help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2005-0983
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
147/6/2886    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
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 PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jethwa, P. H.
Right arrow Articles by Bloom, S. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jethwa, P. H.
Right arrow Articles by Bloom, S. R.
Endocrinology Vol. 147, No. 6 2886-2892
Copyright © 2006 by The Endocrine Society

Neuromedin U Partially Mediates Leptin-Induced Hypothalamo-Pituitary Adrenal (HPA) Stimulation and Has a Physiological Role in the Regulation of the HPA Axis in the Rat

Preeti H. Jethwa1, Kirsty L. Smith1, Caroline J. Small, Caroline R. Abbott, Sarah J. Darch, Kevin G. Murphy, Asha Seth, Nina M. Semjonous, Sejal R. Patel, Jeannie F. Todd, Mohammad A. Ghatei and Stephen R. Bloom

Department of Metabolic Medicine, Imperial College London, London W12 0NN, United Kingdom

Address all correspondence and requests for reprints to: Professor S. R. Bloom, Department of Metabolic Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, United Kingdom. E-mail: s.bloom{at}imperial.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intracerebroventricular (ICV) administration of the hypothalamic neuropeptide neuromedin U (NMU) or the adipostat hormone leptin increases plasma ACTH and corticosterone. The relationship between leptin and NMU in the regulation of the hypothalamo-pituitary adrenal (HPA) axis is currently unknown. In this study, leptin (1 nM) significantly increased the release of CRH from ex vivo hypothalamic explants by 207 ± 8.4% (P < 0.05 vs. basal), an effect blocked by the administration of anti-NMU IgG. The ICV administration of leptin (10 µg, 0.625 nmol) increased plasma ACTH and corticosterone 20 min after injection [plasma ACTH (picograms per milliliter): vehicle, 63 ± 20, leptin, 135 ± 36, P < 0.05; plasma corticosterone (nanograms per milliliter): vehicle, 285 ± 39, leptin, 452 ± 44, P < 0.01]. These effects were partially attenuated by the prior administration of anti-NMU IgG. Peripheral leptin also stimulated ACTH release, an effect attenuated by prior ICV administration of anti-NMU IgG. We examined the diurnal pattern of hypothalamic NMU mRNA expression and peptide content, plasma leptin, and plasma corticosterone. The diurnal changes in hypothalamic NMU mRNA expression were positively correlated with hypothalamic NMU peptide content, plasma corticosterone, and plasma leptin. The ICV administration of anti-NMU IgG significantly attenuated the dark phase rise in corticosterone [corticosterone (nanograms per milliliter): vehicle, 493 ± 38; NMU IgG, 342 ± 47 (P < 0.05)]. These studies suggest that NMU may play a role in the regulation of the HPA axis and partially mediate leptin-induced HPA stimulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ACTIVITY OF THE hypothalamo-pituitary adrenal (HPA) axis is altered by obesity and caloric deprivation. Leptin, the protein product of the Lep (obese) gene, is released into the circulation from adipose tissue and regulates food intake, energy expenditure, and the HPA axis in part via the hypothalamus (1, 2, 3, 4). Leptin influences hypothalamic neurons via the long variant isoform of the leptin receptor, Ob-Rb (1, 5, 6).

The effects of leptin on the HPA axis are complex. The Ob-Rb is abundant on CRH neurons in the parvocellular region of the paraventricular nucleus (PVN) (7). It has been reported that administration of anti-CRH antibodies or the CRH receptor antagonist {alpha}-helical CRH(9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41) [{alpha}-hCRH(9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41)] inhibits the anorectic effects of leptin (8, 9). These authors suggest that leptin partially regulates energy balance via hypothalamic CRH.

However, leptin has also been reported to have an inhibitory effect on the HPA axis. Leptin-deficient (ob/ob) mice are hypercorticosteronemic (10). Administration of leptin to fasted rodents (11), ob/ob mice (11), or immobilization-stressed rodents (12) inhibits plasma ACTH and corticosterone levels. In addition, CRH mRNA expression in the PVN is increased in fasted rats, which have low circulating levels of leptin, and ob/ob mice, in which leptin is absent (11, 12). This apparent inverse relationship between plasma leptin and plasma corticosterone appears to support the hypothesis that leptin inhibits the HPA axis (11). However, recent publications suggest that leptin can stimulate the HPA axis under certain circumstances. Intracerebroventricular (ICV) administration of leptin has been shown to stimulate corticosterone secretion at the onset of the dark phase (13). In addition, a single ICV injection of leptin in the early light phase has shown to dose-dependently increase plasma ACTH and corticosterone secretion (14). ICV injection of leptin also increases the expression of CRH and its receptor, CRH-R2 in the PVN (15). Leptin may therefore have both inhibitory and stimulatory roles in the regulation of the HPA axis. The mechanism by which leptin interacts with the HPA axis is currently unknown.

Neuromedin U (NMU) is a gastrointestinal and central nervous system peptide that has been reported to reduce food intake (16, 17), activate the sympathetic nervous system (18), and increase energy expenditure (17, 19). These effects are similar to those observed after leptin administration (20, 21, 22). NMU also potently stimulates the HPA axis and induces stress-related behaviors (16). Within the hypothalamus, NMU mRNA is predominantly expressed in the arcuate, suprachiasmatic, and dorsomedial nuclei (17, 23), which also express the Ob-Rb (24). NMU immunoreactive fibers project to the PVN (25). ICV or intra-PVN administration of NMU to rats significantly increases plasma ACTH and corticosterone (16, 26). Incubation of ex vivo hypothalamic explants with NMU significantly increases the release of CRH (16). The effects of NMU on the HPA axis are thought to be mediated via the NMU 2 receptor (NMU2R) (16), which is expressed in the ependymal layer of the third ventricle, PVN (17, 19, 23, 27, 28), and arcuate nucleus (23).

