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Obestatin Partially Affects Ghrelin Stimulation of Food Intake and Growth Hormone Secretion in Rodents

Unité Mixte de Recherche, 549 Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine, Université Paris-Descartes, and Institut Fédératif de Recherche Broca-Ste Anne, 75014 Paris, France

Address all correspondence and requests for reprints to: Philippe Zizzari, Unité Mixte de Recherche, 549 Institut National de la Santé et de la Recherche Médicale, 2ter rue d’Alésia, 75014, Paris, France. E-mail: philippe.zizzari{at}broca.inserm.fr.

	Abstract

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Administration of ghrelin, an endogenous ligand for the GH secretagogue receptor 1a (GHSR 1a), induces potent stimulating effects on GH secretion and food intake. However, more than 7 yr after its discovery, the role of endogenous ghrelin remains elusive. Recently, a second peptide, obestatin, also generated from proteolytic cleavage of preproghrelin has been identified. This peptide inhibits food intake and gastrointestinal motility but does not modify in vitro GH release from pituitary cells. In this study, we have reinvestigated obestatin functions by measuring plasma ghrelin and obestatin levels in a period of spontaneous feeding in ad libitum-fed and 24-h fasted mice. Whereas fasting resulted in elevated ghrelin levels, obestatin levels were significantly reduced. Exogenous obestatin per se did not modify food intake in fasted and fed mice. However, it inhibited ghrelin orexigenic effect that were evident in fed mice only. The effects of obestatin on GH secretion were monitored in superfused pituitary explants and in freely moving rats. Obestatin was only effective in vivo to inhibit ghrelin stimulation of GH levels. Finally, the relationship between octanoylated ghrelin, obestatin, and GH secretions was evaluated by iterative blood sampling every 20 min during 6 h in freely moving adult male rats. The half-life of exogenous obestatin (10 µg iv) in plasma was about 22 min. Plasma obestatin levels exhibited an ultradian pulsatility with a frequency slightly lower than octanoylated ghrelin and GH. Ghrelin and obestatin levels were not strictly correlated. In conclusion, these results show that obestatin, like ghrelin, is secreted in a pulsatile manner and that in some conditions; obestatin can modulate exogenous ghrelin action. It remains to be determined whether obestatin modulates endogenous ghrelin actions.

	Introduction

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GHRELIN WAS IDENTIFIED in 1999 as the endogenous ligand for the GH secretagogue receptor 1a (GHSR 1a) (1). Soon after its discovery, in addition to its strong GH releasing activity (2, 3), ghrelin was found to increase food intake, down-regulate energy expenditure and conserve body fat, causing weight gain and adipogenesis (4, 5, 6, 7). However, if exogenous ghrelin actions are well established, the role of endogenous ghrelin is still unclear. Ghrelin antagonists have significant effects on these two functions (8, 9, 10) and active vaccination against ghrelin immunoconjugates decreases feed efficiency, relative adiposity, and body weight gain in mature rats (11). Mice invalidated for the preproghrelin or ghrelin receptor gene do not display a major phenotype in term of body growth (12, 13) but are protected against early-onset obesity (14, 15). The recent identification of obestatin, a new peptide derived from preproghrelin that has been reported to bind to and activates the orphan receptor GPR39 (16, 17), may explain such discrepancies. Indeed, obestatin exhibits opposite effects of ghrelin on energy homeostasis and gastrointestinal function but appears ineffective on GH secretion (17).

In the present work, we further investigated obestatin’s ability to modulate spontaneous or ghrelin-induced food intake and GH secretion. Plasma obestatin, ghrelin, and GH levels were monitored by selective assays. Food intake was measured at the onset of the dark phase in fed and in 24 h-fasted/refed mice. GH secretion was evaluated ex vivo from superfused rat pituitaries and in vivo in freely moving male rats. Finally, the relationship between obestatin (18, 19, 20), ghrelin, and GH secretion was assessed by sampling blood every 20 min for a consecutive 6 h, the first three corresponding to the end of the lights-on period and the last three to the beginning of the lights-off period.

