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GRH-SOMATOSTATIN-GH |
Institut National de la Santé et de la Recherche Médicale U549 (V.T., M.-H.B., P.Z., F.P.-J., J.E., M.-T.B.-P.), Institut Fédératif de Recherche Broca-Sainte Anne, 75014 Paris, France; and Institut de Génétique et de Biologie Moléculaire et Cellulaire (C.T.), Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale U184/Université Louis Pasteur, Illkirch 67404, France
Address all correspondence and requests for reprints to: Jacques Epelbaum, U549 Institut National de la Santé et de la Recherche Médicale, Institut Fédératif de Recherche Broca-Sainte Anne, 2ter rue d’Alésia, 75014, Paris, France. E-mail: . epelbaum{at}broca.inserm.fr
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Abstract |
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Introduction |
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In rodents, ghrelin has recently been shown not only to stimulate GH secretion (11, 12) but also to exert gastroprokinetic (13), orexigenic, and adipogenic effects (14, 15, 16). Effects on GH secretion (17, 18, 19, 20) and feeding behavior (21, 22, 23) are also observed in the human species. As recently reviewed, the dual action on GH secretion and food intake as well as the dual localization of ghrelin and its receptors in hypothalamus and stomach immediately raise the question of the interdependency of these actions (24). Many studies have linked nutrition and episodic GH secretion although these relationships vary from species to species, undernutrition reducing GH pulsatility in the rat, while the converse is true in humans (25). One common property of feeding behavior and GH secretion is their rhythmicity. The pulsatile mode of GH secretion depends primarily on the coordinate actions of hypothalamic GHRH and somatostatin (SRIF) release from median eminence terminals but multiple intra and extra pituitary regulatory signals and diurnally varying neuronal inputs related to the sleep-wake pattern are also involved (26, 27). In return, feeding behavior is also dependent on the interaction of regulatory signals, including sleep-wake patterns, and diurnally neuronal inputs on a complex array of hypothalamic peptidergic neurons (28).
In the present work, we attempted to determine the relationships between ghrelin and the ultradian rhythmicity of GH secretion, feeding behavior, and sleep-wake cycle in freely moving rats sampled for 9 h, the first three corresponding to the end of the lights-on period and the last six to a lights-off period. This schedule was chosen to observe behavioral parameters in both light and dark periods because 1) feeding and sleep-wake patterns differ considerably in these two conditions; and 2) GH secretion in rats is remarkably entrained to the light/dark cycle. In a first series of experiments, we analyzed the effect of repeated ghrelin administrations in the lights-on and lights-off periods on these parameters. In a second series of experiments, we measured ghrelin plasma levels and analyzed its pulsatile and entropic modes in comparison with those of GH, feeding, and sleep-wake states.
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Materials and Methods |
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Surgery and experimental procedures
Two weeks before the experiments, electrodes for recording the electroencephalogram (EEG) and electromyogram (EMG) were implanted. Rats were anesthetized with pentobarbital (50 mg/kg), placed into a stereotaxic frame and maintained at 37 C with an isothermal heating pad. Bipolar electrodes were placed in the right and left dorsal hippocampus (stratum lacunosum moleculare). A stainless steel screw was inserted into the skull above the right parietal cortex. Another screw placed on the occipital crest served as reference. A stainless steel hook soldered to fine flexible insulated silver wires was placed in the neck muscles to record EMG. All recording electrodes were soldered to a connector. A liquid bonding resin (Superbond, Sun Medical Co., Tokyo, Japan) was applied to the cleaned, dried skull surface. The hardened resin was subsequentely covered with a layer of acrylic cement. The connector was embedded in a mound of acrylic cement and firmly jointed to the rat skull. The rat was removed from the stereotaxic frame and allowed to recover from anesthesia. Cephalosporin (cefuroxime 60 mg/kg) was administered im every 2 d during a week.
During the following week, rats were connected to a rotating collector by a flexible cable allowing a maximum amount of freedom and gradually habituated to the recording conditions.
Experiments were performed on freely moving rats. Two days before data collection, an indwelling cannula was inserted into the right atrium under ether anesthesia as previously described (29).
In a preliminary experiment, the half-life of ghrelin in plasma was determined. Ten micrograms of ghrelin were iv injected to four rats and blood samples collected 0, 5, 10, 20, 40, 60, 80, and 120 min after the injection.