NMU mRNA expression in the arcuate nucleus is reduced after a 48-h fast, when leptin concentrations are low, and reduced in ob/ob mice, in which leptin is absent (17). Incubation of ex vivo hypothalamic explants with leptin increases the release of NMU in vitro (16). However there are few other published data on the interaction between leptin and NMU systems with regard to regulation of the HPA axis.

Here we investigate the hypothesis that hypothalamic NMU in part mediates the leptin-induced activation of the HPA axis by using an anti-NMU IgG to block endogenous NMU signaling. We also used anti-NMU IgG to investigate the role of endogenous hypothalamic NMU in the normal diurnal rise in plasma corticosterone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Wistar rats (specific pathogen free; Charles River, Margate, UK), weighing 200–250 g, were maintained in individual cages under controlled temperature (21–23 C) and light (12 h light, 12-h dark cycle, lights on at 0700 h) with ad libitum access to food (RM1 diet; SDS UK Ltd., Witham, UK), unless otherwise described. Food intake and body weight were measured daily throughout the study, and all rats were handled daily to habituate and minimize any stress. All animal experimentation was conducted in accordance with accepted standards of humane animal care and carried out under the 1986 British Animals (Scientific Procedures) Act (license no. 70/5516).

Materials
Recombinant murine leptin was a gift from M. Chiesi and N. Levens (Novartis, Basel, Switzerland). Reagents for the ex vivo hypothalamic explant experiments were supplied by BDH (Poole, Dorset, UK). All peptides were reconstituted at the beginning of each study in vehicle [nonimmune rabbit serum (NIRS)].

Antibody purification
The anti-NMU IgG was produced and purified using methods previously described (29, 30).

ICV cannulation and injection
Animal surgical procedures and handling were carried out as previously described (30). All compounds were injected using a 27-gauge stainless steel injector placed in and projecting 1 mm below the tip of the cannula. Cannula placement was confirmed by a positive dipsogenic response to angiotensin II (150 ng). Only those animals showing a positive dipsogenic response were included in the data analysis (>98%). All animals were habituated to the injection process by a subsequent saline injection.

Leptin and immunoblockade of leptin by anti-NMU IgG on hypothalamic CRH release
A static incubation system was used as described previously (16). Briefly, ad libitum-fed male Wistar rats were killed by decapitation and the whole brain immediately removed. The brain was mounted with the ventral surface uppermost and placed on a vibrating microtome (Microfield Scientific Ltd., Dartmouth, UK). A 1.7-mm slice was taken from the basal hypothalamus and blocked lateral to the Circle of Willis. The hypothalamic slices were incubated in individual chambers containing 1 ml artificial cerebrospinal fluid (aCSF) [20 mM NaHCO3, 126 mM NaCl, 0.09 mM Na2HPO4, 6 mM KCl, 1.4 mM CaCl2, 0.09 mM MgSO4, 5 mM glucose, 0.18 mg/ml ascorbic acid, and 100 µg/ml aprotinin (Bayer, Haywards Heath, UK)] equilibrated with 95% O2 and 5% CO2.

The tubes were placed on a platform in a water bath maintained at 37 C. After an initial 2-h equilibration period, the hypothalami were incubated for 45 min in 600 µl aCSF (basal period) before being challenged with leptin (1, 10, and 100 nM), 1 nM leptin combined with anti-NMU IgG (1:20), anti-NMU IgG alone (1:20), or NIRS (1:20) in 600 µl aCSF for 45 min. The viability of the tissue was verified by 45 min of exposure to aCSF containing 56 mM KCl. Isotonicity was maintained by substituting K+ for Na+. Hypothalamic explants that failed to show peptide release above that of basal in response to hyperkalaemic aCSF were excluded from the data analysis (<10%). The experiment was repeated twice, with eight to 12 hypothalamic slices given each treatment. At the end of each period, aCSF was collected and stored at –20 C until measurement of CRH by RIA. To confirm whether the effect of anti-NMU IgG was specific for leptin, ex vivo hypothalamic explants were incubated in either 1000 nM ghrelin combined with anti-NMU IgG or 1000 nM ghrelin with NIRS. This dose of ghrelin has previously been shown to stimulate CRH release (31).

Anti-NMU IgG immunoblockade on the effects of ICV leptin on the HPA axis
Ad libitum-fed male Wistar rats received a single ICV injection, between 0900 and 1000 h, of vehicle, leptin (10 µg, 0.625 nmol) alone, anti-NMU IgG (5 µl, concentration 150 mg/ml) alone, or leptin (0.625nmol) and anti-NMU IgG (5 µl) together. The same dose of anti-NMU IgG has previously been shown to block the inhibitory actions on food intake of 1 nmol NMU administered ICV (30). Morimoto et al. (14) reported that ICV administration of leptin significantly increased plasma ACTH and corticosterone 20 min after injection. Rats were therefore killed by decapitation 20 min after injection (n = 10–15/group per time point), and trunk blood was collected into plastic lithium heparin tubes containing 0.6 mg aprotinin and plastic tubes containing potassium EDTA (final concentration of 1.2–2 mg EDTA per milliliter of blood). Plasma was separated by centrifugation, frozen, and stored at –20 C for the measurement of ACTH and corticosterone by immunoradiometric assay (IRMA) and RIA, respectively, according to the manufacturer’s protocol.

