Hexarelin decreases slow-wave sleep and stimulates the secretion of GH, ACTH, cortisol and prolactin during sleep in healthy volunteers

Psychoneuroendocrinology (2004) 29, 851–860

www.elsevier.com/locate/psyneuen

Hexarelin decreases slow-wave sleep and stimulates the secretion of GH, ACTH, cortisol and prolactin during sleep in healthy volunteers Ralf-Michael Frieboes, Irina A. Antonijevic, Katja Held, Harald Murck, ¨cher, Manfred Uhr, Axel Steiger* Thomas Pollma Max Planck Institute of Psychiatry, D-80804 Munich, Germany Received 7 February 2003; received in revised form 30 June 2003; accepted 25 July 2003

KEYWORDS Growth hormone; Growth hormone secretagogues; Hexarelin; Corticotropins; TNF-a; Sleep

Summary Ghrelin, the endogenous ligand of the growth hormone (GH) secretagogue (GHS) receptor and some GHSs exert different effects on sleep electroencephalogram (EEG) and sleep-related hormone secretion in humans. Similar to GH-releasing hormone (GHRH) ghrelin promotes slow-wave sleep in humans, whereas GH-releasing peptide-6 (GHRP-6) enhances stage 2 nonrapid-eye movement sleep (NREMS). As GHRP-6, hexarelin is a synthetic GHS. Hexarelin is superior to GHRH and GHRP-6 in stimulating GH release. The influence of hexarelin on sleependocrine activity and the immune system is unknown. We investigated simultaneously the sleep EEG and nocturnal profiles of GH, ACTH, cortisol, prolactin, leptin, tumor necrosis factor (TNF)-a, and soluble TNF-a receptors in seven young normal volunteers after repetitive administration of 4
50 lg hexarelin or placebo at 22.00, 23.00, 24.00 and 01.00 h. Following hexarelin, stage 4 sleep during the first half of the night, and EEG delta power during the total night decreased significantly. Significant increases of the concentrations of GH and prolactin during the total night, and of ACTH and of cortisol during the first half of the night were found. Leptin levels, TNF-a and soluble TNF receptors remained unchanged. We hypothesize that sleep is impaired after hexarelin since the GHRH/corticotropinreleasing hormone (CRH) ratio is changed in favour of CRH. There are no hints for an interaction of hexarelin and the immune system. # 2003 Elsevier Ltd. All rights reserved.

1. Introduction

mone secretion (reviewed in Steiger, 2002a). Two recent studies report promotion of sleep after systemic administration of the endogenous ligand of the GHS receptor, ghrelin. In mice nonrapid eye movement sleep (NREMS) increased after systemic ghrelin (Oba ´l et al., 2003). Similarly slow-wave sleep (SWS) increased in young normal male subjects after repetitive intravenous (iv) injection of ghrelin. Furthermore nocturnal GH, corticotropin

Ligands of the growth hormone (GH) secretagogue (GHS) receptor modulate sleep electroencephalogram (EEG) and sleep-associated hor* Corresponding author. Tel.: +49-89-30622-236; fax: +49-8930622-552. E-mail address: [email protected] (A. Steiger).

0306-4530/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4530(03)00152-5

852

(ACTH), cortisol and prolactin levels increased, whereas leptin levels remained unchanged (Weikel et al., 2003). Recently Cowley et al. (2003) described the expression of ghrelin in a group of neurons adjacent to the third ventricle between the dorsal, central, paraventricular and arcuate hypothalamic nuclei. These authors claimed that the internuclear space occupied by ghrelin-immunoreactive cells overlaps exactly the hypothalamic projections from the suprachiasmatic nucleus and the ventral lateral geniculate body of the thalamus. They hypothesize that the central ghrelin neurons mediates circadian information from these sites to postsynaptic targets. Studies with GH-releasing hormone (GHRH) and several synthetic GHSs showed a variety of changes of sleep EEG and nocturnal hormone secretion after various substances. Hexarelin is a synthetic GHS, which was at daytime more potent than other GHSs compounds of its class to release GH (Ghigo et al., 1999). Synthetic GHSs including hexarelin are thought to be useful in the treatment of GHdeficient children and adults, in frail elderly adults, in obese patients, in patients with congestive heart failure and patients suffering from wasting catabolic disease (Svensson, 2000). The effects of hexarelin on sleep EEG and sleeprelated hormone secretion are unknown so far. We choose hexarelin as a tool to further investigate the role of the GHS receptor in sleep-endocrine regulation. In detail the following effects of GHRH and of synthetic GHSs were reported. The influences of ghrelin on sleep EEG in humans and mice resembled those of GHRH in several species including humans. After systemic and central administration of GHRH NREMS increased in rats, rabbits and mice (reviewed in Oba ´l and Krueger, 2002). In young normal male subjects SWS was enhanced after iv GHRH (Steiger et al., 1992; Kerkhofs et al., 1993; Marshall et al., 1996). It was hypothesised that GHRH is involved in the sleep-promoting effect of ghrelin since ghrelin did not modulate sleep in mice with deficient GHRH receptors (Oba ´l et al., 2003). We investigated changes of sleep EEG after administration of GH-releasing peptide 6 (GHRP-6) by various routes of administration (Frieboes et al., 1999). The most distinct effect of GHRP-6 was found after repetitive iv administration as NREMS stage 2 increased (Frieboes et al., 1995). GHRP-1 had no effect on sleep EEG (Moreno-Reyes et al., 1998). Stage 4 NREMS and rapid eye movement sleep (REMS) increased in young normal male subjects and REMS was enhanced in elderly normal controls after 1 week of oral intake of MK-677 (Copinschi et al., 1997). In contrast to ghrelin GHRH blunted nocturnal cortisol (Steiger et al., 1992; Antonijevic et al.,

R.-M. Frieboes et al.