In a first experiment, rats received iv injection of ghrelin (10 µg/rat) or saline at 1020 h, 1420 h, and 1720 h and GH secretion, feeding behavior and sleep-wake pattern were evaluated. Two hours before the sampling and recording period, the distal extremity of the cannula was connected to a polyethylene catheter filled with 25 IU/ml heparinized saline. Recording electrodes were connected to a polygraph via the rotating collector. From 1000–1900 h, EEG and EMG were continuously recorded and blood samples were withdrawn every 20 min. Blood samples were centrifuged immediately. Red blood cells were resuspended in saline and reinjected every hour to attenuate hemodynamic modifications. Plasma was stored at -20 C until GH assays.
In a second experiment, blood was sampled in the same schedule to determine the endogenous ghrelin secretion in addition to the other parameters.
Analysis of sleep-wake patterns
Polygraph records were scored in 20-sec epochs by visual analysis. Four states of arousal were differentiated: active wakefulness (AW), characterized by cortex low-voltage activity, hippocampal theta activity (6–8 Hz), motor activity with high muscular tone (EMG); quiet wakefulness (QW), characterized by nonmotivated motor activity associated to low voltage EEG and absence of hippocampal theta; slow wave sleep (SWS), characterized by high-amplitude low frequency EEG intermingled with spindles and reduced voltage EMG; rapid eye movement (REM) sleep, identified by hypersynchronous theta waves, loss of muscular tone, jerks of jaw muscles, and vibrissae at the end of the episode.
The total durations of the four states of vigilance were calculated on the basis of the overall session and during either the lights-on or the dark periods. For clarity, AW and QW were represented together as wakefulness. The total number of REM sleep episodes and the duration of each bout were calculated. Temporal relations between the occurrence and length of eating episodes and hormonal parameters (GH, endogenous, and exogenous ghrelin) were determined.
Feeding behavior
Rats were freely fed. Food intake episodes were constantly monitored by two researchers during the course of the experiments. Food intake was measured at the end of the sampling period.
Hormonal determinations
Plasma GH concentrations were measured by EIA as previously described (30). Values are reported in terms of rGH-RP2, with sensitivity of 5 ng/ml and intra and interassay coefficients of variation below 7%.
Plasma ghrelin concentrations were determined using RIA kits (Phoenix Pharmaceuticals, Inc., Belmont, CA). The sensitivity was 53 pg/ml. The intra and interassay coefficients of variation were below 10%.
Statistical analysis
Ghrelin and GH pulse analysis was performed using the Cluster program (31) setting the t value to 2 to maintain false positive rates under 1%. Cluster size was set to two prepeak and two postpeak nadir values. False positive error for peak detection was 7%.
Regularity of GH, ghrelin, and food intake pattern was estimated by determination of approximate entropy (ApEn) and cross-approximate entropy (X-ApEn) (32). The series containing 54 observations, ApEn and X-ApEn were calculated using r (tolerance for detecting pattern recurrence) = 20% and m (pattern length) = 1.
Areas under the GH response curves (AUC) were calculated by mean of trapezoidal analysis.
Values are given as means ± SEM and statistical analysis was performed by ANOVA and paired t test using the statview 4.5 software (Abacus Concepts, Palo Alto, CA).
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Results |
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Ghrelin stimulated GH secretion (Fig. 2), and the amplitude of the response was identical after the different injections (AUC during the 40-min period following the administration: 7476 ± 1788, 9461 ± 1222, and 8027 ± 1826, respectively).
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). Within the 30 min following injection, food intake always lasted longer in ghrelin-treated animals than in controls (Table 1).
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and Table 1
). A decrease in REM duration was observed after ghrelin injection only at 1420 h. In this case, within the hour following ghrelin injection, the mean number of REM episodes was not different (8.4 ± 2.2 vs. 10.0 ± 1.2 in controls), but their mean duration was significantly decreased (43.3 ± 10.7 vs. 88.3 ± 7.4 sec, P < 0.01).
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Relation between endogenous plasma ghrelin levels, GH secretion, feeding behavior, and states of arousal
Individual profiles of GH and ghrelin secretion are displayed on Fig. 5. Plasma ghrelin levels, sampled every 20 min during 9 h, exhibited pulsatile variations of moderate amplitude when compared with those of GH secretion (Figs. 5 and 6).
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Cluster analysis of pulsatility parameters for ghrelin and GH secretion is given on Table 2. Mean interpeak intervals and pulse frequencies were similar, but peak amplitudes were much more important for GH (574% over nadir) than for ghrelin (77%). Moreover, ghrelin secretion was quantitatively more irregular than that of GH, as indicated by ApEn (Fig. 7A).