Anti-NMU IgG immunoblockade on the effects of peripheral leptin on the HPA axis
Ad libitum-fed male Wistar rats received a single ICV injection, between 0900 and 1000 h, of either vehicle or anti-NMU IgG (1 µl). Five minutes after injection, animals received an ip injection of either leptin (1.1 mg/kg) or saline. Animals were killed by decapitation 20 min after ICV injection (n = 10/group) and trunk blood was collected into plastic lithium heparin tubes containing 0.6 mg aprotinin and plastic tubes containing potassium EDTA (final concentration of 1.2–2 mg EDTA per milliliter of blood). Plasma was separated by centrifugation, frozen, and stored at –20 C before the measurement of ACTH and corticosterone by IRMA and RIA, respectively.

Diurnal changes in hypothalamic NMU mRNA expression and peptide content, plasma leptin, and corticosterone levels
Ad libitum-fed male Wistar rats were decapitated at the following time points: 0700 h (lights on), 1100 h, 1500 h, 1700 h, 1900 h (lights off), 2100 h, 2300 h, 0300 h, and 0700 h) (n = 15 per time point). At each time point, hypothalami were dissected out and snap frozen in liquid nitrogen.

For quantification of hypothalamic NMU mRNA expression, hypothalamic mRNA was extracted (n = 10 per time point) using Tri-Reagent (Helena Biosciences, Sunderland, UK) according to the manufacturer’s protocol. Quantification of NMU mRNA expression was performed as previously described (32) using the Ambion RNase protection assay III kit (Ambion Inc., Austin, TX) under conditions optimized within our laboratory. The NMU riboprobe corresponded to nucleotides 121–336 of the full-length rat NMU sequence (accession no. NM_022239), a 215-bp product. Briefly, 5 µg RNA were hybridized overnight at 42 C with 1.3 x 103 Bq 32P[CTP]-labeled riboprobe. Rat ß-actin was used as an internal control (Ambion). Reaction mixtures were digested with RNase A/T1 and the protected fragments precipitated and separated on a 4% polyacrylamide gel. The dried gel was exposed to a PhosphorImager screen overnight and bands quantified by image densitometry using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

For quantification of hypothalamic NMU peptide content, peptide was extracted from hypothalami by boiling in 0.5 M acetic acid (10 ml/g hypothalamus) for 20 min (n = 5 per time point). The hypothalamic extracts were cooled on ice and then stored at –20 C until determination of NMU peptide content by RIA. To normalize the data, the hypothalamic protein content was determined using the Coomassie Plus protein assay kit (Pierce Biotechnology Inc., Rockford, IL).

Trunk blood was collected into plastic lithium heparin tubes containing 0.6 mg aprotinin, separated by centrifugation, frozen, and stored at –20 C until measurement of plasma leptin and corticosterone by RIA according to the manufacturer’s protocol.

The effect of peripheral leptin on hypothalamic NMU mRNA and peptide expression
Ad libitum-fed male Wistar rats received a single ip injection, between 0900 and 1000 h of either saline or leptin (1.1 mg/kg). This dose of leptin has previously been shown to activate hypothalamic neurons (33, 34). Four hours after injection, animals were decapitated, brains removed, and the hypothalamus dissected out and snap frozen in liquid nitrogen. Hypothalamic mRNA was extracted as previously described and hypothalamic NMU mRNA expression was quantified by RNase protection assay. To measure hypothalamic NMU peptide content, peptide was extracted as described above and hypothalamic NMU content was quantified by RIA.

The effect of ICV administration of anti-NMU IgG on the nocturnal rise of corticosterone
Hypothalamic NMU peptide content rose between 1900 and 2100 h (see Fig. 3AGo); therefore, ad libitum-fed male Wistar rats received a single ICV injection of either vehicle or anti-NMU IgG (5 µl) just before the onset of the dark phase (1830 h) (n = 10–15/group per time point) to block the rise in hypothalamic NMU peptide. Plasma corticosterone levels begin to rise from 1500 h (see Fig. 3BGo) and peak at 2100 h. To examine the blockade of endogenous NMU during the nocturnal rise in corticosterone, animals were decapitated at 1930 h. Trunk blood was collected into plastic lithium heparin tubes containing 0.6 mg aprotinin. Plasma was separated by centrifugation, frozen and stored at –20 C until measurement of corticosterone by RIA according to manufacturer’s protocol.


Figure 3
View larger version (29K):
[in this window]
[in a new window]
  		FIG. 3. Diurnal patterns of hypothalamic NMU peptide content, plasma corticosterone, and plasma leptin. Diurnal profiles of hypothalamic NMU mRNA expression (A), hypothalamic NMU peptide content (B), plasma corticosterone (C), and plasma leptin (D) at nine time points: 0700, 1100, 1500, 1700, 1900 (lights out), 2100, 2300, 0300, and 0700 h (n = 10 per time point). Open bar, Light phase; black bar, dark phase.