2000b) and ACTH (Antonijevic et al., 2000b) in young normal males. Similar to ghrelin GHRP-6 increased GH and prolactin during the total night and cortisol and ACTH during the first half of the night (Frieboes et al., 1995). After iv administration at daytime hexarelin shares the capacity of ghrelin (Peino et al., 2000; Takaya et al., 2000) and GHRP-6 (Huhn et al., 1993) to stimulate GH (Ghigo et al., 1994), ACTH, cortisol and prolactin (Arvat et al., 1997a, b; Korbonits et al., 1999). Besides their effects on hormone secretion and sleep GHSs influence food intake and body weight. It is well documented that ghrelin is an orexigenic substance (reviewed in Horvath et al., 2001). Increases of appetite were found in humans after iv ghrelin (Wren et al., 2001; Steiger et al., 2002) and hexarelin (Korbonits et al., 1999) in the morning. In animal models orexigenic effects of hexarelin were found in rats (Torsello et al., 1998) and dogs (Rigamonti et al., 1999). Preclinical studies suggest an interaction of ghrelin and the anorectic leptin in the energy balance (Horvath et al., 2001; Shintani et al., 2001). We did not observe acute changes of leptin levels after iv ghrelin during sleep (Weikel et al., 2003) and at daytime (Steiger et al., 2002). The effect of hexarelin on leptin is unknown. The sleep-modulating effect of GHRH is closely related to the respective effects of inflammatory cytokines (Krueger et al., 1999). To our knowledge the role of the immune system in the influence of GHRH or GHSs on human sleep is unknown so far. Tumor necrosis factor (TNF)-a and soluble TNF receptors are involved in sleep responses to infection and inflammation in humans (Pollma ¨cher et al., 2000). Therefore the determination of their levels after administration of sleep-modulating peptides appears to be useful to clarify this issue. In order to delineate the effects of hexarelin on sleep EEG, nocturnal hormone secretion and cytokine levels we applied a sleep-endocrine protocol with repetitive iv administration of the substance according to previous studies of our laboratory applying various peptides including GHRH (Steiger et al., 1992), GHRP-6 (Frieboes et al., 1999) and ghrelin (Weikel et al., 2003).

2. Materials and methods The study group consisted of seven healthy male paid volunteers of normal weight and height. The mean age of the probands was 24.62.8 (SD) years (range, 20–28). After written informed consent had been obtained, the subjects were inter-

Sleep and hormones after hexarelin

853

Table 1 Sleep-EEG variables after placebo and hexarelin administration

Sleep continuity TIB (min) SPT (min) TST (min) SEI SOL (min) Awakenings

Placebo (meanSD)

Hexarelin (meanSD)

MANOVA

479.70.99 458.317.1 424.454.23 0.930.11 18.413.6 10.96.8

480.01.08 448.233.2 430.434.1 0.960.05 18.913.4 12.711.7

NS NS NS NS NS NS

48.933.1 21.011.1 258.635.8 41.217.7 19.716.0 60.923.13 85.715.9

NS NS NS NS NS NS NS

25.119.2

17.814.9

p<0.05

129.118.2

129.626.4

NS

116.823.0

129.227.7

p<0.1

62.823.9

63.015.7

NS

Sleep architecture, min spent in each stage during TIB Awake 54.552.8 Stage 1 18.63.6 Stage 2 245.636.9 Stage 3 39.613.4 Stage 4 25.119.2 SWS 64.619.7 REM 91.923.1 min, spent during Stage 4: first half of the night (SPT) Stage 2: first half of the night (SPT) Stage 2: second half of the night (SPT) REM: second half of the night (SPT)

Abbreviations: TIB, time in bed; SPT, sleep period time; TST, total sleep time; SEI, sleep efficiency index; SOL, sleep onset latency; SWS, slow wave sleep; REM, rapid eye movement; significance in MANOVA group
treatment effect p < 0:05; trend in MANOVA group
treatment effect p < 0:1; NS, not significant.

viewed by a senior psychiatrist to exclude a personal or family history of psychiatric disorders and recent stressful life events. Shift workers and subjects who had made a recent transmeridian flight were excluded from the study. Abuse of alcohol, drugs, nicotine or caffeine and any history of use of medication during the last 3 months were ruled out. The subjects underwent a thorough physical examination and extensive laboratory tests, including hematology, virology, clinical chemistry, endocrine evaluation, EEG, and electrocardiogram (ECG). The protocol was approved by the Ethics Committee for Human Experiments of the Bavarian Physicians‘ Board. The study consisted of two sessions at an interval of 1 week in which placebo or 50 lg hexarelin (His–D-2-methylTrp–Ala–Trp–D-phe–Lys–NH2; Clinalfa, La ¨ufelfingen, Switzerland) was administered each hour from 22.00 to 01.00 h according to a randomized double-blind schedule. The sleep EEG and sleepassociated hormone secretion were investigated simultaneously as described in detail elsewhere (Holsboer et al., 1988). Each session consisted of two successive nights in our sleep laboratory. The first night of each session served for adaptation to the laboratory setting including application of

