GHRH

0021.972x196/$03.0010 Journal of Clinical Endocrinology and Metabolism Copyright 0 1996 by The Endocrine Society

Vol. 81, No. 3 Printed an U.S.A.

Greater Efficacy of Episodic than Continuous Growth Hormone-Releasing Hormone (GHRH) Administration Promoting Slow-Wave Sleep (SWS)* LISA MARSHALL, HORST L. FEHM,

MATTHIAS MijLLE, JAN BORN

GERHARD

BOSCHEN,

AXEL

in

STEIGER,

AND

Clinical Neuroendocrinology (L.X., M.M., G.B., H.L.F., J.B.), Department of Internal Medicine (H.L.F.), Medical University of Lubeck; Department of Psychiatry (A.S.), Max-Planck-Institute of Psychiatry, Munchen; Department of Physiological Psychology (J.B.), University of Bamberg, Federal Republic of Germany ABSTRACT It has been suggested that growth hormone (GH)-releasing hormone (GHRH) stimulates the surge in GH and enhances slow-wave sleep (SWS), two phenomena that characterize the beginning of nocturnal sleep. However, in human studies the effects of systemic GHRH administration on sleep were not consistent. This may reflect the differential influence of administration procedures being episodic in one of the above studies, but either a continuous infusion or a single bolus in the others. The present study in healthy volunteers compared changes in nocturnal sleep following 200 pg GHRH administered iv either episodically (4 boluses of 50 pg each at 2200, 2300, 2400, and 0100 h) or as a continuous infusion (57 pg/h between 2130 and 0100 h). Time spent in stage 4 of SWS on nights of episodic GHRH ad-

ministration significantly exceeded that on nights of continuous GHRH administration (P < 0.01). Compared with a placebo condition, episodic administration of GHRH enhanced SWS P < 0.01) and rapid eye movement (REM) sleep (P < 0.05) and diminished time spent in wakefulness and sleep stage 1 (P < 0.05). Effects of continuous GHRH infusion on sleep generally remained insignificant compared with placebo. Plasma GH concentrations were enhanced during both conditions of GHRH administration (P < O.Ol), with the increase following episodic administration slightly exceeding that during continuous infusuion (P < 0.05). The results support a greater physiological efficacy of episodic GHRH stimulation in promoting sleep. (J Clin Endocrinol Metab 81: 1009-1013, 1996)

ITHIN the last decade growth hormone (GH)-releasing hormone (GHRH) has been put forward as a putative sleep-enhancing substance (l-3). It has been suggested that GHRH affects in parallel both GH secretion and sleep (4-7), thus explaining the close temporal correlation between slow-wave sleep (SWS) and surge in GH plasma levels in the beginning of nocturnal sleep (8, 9). The effects of GHRH administration on sleep were, however, controversial. Whereas a reduced sleep onset latency, prolonged SWS-time, and an increase in slow-wave amplitude have been reported in rats and rabbits following intracerebroventricular administration of GHRH (1,3), most human studieshave failed to show an effect of GHRH on sleepfollowing iv administration around normal bedtime (4, 10-13). The study by Steiger et nl. (14) presents an exception. In their study an increase in SWS was observed in healthy humans following episodic administration of GHRH, involving four hourly bolusesof 50 pg GHRH between 2200and 0100h. All other investigations in humans had employed either a constant rate infusion or a single bolus. Thus, a possible factor explaining the discrepanciesbetween the findings by Steiger et al. (14) and the remaining investigations in humans could be the temporal pattern of GHRH administration: episodic in

the former, while a continuous infusion or a single bolus in the latter studies. A particular sensitivity to pulsatile stimulation US.continuous perfusion has been found for the response of somatotroph pituitary cells in vitro (15), as well as for gonadotrophs (16). Also, there are indications that, under normal conditions, the sleep related GH surge is invoked by a series of discrete GHRH secretory pulses (17-19). Thus, pulsatile stimulation may be asphysiologically relevent for promoting sleep as it appears to be for stimulating GH release.Therefore, the present experiment investigated the effect of equal doses, but different systemic administration procedures of GHRH on nocturnal sleep: episodic VS.continuous.

Received August 1, 1995. Accepted October 23, 1995. Address correspondence and reprint requests to: L. Marshall, Medical University of Liibeck, Klinische Forschergruppe, Haus 23a, Ratzeburger Allee 160, 23538 Liibeck, Germany. * This study was supported by a grant from the Deutsche Forschungsgemeinschaft to H.L.F. and J.B.