 
RIAs
CRH immunoreactivity (IR) and NMU-IR were measured using established assays (16). The intra- and interassay variations of both assays were less than 10%. Plasma corticosterone (MP Biomedicals, Hampshire, UK) and leptin (Linco Research Inc., St. Louis, MO) levels were measured using commercially available RIA kits. The intra- and interassay coefficients of variation for plasma corticosterone were less than 10%. Plasma ACTH (IDS Ltd., Tyne and Wear, UK) was measured by IRMA. The intra- and interassay coefficients of variation for ACTH were less than 4%.

Statistical analysis
Data are presented as mean ± SEM. Data from ex vivo hypothalamic explant release experiments were compared by paired t test between the basal period and the test period. For the ICV studies, groups were compared by one-way ANOVA followed by a post hoc Fisher’s least significant difference test (Systat, Evanston, IL). Correlation between hypothalamic NMU mRNA and peptide expression, plasma corticosterone, and leptin levels with respect to time was determined by Spearman rank order correlation. In all cases P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Leptin and immunoblockade by anti-NMU IgG on hypothalamic CRH release
Incubation of ex vivo hypothalamic explants with 1, 10, and 100 nM leptin significantly increased CRH-IR [(percentage of basal ± SEM) basal, 100 ± 11; leptin (1 nM), 219 ± 20 (P < 0.05 vs. basal); leptin (10 nM), 270 ± 19 (P < 0.01 vs. basal); leptin (100 nM), 207 ± 18 (P < 0.01 vs. basal) n = 8–12/treatment] (Fig. 1AGo). The increase in CRH release (Fig. 1AGo) from hypothalamic explants after incubation of leptin (1 nM) was attenuated by anti-NMU IgG (1:20) [CRH-IR (percentage of basal ± SEM) basal, 100 ± 7.7; leptin (1 nM), 207 ± 8.4 (P < 0.05 vs. basal); leptin (1 nM)/anti-NMU IgG, 116 ± 23 (p = n.s. vs. basal; P < 0.05 vs. leptin); anti-NMU IgG, 92 ± 6.9 (p = n.s. vs. basal, P < 0.05 vs. leptin), NIRS, 109 ± 7.4 (p = n.s. vs. basal; P < 0.05 vs. leptin), n = 8–12/treatment] (Fig. 1BGo). The effect of leptin on CRH release was not affected by incubation with NIRS [CRH-IR (percentage of basal ± SEM) basal, 100 ± 18.5; leptin (1 nM)/NIRS, 167.4 ± 8.5 (P < 0.05 vs. basal)]. To confirm the specificity of the effect for leptin, hypothalamic explants were incubated with 1000 nM ghrelin and either NIRS or anti-NMU IgG. Ghrelin stimulated CRH release, an effect that remained in the presence of anti-NMU IgG (data not shown).


Figure 1
View larger version (17K):
[in this window]
[in a new window]
  		FIG. 1. Leptin-induced CRH release is attenuated by administration of anti-NMU IgG. CRH release from ex vivo hypothalamic explants (n = 8–12/treatment) after a 45-min basal incubation period followed by 45 min exposure to 1, 10, 100 nM leptin in aCSF (A) and 45 min exposure to 1 nM leptin, leptin (1 nM)/anti-NMU IgG (1:20) in aCSF, anti-NMU IgG (1:20) in aCSF and NIRS (1:20) in aCSF (B). Data are expressed as a percentage of basal ± SEM. Significant values are indicated. *, P < 0.05 vs. basal; **, P < 0.01 vs. basal; #, P < 0.05 leptin/NMU IgG vs. NMU IgG and NIRS.

 
Anti-NMU IgG immunoblockade on the effect of ICV leptin on the HPA axis
ICV administration of leptin significantly increased plasma ACTH and corticosterone at 20 min after injection when compared with the vehicle-injected group [plasma ACTH (picograms per milliliter): vehicle, 73 ± 25; leptin (0.625 nmol), 135 ± 36; P < 0.05 vs. vehicle; plasma corticosterone (nanograms per milliliter): vehicle, 284 ± 39; leptin (0.625 nmol), 452 ± 44; P < 0.01 vs. vehicle) n = 10–15/group] (Fig. 2Go, A and B). The leptin-induced increase in plasma ACTH and corticosterone was attenuated by the administration of anti-NMU IgG [plasma ACTH (picograms per milliliter): leptin/anti-NMU IgG, 43 ± 11; p = n.s. vs. vehicle; P < 0.01 vs. leptin; plasma corticosterone (nanograms per milliliter): leptin/anti-NMU IgG, 319 ± 44; p = n.s. vs. vehicle, P < 0.01 vs. leptin)] (Fig. 2Go, A and B). Anti-NMU IgG alone had no effect on plasma ACTH or corticosterone [plasma ACTH (picograms per milliliter): anti-NMU IgG, 74 ± 27; plasma corticosterone (nanograms per milliliter): anti-NMU IgG, 312 ± 38] (Fig. 2Go, A and B).


