancreas, uterus, lung, and kidney, whereas NMU2R is selectively detected in discrete brain areas such as the paraventricular nucleus and ARC at the hypothalamus (12, 18, 19, 23, 39, 40). Very recently, a neuropeptide structurally related to NMU has been identified and named neuromedin S (NMS) (28). This neuropeptide displays complete sequence homology with the COOH-terminal seven-amino acid region of NMU and is able to bind both NMU1R and NMU2R (28); yet, NMU and NMS are encoded by different genes. Moreover, contrary to NMU, the pattern of tissue distribution of NMS is considerably restricted, with prominent expression at the SCN within the brain, the spleen and testis (28).

Consistent with its ubiquitous expression, NMU has been involved in the control of a wide variety of biological functions, including contraction of smooth muscle, gastrointestinal secretion and ion transport, blood pressure regulation, nociception, cancer formation and metastasis, immunomodulation, stress responses, pituitary hormone secretion, circadian rhythmicity, food intake, and energy homeostasis (3, 5, 13, 14, 16, 17, 19, 26, 27, 29, 30, 44). Although the peripheral actions of NMU have been the subject of intense investigation over the past two decades, its central effects initially have remained poorly characterized. Among the latter, the potential involvement of brain NMU in the physiological control of feeding behavior, body weight, and energy balance has recently received quite some attention, as 1) pharmacological administration of NMU reduced food intake and body weight and increased energy expenditure (19, 21, 31, 43); 2) immunoneutralization of central NMU evoked an increase in food consumption (22); 3) genetic inactivation of the NMU gene resulted in hyperphagia and obesity together with decreased energy expenditure (17); and 4) transgenic overexpression of NMU induced leanness and hypophagia (24). All together, the data above suggest a relevant role of NMU in the brain circuitries controlling food intake and energy balance, whose potential as target for obesity treatment remains to be explored (9). In addition, prominent expression of NMU at the SCN suggested a role of this neuropeptide in the central control of circadian oscillators, whose physiological relevance in the modulation of rhythmic phenomena (from locomotor activities to neurosecretion) is yet to be fully elucidated.

The biological actions of NMU in terms of body weight control and rhythmicity make this neuropeptide ideally posed for the physiological coupling of those functions and reproduction, which is known to be highly sensitive to numerous metabolic hormones with key roles in energy homeostasis (10) as well as to photoperiodic cues and endogenous rhythms (7, 8). On these premises, the ability of NMU to modulate gonadotropin (LH) secretion and puberty has been previously, albeit scarcely, explored in rodents (13, 36, 37). Yet, such initial analyses were conducted mostly in rather unphysiological settings, such as the ovariectomized (OVX) rat and the constitutive NMU-null mouse, where, despite it being a satiety signal putatively activated in conditions of energy abundance, a counterintuitive effect of NMU as inhibitor of LH secretion and puberty onset has been suggested (13, 36, 37). To gain further insight into the physiological roles of NMU as putative regulator of the gonadotropic axis, we report herein a comprehensive series of analyses on the hypothalamic expression of NMU gene and LH responses to central injection of the neuropeptide in different functional states of the reproductive axis in the female rat. Contrary to previous observations in extreme experimental conditions (13, 36, 37), our data are the first to disclose a predominant stimulatory role of NMU in the control of LH secretion, a phenomenon that is coupled to the dynamic regulation of hypothalamic NMU gene expression by number of "reproductive" variables, such as the stage of sexual maturation, the phase of the ovarian cycle, the input of sex steroids, and photic cues with clear-cut influences on the gonadotropic axis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and drugs. Wistar rats bred in the vivarium of the University of Córdoba were used. The day the litters were born was considered day 1 (1-d) of age. Unless otherwise stated, the animals were maintained under regular light-dark cycles (14 h of light, from 0700) and temperature (22°C) and were weaned at day 21 in groups of four rats per cage with free access to pelleted food and tap water. Experimental procedures were approved by the Córdoba University Ethics Committee for Animal Experimentation and were conducted in accordance with the European Union normatives for care and use of experimental animals. In all experiments, the animals were killed by decapitation at the end of the procedures, and in those studies involving mRNA analysis hypothalami were dissected out from the experimental animals as previously described (42). For experiments involving hormonal tests, rat NMU was obtained from Phoenix Pharmaceuticals (Belmont, CA). 17beta-Estradiol (E2) and progesterone (P) were purchased from Sigma Chemical (St. Louis, MO).

Experimental designs. To assess the putative roles of NMU in the control of the gonadotropic system, a combination of expression analyses and hormonal tests was conducted in female rats at different developmental and functional states of the reproductive axis.

In experiment 1, relative expression levels of NMU mRNA were assayed in hypothalamic samples from female rats 1, 5, 10, 15, 20, 30, and 60 days postpartum (n = 5–10 per group); corresponding to the neonatal (1-d and 5-d), infantile (10-d and 15-d), prepuberal (20-d), pubertal (30-d), and early adult (60-d) stages of postnatal development (33). On the latter, adult female rats were monitored for estrous cyclicity by daily vaginal cytology, and only rats with at least two consecutive, regular 4-day estrous cycles were used in expression analyses. In this experiment, hypothalamic tissue sampling was conducted at diestrus 1 (D1).

In experiment 2, pubertal (30-d) female rats (n = 10–12 per group) were subjected to a single intracerebroventricular (icv) injection of 1 nmol NMU (in 10 µl of physiological saline), and serial blood samples were taken via jugular venipuncture under light ether anesthesia 15, 30, and 60 min after injection. For icv administration, the cannulae were lowered to a depth of 3 mm beneath the surface of the skull, with an insert point 1 mm posterior and 1.2 mm lateral to bregma, as described in detail elsewhere (32). Further analyses of the effects of central administration of NMU on the gonadotropic axis at puberty were conducted in experiment 3, where repeated icv administration of NMU was carried out between 29 and 35 days post partum in female rats (n = 10), following previously reported protocols (32). The treatment regimen was set at 1 nmol NMU per animal in 10 µl every 12 h; icv cannulae were implanted on day 28, as described in experiment 2. Age-matched females (n = 10) injected with vehicle served as controls.