electrodes and insertion of an indwelling catheter into a forearm vein at 19.30 h. The catheter was connected to a plastic tube that ran through a soundproof lock into the adjacent room. The polygraphic recordings (EEG, electrooculogram, electromyogram and ECG), obtained in accordance with standard procedure (Rechtschaffen and Kales, 1968), were monitored and the subjects could be observed on a TV screen throughout the night. They were not allowed to sleep until the lights were turned off at 23.00 h. On the second night of each session, beginning at 22.00 h, blood was drawn into heparinized chilled tubes every, 20 min until 07.00 h through the long catheter. Blood samples (4 ml) were immediately cen  trifuged at 4 C and plasma was frozen at 25 C. Plasma concentrations of the hormones GH (Advantage, Nichols Institute, San Juan Capistrano, CA, USA), ACTH (RIA Kit J125 Nichols Institute, San Juan Capistrano, CA, USA), cortisol (RIA Kit J125, ICN Biomedicals, Carson, CA, USA), prolactin (Advantage, Byk-Santec, Dietzenbach, Germany) and leptin (DRG Instruments, Marburg, Germany) were determined by standard commercially available radioimmunoassays. All samples from a given subject were analysed in the same

854

R.-M. Frieboes et al.

3.2. Humoral variables

Fig. 1. Spectral analysis of nonrapid eye movement sleep of the total night showing the differences in the EEG spectral power occurring due to hexarelin administration (22.00–01.00 h). There is a significant decrease in the delta frequency range.

assay. Endocrine variables were calculated as described elsewhere (Holsboer et al., 1988; Steiger et al., 1989). The limit of detection for all hormones was 0.5 ng/ml, and the intra- and interassay coefficients of variation were 7% and 9%, respectively. TNF-a and both TNF receptor levels were measured by enzyme linked immunosorbent assays as described in detail previously (HinzeSelch et al., 2000). Between 23.00 and 07.00 h the sleep EEG was recorded and was scored visually at 30-s epochs (Rechtschaffen and Kales, 1968; Holsboer et al., 1988). EEG spectral analysis was performed as reported elsewhere (Murck et al., 1996). All data are expressed as meansstandard deviation (SD). Differences between the sleep-EEG variables and the hormone parameters after hexarelin versus placebo were tested for significance with multivariate analysis of variance (MANOVA), with a nominal level of significance of a ¼ 0:05. At 08.00 h after each of the experimental nights side effects including changes of appetite were assessed by a semi-structured interview.

3. Results 3.1. Sleep EEG variables Table 1 shows the results of conventional sleepEEG analysis. MANOVA yielded only two significant results. In the first half of the night, there was a significant decrease in sleep stage 4 after hexarelin. Apart from that, sleep continuity, sleep architecture and REMS variables remained unchanged. Spectral analysis of NREMS revealed a significant decrease of delta power during the total night, as depicted in Fig. 1.

The endocrine variables are given in Table 2. The detailed time courses of hormone secretion under hexarelin administration are shown in Fig. 2. GH and prolactin secretion were stimulated during the total night with the major enhancement in the first part of the night (22.00 to 03.00 h). The concentrations of ACTH and cortisol increased during the first part of the night, and additionally, the secretion of cortisol decreased in the second part of the night (03.00 to 07.00 h). Leptin levels did not change after hexarelin. Nor did TNF-a and the soluble TNF-a receptors 1 and 2. Their concentrations are given in Table 3.

3.3. Side effects The substance was well tolerated. No side effects, particularly no change in appetite were reported.

4. Discussion The major findings of our study are decreases of sleep stage 4 during the first half of the night and slow wave activity during the total night and increases of GH and prolactin after repetitive administration of hexarelin to healthy young men. During the first part of the night cortisol and ACTH increased. These hormones were blunted during the second half of the night. No changes of leptin, TNF-a and soluble TNF-receptor levels were found. Whereas the effects on peripheral hormone secretion are similar to those of ghrelin and GHRP-6 in parallel protocols, it is obvious, that the synthetic GHS receptor ligand hexarelin does not mimic the sleep-EEG changes induced by the endogenous ligand of the same receptor, ghrelin. After ghrelin in young normal male subjects SWS increased during the total night and SWA was enhanced during the second half of the night. Separate inspection of stage 4 sleep showed that this component of SWS was also enhanced during the total night (Weikel et al., 2003). Similarly in normal mice NREMS increased after systemic ghrelin, whereas this effect was absent in mice with deficient GHRH receptors (Oba ´l et al., 2003). This observation and the fact that the sleep-EEG changes after ghrelin resemble those after GHRH in humans (Steiger et al., 1992; Kerkhofs et al., 1993; Marshall et al., 1999) and in laboratory animals (Oba ´l and Krueger, 2002) suggest that GHRH is involved in sleep promotion after ghrelin. Interestingly, also oral treatment with the synthetic GHS MK-677 during one week prompted an

Sleep and hormones after hexarelin

855

Table 2 Endocrine variables after placebo and hexarelin administration Placebo (meanSD)

Hexarelin (meanSD)

