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GHRH and sleep
Sleep Medicine Reviews (2004) 8, 367–377
www.elsevier.com/locate/smrv
PHYSIOLOGICAL REVIEW
GHRH and sleep Ferenc Obal Jr.a,1, James M. Kruegerb,* a
¨rgyi Medical Center, University of Szeged, 6720, Szeged, Hungary Department of Physiology, A. Szent-Gyo Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology; Washington State University; PO Box 646520, 205, Wegner Hall, Pullman, WA 99164-6520, USA
b
KEYWORDS Somatotropic axis; Growth hormonereleasing hormone; Growth hormone; Somatostatin; Delta sleep; EEG power; Hypothalamus; Insulinlike growth factor; Interleukin
Summary A significant portion of the total daily growth hormone (GH) secretion is associated with deep non-REM sleep (NREMS). GH secretion is stimulated by the hypothalamic neurohormone, GH-releasing hormone (GHRH). Exogenous GHRH promotes NREMS in various species. Suppression of endogenous GHRH (competitive antagonist, antibodies, somatostatinergic stimulation, high doses of GH or insulin-like growth factor) results in simultaneous inhibition of NREMS. Mutant and transgenic animals with a defect in GHRHergic activity display permanently reduced NREMS which cannot be reversed by means of GH supplementation. GHRH contents and mRNA levels in the hypothalamus correlate with sleep-wake activity during the diurnal cycle and sleep deprivation and recovery sleep. Stimulation of NREMS by GHRH is a hypothalamic action. GABAergic neurons in the anterior hypothalamus/preoptic region are candidates for mediating promotion of NREMS by GHRH. In contrast to NREMS, stimulation of REMS by GHRH is mediated by GH. Simultaneous stimulation of NREMS and GH secretion by GHRH may promote adjustment of tissue anabolism to sleep. q 2004 Elsevier Ltd. All rights reserved.
Introduction There are bidirectional interactions between sleep and the endocrine system. Several hormones have the capacity to affect sleep but the physiological significance of hormonal modulation of sleep is generally unclear. The plasma concentrations of many hormones display sleep-related variations suggesting that sleep influences hormone secretion. However, sleep and hormone levels may correlate without causal relationship between them. For instance, circadian regulation may synchronize these events. Marked sleep-related hormonal *Corresponding author. Tel.: þ1-509-335-2090; fax: þ1-509335-4650. E-mail addresses: [email protected] (J.M. Krueger); [email protected] (F. Obal Jr.). 1 Tel.: þ36-62-545-100; fax: þ 36-62-545-842.
rhythms may also develop as secondary phenomena driven by changes in the activity of the autonomic nervous system during sleep. Growth hormone (GH) is the best documented hormone with a strong sleep-related secretory pattern. GH is produced by anterior pituitary somatotroph cells. Synthesis and secretion of pituitary GH are controlled by two hypothalamic neurohormones; growth hormone-releasing hormone (GHRH), which stimulates, and somatostatin, which inhibits GH. GHRH and somatostatin are secreted into the pituitary portal circulation at the median eminence, and they are carried into the anterior pituitary by the blood. GH secretion is also stimulated by ghrelin. Ghrelin is a hormone released by the endocrine cells of the stomach and it acts via the GH-secretagogue receptor, a receptor different from the GHRH receptor. The somatotropic axis is a fundamental
1087-0792/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.smrv.2004.03.005
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Nomenclature AH/MPO anterior hypothalamus/medial preoptic region EEG electroencephalogram GH growth hormone GHRH growth hormone releasing hormone icv intracerebroventricular IGF-1 insulin-like growth factor-1
anabolic system for the body. It stimulates tissue growth via cell division and via stimulation of protein synthesis. GH acts in part directly and in part via insulin-like growth factor-1 (IGF-1) on target tissues. IGF-1 is both a hormone produced by the liver, and a local paracrine/autocrine substance synthesized in the tissues. The analysis of the interaction between sleep and the somatotropic axis may promote an understanding of the mechanism of coupling body metabolism to sleep, and may provide clues to sleep regulation. In the mouse, the GHRH gene is found within the genomic region linked to encephalographic (EEG) slow wave activity during sleep.1
Sleep and secretion of GH Sleep-related GH secretion was discovered almost 40 years ago.2,3 GH secretion occurs in pulses throughout the day but deep slow wave sleep after sleep onset is associated with particularly large bursts of GH secretion. The GH release during sleep can amount to two thirds of the total GH secreted in 24 h in young males. Sleep deprivation (SD) tends to suppress GH secretion, and large GH releases occur during recovery sleep.3 – 5 Reports from various laboratories confirmed and expanded our understanding of the phenomenon of sleep-related GH secretion (reviewed in Refs.[6,7]). Thus, in addition to the robust sleep-related component, GH secretion has a weaker circadian component. This circadian component may be obvious during SD or when sleep onset is delayed. Calculation of the secretory rate instead of using plasma GH concentration was an important technical development in the analysis of the relationship between sleep and hormone secretions. This technique revealed that GH secretion occurs predominantly during deep slow wave sleep.8,9 The difference in GH secretion between waking and sleep develops at about three-months of age in humans.10 There are gender differences in GH
F. Obal Jr., J.M. Krueger
IL1 iv Mt NREMS REMS SD SDR SEM VIP
interlukin-1 beta intravenous metallothionein non-rapid eye movement sleep rapid eye movement sleep sleep deprivation spontaneous dwarf rat standard error of the mean vasoactive intestinal polypeptide
secretion in both animals and humans with more frequent GH secretory pulses in females than in males. The sleep-related GH secretion is a robust phenomenon in young males. In women, the amount of GH secreted in association with deep NREMS is a much smaller fraction of the total daily GH secretion than in men (reviewed in Ref. [6]). The sleep-associated GH secretion is also age-dependent.11 In males, GH secretion during sleep progressively decreases starting in the third decade and practically disappears above the age of 50. These changes correlate with the decline of deep slow wave sleep. In women, the decrease in sleep-related GH secretion occurs after menopause. Studying the link between sleep and plasma GH is more difficult in animals than in humans because of the short sleep-wake cycles in most laboratory animals such as rats. Nevertheless, correlation between GH secretory rate or plasma GH and sleep occurs in rats,4,12 baboons,13 and lambs.14 The existence of sleep-associated components of GH secretion is also evident in dogs.5 NREMS enhances the responsiveness of GH secretion to secretory stimuli.6 GH may also stimulate sleep.15 Nevertheless, sleep and GH secretion can dissociate. Therefore, a direct causal relationship between sleep and GH secretion cannot explain the coupling between them. It was, therefore, proposed that a common subcortical mechanism may stimulate these events simultaneously.16
GHRH GHRH likely provides the subcortical mechanism synchronizing sleep and GH secretion. GHRH was discovered in 1982.17,18 It is a peptide composed of 40 – 44 amino acid residues. GHRH is a member of the secretin-glucagon peptide family displaying structural homology with vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine,
GHRH and sleep
peptide histidine methionine, pituitary adenylate cyclase activating peptide, gastric inhibitory peptide, secretin, glucagon, and glucagon-like peptide. The homology among some of these peptides is sufficient so that they cross-react on the same receptors. The close relationship between GHRH and VIP was particularly interesting because by the mid 80s, VIP emerged as a peptide for which consistent sleep promoting activity was observed in rats in various laboratories (reviewed in Ref. [19]). Intracerebroventricular (icv) injected VIP enhances both non-rapid eye movement sleep (NREMS) and REMS in rats. However, there were some problems with the NREMS promoting activity of VIP. Thus, inhibition of endogenous VIP selectively suppressed REMS and did not alter NREMS in rats. While VIP stimulated REMS in each species tested, the NREMS response to VIP was not consistent across species. It seemed that VIP might be involved in REMS regulation but the stimulation of NREMS is a non-specific action of exogenous VIP. This may result from VIP mimicking the action of a closely related peptide, i.e. GHRH. The hypothesis has never been tested that stimulation of NREMS by VIP is mediated by GHRH receptor in the rat. Nevertheless, binding characteristics of hypothalamic GHRH receptors demonstrate that these receptors have a very high affinity for VIP in rats.20
Promotion of NREMS by GHRH The NREMS-promoting activity of GHRH was discovered after icv injection of GHRH in rats and rabbits.21 – 23 Perhaps due to the lack of strong circadian regulation, rabbits are particularly responsive to icv GHRH (Fig. 1). For example, in rabbits 1 nmol/kg GHRH induces 53 min of excess NREMS within the first 6 h postinjection but it is half as effective in rats. In both rats and rabbits, GHRH enhances the time spent in NREMS and the intensity of EEG slow wave activity during NREMS. GHRH also stimulates NREMS when it is microinjected directly into the anterior hypothalamus/medial preoptic region (AH/MPO).24 GHRH strongly promotes NREMS after intravenous (iv) injection in rats25 and after intraperitoneal administration in mice.26 Sleep responses to systemic administration of GHRH were first determined in humans. Promotion of sleep by GHRH was reported after iv GHRH from several laboratories, (reviewed in Refs. [6,27]). Briefly, GHRH injected in the morning has no impact on sleep during the subsequent night, and continuous infusion of GHRH is also ineffective.
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Figure 1 Hourly duration of NREMS (mean ^ SEM) in rabbits ðn ¼ 6 – 8Þ after icv injection of artificial cerebrospinal fluid (open symbols) or GHRH (closed symbols). Arrows: injection. Asterisks: significant difference in hour 1 (paired t-test).23
When GHRH enhanced sleep, it was injected prior to sleep, during the first sleep cycle or in the middle of the night. Repeated pulsatile injections28 or a rapid rise in plasma GHRH concentration29 are important for the GHRH-induced NREMS response. GHRH increases the time spent in deep slow wave sleep, it elicits GH secretion and suppresses plasma cortisol concentration in humans. Weak sleep promoting activity is also observed in response to GHRH administered in nasal spray.30 The effects of GHRH on sleep may vary with the time of day. Some observations suggest that GHRH might be less effective at times of very high sleep propensity (beginning of sleep) and may stimulate NREMS more powerfully during periods of decreasing sleep propensity (middle of the night in humans, middle of the light period in the rat).25,31 Finally, the GHRH dose-sleep response curve seems to be bell-shaped for the NREMS-promoting activity of systemic GHRH in the rat.25 The NREMS response decreases then vanishes when the dose of GHRH is increased whereas the GH secretion increases with the dose (Fig. 2). The decline of the NREMS response is attributed to an inhibition of endogenous GHRH by GH. The sleep-promoting activity of GHRH varies with gender and age in humans (reviewed in Ref. [7]). Strong responses are observed in young
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Inhibition of GHRH via endogenous feed back
Figure 2 GH and sleep responses (mean ^ SEM) to iv GHRH in rats ðn ¼ 5 – 10Þ: GHRH was administered in hour 7 of the rest period. GH concentrations are shown at 30 min postinjection. For NREMS and REMS differences from baseline (injection of physiological saline) are depicted during 5 h postinjection. Asterisks: significant difference from baseline (paired t-test).25
male subjects. Instead of enhancing slow wave sleep, GHRH increases stage 2 NREMS and sleep continuity in the elderly. GHRH does not promote sleep in females, in contrast, wake time increases and adrenocorticotropic hormone secretion is stimulated, suggesting a synergism between GHRH and corticotropin releasing hormone in women.