In experiment 4, the LH-releasing effects of NMU were studied in adult cycling female rats, selectively at D1, proestrus, or estrus. Groups of regularly cycling adult female rats (n = 10–12), selected as described for experiment 1, were used. Procedures for icv administration of NMU and serial blood sampling were as described for experiment 2, except for the fact that icv cannulae were lowered to a depth of 4 mm beneath the skull. Hormonal tests were conducted starting at 1000 of D1 and estrous phases of the cycle (when low basal LH levels are expected), as well as at 1800 of proestrus (coinciding with the preovulatory surge of gonadotropins). Blood samples were taken at 15 and 60 min after NMU injection.

In experiment 5, changes in NMU mRNA levels at the hypothalamus were analyzed along the estrous cycle in the rat. Assessment of estrous cyclicity was conducted as described for experiment 1. Groups of cycling females (n = 6), at estrus, D1, diestrus 2 (D2), and proestrus were used. For each phase, tissue sampling was conducted between 0900 and 1000. Additional groups of cycling females (n = 6) were sampled at 1800 and 2100 of proestrus and at 0200 of estrus.

In experiment 6, the expression levels of NMU mRNA were assayed in gonadectomized rats to evaluate the potential regulation of NMU gene expression at the hypothalamus by gonadal signals. Cycling female rats were bilaterally ovariectomized under ether anesthesia at random stages of the estrous cycle, and hypothalamic samples (n = 5 per group) were obtained 1 wk after surgery. Sham-operated females at 1000 proestrus served as controls. Hypothalamic samples (n = 5) were obtained upon decapitation of the animals 1 wk after OVX. Because data from experiment 6 suggested a role of ovarian signals in the modulation of hypothalamic expression of NMU mRNA, in experiment 7 groups of bilaterally OVX females (n = 6) were implanted at the time of surgery with Silastic brand silicon tubing (Dow Corning, Midland, MI) elastomers (20 mm length, inner diameter 0.062 cm, exterior diameter 0.125 cm) containing E2, P, or E2 plus P. Selection of dosage and capsule length was based on previous physiological studies in the OVX female rat (38, 42). A group of OVX rats (n = 6) implanted with empty capsules served as controls. Hypothalamic samples for RNA analyses were excised upon decapitation of the animals 1 wk after surgery/capsule implantation.

In experiment 8, the effects of NMU on (prestimulated) LH levels were explored in OVX rats. Young adult female rats (n = 10–12) were subjected to bilateral gonadectomy as described for experiment 6. Protocols for hormonal tests, involving icv injection of 1 nmol NMU and serial blood sampling at 15, 30, and 60 min, were as in experiment 4 and conducted 1 wk after gonadectomy. In addition, in experiment 9, the effects of icv administration of NMU were explored in female rats after pretreatment with an effective dose of kisspeptin-10 (32). To avoid the potential confounding factor of variations in LH responses to kisspeptin-10 across the cycle (38), pubertal female rats (i.e., before occurrence of first estrus) were used. Groups of female animals (n = 10–12) were sequentially injected icv with 1 nmol kisspeptin-10 (at –15 min) and 1 nmol NMU (at 0 min). Blood sampling was conducted thereafter as described in previous experiments, 15, 30, and 60 min after NMU administration. Vehicle-injected rats served as controls.

Finally, in experiment 10, the hypothalamic levels of NMU mRNAs and LH responses to the neuropeptide were assayed in adult female rats after photoperiodic manipulation. Female rats showing at least two consecutive regular 4-day estrous cycles were submitted to a regimen of constant light exposure, as previously described (4), starting with constant light at D1. The animals were maintained under constant illumination for 10 wk and checked for acyclicity (persistent estrus) by daily vaginal cytology. Hypothalamic tissues (n = 6) were obtained at the end of this period, after confirmation of absence of estrous cyclicity. Cycling female rats at D1 (n = 6), maintained under a regular light-dark regimen, were used as controls. In addition, the ability of NMS to modulate LH secretion was explored under such conditions of photoperiod manipulation. After 10 wk of constant light exposure, groups of females (n = 10–12) were implanted with icv cannulae as described in experiment 4, and hormonal testes were applied as described above: single icv injection of 1 nmol NMU and blood sampling at 15 and 60 min. Regularly cycling rats at D1, maintained under a conventional light-dark regimen, were tested in parallel for reference purposes.

For experiments involving central NMU administration and blood sampling, the animals were injected under conscious conditions after careful handling to avoid any stressful influence. Selection of the dose and time points for blood sampling was based on previous references, testing the neuroendocrine effects of NMU, NMS, and other centrally delivered neuropeptides (31, 32, 34, 36, 42, 43). In all experiments involving icv administration, the correct positioning of icv cannulae was routinely inspected before injection to exclude animals with obvious mislocation or deattachment and checked post mortem by visualization of their inner insert point (after decapitation), as described elsewhere (32).

RNA analysis by semiquantitative RT-PCR. Total RNA was isolated from hypothalamic samples using the single-step, acid guanidinium thiocyanate-phenol-chloroform extraction method. Hypothalamic expression of NMU mRNAs was assessed by RT-PCR, optimized for semiquantitative (semi-Q) detection, using the following primer pair: NMU-forward (5'-CGCCTCAAGATTACCACCAG-3') and NMU-reverse (5'-CTCCATCAGGACACGGCAAAG-3'). These sets of primers were generated on the basis of the published sequence of rat NMU gene (GenBank accession no. NM_022239; amplicon 137 bp), and designed to span intron sequences. As internal control for reverse transcription and reaction efficiency, amplification of a 290-bp fragment of L19 ribosomal protein mRNA was carried out in parallel in each sample by using the primer pair and conditions as reported elsewhere (42).

For amplification of the targets, RT and PCR were run in two separate steps. Thus, equal amounts of total RNA (2 µg) were used as a template for cDNA synthesis by RT with random hexamer priming (42). For semi-Q PCR, 3 µl of RT products was amplified in separate reactions using specific primers for rat NMU and L19. PCR reactions for NMU amplification consisted of a first denaturing cycle at 97°C for 5 min followed by a variable number of cycles of amplification, defined by denaturation at 96°C for 45 sec, annealing for 1 min, and extension at 72 C for 1 min. A final extension cycle of 72°C for 10 min was included. Annealing temperature was adjusted for each target and primer pair: 60 and 55°C for NMU and RP-L19 transcripts, respectively. Different numbers of cycles were tested to optimize amplification in the exponential phase of PCR. On this basis, 30 (NMU) and 25 (RP-L19) cycles were chosen for semi-Q analysis. Identity of PCR products was confirmed by direct sequencing (Central Sequencing Service, University of Córdoba, Spain). Quantification of intensity of RT-PCR signals was carried out by densitometric scanning using an image analysis system (1-D Manager; TDI, Madrid, Spain), and values of the specific target (NMU) were normalized to those of internal controls to express arbitrary units of relative expression. In all assays, liquid controls and reactions without RT resulted in negative amplification.