MANOVA

GH concentration, ng/ml 22.00–07.00 h 22.00–03.00 h 03.00–07.00 h

2.41.3 3.52.0 1.00.6

7.64.9 12.88.3 0.90.6

p<0.05 p<0.05 NS

GH AUC, ng/ml
min 22.00–07.00 h 22.00–03.00 h 03.00–07.00 h

1335705 1103771 232140

42562750 40622634 194132

p<0.05 p<0.05 NS

ACTH concentration, pg/ml 22.00–07.00 h 22.00–03.00 h 03.00–07.00 h

16.75.2 8.73.1 26.28.5

15.03.3 11.11.8 19.45.2

NS p<0.05 NS

ACTH AUC, pg/ml
min 22.00–07.00 h 22.00–03.00 h 03.00–07.00 h

89642809 2562907 64022112

79681714 3368549 46001256

NS p<0.05 NS

Cortisol concentration, ng/ml 22.00–07.00 h 75.124.6 22.00–03.00 h 36.719.7 03.00–07.00 h 121.234.2

88.931.5 77.939.6 100.123.1

NS p<0.05 p<0.05

Cortisol AUC, ng/ml
min 22.00–07.00 h 22.00–03.00 h 03.00–07.00 h

47 48717 157 23 79412 222 23 6925645

NS p<0.05 p<0.05

Prolactin concentration, ng/ml 22.00–07.00 h 16.12.8 22.00–03.00 h 13.93.3 03.00–07.00 h 19.02.7

19.13.5 19.33.7 18.84.5

p<0.05 p<0.05 NS

Prolactin AUC, ng/ml
min 22.00–07.00 h 22.00–03.00 h 03.00–07.00 h

10 3301863 58581107 44721097

p<0.05 p<0.05 NS

Leptin concentration, ng/ml 22.00–07.00 h 3.51.7 22.00–03.00 h 3.71.9 03.00–07.00 h 3.31.6

3.52.3 3.62.4 3.32.3

NS NS NS

Leptin AUC, ng/ml
min 22.00–07.00 h 22.00–03.00 h 03.00–07.00 h

18621240 1069706 793536

NS NS NS

39 87613284 10 6435719 29 2338265

87651552 41971019 4568654

1910937 1119558 791384

Abbreviations: GH, Growth hormone; AUC, area under the curve; ACTH, adreno-corticotropic hormone; significance in MANOVA group
treatment effect p < 0:05; NS, not significant.

increase of SWS in normal young male subjects (Copinschi et al., 1996). It is thought, that besides binding at the GHS receptor, MK-677 facilitates the action of endogenous GHRH (Smith, 1998). Also repetitive iv administration of GHRP-6 promoted sleep in young normal male subjects, however in a different manner. After GHRP-6 NREMS stage 2 increased and SWS including stage 4

remained unchanged (Frieboes et al., 1995). The lack of an effect of single bolus injections of GHRP-1 on sleep EEG (Moreno-Reyes et al., 1998) may be explained by differences in the study protocol. We have argued, that repetitive iv administration is a crucial prerequisite for effects of peptides on sleep (Steiger and Holsboer, 1997). This view is corroborated by the comparison of

856

Fig. 2. Time course of hormone plasma concentration of GH, ACTH, cortisol, prolactin and leptin (means SEM) after hexarelin and after placebo iv administration. Continuous line and asterisk mean interval with significant increase. Dotted line and asterisk mean significant decrease.

pulsatile and continuous administration of GHRH to control subjects. SWS increased after repetitive

R.-M. Frieboes et al.

iv administration, whereas sleep EEG remained unchanged under continuous infusion (Marshall et al., 1996). These methodological issues may also explain the lack of distinct effects of intranasal and sublingual GHRP-6 on sleep EEG (Frieboes et al., 1999). Whereas repetitive iv injections of ghrelin, GHRH and GHRP-6 promoted NREMS, sleep was impaired after hexarelin as stage 4 sleep and SWA decreased. The changes of sleep EEG after hexarelin should be discussed in the line of its effects on peripheral hormone secretion. The pattern of the endocrine response after hexarelin resembles that after repetitive iv GHRP-6 (Frieboes et al., 1995) and after repetitive iv ghrelin (Weikel et al., 2003). GH and prolactin increased throughout the night after GHRP-6, and cortisol increased after ghrelin during the total night. The hormones of the hypothalamic pituitary adrenocortical (HPA) system ACTH and cortisol were elevated during the first part of the night. During the second part of the night HPA hormones were blunted after GHRP-6 and after hexarelin as well, probably due to negative feedback inhibition. A similar response of cortisol was observed after repetitive CRH (Holsboer et al., 1988). Also GHRH augmented the nocturnal GH surge in normal males and females of a wide age range (Steiger et al., 1992; Kerkhofs et al., 1993; Guldner et al., 1997; Marshall et al., 1999; Antonijevic et al., 2000b) and in patients with depression of both sexes (Antonijevic et al., 2000b). However, in normal young male subjects (Antonijevic et al., 2000a,b; Steiger et al., 1992) the increases of NREMS were accompanied by blunted cortisol (Steiger et al., 1992; Antonijevic et al., 2000a,b) and ACTH levels (Antonijevic et al., 2000a,b). In contrast HPA hormones were elevated and sleep was impaired in healthy and depressed women after GHRH (Antonijevic et al., 2000a, 2000b). A more detailed look shows differences in the potency of various GHSs to stimulate GH and cortisol. As reported in detail elsewhere (Weikel et al., 2003), hexarelin was more potent to stimulate GH during the total night than iv GHRH (Steiger et al., 1992), ghrelin (Weikel et al., 2003) and iv GHRP-6 (Frieboes et al., 1995) in our previous studies with complementary protocols. Its effect on cortisol during the first part of the night was lower than the stimulation by GHRP-6 and ghrelin. Given the hints that GHRH is involved in the sleep-promoting effect of ghrelin (Oba ´l et al., 2003) one might expect a distinct increase of SWS after hexarelin. Obviously our results contradict a sleep-promoting effect of hexarelin. A reciprocal interaction of GHRH and CRH in sleep regulation was submitted first by Ehlers and