Acute inhibition of GHRH If endogenous GHRH is a physiological NREMS-regulatory substance then suppression of endogenous GHRH should be associated with decreases in NREMS. Thus, a competitive GHRH antagonist32 elicits dose-dependent decreases in the duration and intensity of NREMS and increases sleep latency. Further, icv administration of antibodies to GHRH decreases NREMS.33 The intensity of EEG slow wave activity also decreases during NREMS after antagonist or antibody treatment. Finally, immunoneutralization of GHRH blocks the sleep response to short (3 h) SD including the increases in the duration and intensity of NREMS in rats.33
The somatotropic axis includes several negative feed back loops inhibiting GHRH. In the ultrashort feed back loop, GHRH inhibits its own secretion in the hypothalamus. The ultrashort loop might also involve somatostatin: GHRH stimulates somatostatinergic neurons which inhibit GHRHergic activity. GH inhibits GHRH through a short loop negative feed back, which is also mediated by somatostatin. GH stimulates hypothalamic somatostatinergic neurons. Somatostatin inhibits hypothalamic GHRHergic neurons, and somatostatin is also released into the pituitary circulation and inhibits GH secretion directly in the pituitary. Finally, IGF-1 inhibits GHRH via a long loop feed back. The long loop feed back is in part a direct action and in part it may involve somatostatin. In conclusion, IGF-1, GH, GHRH itself, and somatostatin can inhibit GHRH and somatostatin seems to have a major significance in mediating the various negative feed backs. Both IGF-1 and GH are capable of inhibiting NREMS. Thus, an icv dose of IGF-1, which causes prompt suppression of GH secretion (i.e. it inhibits GHRH), also elicits rapid suppression of sleep in rats and rabbits;34 sleep and GH secretion recover after 1 h. Decreases in NREMS were reported in response to systemic injection of a high dose of GH in humans35 and cats became restless after a high dose of GH.36 Preliminary studies in our laboratory also demonstrate decreases in NREMS time in response to a high dose of icv injected GH in rats (unpublished). Physiological GH concentrations occurring during normal GH surges may also impair slow wave sleep if these surges precede sleep onset in humans.37 Somatostatin has five receptors named sst1 to sst5 (reviewed in Ref. [38]). Inhibition of the somatotropic axis is mediated by the sst2 receptors. Within tissues, the half-life of somatostatin is only a few minutes. Therefore, hydrolysisresistant analogs, such as octreotide, are often used experimentally. Octreotide stimulates sst2 and sst5 receptors, has a low affinity for sst3 receptors, and does not bind the sst1 and sst4 receptors. Icv or systemic injection of octreotide elicits dose-dependent, prompt and simultaneous suppression of NREMS and GH secretion.39 The inhibition lasts for 1 – 2 h then GH secretion recurs and sleep time normalizes or tends to increase above baseline, and a dose-dependent and strong enhancement in EEG slow wave activity occurs during NREMS. Octreotide-induced impairment of
GHRH and sleep NREMS was also verified in humans.40 Octreotide promptly inhibits hypothalamic GHRH release coinciding with sleep suppression.41 After 1 – 3 h postinjection, when intensity of NREMS increases, the accumulated GHRH gradually is released from the hypothalamus. Therefore, a possible explanation for the biphasic sleep response to octreotide is that octreotide suppresses sleep via inhibiting GHRH release, and this period is followed by excessive GHRH release and deep NREMS. Somatostatin is, however, a ubiquitous neurotransmitter in the central nervous system. Octreotide injected icv may interfere with various functions which themselves can influence sleep. In fact, icv octreotide elicits angiotensin-like responses such as prompt drinking, vasopressin secretion and rises in blood pressure followed by vigorous eating. 39 The role of intracerebral angiotensin was verified in the actions of somatostatin by showing that these responses are inhibited by the angiotensin convertase inhibitor, captopril, and by angiotensin receptor blockers (saralasin, losartan) (reviewed in Ref. [42]). In contrast, inhibition of angiotensin convertase does not alter the sleep response to octreotide suggesting that angiotensin is not involved in octreotide-induced sleep suppression.39 If octreotide is injected into mice with decreased GHRH production, the sleep-suppressive action of octreotide is significantly attenuated.43 Further, octreotide completely fails to inhibit sleep in mice with non-functional GHRH receptors though these mice continue to drink in response to octreotide.26 These findings strongly suggest that the sleep-suppressive action of octreotide requires GHRH, i.e. octreotide inhibits sleep via inhibiting GHRHergic activity. Further support for this hypothesis came from experiments with microinjections of octreotide into various structures of the brain the rat.42 Octreotide elicits drinking when injected into the subfornical organ or the paraventricular nucleus, which are parts of the known intracerebral angiotensinergic dipsogenic circuit. These microinjections do not have consistent effects on sleep. In contrast, NREMS is promptly inhibited when octreotide is microinjected into the arcuate nucleus or the AH/ MPO (Fig. 3), where GHRHergic neurons and terminals reside; these injections fail to elicit drinking. The initial NREMS suppression is followed by increases in NREMS intensity starting in hour 2 – 3 postinjection, i.e. the sleep response is similar to that observed after icv administration of octreotide. We conclude that somatostatinergic stimulation suppresses NREMS via inhibiting GHRHergic activity.
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Figure 3 NREMS responses (mean ^ SEM) to microinjection of octreotide (0.02 mg in 0.2 ml, closed symbol) or physiological saline/vehicle (0.2 ml open symbols) into the paraventricular nucleus (PVN), arcuate nucleus (ARC), and anterior hypothalamus/medial preoptic region (AH/MPO) in rats ðn ¼ 7 – 10Þ: Arrows: injections. Asterisks: significant differences in hour 1 (paired t-test).42
Chronic alterations in GHRHergic activity Mutant and transgenic animals provide excellent models for well-defined chronic alterations in the somatotropic axis.44 Lit/lit mice have a point mutation in the GHRH receptor gene and their receptor protein does not bind GHRH. Because of the lack of stimulation of the pituitary somatotroph cells, the lit/lit mice are deficient in GH and IGF-1, and they are dwarfs. The dw/dw dwarf rat is similar to the lit/lit mouse in that the defect is in the GHRH receptors but the exact cause of the malfunction of the receptors is currently not known. GH and IGF-1 productions are greatly decreased in the dw/dw rats. Spontaneous NREMS is significantly reduced in both the lit/lit mice26 and the dw/dw rats.45 Chronic GH replacement (8 – 9 days) in the lit/lit mice increases plasma IGF-1 concentration but fails to increase NREMS (Table 1).26 The TH-hGH transgenic mouse provides another model of decreased GHRHergic activity. In these mice, the promoter region of the tyrosine hydroxylase gene stimulates the transcription of the human (h) GH transgene. Therefore, tyrosine hydroxylase positive catecholaminergic neurons produce and release hGH. GH in the hypothalamus provides a negative feed back signal and suppresses GHRH production and release. Because of the GHRH deficiency pituitary GH is not stimulated, systemic GH concentration is low,
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Table 1 Duration of NREMS and REMS in the 12 h light period in control mice (heterozygous C57BL/6J mice with normal GHRH receptors, n ¼ 12), in lit/lit mice ðn ¼ 12Þ; and in lit/lit mice infused with 24 mg rat GH/day for 8– 9 days ðn ¼ 12Þ:26
Control Lit/lit Lit/lit þ GH a
NREMS (min/12 h)
REMS (min/12 h)
347.1 ^ 10.2 235.3 ^ 12.7a 243.6 ^ 10.5a
62.6 ^ 2.0 49.6 ^ 3.8a 61.8 ^ 2.1
Significantly different from control (ANOVA followed by Student–Neuman–Keuls test).
and the mice are dwarfs. Like the lit/lit mice and dw/dw rats, the TH-hGH mice also display less NREMS than their normal littermates.46 GHRH deficiency is always associated with GH and IGF-1 deficiencies (and dwarf phenotype), and therefore, it might not be obvious whether the alterations in NREMS result from changes in GHRH, GH, or IGF-1. The GH-replacement experiments in the lit/lit mice suggest that it is not GH/IGF-1 which is responsible for the NREMS deficit.26 However, GH/IGF-1 deficiencies may cause alterations in brain development resulting in changes in NREMS, and those changes might be resistant to GH replacements in adult rats. It was, therefore, important to study sleep in animal models where the genetic defect is downstream from the GHRH receptors. This model is the spontaneous dwarf rat (SDR) with a mutation of the GH gene causing practically total loss of GH and dwarfism. Preliminary results demonstrate that NREMS is not reduced in the SDRs, in fact the SDRs display more NREMS during the rest period than normal Sprague – Dawley rats, the strain of rats from which the SDRs are derived. These findings suggest that reduction in spontaneous NREMS in models with chronic GHRH or GHRH receptor deficiencies is not related to the lack of GH/IGF-1 but it is caused by the decreased GHRHergic activity. The response to SD (4 h) was also studied in the lit/lit mice and the dw/dw rats, and a difference was noted between the two species. In the dw/dw rat, SD-induced enhancement of EEG delta activity during recovery sleep is only half that observed in control Lewis rats.45 In contrast, the duration of recovery sleep is slightly decreased but EEG slow wave activity is normal during recovery in the lit/lit mice. 26 The explanation for this difference is currently not clear. A finding, which is seemingly at variance with the anticipated changes in NREMS in GHRH deficiency, was obtained in the Mt-rGH transgenic mouse, also
called the Supermouse.43 In the Mt-rGH mice, the transgene is composed of the promoter region of the metallothionein (Mt) gene and the coding region of the rat (r) GH gene. Supermice produce large quantities of rGH in various tissues and because of the excess GH their body size is greatly increased. The high GH concentration is supposed to suppress GHRH production. However, behavioral observations suggested that the Mt-rGH mice slept more than normal mice.47 Sleep recording verified modest increases in NREMS time (42 min in the 12 h light period). The Supermice also responded to SD with normal sleep rebound. We speculate that the high concentration of GH/IGF-1 stimulated spontaneous NREMS independently of GHRH.
Sleep-related variation in hypothalamic GHRH Synthesis and release of a physiological sleep regulatory substance are anticipated to vary with sleep-wake activity. The GH pulses associated with deep NREMS suggest an activation of the GHRHergic neurons during NREMS. To gain further insight into the dynamics of GHRHergic activity, we determined the diurnal rhythm and SD-induced changes in hypothalamic GHRH mRNA levels and GHRH contents in rats. In the rat, the highest amounts of NREMS occur in the first portion of the light period. As assessed by EEG delta power, NREMS intensity also peaks in the first hour of the light period. Then, the time spent in NREMS and NREMS intensity decrease steadily until dark onset. Rats sleep little during the dark period. The intensity of the short nocturnal sleep bouts increases progressively towards the end of the night. GHRH mRNA levels peak around light onset, decrease towards the end of the light period and stay at very low levels at night.48 The light onset is followed by a short rise in GHRH contents suggesting that the transcribed mRNA is translated into protein very rapidly.49 However, after the transient rise, GHRH contents drop to low levels indicating robust GHRH release. GHRH release declines and thus hypothalamic GHRH content increases towards the end of the dark period. GHRH content decreases at night, which may indicate release or degradation. The major conclusion of the variations in GHRH mRNA levels and GHRH contents is that the time of the day where maximum GHRH synthesis and release occur corresponds to the period of deepest NREMS. Experiments with SD support this conclusion. SD for 8 h results in excessive depletion of
GHRH and sleep
hypothalamic GHRH with very low GHRH contents at the termination of the deprivation. 49 Simultaneously, however, GHRH mRNA levels increase significantly50 suggesting that transcription is stimulated, perhaps because of the high rate of release. It seems that the release continues during recovery because GHRH contents remain low for 1 –2 h. In situ hybridization studies also demonstrate diurnal and SD-induced variations in GHRH mRNA levels.51 Finally, the rise in GHRH mRNA levels after SD is associated with simultaneous decreases in hypothalamic somatostatin mRNA levels.50 An interesting finding, which indirectly supports SD-induced excessive GHRH release, was obtained by determining hypothalamic and pituitary GHRH binding and GHRH receptor mRNA levels in sleep deprived rats.20 Compared to controls, GHRH receptor mRNA and GHRH binding were reduced by 50% in the hypothalamus after 8 h of SD in rats whereas no changes were observed in pituitary GHRH receptors. It is characteristic of GHRH receptors that massive exposure to GHRH elicits down-regulation.52 Therefore a robust intrahypothalamic GHRH release is a likely explanation for the down-regulation of hypothalamic GHRH receptors. The unaltered pituitary GHRH receptor mRNA and binding suggest that massive GHRH release into the pituitary vessels does not occur during SD, i.e. GHRH secretion at the median eminence is inhibited. The inhibition of GHRH secretion at the median eminence is likely the function of somatostatinergic neurons.51
The hypothalamic GHRH network; a sleep-promoting site of action The findings above suggest that the NREMS-promoting activity of GHRH is not mediated by the GH-IGF-1 axis. This hypothesis was verified in hypophysectomized rats.25 GHRH elicits increases in the duration and intensity of NREMS in both intact and hypophysectomized animals showing that GH is not necessary for the GHRH-induced increases in NREMS. Stimulation of NREMS, therefore, is a central action of GHRH. Unlike many other neuropeptides, GHRH that is detectable by means of immunocytochemistry, is confined to a very restricted area in the brain.53 The majority of the GHRH-containing neurons reside in the arcuate nucleus. Smaller subgroups of GHRHcontaining neurons are found around the ventral pole of the ventromedial nucleus and in the parvicellular portion of the paraventricular nucleus. The GHRHergic neurons in the arcuate nucleus are the major source of GHRH released at the median eminence.