	Materials and Methods

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Animals
Four weeks before experiments, adult male C57Bl/6 mice and Sprague Dawley rats (Charles River Laboratories, Inc., L’Arbresle, France) weighing respectively between 20 and 25 g and 100 and 125 g at the onset of the experiment, were housed individually in transparent plastic containers placed in a sound-proof room with controlled temperature (22–24 C) and illumination (12-h light, 12-h dark schedule with lights off at 1700 h). They had free access to food and water and they were regularly weighed and handled to minimize manipulation stress. Animal experiments were performed according to the guidelines of the French Act of Animal Care and Experimentation (1990; registration no. 75-343). All efforts were made to minimize pain and suffering and to reduce the number of animals used.

In vivo experiments
Mouse experiments.
For the food intake experiments, C57Bl/6 mice had free access to food and water until the fasting period. Every other day during the 2 wk before the experiments, mice received ip saline injections to minimize stress. They were assigned randomly to the fast and fed groups. One day before the experiment, a group was fasted for 24 h. On the day of the experiment, animals received an ip injection of saline, ghrelin, obestatin, or obestatin + ghrelin (1 µmol/kg each; NeoMPS Strasbourg, France) 1 h before the onset of the dark period. Fasted animals were given access to food just after the injection. In a first set of experiments, blood samples were withdrawn from the jugular vein at 1600 h after anesthesia with ketamine-xylazine to determine endogenous levels of ghrelin, obestatin, and glucose at injection time. In a second one, food intake was monitored 1, 3, 5, and 18 h after the injection. Finally, in a third one, animals were decapitated 1 h after peptide injection and blood was sampled to determine glucose levels (measured by glucose oxydase; Beckman analyzer II; Beckman Coulter, Fullerton, CA).

Rat experiments.
Two days before blood sampling, an indwelling cannula was inserted into the right atrium as previously described (21). Two hours before the sampling period, the distal extremity of the cannula was connected to a polyethylene catheter filled with 25 IU/ml heparinized saline. Blood samples were collected on EDTA (1 mg/ml) and p-hydroxy-mercuribenzoic acid (Sigma, Saint Quentin Fallavier, France) (0.36 mg/ml) to avoid ghrelin degradation, immediately centrifuged, and plasma was stored at –20 C until hormone assays. Blood from donor rats was regularly reinjected to attenuate hemodynamic modifications.

In experiment 2, saline, obestatin (10 µg/rat), ghrelin (10 µg/rat), or obestatin plus ghrelin were administered iv at 1000 h and blood samples withdrawn just before and 5, 10, 20, 30, 45, and 60 min after injection for GH determination.

In experiment 3, the half-life of obestatin in plasma was determined. Blood samples were collected 0, 5, 10, 20, 30, 45, and 60 min after iv injection of 10 µg of obestatin and plasma obestatin levels were measured. Half-life was calculated as t[1/2] = ln(2)/Kel where Kel = –slope.

In experiment 4, blood was sampled every 20 min from 1400–2000 h, to compare endogenous obestatine, octanoylated ghrelin, and GH secretory parameters.

Ex vivo experiments
Rats were killed by decapitation. Pituitaries were rapidly dissected, washed for 30 min in oxygenated DMEM (with 2.5 mM L-glutamine, 17.51 mM D-glucose, and 25 mM HEPES) containing 0.1% BSA, placed in perifusion chambers (vol 0.3 ml) and superfused at a rate of 0.1 ml/min with the same medium. After a 120-min equilibration period, effluents were collected every 5 min. Peptides were added to the medium during 15-min periods. Samples were frozen until GH determinations.

Hormone assays
Octanoylated ghrelin was measured by an in-house immunoenzymatic assay using polyclonal rabbit antibodies made against N-terminal rat ghrelin (kindly provided by Dr. Hosoda, Osaka, Japan), and human ghrelin coupled to acetylcholinesterase (Spibio, Saclay, France) as tracer. The sensitivity was 6 pmol/liter and the intraassay coefficient of variation was 7%.