Sleep and hormones after hexarelin

857

Table 3 TNF-a and soluble TNF-a receptors after placebo and hexarelin administration Placebo (meanSD)

Hexarelin (meanSD)

MANOVA

TNF-a, concentration, pg/ml 22.00–07.00 h 7.33.5 22.00–03.00 h 7.43.8 03.00–07.00 h 7.33.5

7.33.3 7.53.4 7.43.9

NS NS NS

TNF-a, AUC, pg/ml
min 22.00–07.00 h 22.00–03.00 h 03.00–07.00 h

31551458 1814817 1328690

NS NS NS

TNF-a receptor 1, concentration, pg/ml 22.00–07.00 h 1.40.4 22.00–03.00 h 1.40.4 03.00–07.00 h 1.40.4

1.50.4 1.40.3 1.50.4

NS NS NS

TNF-a receptor 1, AUC, pg/ml
min 22.00–07.00 h 607158 22.00–03.00 h 34785 03.00–07.00 h 26074

618149 34382 27468

NS NS NS

TNF-a receptor 2, concentration, pg/ml 22.00–07.00 h 3.50.6 22.00–03.00 h 3.40.5 03.00–07.00 h 3.60.7

3.50.4 3.40.4 3.50.5

NS NS NS

TNF-a receptor 2, AUC, pg/ml
min 22.00–07.00 h 1473242 22.00–03.00 h 822121 03.00–07.00 h 653117

1471169 82988 64181

NS NS NS

31001473 1770873 1331624

Abbreviations: TNF-a, tumor necrosis factor alpha; AUC, area under the curve; significance in MANOVA group
treatment effect p < 0:05; NS, not significant.

Kupfer (1987). This view is corroborated by various studies, at least in male subjects (reviewed in Steiger, 2002b). In short it was shown, that GHRH promotes SWS and GH and inhibits HPA hormones, whereas CRH has opposite effects. Changes of the balance between these peptides are thought to be associated with changes in sleep-endocrine activity. If GHRH preponderates NREMS is promoted, if the balance is changed in favour of CRH, more shallow sleep occurs. We suggest, that after hexarelin the latter change occurred in our study. The decrease of stage 4 sleep and SWA after hexarelin may be explained by 1. feedback inhibition of endogenous GHRH due to the elevation of peripheral GH; 2. by the increase of central CRH; or 3. the combination of both facts. Given the distinct effect of hexarelin on GH secretion a major role of the first explanation appears to be most likely. Peripheral administration of GH was shown to decrease SWS in humans (Mendelson et al., 1980) and laboratory animals (Stern et al., 1975). CRH

decreased SWS in young normal subjects (Holsboer et al., 1988) and impaired sleep and promoted wakefulness in laboratory animals (reviewed in Opp, 1995; Steiger, 2002b). The response of HPA hormones to hexarelin may be mediated by CRH. Alternatively and/or additionally arginine vasopressin is thought to contribute to this effect (Korbonits et al., 1999). The effects of arginine vasopressin on sleep are less clear than those of CRH. ICV vasopressin increased wakefulness in rats (Arnauld et al., 1989). After intranasal vasopressin SWS and REMS decreased and stage 2 sleep increased in control subjects (Timsit-Berthier et al., 1982). REMS decreased after infusion of the peptide to healthy volunteers (Born et al., 1992). Intranasal administration of the substance for 3 months improved sleep in elderly subjects (Perras et al., 1999). Studies with repetitive iv administration of arginine vasopressin are lacking. It is unlikely that the increase of prolactin contributed to the sleep-EEG changes. Animal studies suggest a role of prolactin in the promotion of REMS (reviewed in Roky et al., 1995). In patients with

858

excessive prolactin levels due to prolactinoma SWS was increased (Frieboes et al., 1998). It should be kept in mind that there might be more than one subtype of the GHS receptor, and there might be differences between the ligands in the affinity to these receptors (Chen, 2000). Furthermore it is likely that systemically administered GHSs have to enter the brain in order to promote sleep (though GHSs also have various peripheral actions which may influence sleep EEG and may contribute to the differences between the actions of GHSs and GHRH). There might be differences among GHSs in the ability to enter the brain. Although the presence of GHRH seems to be necessary for GH release by GHSs in vivo the pituitary does express GHS receptors and GHSs might be capable of directly stimulating pituitary hormones; these might be dependent on the dosage and the potency. Neither appetite nor leptin levels were changed after hexarelin in our study, whereas orexigenic effects of hexarelin and ghrelin are well known. Similarly we failed to find changes of leptin levels after nocturnal iv injection of ghrelin (Weikel et al., 2003) and after a higher dose of ghrelin in the morning which augmented appetite (Steiger et al., 2002). Also TNF-a levels and the concentration of soluble TNF-a receptors remained unchanged after hexarelin. Because the sleep-promoting effects of GHRH are closely related to the respective effect of inflammatory cytokines (Krueger et al., 1999) a change of TNF-a appeared possible. Furthermore a single injection of GH in children stimulated TNFa (Bozzola et al., 1998). Our negative findings suggest that neither TNF-a nor soluble TNF receptors, also well known to be involved in humans sleep responses to infection and inflammation (Pollma ¨cher et al., 2000) do not mediate the effects of hexarelin on sleep observed in the present study. Taken together our study shows that hexarelin does not mimic the sleep-promoting effect of ghrelin. We suggest that the distinct release of GH after hexarelin contributes to a change of the central GHRH:CRH balance in favour of CRH resulting in decreases of stage 2 sleep and SWA.