Hormone measurements. Serum LH levels were measured in a volume of 50 µl using a double-antibody method and radioimmunoassay kits supplied by the National Institutes of Health (Dr. A. F. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Peptide Program, Torrance, CA). Rat LH-I-9 was labeled with 125I using Iodo-gen tubes, following the instructions of the manufacturer (Pierce, Rockford, IL). Hormone concentrations were expressed using reference preparations LH-RP-3 as standard. Intra- and interassay coefficients of variation were below 8 and 10%, respectively. The sensitivity of the assay was 5 pg/tube. Accuracy of hormone determinations was confirmed by assessment of rat serum samples of known hormone concentrations used as external controls.

Presentation of data and statistics. Hormonal determinations were conducted in duplicate with a minimal total number of 10 samples per group. When appropriate, in addition to individual time point measurements, integrated LH-secretory responses are presented as the area under the curve (AUC), calculated following the trapezoidal rule, over the 60-min period following administration of NMU. Semi-Q RT-PCR analyses were carried out in duplicate from at least four to five independent RNA samples of each experimental group. Semi-Q RNA and hormonal data are presented as means ± SE. Results were analyzed for statistically significant differences by using Student's t-test or ANOVA followed by the Student-Newman-Keuls multiple range test (SigmaStat 2.0; Jandel, San Rafael, CA). P ≤ 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To gain further knowledge on the putative roles of NMU in the control of the gonadotropic axis, a series of analyses on the hypothalamic expression of NMU gene and LH responses to central injection of the neuropeptide was conducted in female rats at different functional states of the reproductive axis. As a first approach, in experiment 1, expression of NMU gene was assayed in whole hypothalamic fragments from female rats throughout postnatal maturation. These analyses revealed the persistent expression of NMU mRNA at the hypothalamus of the female rat, from the neonatal period to adulthood, with low relative levels of NMU mRNA during the neonatal-infantile period (≤10 days postpartum) that progressively increased thereafter and attained peak expression levels in pubertal (30-d) and adult (60-d) cycling animals (Fig. 1).


Figure 1
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 1. Semiquantitative data on relative expression levels of neuromedin U (NMU) mRNA in the hypothalamus of female rats at different stages of postnatal development. For each experimental group, values are means ± SE of ≥4–6 independent determinations of the specific signal (NMU) normalized to that of internal control (RP-L19). Groups with different superscript letters are statistically different (P < 0.05, ANOVA followed by Student-Newman-Keuls multiple range test).

 
The prominent hypothalamic expression of NMU gene at puberty and adulthood prompted us to explore the potential influence of the neuropeptide on gonadotropin (LH) secretion at these stages of sexual maturation. Hormonal tests assessing LH responses to central administration of an effective dose of NMU were initially conducted in pubertal (30-d) female rats (experiment 2), where acute icv injection of 1 nmol NMU (single bolus) induced a significant increase (~2-fold) in serum LH levels at 15 min; a stimulatory effect that was maintained 30 min after injection, whereas at 60 min, LH concentrations had returned to levels of vehicle-injected rats (Fig. 2A). Accordingly, the integrated LH secretion in response to NMU (over the 60-min period after icv injection) was ~60% higher than in controls (Fig. 2A, inset). In addition, in experiment 3, repeated icv administration of 1 nmol NMU to immature female rats (twice daily every 12 h, between 29-d and 35-d postpartum) tended to advance the mean age of vaginal opening (VO), as an external index of puberty onset, without inducing major changes in body weight gain along the study period (Fig. 2B). Yet, the above difference in the timing of VO did not reach statistical significance. Likewise, no significant differences were detected in terms of ovarian and uterine weights, as well as serum estradiol levels, between vehicle- and NMU-injected rats at the end of the chronic treatments (data not shown).


Figure 2
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 2. Effects of acute and repeated administration of NMU on the gonadotropic axis at puberty in the female rat. A: pubertal female rats were acutely injected icv with 1 nmol NMU or vehicle (denoted by arrow), and blood samples for LH determination were obtained before (0 min) and 15, 30, and 60 min after injection. In addition to time course profiles, integrated secretory responses to central administration of NMU (calculated as AUC over the 60-min study period) are shown in the inset. Hormonal values are means ± SE of 10–12 independent determinations per group. **P < 0.01 vs. corresponding control values (ANOVA followed by Student-Newman-Keuls multiple range test or Student's t-test for AUC data). B: pubertal female rats were repeatedly injected icv with 1 nmol NMU or vehicle (twice daily every 12 h from days 29 to 35 post partum), and body weights (BW) and cumulative percentage of vaginal opening (VO) were recorded in both groups. Mean ± SE data of age at vaginal opening are also provided.

 
As was the case in pubertal rats, hormonal tests in adult cycling female rats revealed that central administration of NMU was able to induce stimulatory LH responses (experiment 4). Of note, however, although significant stimulation of LH secretion in response to NMU was detected predominantly in cycling female rats, the magnitude of such responses appeared clearly dependent on the stage of the cycle. Thus, in females at diestrus (1000), 1 nmol NMU elicited a modest, but significant, twofold elevation of serum LH levels at 15 min after injection (which did not persist at 60 min), whereas at estrus a similar dose of NMU evoked very potent LH-secretory responses, with ~4.0- and 3.0-fold increases over corresponding control levels 15 and 60 min after injection, respectively. In contrast, icv injection of 1 nmol NMU at 1800 proestrus (i.e., coincident with the preovulatory surge of gonadotropins) failed to significantly modify serum LH levels 15 and 60 min after administration (Fig. 3).


Figure 3
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 3. Effects of NMU on LH secretion in adult female rats at different stages of the estrous cycle. Three representative stages of the cycle were tested: morning of diestrus-1 (D1 at 1000), afternoon of proestrus (P at 1800), and morning of estrus (E at 1000). Experimental animals were injected icv with a single dose of 1 nmol NMS or vehicle, and blood samples for LH determination were obtained 15 and 60 min after injection. Hormonal values are means ± SE of 10–12 independent determinations per group. **P < 0.01 vs. corresponding values in vehicle-injected controls (ANOVA followed by Student-Newman-Keuls multiple range test). Note that scales of y-axes in the different panels are different.

