J. Sleep Res.

(2005) 14, 329336

Nocturnal ghrelin levels relationship to sleep EEG, the levels of growth hormone, ACTH and cortisol and gender dierences
PETRA SCHUESSLER, MANFRED UHR, MARCUS ISING, DAGMAR S C H M I D , J O T T A W E I K E L and A X E L S T E I G E R
Max Planck Institute of Psychiatry, Munich, Germany

Accepted in revised form 22 April 2005; received 31 January 2005

SUMMARY

Ghrelin, an endogenous ligand of the growth hormone (GH) secretagogue receptor, stimulates sleep, appetite and weight gain as well as the secretion of GH, adrenocorticotropic hormone (ACTH), cortisol in humans and rodents. The interaction between nocturnal ghrelin levels, sleep EEG and the secretion of these hormones was not investigated systematically so far. Furthermore conicting data exist on gender dierences in nocturnal ghrelin secretion. We examined simultaneously sleep EEG and the nocturnal levels of ghrelin, GH, ACTH and cortisol in young and middle-aged normal human subjects (eight males, eight females). A signicant interaction between gender and the course of ghrelin concentration was observed to the interval between 20:00 and 23:00 hours. In males a continuous increase of ghrelin levels before sleep onset was found. In females, however, a rise of ghrelin during the night was missed. We found a trend suggesting a lower time spent in stage I sleep in subjects with high nocturnal ghrelin levels. Other systematic interactions between plasma ghrelin, sleep EEG and other hormones were not found. No peak in plasma ghrelin levels resembling the GH surge was observed. We suggest that under naturalistic conditions plasma ghrelin levels show no distinct interaction with sleep. keywords adrenocorticotrophic hormone, cortisol, gender, ghrelin, growth hormone, sleep endocrinology

INTRODUCTION Ghrelin is a peptide hormone acting at the growth hormone (GH) secretagogue (GHS) receptor. It was isolated from the stomach and CNS of rats and humans (Kojima et al., 1999). The substance participates in the regulation of endocrine activity, energy balance and sleep. Since the sleeping period is a time of considerable activity in various endocrine systems, we were interested in the sleep-related pattern of ghrelin levels and its interaction with sleep EEG and other hormones. Given the fact that levels of leptin, which appears to counteract the inuence of ghrelin on the energy balance, dier distinctly between women and men, we also investigated gender dierences in nocturnal ghrelin concentrations (Antonijevic et al., 1998; Saad et al., 1997).
Correspondence: Axel Steiger, Max Planck Institute of Psychiatry, Kraepelinstrasse 10, 80804 Munich, Germany. Tel.: +49 89 30622 236; fax: +49 89 30622 552; e-mail: steiger@mpipsykl.mpg.de 2005 European Sleep Research Society

Besides GH-releasing hormone (GHRH) and somatostatin, ghrelin appears to be another endogenous factor in the regulation of GH release. After ghrelin administration, GH secretion was enhanced in rats (Kojima et al., 1999) and humans at daytime (Peino et al., 2000) and around sleep onset (Weikel et al., 2003). Obviously ghrelin shares the capacity of GHRH to stimulate GH. A synergism between these peptides is suggested in this action, whereas they were also shown to be partly independent from each other (Horvath et al., 2001). GHRH and ghrelin dier in their eect on the hypothalamo-pituitary-adrenocortical (HPA) system. In male subjects after repetitive i.v. administration of GHRH around sleep onset HPA hormones are blunted (Antonijevic et al., 2000; Steiger et al., 1992), whereas after ghrelin in an analogue protocol cortisol and adrenocorticotrophic hormone (ACTH) are increased in young males (Weikel et al., 2003). Similarly cortisol and ACTH are elevated in humans after ghrelin at daytime (Arvat et al., 2001). We suggest that ghrelin may act as an interface