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The extraarcuate GHRHergic neurons and in part the intraarcuate GHRHergic neurons project to various regions in the basal forebrain, predominantly to the AH/MPO. In situ hybridization experiments suggest that the diurnal variation in GHRH mRNA with light onset maximum occurs in the arcuate nucleus whereas SD stimulates GHRH mRNA in the paraventricular nucleus.51 It is possible, therefore, that the GHRHergic neuronal pools differ in their participation in the diurnal regulation of sleep and the sleep response to deprivation. Somatostatinergic inhibitory interneurons are found throughout the hypothalamus. The somatostatinergic neurons which are implicated in the somatotropic system reside in the arcuate and the periventricular nuclei.54 The arcuate somatostatinergic neurons inhibit GHRHergic (and other) neurons locally. Expression of the Fos protein, a marker of neuronal activity, is increased in the arcuate nucleus in response to SD and also during the active period of the day in the rat.55 In situ hybridization shows that SD induces increases in somatostatin mRNA expression in the arcuate nucleus.51 Enhanced intraarcuate somatostatinergic activity may explain the suppression of GH secretion during SD (see above). The hypophyseotropic somatostatinergic neurons, i.e. the somatostatinergic neurons which project to the median eminence and secrete their contents into the pituitary circulation, are in the periventricular nucleus. The periventricular somatostatinergic neurons also seem to inhibit the intraarcuate GHRHergic neurons. 56 The somatostatinergic inhibition of the GHRHergic neurons is mediated via sst2 receptors and explains the sleep suppression following intraarcuate injection of octreotide42 (Fig. 3). Inhibition of sleep by octreotide in the AH/MPO suggests that there are either presynaptic sst2 receptors on the GHRHergic terminals projecting to this area or the neurons modulated by GHRH also express sst2 receptors. The AH/MPO is a well-documented area involved in sleep regulation (reviewed in Ref. [57]). Microinjection of GHRH into the AH/MPO elicits robust, dose-dependent increases in NREMS duration and intensity.24 After 0.1 nmol/kg GHRH, NREMS is enhanced for 7–8 h, and EEG delta power during NREMS is also increased. Local administration of a GHRH receptor antagonist into the same injection sites decreases spontaneous NREMS and delta power during NREMS, and attenuates the NREMS response to SD. These observations suggest that GHRH acts in the AH/MPO. The possible importance of the connections between the arcuate nucleus and the AH/MPO was studied in rats by means of transections at the frontal pole of the arcuate
374 nucleus.58 The transection was followed by significant decreases in NREMS duration during the light period, and the diurnal rhythm of EEG delta power during NREMS was almost abolished. The rats were recorded for 8 weeks after transection, and the sleep alterations persisted. In hypothalamic neuronal cultures prepared from fetal rats, 7.6% of the neurons responded promptly with a rise of intracellular calcium concentration after exposure to GHRH.59 Almost all of the GHRH-responsive neurons (96%) displayed immunoreactivity for glutamate decarboxylase (GAD), the enzyme producing GABA. Differences, of course, may exist between fetal and adult neurons but there is now a significant possibility that the AH/MPO neurons mediating the NREMS-promoting activity of GHRH are GABAergic.
GHRH and REMS GHRH often stimulates REMS in animals but this action is not consistent. Icv injection of GHRH weakly stimulates REMS in the rat.23 In contrast, when GHRH is injected into the AH/MPO, REMS does not change.24 In rabbits, the REMS response to icv GHRH is significantly delayed compared to the NREMS response.23 Systemic administration of GHRH enhances REMS in the rat but the effect does not depend on the dose (Fig. 2).25 Systemic injection of GHRH fails to alter REMS in the mouse.26 Finally, no change28 and increases29,31 in REMS were both reported after systemic administration of GHRH in humans. The threshold dose of the icv-injected competitive GHRH antagonist decreasing REMS, is much higher than the dose required for NREMS suppression.32 This high dose also suppresses GH secretion. A very high dose of the competitive antagonist causes only small decreases in REMS when injected into the AH/MPO.24 Administration of antibodies to GHRH simultaneously decreases both REMS and GH secretion.33 These observations suggest that promotion of REMS by GHRH is an indirect action. That the REMS response to GHRH requires intact pituitary functions, possibly GH, was demonstrated by the fact that hypophysectomy prevented stimulation of REMS by systemic GHRH.25 The role of GH was further supported by experiments in the animal models with chronic alterations in the somatotropic axis. During the rest period, REMS time is almost twice that of normal mice in the Supermouse with excess GH.43 In contrast, REMS decreases significantly in the models with GHRH receptor deficiency (dw/dw rats, lit/lit mice) (Table 1).26,45 REMS is markedly reduced in the SDRs,
F. Obal Jr., J.M. Krueger
which do not produce GH but have normal GHRH and GHRH receptors (unpublished). The continuous GH infusion, which fails to alter NREMS, is capable of normalizing REMS in the lit/lit mice (Table 1).26 A particularly interesting finding was obtained in the TH-hGH transgenic mouse. This model is characterized by peripheral GH deficiency but produces hGH in the brain, and the mice display normal REMS.46 In conclusion, promotion of REMS by GHRH seems to depend on GH. Stimulation of REMS by acute rises in GH has been documented in rats,60 cats36 and humans.35 The mechanism of stimulation of REMS by GH is not known. It likely does not involve IGF-1 because acute administration of IGF-1 does not promote REMS.34 GH stimulates hypothalamic somatostatin, and somatostatin has been implicated in REMS regulation. However, intrahypothalamic injection of octreotide does not increase REMS,42 and previous experiments indicate that the brainstem is the site of action for REMS somatostatin promotion.61 It is unclear whether GH modulates somatostatinergic transmission in the brainstem. The latency of the REMS response to GH takes several hours, and during this period, GH stimulates the synthesis of some proteins in the brainstem.60 It is possible that GH regulates the production of an enzyme which is involved in the synthesis of some neurotransmitters involved in the regulation of REMS. (REMS may increase after 1 h latency in response to GHRH.31 Instead of the transporters in the choroid plexus through which systemic GH enters the brain, GH released by GHRH in the pituitary has direct access to the hypothalamus through the window in the bloodbrain barrier at the median eminence. The difference in entering the brain may explain that the REMS response has longer latency after systemic administration of GH than after GHRH).
Interactions with IL1 Theoretically, stimuli, which induce hypothalamic GHRH release, should also elicit or enhance NREMS. However, these stimuli are currently not known. Intraarcuate GHRHergic neurons may receive input from the suprachiasmatic nucleus,62 and through this route they may contribute to the circadian regulation of NREMS. Ghrelin, the GH secretagogue hormone produced by the stomach, stimulates GHRH-containing neurons in the arcuate nucleus. Ghrelin has a NREMS promoting activity63 which is absent in the lit/ lit mice with a deficiency of GHRH receptors.26 GHRH may contribute to the sleep-promoting activity of the cytokine, interleukin-1b (IL1). Icv injection of a sleep-promoting dose of IL1 elicits
GHRH and sleep GH secretion.64 Immunoneutralization of GHRH blocks the IL1-induced GH response and attenuates the sleep response to icv IL1.65 We proposed that IL1 may stimulate GHRH release. Although IL1 has in fact a weak GHRH-releasing activity, recent findings in vitro and in vivo suggest that the major action of IL1 is an up-regulation of the hypothalamic GHRH receptors.66 The majority of the fetal hypothalamic neurons that respond with rises in intracellular calcium to GHRH also produce calcium signals when exposed to IL1.59 It seems, therefore, that IL1 upregulates the GHRH receptors in AH/MPO GABAergic neurons enhancing the sensitivity of these neurons to GHRH. There are neurons dispersed in the AH/MPO that enhance their firing during sleep (reviewed in Ref. [57]); it is assumed that these sleep-active neurons are involved in the promotion of sleep. IL1 is capable of stimulating AH/MPO sleep-active neurons67 but currently it is not known whether these neurons are the same as the GABAergic neurons responsive to both IL1 and GHRH in cell culture. Enhancement in NREMS associated with the acute phase response to infectious diseases is a model of excessive sleep induction by endogenous cytokines, including IL1. By means of a quantitative trait loci analysis, the GHRH receptor gene has been identified as one of three candidate genes that might be involved in the mediation of enhanced NREMS in mice infected with influenza virus.68 Influenza virus fails to stimulate NREMS in the lit/lit mice with GHRH receptor deficiency; in fact, NREMS decreases in these mice.69 It is assumed that the mechanism of the sleep response to influenza includes stimulation of GHRHergic action by IL1 through an up-regulation of the hypothalamic GHRH receptors. Expression of GHRH receptor mRNA is increased in intact mice infected with influenza virus (unpublished observation).
The functional significance of GHRH in sleep regulation GHRHergic neurons are part of the hypothalamic circuit regulating sleep-wake activity. GHRH is one of the many inputs promoting NREMS. Although chronic withdrawal of GHRH is associated with a permanent reduction of NREMS, this decrease is modest (20 – 25% of NREMS time), thus, GHRH deficiency does not result in total sleep loss. The possible significance of GHRH in sleep regulation might be associated with the feature that this peptide simultaneously promotes NREMS and GH secretion. GH is a major anabolic hormone of the body. Simultaneous stimulation of sleep and GH secretion synchronizes the anabolic processes of the body to periods of rest provided by sleep.
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Practice points † Secretion of GH has a marked NREMSassociated component suggesting an activation of GHRH during deep NREMS. † GHRH promotes NREMS after various routes of administration in every species tested (rat, rabbit, mouse, human). † Acute inhibition of endogenous GHRH causes decreases in NREMS. † Activation of the GHRH inhibiting negative feed back mechanisms within the somatotropic system results in inhibition of NREMS. † Experimental genetic alterations, which compromise GHRHergic transmission, are associated with reduced NREMS. † Hypothalamic GHRH displays diurnal and sleep-related variations. † GH and IGF-1 are not involved in the NREMSpromoting activity of GHRH. † Promotion of NREMS by GHRH is a hypothalamic action mediated by GABAergic neurons in the AH/MPO. † Stimuli that increase GHRHergic activity also enhance NREMS. † Promotion of REMS by GHRH is indirectly mediated by GH.
Research agenda † Elucidation of the hypothalamic GHRHergic and somatostatinergic network involved in sleep regulation. † Identification of further stimuli that modulate sleep via GHRH. † Studies of the mechanism of desynchronization between sleep and GH secretion. † Investigation of the relationships between gender differences in sleep and GHRHergic activity. † Understanding the role of GHRH in agingassociated sleep alterations.
Acknowledgements This work was supported, in part, by grants from the National Institutes of Health, USA (Grant numbers NS 25378 and NS27250) and the Hungarian National
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Foundation and Ministry of Health (OTKA-T-043156, ETT 103 04/2003).