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). Ghrelin concentrations were correlated with the duration of food intake, during the dark period only (Fig. 8), and plasma ghrelin concentrations decreased by 26% in the 20 min following the end of a food intake period (991 ± 170 vs. 1338 ± 222 pg/ml, P = 0.0032 paired t test) whatever the period of sampling. Total amount of GH secreted was also correlated with the duration of food intake (r= 0.830, P < 0.05). Cross-ApEn analysis revealed that GH/food intake and ghrelin/food intake rhythms were more synchronized than GH/ghrelin secretory patterns (Fig. 7B).
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Discussion |
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As previously described (12), repeated administrations of ghrelin at 3- to 4-h intervals elicited an increase in GH release without desensitization. The immediate response is consistent with the estimated half-life of exogenous ghrelin (30 min in rat plasma) and with the fact that GHRH neurons would be activated after GH-secretagogues (33, 34) or ghrelin injection (35, 36), whereas SRIF neurons would be inhibited (12).
The constant observation of rat activity indicated an immediate modification in feeding behavior. The successive ghrelin injections corresponded to three different spontaneous states: at 1020 h, control rats do not eat; at 1420 h they have just eaten; and at 1720 h they are eating. In every instance, rats began searching for food immediately after ghrelin injection, then ate in the first 10 min following ghrelin administration and stopped from 30–50 min, depending on the light-dark period. Wren et al. (15) already reported an increase of ingested food 1 h after two ip injections of ghrelin separated of 4 h. The time-course of ghrelin effect on food intake is similar to that observed for ghrelin-induced GH release. Nevertheless, ghrelin effect on feeding seems independent of GH because Tschöp et al. (14) have shown that ghrelin induced a similar food intake increase in control and GH-deficient dwarf rats. Moreover, KP 102, a pharmacology designed GH secretagogue, is still effective on food ingestion after GHRH passive immunization (37), thus providing further evidence that ghrelin-induced food intake is independent from ghrelin-mediated GHRH release. The induction of food intake episodes by ghrelin injections, even when control animals were already engaged in normal feeding, suggests that the mechanisms implicated in the orexigenic action of ghrelin are complementary to those of spontaneous food intake. Activation of NPY/AGRP neurons could be implicated in the induction of feeding behavior because NPY injection into the hypothalamus stimulates food intake (28). GHS-R receptors are present on these neurons (38) and ghrelin-induced feeding is inhibited by an anti-NPY IgG and an anti-AGRP IgG (39). Exogenous ghrelin could antagonize the negative effect of leptin on NPY/AGRP neurons, thereby increasing food intake, because ghrelin is able to block leptin-induced feeding reduction (40). Interestingly, the quantity of food ingested during the 9-h period observation was not different between treated and control animals as already described (40). This can be explained by a compensatory decrease of food intake in ghrelin-treated rats between the periods of stimulation. It cannot be excluded that ghrelin could induce a rapid craving for food followed by satiety.
The sleep-wake pattern was also modified by ghrelin injections. Wakefulness was only increased and SWS decreased when ghrelin was administered when rats were sleeping (at 1020 h mostly SWS and at 1420 h, a lot of REM sleep). When injected at 1720 h (rats are awake and eating), ghrelin was ineffective. Nevertheless, and as for food intake, on the 9-h period of monitoring, total wakefulness, and SWS durations were unchanged in the ghrelin-treated group. The immediate and temporary effect of ghrelin on wakefulness and SWS could be an indirect consequence of its orexigenic effect. In contrast, REM sleep decrease was observed on the 9 h of testing, suggesting that it is independent of the orexigenic effect of ghrelin. Ghrelin-induced GH or GHRH increases are probably not implicated because GH (41) and GHRH (42) induce REM sleep in rats. On the other hand, ghrelin inhibition of REM sleep would be compatible with its antagonistic function on SRIF activity because SRIF facilitates REM sleep in rats (43, 44). Microinjection of a SRIF antagonist in the locus coeruleus decreases REM sleep (45). During REM sleep, there is an inhibition of locus coeruleus neurons that may, at least in part, be due to SRIF release from fibers originating in the hypothalamus (46, 47, 48). One might suggest that ghrelin, by reducing the inhibitory influence of SRIF, modifies the monoaminergic mechanisms that control REM sleep onset and duration. Finally, cholinergic neurons in the pontomesencephalic tegmental nuclei play an important role in REM sleep genesis. It is likely that SRIF facilitates REM sleep by increasing acetylcholine release by mesopontine tegmental neurons (49). Ghrelin might reduce acetylcholine release via its effect on SRIF transmission and, indirectly, alters REM sleep.