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around midnight resembling the nocturnal GH peak. Ghrelin fell to trough levels within 1 h after eating. Intermeal ghrelin levels are found to rise throughout the day to zenith at 01:00 hours, then falling overnight to a nadir at 09:00 hours. Dzaja et al. (2004) compared intra-individually during 24-h ghrelin concentrations assessed hourly in young normal male subjects who were in supine position under two conditions. They either slept at night or were sleep deprived. In contrast to Cummings et al. (2001), in the sleeping condition there is no continuous rise throughout the day, but a distinct increase in ghrelin concentration at 01:00 hours is observed which is followed by a steady decline until the morning. Ghrelin secretion is blunted, however, during sleep deprivation and ghrelin levels increase only slightly until the early morning. From their data, Dzaja et al. (2004) suggested that sleep is a stimulus for ghrelin release, whereas Cummings et al. (2001) hypothesised that ghrelin displays a diurnal rhythm which is positively regulated by uxes in the overall energy balance. In rats, no signicant correlation between GH and ghrelin circulating levels is found, whereas mean interpeak intervals and pulse frequencies are close for the two hormones (Tolle et al., 2002). In another study in rats, plasma ghrelin displays diurnal rhythms with a peak preceding the major daily feeding peak after dark onset (Bodosi et al., 2004). Two recent studies report relationship between sleep duration and ghrelin levels. Spiegel et al. (2004) compared nights with sleep restriction and nights with sleep extension in normal male controls. Sleep restriction is associated with increases in ghrelin levels during daytime from 09:00 to 21:00 hours. Similarly, in a sample of 1024 volunteers an association between short sleep and elevated morning ghrelin levels is found (Taheri et al., 2004). There exists no simultaneous investigation of sleep EEG and nocturnal levels of ghrelin and hormones which may interact with its secretion, e.g. GH and HPA hormones employing a sampling rate of specimens below 1 h. Finally, studies on gender dierences in ghrelin activity show conicting results. In healthy and obese children morning ghrelin levels are independent of gender (Bellone et al., 2002). In 60 adult men and women of widely varying ages and weights (Purnell et al., 2003) and in healthy elderly women and men (Rigamonti et al., 2002) ghrelin levels do not dier between sexes. Espelund et al. (2005) sampled ghrelin concentrations every 3 h from 12 to 84 h of fasting. They describe diurnal changes with a nadir in the morning, peak levels in the afternoon and a gradual decline during the night. Ghrelin levels remain higher in women than in men throughout the study. Glucose load decreases ghrelin levels in adult subjects. This response is modulated by sex, in that levels are higher in females (Greenman et al., 2004). In young rats stomach ghrelin mRNA levels are not gender dependent (Gualillo et al., 2001). In contrast, in older female mice ghrelin mRNA is higher than in males (Liu et al., 2002). We wanted to investigate interactions between ghrelin, sleep EEG and other hormones and to elucidate gender dierences in these variables. Therefore, we examined sleep EEG and the
2005 European Sleep Research Society, J. Sleep Res., 14, 329336

between the HPA and the hypothalamo-pituitary-somatotrophic (HPS) systems (Weikel et al., 2003). The key role of ghrelin in the energy balance is well documented (Horvath et al., 2001). After ghrelin food intake in rodents (Tscho p et al., 2000; Wren et al., 2001b) and humans (Wren et al., 2001a), appetite in humans (Schmid et al., 2005; Wren et al., 2001a) and body weight in rodents (Tscho p et al., 2000) increase. Ghrelin levels are blunted in obese patients (Tscho p et al., 2001) and are elevated in patients with anorexia (Otto et al., 2001). Weight gain during antidepressive drug treatment is related to a decrease of ghrelin levels (Steiger et al., 2003). It is thought that a reciprocal interaction exists between the orexigenic ghrelin and the anorexigenic leptin (Horvath et al., 2001). Additionally, ghrelin promotes sleep. Non-REM sleep l et al., 2003). increased after systemic ghrelin in mice (Oba Similarly, after repetitive i.v. ghrelin slow wave sleep (SWS) increases in young normal males (Weikel et al., 2003). These data show that ghrelin shares the sleep-promoting eect of l and Krueger, 2002). In mice, intact GHRH GHRH (Oba l et al., receptors show to be the prerequisite for this eect (Oba 2003). In humans, the sleep EEG and the nocturnal secretion of various hormones show distinct patterns. During the rst half of the night the GH peak and the major portion of SWS preponderate, whereas HPA activity is low. In contrast during the second half of the night the amounts of GH and SWS are low and the major amounts of cortisol, ACTH and rapid eye movement (REM) sleep occur. There is good evidence that neuropeptides are common regulators of the neurophysiological and the endocrine components of sleep. It is thought that GHRH promotes SWS and GH around sleep onset, which is followed by corticotropin-releasing hormone in the early morning hours (Steiger, 2003). Leptin also shows variations throughout the night with a maximum between 00:00 and 04:00 hours (Licinio et al., 1997). Leptin levels are about threefold higher in females than in males (Antonijevic et al., 1998; Deuschle et al., 1996; Saad et al., 1997). The knowledge accumulated on ghrelin so far points to its role in the regulation of rhythms. This view is further supported by the observation that ghrelin containing neurons are located adjacent to the third ventricle between the dorsal, central, paraventricular and arcuate hypothalamic nuclei. It is claimed that the space occupied by ghrelin immunoreactive cells overlaps the hypothalamic projections from the suprachiasmatic nucleus and the central lateral geniculate body of the thalamus. The authors hypothesised that central ghrelin mediates circadian information from these sites of the hypothalamus (Cowley et al., 2003). Little is known about the sleep-related levels of ghrelin. Cummings et al. (2001) investigated ghrelin levels in humans (nine women, one man, 2963 years old) over 24 h. Specimens were collected at 30-min intervals from 08:00 to 21:00 hours, then hourly until 08:00 hours the next morning. These authors reported increases of ghrelin before meals and another peak