References 1. Franken P, Chollet D, Tafti M. The homeostatic regulation of sleep need is under genetic control. J Neurosci 2001; 21: 2610—2621. 2. Takahashi Y, Kipnis DM, Daughaday WH. Growth hormone secretion during sleep. J Clin Invest 1968; 47: 2079—2090. 3. Sassin JF, Parker DC, Mace JW et al. Human growth hormone release: relation to slow-wave sleep and sleep-waking cycles. Science 1969; 165: 513—515. 4. Kimura F, Tsai C-W. Ultradian rhythm of growth hormone secretion and sleep in the adult male rat. J Physiol (Lond) 1984; 353: 305—3315. 5. Takahashi Y, Ebihara S, Nakamura Y. A model of human sleep-related growth hormone secretion in dogs: effects of 3, 6, and 12 hours of forced wakefulness on plasma growth hormone, cortisol, and sleep stages. Endocrinology 1981; 109: 262—272. *6. Van Cauter E, Plat L, Copinschi G. Interrelations between sleep and the somatotropic axis. Sleep 1998; 21: 553—565. *7. Steiger A. Sleep and endocrine regulation. Frontiers Biosci 2003; 8: s358—s376. 8. Holl RW, Hartman ML, Veldhuis JD et al. Thirty-second sampling of plasma growth hormone in man: correlation with sleep stages. J Clin Endocrinol Metab 1991; 72: 854—861. 9. Gronfier C, Luthringer R, Follenius M et al. A quantitative evaluation of the relationships between growth hormone secretion and delta wave electroencephalographic activity during normal sleep and after enrichment in delta waves. Sleep 1996; 19: 817—824. 10. Vigneri R, D’Agata R. Growth hormone release during the first year of life in relation to sleep-wake periods. J Clin Endocrinol Metab 1971; 33: 561—563. 11. Van Cauter E, Leproult R, Plat L. Age-related changes in slow wave sleep and REM sleep and relationship with growth hormone and cortisol levels in healthy men. JAMA 2000; 284: 861—868. 12. Mitsugi N, Kimura F. Simultaneous determination of blood levels of corticosterone and growth hormone in the male rat: relation to sleep-wakefulness cycle. Neuroendocrinology 1985; 41: 125—130. 13. Parker DC, Morishima M, Koerker JD et al. Pilot study of growth hormone release in sleep of the chair- adapted baboon: potential as model of human sleep release. Endocrinology 1972; 91: 1462—1467. 14. Laurentie MP, Barenton B, Charrier J et al. Instantaneous secretion rate of growth hormone in lambs: relationships with sleep, food intake, and posture. Endocrinology 1989; 125: 642—651. 15. Astro ¨m C. Interaction between sleep and growth hormone. Acta Neurol Scand 1997; 92: 281—296. 16. Parker DC, Sassin JF, Mace JW et al. Human growth hormone release during sleep: electroencephalograhic correlation. J Clin Endocrinol Metab 1969; 29: 871—874. 17. Rivier J, Spiess J, Thorner M et al. Characterization of a growth hormone-releasing factor from a human pancreatic islet tumour. Nature 1982; 300: 326—329. * The most important references are denoted by an asterisk.
F. Obal Jr., J.M. Krueger
18. Guillemin R, Brazeau P, Bo ¨hlen P et al. Growth-hormone releasing factor from a human pancreatic tumor that caused acromegaly. Science 1982; 218: 585—587. 19. Obal Jr F, Krueger JM. Biochemical regulation of non-rapideye-movement sleep. Frontiers Biosci 2003; 8: 520—550. 20. Gardi J, Taishi P, Speth R et al. Sleep loss alters hypothalamic growth hormone-releasing hormone receptors in rats. Neurosci Lett 2002; 329: 69—72. *21. Ehlers C, Reed TK, Henriksen SJ. Effects of corticotropinreleasing factor and growth hormone-releasing factor on sleep and activity in rats. Neuroendocrinology 1986; 42: 467—474. 22. Nistico G, DeSarro GB, Bagetta G et al. Behavioural and electrocortical spectrum power effects of growth hormone releasing factor in rats. Neuropharmacology 1987; 26: 75—78. *23. Obal Jr F, Alfo ¨ldi P, Cady AB et al. Growth hormone-releasing factor enhances sleep in rats and rabbits. Am J Physiol 1988; 255: R310—R316. *24. Zhang J, Obal Jr F, Zheng T et al. Intrapreoptic microinjection of GHRH or its antagonist alters sleep in rats. J Neurosci 1999; 19: 2187—2194. *25. Obal Jr F, Floyd R, Kapas L et al. Effects of systemic GHRH on sleep in intact and hypophysectomized rats. Am J Physiol 1996; 270: E230—E237. *26. Obal Jr F, Alt J, Taishi P et al. Sleep in mice with nonfunctional growth hormone-releasing hormone receptors. Am J Physiol 2003; 284: R131—R139. 27. Steiger A, Antonijevic IA, Bohlhalter S et al. Effects of hormones on sleep. Horm Res 1998; 49: 125—130. 28. Steiger A, Guldner J, Hemmeter U et al. Effects of growth hormone-releasing hormone and somatostatin on sleep EEG and nocturnal hormone secretion in male controls. Neuroendocrinology 1992; 56: 566—573. 29. Marshall L, Derad I, Strasburger CJ et al. A determinant factor in the efficacy of GHRH administration in promoting sleep: high peak concentration versus recurrent increasing slopes. Psychoneuroendocrinology 1999; 24: 363—370. 30. Perras B, Marshall L, Ko ¨hler G et al. Sleep and endocrine changes after intranasal administration of growth hormonereleasing hormone in young and aged humans. Psychoneuroendocrinology 1999; 24: 743—757. 31. Kerkhofs M, Van Cauter E, Van Onderbergen A et al. Sleeppromoting effect of growth hormone-releasing hormone in normal men. Am J Physiol 1993; 264: E594—E598. 32. Obal Jr F, Payne L, Kapas L et al. Inhibition of growth hormone-releasing factor suppresses both sleep and growth hormone secretion in the rat. Brain Res 1991; 557: 149—153. 33. Obal Jr F, Payne L, Opp M et al. Growth hormone-releasing hormone antibodies suppress sleep and prevent enhancement of sleep after sleep deprivation. Am J Physiol 1992; 263: R1078—R1085. 34. Obal Jr F, Kapas L, Gardi J et al. Insulin-like growth factor-1 (IGF-1)-induced inhibition of growth hormone secretion is associated with sleep suppression. Brain Res 1999; 818: 267—274. 35. Mendelson WB, Slater S, Gold P et al. The effect of growth hormone administration on human sleep: a dose response study. Biol Psychiatry 1980; 15: 613—618. 36. Stern WC, Jalowiec JE, Shabshelowitz H et al. Effects of growth hormone on sleep-waking patterns in cats. Horm Behav 1975; 6: 189—196. 37. Spiegel K, Leproult R, Colecchia EF et al. Adaptation of the 24-h growth hormone profile to a state of sleep debt. Am J Physiol 2000; 279: R874—R883.
GHRH and sleep
38. Reisine T. Somatostatin receptors. Am J Physiol 1995; 269: G813—G820. 39. Beranek L, Hajdu I, Gardi J et al. Central administration of the somatostatin analog octreotide induces captopril-insensitive sleep responses. Am J Physiol 1999; 277: R1297—R1304. 40. Ziegenbein M, Held K, Kuenzel HE et al. The somatostatin analogue octreotide impairs sleep and decreases EEG sigma power in young male subjects. Neuropsychopharmacology 2004; 29: 146—151. 41. Gardi J, Szentirmai E, Hajdu I et al. The somatostatin analog, octreotide, causes accumulation of growth hormone-releasing hormone and depletion of angiotensin in the rat hypothalamus. Neurosci Lett 2001; 315: 37—40. *42. Hajdu I, Szentirmai E, Obal Jr F et al. Different brain structures mediate drinking and sleep suppression elicited by the somatostatin analog, octreotide, in rats. Brain Res 2003; 994: 115—123. 43. Hajdu I, Obal Jr F, Fang J et al. Sleep of transgenic mice producing excess rat growth hormone. Am J Physiol 2002; 282: R70—R76. 44. Frohman LA. New insights into the regulation of somatotrope function using genetic and transgenic models. Metabolism 1996; 45: S1:1—S1:3. 45. Obal Jr F, Fang J, Taishi P et al. Deficiency of growth hormone-releasing hormone signaling is associated with sleep alterations in the dwarf rat. J Neurosci 2001; 21: 2912—2918. 46. Zhang J, Obal Jr F, Fang J et al. Non-rapid eye movement sleep is suppressed in transgenic mice with a deficiency in the somatotropic system. Neurosci Lett 1996; 220: 97—100. 47. Lachmansingh E, Rollo CD. Evidence for a trade-off between growth and behavioural activity in giant Supermice genetically engineered with extra growth hormone genes. Can J Zool 1994; 72: 2158—2168. 48. Bredow S, Taishi P, Obal Jr F et al. Hypothalamic growth hormone-releasing hormone mRNA varies across the day in rats. NeuroReport 1996; 7: 2501—2505. *49. Gardi J, Obal Jr F, Fang J et al. Diurnal variations and sleep deprivation-induced changes in rat hypothalamic GHRH and somatostatin contents. Am J Physiol 1999; 277: R1339—R1344. 50. Zhang J, Chen Z, Taishi P et al. Sleep deprivation increases rat hypothalamic growth hormone-releasing hormone mRNA. Am J Physiol 1999; 275: R1755—R1761. 51. Toppila J, Alanko L, Asikainen M et al. Sleep deprivation increases somatostatin and growth hormone-releasing hormone messenger RNA in the rat hypothalamus. J Sleep Res 1997; 6: 171—178. 52. Bilezikjian LM, Seifert H, Vale W. Desensitization to growth hormone-releasing factor (GRF) is associated with downregulation of GRF-binding sites. Endocrinology 1986; 118: 2045—2052. *53. Sawchenko PE, Swanson LW, Rivier J et al. The distribution of growth-hormone-releasing factor (GRF) immunoreactivity
377
54.
55.
56.
57.
58.
59.
60.
61.
62.
63. 64.
65.
66.
67.
68.
69.
in the central nervous system of the rat: an immunohistochemical study using antisera directed against rat hypothalamic GRF. J Comp Neurol 1985; 237: 100—115. Bertherat J, Bluet-Pajot M-T, Epelbaum J. Neuroendocrine regulation of growth hormone. Eur J Endocrinol 1995; 132: 12—24. Peterfi Z, Churchill L, Hajdu I et al. Fos-immunoreactivity in the hypothalamus: dependency on the diurnal rhythm, sleep, gender, and estrogen. Neuroscience 2004; 124: 695—707. Lanneau C, Peineau S, Petit F et al. Somatostatin modulation of excitatory synaptic transmission between periventricular and arcuate hypothalamic nuclei in vitro. J Neurophysiol 2000; 84: 1464—1474. McGinty D, Szymusiak R. Brain structures and mechanisms involved in the generation of NREM sleep: focus on the preoptic hypothalamus. Sleep Med Rev 2001; 5: 323—342. Obal Jr F. The role of growth hormone-releasing hormone in sleep regulation. In: AA Borbe ´ly, O Hayaishi, TJ Sejnowski, JS Altman (eds.) The regulation of sleep. Strasbourg: HFSP 2000; 112—121. De A, Churchill L, Obal Jr. F et al. GHRH and IL1b increase cytoplasmic Ca2 þ levels in cultured hypothalamic GABAergic neurons. Brain Res 2002; 949: 209—212. Drucker-Colin RR, Spanis CW, Hunyadi J et al. Growth hormone effects on sleep and wakefulness in the rat. Neuroendocrinology 1975; 18: 1—8. Danguir J, De Saint-Hilaire-Kafi Z. Somatostatin antiserum blocks carbachol-induced increase of paradoxical sleep in the rat. Brain Res Bull 1988; 20(1): 9—12. Saeb-Parsy K, Lombardelli S, Khan FZ et al. Neural connections of hypothalamic neuroendocrine nuclei in the rat. J Neuroendocrinol 2000; 12: 635—648. Weikel JC, Wichniak A, Ising M et al. Ghrelin promotes slowwave sleep in humans. Am J Physiol 2003; 284: E407—E415. Payne LC, Obal Jr F, Opp MR et al. Stimulation and inhibition of growth hormone secretion by interleukin-1b: the involvement of growth hormone-releasing hormone. Neuroendocrinology 1992; 56: 118—123. Obal Jr F, Fang J, Payne LC et al. Growth hormone-releasing hormone mediates the sleep-promoting activity of interleukin-1 in rats. Neuroendocrinology 1995; 61: 559—565. Taishi P, De A, Alt J et al. Interleukin-1b stimulates growth hormone-releasing hormone mRNA expression in the rat hypothalamus in vitro and in vivo. J Neuroendocrinol 2004; 16: 1—6. Alam N, McGinty D, Imeri L et al. Effects of interleukin-1 beta on sleep- and wake-related preoptic anterior hypothalamic neurons in unrestrained rats. Sleep 2001; 24: A59. Toth LA, Williams RW. A quantitative genetic analysis of slow-wave sleep in influenza-infected CXB recombinant inbred mice. Behav Genet 1999; 29: 339—348. Alt JA, Obal Jr F, Traynor TR et al. Alterations in EEG activity and sleep after influenza viral infection in GHRH receptordeficient mice. J Appl Physiol 2003; 95: 460—468.
www.elsevier.com/locate/smrv
PHYSIOLOGICAL REVIEW
GHRH and sleep Ferenc Obal Jr.a,1, James M. Kruegerb,* a
¨rgyi Medical Center, University of Szeged, 6720, Szeged, Hungary Department of Physiology, A. Szent-Gyo Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology; Washington State University; PO Box 646520, 205, Wegner Hall, Pullman, WA 99164-6520, USA
b
KEYWORDS Somatotropic axis; Growth hormonereleasing hormone; Growth hormone; Somatostatin; Delta sleep; EEG power; Hypothalamus; Insulinlike growth factor; Interleukin
Summary A significant portion of the total daily growth hormone (GH) secretion is associated with deep non-REM sleep (NREMS). GH secretion is stimulated by the hypothalamic neurohormone, GH-releasing hormone (GHRH). Exogenous GHRH promotes NREMS in various species. Suppression of endogenous GHRH (competitive antagonist, antibodies, somatostatinergic stimulation, high doses of GH or insulin-like growth factor) results in simultaneous inhibition of NREMS. Mutant and transgenic animals with a defect in GHRHergic activity display permanently reduced NREMS which cannot be reversed by means of GH supplementation. GHRH contents and mRNA levels in the hypothalamus correlate with sleep-wake activity during the diurnal cycle and sleep deprivation and recovery sleep. Stimulation of NREMS by GHRH is a hypothalamic action. GABAergic neurons in the anterior hypothalamus/preoptic region are candidates for mediating promotion of NREMS by GHRH. In contrast to NREMS, stimulation of REMS by GHRH is mediated by GH. Simultaneous stimulation of NREMS and GH secretion by GHRH may promote adjustment of tissue anabolism to sleep. q 2004 Elsevier Ltd. All rights reserved.
