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Am J Physiol Endocrinol Metab 292: E913-E919, 2007. First published November 28, 2006; doi:10.1152/ajpendo.00346.2006
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Hypothalamic mapping of orexigenic action and Fos-like immunoreactivity following relaxin-3 administration in male Wistar rats

B. M. McGowan,* S. A. Stanley,* N. E. White, A. Spangeus, M. Patterson, E. L. Thompson, K. L. Smith, J. Donovan, J. V. Gardiner, M. A. Ghatei, and S. R. Bloom

Department of Metabolic Medicine, Imperial College London, Hammersmith Hospital, London, United Kingdom

Submitted 14 July 2006 ; accepted in final form 22 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The insulin superfamily, characterized by common disulphide bonds, includes not only insulin but also insulin-like peptides such as relaxin-1 and relaxin-3. The actions of relaxin-3 are largely unknown, but recent work suggests a role in regulation of food intake. Relaxin-3 mRNA is highly expressed in the nucleus incertus, which has extensive projections to the hypothalamus, and relaxin immunoreactivity is present in several hypothalamic nuclei. In the rat, relaxin-3 binds and activates both relaxin family peptide receptor 1, which also binds relaxin-1, and a previously orphaned G protein-coupled receptor, RXFP3. These receptors are extensively expressed in the hypothalamus. The aims of these studies were twofold: 1) map the hypothalamic site(s) of the orexigenic action of relaxin-3 and 2) examine the site(s) of neuronal activation following central relaxin-3 administration. After microinjection into hypothalamic sites, human relaxin-3 (H3; 180 pmol) significantly stimulated 0- to 1-h food intake in the supraoptic nucleus (SON), arcuate nucleus (ARC), and the anterior preoptic area (APOA) [SON 0.4 ± 0.2 (vehicle) vs. 2.9 ± 0.5 g (H3), P < 0.001; ARC 0.7 ± 0.3 (vehicle) vs. 2.7 ± 0.2 g (H3), P < 0.05; and APOA 0.8 ± 0.1 (vehicle) vs. 2.2 ± 0.2 g (H3), P < 0.05]. Cumulative food intake was significantly increased ≤8 h following administration into the SON and 4 h into the APOA. A significant increase in Fos-like immunoreactivity was seen in the SON following central relaxin-3 administration. Relaxin-3 stimulates feeding in several hypothalamic nuclei, and these studies provide additional support for relaxin-3 as an important peptide in appetite regulation.

relaxin family peptide receptor 3; appetite; supraoptic nucleus; arcuate nucleus

THE INSULIN SUPERFAMILY of peptides is characterized by a common structure typified by one intrachain and two interchain disulphide bridges (20). In addition to insulin and insulin-like growth factors, the family also includes several insulin-like peptides: relaxin-1, relaxin-3, and insulin-like peptide-3, -4, -5, and -6 (5). Classical relaxin (H2 in humans, M1 in mice, and R1 in rats, referred to as relaxin-1 for clarity) is highly expressed in the female reproductive tract (36). Its primary roles appear to be in reproduction and parturition, but peripheral relaxin-1 also has marked effects on water intake (37, 45). The physiological role of the more recently described relaxin-3 remains largely unknown. In contrast to relaxin-1, relaxin-3 is expressed at the greatest levels in the central nervous system and primarily in the nucleus incertus (NI) of the brainstem (9, 44). This area has extensive projections to the forebrain, in particular to the hypothalamus, including the lateral hypothalamic and preoptic areas (18, 44). In addition, relaxin-3 immunoreactivity has been described in both the arcuate nucleus (ARC) and paraventricular nucleus (PVN) (5, 44).

Relaxin-1 and relaxin-3 receptors are highly expressed in the central nervous system. Unlike insulin, which signals via tyrosine kinase receptors, relaxin-1 interacts with the G protein-coupled receptors (GPCRs) relaxin family peptide receptor (RXFP)1 (formerly LGR7) and RXFP2 (formerly LGR8) to increase intracellular cAMP (19). Autoradiography studies using [32P]relaxin-1 have shown high-affinity relaxin-1 binding sites in several hypothalamic areas, particularly the PVN, ARC, and supraoptic nucleus (SON) (32). RXFP1 mRNA and RXFP1-like immunoreactivity have been detected in the SON, PVN, and ARC (10). RXFP2 mRNA has been detected in the brain, mainly in the thalamus (35).

Relaxin-3 also binds and activates several GPCRs: RXFP1 (in common with relaxin-1) (40) and the previously orphaned GPCRs RXFP3 (formerly GPCR135) and RXFP4 (formerly GPCR142). In contrast to relaxin-1 signaling, relaxin-3 binding to RXFP3 and RXFP4 inhibit intracellular cAMP production (25, 26). Although RXFP4 is a pseudogene and absent in the rat (11), RXFP3 is expressed within the rat hypothalamus (10, 11, 26). In situ hybridization for RXFP3 demonstrates expression in both the PVN and SON with additional expression in preoptic and lateral hypothalamic areas (42, 43). In agreement with this, the RXFP3-specific agonist relaxin-3/insulin-like peptide-5 chimeric peptide binds in the PVN (24, 43).