Ghrelin during sleep

sleep-related levels of ghrelin, GH, ACTH and cortisol in young and middle-aged normal controls of both sexes.

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The subjects were observed on a television screen in the adjacent room. Sleep EEG recordings Electrodes for polysomnographic recordings (Comlab 32 Digital Sleep Lab, Brainlab V 3.3 Software, Schwarzer GmbH, Munich, Germany) were xed between 21:00 and 22:00 hours. The subjects were not allowed to sleep until the lights were turned o at 23:00 hours. Polysomnographic recordings were obtained from 23:00 to 07:00 hours and consisted of two EEGs (C3A2, C4A1; time constant 0.3 s, low-pass ltering 70 Hz), vertical and horizontal electro-oculograms (EOG) and electromyogram (EMG). Conventional sleep EEG analysis Sleep stages [wakefulness, stages IIV sleep (stages III and IV sleep is SWS) and REM sleep] were scored visually in all subjects o-line according to conventional criteria (Rechtschaen and Kales, 1968) by a rater who was unaware of the aim of the study. Calculations of sleep parameters included time in bed (TIB), sleep onset latency (time between lights o and the rst occurrence of stage II sleep), SWS latency (interval between sleep onset and the rst 30-s epoch including stage III sleep), REM latency (interval between sleep onset and the rst 30-s epoch including REM sleep), number of awakenings and the time spent in the dierent sleep stages with reference to TIB. Sleep-EEG parameters were analysed for the TIB (480 min; identical to the total night) and the both halves of the night (each 240 min). Endocrine analysis Plasma ghrelin (Phoenix, Belmont, CA, USA; intra- and interassay coecients of variation <13%), plasma ACTH (Ria Kit J125; Nichols Institute, San Juan Capistrano, CA, USA; intra- and interassay coecients of variation <8%) and cortisol (Ria Kit J125; ICN Biomedicals, Carson, CA, USA; intra- and interassay coecients of variation <7%) concentrations were measured by radio-immunoassay; GH (Advantage; Nichols Institute, San Juan Capistrano, CA, USA; intra- and interassay coecients of variation <10%) concentrations were determined by chemiluminescence. Random samples for each hormone were analysed in duplicate. According to standard procedures for time series the remaining specimens were analysed only once. Statistical analysis Areas under the plasma concentration curves (AUC) were calculated for the intervals 20:0023:00 (evening), 23:0003:00 (rst half of the night), 03:0007:00 (second half of the night) and 23:0007:00 hours (total night) by applying the trapezoid rule (Yeh and Kwan, 1978). AUC scores were log-transformed to reduce the skewness of the distribution. Additionally,