Subjects

and

Methods

Twelve male volunteers (aged 22-28 yr) taking no medication and within 2 10% of ideal body weight were studied after having given informed written consent. All men were healthy nonsmoking students, and none had a history of sleep disturbances. The studies took place in a sleep laboratory at the Medical University of Liibeck, and the experimental protocol was approved by the university’s Ethics Committee. Subjects were obliged to get up between 0700 and 0800 h on the days before experimental nights, not to take any naps on the day of the experiment, and not to participate in any exerting physical activity. On experimental days they ate their dinner before 1800 h. To become accustomed to the experimental conditions subjects spent one preexperimental night in the laboratory, including the placement of a catheter, the collection of blood samples, and the attachement of electrodes for polysomnographical recordings. For the experiment proper subjects arrived at the laboratory at 1900 h, and were prepared for polysomnographic recordings and blood sampling. Subjects were assigned to bed at 2000 h. Lights were turned off

1009

1010

hIARSHALL

at 2300 h (bedtime) and continues recordings were obtained until 0700 h, when the subjects were awakened. Experiments were designed according to a double blind within-subject cross-over comparison. Each subject participated in three experimental sessions, separated by at least seven days. They received either (i) a continuous infusion of 250 mL saline solution betweeen 2130 and 0100 h and four 5 mL bolus injections of saline solution at 2200, 2300, 2400, and 0100 h (placebo); (ii) a continuous infusion of 200 pg h-GHRH (Ferring GmbH, Kiel, Germany) dissolved in 250 mL saline solution between 2130 and 0100 h and four 5 mL bolus injections of saline solution at 2200,2300,2400, and 0100 h (Continuous GHRH); or (iii) a continuous infusion of 250 mL saline solution betweeen 2130 and 0100 h and four 5 mL bolus injections, each of 50 pg h-GHRH dissolved in 5 mL saline solution at 2200, 2300,2400, and 0100 h (Episodic GHRH). Rate of the continuous infusion was 72 mL/h. To each 250 mL infusion 2 mL 2OOg/L h-albumin (Serapharm, Miinster, Germany) were added. The order of treatment administration was randomized among subjects, who were blinded to whether they would receive placebo, episodic, or continuous GHRH. For analysis of GH and cortisol, blood samples were drawn at 30-min intervals from 2000 until 2130 h, and then at 15-min intervals until 0700 h, through an indwelling catheter inserted at 1900 h in an antecubital vein. The catheter was connected to a long thin tube (volume 1.5 mL), which enabled blood collection from an adjacent room without disturbing the subject’s sleep. From 2000 to 2130 h and from 0100 to 0700 h the iv line was kept patent with a slow drip of saline solution (30 mL/h). Polysomnographic recordings were scored visually, off-line according to the criteria of Rechtschaffen and Kales (20). Each record was

scored by two experimenters who were blind to the treatment conditions. Sleep stages l-4, rapid eye movement (REM) sleep, and wakefulness were scored for 30-s intervals. SWS was defined by the sum of sleep stages 3 and 4. For each night sleep onset latency with reference to 2300 h, sleep time, percentages of time spent in the different sleep stages and in wakefulness, and latencies of the sleep stages with reference to sleep onset were determined. The percentages of time spent in the different sleep stages and the average plasma concentrations of GH and cortisol were calculated for total sleep time as well as for the first and second half of each subject’s sleep time. GH and cortisol were measured by commercial RIAs (Biermann, Bad Nauheim, Germany and Boeringer Mannheim, Mannheim, Germany, respectively). The sensitivity of the GH assay was 0.9 pg/L. The intraassay coefficient of variation was below 5% for the relevant range of concentrations, and the interassay coefficient below 9%. The sensitivity of the cortisol assay was 1.0 &L, the intraassay and interassay coefficients of variation were below 4 and 6%, respectively. Differences among the effects of the treatments and the time spent in each sleep stage were analyzed using a multivariate analysis of variance including sleep stages as variates and the treatments as within factors. Differences between the effects of any two treatments were further specified by contrasts. Multivariate analysis of variances and subsequent contrasts were similarily employed for the parameters of GH and cortisol. Contrasts, shown in Tables 1 and 2, were only calculated when the multivariate analysis of variance indicated significance or a trend regarding the within effect for a variable. A Greenhouse-Geisser corrected P-value of less than 0.05 was considered significant.