Acknowledgements This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Ste 486/5-1).

R.-M. Frieboes et al.

References Antonijevic, I.A., Murck, H., Frieboes, R.M., Barthelmes, J., Steiger, A., 2000a. Sexually dimorphic effects of GHRH on sleep-endocrine activity in patients with depression and normal controls–part I: the sleep EEG. Sleep Research Online 3, 5–13. Antonijevic, I.A., Murck, H., Frieboes, R.M., Steiger, A., 2000b. Sexually dimorphic effects of GHRH on sleep-endocrine activity in patients with depression and normal controls– part II: hormone secretion. Sleep Research Online 3, 15–21. Arnauld, E., Bibene, V., Meynard, J., Rodriguez, F., Vincent, J.D., 1989. Effects of chronic icv infusion of vasopressin on sleep-waking cycle of rats. Am. J. Physiol. 256, R674–R684. Arvat, E., Maccagno, B., Ramunni, J., Di Vito, L., Broglio, F., Deghenghi, R., Camanni, F., Ghigo, E., 1997a. Hexarelin, a synthetic growth-hormone releasing peptide, shows no interaction with corticotropin-releasing hormone and vasopressin on adrenocorticotropin and cortisol secretion in humans. Neuroendocrinology 66, 432–438. Arvat, E., Ramunni, J., Bellone, J., Di Vito, L., Baffoni, C., Broglio, F., Deghenghi, R., Bartolotta, E., Ghigo, E., 1997b. The GH, prolactin, ACTH and cortisol responses to Hexarelin, a synthetic hexapeptide, undergo different age-related variations. Eur. J. Endocrinol. 137, 635–642. Born, J., Kellner, C., Uthgenannt, D., Kern, W., Fehm, H.L., 1992. Vasopressin regulates human sleep by reducing rapideye-movement sleep. Am. J. Physiol. 262, E295–E300. Bozzola, M., De Amici, M., Zecca, M., Schimpff, R.M., Rapaport, R., 1998. Modulating effect of human growth hormone on tumour necrosis factor-alpha and interleukin-1beta. Eur. J. Endocrinol. 138, 640–643. Chen, C., 2000. Growth hormone secretagogue actions on the pituitary gland: multiple receptors for multiple ligands? Clin. Exp. Pharmacol. Physiol. 27, 323–329. Copinschi, G., Leproult, R., Van Onderbergen, A., Caufriez, A., Cole, K.Y., Schilling, L.M., Mendel, C.M., De Lepeleire, I., Bolognese, J.A., Van Cauter, E., 1997. Prolonged oral treatment with MK-677, a novel growth hormone secretagogue, improves sleep quality in man. Neuroendocrinology 66, 278– 286. Copinschi, G., Van Onderbergen, A., L’Hermite-Bale ´riaux, M., Mendel, C.M., Caufriez, A., Leproult, R., Bolognese, J.A., De Smet, M., Thorner, M.O., Van Cauter, E., 1996. Effects of a-day treatment with a novel orally active nonpeptide growth hormone secretagogue, MK-677, on 24-hour growth hormone profiles, insulin-like growth factor-I and adrenocortical function in normal young men. J. Clin. Endocrinol. Metab. 81, 2776–2782. Cowley, M.A., Smith, R.G., Diano, S., Tscho ¨p, M., Pronchuk, N., Grove, K.L., Strasburger, C.J., Bidlingmaier, M., Esterman, M., Heiman, M.L., Garcia-Segura, L.M., Nillni, E.A., Mendez, P., Low, M.J., Sotonyi, P., Friedman, J.M., Liu, H.Y., Pinto, S., Colmers, W.F., Cone, R.D., Horvath, T.L., 2003. The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron 37, 649–661. Ehlers, C.L., Kupfer, D.J., 1987. Hypothalamic peptide modulation of EEG sleep in depression: a further application of the S-process hypothesis. Biol. Psychiat. 22, 513–517. Frieboes, R.M., Murck, H., Antonijevic, I.A., Steiger, A., 1999. Effects of growth hormone-releasing peptide-6 on the nocturnal secretion of GH, ACTH and cortisol and on the sleep EEG in man: role of routes of administration. J. Neuroendocrinol. 11, 473–478. Frieboes, R.M., Murck, H., Maier, P., Schier, T., Holsboer, F., Steiger, A., 1995. Growth hormone-releasing peptide-6 sti-