METHODS Subjects The subjects were 16 healthy volunteers, eight males (mean age SD: 28.5 8.2 years, range 1946) and eight females (30.8 8.3 years, range 2249). Age distribution did not dier between male and female subjects (P 0.593). All subjects were free of any psychiatric or medical conditions. The body mass index (BMI) of the male subjects (23.0 0.7) was signicantly higher (P < 0.05) than the BMI of the female subjects (20.5 2.1). All women had regular menstrual cycles and were not taking hormonal contraceptives. They were examined in the follicular phase (between days 4 and 6). The experiment was approved by the Ethics Committee for Human Experiments at the University of Munich. After the purpose of the study had been explained to the subjects, all of them gave their informed consent according to the tenets of the declaration of Helsinki. They underwent extensive psychiatric, physical and laboratory (hematology, virology, clinical chemistry, endocrinology, EEG and electrocardiographic) examinations. Individuals with a personal or family history of psychiatric disorders or a recent stressful life event were excluded, as were shift workers and persons who had recently made a transmeridian ight. Subjects were interviewed about their usual sleep habits and were included in the study, when these were similar to the bed times (23:00 lights o, 07:00 lights on) in the sleep laboratory. Further, they were asked to keep this bed time habits in the week before the experimental procedure. Other exclusion factors were abuse of drugs, nicotine (more than two cigarettes per day), alcohol and caeine. Consumption of alcohol was not allowed throughout the study period starting 1 week prior to the adaptation night. Caeine was restricted to 200 ml coee in the morning. The subjects had standardised meals at 08:00, 12:00 and 18:00 hours with a macronutrient composition about 50% carbohydrates, 20% proteins and 30% fat. The median energy intake per day was about 30 kcal kg)1 bodyweight). It was ensured that there was no history of drug treatment during the previous 3 months. Experimental procedure The experimental sessions consisted of two successive nights in our sleep laboratory. The rst night served for adaptation to the laboratory setting. On the second night, an indwelling i.v. catheter was inserted at 19:30 hours and connected to plastic tubing that ran through a soundproof lock into the adjacent room. This allowed repeated blood collection in the adjacent laboratory without disturbing the study subject. All subjects were in a supine position from 20:00 hours. Blood samples were collected every 30 min between 20:00 and 22:00 hours and every 20 min between 22:00 and 07:00 hours.
2005 European Sleep Research Society, J. Sleep Res., 14, 329336

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nocturnal course of the ghrelin concentrations is depicted separately for male and female subjects in Fig. 1a. We observed a signicant interaction between gender and the course of the ghrelin secretion (F31,434 2.92; P 0.025, GreenhouseGeisser corrected) suggesting a nocturnal increase in male subjects (indicated by the positive 95% condence interval for the mean slope ranging from 73 to 417) and stable concentrations in female subjects (indicated by the slope 95% condence interval around zero; )403 to 131). This interaction eect can be considered as a large eect according to the eect size measure (partial g2 0.17). Fig. 1bd displays overnight secretion of GH, ACTH and cortisol in male and female subjects. We found signicantly higher maximum GH scores between 20:00 and 23:00 hours in female subjects (P 0.046). This eect could not be shown for the AUC (P 0.115). Although GH secretion appears to be higher in male subjects during the rst half of the night, a statistically signicant eect was not detected (AUC: P 0.208; maximum scores: P 0.247), presumably due to the large interindividual variability of the GH secretion (as indicated by the standard errors). Gender eects on sleep Besides a trend towards higher latency until stage IV sleep and a higher amount of stage I sleep in female subjects, we did not observe gender eects on polysomnographic sleep parameter (Table 2a). When the rst and second halves of the night are separately analysed we could replicate the higher amount of shallow stage I sleep in women (Table 2b). Eect size measures (partial eta-squared) suggest a large eect of gender on stage I sleep (total night: partial g2 0.41; 1st half: partial g2 0.29; 2nd half: partial g2 0.27). Associations between ghrelin secretion and sleep We observed a trend (P 0.095) suggesting a lower time spent in stage I sleep during the total night in subjects with high ghrelin AUC scores during this interval (r )0.31; P 0.095), which achieves signicance when only the second half of the night is considered (r )0.42;P 0.024). Interestingly, we observed a signicant gender eect suggesting less stage I sleep in the second half of the night in female subjects (P 0.040). However, the negative association between ghrelin secretion and time in stage I in the second half of the night remained signicant when a partial correlation was applied controlling for the eects of gender (AUC: r )0.55, P 0.030; Peak: r )0.54, P 0.036). No further associations were found. Associations between GH secretion and sleep We additionally calculated the associations between GH secretion and sleep parameters. We found a negative association between the amount of REM sleep and low GH secretion during the total night (AUC: r )0.39, P 0.038; maximum:
2005 European Sleep Research Society, J. Sleep Res., 14, 329336