Figure 2
View larger version (15K):
[in this window]
[in a new window]
  		FIG. 2. The stimulatory effects of leptin on the HPA axis are blocked by ICV administration of anti-NMU IgG. Plasma ACTH (A) and corticosterone (B) 20 min after injection of ICV vehicle, leptin (0.625 nmol), or anti-NMU IgG (5 µl) (n = 10–12/group). Plasma ACTH (C) 20 min after ICV injection of either anti-NMU IgG (5 µl) or vehicle and peripheral administration of either leptin (1.1 mg/kg) or saline. Plasma ACTH: a, P < 0.05 vs. vehicle; b, P < 0.05 vs. leptin/anti-NMU IgG; c, P < 0.05 vs. anti-NMU IgG; plasma corticosterone: a, P < 0.01 vs. vehicle; b, P < 0.05 vs. leptin/anti-NMU IgG; c, P < 0.05 vs. anti-NMU IgG.

 
Anti-NMU IgG immunoblockade on the effect of peripheral leptin on the HPA axis
Intraperitoneal administration of leptin significantly increased plasma ACTH at 20 min after injection when compared with the vehicle-injected control group. The leptin-induced increase in plasma ACTH was attenuated by ICV administration of anti-NMU IgG. Anti-NMU IgG alone had no effect on plasma ACTH [plasma ACTH (picograms per milliliter): saline/vehicle, 86.4 ± 12.2; leptin/vehicle, 135.6 ± 24.4; saline/anti-NMU IgG, 59.8 ± 7.0; leptin/anti-NMU IgG, 66.8 ± 13.7 (P < 0.05 leptin/vehicle vs. saline/vehicle, saline/anti-NMU IgG, and leptin/anti-NMU IgG) n = 10/group] (Fig. 2CGo). Intraperitoneal injection of leptin caused a nonsignificant increase in plasma corticosterone 20 min after injection when compared with the vehicle-injected control group. There was a slight attenuation of this effect by ICV administration of anti-NMU IgG, although, again, this was not significant [plasma corticosterone (nanograms per milliliter): saline/vehicle, 279.3 ± 36.6; leptin/vehicle, 387.6 ± 53.2; saline/anti-NMU IgG, 242.4 ± 46.0; leptin/anti-NMU IgG, 296.1 ± 45.2, n = 10/group].

Diurnal changes in hypothalamic NMU mRNA expression and peptide content, plasma corticosterone, and plasma leptin
Hypothalamic NMU mRNA expression showed a diurnal rhythm with levels peaking at 2300 h, 4 h after lights off (Fig. 3AGo). Hypothalamic NMU peptide content displayed a diurnal variation reaching a nadir at 1100 h, 4 h after lights on. NMU peptide content subsequently increased throughout the day, peaking at the onset of the dark phase (1900 h) (Fig. 3BGo). Hypothalamic NMU mRNA expression was positively correlated with NMU peptide content (r2 = 0.514, P < 0.02).

As expected, plasma corticosterone displayed a diurnal variation, with levels rising toward the end of the light phase (1700 h) and peaking at 2100 h (Fig. 3AGo). A similar diurnal pattern existed between plasma corticosterone and hypothalamic NMU peptide. However, this correlation did not achieve statistical significance (r2 = 0.303, P = 0.062).

Plasma leptin exhibited a diurnal pattern, reaching a nadir at 1100 h, 4 h after lights on. Plasma leptin began to rise at the onset of the dark phase (1900 h) and peaked at 2100 h (Fig. 3CGo). Plasma leptin levels were positively correlated with hypothalamic NMU mRNA expression (r2 = 0.321, P < 0.05) and peptide content (r2 = 0.588, P < 0.01) (see supplemental figures for regression plots, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).

The effect of peripheral leptin on hypothalamic NMU mRNA and peptide expression
A single ip injection of leptin had no effect on hypothalamic NMU mRNA expression (saline, 24.9 ± 1.2 arbitrary units, 1.1 mg/kg; leptin, 22.4 ± 1.2, n = 10) or NMU peptide content (saline, 5.6 ± 0.5 fmol/µg protein, 1.1 mg/kg; leptin, 4.9 ± 0.4 fmol/µg protein, n = 10) 4 h after injection.

ICV administration of anti-NMU IgG on the nocturnal rise of plasma corticosterone
Blocking endogenous hypothalamic NMU signaling with an ICV injection of anti-NMU IgG at the onset of the dark phase attenuated the nocturnal rise in plasma corticosterone at 1930 h [plasma corticosterone (nanograms per milliliter): vehicle, 493 ± 36; anti-NMU IgG, 342 ± 48 (P < 0.05 vs. vehicle), n = 10–15/group] (Fig. 4Go).


Figure 4
View larger version (15K):
[in this window]
[in a new window]
  		FIG. 4. Immunoblockade of hypothalamic NMU attenuates the nocturnal rise in corticosterone. Ad libitum-fed male Wistar rats received an injection of either anti-NMU IgG (5 µl) or vehicle in the early dark phase. Plasma corticosterone was measured at 1930 h (n = 10–12). Significance values are as indicated. *, P < 0.05 vs. vehicle.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The evidence for the role of leptin in the regulation of the HPA axis is conflicting. Models of leptin deficiency suggest leptin inhibits the HPA axis (11, 35), and leptin administration has been reported to suppress HPA axis activity (12, 36). However, there is also evidence that leptin can stimulate the HPA axis (13, 14, 15). These current studies suggest that leptin stimulates the release of CRH from ex vivo hypothalamic explants. This is in accord with studies showing that leptin increases CRH release, peptide concentration, and mRNA expression in the PVN (14, 37). In our current experiments, ICV administration of leptin increased plasma ACTH and corticosterone 20 min after injection, in agreement with published data (14). Leptin therefore appears to stimulate the HPA axis via the release of CRH under these specific conditions. A number of hypothalamic circuits are thought to regulate different aspects of stress and HPA axis activation. It is therefore quite possible that leptin can play both stimulatory and inhibitory roles in the regulation of the neuroendocrine response to stress. For example, both neuropeptide Y (NPY) and CRH neurons are leptin responsive, although leptin inhibits NPY release while stimulating CRH (1). Both of these neuropeptides stimulate the HPA axis but may be differentially regulated by different types of stress (13).