 
The above-mentioned changes in LH responsiveness to central administration of NMU moved us to explore potential changes in its hypothalamic gene expression across the estrous cycle (experiment 5). Analyses conducted in cycling female rats revealed that hypothalamic NMU mRNA levels fluctuate in a cycle-dependent manner, with a progressive decline being observed between the morning and evening of proestrus (1000, 1800, and 2100) and the early morning of estrus (0200), when the lowest levels of NMU mRNA levels were detected, the latter being coincident with the time of ovulation. Thereafter, NMU mRNA levels increased and reached peak expression values in the morning of estrus (1000), and remained elevated at the D1 and D2 phases of the cycle (Fig. 4). This cyclic profile of expression was suggestive of its potential regulation by ovarian factors along the cycle. In line with this hypothesis, results from experiments 6 and 7 demonstrated that hypothalamic expression of NMU mRNA is significantly decreased in OVX rats (Fig. 5A), whereas replacement with effective doses of E2 and/or P potently enhanced (3- to 4-fold increase) NMU mRNA levels in OVX animals (Fig. 5B).


Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 4. Expression levels of NMU mRNA in the hypothalamus of adult female rats at different stages of the estrous cycle. For each phase, semiquantitative values are means ± SE of ≥4–6 independent determinations of specific signal (NMU) normalized to that of internal control (RP-L19). Samples from female rats at 1000, 1800, and 2100 of proestrus (P), at 0200 and 1000 of estrus (E), and at 1000 of D1 and D2 were assayed .In addition to semiquantitative data, representative RT-PCR assays of NMU and RP-L19 targets at the different stages of the cycle are also presented. Groups with different superscript letters are statistically different (P < 0.05, ANOVA followed by Student-Newman-Keuls multiple range test).

 

Figure 5
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 5. Expression of levels of NMU mRNA in the hypothalamus of female rats 1 wk after bilateral ovariectomy (OVX) with or without hormonal replacement. A: representative RT-PCR assays and semiquantitative data are presented of hypothalamic NMU mRNA expression in cycling females at proestrus (P at 1000; controls) and OVX rats. B: in addition, effects of replacement with estradiol (E2), progesterone (P) or E2 + P on expression levels of NMU mRNA (representative RT-PCRs and semiquantitative data) are shown. Semiquantitative values of NMU mRNA levels are means ± SE of ≥4–6 independent determinations. For each figure, groups with different superscript letters are statistically different (P < 0.05, A: Student's t-test; B: ANOVA followed by Student-Newman-Keuls multiple range test).

 
Hormonal tests were also conducted in OVX females to assess the influence of gonadal factors and prevailing gonadotropin levels on the pattern of LH responses to NMU. In contrast to intact, cycling animals, central injection of 1 nmol NMU to OVX rats induced a sustained reduction of (preelevated) serum LH levels, which was detected 15, 30, and 60 min after icv administration (experiment 8). Accordingly, the integrated LH secretion during the 1-h period following NMU injection to OVX rats was ~40% lower than in gonadectomized controls injected with vehicle (Fig. 6). Similarly, in experiment 9, LH responses to NMU were monitored in pubertal female rats after preinjection of the potent elicitor of gonadotropin secretion kisspeptin-10 (32). Again, significant inhibitory responses to NMU were detected in female rats with elevated LH levels due to pretreatment with an effective dose of kisspeptin-10, an effect that reached statistical significance at 30 min and resulted in a mean 30% reduction in the integrated LH secretion elicited by central administration of kisspeptin (Fig. 6B).


Figure 6
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 6. Effects of NMU on LH secretion in conditions of preelevated gonadotropin levels. A: LH responses to central injection of NMU were tested in female rats 1 wk after bilateral OVX. B: likewise, LH secretion in response to icv injection of NMU is shown in female rats pretreated (at –15 min, icv) with an effective dose of kisspeptin-10 (1 nmol). In both settings, animals were injected icv with 1 nmol NMU or vehicle (denoted by arrow), and blood samples for LH determination were obtained 15, 30, and 60 min after administration. In addition to time course profiles, integrated secretory responses to central administration of NMU (calculated as AUC over the 60-min study period) are shown in insets. Hormonal values are means ± SE of 10–12 independent determinations per group. *P < 0.05, **P < 0.01 vs. corresponding control values (ANOVA followed by Student-Newman-Keuls multiple range test or Student's t-test for AUC data).

 
Finally, in addition to developmental, estrous cycle, and gonadal influences, regulation of hypothalamic expression of NMU by photoperiodic cues and their impact on LH responses to this neuropeptide were explored in experiment 10 by use of a model of constant light exposure. Cycling female rats subjected to a regimen of constant illumination for 10 wk displayed acyclicity in vaginal smears (persistent estrus) and significantly decreased LH levels (0.89 ± 0.09 vs. 2.81 ± 0.17 ng/ml in reference D1 controls housed under standard light-dark cycle conditions). In this model, relative levels of NMU mRNA at the hypothalamus were significantly lower than in cycling diestrous females (Fig. 7A). Nevertheless, such a decrease in basal LH levels and hypothalamic expression of NMU was associated with sustained LH responses to icv administration of NMU that peaked 15 min after administration, with absolute values similar to those obtained in control females. In fact, due to the prevailing reduction of serum LH levels, such responses (at 15 min after NMU injection) accounted for a more than fivefold increase over corresponding control values, whereas a modest, less than twofold elevation in LH concentrations was detected in cycling female rats at diestrus (Fig. 7B).