hormonal maximum concentrations were identied as the individually highest concentration of all samples collected during the evening (20:0023:00 hours), during the rst (23:00 03:00 hours) and second half (03:0007:00 hours) of the night and during the total night (23:0007:00 hours). For correlation analyses the Kendall Tau (b) coecients were applied. Kendall Tau (b) is a non-parametric correlation coecient similar to Spearmans rho, but regarded as superior especially when tied values occur. In case of a signicant association a partial correlation controlling for gender was additionally calculated to rule out that gender eects interfere with the result of the non-parametric analysis. Dierences between male and female subjects were evaluated by anova. The level of signicance was set to P 0.05. Mean values and standard deviations are reported.

RESULTS Associations between age or BMI and ghrelin levels We did not observe any signicant associations between age or BMI and ghrelin concentrations (data not shown). A parametric partial correlation analysis controlling for gender did not reveal signicant associations either. Gender eects on ghrelin levels Gender eects in ghrelin levels were assessed by AUC and maximum values were evaluated by means of an anova. We observed a trend suggesting higher AUC ghrelin concentrations between 20:00 and 23:00 hours in female subjects (P 0.075), which was signicant when maximum values during the interval are considered. The results are presented in Table 1. No gender eects were observed for the ghrelin secretion between 23:00 and 07:00 hours. Furthermore, we conducted a repeated measures analysis with the single ghrelin concentrations as a within-subjects factor and gender as a between-subjects factor. The mean
Table 1 Gender eects on ghrelin baseline and overnight secretion assessed by AUC and peak values Male subjects (n 8) Ghrelin AUC 20:0023:00 23:0003:00 03:0007:00 23:0007:00 Ghrelin maximum 20:0023:00 23:0003:00 03:0007:00 23:0007:00 10.70 (0.62) 11.38 (0.46) 11.45 (0.31) 12.11 (0.38) levels (pg ml)1) 346 (186) 536 (251) 546 (233) 628 (279) Female subjects (n 8) 11.37 11.73 11.68 12.40 734 736 638 740 (0.77) (0.62) (0.52) (0.57) (418) (453) (322) (451) P 0.075 0.215 0.307 0.253 0.031 0.294 0.527 0.558

AUC values are log-transformed; P-values indicate eects of gender in a univariate analysis of variance. The values are expressed as mean (SD). AUC, areas under the plasma concentration curves.

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Figure 1. (ad) Nocturnal ghrelin, GH, ACTH and cortisol secretion in male (dark line with triangles) and female (light line with circles) subjects. Means and standard errors of the mean (SEM) are presented. ACTH levels were not determined in samples collected before lights were switched o (23:00 hours).