Sleep and hormones after hexarelin

mulates sleep, growth hormone, ACTH and cortisol release in normal man. Neuroendocrinology 61, 584–589. Frieboes, R.M., Murck, H., Stalla, G.K., Antonijevic, I.A., Steiger, A., 1998. Enhanced slow wave sleep in patients with prolactinoma. J. Clin. Endocrinol. Metab. 83, 2706–2710. Ghigo, E., Arvat, E., Broglio, F., Giordano, R., Gianotti, L., Muccioli, G., Papotti, M., Graziani, A., Bisi, G., Deghenghi, R., Camanni, F., 1999. Endocrine and non-endocrine activities of growth hormone secretagogues in humans. Horm. Res. 51 (Suppl. 3), 9–15. Ghigo, E., Arvat, E., Gianotti, L., Imbimbo, B.P., Lenaerts, V., Deghenghi, R., Camanni, F., 1994. Growth hormone-releasing activity of hexarelin, a new synthetic hexapeptide, after intravenous, subcutaneous, intranasal, and oral administration in man. J. Clin. Endocrinol. Metab. 78, 693–698. Guldner, J., Schier, T., Friess, E., Colla, M., Holsboer, F. Steiger, A., 1997. Reduced efficacy of growth hormonereleasing hormone in modulating sleep endocrine activity in the elderly. Neurobiol. Aging 18, 491–495. Hinze-Selch, D., Schuld, A., Kraus, T., Kuhn, M., Uhr, M. Pollma ¨cher, T., 2000. Effects of antidepressants on weight and on the plasma levels of leptin, TNF-alpha and soluble TNF receptors: A longitudinal study in patients treated with amitriptyline or paroxetine. Neuropsychopharmacology 23, 13–19. Holsboer, F., von Bardeleben, U., Steiger, A., 1988. Effects of intravenous corticotropin-releasing hormone upon sleeprelated growth hormone surge and sleep EEG in man. Neuroendocrinology 48, 32–38. Horvath, T.L., Diano, S., Sotonyi, P., Heiman, M., Tscho ¨p, M., 2001. Minireview: ghrelin and the regulation of energy balance–a hypothalamic perspective. Endocrinology 142, 4163– 4169. Huhn, W.C., Hartman, M.L., Pezzoli, S.S., Thorner, M.O., 1993. Twenty-four-hour growth hormone (GH)-releasing peptide (GHRP) infusion enhances pulsatile GH secretion and specifically attenuates the response to a subsequent GHRP bolus. J. Clin. Endocrinol. Metab. 76, 1202–1208. Kerkhofs, M., Van Cauter, E., Van Onderbergen, A., Caufriez, A., Thorner, M.O., Copinschi, G., 1993. Sleep-promoting effects of growth hormone-releasing hormone in normal men. Am. J. Physiol. 264, E594–E598. Korbonits, M., Kaltsas, G., Perry, L.A., Putignano, P. Grossman, A.B., Besser, G.M., Trainer, P.J., 1999. The growth hormone secretagogue hexarelin stimulates the hypothalamo-pituitary-adrenal axis via arginine vasopressin. J. Clin. Endocrinol. Metab. 84, 2489–2495. Krueger, J.M., Oba ´l, Jr.., F., Fang, J., 1999. Humoral regulation of physiological sleep: cytokines and GHRH. J. Sleep Res. 8, 53–59. Marshall, L., Derad, L., Starsburger, C.J., Fehm, H.L., Born, J., 1999. A determinant factor in the efficacy of GHRH administration in the efficacy of GHRH administration in promoting sleep: high peak concentration versus recurrent increasing slopes. Psychoneuroendocrinology 24, 363–370. Marshall, L., Mo ¨lle, M., Bo ¨schen, G., Steiger, A., Fehm, H.L., Born, J., 1996. Greater efficacy of episodic than continuous growth hormone releasing hormone (GHRH) administration in promoting slow wave sleep (SWS). J. Clin. Endocrinol. Metab. 81, 1009–1013. Mendelson, W.B., Slater, S., Gold, P., Gillin, J.C., 1980. The effect of growth hormone administration on human sleep: a dose-response study. Biol. Psychiat. 15, 613–618. Moreno-Reyes, R., Kerkhofs, M., L’Hermite-Bale ´riaux, M., Thorner, M.O., Van Cauter, E., Copinschi, G., 1998. Evidence against a role for the growth hormone-releasing peptide