Figure 7
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 7. Expression of NMU mRNA in the hypothalamus and LH responses to the neuropeptide in female rats after photoperiod manipulation by means of 10-wk constant light exposure (light-light regimen, L/L). A: representative RT-PCR assays and semiquantitative data are presented of hypothalamic NMU mRNA levels in control female rats at D1 (standard light-dark regimen, L/D) vs. L/L females. Semiquantitative data of NMU mRNA are means ± SE of ≥4–6 independent determinations. Groups with different superscript letters are statistically different (P < 0.05, Student's t-test). B: effects of NMU on LH secretion in L/L female rats. Cycling female controls (L/D) at D1 were used as reference controls. Experimental animals were injected icv with a single dose of 1 nmol NMU or vehicle (denoted by arrow), and blood samples for LH determination were obtained before (0 min) and 15 and 60 min after administration. Hormonal values are means ± SE of 10–12 independent determinations per group. **P < 0.01 vs. corresponding control values; aP < 0.01 vs. corresponding values in L/D groups (ANOVA followed by Student-Newman-Keuls multiple range test).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although NMU was identified as a brain-gut peptide more than two decades ago (26), its physiological relevance as a neuroendocrine signal controlling food intake and energy homeostasis has been recognized only recently (17, 19, 22, 43). Similarly, the potential involvement of NMU, and the related peptide NMS, in the regulation of circadian rhythms has emerged lately (14, 28, 30). This biological profile led us to hypothesize that NMU might also participate in the central control of the gonadotropic axis, thus serving as an integrator for the joint control of energy balance, endogenous rhythms, and reproductive function. Our current data support such a reproductive "dimension" of the NMU system, as 1) expression of NMU gene at the hypothalamus was influenced by the stage of postnatal sexual development and under the control of gonadal hormones and photoperiodic cues; and 2) central administration of NMU resulted in clear-cut changes in LH secretion, with predominant stimulatory responses at different functional states of the gonadotropic axis that were modulated by gonadal and environmental (photic) influences. These observations complement our recent findings on the gonadal modulation of NMU2R and NMS gene expression at the hypothalamus, as well as on the effects of NMS on LH secretion, in the female rat (42), thus suggesting that the NMU-NMS-NMU2R system does play a role in the dynamic control of the gonadotriopic axis and, putatively, in its functional coupling with the systems controlling energy homeostasis and circadian rhythms.

Admittedly, the ability of NMU to influence LH secretion had been previously, albeit scarcely, evaluated. Of note, however, most of those studies had been conducted in rather unphysiological settings, such as the OVX female rat (36, 37). Although the validity of such a model for the analysis of factors affecting specific aspects of pulsatile LH release is not questioned, it is also obvious that it fails to consider the potential impact of elimination of sex steroids and elevation of prevailing LH levels on the observed hormonal responses. In fact, by using OVX rats, inhibitory effects of NMU on LH secretion were initially observed, a finding similar to that reported herein for gonadectomized female rats. Similarly, our data demonstrate that central administration of NMU is able to moderately, but significantly, attenuate LH-secretory responses to the potent secretagogue kisspeptin-10. Nevertheless, in a wide diversity of physiological settings (from pubertal females to adult cycling rats), NMU was proved to significantly stimulate LH secretion, observations that are in keeping with the expected stimulatory/permissive effects on the gonadotropic axis of satiety signals such as leptin (6, 10). All together, the data above strongly suggest a dual mode of action of NMU on the centers governing gonadotropin secretion, with predominant stimulatory LH responses but detectable inhibitory effects being selectively observed in conditions of elevated prevailing LH levels, such as gonadectomy or kisspeptin pretreatment. The neuroendocrine substrate and potential physiological relevance of this bimodal mode of action of NMU on LH secretion merit further investigation.

Very recently, the putative role of NMU as a negative modulator of the gonadotropic axis has been further supported by the observation that NMU-null mice showed advanced VO, as an external index of puberty onset, whereas a challenge to rat pituitary tissue with NMU resulted in a modest, but significant, inhibition of LH release ex vivo (13). Worthy of note, however, NMU knockout mice also show early-onset obesity (17), which might contribute to the modest precocity in puberty onset observed in this model (13). Moreover, the constitutive lack of NMU during development might have activated compensatory mechanisms, such as NMS overexpression, NMS being a stimulatory signal for LH secretion (42). Indeed, in our present study, repeated intracerebral administration of NMU to immature female rats did not result in a detectable delay, but rather in a trend toward advancement, in the age of VO, suggesting that NMU is not a physiological inhibitor of puberty onset. Curiously enough, despite its reported anorexigenic effects in adult rodents (22, 31, 43), at doses similar to those of present study chronic central injections of NMU did not evoke significant changes in body weight gain across puberty, which is suggestive of a lower sensitivity to the putative anorectic effects of NMU at this stage of postnatal development. On the other hand, the observation that, at very high concentrations (10–5 M), NMU can evoke a moderate decrease in LH secretion directly at the pituitary level (13) is not at odds with our present results on the predominant stimulatory effect of centrally administered NMU on LH secretion in vivo, a dichotomy between central and pituitary effects that has been previously reported for other regulators of LH release, such as ghrelin (11).

Our studies in the female rat have demonstrated that hypothalamic expression of NMU gene is sensitive not only to developmental influences but also to ovarian (sex steroid) signals. Indeed, ovariectomy resulted in a significant suppression of NMU mRNA levels at the hypothalamus, which was prevented by E2 and P treatments. The positive effects of estrogen and progestagen on hypothalamic expression of NMU might explain the observed increase in NMU mRNA from puberty onward in the female. Likewise, fluctuations in NMU mRNA levels observed in adult female rats across the estrous cycle might, at least partially, derive from changes in circulating levels of E2 (i.e., cessation of the proestrous surge of estrogen may contribute to the decline of NMU mRNA levels during the proestrus-to-estrus transition) and P (i.e., high levels of P at diestrus may be causative for enhanced NMU expression at this stage). Admittedly, however, some of the observed changes in hypothalamic NMU levels along the ovarian cycle (e.g., their rise between 0200 and 1000 estrus) cannot be explained solely by variations in serum sex steroid concentrations, which suggest additional regulatory cues, whose nature is yet to be elucidated. Nevertheless, considering the reported stimulatory effects of NMU on LH secretion in the cycling female rat (see Fig. 3), it is tempting to propose that the significant drop in hypothalamic NMU levels from proestrus to estrus, with elevated expression preceding and during the LH surge (proestrus 1000 and 1800) and a decline thereafter (proestrus 2100 and estrus 0200), might be mechanistically relevant, as 1) the lack of further stimulation of LH secretion of at the time of the surge (proestrus 1800) might be indicative of the activation of the endogenous NMU system at this period, and 2) the decrease in NMU expression at early estrus (0200) might contribute to the cessation of the surge (see Fig. 3: potent stimulatory effect of NMU on LH secretion at estrus). This hypothesis is based on the demonstration of a predominant stimulatory effect of NMU on LH secretion across the ovarian cycle. Yet, given its ability to induce inhibitory LH responses at certain experimental conditions, further analyses on the putative neuroendocrine pathways for the central regulation of the GnRH-LH axis by NMU in the cycling female, and on the effects of this neuropeptide in experimental models of steroid-induced LH secretion, are needed to fully support this hypothesis.