r )0.41, P 0.030), which could be conrmed after controlling for the eects of gender (AUC: r )0.61, P 0.015; peak: r )0.58, P 0.022). We observed a trend towards a positive association between the total night amount of SWS (stage III + stage IV) and GH maximum values (r 0.34, P 0.071), which could not be replicated after gender eects were controlled. We repeated this analysis separately for both genders and found a signicant association in male subjects (r 0.41, P 0.030) but not in female subjects (r 0.30, P 0.315), suggesting a more pronounced association in males. Associations between ghrelin, GH, ACTH and cortisol concentrations Besides a positive association between ACTH and cortisol during the rst half of the night (AUC: r 0.38, P 0.038) we did not observe any correlations between ghrelin, GH, ACTH and cortisol, neither for the total nor for the two halves of the night (data not shown). DISCUSSION The major ndings of our study are (i) higher ghrelin levels in young and middle aged normal females than in males during the interval between 20:00 and 23:00 hours, but the lack of
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signicant gender dierences between 23:00 and 07:00 hours, (ii) a negative correlation between time spent in stage I sleep and ghrelin levels during the second half of the night and (iii) the absence of other interactions between nocturnal ghrelin levels and sleep EEG, GH, ACTH and cortisol secretion. During the investigated interval from 20:00 to 07:00 hours mean ghrelin levels in females did not display major variations. In males, however, the lowest mean value was found at 20:00 hours. It was followed by an arbitrarily continuous increase until 01:00 hours. Later on the values in males remained, by trend, lower than in females. Similar to the females, the following course in males remained widely stable except for a small peak at 04:20 hours in the males. No major nocturnal peak of ghrelin levels was detectable either in females or males. This nding corresponds to the individual inspection of the timing of the ghrelin maxima, which revealed a widely spread distribution throughout the night. A sleepdependent rise of ghrelin around midnight as observed in males by Dzaja et al. (2004) was absent. In contrast, ghrelin levels in females showed, from the start of the observation period at 20:00 hours, similar values to those at midnight and beyond, up to the last sampling at 07:00 hours. Whereas ghrelin levels in males started to increase already at 20:00 hours, i.e. 3 h before lights were switched o and subjects were allowed to sleep, we did not observe a sleeprelated increase. One may argue that the observed gender

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the peptide. The sharp increases before meals may promote hunger and food intake, whereas the nocturnal rise observed in our study in males and in previous reports (Cummings et al., 2001, Dzaja et al., 2004) maybe related to sleep. It is also possible that depending on the threshold higher concentrations induce hunger. This may be the mechanism of night-eating disorder. In a single patient with this disorder very high nocturnal ghrelin levels were observed (Rosenhagen et al., 2005). Since we started to examine ghrelin levels on 20:00 hours, when values in females did not dier from those later on during the night, it remains unclear whether we missed an increase prior to 20:00 hours, or whether major uctuations were absent in the females of our sample throughout the 24 h. A limitation of our study might be that ghrelin levels appear to be aected by sleep duration. The night adaptation can be associated with reduced total sleep time where elevated ghrelin levels are reported (Spiegel et al., 2004; Taheri et al., 2004). This may inuence the ghrelin levels during the examination night. As summarised in the Introduction only a few studies have addressed gender dierences in ghrelin activity and have reported conicting results. Furthermore, an inuence of sexual steroids on morning ghrelin levels has been suggested recently. In male patients with hypogonadism ghrelin concentration was lower than in controls. After testosterone replacement therapy ghrelin levels increased in the patients (Pagotto et al., 2003). A study in obese female patients with and without polycystic ovary syndrome showed a negative correlation between ghrelin and androstendione levels (Pagotto et al., 2002). Leptin is thought to oppose the action of ghrelin on the energy balance. In women, leptin levels are about three times higher than in men (Antonijevic et al., 1998; Deuschle et al. 1996; Saad et al., 1997). Given these observations we expected a major inuence of gender on ghrelin levels. This was restricted to the evening hours, however, in our study. The negative correlation between stage I sleep, which corresponds to shallow sleep and ghrelin levels is compatible with its role as a sleep promoting factor. Exogenous ghrelin l et al., 2003) and enhanced non-REM sleep in mice (Oba humans (Weikel et al., 2003). Intact GHRH receptors have been determined to be the prerequisite for this eect in mice l et al., 2003). Both GHRH and ghrelin promote sleep (Oba l and Krueger, 2002). A partly and stimulate GH (Oba synergistic action of both peptides in the release of GH has been suggested (Horvath et al., 2001). Similarly it appears likely that ghrelin and GHRH are cofactors in sleep regulation. A coincidence of the highest amounts of GH and SWS is well established. As expected in our study, there was a correlation between SWS and GH secretion (Van Cauter and Copinschi, 2000). In the rat GHRH synthesis and release peak at the time of highest sleep propensity at the onset of the light period (Bredow et al., 1996; Gardi et al., 1999). Therefore one might expect a peak of ghrelin overlapping the GH peak. This is also suggested by the observation of Cummings et al. (2001). Similarly Dzaja et al. (2004) reported a positive correlation between the nocturnal GH peak and ghrelin levels between
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Table 2 Gender eects on (a) total night sleep parameters and (b) sleep separately for the rst and the second half of the night Male subjects (n 8) (a) Time in bed Sleep period time Total sleep time Sleep eciency Sleep onset latency Stage III latency Stage IV latency REM latency Time in stage I (min) Time in stage II (min) Time in stage III (min) Time in stage IV (min) Time in REM (min) Time awake (min) (b) First half of the night Time in stage I (min) Time in stage II (min) Time in stage III (min) Time in stage IV (min) Time in REM (min) Time awake (min) Second half of the night Time in stage I (min) Time in stage II (min) Time in stage III (min) Time in stage IV (min) Time in REM (min) Time awake (min) 482.6 449.7 411.7 85.3 32.1 24.3 9.9 89.3 27.8 229.1 30.4 36.1 81.6 42.3 (4.8) (17.9) (35.5) (7.3) (15.4) (35.7) (6.3) (65.5) (6.6) (40.5) (10.6) (25.2) (18.5) (39.6) Female subjects (n 8) 484.0 430.6 413.9 85.5 48.1 26.8 33.6 77.1 16.3 226.8 33.3 41.6 88.0 21.1 (4.5) (47.1) (54.9) (11.4) (41.3) (28.8) (31.3) (19.0) (8.2) (38.1) (11.0) (27.0) (28.1) (18.8) P 0.565 0.302 0.926 0.963 0.321 0.883 0.054 0.621 0.008 0.910 0.603 0.680 0.601 0.194