TABLE 1. Mean (? SEM) of sleep onset latency (with reference to lights off at 2300 h), total wakefulness (W) and in the different sleep stages 1 (Sl), 2 (S2), 3 (S3), 4 (S4), in REM sleep episodic (E), and continuous (C) GHRH administration. P

Parameter Sleep Sleep

onset time

(min) (min)

%, total W Sl s2 s3 s4 sws REM %, 1st half W Sl s2 s3 s4

sws

REM %, 2nd half W E Ei sws REM Latency s2 sws REM

18.8 (4.1) 461.7 (4.1)

JCE C M . 1996 Vol81 . No 3

ET AL.

sleep time, and percentage of time spent in and SWS for the three conditions: Placebo (P),

E 13.3 (3.0) 467.2 (3.0)

C 14.4 (3.4) 466.1 (3.4)

Significance NS NS

6.4 10.3 52.7 9.2 4.5 13.7 16.9

(2.2) (1.6) (2.0) (0.9) (0.5) (0.9) (2.3)

2.6 (1.7) 6.9 (1.9) 50.1(1.9) lO.l(l.0) 9.3 (1.2) 19.4 (1.9) 21.0 (1.5)

5.4 8.8 52.3 10.0 5.1 15.1 18.4

(2.4) (2.1) (2.7) (1.5) (0.6) (1.7) (1.9)

P-E” NS NS NS P-Eb E-C’ P-E’ E-C” P-E” E-C”

6.6 11.5 53.6 12.3 6.5 18.8 9.4

(2.6) (2.3) (3.0) (1.5) (0.9) (1.5) (2.0)

1.8 (1.2) 5.9 (1.8) 50.1 (2.5) 14.1(1.7) 15.0 (1.8) 29.1 (2.5) 13.2 (1.2)

5.1(2.2) 8.2 (2.6) 51.5 (3.6) 15.2 (2.5) 7.5 (1.2) 22.7 i2.8j 12.5 (1.9)

P-E” E-C” P-E” NS NS P-Eb E-C’ P-E* E-C” P-E”

3.2 8.0 50.2 7.2 2.6 9.8 28.8

5.5 9.6 53.1 5.8 1.7 7.5 24.4

NS NS NS NS NS NS NS

6.1(2.4) 9.0 (1.5) 51.9 (2.2) 7.1(1.0) 1.5 (0.4) 8.6 (1.2) 24.4 (3.1)

(2.3) (2.3) (1.8) (1.1) (1.1) (1.8) (3.0)

(2.9) (1.8) (2.5) (1.1) (0.4) (1.4) (2.7)

(min) 6.4 (1.2) 22.5 (4.7) 122.2 (17.5)

5.8 (0.7) 20.7 (3.2) 102.5 (12.7)

“P < 0.1. h P < 0.01 c P < 0.05. Percentages are provided for the entire night as well as separately for the first and second (with reference to sleep onset) of 52, SWS, and REM sleep. Right column indicates significant NS: nonsignificant.

6.7 (1.0) 31.4 (8.3) 102.0 (15.1)

NS NS NS

half of sleep time. Bottom rows indicate latencies differences between any two of the conditions.

EPISODIC

,

I

1

VS.

CONTINUOUS

r-w

I

GHRH

ADMINISTRATION

I

1011

r-W

1 I I, 20.00 21.00 2X4 100

r 20

I I I I I I 2Q.M 21M 22.W 22.00 0.M 1.W ZM &MI 4.W 61

S.00 7.M th’

Cortisol

20.00 2t.w 220428.00

I1 I I I c 0.00 1.M 2dO 3.00 4.00 SW 6.00 7.M ‘lrno

r

20

r20

0.00 1.M 2.M 3.w 4.00 5.w ml 7JN th’

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t

-ima

20.00 21.00 22w 2200 0.04 1.00 2.a a.ca 4.00 5.00 6.00 7.00

I I I I NLM2lM2ZM22MOMl.M

I

I

Cortlsol

I I1 I, 2.M 900 4.M 61

8.007.M

r 20 16

I I I I I I I I I 2050 21.00 ZZ.WZSM 0.00 1.M 2.M 2Q4 4.00 6110 IM 7.QU FIG.

panel, turned

1. Profiles of sleep (top), cortisol (middle), Placebo condition. Middlepanel, Episodic off at 2300 h, subjects were awakened