Relaxin-3 has recently been shown (29) to stimulate food intake when administered into the third ventricle or PVN of male rats. This effect may be mediated by RXFP3. However, there are several additional hypothalamic areas where relaxin immunoreactivity is present and/or relaxin-3 binding sites have been demonstrated (44). Many of these nuclei are known to influence energy balance. We sought to map the hypothalamic areas where relaxin-3 influences energy intake by injection of relaxin-3 into regions demonstrating RXFP3 expression or relaxin binding and which have previously been implicated in regulation of food intake. To investigate which hypothalamic areas are activated in response to relaxin-3, immunohistochemistry for the early gene c-fos was examined following administration of relaxin-3 into the lateral ventricle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Human relaxin-3 (H3) was purchased from Phoenix Pharmaceuticals (Belmont, CA) and synthesized by Phoenix using solid phase synthesis. H3 was dissolved in vehicle (10% acetonitrile in 0.9% saline) for all studies.

Animals. Male Wistar rats (Specific Pathogen Free; Charles River) weighing 200–250 g were maintained in individual cages for all studies. Animals were housed in conditions of controlled light (12:12-h light-dark, lights on at 0700) and temperature (21°C) with ad libitum access to water and food (RM1 diet; Special Diet Service). All studies undertaken were approved under the British Home Office Animals (Scientific Procedures) Act of 1986 (project license 70/5516).

Intranuclear cannulation and injection. Surgical procedures and handling were carried out as previously described (29). Briefly, animals were implanted with permanent 26-gauge stainless steel guide cannulae (Plastics One, Roanoke, VA) projecting into the anterior preoptic area (APOA), medial preoptic area (MPOA), SON, dorsomedial hypothalamic nucleus (DMH), ARC, and lateral hypothalamic area (LHA) according to the coordinates based on the atlas of Paxinos and Watson (Table 1 and Fig. 1) (33). Animals were allowed to recover for 7 days before studies commenced and were habituated to handling and the injection procedure.


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  		Table 1. Coordinates of intranuclear injection

 

Figure 1
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  		Fig. 1. Schematic representation of coronal sections through the rat hypothalamus [Paxinos G and Watson C (33)] depicting the injection site and position from bregma with respect to each hypothalamic nucleus cannulated in the present study. The injection site is magnified on the right of each coronal section. APOA, anterior preoptic area; MPOA, medial preoptic area; SON, supraoptic nucleus; DMH, dorsomedial hypothalamic nucleus; ARC, arcuate nucleus; LHA, lateral hypothalamic area. +Rostral distance from bregma; –caudal distance from bregma. Reprinted from The Rat Brain in Stereotaxic Coordinates (4th ed.) (Figs. 16, 21, 23, 32, 33, and 34), with permission from Elsevier.

 
Intranuclear injections of H3 (180 pmol) or vehicle were administered in 1 µl for 1 min by a 33-gauge stainless steel injector placed in and projecting 1 mm below the end of the guide cannula. The dose of H3 used results in a submaximal stimulation of food intake in intraparaventricular (i)PVN dose-response studies (28). An injection of 1 µl is reported to diffuse 1 mm3 in the hypothalamic PVN (47). H3 was dissolved in vehicle (as described above), and studies were performed in the early light phase (0900–1000) in ad libitum-fed rats. Animals were returned to their home cage following injection with preweighed chow, and food was weighed at 1, 2, 4, 8, and 24 h after injection.

Cannula position was verified histologically at the end of the study, as previously described (48). Briefly, immediately following death, 1 µl of india ink was injected intranuclearly and guide cannulae removed. The brains were rapidly dissected, fixed in 4% paraformaldahyde, dehydrated in 40% sucrose, and frozen in liquid nitrogen. A freezing sled microtome (Shandon Southern Products, Cheshire, UK) was used to take 20-µm coronal sections. Sections were compared with the corresponding section from the rat brain atlas (33). Data from animals were excluded if the injection site extended >0.2 mm outside the intended injection site or if any ink was detected in the cerebral ventricular system. The final numbers of animals with correct cannula placement, and therefore included in the analysis, were APOA (n = 9–10/group), MPOA (n = 8–10/group), SON (n = 7/group), DMH (n = 8–10/group), ARC (n = 7/group), and LHA (n = 9–11/group).

Lateral ventricle cannulation and injection. Surgical procedures and handling were carried out as previously described (29). Briefly, animals were implanted with 22-gauge stainless steel cannulae (Plastics One) projecting into the lateral ventricle (LV). Animals were allowed to recover for 7 days before studies commenced and were habituated to handling and the injection procedure. LV injections of 5 µl were administered for 1 min via a 28-gauge stainless steel injector. Correct cannula position was verified by a positive orexigenic response to neuropeptide Y (NPY; 5 nmol) given in the early light phase. Animals consuming <2 g of food in the 2 h following NPY administration were excluded from the study. Administration of vehicle or H3 to determine Fos-like immunoreactivity was performed in the early light phase (0900–1000) in ad libitum-fed rats.

Immunohistochemical measurement of Fos-like immunoreactivity following relaxin-3 administration. Fos-like immunoreactivity (FLI), as a marker of neuronal activation, was determined in response to H3, as previously described (31). Briefly, LV-cannulated rats received either vehicle or H3 (540 pmol; n = 5/group) into the LV. They were returned to their home cage with food removed. The LV dose of H3 administered was three times higher than the dose used for the intranuclear studies and based on pilot data (not shown). Ninety minutes after injection, animals were terminally anesthetized and transcardially perfused with phosphate buffered saline (PBS; 0.1 M, pH 7.4) followed by 4% paraformaldehyde in PBS. Brains were immediately removed and placed in 4% paraformaldehyde in 0.1 M PBS and transferred to 40% sucrose 24 h later. They were frozen at –80°C and cut into 40-µm sections. For each brain, sections ~120 µm apart, covering a region extending from the rostral aspect of the APOA to the caudal aspect of the LH, were stained and examined as described below.

Sections were immersed in 0.6% hydrogen peroxide in methanol (vol/vol) for 30 min to inactivate endogenous peroxidase activity, followed by incubation with 3% normal goat serum for 2 h to block nonspecific binding. The primary antibody, rabbit anti-c-fos (Ab-5; Calbiochem, San Diego, CA), was applied in a 1:20,000 dilution overnight at room temperature. Slides were then incubated for 2 h with biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA), dilution 1:400, followed by avidin-biotin-peroxidase complex (1:220) for 1 h (Dako Cytomation, Glostrup, Denmark). The antigen-antibody complex was visualized with 3,3'-diaminobenzidine in 0.01% hydrogen peroxide. Sections were then mounted. Quantification of FLI was performed manually directly from the microscope image by an observer blinded to the treatment group, using a Nikon Eclipse E800 microscope. The number of matched sections examined per animal for the reported nuclei are as follows: SON 3–4 sections, PVN 2–3 sections, and ARC 5–7 sections.

Statistical analysis. Food intake data are shown as means ± SE. The effect of relaxin-3 on food intake following administration into a hypothalamic nucleus was compared with vehicle by unpaired Student's t-test at each time point. Fos-like immunoreactivity data, expressed as the mean number of FLI-positive neurons per section, was nonparametric and compared using Kruskal-Wallis one-way ANOVA on ranks with post hoc Student-Newman-Keuls method. In all cases, P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Localization of the hypothalamic sites of the orexigenic action of relaxin-3. Animals received either vehicle or H3 (180 pmol) in one of six different hypothalamic nuclei in the early light phase. H3 significantly increased food intake in the first hour following injection into the SON, ARC, and the APOA [0- to 1-h food intake: SON 0.4 ± 0.2 (vehicle) vs. 2.9 ± 0.5 g (H3), P < 0.001; ARC 0.7 ± 0.3 (vehicle) vs. 2.7 ± 0.2 g (H3), P < 0.05; and APOA 0.8 ± 0.1 (vehicle) vs. 2.2 ± 0.2 g (H3), P < 0.05] (Fig. 2A). There was no significant difference in interval food intake between control and treated groups at later time points.


Figure 2
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  		Fig. 2. Effect of human relaxin-3 (H3; 180 pmol) on food intake in the 6 different sites cannulated as %vehicle control, with control food intake (100%) indicated by dotted line. A: 0- to 1-h food intake. B: 24-h food intake. *P < 0.05; ***P < 0.001 vs. vehicle.

 
The greatest orexigenic response in the first hour was seen in the SON, where cumulative food intake was also significantly increased ≤8 h following peptide administration [0- to 8-h food intake: SON 1.8 ± 0.6 (vehicle) vs. 4.5 ± 0.6 g (H3), P < 0.01]. (Fig. 3C). A significant increase in cumulative food intake was also observed in the APOA ≤4 h following administration [0- to 4-h food intake: APOA 1.1 ± 0.2 (vehicle) vs. 3.2 ± 0.3 g (H3), P < 0.05] (Fig. 3A). In the ARC, food intake was significantly increased in the first hour following administration, with no significant difference in cumulative food intake at later time points (Fig. 3E).


Figure 3
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  		Fig. 3. Effect of H3 (180 pmol; {blacktriangleup}) on 0- to 8-h food intake (g) in the 6 different sites cannulated compared with vehicle ({blacksquare}). A: APOA. B: MPOA. C: SON. D: DMH. E: ARC. F: LHA. *P < 0.05; **P < 0.01; ***P < 0.001 vs. vehicle.

 
There was no significant difference in food intake following injection of H3 in the MPOA (Fig. 3B), DMH (Fig. 3D), or LHA (Fig. 3F) at any time point. Twenty-four-hour food intake was not significantly altered following H3 injection into any area (Fig. 2B).

FLI in the hypothalamus in response to relaxin-3. Hypothalamic FLI was examined following intracerebroventricular (ICV) administration of H3. A 130% increase in FLI was observed in the SON in response to H3 compared with vehicle-treated controls [FLI-positive neurons per section (SON): vehicle 8.1 ± 2.0 vs. H3 18.6 ± 3.2, P < 0.05; Fig. 4, A and B]. However, there were no significant differences in FLI between the control and treatment groups in the PVN [FLI-positive neurons per section (PVN): vehicle 120.4 ± 22.0 vs. H3 101.0 ± 5.5] or ARC [FLI-positive neurons per section (ARC): vehicle 56.9 ± 3.0 vs. H3 52.6 ± 6.5; Fig. 4, C and D]. Although not formally quantified, there was no apparent difference in FLI in the MPOA, APOA, DMH, and LHA between vehicle and H3-treated animals.


Figure 4
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  		Fig. 4. Representative photomicrograph showing Fos-like immunoreactivity (FLI) in response to administration into the lateral ventricle. A: FLI in the SON (vehicle; x200 magnification). B: FLI in the SON (H3, 540 pmol; x200 magnification). C: FLI in the ARC (vehicle; x100 magnification). D: FLI in the ARC (H3; 540 pmol x100 magnification). Arrow shows localization of FLI. 3V, 3rd ventricle; OX, optic chiasma.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Relaxin-3 is a recently described member of the insulin superfamily, and its roles and sites of action are largely unknown. Our previous work (28, 29) demonstrated a significant orexigenic effect of H3 following ICV and PVN administration. These studies extend previous data to demonstrate that H3 is a potent orexigen following administration into several hypothalamic nuclei.

Relaxin-3 binds to and activates at least two hypothalamic GPCRs: RXFP1 and RXFP3. Evidence from binding studies (with nonspecific ligands such as relaxin-1 and more specific ligands such as relaxin-3/insulin-like peptide-5 chimeric peptide) and in situ hybridization suggests that these receptors are widespread in the hypothalamus, PVN, SON, ARC, LHA, and preoptic areas (32, 42, 43).

In this study, the SON is the hypothalamic area most sensitive to the orexigenic actions of H3. Food intake increased more than sevenfold compared with control following injection in this area. In keeping with previous studies (29), the orexigenic response does not extend beyond the first hour, although cumulative food intake is increased for ≤8 h. Administration of several neuropeptides into the SON has been demonstrated to influence food intake in previous studies. Ghrelin (48) and cocaine- and amphetamine-regulated transcript (CART) (2) significantly stimulate food intake, whereas cholecystokinin (6) inhibits food intake after SON administration. Numerous neuropeptides are expressed within the SON, including those known to modulate food intake, such as NPY (23), galanin (17), and CART (15). In addition, the neurons in the SON respond to peripheral signals such as leptin (21). However, no leptin receptors have been demonstrated in this region (16), and therefore, these actions may be indirect.

In keeping with the orexigenic action of H3 seen in the SON, central administration of H3 induced a small but significant increase in FLI in this area. Relaxin-3 immunoreactive fibers (44) and RXFP-3 receptors are both present in the SON, and thus increased FLI may be a consequence of H3 binding to RXFP-3. RXFP-1 is also expressed in the SON, and although it is the cognate receptor for relaxin-1, it also binds relaxin-3 with lower affinity. Previous work (30, 41) has demonstrated peripheral and ICV administration of relaxin-1 to stimulate water intake and induce FLI in the magnocellular neurons of the SON. It has been shown recently (4) that central administration of relaxin-3 results in a significant increase in water intake. Thus FLI in the SON may be in keeping with binding and stimulation of RXFP-1 receptors by H3. It would be interesting to determine whether relaxin-3-induced FLI and relaxin-3 receptors are colocalized with vasopressin neurons, indicating a role in water homeostasis or with an alternative cell population that may act to modulate food intake. However, relaxin-3 in the SON may also be part of a stress "circuit." Relaxin-3-expressing neurons in the NI exhibit FLI in response to stress (44), and therefore, projections from the NI to the SON may possibly play a role in stress-related vasopressin release. Relaxin-3 immunoreactive fibers in the SON also raise the possibility of an autocrine role for relaxin-3 in this nucleus (9). Whether relaxin-3 receptors are localized to these immunoreactive fibers, and if so, whether they play an inhibitory or excitatory role, would also be of great interest.

Binding to either RXFP-3 or RXFP-1 may result in FLI as a direct effect of H3 within the SON. However, an indirect induction of FLI by H3 may also be possible. The major inputs into the SON are from extrahypothalamic areas, the subfornical organ, medial and lateral septum, and the median preoptic nuclei within the forebrain and from the A1 region and nucleus of the solitary tract within the brain stem (46). However, the NI projects to the ventral tegmental area and the zona incerta (18) that provide inputs to the SON (46) and may provide an indirect pathway for relaxin-3 induced activation of the SON.

ARC administration of H3 led to a fourfold increase in food intake in the first hour following administration. The ARC has long been known to influence energy homeostasis. Lesions of the ARC by neonatal monosodium glutamate administration produce obesity (8). Intra-arcuate administration of many peptides alters feeding, including NPY (7), melanin-concentrating hormone (1), neuromedin U (49), and ghrelin (48). The ARC also expresses several neuropeptides that regulate energy balance (38) and are modulated by peripheral signals such as leptin (12).

We have also shown that H3 administration into the APOA significantly increases food intake. Although the preoptic area is not traditionally associated with regulation of energy balance, leptin and estrogen administration in this area modulate energy balance (3, 13), and lesioning of preoptic areas alters food intake and body weight (27). However, neither RXFP3 nor RXFP1 receptor expression or binding have been reported in the preoptic area in rats, although RXFP3 expression is present in the preoptic area of the mouse (42).

Interestingly, no FLI was observed in either the ARC or preoptic area despite significant increases in food intake following relaxin-3 administration. Although statistically significant, the increased FLI in the SON following relaxin-3 administration is small, and since it is absent in both the ARC and preoptic area, it is possible that c-fos expression is not a marker of neurons activated by relaxin-3 feeding pathways. Dissociation between c-fos expression and neuronal depolarization has been described previously (22). One of the principal pathways for c-fos induction is via increased intracellular cAMP. The second messenger pathway of RXFP-3, the cognate receptor for relaxin-3, is not well understood. However, in contrast to RXFP-1, binding and stimulation of RXFP-3 decreases intracellular cAMP. Other immediate early gene products have also been used to monitor neuronal activity, although these tend to be less reliable markers than c-fos (22). It may be interesting to examine the expression of these markers, but electrophysiological recording from RXFP-3-expressing cells would provide more information regarding neuronal firing in response to relaxin-3. Both electrophysiological (39) and tracing studies (14) have demonstrated that extensive connectivity exists between hypothalamic nuclei, including pathways that are leptin-responsive. For example, reciprocal connections exist between the ARC and suprachiasmatic nucleus and between the suprachiasmatic nucleus and the SON (34). Thus relaxin-3-induced neuronal firing in one hypothalamic region, such as the SON, may influence activity in several additional hypothalamic nuclei.

The receptor mediating the actions of H3 on food intake is unknown. Previous work (29) has shown that relaxin-1, which binds RXFP-1, does not significantly increase food intake, suggesting that the orexigenic actions of H3 may be mediated by RXFP-3. H3 significantly increased food intake in both the ARC and APOA, but in the rat, the RXFP-3-specific ligand relaxin-3/insulin-like peptide-5 chimeric peptide does not bind in these regions, and no RXFP-3 expression has been reported (24, 32, 43). Similarly, administration of H3 into the LHA had no effect on food intake, yet low-level RXFP-3 expression has been described here (43). Therefore, it is possible that the orexigenic actions of relaxin-3 may be mediated by a novel receptor. It would be interesting to examine the effect of the RXFP-3-specific ligand relaxin-3/insulin-like peptide-5 chimeric peptide administration on energy intake.

Relaxin-3 immunoreactivity has been demonstrated within fibers in the LH, ARC, and sparsely in the PVN (44). This raises the possibility of an autocrine role for relaxin-3. However, as described above, the cognate receptor for relaxin-3, RXFP-3, is not found within the ARC (11, 26, 42, 43), although RXFP-1 is expressed here (10). Therefore, in the ARC, relaxin-3 may have an autocrine role on RXFP-1 receptors. Both RXFP-3 receptors and relaxin-3 immunopositive fibers are expressed in the PVN, and here it may act in an autocrine fashion to modulate either feeding or endocrine function (29). Dual in situ hybridization for RXFP-3 and immunohistochemistry for relaxin-3 would be needed to confirm whether RXFP-3 mRNA and relaxin-3 immunoreactivity are localized to the same neurons.

The dose of H3 administered in this study was submaximal to minimize diffusion between hypothalamic nuclei, but this possibility cannot be excluded. If diffusion from the administration site to a responsive area, such as the PVN, were responsible for the orexigenic effects seen, one might expect an increase in food intake following injection into those areas closest to the PVN. However, there was no acute orexigenic effect following either MPOA or LHA administration of H3 even though these areas are anatomically closer to the responsive PVN than the SON, APOA, or ARC. In addition, diffusion might be expected to result in a delayed feeding response, but relaxin-3 administration in the SON, APOA, and ARC results in a prompt increase in food intake. The acute results obtained here are therefore unlikely to be due to diffusion.

In summary, we have shown that relaxin-3 significantly stimulates feeding in several hypothalamic nuclei, the SON, ARC, and APOA, in addition to its known orexigenic effects when administered into the PVN. The orexigenic actions in the ARC, in the absence of reported RXFP-3 receptor expression, raise the possibility of an additional relaxin receptor. We have also demonstrated a small increase in FLI in the SON in response to relaxin-3 but no change in FLI with relaxin-3 in the ARC or preoptic area despite significant orexigenic actions. It would be interesting to examine the electrophysiological effects of relaxin-3 on RXFP-3-expressing cells and characterize the responsive neurons in these areas.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work has been supported by a Medical Research Council (MRC) grant. B. M. McGowan is an MRC research fellow. S. A. Stanley is an MRC clinician scientist. N. E. White is funded by the Biotechnology and Biological Sciences Research Council (BBSRC). A. Spangeus is supported by Futurum Research Council, Jönköping County Council, the Swedish Society of Medicine, and Henning and Johan Throne-Holst's foundation. E. L. Thompson is supported by BBSRC/GlaxoSmithkline Case studentship. M. Patterson is supported by the BBSRC.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Bloom, Dept. of Metabolic Medicine, Imperial College London, 6th Floor Commonwealth Bldg., Hammersmith Hospital, Du Cane Road, London W12 0NN, UK (e-mail: s.bloom{at}imperial.ac.uk)

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.

* These authors contributed equally to the studies. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abbott CR, Kennedy AR, Wren AM, Rossi M, Murphy KG, Seal LJ, Todd JF, Ghatei MA, Small CJ, Bloom SR. Identification of hypothalamic nuclei involved in the orexigenic effect of melanin-concentrating hormone. Endocrinology 144: 3943–3949, 2003.[Abstract/Free Full Text]
  2. Abbott CR, Rossi M, Wren AM, Murphy KG, Kennedy AR, Stanley SA, Zollner AN, Morgan DG, Morgan I, Ghatei MA, Small CJ, Bloom SR. Evidence of an orexigenic role for cocaine- and amphetamine-regulated transcript after administration into discrete hypothalamic nuclei. Endocrinology 142: 3457–3463, 2001.[Abstract/Free Full Text]
  3. Bagnasco M, Dube MG, Kalra PS, Kalra SP. Evidence for the existence of distinct central appetite, energy expenditure, and ghrelin stimulation pathways as revealed by hypothalamic site-specific leptin gene therapy. Endocrinology 143: 4409–4421, 2002.[Abstract/Free Full Text]
  4. Bathgate RA, Lin F, Hanson NF, Otvos L Jr, Guidolin A, Giannakis C, Bastiras S, Layfield SL, Ferraro T, Ma S, Zhao C, Gundlach AL, Samuel CS, Tregear GW, Wade JD. Relaxin-3: improved synthesis strategy and demonstration of its high-affinity interaction with the relaxin receptor LGR7 both in vitro and in vivo. Biochemistry 45: 1043–1053, 2006.[CrossRef][Medline]
  5. Bathgate RA, Samuel CS, Burazin TC, Gundlach AL, Tregear GW. Relaxin: new peptides, receptors and novel actions. Trends Endocrinol Metab 14: 207–213, 2003.[CrossRef][Web of Science][Medline]
  6. Blevins JE, Stanley BG, Reidelberger RD. Brain regions where cholecystokinin suppresses feeding in rats. Brain Res 860: 1–10, 2000.[CrossRef][Web of Science][Medline]
  7. Bouali SM, Fournier A, St-Pierre S, Jolicoeur FB. Effects of NPY and NPY2–36 on body temperature and food intake following administration into hypothalamic nuclei. Brain Res Bull 36: 131–135, 1995.[CrossRef][Web of Science][Medline]
  8. Bunyan J, Murrell EA, Shah PP. The induction of obesity in rodents by means of monosodium glutamate. Br J Nutr 35: 25–39, 1976.[CrossRef][Web of Science][Medline]
  9. Burazin TC, Bathgate RA, Macris M, Layfield S, Gundlach AL, Tregear GW. Restricted, but abundant, expression of the novel rat gene-3 (R3) relaxin in the dorsal tegmental region of brain. J Neurochem 82: 1553–1557, 2002.[CrossRef][Web of Science][Medline]
  10. Burazin TC, Johnson KJ, Ma S, Bathgate RA, Tregear GW, Gundlach AL. Localization of LGR7 (relaxin receptor) mRNA and protein in rat forebrain: correlation with relaxin binding site distribution. Ann NY Acad Sci 1041: 205–210, 2005.[CrossRef][Web of Science][Medline]
  11. Chen J, Kuei C, Sutton SW, Bonaventure P, Nepomuceno D, Eriste E, Sillard R, Lovenberg TW, Liu C. Pharmacological characterization of relaxin-3/INSL7 receptors GPCR135 and GPCR142 from different mammalian species. J Pharmacol Exp Ther 312: 83–95, 2005.[Abstract/Free Full Text]
  12. Cone RD, Cowley MA, Butler AA, Fan W, Marks DL, Low MJ. The arcuate nucleus as a conduit for diverse signals relevant to energy homeostasis. Int J Obes Relat Metab Disord 25, Suppl 5: S63–S67, 2001.
  13. Dagnault A, Richard D. Involvement of the medial preoptic area in the anorectic action of estrogens. Am J Physiol Regul Integr Comp Physiol 272: R311–R317, 1997.[Abstract/Free Full Text]
  14. DeFalco J, Tomishima M, Liu H, Zhao C, Cai X, Marth JD, Enquist L, Friedman JM. Virus-assisted mapping of neural inputs to a feeding center in the hypothalamus. Science 291: 2608–2613, 2001.[Abstract/Free Full Text]
  15. Elias CF, Lee CE, Kelly JF, Ahima RS, Kuhar M, Saper CB, Elmquist JK. Characterization of CART neurons in the rat and human hypothalamus. J Comp Neurol 432: 1–19, 2001.[CrossRef][Web of Science][Medline]
  16. Elmquist JK, Bjorbaek C, Ahima RS, Flier JS, Saper CB. Distributions of leptin receptor mRNA isoforms in the rat brain. J Comp Neurol 395: 535–547, 1998.[CrossRef][Web of Science][Medline]
  17. Giorgi S, Forloni G, Amoroso D, Consolo S. Expression of GAL mRNA in rat hypothalamus: effect of frontal deafferentation and colchicine treatment. Brain Res Mol Brain Res 14: 277–281, 1992.[Medline]
  18. Goto M, Swanson LW, Canteras NS. Connections of the nucleus incertus. J Comp Neurol 438: 86–122, 2001.[CrossRef][Web of Science][Medline]
  19. Hsu SY, Nakabayashi K, Nishi S, Kumagai J, Kudo M, Sherwood OD, Hsueh AJ. Activation of orphan receptors by the hormone relaxin. Science 295: 671–674, 2002.[Abstract/Free Full Text]
  20. Hudson P, Haley J, John M, Cronk M, Crawford R, Haralambidis J, Tregear G, Shine J, Niall H. Structure of a genomic clone encoding biologically active human relaxin. Nature 301: 628–631, 1983.[CrossRef][Medline]
  21. Isse T, Ueta Y, Serino R, Noguchi J, Yamamoto Y, Nomura M, Shibuya I, Lightman SL, Yamashita H. Effects of leptin on fasting-induced inhibition of neuronal nitric oxide synthase mRNA in the paraventricular and supraoptic nuclei of rats. Brain Res 846: 229–235, 1999.[CrossRef][Web of Science][Medline]
  22. Kong WM, Martin NM, Smith KL, Gardiner JV, Connoley IP, Stephens DA, Dhillo WS, Ghatei MA, Small CJ, Bloom SR. Triiodothyronine stimulates food intake via the hypothalamic ventromedial nucleus independent of changes in energy expenditure. Endocrinology 145: 5252–5258, 2004.[Abstract/Free Full Text]
  23. Larsen PJ, Mikkelsen JD, Jessop DS, Lightman SL, Chowdrey HS. Neuropeptide Y mRNA and immunoreactivity in hypothalamic neuroendocrine neurons: effects of adrenalectomy and chronic osmotic stimulation. J Neurosci 13: 1138–1147, 1993.[Abstract]
  24. Liu C, Chen J, Kuei C, Sutton S, Nepomuceno D, Bonaventure P, Lovenberg TW. Relaxin-3/insulin-like peptide 5 chimeric peptide, a selective ligand for G protein-coupled receptor (GPCR)135 and GPCR142 over leucine-rich repeat-containing G protein-coupled receptor 7. Mol Pharmacol 67: 231–240, 2005.[Abstract/Free Full Text]
  25. Liu C, Chen J, Sutton S, Roland B, Kuei C, Farmer N, Sillard R, Lovenberg TW. Identification of relaxin-3/INSL7 as a ligand for GPCR142. J Biol Chem 278: 50765–50770, 2003.[Abstract/Free Full Text]
  26. Liu C, Eriste E, Sutton S, Chen J, Roland B, Kuei C, Farmer N, Jornvall H, Sillard R, Lovenberg TW. Identification of relaxin-3/INSL7 as an endogenous ligand for the orphan G-protein-coupled receptor GPCR135. J Biol Chem 278: 50754–50764, 2003.[Abstract/Free Full Text]
  27. Mascarenhas JF. Role of medial preoptic area (MPOA) in the reproductive function and feeding behavior in rats. Indian J Physiol Pharmacol 30: 232–240, 1986.[Medline]
  28. McGowan BM, Stanley SA, Smith KL, Minnion JS, Donovan J, Thompson EL, Patterson M, Connolly MM, Abbott CR, Small CJ, Gardiner JV, Ghatei MA, Bloom SR. Effects of acute and chronic relaxin-3 on food intake and energy expenditure in rats. Regul Pept 136: 72–77, 2006.[CrossRef][Web of Science][Medline]
  29. McGowan BM, Stanley SA, Smith KL, White NE, Connolly MM, Thompson EL, Gardiner JV, Murphy KG, Ghatei MA, Bloom SR. Central relaxin-3 administration causes hyperphagia in male Wistar rats. Endocrinology 146: 3295–3300, 2005.[Abstract/Free Full Text]
  30. McKinley MJ, Burns P, Colvill LM, Oldfield BJ, Wade JD, Weisinger RS, Tregear GW. Distribution of Fos immunoreactivity in the lamina terminalis and hypothalamus induced by centrally administered relaxin in conscious rats. J Neuroendocrinol 9: 431–437, 1997.[CrossRef][Web of Science][Medline]
  31. Neary NM, Small CJ, Druce MR, Park AJ, Ellis SM, Semjonous NM, Dakin CL, Filipsson K, Wang F, Kent AS, Frost GS, Ghatei MA, Bloom SR. Peptide YY3-36 and glucagon-like peptide-17-36 inhibit food intake additively. Endocrinology 146: 5120–5127, 2005.[Abstract/Free Full Text]
  32. Osheroff PL, Phillips HS. Autoradiographic localization of relaxin binding sites in rat brain. Proc Natl Acad Sci USA 88: 6413–6417, 1991.[Abstract/Free Full Text]
  33. Paxinos G, Watson C. The Rat Brain in Stereotactic Coordinates (4th ed.). New York: 1998.
  34. Saeb-Parsy K, Lombardelli S, Khan FZ, McDowall K, Au-Yong IT, Dyball RE. Neural connections of hypothalamic neuroendocrine nuclei in the rat. J Neuroendocrinol 12: 635–648, 2000.[CrossRef][Web of Science][Medline]
  35. Shen PJ, Fu P, Phelan KD, Scott DJ, Layfield S, Tregear GW, Bathgate RA, Gundlach AL. Restricted expression of LGR8 in intralaminar thalamic nuclei of rat brain suggests a role in sensorimotor systems. Ann NY Acad Sci 1041: 510–515, 2005.[CrossRef][Web of Science][Medline]
  36. Sherwood OD. Relaxin's physiological roles and other diverse actions. Endocr Rev 25: 205–234, 2004.[Abstract/Free Full Text]
  37. Sinnayah P, Burns P, Wade JD, Weisinger RS, McKinley MJ. Water drinking in rats resulting from intravenous relaxin and its modification by other dipsogenic factors. Endocrinology 140: 5082–5086, 1999.[Abstract/Free Full Text]
  38. Stanley S, Wynne K, McGowan B, Bloom S. Hormonal regulation of food intake. Physiol Rev 85: 1131–1158, 2005.[Abstract/Free Full Text]
  39. Sternson SM, Shepherd GM, Friedman JM. Topographic mapping of VMH -> arcuate nucleus microcircuits and their reorganization by fasting. Nat Neurosci 8: 1356–1363, 2005.[CrossRef][Web of Science][Medline]
  40. Sudo S, Kumagai J, Nishi S, Layfield S, Ferraro T, Bathgate RA, Hsueh AJ. H3 relaxin is a specific ligand for LGR7 and activates the receptor by interacting with both the ectodomain and the exoloop 2. J Biol Chem 278: 7855–7862, 2003.[Abstract/Free Full Text]
  41. Sunn N, Egli M, Burazin TC, Burns P, Colvill L, Davern P, Denton DA, Oldfield BJ, Weisinger RS, Rauch M, Schmid HA, McKinley MJ. Circulating relaxin acts on subfornical organ neurons to stimulate water drinking in the rat. Proc Natl Acad Sci USA 99: 1701–1706, 2002.[Abstract/Free Full Text]
  42. Sutton SW, Bonaventure P, Kuei C, Nepomuceno D, Wu J, Zhu J, Lovenberg TW, Liu C. G-protein-coupled receptor (GPCR)-142 does not contribute to relaxin-3 binding in the mouse brain: further support that relaxin-3 is the physiological ligand for GPCR135. Neuroendocrinology 82: 139–150, 2005.[CrossRef][Web of Science][Medline]
  43. Sutton SW, Bonaventure P, Kuei C, Roland B, Chen J, Nepomuceno D, Lovenberg TW, Liu C. Distribution of G-protein-coupled receptor (GPCR)135 binding sites and receptor mRNA in the rat brain suggests a role for relaxin-3 in neuroendocrine and sensory processing. Neuroendocrinology 80: 298–307, 2004.[CrossRef][Web of Science][Medline]
  44. Tanaka M, Iijima N, Miyamoto Y, Fukusumi S, Itoh Y, Ozawa H, Ibata Y. Neurons expressing relaxin 3/INSL 7 in the nucleus incertus respond to stress. Eur J Neurosci 21: 1659–1670, 2005.[Web of Science][Medline]
  45. Thornton SM, Fitzsimons JT. The effects of centrally administered porcine relaxin on drinking behaviour in male and female rats. J Neuroendocrinol 7: 165–169, 1995.[CrossRef][Web of Science][Medline]
  46. Tribollet E, Armstrong WE, Dubois-Dauphin M, Dreifuss JJ. Extra-hypothalamic afferent inputs to the supraoptic nucleus area of the rat as determined by retrograde and anterograde tracing techniques. Neuroscience 15: 135–148, 1985.[CrossRef][Web of Science][Medline]
  47. Ward H, Gardiner J, Kong WM, Murphy K, Bloom S. Distribution of fluorescence following injection of recombinant adeno-associated virus encoding green fluorescent protein into the paraventricular nucleus. Neuroendocrinology 77: 100–104, 2003.[CrossRef][Web of Science][Medline]
  48. Wren AM, Small CJ, Abbott CR, Dhillo WS, Seal LJ, Cohen MA, Batterham RL, Taheri S, Stanley SA, Ghatei MA, Bloom SR. Ghrelin causes hyperphagia and obesity in rats. Diabetes 50: 2540–2547, 2001.[Abstract/Free Full Text]
  49. 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]



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B. M. McGowan, S. A. Stanley, J. Donovan, E. L. Thompson, M. Patterson, N. M. Semjonous, J. V. Gardiner, K. G. Murphy, M. A. Ghatei, and S. R. Bloom
Relaxin-3 stimulates the hypothalamic-pituitary-gonadal axis
Am J Physiol Endocrinol Metab, August 1, 2008; 295(2): E278 - E286.
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