8.7 101.1 17.8 27.2 28.1 25.1 19.1 128.0 12.6 8.9 53.5 17.2

(5.6) (18.9) (8.4) (19.2) (15.7) (31.4) (3.7) (26.0) (9.3) (9.7) (10.7) (11.4)

3.6 92.0 22.1 30.6 33.6 10.6 12.7 134.8 11.2 10.9 54.4 10.4

(2.2) (25.9) (7.8) (22.8) (14.0) (19.2) (7.1) (18.4) (7.5) (12.6) (18.3) (5.7)

0.032 0.438 0.311 0.749 0.472 0.286 0.040 0.555 0.749 0.719 0.909 0.155

The values are expressed as mean (SD). REM, rapid eye movement.

dierences can be explained by dierences in BMI between male and female subjects. However, all subjects were within a normal weight range and we did not observe any signicant association between the individual BMI and ghrelin levels. The discrepancy between our observation and the report by Dzaja et al. (2004) may be explained by methodological dierences. In the latter study subjects were supine for 24 h, whereas our subjects performed their normal activities until 19:00 hours. The pattern found in our sample resembles that reported by Cummings et al. (2001). These authors, however, investigated mainly females, who were older and had a higher BMI than our sample. Sleep EEG was not recorded in their study. Our nding that ghrelin levels in males already increase prior to sleep onset resembles our previous report that GH levels in a subgroup of healthy young men increased before sleep onset (Steiger et al., 1987). Otherwise it must be considered that a postprandial rise after dinner at 18:00 hours of the ghrelin levels (Cummings et al., 2001) in males may play a role, whereas females had their meals at the same time without a change of ghrelin levels before sleep onset. It appears possible, however, that depending on the time of the day changes of ghrelin levels reect dierent physiological roles of

Ghrelin during sleep

23:00 and 03:00 hours. In our study, however, a ghrelin peak around sleep onset was missing. This does not exclude an enhanced activity of central ghrelin around sleep onset and the GH peak. In rats, interpeak intervals and pulse frequencies were close for ghrelin and GH, whereas no signicant correlation between these hormones was found (Tolle et al., 2002); Bodosi et al. (2004), however, reported a peak of ghrelin after dark onset in rats prior to their feeding peak. Probably the major surge of plasma ghrelin analysed in our study derived from the stomach (Ariyasu et al., 2001). The exact pattern of central ghrelin activity related to sleep remains to be elucidated by preclinical studies. Our study suggests gender dierences in the pattern of peripheral ghrelin levels in the interval between 20:00 and 23:00 hours in young and middle-aged healthy subjects. No major interaction between ghrelin and sleep EEG and between ghrelin and the other investigated hormones were found. ACKNOWLEDGEMENTS This study was supported by a grant from the Deutsche Forschungsgemeinschaft (STE 485/5-3). REFERENCES
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