One of the main findings of the present study is the demonstration that ghrelin secretion is pulsatile and displays an ultradian rhythmicity. Interestingly, the number of peak and the interval between peaks of ghrelin are similar to those observed for GH secretion, whereas peak amplitudes are much more important for GH. However, plasma ghrelin and GH levels are not strictly correlated, and cross-ApEn did not reveal a great synchronism between these two parameters. This result might have been expected because, based on hypophyseal portal rat blood sampling data, GHRH and SRIF fluctuations are also involved in this phenomenon (25, 26), with SRIF withdrawal being a prerequisite for GHRH action (50). It remains to be demonstrated directly, on rat portal blood measurements, whether ghrelin does inhibit SRIF release as already observed in vitro (12). In the sheep, only a minor and nonsignificant inhibition of SRIF portal levels was observed after iv injection of a synthetic GHS, hexarelin, whereas GHRH levels were markedly stimulated (34). On the other hand, a recent report even questions the role of GHRH in the generation of endogenous GH pulses in humans because GH secretion, though markedly diminished, is still pulsatile in patients with an inactivating defect of the GHRH receptor (51). So, a direct implication of endogenous ghrelin in the control of pulsatile GH release in the rat remains to be demonstrated. In that respect, it is noteworthy that rats presenting lower GH amplitudes also displayed lower ghrelin levels.
The positive correlation between the AUC of endogenous ghrelin levels and the duration of food intake episodes as well as the low X-ApEn value indicated that ghrelin and feeding behavior are closely linked. However, it is not clear whether endogenous ghrelin levels influence food intake or whether ghrelin levels are modified after a change in feeding activity. The decrease of food intake observed after intracerebroventricular administration of antighrelin IgG (39) does suggest that the endogenous peptide regulates feeding activity. Indeed, circulating ghrelin fell by 26% within the 20 min following the end of food intake and a comparable observation has been reported by Tschöp et al. (22) and Cummings et al. (52) in humans in which plasma ghrelin levels are decreased 2-fold within 1 h following each meal. Moreover, fasting increases plasma ghrelin levels (14), which are diminished in obese patients (23), some of whom even bearing mutations in the preproghrelin gene (53). Taken together, these data suggest that endogenous ghrelin influences food intake which in turn could modify ghrelin levels.
In our study on freely feeding undisturbed rats, correlation between endogenous ghrelin levels and sleep-wake pattern was only detected in the active wake during the first 3 h of the dark period. In previous studies, sleep-wake pattern, particularly REM sleep, and feeding activity were only related when the animals are put in drastic conditions such as food restriction and sleep deprivation (54). Indeed, when rats were food restricted during the light period, the diurnal distribution of REM sleep is reversed (55). Interestingly, the inhibitory effects of leptin on REM sleep are no more observed in rats food-deprived for 18 h (56). It remains to be determined whether plasma ghrelin levels would be modified in such conditions. Alternately, exogenous ghrelin injection always elicited an immediate and short lasting decrease in REM sleep, concomitant with the induction of feeding behavior. Thus, it might be hypothesized that the feeding behavior modification induced by exogenous ghrelin is related to a direct effect on REM sleep.
Sleep-wake pattern has also been related to GH ultradian rhythmicity in humans (27). Maximal GH release occurred within minutes of the onset of stage 3 or 4 sleep (57). In the adult male rat, conflicting results were reported. Original studies by Willoughby et al. (58) were negative, while other investigators found a definite correlation with each GH peak occurring with a consistent time lag 40–70 min after the onset of a sleep cycle (59). However, these studies did not differentiate between SWS and REM sleep episodes. A role of endogenous ghrelin in the regulation of REM sleep cannot be excluded but requires new investigations during longer recording periods.
In conclusion, ghrelin, in addition to affect GH secretion and feeding behavior, also modifies sleep-wake patterns. This last action is probably related to its effects on feeding behavior. Moreover, ghrelin secretion is pulsatile and directly correlated to feeding behavior only in accordance with its gastroprokinetic activity (13) and structural resemblance to motilin (2).
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Acknowledgments |
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Footnotes |
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Abbreviations: ApEn, Approximate entropy; AUC, area under the curve; AW, active wakefulness; EEG, electroencephalogram; EMG, electromyogram; GHS-R, GH secretagogue receptor; QW, quiet wakefulness; REM, rapid eye movement; SRIF, somatostatin; SWS, slow wave sleep; X-ApEn, cross-approximate entropy.
Received October 22, 2001.
Accepted for publication December 4, 2001.
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in rats are reversed by food deprivation. J Sleep Res 8:197–203[CrossRef][Medline]
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