As further evidence for the potential involvement of NMU in the control of the female gonadotropic system, disruption of estrous cyclicity by means of photoperiodic manipulation (constant light exposure) resulted in a concomitant decrease in NMU gene expression at the hypothalamus and circulating levels of LH. Yet, relative stimulatory responses to NMU (in terms of LH secretion) were not only preserved, but rather enhanced in noncycling females under constant illumination. Of note, an increase in hypothalamic NMU2R gene expression has been detected by our group in this model (unpublished data), which is likely derived from a primary decrease in the endogenous tone of NMU and might provide the potential basis for the reported state of hyperresponsiveness to the exogenous neuropeptide. Taken together, these observations are strongly suggestive of a causative association between the suppression of NMU gene expression at the hypothalamus and the disruption of estrous cyclicity and basal gonadotropin secretion following photoperiodic manipulation. Indeed, considering the reported changes in NMU expression at the SCN according to the light cycle in the mouse (14), and its effect on LH secretion in the female rat described herein, it is tempting to hypothesize that NMU might operate as physiological neuropeptide effector for the SCN pathways projecting to the centers governing the GnRH-gonadotropic axis. Interestingly, NMU mRNA has been detected in neurons of the SCN expressing arginine vasopressin (AVP), with a coincident rhythm of expression (14). Of note, AVP has been proposed as the primary efferent signal from the SCN involved in the generation of the LH surge by projecting to the hypothalamic-anteroventral-periventricular nucleus (AVPV) (1, 35). It is therefore possible, although yet to be proved, that NMU might contribute in such a pathway (AVP/NMU neurons from SCN) to the cyclic regulation of LH secretion in the female.

Nevertheless, given the effects of sex steroids on hypothalamic NMU expression described herein, the possibility that changes in ovarian hormone secretion after constant illumination might partially contribute to the observed alterations in NMU mRNA levels cannot be completely ruled out on the basis of our present data. Yet, several reasons make it extremely unlikely that the reported changes in NMU signaling following photoperiod manipulation might derive solely from alterations in the circulating levels of sex steroids. First, although the observed decrease in hypothalamic NMU mRNA levels after constant illumination might be compatible with a complete suppression of circulating estrogen (see Fig. 5 data from OVX rats), previously published data evidenced that serum E2 levels are not depressed but rather enhanced or temporally shifted in female rats exposed to constant light (41). In fact, in our experiments, uterine weights in females under constant illumination were not significantly lower than those of control cycling females at diestrus (data not shown), an observation that argues against the possibility that the dramatic suppression of NMU mRNA levels seen after photoperiodic manipulation is derived merely from suppression of E2 levels. Second, from a functional standpoint, our hormone tests demonstrate that LH responses to NMU in females under constant illumination are not similar to (but rather opposite) those observed in OVX rats, as augmented stimulatory responses were observed in the former, whereas in gonadectomized females NMU inhibited LH secretion after its central administration. Overall, on the basis of our current data, it is highly plausible that changes in photoperiodic cues have a primary impact on hypothalamic NMU signaling, which might contribute to the central dysregulation of GnRH secretion caused by constant illumination in the female rat (25). Nonetheless, the relative importance of additional peripheral events, such as changes in ovarian sex steroid secretion, in this phenomenon merits further investigation.

Very recent studies from our group have disclosed that, similarly to NMU, hypothalamic expression of NMS mRNA appears regulated by developmental cues and gonadal signals and that NMS is capable of modulating LH secretion after its central administration in the female rat (42); yet, some (minor) divergences in terms of developmental expression and gonadal modulation were also noticed. These commonalities in the patterns of expression and functional LH responses to both neuromedins might reflect some degree of redundancy between NMU and NMS, which was previously demonstrated for some of their central actions (food intake, circadian rhythms) (14, 20, 28, 31). Admittedly, the fact that both NMU and NMS act centrally via the same receptor (NMU2R) makes it difficult to dissect out the relative importance of these two neuropeptides on the basis of pharmacological tests. However, the fact that NMU gene expression at the hypothalamus was overtly regulated by such reproductive cues as the stage of sexual maturation and estrous cycle, ovarian steroids, and photoperiod strongly suggests a genuine role of central NMU pathways in the control of the gonadotropic axis in the rat.

In sum, our present data provide evidence that hypothalamic expression of the gene encoding NMU, a brain-gut peptide previously known to be involved in the control of food intake and circadian rhythms, is under the regulation of reproductive signals and that NMU is likely to operate as central modulator (mostly as elicitor) of LH secretion in different functional states of rat reproductive axis. The potential contribution and physiological relevance of this neuropeptide, and the structurally related NMS, in the integral control of energy balance, endogenous rhythmicity, and reproductive function merit further investigation.


   ACKNOWLEDGMENTS
 
This work was supported by Grant BFU 2005-07446 (Ministerio de Educacion y Ciencia, Spain), funds from Instituto de Salud Carlos III (Red de Centros RCMN C03/08 and Project PI042082, Ministerio de Sanidad, Spain), and European Union Research Contract EDEN QLK4-CT-2002-00603.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Tena-Sempere, Physiology Section, Dept. of Cell Biology, Physiology and Immunology. Faculty of Medicine, Univ. of Córdoba, Avda. Menéndez Pidal s/n, 14004 Cordoba, Spain (e-mail: fi1tesem{at}uco.es)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Abrahamson EE, Moore RY. Suprachiasmatic nucleus in the mouse: retinal innervation, intrinsic organization and efferent projections. Brain Res 916: 172–191, 2001.[CrossRef][Web of Science][Medline]
  2. Augood SJ, Keast JR, Emson PC. Distribution and characterisation of neuromedin U-like immunoreactivity in rat brain and intestine and in guinea pig intestine. Regul Pept 20: 281–292, 1988.[CrossRef][Web of Science][Medline]
  3. Brighton PJ, Szekeres PG, Willars GB. Neuromedin U and its receptors: structure, function, and physiological roles. Pharmacol Rev 56: 231–248, 2004.[Abstract/Free Full Text]
  4. Campbell C, Schwartz N. The impact of constant light on the estrous cycle of the rat. Endocrinology 106: 1230–1238, 1980.[Abstract/Free Full Text]
  5. Cao CQ, Yu XH, Dray A, Filosa A, Perkins MN. A pro-nociceptive role of neuromedin U in adult mice. Pain 104: 609–616, 2003.[CrossRef][Web of Science][Medline]
  6. Casanueva FF, Dieguez C. Neuroendocrine regulation and actions of leptin. Front Neuroendocrinol 20: 317–363, 1999.[CrossRef][Web of Science][Medline]
  7. Chappell P. Clocks and the black box: circadian influences on gonadotropin-releasing hormone secretion. J Neuroendocrinol 17: 119–130, 2005.[CrossRef][Web of Science][Medline]
  8. de la Iglesia H, Schwartz W. Timely ovulation: circadian regulation of the female hypothalamo-pituitary-gonadal axis. Endocrinology 147: 1148–1153, 2006.[Abstract/Free Full Text]
  9. Doggrell SA. Neuromedin U—a new target in obesity. Expert Opin Ther Targets 9: 875–877, 2005.[CrossRef][Web of Science][Medline]
  10. Fernandez-Fernandez R, Martini AC, Navarro VM, Castellano JM, Dieguez C, Aguilar E, Pinilla L, Tena-Sempere M. Novel signals for the integration of energy balance and reproduction. Mol Cell Endocrinol 254–255: 127–132, 2006.[CrossRef][Web of Science][Medline]
  11. Fernandez-Fernandez R, Tena-Sempere M, Navarro VM, Barreiro ML, Castellano JM, Aguilar E, Pinilla L. Effects of ghrelin upon gonadotropin-releasing hormone and gonadotropin secretion in adult female rats: in vivo and in vitro studies. Neuroendocrinology 82: 245–255, 2005.[CrossRef][Web of Science][Medline]
  12. Fujii R, Hosoya M, Fukusumi S, Kawamata Y, Habata Y, Hinuma S, Onda H, Nishimura O, Fujino M. Identification of neuromedin U as the cognate ligand of the orphan G protein-coupled receptor FM-3. J Biol Chem 275: 21068–21074, 2000.[Abstract/Free Full Text]
  13. Fukue Y, Sato T, Teranishi H, Hanada R, Takahashi T, Nakashima Y, Kojima M. Regulation of gonadotropin secretion and puberty onset by neuromedin U. FEBS Lett 580: 3485–3488, 2006.[CrossRef][Web of Science][Medline]
  14. Graham ES, Littlewood P, Turnbull Y, Mercer JG, Morgan PJ, Barrett P. Neuromedin-U is regulated by the circadian clock in the SCN of the mouse. Eur J Neurosci 21: 814–819, 2005.[CrossRef][Web of Science][Medline]
  15. Graham ES, Turnbull Y, Fotheringham P, Nilaweera K, Mercer JG, Morgan PJ, Barrett P. Neuromedin U and neuromedin U receptor-2 expression in the mouse and rat hypothalamus: effects of nutritional status. J Neurochem 87: 1165–1173, 2003.[CrossRef][Web of Science][Medline]
  16. Hanada R, Nakazato M, Murakami N, Sakihara S, Yoshimatsu H, Toshinai K, Hanada T, Suda T, Kangawa K, Matsukura S, Sakata T. A role for neuromedin U in stress response. Biochem Biophys Res Commun 289: 225–228, 2001.[CrossRef][Web of Science][Medline]
  17. Hanada R, Teranishi H, Pearson JT, Kurokawa M, Hosoda H, Fukushima N, Fukue Y, Serino R, Fujihara H, Ueta Y, Ikawa M, Okabe M, Murakami N, Shirai M, Yoshimatsu H, Kangawa K, Kojima M. Neuromedin U has a novel anorexigenic effect independent of the leptin signaling pathway. Nat Med 10: 1067–1073, 2004.[CrossRef][Web of Science][Medline]
  18. 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. Identification and functional characterization of a novel subtype of neuromedin U receptor. J Biol Chem 275: 29528–29532, 2000.[Abstract/Free Full Text]
  19. Howard AD, Wang R, Pong SS, Mellin TN, Strack A, Guan XM, Zeng Z, Williams DL Jr, Feighner SD, Nunes CN, Murphy B, Stair JN, Yu H, Jiang Q, Clements MK, Tan CP, McKee KK, Hreniuk DL, McDonald TP, Lynch KR, Evans JF, Austin CP, Caskey CT, Van der Ploeg LH, and Liu Q. Identification of receptors for neuromedin U and its role in feeding. Nature 406: 70–74, 2000.[CrossRef][Medline]
  20. Ida T, Mori K, Miyazato M, Egi Y, Abe S, Nakahara K, Nishihara M, Kangawa K, Murakami N. Neuromedin S is a novel anorexigenic hormone. Endocrinology 146: 4217–4223, 2005.[CrossRef][Web of Science][Medline]
  21. Ivanov TR, Lawrence CB, Stanley PJ, Luckman SM. Evaluation of neuromedin U actions in energy homeostasis and pituitary function. Endocrinology 143: 3813–3821, 2002.[Abstract/Free Full Text]
  22. Jethwa PH, Small CJ, Smith KL, Seth A, Darch SJ, Abbott CR, Murphy KG, Todd JF, Ghatei MA, Bloom SR. Neuromedin U has a physiological role in the regulation of food intake and partially mediates the effects of leptin. Am J Physiol Endocrinol Metab 289: E301–E305, 2005.[Abstract/Free Full Text]
  23. Kojima M, Haruno R, Nakazato M, Date Y, Murakami N, Hanada R, Matsuo H, Kangawa K. Purification and identification of neuromedin U as an endogenous ligand for an orphan receptor GPR66 (FM3). Biochem Biophys Res Commun 276: 435–438, 2000.[CrossRef][Web of Science][Medline]