Introduction There are bidirectional interactions between sleep and the endocrine system. Several hormones have the capacity to affect sleep but the physiological significance of hormonal modulation of sleep is generally unclear. The plasma concentrations of many hormones display sleep-related variations suggesting that sleep influences hormone secretion. However, sleep and hormone levels may correlate without causal relationship between them. For instance, circadian regulation may synchronize these events. Marked sleep-related hormonal *Corresponding author. Tel.: þ1-509-335-2090; fax: þ1-509335-4650. E-mail addresses: [email protected] (J.M. Krueger); [email protected] (F. Obal Jr.). 1 Tel.: þ36-62-545-100; fax: þ 36-62-545-842.
rhythms may also develop as secondary phenomena driven by changes in the activity of the autonomic nervous system during sleep. Growth hormone (GH) is the best documented hormone with a strong sleep-related secretory pattern. GH is produced by anterior pituitary somatotroph cells. Synthesis and secretion of pituitary GH are controlled by two hypothalamic neurohormones; growth hormone-releasing hormone (GHRH), which stimulates, and somatostatin, which inhibits GH. GHRH and somatostatin are secreted into the pituitary portal circulation at the median eminence, and they are carried into the anterior pituitary by the blood. GH secretion is also stimulated by ghrelin. Ghrelin is a hormone released by the endocrine cells of the stomach and it acts via the GH-secretagogue receptor, a receptor different from the GHRH receptor. The somatotropic axis is a fundamental
1087-0792/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.smrv.2004.03.005
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Nomenclature AH/MPO anterior hypothalamus/medial preoptic region EEG electroencephalogram GH growth hormone GHRH growth hormone releasing hormone icv intracerebroventricular IGF-1 insulin-like growth factor-1
anabolic system for the body. It stimulates tissue growth via cell division and via stimulation of protein synthesis. GH acts in part directly and in part via insulin-like growth factor-1 (IGF-1) on target tissues. IGF-1 is both a hormone produced by the liver, and a local paracrine/autocrine substance synthesized in the tissues. The analysis of the interaction between sleep and the somatotropic axis may promote an understanding of the mechanism of coupling body metabolism to sleep, and may provide clues to sleep regulation. In the mouse, the GHRH gene is found within the genomic region linked to encephalographic (EEG) slow wave activity during sleep.1
Sleep and secretion of GH Sleep-related GH secretion was discovered almost 40 years ago.2,3 GH secretion occurs in pulses throughout the day but deep slow wave sleep after sleep onset is associated with particularly large bursts of GH secretion. The GH release during sleep can amount to two thirds of the total GH secreted in 24 h in young males. Sleep deprivation (SD) tends to suppress GH secretion, and large GH releases occur during recovery sleep.3 – 5 Reports from various laboratories confirmed and expanded our understanding of the phenomenon of sleep-related GH secretion (reviewed in Refs.[6,7]). Thus, in addition to the robust sleep-related component, GH secretion has a weaker circadian component. This circadian component may be obvious during SD or when sleep onset is delayed. Calculation of the secretory rate instead of using plasma GH concentration was an important technical development in the analysis of the relationship between sleep and hormone secretions. This technique revealed that GH secretion occurs predominantly during deep slow wave sleep.8,9 The difference in GH secretion between waking and sleep develops at about three-months of age in humans.10 There are gender differences in GH
F. Obal Jr., J.M. Krueger
IL1 iv Mt NREMS REMS SD SDR SEM VIP
interlukin-1 beta intravenous metallothionein non-rapid eye movement sleep rapid eye movement sleep sleep deprivation spontaneous dwarf rat standard error of the mean vasoactive intestinal polypeptide
secretion in both animals and humans with more frequent GH secretory pulses in females than in males. The sleep-related GH secretion is a robust phenomenon in young males. In women, the amount of GH secreted in association with deep NREMS is a much smaller fraction of the total daily GH secretion than in men (reviewed in Ref. [6]). The sleep-associated GH secretion is also age-dependent.11 In males, GH secretion during sleep progressively decreases starting in the third decade and practically disappears above the age of 50. These changes correlate with the decline of deep slow wave sleep. In women, the decrease in sleep-related GH secretion occurs after menopause. Studying the link between sleep and plasma GH is more difficult in animals than in humans because of the short sleep-wake cycles in most laboratory animals such as rats. Nevertheless, correlation between GH secretory rate or plasma GH and sleep occurs in rats,4,12 baboons,13 and lambs.14 The existence of sleep-associated components of GH secretion is also evident in dogs.5 NREMS enhances the responsiveness of GH secretion to secretory stimuli.6 GH may also stimulate sleep.15 Nevertheless, sleep and GH secretion can dissociate. Therefore, a direct causal relationship between sleep and GH secretion cannot explain the coupling between them. It was, therefore, proposed that a common subcortical mechanism may stimulate these events simultaneously.16
GHRH GHRH likely provides the subcortical mechanism synchronizing sleep and GH secretion. GHRH was discovered in 1982.17,18 It is a peptide composed of 40 – 44 amino acid residues. GHRH is a member of the secretin-glucagon peptide family displaying structural homology with vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine,
GHRH and sleep
peptide histidine methionine, pituitary adenylate cyclase activating peptide, gastric inhibitory peptide, secretin, glucagon, and glucagon-like peptide. The homology among some of these peptides is sufficient so that they cross-react on the same receptors. The close relationship between GHRH and VIP was particularly interesting because by the mid 80s, VIP emerged as a peptide for which consistent sleep promoting activity was observed in rats in various laboratories (reviewed in Ref. [19]). Intracerebroventricular (icv) injected VIP enhances both non-rapid eye movement sleep (NREMS) and REMS in rats. However, there were some problems with the NREMS promoting activity of VIP. Thus, inhibition of endogenous VIP selectively suppressed REMS and did not alter NREMS in rats. While VIP stimulated REMS in each species tested, the NREMS response to VIP was not consistent across species. It seemed that VIP might be involved in REMS regulation but the stimulation of NREMS is a non-specific action of exogenous VIP. This may result from VIP mimicking the action of a closely related peptide, i.e. GHRH. The hypothesis has never been tested that stimulation of NREMS by VIP is mediated by GHRH receptor in the rat. Nevertheless, binding characteristics of hypothalamic GHRH receptors demonstrate that these receptors have a very high affinity for VIP in rats.20
Promotion of NREMS by GHRH The NREMS-promoting activity of GHRH was discovered after icv injection of GHRH in rats and rabbits.21 – 23 Perhaps due to the lack of strong circadian regulation, rabbits are particularly responsive to icv GHRH (Fig. 1). For example, in rabbits 1 nmol/kg GHRH induces 53 min of excess NREMS within the first 6 h postinjection but it is half as effective in rats. In both rats and rabbits, GHRH enhances the time spent in NREMS and the intensity of EEG slow wave activity during NREMS. GHRH also stimulates NREMS when it is microinjected directly into the anterior hypothalamus/medial preoptic region (AH/MPO).24 GHRH strongly promotes NREMS after intravenous (iv) injection in rats25 and after intraperitoneal administration in mice.26 Sleep responses to systemic administration of GHRH were first determined in humans. Promotion of sleep by GHRH was reported after iv GHRH from several laboratories, (reviewed in Refs. [6,27]). Briefly, GHRH injected in the morning has no impact on sleep during the subsequent night, and continuous infusion of GHRH is also ineffective.
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Figure 1 Hourly duration of NREMS (mean ^ SEM) in rabbits ðn ¼ 6 – 8Þ after icv injection of artificial cerebrospinal fluid (open symbols) or GHRH (closed symbols). Arrows: injection. Asterisks: significant difference in hour 1 (paired t-test).23
When GHRH enhanced sleep, it was injected prior to sleep, during the first sleep cycle or in the middle of the night. Repeated pulsatile injections28 or a rapid rise in plasma GHRH concentration29 are important for the GHRH-induced NREMS response. GHRH increases the time spent in deep slow wave sleep, it elicits GH secretion and suppresses plasma cortisol concentration in humans. Weak sleep promoting activity is also observed in response to GHRH administered in nasal spray.30 The effects of GHRH on sleep may vary with the time of day. Some observations suggest that GHRH might be less effective at times of very high sleep propensity (beginning of sleep) and may stimulate NREMS more powerfully during periods of decreasing sleep propensity (middle of the night in humans, middle of the light period in the rat).25,31 Finally, the GHRH dose-sleep response curve seems to be bell-shaped for the NREMS-promoting activity of systemic GHRH in the rat.25 The NREMS response decreases then vanishes when the dose of GHRH is increased whereas the GH secretion increases with the dose (Fig. 2). The decline of the NREMS response is attributed to an inhibition of endogenous GHRH by GH. The sleep-promoting activity of GHRH varies with gender and age in humans (reviewed in Ref. [7]). Strong responses are observed in young
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Inhibition of GHRH via endogenous feed back
Figure 2 GH and sleep responses (mean ^ SEM) to iv GHRH in rats ðn ¼ 5 – 10Þ: GHRH was administered in hour 7 of the rest period. GH concentrations are shown at 30 min postinjection. For NREMS and REMS differences from baseline (injection of physiological saline) are depicted during 5 h postinjection. Asterisks: significant difference from baseline (paired t-test).25
male subjects. Instead of enhancing slow wave sleep, GHRH increases stage 2 NREMS and sleep continuity in the elderly. GHRH does not promote sleep in females, in contrast, wake time increases and adrenocorticotropic hormone secretion is stimulated, suggesting a synergism between GHRH and corticotropin releasing hormone in women.