I I 1 I 20.00 21.00 22.0022Ml

I I I I 9 I I rtbno 0.00 1.M 2M 3.00 4.00 6.00 6.00 760

and GH (bottom) plasma GHRH condition (arrows). at 0700 h. W, Wakefulness;

Results Sleep

Results for sleep parameters are summarized in Table 1. Figure 1 profiles of sleep, cortisol, and GH concentrations for two individuals during the three experimental nights. Sleep patterns during placebo nights were comparable with those typically obtained under laboratory conditions, except that time in sleepstage 1 appeared to be somewhat increased and time in SWS slightly shorter than usual. This may reflect the fact that sleep experiments were carried out during long midsummer days. In fact, half of the subjects reported that during the last weeks they were accustomed to going to bed later than 2400 h. The most consistenteffect of GHRH was an increasein SWS

I 8 I I I I 21LOO21.00 22.M 22.W 0.00 1M

I , , I I r 200 IOQ 4.00 6.00 6.00 7.M ‘Ime

levels from two representative subjects (upper part, lower part). Left Rightpanel, Continuous GHRH condition (hatched bar). Lights were Sl,S2,S3,S4, sleep stages 1 to 4; REM, REM sleep.

and sleepstage4 during episodic administration of the peptide in comparisonto placeboduring the first half of the night aswell asover the entire night. Significantly, more time was alsospent in sleepstage 4 during episodic sleepthan during continuous GHRH administration (refer to Table 1 for statistical significances).Wakefulness and stage 1 sleep were reduced during episodic administration of GHRH in the first half of the night. There was a trend towards reduced time spent awake with episodic GHRH administration, compared with the effects of the continuous infusion for the first half of the night. REM sleep also increasedsignificantly during episodic administration of GHRH over the first half of the night and revealed a tendency over the entire night to be higher than in both the placebo and continuous GHRH conditions. Continuous infusion of GHRH

1012

MARSHALL

ET AL.

TABLE 2. GH and cortisol peak concentrations (means 5 SEM) and peak latencies conditions: Placebo (P), episodic (El, and continuous (C) GHRH administration Parameter

P

GH (ng/mL) Peat Peak latency (min) Total sleep time Average 1st half average 2nd half average

(with

E

11.1 (3.2) 159.3 (24.9) 2.8 (0.5) 4.0 (1.1) 1.6 (0.3)

Cortisol (pg/dL) Peak Peak latency (min) Total sleep time Average 1st half average 2nd half average

JCE & M . 1996 Vol81 . No 3

15.2 (0.9) 494.3 (14.5)

reference

to 2200

h) for the three

C

experimental

Significance

31.8 (4.8) 100.5 (11.4)

27.7 (4.7) 123.0 (11.1)

P-E” P-E”

P-C”

8.7 (1.4) 15.7 (2.8) 1.7 (0.3)

7.4 (1.4) 12.5 (2.8) 2.4 (0.6)

P-E” P-E” NS

P-C” P-C”

16.7 (1.2) 498.0 (11.4)

14.9 (0.9) 495.5 (15.7)

NS NS

5.1(0.5) 2.2 (0.4) 7.9 (0.7)

5.4 (0.4) 2.0 (0.3) 8.7 (0.5)

4.9 (0.5) 2.1 (0.2) 7.7 (0.8)

E-C

E-Ch

NS NS NS

n P < 0.01 bP < 0.1 c P < 0.05 Average

GH and

cortisol

plasma

concentrations

are provided

for the total

sleep

time,

and separately

for the first

and second

half

of sleep

time. Right columnindicatessimificant differencesbetweenanv two of the conditions. NS: nonsignificant.

-

remained without any significant effect on sleepparameters,as compared with placebo. GH and cortisol

Mean plasma GH and cortisol concentrations during the three experimental nights, plus statistical findings, are given in Table 2. As expected, episodic and continuous GHRH administrations both markedly increased peak GH concentrations and mean GH levels during the first half as well as over the entire night in comparison with placebo administration. During episodic administration, the average GH level in the first half of the night was higher than continuous administration. For cortisol, the peak concentrations during episodic administration showed a tendency to be higher than during continuous administration or placebo. Discussion