Hypothalamic release of NMU from ex vivo hypothalamic explants is stimulated by leptin (16), and hypothalamic NMU mRNA expression is reduced in models of low or absent circulating leptin (17). NMU increases the release of CRH from ex vivo hypothalamic explants (16), whereas intra-PVN administration of NMU increases plasma ACTH and corticosterone (16). The increase in plasma ACTH and corticosterone after ICV administration of NMU is attenuated in CRH-deficient mice and rodents pretreated with {alpha}-hCRH(9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41) (38, 39). Although it is currently unknown whether NMU2R is coexpressed with CRH neurons, NMU2R is expressed in the PVN (17, 27, 40), and ICV administration of NMU increases c-fos, a marker of neuronal activation, in CRH neurons in the PVN (41, 42). The authors of these reports have suggested that the stimulatory effect of NMU on the HPA axis is mediated via CRH.

A number of leptin-responsive neuropeptides have been shown to regulate the HPA axis, including NPY (43). Our data suggest that the actions of leptin on the HPA axis may be partially mediated by the NMU/CRH pathway. Coadministration of anti-NMU IgG with leptin blocks leptin-induced CRH release from ex vivo hypothalamic explants. Furthermore, ICV administration of anti-NMU IgG partially attenuates the leptin-induced increase in plasma ACTH and corticosterone in vivo after ICV administration. Leptin is produced by adipose tissue, circulates, and enters the hypothalamus via the blood brain barrier. The effect of peripheral administration of leptin on the HPA axis is therefore perhaps more relevant than ICV administration. Peripheral administration of leptin caused a significant increase in plasma ACTH at 20 min after injection. This effect is attenuated by ICV pretreatment with anti-NMU IgG. These results suggest that NMU may mediate some of the effects of leptin on the HPA axis via CRH. The arcuate nucleus is incompletely isolated by the blood brain barrier and is thus in direct communication with peripheral signals from the blood and the CSF (44). It is therefore possible that arcuate NMU neurons respond to circulating leptin, signaling to CRH neurons in the PVN leading to activation of the HPA axis.

Our results suggest that both hypothalamic NMU peptide content and mRNA expression vary diurnally in rhythms that correlate with the diurnal variation in plasma leptin and corticosterone. Furthermore, ICV administration of anti-NMU IgG partly attenuated the normal night-time rise in plasma corticosterone suggesting that NMU may play a role in regulating the diurnal changes in plasma corticosterone. While indicating hypothalamic NMU mRNA expression and peptide content at specific time points, the diurnal patterns do not give an indication of the dynamics of peptide synthesis and release. For example, it is unclear whether high levels of hypothalamic NMU peptide content indicate increased peptide release or replenishment of neuronal peptide levels after release. The hypothalamus expresses low levels of NMU, making it difficult to carry out a detailed dynamic investigation. In this current study, samples were taken only every 4 h. More frequent sampling is required to determine the exact temporal relationship between hypothalamic NMU mRNA expression and peptide content, plasma corticosterone, and plasma leptin. It is possible that NMU may not mediate the initial rise in corticosterone but may be involved in the maintenance of high corticosterone levels at the beginning of the dark phase.

The hypothalamic pathways that control the HPA axis are complex and are not fully characterized. The results presented in this manuscript suggest that leptin may mediate a stimulatory action on the HPA axis by altering hypothalamic NMU expression and release.


   Acknowledgments
 
The authors thank David Robinson (Senior Consultant, Statistical Services Unit, University of Sheffield, Sheffield, UK) for statistical advice.


   Footnotes
 
This work was supported by the Medical Research Council. K.G.M. is supported by a Biotechnology and Biological Sciences Research Council New Investigator Award.

Disclosure summary: P.H.J., K.L.S., C.J.S., C.R.A., S.J.D., K.G.M., A.S., N.M.S., S.R.P., J.F.T., M.A.G., and S.R.B. have nothing to declare.

First Published Online March 23, 2006

1 P.H.J. and K.L.S. contributed equally to this work. Back

Abbreviations: aCSF, Artificial cerebrospinal fluid; HPA, hypothalamo-pituitary-adrenal; ICV, intracerebroventricular; IR, immunoreactivity; IRMA, immunoradiometric assay; NIRS, nonimmune rabbit serum; NMU, neuromedin U; NMU2R, NMU 2 receptor; NPY, neuropeptide Y; PVN, paraventricular nucleus.

Received August 2, 2005.