Acute inhibition of GHRH If endogenous GHRH is a physiological NREMS-regulatory substance then suppression of endogenous GHRH should be associated with decreases in NREMS. Thus, a competitive GHRH antagonist32 elicits dose-dependent decreases in the duration and intensity of NREMS and increases sleep latency. Further, icv administration of antibodies to GHRH decreases NREMS.33 The intensity of EEG slow wave activity also decreases during NREMS after antagonist or antibody treatment. Finally, immunoneutralization of GHRH blocks the sleep response to short (3 h) SD including the increases in the duration and intensity of NREMS in rats.33
The somatotropic axis includes several negative feed back loops inhibiting GHRH. In the ultrashort feed back loop, GHRH inhibits its own secretion in the hypothalamus. The ultrashort loop might also involve somatostatin: GHRH stimulates somatostatinergic neurons which inhibit GHRHergic activity. GH inhibits GHRH through a short loop negative feed back, which is also mediated by somatostatin. GH stimulates hypothalamic somatostatinergic neurons. Somatostatin inhibits hypothalamic GHRHergic neurons, and somatostatin is also released into the pituitary circulation and inhibits GH secretion directly in the pituitary. Finally, IGF-1 inhibits GHRH via a long loop feed back. The long loop feed back is in part a direct action and in part it may involve somatostatin. In conclusion, IGF-1, GH, GHRH itself, and somatostatin can inhibit GHRH and somatostatin seems to have a major significance in mediating the various negative feed backs. Both IGF-1 and GH are capable of inhibiting NREMS. Thus, an icv dose of IGF-1, which causes prompt suppression of GH secretion (i.e. it inhibits GHRH), also elicits rapid suppression of sleep in rats and rabbits;34 sleep and GH secretion recover after 1 h. Decreases in NREMS were reported in response to systemic injection of a high dose of GH in humans35 and cats became restless after a high dose of GH.36 Preliminary studies in our laboratory also demonstrate decreases in NREMS time in response to a high dose of icv injected GH in rats (unpublished). Physiological GH concentrations occurring during normal GH surges may also impair slow wave sleep if these surges precede sleep onset in humans.37 Somatostatin has five receptors named sst1 to sst5 (reviewed in Ref. [38]). Inhibition of the somatotropic axis is mediated by the sst2 receptors. Within tissues, the half-life of somatostatin is only a few minutes. Therefore, hydrolysisresistant analogs, such as octreotide, are often used experimentally. Octreotide stimulates sst2 and sst5 receptors, has a low affinity for sst3 receptors, and does not bind the sst1 and sst4 receptors. Icv or systemic injection of octreotide elicits dose-dependent, prompt and simultaneous suppression of NREMS and GH secretion.39 The inhibition lasts for 1 – 2 h then GH secretion recurs and sleep time normalizes or tends to increase above baseline, and a dose-dependent and strong enhancement in EEG slow wave activity occurs during NREMS. Octreotide-induced impairment of
GHRH and sleep NREMS was also verified in humans.40 Octreotide promptly inhibits hypothalamic GHRH release coinciding with sleep suppression.41 After 1 – 3 h postinjection, when intensity of NREMS increases, the accumulated GHRH gradually is released from the hypothalamus. Therefore, a possible explanation for the biphasic sleep response to octreotide is that octreotide suppresses sleep via inhibiting GHRH release, and this period is followed by excessive GHRH release and deep NREMS. Somatostatin is, however, a ubiquitous neurotransmitter in the central nervous system. Octreotide injected icv may interfere with various functions which themselves can influence sleep. In fact, icv octreotide elicits angiotensin-like responses such as prompt drinking, vasopressin secretion and rises in blood pressure followed by vigorous eating. 39 The role of intracerebral angiotensin was verified in the actions of somatostatin by showing that these responses are inhibited by the angiotensin convertase inhibitor, captopril, and by angiotensin receptor blockers (saralasin, losartan) (reviewed in Ref. [42]). In contrast, inhibition of angiotensin convertase does not alter the sleep response to octreotide suggesting that angiotensin is not involved in octreotide-induced sleep suppression.39 If octreotide is injected into mice with decreased GHRH production, the sleep-suppressive action of octreotide is significantly attenuated.43 Further, octreotide completely fails to inhibit sleep in mice with non-functional GHRH receptors though these mice continue to drink in response to octreotide.26 These findings strongly suggest that the sleep-suppressive action of octreotide requires GHRH, i.e. octreotide inhibits sleep via inhibiting GHRHergic activity. Further support for this hypothesis came from experiments with microinjections of octreotide into various structures of the brain the rat.42 Octreotide elicits drinking when injected into the subfornical organ or the paraventricular nucleus, which are parts of the known intracerebral angiotensinergic dipsogenic circuit. These microinjections do not have consistent effects on sleep. In contrast, NREMS is promptly inhibited when octreotide is microinjected into the arcuate nucleus or the AH/ MPO (Fig. 3), where GHRHergic neurons and terminals reside; these injections fail to elicit drinking. The initial NREMS suppression is followed by increases in NREMS intensity starting in hour 2 – 3 postinjection, i.e. the sleep response is similar to that observed after icv administration of octreotide. We conclude that somatostatinergic stimulation suppresses NREMS via inhibiting GHRHergic activity.
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Figure 3 NREMS responses (mean ^ SEM) to microinjection of octreotide (0.02 mg in 0.2 ml, closed symbol) or physiological saline/vehicle (0.2 ml open symbols) into the paraventricular nucleus (PVN), arcuate nucleus (ARC), and anterior hypothalamus/medial preoptic region (AH/MPO) in rats ðn ¼ 7 – 10Þ: Arrows: injections. Asterisks: significant differences in hour 1 (paired t-test).42
Chronic alterations in GHRHergic activity Mutant and transgenic animals provide excellent models for well-defined chronic alterations in the somatotropic axis.44 Lit/lit mice have a point mutation in the GHRH receptor gene and their receptor protein does not bind GHRH. Because of the lack of stimulation of the pituitary somatotroph cells, the lit/lit mice are deficient in GH and IGF-1, and they are dwarfs. The dw/dw dwarf rat is similar to the lit/lit mouse in that the defect is in the GHRH receptors but the exact cause of the malfunction of the receptors is currently not known. GH and IGF-1 productions are greatly decreased in the dw/dw rats. Spontaneous NREMS is significantly reduced in both the lit/lit mice26 and the dw/dw rats.45 Chronic GH replacement (8 – 9 days) in the lit/lit mice increases plasma IGF-1 concentration but fails to increase NREMS (Table 1).26 The TH-hGH transgenic mouse provides another model of decreased GHRHergic activity. In these mice, the promoter region of the tyrosine hydroxylase gene stimulates the transcription of the human (h) GH transgene. Therefore, tyrosine hydroxylase positive catecholaminergic neurons produce and release hGH. GH in the hypothalamus provides a negative feed back signal and suppresses GHRH production and release. Because of the GHRH deficiency pituitary GH is not stimulated, systemic GH concentration is low,
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Table 1 Duration of NREMS and REMS in the 12 h light period in control mice (heterozygous C57BL/6J mice with normal GHRH receptors, n ¼ 12), in lit/lit mice ðn ¼ 12Þ; and in lit/lit mice infused with 24 mg rat GH/day for 8– 9 days ðn ¼ 12Þ:26
Control Lit/lit Lit/lit þ GH a
NREMS (min/12 h)
REMS (min/12 h)
347.1 ^ 10.2 235.3 ^ 12.7a 243.6 ^ 10.5a
62.6 ^ 2.0 49.6 ^ 3.8a 61.8 ^ 2.1
Significantly different from control (ANOVA followed by Student–Neuman–Keuls test).
and the mice are dwarfs. Like the lit/lit mice and dw/dw rats, the TH-hGH mice also display less NREMS than their normal littermates.46 GHRH deficiency is always associated with GH and IGF-1 deficiencies (and dwarf phenotype), and therefore, it might not be obvious whether the alterations in NREMS result from changes in GHRH, GH, or IGF-1. The GH-replacement experiments in the lit/lit mice suggest that it is not GH/IGF-1 which is responsible for the NREMS deficit.26 However, GH/IGF-1 deficiencies may cause alterations in brain development resulting in changes in NREMS, and those changes might be resistant to GH replacements in adult rats. It was, therefore, important to study sleep in animal models where the genetic defect is downstream from the GHRH receptors. This model is the spontaneous dwarf rat (SDR) with a mutation of the GH gene causing practically total loss of GH and dwarfism. Preliminary results demonstrate that NREMS is not reduced in the SDRs, in fact the SDRs display more NREMS during the rest period than normal Sprague – Dawley rats, the strain of rats from which the SDRs are derived. These findings suggest that reduction in spontaneous NREMS in models with chronic GHRH or GHRH receptor deficiencies is not related to the lack of GH/IGF-1 but it is caused by the decreased GHRHergic activity. The response to SD (4 h) was also studied in the lit/lit mice and the dw/dw rats, and a difference was noted between the two species. In the dw/dw rat, SD-induced enhancement of EEG delta activity during recovery sleep is only half that observed in control Lewis rats.45 In contrast, the duration of recovery sleep is slightly decreased but EEG slow wave activity is normal during recovery in the lit/lit mice. 26 The explanation for this difference is currently not clear. A finding, which is seemingly at variance with the anticipated changes in NREMS in GHRH deficiency, was obtained in the Mt-rGH transgenic mouse, also
called the Supermouse.43 In the Mt-rGH mice, the transgene is composed of the promoter region of the metallothionein (Mt) gene and the coding region of the rat (r) GH gene. Supermice produce large quantities of rGH in various tissues and because of the excess GH their body size is greatly increased. The high GH concentration is supposed to suppress GHRH production. However, behavioral observations suggested that the Mt-rGH mice slept more than normal mice.47 Sleep recording verified modest increases in NREMS time (42 min in the 12 h light period). The Supermice also responded to SD with normal sleep rebound. We speculate that the high concentration of GH/IGF-1 stimulated spontaneous NREMS independently of GHRH.
Sleep-related variation in hypothalamic GHRH Synthesis and release of a physiological sleep regulatory substance are anticipated to vary with sleep-wake activity. The GH pulses associated with deep NREMS suggest an activation of the GHRHergic neurons during NREMS. To gain further insight into the dynamics of GHRHergic activity, we determined the diurnal rhythm and SD-induced changes in hypothalamic GHRH mRNA levels and GHRH contents in rats. In the rat, the highest amounts of NREMS occur in the first portion of the light period. As assessed by EEG delta power, NREMS intensity also peaks in the first hour of the light period. Then, the time spent in NREMS and NREMS intensity decrease steadily until dark onset. Rats sleep little during the dark period. The intensity of the short nocturnal sleep bouts increases progressively towards the end of the night. GHRH mRNA levels peak around light onset, decrease towards the end of the light period and stay at very low levels at night.48 The light onset is followed by a short rise in GHRH contents suggesting that the transcribed mRNA is translated into protein very rapidly.49 However, after the transient rise, GHRH contents drop to low levels indicating robust GHRH release. GHRH release declines and thus hypothalamic GHRH content increases towards the end of the dark period. GHRH content decreases at night, which may indicate release or degradation. The major conclusion of the variations in GHRH mRNA levels and GHRH contents is that the time of the day where maximum GHRH synthesis and release occur corresponds to the period of deepest NREMS. Experiments with SD support this conclusion. SD for 8 h results in excessive depletion of
GHRH and sleep
hypothalamic GHRH with very low GHRH contents at the termination of the deprivation. 49 Simultaneously, however, GHRH mRNA levels increase significantly50 suggesting that transcription is stimulated, perhaps because of the high rate of release. It seems that the release continues during recovery because GHRH contents remain low for 1 –2 h. In situ hybridization studies also demonstrate diurnal and SD-induced variations in GHRH mRNA levels.51 Finally, the rise in GHRH mRNA levels after SD is associated with simultaneous decreases in hypothalamic somatostatin mRNA levels.50 An interesting finding, which indirectly supports SD-induced excessive GHRH release, was obtained by determining hypothalamic and pituitary GHRH binding and GHRH receptor mRNA levels in sleep deprived rats.20 Compared to controls, GHRH receptor mRNA and GHRH binding were reduced by 50% in the hypothalamus after 8 h of SD in rats whereas no changes were observed in pituitary GHRH receptors. It is characteristic of GHRH receptors that massive exposure to GHRH elicits down-regulation.52 Therefore a robust intrahypothalamic GHRH release is a likely explanation for the down-regulation of hypothalamic GHRH receptors. The unaltered pituitary GHRH receptor mRNA and binding suggest that massive GHRH release into the pituitary vessels does not occur during SD, i.e. GHRH secretion at the median eminence is inhibited. The inhibition of GHRH secretion at the median eminence is likely the function of somatostatinergic neurons.51
The hypothalamic GHRH network; a sleep-promoting site of action The findings above suggest that the NREMS-promoting activity of GHRH is not mediated by the GH-IGF-1 axis. This hypothesis was verified in hypophysectomized rats.25 GHRH elicits increases in the duration and intensity of NREMS in both intact and hypophysectomized animals showing that GH is not necessary for the GHRH-induced increases in NREMS. Stimulation of NREMS, therefore, is a central action of GHRH. Unlike many other neuropeptides, GHRH that is detectable by means of immunocytochemistry, is confined to a very restricted area in the brain.53 The majority of the GHRH-containing neurons reside in the arcuate nucleus. Smaller subgroups of GHRHcontaining neurons are found around the ventral pole of the ventromedial nucleus and in the parvicellular portion of the paraventricular nucleus. The GHRHergic neurons in the arcuate nucleus are the major source of GHRH released at the median eminence.