The present study compared the effects on sleep and endocrine responsesof 200 pg GHRH administered either as a continuous infusion or episodically, as 4 hourly bolus injections. In comparison with the placebo night, both procedures of administration markedly increased the endogenous sleeprelated nocturnal plasma GH level in humans, as has been reported previously by several authors (e.g. 4,11,12,14,21). The main finding was that SWS, specifically time spent in sleep stage 4, was only promoted significantly by the episodic administration of GHRH. In contrast, following continuous administration of the same dose of GHRH, changes in SWS and sleep stage 4 were substantially smaller and too inconsistent to yield any statistical significance when compared with the effects of placebo. These results confirm the occurrence of a similar increase in SWS after episodic administration of GHRH in a previous study (141, and it simultaneously explains negative outcomes of other human studies (11, 121,in which GHRH (at a comparable dosage) was administered at a constant rate infusion. Also, only following episodic administration, indications of decreased

wakefulness and enhanced REM sleep were found. An increase in REM sleep with GHRH administration has been likewise reported in animals (5) and man (131,but was not found in another study with a GHRH regimen most similar to ours (14). The response of the pituitary in the present experiment revealed only a weak dependence upon the kind of administration. Possibly the pituitary response, asindicated by the supra physiological GH concentrations, was nearly exhausted with both administration procedures. At this point it should be mentioned that experiments took place during long midsummer days, and subjects,required to go to sleepat 2300h, appeared to sleepsomewhat poorer under placebo (with increasedtime in sleepstage1 and reduced SWS) than in comparablestudies (e.g. 12).Thus, it may be argued that improved sleep under GHRH administration is especially evident under suboptimal sleepingconditions. In fact, Kerkhofs et al. (13) concluded from their experiments comparing the effects before and after the occurrenceof the main epochsof nocturnal SWS that the promoting effect of GHRH on SWS is enhanced under conditions of diminished SWS propensity, i.e. after the main epochs of SWS have passed. Becauseof the seasonal conditions, the endogenoussleeppressureof our subjectsmay have likewise been reduced. This line of reasoning is contradicted, however, by experiments in which GHRH has promoted SWS under optimal sleepconditions (141,aswell asby negative findings in experiments in which GHRH did not develop its sleep promoting efficacy when administered during light sleep (22). The efficacy of episodic neuropeptide administration finds support in the current findings of a greater sleep-promoting effect with repetitive bolus injections than with a continuous infusion of GHRH. In response to continuous infusions, adaptation processesand reduced efficacy of stimulation have been considered at the cellular level, e.g. receptor downregulation and desensitization (21,231 or via inhibitory hormonal processes(24). On the other hand, with episodic ad-

EPISODIC VS. CONTINUOUS ministration, a time interval of 60 min between pulses allows for hormone degradation (25), thus excluding the occurrence of adaptive processes. Furthermore, episodic administration invokes temporarily very high plasma concentrations of GHRH allowing for cumulative effects at receptor sites mediating the central nervous and endocrine effects of the peptide (7, 26). Along this line of argumentation also, recent findings by Kerkhofs et al. (13) could be explained. Whereas in that study an iv bolus of GHRH enhanced SWS when administered during the third REM sleep epoch of one night, it failed to effect SWS when administerd during the first SWS epoch. Possibly, with this latter administration exogenous GHRH interfered with endogenous GHRH release, supposed to be at a maximum in the beginning of sleep. The receptor sites mediating the effects of GHRH on sleep are at present unknown. Central nervous GHRH receptors appear to be located primarily in the hypothalamus (27). Also, the normal distribution of GHRH cells and fibers in the brain, as shown by GHRH immunoreactivity, appears to be strictly limited to the hypothalamus and adjoining tissue, including the basal forebrain (28), which had previously been suggested as a target structure of NREM sleep promoting GHRH actions (6, 29). Assuming that GHRH after systemic administration does not have free access to the brain receptors, the mechanisms responsible for the greater efficacy of episodic than continuous GHRH administration in promoting SWS could also primarily involve the blood-brain barrier, rather than processes at the receptor site itself. The lack of a strong difference in pituitary response suggests the mechanism underlying the SWS promoting effect of episodic GHRH administration does not rely on the pituitary GH response. Although the mediating mechanisms are obscure at present, it is important to emphasize that the episodic administration of GHRH mimicked the pulsatile model of secretory activity under normal physiological conditions. In this regard, the present findings of a sleep promoting effect of GHRH being selectively present after episodic administration provide further support for the notion of a physiological role of GHRH in the regulation of sleep and SWS in humans. Acknowledgments The authors Anja Otterbein

thank Stefanie Baxmann for preparing the figures.

for

technical

assistence

and

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