Accepted for publication March 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG 1996 Identification of targets of leptin action in rat hypothalamus. J Clin Invest 98:1101–1106[Medline]
  2. Porte Jr D, Baskin DG, Schwartz MW 2002 Leptin and insulin action in the central nervous system. Nutr Rev 60:S20–S29
  3. Bjorbaek C, Hollenberg AN 2002 Leptin and melanocortin signaling in the hypothalamus. Vitam Horm 65:281–311[Medline]
  4. Casanueva FF, Dieguez C 1999 Neuroendocrine regulation and actions of leptin. Front Neuroendocrinol 20:317–363[CrossRef][Medline]
  5. Sahu A 1998 Evidence suggesting that galanin (GAL), melanin-concentrating hormone (MCH), neurotensin (NT), proopiomelanocortin (POMC) and neuropeptide Y (NPY) are targets of leptin signaling in the hypothalamus. Endocrinology 139:795–798[Abstract/Free Full Text]
  6. Baskin DG, Breininger JF, Schwartz MW 1999 Leptin receptor mRNA identifies a subpopulation of neuropeptide Y neurons activated by fasting in rat hypothalamus. Diabetes 48:828–833[Abstract]
  7. Hakansson ML, Brown H, Ghilardi N, Skoda RC, Meister B 1998 Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus. J Neurosci 18:559–572[Abstract/Free Full Text]
  8. Uehara Y, Shimizu H, Ohtani K, Sato N, Mori M 1998 Hypothalamic corticotropin-releasing hormone is a mediator of the anorexigenic effect of leptin. Diabetes 47:890–893[Abstract]
  9. Masaki T, Yoshimichi G, Chiba S, Yasuda T, Noguchi H, Kakuma T, Sakata T, Yoshimatsu H 2003 Corticotropin-releasing hormone-mediated pathway of leptin to regulate feeding, adiposity, and uncoupling protein expression in mice. Endocrinology 144:3547–3554[Abstract/Free Full Text]
  10. Edwardson JA, Hough CA 1975 The pituitary-adrenal system of the genetically obese (ob/ob) mouse. J Endocrinol 65:99–107[Abstract/Free Full Text]
  11. Ahima RS, Prabakaran D, Flier JS 1998 Postnatal leptin surge and regulation of circadian rhythm of leptin by feeding. Implications for energy homeostasis and neuroendocrine function. J Clin Invest 101:1020–1027[Medline]
  12. Heiman ML, Ahima RS, Craft LS, Schoner B, Stephens TW, Flier JS 1997 Leptin inhibition of the hypothalamic-pituitary-adrenal axis in response to stress. Endocrinology 138:3859–3863[Abstract/Free Full Text]
  13. van DG, Donahey JC, Thiele TE, Scheurink AJ, Steffens AB, Wilkinson CW, Tenenbaum R, Campfield LA, Burn P, Seeley RJ, Woods SC 1997 Central leptin stimulates corticosterone secretion at the onset of the dark phase. Diabetes 46:1911–1914[Abstract]
  14. Morimoto I, Yamamoto S, Kai K, Fujihira T, Morita E, Eto S 2000 Centrally administered murine-leptin stimulates the hypothalamus-pituitary-adrenal axis through arginine-vasopressin. Neuroendocrinology 71:366–374[CrossRef][Medline]
  15. Nishiyama M, Makino S, Asaba K, Hashimoto K 1999 Leptin effects on the expression of type-2 CRH receptor mRNA in the ventromedial hypothalamus in the rat. J Neuroendocrinol 11:307–314[CrossRef][Medline]
  16. Wren AM, Small CJ, Abbott CR, Jethwa PH, Kennedy AR, Murphy KG, Stanley SA, Zollner AN, Ghatei MA, Bloom SR 2002 Hypothalamic actions of neuromedin U. Endocrinology 143:4227–4234[Abstract/Free Full Text]
  17. Howard AD, Wang R, Pong SS, Mellin TN, Strack A, Guan XM, Zeng Z, Williams Jr DL, 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, Liu Q 2000 Identification of receptors for neuromedin U and its role in feeding. Nature 406:70–74[CrossRef][Medline]
  18. Chu C, Jin Q, Kunitake T, Kato K, Nabekura T, Nakazato M, Kangawa K, Kannan H 2002 Cardiovascular actions of central neuromedin U in conscious rats. Regul Pept 105:29–34[CrossRef][Medline]
  19. Ivanov TR, Lawrence CB, Stanley PJ, Luckman SM 2002 Evaluation of neuromedin U actions in energy homeostasis and pituitary function. Endocrinology 143:3813–3821[Abstract/Free Full Text]
  20. Seeley RJ, van DG, Campfield LA, Smith FJ, Burn P, Nelligan JA, Bell SM, Baskin DG, Woods SC, Schwartz MW 1996 Intraventricular leptin reduces food intake and body weight of lean rats but not obese Zucker rats. Horm Metab Res 28:664–668[Medline]
  21. Commins SP, Watson PM, Levin N, Beiler RJ, Gettys TW 2000 Central leptin regulates the UCP1 and ob genes in brown and white adipose tissue via different ß-adrenoceptor subtypes. J Biol Chem 275:33059–33067[Abstract/Free Full Text]
  22. Fox AS, Olster DH 2000 Effects of intracerebroventricular leptin administration on feeding and sexual behaviors in lean and obese female Zucker rats. Horm Behav 37:377–387[CrossRef][Medline]
  23. Graham ES, Turnbull Y, Fotheringham P, Nilaweera K, Mercer JG, Morgan PJ, Barrett P 2003 Neuromedin U and neuromedin U receptor-2 expression in the mouse and rat hypothalamus: effects of nutritional status. J Neurochem 87:1165–1173[CrossRef][Medline]
  24. Meister B 2000 Control of food intake via leptin receptors in the hypothalamus. Vitam Horm 59:265–304[Medline]