  24. Kowalski TJ, Spar BD, Markowitz L, Maguire M, Golovko A, Yang S, Farley C, Cook JA, Tetzloff G, Hoos L, Del Vecchio RA, Kazdoba TM, McCool MF, Hwa JJ, Hyde LA, Davis H, Vassileva G, Hedrick JA, Gustafson EL. Transgenic overexpression of neuromedin U promotes leanness and hypophagia in mice. J Endocrinol 185: 151–164, 2005.[Abstract/Free Full Text]
  25. Leranth C, Palkovits M, MacLusky NJ, Shanabrough M, Naftolin F. Reproductive failure due to experimentally induced constant estrus does not alter the LH-RH fiber density in the median eminence of the rat. Neuroendocrinology 43: 526–532, 1986.[Web of Science][Medline]
  26. Minamino N, Sudoh T, Kangawa K, Matsuo H. Neuromedins: novel smooth-muscle stimulating peptides identified in porcine spinal cord. Peptides 6, Suppl 3: 245–248, 1985.
  27. Mondal MS, Date Y, Murakami N, Toshinai K, Shimbara T, Kangawa K, Nakazato M. Neuromedin U acts in the central nervous system to inhibit gastric acid secretion via CRH system. Am J Physiol Gastrointest Liver Physiol 284: G963–G969, 2003.[Abstract/Free Full Text]
  28. Mori K, Miyazato M, Ida T, Murakami N, Serino R, Ueta Y, Kojima M, Kangawa K. Identification of neuromedin S and its possible role in the mammalian circadian oscillator system. EMBO J 24: 325–335, 2005.[CrossRef][Web of Science][Medline]
  29. Moriyama M, Matsukawa A, Kudoh S, Takahashi T, Sato T, Kano T, Yoshimura A, Kojima M. The neuropeptide neuromedin U promotes IL-6 production from macrophages and endotoxin shock. Biochem Biophys Res Commun 341: 1149–1154, 2006.[CrossRef][Web of Science][Medline]
  30. Nakahara K, Hanada R, Murakami N, Teranishi H, Ohgusu H, Fukushima N, Moriyama M, Ida T, Kangawa K, Kojima M. The gut-brain peptide neuromedin U is involved in the mammalian circadian oscillator system. Biochem Biophys Res Commun 318: 156–161, 2004.[CrossRef][Web of Science][Medline]
  31. Nakazato M, Hanada R, Murakami N, Date Y, Mondal MS, Kojima M, Yoshimatsu H, Kangawa K, Matsukura S. Central effects of neuromedin U in the regulation of energy homeostasis. Biochem Biophys Res Commun 277: 191–194, 2000.[CrossRef][Web of Science][Medline]
  32. Navarro VM, Fernandez-Fernandez R, Castellano JM, Roa J, Mayen A, Barreiro ML, Gaytan F, Aguilar E, Pinilla L, Dieguez C, Tena-Sempere M. Advanced vaginal opening and precocious activation of the reproductive axis by KiSS-1 peptide, the endogenous ligand of GPR54. J Physiol 561: 379–386, 2004.[Abstract/Free Full Text]
  33. Ojeda SR, Skinner MK. Puberty in the rat. In: The Physiology of Reproduction, edited by Neill JD. San Diego, CA: Academic-Elsevier, 2005, p. 2061–2126.
  34. Ozaki Y, Onaka T, Nakazato M, Saito J, Kanemoto K, Matsumoto T, Ueta Y. 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, 2002.[Abstract/Free Full Text]
  35. Palm IF, Van Der Beek EM, Wiegant VM, Buijs RM, Kalsbeek A. Vasopressin induces a luteinizing hormone surge in ovariectomized, estradiol-treated rats with lesions of the suprachiasmatic nucleus. Neuroscience 93: 659–666, 1999.[CrossRef][Web of Science][Medline]
  36. Quan H, Funabashi T, Furuta M, Kimura F. Effects of neuromedin U on the pulsatile LH secretion in ovariectomized rats in association with feeding conditions. Biochem Biophys Res Commun 311: 721–727, 2003.[CrossRef][Web of Science][Medline]
  37. Quan H, Funabashi T, Kimura F. Intracerebroventricular injection of corticotropin-releasing hormone receptor antagonist blocks the suppression of pulsatile luteinizing hormone secretion induced by neuromedin U in ovariectomized rats after 48 hours of fasting. Neurosci Lett 369: 33–38, 2004.[CrossRef][Web of Science][Medline]
  38. Roa J, Vigo E, Castellano JM, Navarro VM, Fernandez-Fernandez R, Casanueva FF, Dieguez C, Aguilar E, Pinilla L, Tena-Sempere M. Hypothalamic expression of KiSS-1 system and gonadotropin-releasing effects of kisspeptin in different reproductive states of the female rat. Endocrinology 147: 2864–2878, 2006.[Abstract/Free Full Text]
  39. Shan L, Qiao X, Crona JH, Behan J, Wang S, Laz T, Bayne M, Gustafson EL, Monsma FJ Jr, Hedrick JA. Identification of a novel neuromedin U receptor subtype expressed in the central nervous system. J Biol Chem 275: 39482–39486, 2000.[Abstract/Free Full Text]
  40. Szekeres PG, Muir AI, Spinage LD, Miller JE, Butler SI, Smith A, Rennie GI, Murdock PR, Fitzgerald LR, Wu H, McMillan LJ, Guerrera S, Vawter L, Elshourbagy NA, Mooney JL, Bergsma DJ, Wilson S, Chambers JK. Neuromedin U is a potent agonist at the orphan G protein-coupled receptor FM3. J Biol Chem 275: 20247–20250, 2000.[Abstract/Free Full Text]
  41. Takeo Y. Influence of continuous illumination on estrous cycle of rats: time course of changes in levels of gonadotropins and ovarian steroids until occurrence of persistent estrus. Neuroendocrinology 39: 97–104, 1984.[Web of Science][Medline]
  42. Vigo E, Roa J, Lopez M, Castellano JM, Fernandez-Fernandez R, Navarro VM, Pineda R, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M. Neuromedin S as novel putative regulator of luteinizing hormone secretion. Endocrinology 148: 813–823, 2007.[Abstract/Free Full Text]
  43. Wren AM, Small CJ, Abbott CR, Jethwa PH, Kennedy AR, Murphy KG, Stanley SA, Zollner AN, Ghatei MA, Bloom SR. Hypothalamic actions of neuromedin U. Endocrinology 143: 4227–4234, 2002.[Abstract/Free Full Text]
  44. Wu Y, McRoberts K, Berr SS, Frierson HF Jr, Conaway M, Theodorescu D. Neuromedin U is regulated by the metastasis suppressor RhoGDI2 and is a novel promoter of tumor formation, lung metastasis and cancer cachexia. Oncogene 26: 765–773, 2007.[CrossRef][Web of Science][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/5/E1265    most recent
00425.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vigo, E.
Right arrow Articles by Tena-Sempere, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vigo, E.
Right arrow Articles by Tena-Sempere, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.