Obestatin levels were determined with a commercial RIA kit (Phoenix, Belmont, CA). We have verified that rat pre-proghrelin 52–85 and 86–117 (Phoenix) do not cross-react up to the concentration of 3973 pmol/liter. The sensitivity of this assay was 4 pmol/liter and the intraassay coefficient of variation was 8%.

Plasma GH concentrations were evaluated by EIA as previously described (22). Values are reported in terms of rGH-RP2, with sensitivity of 0.6 ng/ml and intra- and interassay coefficients of variation were 4 and 14%, respectively

Statistical analysis
Acylated ghrelin, obestatin, and GH pulse analysis was performed using the Cluster 8 software (23) with the t value set to 2 to maintain false-positive rates under 1%. Number of point for a peak and number of point for a nadir were set to 1 and 2, respectively.

Approximative entropy was calculated using the MC-ApEn software using R-value set to 0.2 and number of MC cycle set to 1000.

These programs are available from http://mljohnson.pharm.virginia.edu/home.html.

Values are given as means ± SEM, and statistical analysis was performed by ANOVA and paired t test using the JMP IN 5.1 software (SAS Institute Inc., Cary, NC).

	Results

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Twenty-four hours of fasting significantly reduced glycemia (131 ± 18 vs. 313 ± 44 mg/dl in ad libitum-fed mice; P < 0.01) and obestatin levels (256 ± 6 vs. 320 ± 12 pmol/liter; P < 0.01), whereas it increased octanoylated ghrelin (787 ± 225 vs. 279 ± 105 pmol/liter; P = 0.087) (n = 4).

Obestatin effects on spontaneous and ghrelin-induced food intake at the onset of the dark period, in fed and 24-h fasted mice
As shown in Fig. 1, during the lights-off period, spontaneous cumulative food consumption (after 1, 3, 5, and 18 h) was lower in ad libitum-fed than in fasted mice given access to food. Administration of obestatin (1 µmol/kg body weight ip) 1 h before lights-off was ineffective to modify food intake in fed (Fig. 1, B and C) or fasted/refed (Fig. 1, A and C) mice during the following 18 h. In the same conditions, ghrelin (1 µmol/kg body weight ip) significantly stimulated food intake in fed mice only and coadministration of obestatin inhibited this effect. In fasted/refed mice, cumulative food intake was similar to that of ghrelin-treated fed mice.

View larger version (17K): [in this window] [in a new window]   FIG. 1. Obestatin effects on spontaneous and ghrelin-induced food intake, in fed and 24-h fasted mice, during the dark period. Cumulative food intake 1, 3, and 5 h after injection of saline (blue square), 1 µmol/kg ghrelin (green square), 1 µmol/kg obestatin (dark triangle), or 1 µmol/kg obestatin + ghrelin (red triangle). A, In 24 h refeeding fasted mice (n = 20, 5 in each group). B, In ad libitum-fed mice (n = 20, 5 in each group). a, Ghrelin vs. saline, P < 0.01; b, ghrelin vs. obestatin, P < 0.01; c, ghrelin vs. obestatin + ghrelin, P < 0.05; d, ghrelin vs. obestatin, P < 0.05. C, Cumulative food intake 18 h after injection in 24 h fasted (left) and ad libitum-fed (right) mice receiving saline (blue), 1 µmol/kg ghrelin (green), 1 µmol/kg obestatin (dark), or 1 µmol/kg obestatin + ghrelin (red). a, Ghrelin vs. saline, P < 0.05; b, ghrelin vs. obestatin, P = 0.06

Ex vivo and in vivo effects of obestatin on spontaneous and ghrelin-induced GH release
As shown on Fig. 2A, ghrelin (10^–7 M) rapidly stimulated GH release from superfused pituitaries ex vivo. Obestatin (10^–7 and 10^–6 M) did not affect spontaneous or ghrelin-induced GH release.