  25. Ballesta J, Carlei F, Bishop AE, Steel JH, Gibson SJ, Fahey M, Hennessey R, Domin J, Bloom SR, Polak JM 1988 Occurrence and developmental pattern of neuromedin U-immunoreactive nerves in the gastrointestinal tract and brain of the rat. Neuroscience 25:797–816[CrossRef][Medline]
  26. Malendowicz LK, Nussdorfer GG, Nowak KW, Mazzocchi G 1993 Effects of neuromedin U-8 on the rat pituitary-adrenocortical axis. In Vivo 7:419–422[Medline]
  27. Raddatz R, Wilson AE, Artymyshyn R, Bonini JA, Borowsky B, Boteju LW, Zhou S, Kouranova EV, Nagorny R, Guevarra MS, Dai M, Lerman GS, Vaysse PJ, Branchek TA, 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[Abstract/Free Full Text]
  28. 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 2000 Neuromedin U is a potent agonist at the orphan G protein-coupled receptor FM3. J Biol Chem 275:20247–20250[Abstract/Free Full Text]
  29. Domin J, Ghatei MA, Chohan P, Bloom SR 1987 Neuromedin U—a study of its distribution in the rat. Peptides 8:779–784[CrossRef][Medline]
  30. Jethwa PH, Small CJ, Smith KL, Seth A, Darch SJ, Abbott CR, Murphy KG, Todd JF, Ghatei MA, Bloom SR 2005 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
  31. Wren AM, Small CJ, Fribbens CV, Neary NM, Ward HL, Seal LJ, Ghatei MA, Bloom SR 2002 The hypothalamic mechanisms of the hypophysiotropic action of ghrelin. Neuroendocrinology 76:316–324[CrossRef][Medline]
  32. Kong WM, Stanley S, Gardiner J, Abbott C, Murphy K, Seth A, Connoley I, Ghatei M, Stephens D, Bloom S 2003 A role for arcuate cocaine and amphetamine-regulated transcript in hyperphagia, thermogenesis, and cold adaptation. FASEB J 17:1688–1690[Abstract/Free Full Text]
  33. Elmquist JK, Ahima RS, Maratos-Flier E, Flier JS, Saper CB 1997 Leptin activates neurons in ventrobasal hypothalamus and brainstem. Endocrinology 138:839–842[Abstract/Free Full Text]
  34. Andersson U, Filipsson K, Abbott CR, Woods A, Smith K, Bloom SR, Carling D, Small CJ 2004 AMP-activated protein kinase plays a role in the control of food intake. J Biol Chem 279:12005–12008[Abstract/Free Full Text]
  35. Stephens TW, Basinski M, Bristow PK, Bue-Valleskey JM, Burgett SG, Craft L, Hale J, Hoffmann J, Hsiung HM, Kriauciunas A 1995 The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature 377:530–532[CrossRef][Medline]
  36. Glasow A, Haidan A, Hilbers U, Breidert M, Gillespie J, Scherbaum WA, Chrousos GP, Bornstein SR 1998 Expression of Ob receptor in normal human adrenals: differential regulation of adrenocortical and adrenomedullary function by leptin. J Clin Endocrinol Metab 83:4459–4466[Abstract/Free Full Text]
  37. Huang Q, Rivest R, Richard D 1998 Effects of leptin on corticotropin-releasing factor (CRF) synthesis and CRF neuron activation in the paraventricular hypothalamic nucleus of obese (ob/ob) mice. Endocrinology 139:1524–1532[Abstract/Free Full Text]
  38. 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[CrossRef][Medline]
  39. 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[CrossRef][Medline]
  40. Shan L, Qiao X, Crona JH, Behan J, Wang S, Laz T, Bayne M, Gustafson EL, Monsma Jr FJ, Hedrick JA 2000 Identification of a novel neuromedin U receptor subtype expressed in the central nervous system. J Biol Chem 275:39482–39486[Abstract/Free Full Text]
  41. Yokota M, Ozaki Y, Sakamoto F, Yamada S, Saito J, Fujihara H, Ueta Y 2004 Fos expression in CRF-containing neurons in the rat paraventricular nucleus after central administration of neuromedin U. Stress 7:109–112[Medline]
  42. 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[Abstract/Free Full Text]
  43. Akabayashi A, Levin N, Paez X, Alexander JT, Leibowitz SF 1994 Hypothalamic neuropeptide Y and its gene expression: relation to light/dark cycle and circulating corticosterone. Mol Cell Neurosci 5:210–218[CrossRef][Medline]
  44. Cone RD, Cowley MA, Butler AA, Fan W, Marks DL, Low MJ 2001 The arcuate nucleus as a conduit for diverse signals relevant to energy homeostasis. Int J Obes Relat Metab Disord 25(Suppl 5):S63–S67



This article has been cited by other articles:


Home page
 EndocrinologyHome page
J. Gay, E. Kokkotou, M. O'Brien, C. Pothoulakis, and K. P. Karalis
Corticotropin-Releasing Hormone Deficiency Is Associated with Reduced Local Inflammation in a Mouse Model of Experimental Colitis
Endocrinology, July 1, 2008; 149(7): 3403 - 3409.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
147/6/2886    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
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 PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jethwa, P. H.
Right arrow Articles by Bloom, S. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jethwa, P. H.
Right arrow Articles by Bloom, S. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals