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Central Relaxin-3 Administration Causes Hyperphagia in Male Wistar Rats
     Endocrine Unit, Imperial College School of Medicine, Hammersmith Hospital, London W12 ONN, United Kingdom

    Address all correspondence and requests for reprints to: Stephen Bloom, Department of Metabolic Medicine, Imperial College, Hammersmith Campus, 6th Floor Commonwealth Building, Du Cane Road, London W12 0NN, United Kingdom. E-mail: s.bloom@imperial.ac.uk.

    Abstract

    Relaxin-3 (INSL-7) is a recently discovered member of the insulin superfamily. Relaxin-3 mRNA is expressed in the nucleus incertus of the brainstem, which has projections to the hypothalamus. Relaxin-3 binds with high affinity to the LGR7 receptor and to the previously orphan G protein-coupled receptor GPCR135. GPCR135 mRNA is expressed predominantly in the central nervous system, particularly in the paraventricular nucleus (PVN). The presence of relaxin-3 and these receptors in the PVN led us to investigate the effect of central administration of relaxin-3 on food intake in male Wistar rats. The receptor involved in mediating these effects was also investigated. Intracerebroventricular injections of human relaxin-3 (H3) to satiated rats significantly increased food intake 1 h post administration in the early light phase [0.96 ± 0.16 g (vehicle) vs. 1.81 ± 0.21 g (180 pmol H3), P < 0.05] and the early dark phase [2.95 ± 0.45 g (vehicle) vs. 4.39 ± 0.39 g (180 pmol H3), P < 0.05]. Intra-PVN H3 administration significantly increased 1-h food intake in satiated rats in the early light phase [0.34 ± 0.16 g (vehicle) vs. 1.23 ± 0.30 g (18 pmol H3), P < 0.05] and the early dark phase [4.43 ± 0.32 g (vehicle) vs. 6.57 ± 0.42 g (18 pmol H3), P < 0.05]. Feeding behavior increased after intra-PVN H3. Equimolar doses of human relaxin-2, which binds the LGR7 receptor but not GPCR135, did not increase feeding. Hypothalamic neuropeptide Y, proopiomelanocortin, or agouti-related peptide mRNA expression did not change after acute intracerebroventricular H3. These results suggest a novel role for relaxin-3 in appetite regulation.

    Introduction

    THE RELAXIN PEPTIDES belong to the insulin superfamily, a group of structurally related hormones typified by the presence of an A and B chain linked by disulfide bridges and an intra-chain disulfide bond (1). Until recently, a single relaxin gene, named M1 and R1 in mice and rats, respectively, (2, 3) and H2 in humans (4), had been described in most mammalian species. The gene product is secreted by the corpus luteum in early pregnancy and is primarily associated with female reproductive physiology, as well as having a dipsogenic effect when administered peripherally or centrally (5, 6). However, a further relaxin gene, relaxin-3, has now been identified in humans (H3) (7), mice (M3) (7), and most recently in rats (R3) (8). The gene products of H3, M3, and R3 retain their insulin-like peptide structure and are highly homologous. R1 and M1 mRNA are widely expressed, but R3 and M3 mRNA expression is localized to the nucleus incertus of the brainstem (8), which projects extensively to hypothalamic regions including the lateral mammillary nucleus, the supramammillary nucleus, the posterior hypothalamic nucleus, and the lateral hypothalamic zone. There are also weaker projections to the medial and periventricular zones (9). Relaxin-like immunoreactivity has been described in the hypothalamic arcuate and paraventricular nuclei (PVN) (10).

    Unlike insulin, relaxin peptides signal via G protein-coupled receptors to modulate intracellular cAMP. The gene products of R1 and M1 act via two leucine-rich repeat-containing receptors, LGR7 and LGR8 (11). More recent studies suggest that relaxin-1 may be the endogenous ligand for LGR7 and another insulin-like peptide, INSL3, may be the physiological ligand for LGR8 (12). LGR7, expressed predominantly in reproductive tissues but also in the central nervous system (11), binds relaxin-3 with high affinity (13). However, relaxin-3 is the cognate ligand for two previously orphan G protein-coupled receptors, GPCR135 and GPCR142 (14, 15). GPCR142 is not expressed in the rat, but GPCR135 mRNA is highly expressed in the rat brain, particularly the PVN and the supraoptic nucleus (14, 16). The distribution of relaxin-3 and its receptors suggest this system could play a role in the regulation of appetite. The aims of these studies were to investigate the effects of relaxin-3 on food intake and to examine which receptor may mediate these effects.

    Materials and Methods

    Materials

    Human relaxin-3 (H3) was purchased from Phoenix Pharmaceuticals (Belmont, CA) and synthesized by the company using solid phase synthesis. Recombinant human relaxin-2 (H2) was purchased from Dr. A. Parlow, National Hormone and Peptide Program (Torrance, CA). Reagents for ribonuclease protection assay studies were purchased from Ambion (Austin, TX).

    Animal studies

    Male Wistar rats (specific pathogen-free; Charles River Laboratories UK Ltd., Margate, Kent, UK) weighing 250–300 g were maintained in individual cages for all studies. All animals were kept under controlled temperature (21–23 C) and light (12 h light, 12 h dark, lights on at 0700 h) with ad libitum access to food (pelleted RM1 chow diet; SDS Ltd., Witham, UK) and water. All procedures undertaken were approved by the British Home Office Animals Scientific Procedures Act 1986 (project license 70/5516).

    Food and water intake studies

    Male Wistar rats underwent third ventricle [intracerebroventricular (ICV)] or unilateral intra-PVN (iPVN) cannulation 7–10 d before feeding studies and were habituated to regular handling and injection, as previously described (17). Central injections [5 μl (ICV) or 1 μl (iPVN)] were administered over 1 min via stainless steel injectors [27-gauge (ICV) or 31-gauge (iPVN)], placed in and projecting 1 mm below the end of the cannula. Spread of a 1-μl injection into the PVN is reported to be limited to 1 mm3 (18). All compounds were dissolved in vehicle (10% acetonitrile in 0.9% saline), and studies were performed in satiated rats (n = 10–12) in the early light phase (0900–1000 h) unless otherwise stated. After injection, animals were returned to their home cage with preweighed chow. Food intake was measured at 1, 2, 4, 8, and 24 h post injection. Neuropeptide Y (NPY) was administered as a positive control in food intake studies (5 nmol/animal ICV or 0.5 nmol/animal iPVN). Water intake was measured at 1 and 2 h postinjection. Angiotensin II was administered ICV as a positive control (150 ng/rat) in water intake studies.

    iPVN cannula position was verified histologically at the end of the study (17). Immediately after decapitation, 1 μl India ink was injected into the cannula. The brains were removed and fixed in 4% paraformaldehyde, dehydrated in 40% sucrose and frozen in liquid nitrogen and stored at –70 C. Brains were sliced on a cryostat (Bright, Huntingdon, UK) into 15-μm coronal sections, and correct PVN placement determined by microscopy according to the position of the India ink. ICV cannula position was verified by a positive dipsogenic response to angiotensin II (150 ng/rat). Only those animals with correct cannula placement were included in the data analysis.

    Behavioral response after ICV and iPVN administration of relaxin-3

    Behavioral responses were monitored after ICV administration of vehicle or 180 pmol H3 and iPVN administration of vehicle, 18 or 180 pmol H3 (n = 10). Animals were immediately returned to their home cages and observed for 1 h after injection by an investigator blinded to the experimental treatment. Behavior was classified into one of nine categories: feeding, drinking, grooming, burrowing, rearing, locomotion, sleep, head down, and tremor. Each rat was observed for three 3-sec periods every 6 min, and the behavior in each period was scored as previously described (19).

    Hypothalamic neuropeptide expression after relaxin-3 administration

    Hypothalamic neuropeptide mRNA expression was measured after ICV administration of vehicle or H3 (180 pmol) (n = 10–12). Food was removed immediately after injection, and at 4 h, animals were killed and hypothalami were dissected and snap frozen. Hypothalamic NPY, agouti-related peptide (AgRP) and proopiomelanocortin (POMC) mRNA expression were determined by ribonuclease protection assay. Briefly, RNA was extracted using Tri-Reagent (Helena Biosciences, Sunderland, UK) according to the manufacturer’s protocol. Rat ?-actin (Ambion) was used to correct for RNA loading. RNA was hybridized overnight at 42 C with 1.3 x 103 Bq of [32P]CTP-labeled riboprobe. Reaction mixtures were digested with RNase A/T1, and the protected fragments were 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) (19).

    Statistical analysis

    Results are shown as mean ± SEM. Data from feeding and water intake studies were compared by ANOVA with post hoc least significant difference test (Systat, Evanston, IL). Neuropeptide expression data were compared by unpaired Student’s t test between control and treated groups. Behavioral data were nonparametric and are expressed as median number of occurrences of behavior (interquartile ranges are expressed in parentheses in Table 1). Comparison between groups was made by Mann-Whitney U test. In all cases, P < 0.05 was considered to be statistically significant.

    TABLE 1. Effect of iPVN administration of H3 (18 or 180 pmol) on behavior in the first hour following injection

    Results

    Feeding studies

    To investigate the hypothesis that relaxin-3 is involved in regulation of appetite, food intake was determined after central relaxin-3 administration.

    Study 1: effect of ICV relaxin-3 on food intake in rats in early light phase and early dark phase

    Animals received an ICV injection of either vehicle or H3 (18, 54, or 180 pmol) in the early light phase. Doses used were based on previously reported effects of porcine relaxin-1 on water intake (6). ICV H3 significantly increased food intake in the first hour at both 54 and 180 pmol [0.96 ± 0.16 g (vehicle) vs. 1.80 ± 0.27 g (54 pmol H3) and 1.81 ± 0.21 g (180 pmol H3), P < 0.05] (Fig. 1A). There was no significant difference in interval food intake between control and treated groups at later time points. However, cumulative food intake was significantly increased at all doses of H3 at 2 and 4 h (Fig. 1B).

    FIG. 1. Effect of ICV administration of relaxin-3 in satiated male Wistar rats. A, Effect of H3 (18–180 pmol) on 1-h food intake. *, P < 0.05 vs. vehicle in early light phase. B, Effect of H3 (18–180 pmol) on cumulative food intake over 4 h in early light phase. &, P < 0.05 at 18 pmol vs. vehicle; *, P < 0.05 at 54 pmol vs. vehicle; #, P < 0.05 at 180 pmol vs. vehicle. C, Effect of H3 (180 pmol) on 1-h food intake in early dark phase. *, P < 0.05 vs. vehicle.

    Rats received an ICV injection of either vehicle or H3 (180 pmol) at the beginning of the dark phase. Nocturnal food intake was significantly increased in the first hour after H3 administration [2.95 ± 0.45 g (vehicle) vs. 4.39 ± 0.39 g (180 pmol H3), P < 0.05], (Fig. 1C). There was no significant effect on interval food intake at later time points or in cumulative food intake.

    Study 2: effect of ICV relaxin-2 on food intake in satiated rats in early light phase

    To differentiate the receptor mediating the effects of H3 on food intake, the feeding response to H3, which binds both LGR7 and GPCR135 receptors, was compared with that after administration of relaxin-2 (H2), which binds LGR7 but not GPCR135. Satiated rats received an ICV injection of either 180 pmol H3 or 180 pmol H2 in the early light phase. After ICV administration of equimolar doses, H3 stimulated 1-h food intake as previously shown [0.21 ± 0.09 g (vehicle) vs. 1.50 ± 0.40 g (180 pmol H3), P < 0.05] (Fig. 2), as well as cumulative food intake up to 4 h [0.75 ± 0.27 g (vehicle) vs. 2.18 ± 0.46 g (180 pmol H3), P < 0.05]. In contrast, H2 had no significant effect on food intake at any time point [0.21 ± 0.09 g (vehicle) vs. 0.43 ± 0.13 g (180 pmol H2) at 1 h], (Fig. 2).

    FIG. 2. Effect of ICV administration of H3 (180 pmol) and H2 (180 pmol) on 1-h food intake in early light phase in satiated male Wistar rats. *, P < 0.05 vs. vehicle.

    Study 3: effect of ICV relaxin-2 and relaxin-3 on water intake in satiated rats in early light phase

    To confirm the bioactivity of H2, the water intake response was assessed in water-replete, satiated male Wistar rats in the early light phase. Administration of ICV H2 (180 pmol) significantly increased water intake in the first hour [0.43 ± 0.26 ml (vehicle) vs. 2.50 ± 0.81 ml (180 pmol H2), P < 0.05] (Fig. 3) and at 2 h [0.63 ± 0.26 ml (vehicle) vs. 2.87 ± 0.79 ml (180 pmol H2), P < 0.05]. ICV H3 (180 pmol) increased water intake in the first hour, but this did not reach statistical significance [0.43 ± 0.26 ml (vehicle) vs. 2.11 ± 0.67 ml (180 pmol H3), P = 0.064], (Fig. 3).

    FIG. 3. Effect of ICV administration of H3 (180 pmol) and H3 (180 pmol) on water intake in satiated male Wistar rats. *, P < 0.05 vs. vehicle.

    Study 4: effect of iPVN relaxin-3 on food intake in satiated rats in early light phase and early dark phase

    Animals received an iPVN injection of either vehicle or H3 (1.8, 5.4, or 18 pmol) in the early light phase. Doses used were 10-fold less than those eliciting a feeding response after ICV administration (17). iPVN H3 administration significantly increased food intake in the first hour at 18 pmol [0.34 ± 0.16 g (vehicle) vs. 1.23 ± 0.30 g (18 pmol H3), P < 0.05] (Fig. 4A). There was no significant difference in interval food intake at later time points, but cumulative food intake was significantly increased at 2 and 4 h after iPVN administration of 18 pmol H3 [0.38 ± 0.18 g (vehicle) vs. 1.49 ± 0.31 g (18 pmol H3) at 2 h and 0.63 ± 0.27 g (vehicle) vs. 1.61 ± 0.35 g (18 pmol H3) at 4 h, P < 0.05].

    FIG. 4. Effect of iPVN administration of H3 in male Wistar rats. A, Effect of H3 (1.8–18 pmol) on 1-h food intake in early light phase. B, Effect of H3 (18 pmol) on 1-h food intake in early dark phase. *, P < 0.05 vs. vehicle.

    Rats received an iPVN injection of either vehicle or H3 (18 pmol) at the beginning of the dark phase. Food intake was significantly increased in the first hour after H3 administration [4.43 ± 0.32 g (vehicle) vs. 6.57 ± 0.42 g (18 pmol H3), P < 0.05] (Fig. 4B). There was no significant effect on interval food intake at later time points, but cumulative food intake was significantly increased in H3-treated animals for 4 h after administration in the early dark phase [9.68 ± 0.60 g (vehicle) vs. 12.28 ± 0.76 g (18 pmol H3), P < 0.05].

    Study 5: effect of iPVN relaxin-2 on food intake in satiated rats in early light phase

    To investigate further the receptor through which relaxin-3 mediates its orexigenic action, satiated rats received an iPVN injection of either 1.8–18 pmol H3 or 1.8–18 pmol H2. After an iPVN administration of equimolar doses, H3 stimulated 1-h food intake as previously shown [0.27 ± 0.11 g (vehicle) vs. 1.52 ± 0.51 g (18 pmol H3), P < 0.05]. In contrast, H2 had no significant effect on food intake at any time point after administration [0.27 ± 0.11 g (vehicle) vs. 0.14 ± 0.04 g (18 pmol H2) at 1 h], (Fig. 5).

    FIG. 5. Effect of iPVN administration of equimolar doses of H3 (18 pmol) and H2 (18 pmol) on 1-h food intake in satiated male Wistar rats. *, P < 0.05 vs. vehicle.

    Study 6: behavioral response after ICV and iPVN administration of relaxin-3

    There were no significant differences in feeding or drinking behaviors after an ICV injection of H3 (180 pmol) to satiated rats in the early light and dark phase, and no abnormal behaviors were observed. Feeding behavior was significantly increased after iPVN administration of H3 (180 pmol) to satiated rats in the early light phase. There were no other significant behavioral differences, and no abnormal behaviors were observed (Table 1).

    Study 7: hypothalamic neuropeptide mRNA expression

    After an ICV injection of H3 (180 pmol), there was no difference in hypothalamic NPY, AgRP, or POMC mRNA expression 4 h postinjection compared with vehicle-treated animals [NPY, 26.8 ± 1.26 (vehicle) vs. 27.8 ± 2.90 (180 pmol H3); AgRP, 13.1 ± 1.35 (vehicle) vs. 13.0 ± 0.78 (180 pmol H3); POMC, 1.90 ± 0.17 (vehicle) vs. 1.85 ± 0.24 (180 pmol H3); units are arbitrary].

    Discussion

    The insulin superfamily comprises functionally diverse peptides with a common structure: A and B chains with interchain disulfide bridges. Relaxin-1 in mice and rats and the human homologue, relaxin-2, were among the first members described, but it is only recently that an additional relaxin peptide, relaxin-3, and its receptors have been identified. Unlike relaxin-1, relaxin-3 mRNA is expressed in few peripheral tissues and only at low levels. There is less than 50% homology between relaxin-1 and relaxin-3 peptides (7). The dominant brainstem expression of relaxin-3, the extensive projections from the nucleus incertus to several hypothalamic nuclei and the rich expression of GPCR135 receptors in the hypothalamic PVN and supraoptic nucleus suggest that this ligand and its receptor may play an important role in the central nervous system. The PVN is crucial in the hypothalamic regulation of appetite and this led us to investigate the role of relaxin-3 in food intake.

    We have shown for the first time that ICV H3 significantly increased food intake in satiated animals in the early light phase and at the beginning of the dark phase. Similarly, H3 injection into the PVN, an area with a high level of expression of GPCR135, also stimulated food intake in the early light phase and increased nocturnal feeding. These studies were performed using human relaxin-3. However, there is a high level of homology among the relaxin-3 peptides of different species; the mouse and rat peptides are identical and share 92% sequence identity to human relaxin-3 (16). At the present time, only human relaxin-3 is commercially available, but this binds with high affinity to rat GPCR135 (16).

    Some orexigenic neuropeptides, for example orexin A, may increase food intake as a consequence of increased spontaneous physical activity and arousal (20). After iPVN H3 administration, the only behavioral change observed was an increase in feeding episodes. This suggests that the orexigenic effect of H3 is a specific feeding effect.

    The doses of relaxin-3 required to elicit a significant feeding response are in the picomolar range and similar to effective doses of other orexigenic peptides, such as ghrelin. For example, a significant orexigenic response with iPVN ghrelin has been seen at 30 pmol (17) compared with 18 pmol of H3 relaxin. Similarly, the lowest dose of the potent orexigenic peptide NPY to significantly stimulate feeding in the PVN is 24 pmol (21). As with NPY and ghrelin, the effect of relaxin-3 occurs in the first hour after administration, but cumulative food intake remains elevated for several hours.

    Centrally administered porcine relaxin has been shown to increase water intake in male and female rats (6). We have shown that human relaxin-2 (H2), which binds to the LGR7 receptor with a similar affinity to porcine relaxin (13), significantly increases water intake after ICV administration in male Wistar rats (Fig. 3). This indicates that the commercially available H2 used was biologically active. H3 also increased water intake at 1 h, although this did not reach statistical significance. The effect of H3 on water intake may be mediated via LGR7 receptors in the subfornical organs and related circuits (22).

    In contrast to H3, equimolar doses of H2 did not elicit any increase in feeding after ICV or iPVN administration to satiated animals in the early light phase. Whereas both relaxin-2 and relaxin-3 bind to the LGR7 receptor with high affinity, only relaxin-3 binds to GPCR135 with similarly high affinity. This suggests that the GPCR135 receptor may mediate the effects of relaxin-3 on food intake. In keeping with this, neither relaxin-1 null mice nor LGR7 null mice have any reported obesity or feeding phenotype (23).

    The action of some orexigenic peptides, for example ghrelin are mediated via NPY, AgRP, and/or the melanocortin system (24). Central administration of ghrelin up-regulates the expression of NPY and AgRP mRNA in the hypothalamus 4 h after injection (25, 26). In contrast, H3 (180 pmol) did not alter hypothalamic NPY, POMC, or AgRP mRNA expression 4 h after ICV administration. However, it is possible that expression of these neuropeptides might change after administration of relaxin-3 at different doses and/or at different time points. Nevertheless, these studies suggest that altered NPY, POMC, or AgRP mRNA expression may not be required in the orexigenic action of relaxin-3. It would be of interest to determine the effects of relaxin-3 on the expression of other hypothalamic neuropeptides.

    In summary, these results demonstrate that ICV and PVN administration of relaxin-3 stimulates feeding in male rats and suggest that this effect is mediated via the GPCR135 receptor. The mechanism for the orexigenic action of relaxin-3 remains to be established and research is currently limited by the absence of specific antagonists for relaxin receptors or antisera for rat relaxin-3. Further work is required to determine whether relaxin-3 plays a physiological role in regulation of appetite and body weight.

    Acknowledgments

    We express our thanks to the hypothalamic group for their assistance with the in vivo experiments and the Medical Research Council for funding this program of research.

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