View larger version (26K): [in this window] [in a new window]   FIG. 2. Obestatin effects on spontaneous or ghrelin-induced GH secretion in the rat. A, Mean profile of GH release from superfused rat pituitaries in basal condition and after infusion of obestatin 10–6 M (dark triangle), ghrelin 10–7 M (green square), obestatin 10–7 M + ghrelin 10–7 M (pink triangle), obestatin 10–6 M + ghrelin 10–7 M (red triangle). The different peptides were infused during 15 min (materialized by dotted line). Each point and vertical bar indicates mean ± SEM of four chambers. B, Effect of 10 µg obestatin (dark triangle), 10 µg ghrelin (green square), or 10 µg obestatin + ghrelin (red triangle) on GH secretion in freely moving rats. The peptides were injected at t = 0, and blood were collected preinjection and 5, 10, 20, 30, 45, and 60 min after injection. Data are means ± SEM. Number of animals are indicated in parentheses. *, Ghrelin vs. Obestatin + Ghrelin, P < 0.05; #, Ghrelin vs. Obestatin, P < 0.05

Obestatin, ghrelin, and GH levels in freely moving rats
Determination of plasma obestatin concentrations from 5–60 min after synthetic obestatin administration (10 µg, iv) in freely moving rats showed that the peptide half-life in plasma was 22 ± 2 min (n = 6) (Fig. 3). This permitted to use a 20-min sampling periodicity during 6 h to compare obestatin and ghrelin secretory profiles.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Determination of exogenous obestatin half-life in rat plasma. Obestatin immunoreactivity was detected by RIA in rat plasma after iv injection of 10 µg synthetic peptide. Data are expressed as the percentage of obestatin concentrations measured immediately after the injection. Values are the means ± SEM of six determinations. A semilogarithmic plot of the data are given in the inset.

View larger version (18K): [in this window] [in a new window]   FIG. 4. Ghrelin, obestatin, and GH profiles in freely moving rats. Representative octanoylated ghrelin (triangle), obestatin (red square), and GH (diamond) secretory patterns during a 6-h sampling period (3-h during the light period and 3-h during the dark period) in four freely moving male rats.

	Discussion

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In the present study we observe that, in some conditions, obestatin can inhibit ghrelin effects on food intake, depending on the feeding status, and on GH secretion. However, it is ineffective per se on these parameters.

Our results on food intake are slightly different from those originally reported by Zhang et al. (17), who observed an inhibitory effect of obestatin on spontaneous food intake in refed mice. In their study, obestatin effects were only observed in refed animals 2 h after light onset, whereas our data were obtained just after the onset of the lights-off period when animals begin to eat spontaneously (24). More recently, the vast majority of studies (18, 20, 25, 26, 27, 28, 29) with one exception (30), reported no effect of obestatin per se or on ghrelin-induced food intake in rats and mice (fed or fasted/refed), whatever the route of administration and mode of obestatin dilution. Effects of orexigenic/anorexigenic peptides such as ghrelin and obestatin likely depends upon the relative concentrations of other orexigenic/anorexigenic factors at the time of injection. Indeed, we observed that, injected 1 h before light off, ghrelin stimulates food intake in fed animals but is ineffective at the same dose in fasted ones given access to food. Similar results were reported when experiments were performed during the lights-on period with a very low increase of food intake in refed animals compared with that of ad libitum ones (30% vs. 320%) (17, 31). Endogenous ghrelin concentrations are markedly increased during fasting (125% after 24 h fasting) in mice (32) and to a lesser extent just before light off (20–30%) in rats (Ref. 33 ; and Zizzari, P., and M. T. Bluet-Pajot, unpublished data). The fact that ghrelin is effective only in fed mice and that in refed mice cumulative food intake is of the same magnitude than in ghrelin-treated fed ones suggests that, beyond a certain threshold, additional exogenous ghrelin cannot further increase an already elevated food consumption. Reciprocally, the inefficiency of obestatin to alter spontaneous food intake at the beginning of the lights-off period is probably not due to plasma ghrelin level increases because obestatin is able to counteract the effect of very high doses of exogenous ghrelin (1 µmol/kg). Other orexigenic/anorexigenic factors exhibit circadian variations which could modify obestatin or ghrelin responses. Numerous studies show modifications of neuropeptide Y, galanin, ß-endorphin, and proopiomelanocortin gene expression at the onset of the dark phase (34, 35, 36), and it will be of interest to test obestatin and ghrelin responses in presence of variable concentrations of these factors.

It is generally believed that ghrelin exerts its orexigenic effect mainly by activating GHSR1a on hypothalamic arcuate nucleus neuropeptide Y/agouti-related protein neurons (37, 38, 39, 40), but the mechanisms by which obestatin could modulate food intake are not yet known. Obestatin exerts its anorectic effect after intracerebroventricular administration at a low dose (8 nmol/kg), suggesting a central action (17). Obestatin was originally reported to bind to GPR39, an orphan receptor, which shares similarities with GHSR1a (16, 17). GPR39 mRNA has been detected by RT-PCR in the hypothalamus and ¹²⁵I obestatin binding sites were reported in the same region (16, 17). Nevertheless, recent studies failed to confirm the presence of specific radiolabeled obestatin binding on GPR39 or activation of this receptor by obestatin (18, 19, 20). In preliminary experiments, we also observed that obestatin did not modify several signal transduction pathways in GPR-39 transfected cells (Pantel, J., unpublished data). It remains to be determined which is the receptor for obestatin and how obestatin interferes with GHSR 1a to inhibit ghrelin stimulated food consumption. Because obestatin inhibits jejunal contractile activity and suppress gastric emptying activity (17), it cannot be excluded that its anorectic effect relies mostly on peripheral sites of action. The inhibition of jejunal contraction could generate an afferent vagus signal to induce satiety in the central nervous system.

As already reported (17, 27), we did not observe any effect of obestatin on spontaneous or ghrelin-induced GH release ex vivo. In contrast, when administered iv to rats, obestatin significantly inhibited ghrelin stimulation of GH secretion. Ghrelin increases plasma GH levels by acting directly on pituitary but also indirectly at the hypothalamic level where it stimulates GHRH secretion (41) and decreases somatostatin release (3). Alternately, gastric vagal afferents are also an important pathway conveying ghrelin signals for GH secretion to the brain (42).

The present study shows, for the first time, that obestatin secretion is pulsatile and displays an ultradian rhythmicity, very similar to ghrelin and GH secretion. Interestingly however, plasma ghrelin and obestatin levels are not strictly correlated and the number of obestatin pulsatile episodes may seem slightly lower than the one observed for ghrelin and GH secretion. Because obestatin and ghrelin are derived from the same gene, this lack of strict correlation supports the notion that obestatin is a physiologically relevant peptide and not only a nonfunctional connective peptide. Such a hypothesis is further substantiated by the differential effect of fasting on ghrelin and obestatin levels, ghrelin being markedly increased and obestatin slightly decreased after 24 h of fasting. This strongly suggests that the secretion of the two peptides is regulated by the nutritional status in an opposite manner. It was previously shown that proopiomelanocortin, another multipeptide precursor, is cleaved into several bioactive fragments that include the anorectic - and ß-MSH and the orexigenic ß-endorphin and that tissue-specific enzymes determine which of those peptides are generated (43). It remains to be determined whether a differential tissue-specific posttranslational process exists in the case of proghrelin and obestatin processing.

	Acknowledgments

 
We thank A. Cougnon for technical assistance. We are grateful to the National Institute of Diabetes and Digestive and Kidney Diseases for providing us with GH assay reagents.

	Footnotes

 
Disclosure Statement: The authors have nothing to disclose.

First Published Online January 4, 2007

Abbreviation: GHSR 1a, GH secretagogue receptor 1a.

Received September 7, 2006.

Accepted for publication December 27, 2006.

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