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The extraarcuate GHRHergic neurons and in part the intraarcuate GHRHergic neurons project to various regions in the basal forebrain, predominantly to the AH/MPO. In situ hybridization experiments suggest that the diurnal variation in GHRH mRNA with light onset maximum occurs in the arcuate nucleus whereas SD stimulates GHRH mRNA in the paraventricular nucleus.51 It is possible, therefore, that the GHRHergic neuronal pools differ in their participation in the diurnal regulation of sleep and the sleep response to deprivation. Somatostatinergic inhibitory interneurons are found throughout the hypothalamus. The somatostatinergic neurons which are implicated in the somatotropic system reside in the arcuate and the periventricular nuclei.54 The arcuate somatostatinergic neurons inhibit GHRHergic (and other) neurons locally. Expression of the Fos protein, a marker of neuronal activity, is increased in the arcuate nucleus in response to SD and also during the active period of the day in the rat.55 In situ hybridization shows that SD induces increases in somatostatin mRNA expression in the arcuate nucleus.51 Enhanced intraarcuate somatostatinergic activity may explain the suppression of GH secretion during SD (see above). The hypophyseotropic somatostatinergic neurons, i.e. the somatostatinergic neurons which project to the median eminence and secrete their contents into the pituitary circulation, are in the periventricular nucleus. The periventricular somatostatinergic neurons also seem to inhibit the intraarcuate GHRHergic neurons. 56 The somatostatinergic inhibition of the GHRHergic neurons is mediated via sst2 receptors and explains the sleep suppression following intraarcuate injection of octreotide42 (Fig. 3). Inhibition of sleep by octreotide in the AH/MPO suggests that there are either presynaptic sst2 receptors on the GHRHergic terminals projecting to this area or the neurons modulated by GHRH also express sst2 receptors. The AH/MPO is a well-documented area involved in sleep regulation (reviewed in Ref. [57]). Microinjection of GHRH into the AH/MPO elicits robust, dose-dependent increases in NREMS duration and intensity.24 After 0.1 nmol/kg GHRH, NREMS is enhanced for 7–8 h, and EEG delta power during NREMS is also increased. Local administration of a GHRH receptor antagonist into the same injection sites decreases spontaneous NREMS and delta power during NREMS, and attenuates the NREMS response to SD. These observations suggest that GHRH acts in the AH/MPO. The possible importance of the connections between the arcuate nucleus and the AH/MPO was studied in rats by means of transections at the frontal pole of the arcuate
374 nucleus.58 The transection was followed by significant decreases in NREMS duration during the light period, and the diurnal rhythm of EEG delta power during NREMS was almost abolished. The rats were recorded for 8 weeks after transection, and the sleep alterations persisted. In hypothalamic neuronal cultures prepared from fetal rats, 7.6% of the neurons responded promptly with a rise of intracellular calcium concentration after exposure to GHRH.59 Almost all of the GHRH-responsive neurons (96%) displayed immunoreactivity for glutamate decarboxylase (GAD), the enzyme producing GABA. Differences, of course, may exist between fetal and adult neurons but there is now a significant possibility that the AH/MPO neurons mediating the NREMS-promoting activity of GHRH are GABAergic.
GHRH and REMS GHRH often stimulates REMS in animals but this action is not consistent. Icv injection of GHRH weakly stimulates REMS in the rat.23 In contrast, when GHRH is injected into the AH/MPO, REMS does not change.24 In rabbits, the REMS response to icv GHRH is significantly delayed compared to the NREMS response.23 Systemic administration of GHRH enhances REMS in the rat but the effect does not depend on the dose (Fig. 2).25 Systemic injection of GHRH fails to alter REMS in the mouse.26 Finally, no change28 and increases29,31 in REMS were both reported after systemic administration of GHRH in humans. The threshold dose of the icv-injected competitive GHRH antagonist decreasing REMS, is much higher than the dose required for NREMS suppression.32 This high dose also suppresses GH secretion. A very high dose of the competitive antagonist causes only small decreases in REMS when injected into the AH/MPO.24 Administration of antibodies to GHRH simultaneously decreases both REMS and GH secretion.33 These observations suggest that promotion of REMS by GHRH is an indirect action. That the REMS response to GHRH requires intact pituitary functions, possibly GH, was demonstrated by the fact that hypophysectomy prevented stimulation of REMS by systemic GHRH.25 The role of GH was further supported by experiments in the animal models with chronic alterations in the somatotropic axis. During the rest period, REMS time is almost twice that of normal mice in the Supermouse with excess GH.43 In contrast, REMS decreases significantly in the models with GHRH receptor deficiency (dw/dw rats, lit/lit mice) (Table 1).26,45 REMS is markedly reduced in the SDRs,
F. Obal Jr., J.M. Krueger
which do not produce GH but have normal GHRH and GHRH receptors (unpublished). The continuous GH infusion, which fails to alter NREMS, is capable of normalizing REMS in the lit/lit mice (Table 1).26 A particularly interesting finding was obtained in the TH-hGH transgenic mouse. This model is characterized by peripheral GH deficiency but produces hGH in the brain, and the mice display normal REMS.46 In conclusion, promotion of REMS by GHRH seems to depend on GH. Stimulation of REMS by acute rises in GH has been documented in rats,60 cats36 and humans.35 The mechanism of stimulation of REMS by GH is not known. It likely does not involve IGF-1 because acute administration of IGF-1 does not promote REMS.34 GH stimulates hypothalamic somatostatin, and somatostatin has been implicated in REMS regulation. However, intrahypothalamic injection of octreotide does not increase REMS,42 and previous experiments indicate that the brainstem is the site of action for REMS somatostatin promotion.61 It is unclear whether GH modulates somatostatinergic transmission in the brainstem. The latency of the REMS response to GH takes several hours, and during this period, GH stimulates the synthesis of some proteins in the brainstem.60 It is possible that GH regulates the production of an enzyme which is involved in the synthesis of some neurotransmitters involved in the regulation of REMS. (REMS may increase after 1 h latency in response to GHRH.31 Instead of the transporters in the choroid plexus through which systemic GH enters the brain, GH released by GHRH in the pituitary has direct access to the hypothalamus through the window in the bloodbrain barrier at the median eminence. The difference in entering the brain may explain that the REMS response has longer latency after systemic administration of GH than after GHRH).
Interactions with IL1 Theoretically, stimuli, which induce hypothalamic GHRH release, should also elicit or enhance NREMS. However, these stimuli are currently not known. Intraarcuate GHRHergic neurons may receive input from the suprachiasmatic nucleus,62 and through this route they may contribute to the circadian regulation of NREMS. Ghrelin, the GH secretagogue hormone produced by the stomach, stimulates GHRH-containing neurons in the arcuate nucleus. Ghrelin has a NREMS promoting activity63 which is absent in the lit/ lit mice with a deficiency of GHRH receptors.26 GHRH may contribute to the sleep-promoting activity of the cytokine, interleukin-1b (IL1). Icv injection of a sleep-promoting dose of IL1 elicits
GHRH and sleep GH secretion.64 Immunoneutralization of GHRH blocks the IL1-induced GH response and attenuates the sleep response to icv IL1.65 We proposed that IL1 may stimulate GHRH release. Although IL1 has in fact a weak GHRH-releasing activity, recent findings in vitro and in vivo suggest that the major action of IL1 is an up-regulation of the hypothalamic GHRH receptors.66 The majority of the fetal hypothalamic neurons that respond with rises in intracellular calcium to GHRH also produce calcium signals when exposed to IL1.59 It seems, therefore, that IL1 upregulates the GHRH receptors in AH/MPO GABAergic neurons enhancing the sensitivity of these neurons to GHRH. There are neurons dispersed in the AH/MPO that enhance their firing during sleep (reviewed in Ref. [57]); it is assumed that these sleep-active neurons are involved in the promotion of sleep. IL1 is capable of stimulating AH/MPO sleep-active neurons67 but currently it is not known whether these neurons are the same as the GABAergic neurons responsive to both IL1 and GHRH in cell culture. Enhancement in NREMS associated with the acute phase response to infectious diseases is a model of excessive sleep induction by endogenous cytokines, including IL1. By means of a quantitative trait loci analysis, the GHRH receptor gene has been identified as one of three candidate genes that might be involved in the mediation of enhanced NREMS in mice infected with influenza virus.68 Influenza virus fails to stimulate NREMS in the lit/lit mice with GHRH receptor deficiency; in fact, NREMS decreases in these mice.69 It is assumed that the mechanism of the sleep response to influenza includes stimulation of GHRHergic action by IL1 through an up-regulation of the hypothalamic GHRH receptors. Expression of GHRH receptor mRNA is increased in intact mice infected with influenza virus (unpublished observation).
The functional significance of GHRH in sleep regulation GHRHergic neurons are part of the hypothalamic circuit regulating sleep-wake activity. GHRH is one of the many inputs promoting NREMS. Although chronic withdrawal of GHRH is associated with a permanent reduction of NREMS, this decrease is modest (20 – 25% of NREMS time), thus, GHRH deficiency does not result in total sleep loss. The possible significance of GHRH in sleep regulation might be associated with the feature that this peptide simultaneously promotes NREMS and GH secretion. GH is a major anabolic hormone of the body. Simultaneous stimulation of sleep and GH secretion synchronizes the anabolic processes of the body to periods of rest provided by sleep.
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Practice points † Secretion of GH has a marked NREMSassociated component suggesting an activation of GHRH during deep NREMS. † GHRH promotes NREMS after various routes of administration in every species tested (rat, rabbit, mouse, human). † Acute inhibition of endogenous GHRH causes decreases in NREMS. † Activation of the GHRH inhibiting negative feed back mechanisms within the somatotropic system results in inhibition of NREMS. † Experimental genetic alterations, which compromise GHRHergic transmission, are associated with reduced NREMS. † Hypothalamic GHRH displays diurnal and sleep-related variations. † GH and IGF-1 are not involved in the NREMSpromoting activity of GHRH. † Promotion of NREMS by GHRH is a hypothalamic action mediated by GABAergic neurons in the AH/MPO. † Stimuli that increase GHRHergic activity also enhance NREMS. † Promotion of REMS by GHRH is indirectly mediated by GH.
Research agenda † Elucidation of the hypothalamic GHRHergic and somatostatinergic network involved in sleep regulation. † Identification of further stimuli that modulate sleep via GHRH. † Studies of the mechanism of desynchronization between sleep and GH secretion. † Investigation of the relationships between gender differences in sleep and GHRHergic activity. † Understanding the role of GHRH in agingassociated sleep alterations.
Acknowledgements This work was supported, in part, by grants from the National Institutes of Health, USA (Grant numbers NS 25378 and NS27250) and the Hungarian National
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Foundation and Ministry of Health (OTKA-T-043156, ETT 103 04/2003).
References 1. Franken P, Chollet D, Tafti M. The homeostatic regulation of sleep need is under genetic control. J Neurosci 2001; 21: 2610—2621. 2. Takahashi Y, Kipnis DM, Daughaday WH. Growth hormone secretion during sleep. J Clin Invest 1968; 47: 2079—2090. 3. Sassin JF, Parker DC, Mace JW et al. Human growth hormone release: relation to slow-wave sleep and sleep-waking cycles. Science 1969; 165: 513—515. 4. Kimura F, Tsai C-W. Ultradian rhythm of growth hormone secretion and sleep in the adult male rat. J Physiol (Lond) 1984; 353: 305—3315. 5. Takahashi Y, Ebihara S, Nakamura Y. A model of human sleep-related growth hormone secretion in dogs: effects of 3, 6, and 12 hours of forced wakefulness on plasma growth hormone, cortisol, and sleep stages. Endocrinology 1981; 109: 262—272. *6. Van Cauter E, Plat L, Copinschi G. Interrelations between sleep and the somatotropic axis. Sleep 1998; 21: 553—565. *7. Steiger A. Sleep and endocrine regulation. Frontiers Biosci 2003; 8: s358—s376. 8. Holl RW, Hartman ML, Veldhuis JD et al. Thirty-second sampling of plasma growth hormone in man: correlation with sleep stages. J Clin Endocrinol Metab 1991; 72: 854—861. 9. Gronfier C, Luthringer R, Follenius M et al. A quantitative evaluation of the relationships between growth hormone secretion and delta wave electroencephalographic activity during normal sleep and after enrichment in delta waves. Sleep 1996; 19: 817—824. 10. Vigneri R, D’Agata R. Growth hormone release during the first year of life in relation to sleep-wake periods. J Clin Endocrinol Metab 1971; 33: 561—563. 11. Van Cauter E, Leproult R, Plat L. Age-related changes in slow wave sleep and REM sleep and relationship with growth hormone and cortisol levels in healthy men. JAMA 2000; 284: 861—868. 12. Mitsugi N, Kimura F. Simultaneous determination of blood levels of corticosterone and growth hormone in the male rat: relation to sleep-wakefulness cycle. Neuroendocrinology 1985; 41: 125—130. 13. Parker DC, Morishima M, Koerker JD et al. Pilot study of growth hormone release in sleep of the chair- adapted baboon: potential as model of human sleep release. Endocrinology 1972; 91: 1462—1467. 14. Laurentie MP, Barenton B, Charrier J et al. Instantaneous secretion rate of growth hormone in lambs: relationships with sleep, food intake, and posture. Endocrinology 1989; 125: 642—651. 15. Astro ¨m C. Interaction between sleep and growth hormone. Acta Neurol Scand 1997; 92: 281—296. 16. Parker DC, Sassin JF, Mace JW et al. Human growth hormone release during sleep: electroencephalograhic correlation. J Clin Endocrinol Metab 1969; 29: 871—874. 17. Rivier J, Spiess J, Thorner M et al. Characterization of a growth hormone-releasing factor from a human pancreatic islet tumour. Nature 1982; 300: 326—329. * The most important references are denoted by an asterisk.
F. Obal Jr., J.M. Krueger
18. Guillemin R, Brazeau P, Bo ¨hlen P et al. Growth-hormone releasing factor from a human pancreatic tumor that caused acromegaly. Science 1982; 218: 585—587. 19. Obal Jr F, Krueger JM. Biochemical regulation of non-rapideye-movement sleep. Frontiers Biosci 2003; 8: 520—550. 20. Gardi J, Taishi P, Speth R et al. Sleep loss alters hypothalamic growth hormone-releasing hormone receptors in rats. Neurosci Lett 2002; 329: 69—72. *21. Ehlers C, Reed TK, Henriksen SJ. Effects of corticotropinreleasing factor and growth hormone-releasing factor on sleep and activity in rats. Neuroendocrinology 1986; 42: 467—474. 22. Nistico G, DeSarro GB, Bagetta G et al. Behavioural and electrocortical spectrum power effects of growth hormone releasing factor in rats. Neuropharmacology 1987; 26: 75—78. *23. Obal Jr F, Alfo ¨ldi P, Cady AB et al. Growth hormone-releasing factor enhances sleep in rats and rabbits. Am J Physiol 1988; 255: R310—R316. *24. Zhang J, Obal Jr F, Zheng T et al. Intrapreoptic microinjection of GHRH or its antagonist alters sleep in rats. J Neurosci 1999; 19: 2187—2194. *25. Obal Jr F, Floyd R, Kapas L et al. Effects of systemic GHRH on sleep in intact and hypophysectomized rats. Am J Physiol 1996; 270: E230—E237. *26. Obal Jr F, Alt J, Taishi P et al. Sleep in mice with nonfunctional growth hormone-releasing hormone receptors. Am J Physiol 2003; 284: R131—R139. 27. Steiger A, Antonijevic IA, Bohlhalter S et al. Effects of hormones on sleep. Horm Res 1998; 49: 125—130. 28. Steiger A, Guldner J, Hemmeter U et al. Effects of growth hormone-releasing hormone and somatostatin on sleep EEG and nocturnal hormone secretion in male controls. Neuroendocrinology 1992; 56: 566—573. 29. Marshall L, Derad I, Strasburger CJ et al. A determinant factor in the efficacy of GHRH administration in promoting sleep: high peak concentration versus recurrent increasing slopes. Psychoneuroendocrinology 1999; 24: 363—370. 30. Perras B, Marshall L, Ko ¨hler G et al. Sleep and endocrine changes after intranasal administration of growth hormonereleasing hormone in young and aged humans. Psychoneuroendocrinology 1999; 24: 743—757. 31. Kerkhofs M, Van Cauter E, Van Onderbergen A et al. Sleeppromoting effect of growth hormone-releasing hormone in normal men. Am J Physiol 1993; 264: E594—E598. 32. Obal Jr F, Payne L, Kapas L et al. Inhibition of growth hormone-releasing factor suppresses both sleep and growth hormone secretion in the rat. Brain Res 1991; 557: 149—153. 33. Obal Jr F, Payne L, Opp M et al. Growth hormone-releasing hormone antibodies suppress sleep and prevent enhancement of sleep after sleep deprivation. Am J Physiol 1992; 263: R1078—R1085. 34. Obal Jr F, Kapas L, Gardi J et al. Insulin-like growth factor-1 (IGF-1)-induced inhibition of growth hormone secretion is associated with sleep suppression. Brain Res 1999; 818: 267—274. 35. Mendelson WB, Slater S, Gold P et al. The effect of growth hormone administration on human sleep: a dose response study. Biol Psychiatry 1980; 15: 613—618. 36. Stern WC, Jalowiec JE, Shabshelowitz H et al. Effects of growth hormone on sleep-waking patterns in cats. Horm Behav 1975; 6: 189—196. 37. Spiegel K, Leproult R, Colecchia EF et al. Adaptation of the 24-h growth hormone profile to a state of sleep debt. Am J Physiol 2000; 279: R874—R883.
GHRH and sleep
38. Reisine T. Somatostatin receptors. Am J Physiol 1995; 269: G813—G820. 39. Beranek L, Hajdu I, Gardi J et al. Central administration of the somatostatin analog octreotide induces captopril-insensitive sleep responses. Am J Physiol 1999; 277: R1297—R1304. 40. Ziegenbein M, Held K, Kuenzel HE et al. The somatostatin analogue octreotide impairs sleep and decreases EEG sigma power in young male subjects. Neuropsychopharmacology 2004; 29: 146—151. 41. Gardi J, Szentirmai E, Hajdu I et al. The somatostatin analog, octreotide, causes accumulation of growth hormone-releasing hormone and depletion of angiotensin in the rat hypothalamus. Neurosci Lett 2001; 315: 37—40. *42. Hajdu I, Szentirmai E, Obal Jr F et al. Different brain structures mediate drinking and sleep suppression elicited by the somatostatin analog, octreotide, in rats. Brain Res 2003; 994: 115—123. 43. Hajdu I, Obal Jr F, Fang J et al. Sleep of transgenic mice producing excess rat growth hormone. Am J Physiol 2002; 282: R70—R76. 44. Frohman LA. New insights into the regulation of somatotrope function using genetic and transgenic models. Metabolism 1996; 45: S1:1—S1:3. 45. Obal Jr F, Fang J, Taishi P et al. Deficiency of growth hormone-releasing hormone signaling is associated with sleep alterations in the dwarf rat. J Neurosci 2001; 21: 2912—2918. 46. Zhang J, Obal Jr F, Fang J et al. Non-rapid eye movement sleep is suppressed in transgenic mice with a deficiency in the somatotropic system. Neurosci Lett 1996; 220: 97—100. 47. Lachmansingh E, Rollo CD. Evidence for a trade-off between growth and behavioural activity in giant Supermice genetically engineered with extra growth hormone genes. Can J Zool 1994; 72: 2158—2168. 48. Bredow S, Taishi P, Obal Jr F et al. Hypothalamic growth hormone-releasing hormone mRNA varies across the day in rats. NeuroReport 1996; 7: 2501—2505. *49. Gardi J, Obal Jr F, Fang J et al. Diurnal variations and sleep deprivation-induced changes in rat hypothalamic GHRH and somatostatin contents. Am J Physiol 1999; 277: R1339—R1344. 50. Zhang J, Chen Z, Taishi P et al. Sleep deprivation increases rat hypothalamic growth hormone-releasing hormone mRNA. Am J Physiol 1999; 275: R1755—R1761. 51. Toppila J, Alanko L, Asikainen M et al. Sleep deprivation increases somatostatin and growth hormone-releasing hormone messenger RNA in the rat hypothalamus. J Sleep Res 1997; 6: 171—178. 52. Bilezikjian LM, Seifert H, Vale W. Desensitization to growth hormone-releasing factor (GRF) is associated with downregulation of GRF-binding sites. Endocrinology 1986; 118: 2045—2052. *53. Sawchenko PE, Swanson LW, Rivier J et al. The distribution of growth-hormone-releasing factor (GRF) immunoreactivity
377
54.
55.
56.
57.
58.
59.
60.
61.
62.
63. 64.
65.
66.
67.
68.
69.
in the central nervous system of the rat: an immunohistochemical study using antisera directed against rat hypothalamic GRF. J Comp Neurol 1985; 237: 100—115. Bertherat J, Bluet-Pajot M-T, Epelbaum J. Neuroendocrine regulation of growth hormone. Eur J Endocrinol 1995; 132: 12—24. Peterfi Z, Churchill L, Hajdu I et al. Fos-immunoreactivity in the hypothalamus: dependency on the diurnal rhythm, sleep, gender, and estrogen. Neuroscience 2004; 124: 695—707. Lanneau C, Peineau S, Petit F et al. Somatostatin modulation of excitatory synaptic transmission between periventricular and arcuate hypothalamic nuclei in vitro. J Neurophysiol 2000; 84: 1464—1474. McGinty D, Szymusiak R. Brain structures and mechanisms involved in the generation of NREM sleep: focus on the preoptic hypothalamus. Sleep Med Rev 2001; 5: 323—342. Obal Jr F. The role of growth hormone-releasing hormone in sleep regulation. In: AA Borbe ´ly, O Hayaishi, TJ Sejnowski, JS Altman (eds.) The regulation of sleep. Strasbourg: HFSP 2000; 112—121. De A, Churchill L, Obal Jr. F et al. GHRH and IL1b increase cytoplasmic Ca2 þ levels in cultured hypothalamic GABAergic neurons. Brain Res 2002; 949: 209—212. Drucker-Colin RR, Spanis CW, Hunyadi J et al. Growth hormone effects on sleep and wakefulness in the rat. Neuroendocrinology 1975; 18: 1—8. Danguir J, De Saint-Hilaire-Kafi Z. Somatostatin antiserum blocks carbachol-induced increase of paradoxical sleep in the rat. Brain Res Bull 1988; 20(1): 9—12. Saeb-Parsy K, Lombardelli S, Khan FZ et al. Neural connections of hypothalamic neuroendocrine nuclei in the rat. J Neuroendocrinol 2000; 12: 635—648. Weikel JC, Wichniak A, Ising M et al. Ghrelin promotes slowwave sleep in humans. Am J Physiol 2003; 284: E407—E415. Payne LC, Obal Jr F, Opp MR et al. Stimulation and inhibition of growth hormone secretion by interleukin-1b: the involvement of growth hormone-releasing hormone. Neuroendocrinology 1992; 56: 118—123. Obal Jr F, Fang J, Payne LC et al. Growth hormone-releasing hormone mediates the sleep-promoting activity of interleukin-1 in rats. Neuroendocrinology 1995; 61: 559—565. Taishi P, De A, Alt J et al. Interleukin-1b stimulates growth hormone-releasing hormone mRNA expression in the rat hypothalamus in vitro and in vivo. J Neuroendocrinol 2004; 16: 1—6. Alam N, McGinty D, Imeri L et al. Effects of interleukin-1 beta on sleep- and wake-related preoptic anterior hypothalamic neurons in unrestrained rats. Sleep 2001; 24: A59. Toth LA, Williams RW. A quantitative genetic analysis of slow-wave sleep in influenza-infected CXB recombinant inbred mice. Behav Genet 1999; 29: 339—348. Alt JA, Obal Jr F, Traynor TR et al. Alterations in EEG activity and sleep after influenza viral infection in GHRH receptordeficient mice. J Appl Physiol 2003; 95: 460—468.