859

axis in human slow-wave sleep regulation. Am. J. Physiol. 274, E779–E784. Murck, H., Guldner, J., Colla-Mu ¨ller, M., Frieboes, R.M., Schier, T., Wiedemann, K., Holsboer, F., Steiger, A., 1996. VIP decelerates non-REM–REM cycles and modulates hormone secretion during sleep in men. Am. J. Physiol. 271, R905–R911. Oba ´l, Jr.., F., Alt, J., Taishi, P., Gardi, J., Krueger, J.M., 2003. Sleep in mice with non-functional growth hormonereleasing hormone receptors. Am. J. Physiol. Regulatroy Integrative Comp. Physiol. 284, R131–R139. Oba ´l, F., Krueger, J.M., 2002. Biochemical regulation of nonrapid-eye-movement sleep. Frontiers Biosci. 8, s520–s550. Opp, M.R., 1995. Corticotropin-releasing hormone involvement in stressor-induced alterations in sleep and in the regulation of waking. Adv. Neuroimmunol. 5, 127–143. Peino, R., Baldelli, R., Rodriguez-Garcia, J., Rodriguez-Segade, S., Kojima, M., Kangawa, K., Arvat, E., Ghigo, E., Dieguez, C., Casanueva, F.F., 2000. Ghrelin-induced growth hormone secretion in humans. Eur. J. Endocrinol. 143, R11–R14. Perras, B., Pannenborg, H., Marshall, L., Pietrowsky, R., Born, J., Fehm, H.L., 1999. Beneficial treatment of age-related sleep disturbances with prolonged intranasal vasopressin. J. Clin. Psychopharmacol. 19, 28–36. Pollma ¨cher, T., Schuld, A., Kraus, T., Haack, M., Hinze-Selch, D., Mullington, J., 2000. Experimental immunomodulation, sleep and sleepiness in humans. Ann. N. Y. Acad. Sci. 917, 488–499. Rechtschaffen, A., Kales, A., 1968. A Manual of Standardized Terminology, Techniques and Scoring System for Sleep Stages of Human Subjects. . US Department of Health, Education & Welfare, Neurological Information Network, Bethesda, MD. Rigamonti, A.E., Cella, S.G., Marazzi, N., Muller, E.E., 1999. Six-week treatment with hexarelin in young dogs: evaluation of the GH responsiveness to acute hexarelin or GHRH administration, and of the orexigenic effect of hexarelin. Eur. J. Endocrinol 141, 313–320. Roky, R., Oba ´l, Jr.., F., Valatx, J.L., Bredow, S., Fang, J., Pagano, L.P., Krueger, J.M., 1995. Prolactin and rapid eye movement sleep regulation. Sleep 18, 536–542. Shintani, M., Ogawa, Y., Ebihara, K., Aizawa-Abe, M., Miyanaga, F., Takaya, K., Hayashi, T., Inoue, G., Hosoda, K., Kojima, M., Kangawa, K., Nakao, K., 2001. Ghrelin, an endogenous growth hormone secretagogue, is a novel orexigenic peptide that antagonizes leptin action through the activation of hypothalamic neuropeptide Y/Y1 receptor pathway. Diabetes 50, 227–232. Smith, R.G., 1998. Unifying mechanism of a non-GHRH growth hormone secretagogue’s action. In: Bercu, B.B., Walker, R.F. (Eds.), Growth Hormone Secretagogues in Clinical Practice. Marcel Dekker, New York, pp. 37–56. Steiger, A., 2002a. Sleep and endocrine regulation. Frontiers in Bioscience 8, s358–s376. Steiger, A., 2002b. Sleep and the hypothalamo-pituitary-adrenocortical system. Sleep Med. Rev. 6, 125–138. Steiger, A., Guldner, J., Hemmeter, U., Rothe, B., Wiedemann, K., Holsboer, F., 1992. Effects of growth hormonereleasing hormone and somatostatin on sleep EEG and nocturnal hormone secretion in male controls. Neuroendocrinology 56, 566–573. Steiger, A., Holsboer, F., 1997. Neuropeptides and human sleep. Sleep 20, 1038–1052. Steiger, A., Schmid, D.A., Held, K., Weikel, J., 2002. Ghrelin stimulates appetite in normal controls. Naunyn Schmiedebergs Arch. Pharmacol. 365 (Suppl. 1), R107.

860

Steiger, A., von Bardeleben, U., Herth, T., Holsboer, F., 1989. Sleep EEG and nocturnal secretion of cortisol and growth hormone in male patients with endogenous depression before treatment and after recovery. J. Affect. Disorder 16, 189–195. Stern, W.C., Jalowiec, J.E., Shabshelowitz, H., Morgane, P.J., 1975. Effects of growth hormone on sleep-waking patterns in cats. Horm. Behav. 6, 189–196. Svensson, J., 2000. Growth hormone secretagogues. Expert Opinion Ther. Patents 10, 1071–1080. Takaya, K., Ariyasu, H., Kanamoto, N., Iwakura, H., Yoshimoto, A., Harada, M., Mori, K., Komatsu, Y., Usui, T., Shimatsu, A., Ogawa, Y., Hosoda, K., Akamizu, T., Kojima, M., Kangawa, K., Nakao, K., 2000. Ghrelin strongly stimulates growth hormone (GH) release in humans. J. Clin. Endocrinol. Metab. 85, 4908–4911. Timsit-Berthier, M., Mantanus, H., Devos, J.E., Spiegel, R., 1982. Action of lysine-vasopressin on human electro-

R.-M. Frieboes et al.

encephalographic activity. Night sleep pattern, auditory evoked potential, contingent negative variation. Neuropsychobiology 8, 248–258. Torsello, A., Luoni, M., Schweiger, F., Grilli, R., Guidi, M., Bresciani, E., Deghenghi, R., Muller, E.E., Locatelli, V., 1998. Novel hexarelin analogs stimulate feeding in the rat through a mechanism not involving growth hormone release. Eur. J. Pharmacol. 360, 123–129. Weikel, J.C., Wichniak, A., Ising, M., Brunner, H., Friess, E., Held, K., Mathias, S., Schmid, D.A., Uhr, M., Steiger, A., 2003. Ghrelin promotes slow-wave sleep in humans. Am. J. Physiol. -Endoc. M. 284, E407–E415. Wren, A.M., Seal, L.J., Cohen, M.A., Brynes, A.E., Frost, G.S., Murphy, K.G., Dhillo, W.S., Ghatei, M.A., Bloom, S.R., 2001. Ghrelin enhances appetite and increases food intake in humans. J. Clin. Endocrinol. Metab. 86, 5992–5995.