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Original Article |
Departments of General Internal Medicine (S.W.K., M.F., A.E.M., H.P.), Endocrinology (F.R.), and Neurology (S.O., G.J.L.), Leiden University Medical Center, 2300 RC Leiden, The Netherlands; and Department of Clinical Neurophysiology, University Hospital Vrije Universiteit (R.L.S.), 1094 LM Amsterdam, The Netherlands
Address all correspondence and requests for reprints to: S. W. Kok, M.D., Department of General Internal Medicine (C1-R38), Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail: s.w.kok{at}lumc.nl.
Abstract
Narcolepsy is a sleep disorder caused by disruption of hypocretin (orexin) neurotransmission. It has been suggested that anomalous timing by the biological clock contributes to the symptomatology. Hypocretins stimulate the pituitary-adrenal (PA) axis in rodents. We explored whether hypocretin deficiency disrupts circadian timing and blunts PA hormone release. We deconvolved 24-h plasma profiles of ACTH and cortisol, and determined their circadian rhythm by cosinor analysis in seven hypocretin-deficient narcoleptic males and seven matched controls. Basal and total ACTH production were blunted in narcoleptics [310 ± 86 vs. 760 ±160 ng/liter·24 h (P = 0.02) and 920 ± 147 vs. 1460 ± 220 ng/liter·24 h (P = 0.04), respectively], whereas pulsatile release did not differ between groups. In contrast, basal, pulsatile and total cortisol secretion were similar in both groups. The cross-approximate entropy of the joint ACTH/cortisol time series was higher in narcoleptics (1.26 ± 0.07 vs. 1.07 ± 0.04; P = 0.04), reflecting reduced secretory process regularity. The acrophases of both ACTH and cortisol occurred at similar clock times (
0830 h) in patients and controls, which supports the idea that the master pacemaker is intact in narcolepsy. The reduced (basal) ACTH secretion and the diminished secretory process regularity of the ACTH/cortisol ensemble conjointly suggest that hypocretin deficiency induces changes in the interplay between PA hormones.
NARCOLEPSY IS CHARACTERIZED by excessive daytime sleepiness, cataplexy, hypnagogic hallucinations, and sleep paralysis (1). It was recently established that disruption of hypocretin (orexin) neurotransmission underlies the disease in animals and man (2, 3, 4). A selective autoimmune-mediated destruction of hypocretin neurons is presumed to cause narcolepsy in humans (5, 6). The hypocretin peptides (hypocretin-1 and -2, also called orexin A and B) are produced by a small group of neurons in the lateral hypothalamus that project widely throughout the central nervous system (7, 8, 9). The latter observation suggests that hypocretins may be involved in a range of neuronal regulatory actions. Indeed, numerous studies show that these peptides affect arousal, energy balance, and a variety of neuroendocrine ensembles (1, 10, 11). The paraventricular nucleus (PVN) is among the various brain sites that receive hypocretin inputs (12). The PVN harbors corticotropin-releasing hormone (CRH) neurons that govern the pituitary-adrenal (PA) axis, and intracerebroventricular injection of hypocretin-1 enhances the activity of this neuroendocrine ensemble in rats (13). Thus, we hypothesized that ACTH and cortisol secretion would be blunted in hypocretin-deficient narcoleptic humans.
Early reports suggested that erroneous timing by the biological clock underlies the disrupted circadian organization of sleep and wakefulness in narcoleptic patients (14, 15). However, more recent data do not support this concept (16) or indicate a subtle circadian phase shift (17). Moreover, we recently showed that the circadian fluctuation of plasma leptin concentrations is abrogated in narcoleptic patients (18), which could be due to disruption of pacemaker function (19). As the activity of the biological clock is accurately reflected by the diurnal fluctuations of PA hormones in plasma (20, 21), we further aimed to establish whether the circadian rhythmicity of plasma ACTH and cortisol concentration time series is perturbed in narcolepsy.
To test our hypotheses, we deconvolved 24-h plasma ACTH and cortisol concentration profiles in patients with narcolepsy and in healthy, age-, sex-, and body weight-matched controls. Circadian rhythmicity of the plasma PA hormone time series was analyzed by cosinor analysis.
Subjects and Methods
Subjects
Seven male narcoleptic patients and seven healthy controls, matched for age, sex, and body mass index, were enrolled in the study. The diagnosis of narcolepsy with cataplexy was made on clinical grounds by a physician experienced with narcolepsy (G.J.L.). Multiple sleep latency testing showed results typical for narcolepsy (22). All patients were HLA-DR2/DQB1*0602 positive and lacked hypocretin-1 in their cerebrospinal fluid [measurements as previously described (4)]. All subjects were free of medication or (in three patients) discontinued medication for at least 2 wk before study. Subjects were eligible for the study after exclusion of hypertension (defined as a repeated blood pressure measurement of systolic >160 mm Hg or diastolic >90 mm Hg), any known (history of) pituitary disease, recent weight change (>3 kg weight gain or loss within the last 3 months), and fasting blood glucose greater than 7.0 mmol/liter. Written informed consent was obtained from all subjects. The study was approved by the ethics committee of Leiden University Medical Center.
Clinical protocol
Subjects were admitted to the Clinical Research Center on the morning of the 24-h study occasion. An iv cannula was inserted in an antecubital vein 1 h before the start of blood sampling. Blood samples were collected with S-monovetten (Sarstedt, Etten-Leur, The Netherlands) from a three-way stopcock that was attached to a 0.9% NaCl and heparin (1 U/ml) infusion (500 ml/24 h) to keep the cannula from clotting. Sampling was performed through a long line to prevent sleep disturbance by investigative manipulations. Blood was collected in EDTA tubes at 10-min intervals for 24 h. The tubes were immediately put on ice and centrifuged at 4 C for 20 min, and plasma was frozen at -20 C until assay. Three standardized meals were served at 0900, 1300, and 1800 h (Nutridrink, 1.5 kcal/ml, 1500–1800 kcal/d; macronutrient composition per 100 ml: protein, 5 g; fat, 6.5 g; carbohydrate, 17.9 g; Nutricia, Zoetermeer, The Netherlands). Subjects remained sedentary except for bathroom visits; at 2300 h lights were switched off.
Assays
Plasma ACTH concentrations were determined by RIA (Nichol Institute Diagnostics, San Juan Capistrano, CA); the detection limit of the assay was 2 ng/liter. The interassay variation was 2.8–7.5%. Plasma cortisol was also measured by RIA (Medgenix, Fleurus, Belgium). The detection limit of the cortisol assay was 20 nmol/liter, and the interassay variation was 2–4%. All samples of one 24-h profile were processed in the same assay procedure.
Sleep recording and analysis
Polygraphic sleep recordings were made using a Porti-5 ambulant electroencephalogram recording system (Twente Medical Systems International, Enschede, NL) and were scored using EEG-2100 review software (Nihon Kohden Corp., Tokyo, Japan). An experienced technician, blinded for the subject under study, visually scored the sleep recordings at 30-sec epochs using standardized criteria (23). Sleep onset was identified as the first epoch of stage II, III, IV, or rapid eye movement sleep, provided that the subsequent interval was not scored as awake. The sleep period was defined as the time between nocturnal sleep onset and the moment of final awakening in the morning. Sleep efficiency was calculated as the total sleep time (sleep period minus duration of intrasleep wake periods), expressed as a percentage of the sleep period.
Calculations and statistics
Deconvolution analysis. Multiple parameter deconvolution was used to estimate various specific measures of secretion and the plasma disappearance rates of ACTH and cortisol, considering all plasma hormone concentrations and their dose-dependent intrasample variance simultaneously (24, 25). The following parameters were estimated in each ACTH and cortisol series: number of secretory bursts, secretory burst half-duration (duration at half-maximal amplitude), burst amplitude, mean mass secreted per burst, hormone half-life, pulsatile secretion rate, basal secretion rate, and total secretion rate per 24 h.
Cosinor analysis. Twenty-four-hour variation in ACTH and cortisol plasma concentrations was analyzed using cosinor analysis, an algorithm that fits to the sum of a cosine wave, and a straight line using repeated nonlinear regression analysis (26). This analysis defines an acrophase, which is the clock time during 24 h at which the concentration of the investigated hormone is maximal; a mesor, which is the average value about which the diurnal rhythm oscillates; and an amplitude, which is half the absolute difference between the nadir and peak value of the 24-h concentration series.
Approximate entropy (ApEn). ApEn is a model-independent statistic used to quantify the orderliness of a time series, in which is measured, within a predefined tolerance r given a pattern of window length m, the likelihood of a similar pattern in the next incremental window (27, 28). Greater regularity yields smaller ApEn values, whereas greater independence among sequential values of a time series yields larger ApEn values. ApEn parameters of m = 1 and r = 20% of the intraseries SD were used, the statistical suitability of which has been established (27). Cross-ApEn was used to quantify the synchrony of the joint ACTH-cortisol time series (28, 29), which reflects hormonal interactions within neuroendocrine ensembles. Larger cross-ApEn values indicate less joint signal synchrony.
Statistical analysis. Results are expressed as the mean ± SEM unless stated otherwise. An unpaired, one-tailed t test (evaluating the a priori hypothesis of reduced PA activity in narcoleptics) was used to compare groups with regard to ACTH and cortisol plasma concentrations and basal/pulsatile secretion rates. Two-tailed tests were used to compare all other variables (i.e. burst area, amplitude, half-duration, and half-life). Differences were considered significant for P < 0.05. Statistical analysis was performed using SPSS for Windows (release 9.0, SPSS, Inc., Chicago, IL).
Results
Subjects
Narcoleptic patients (N) and controls (C) did not differ with respect to gender (male), age [46.1 ± 15.9 (N) vs. 46.9 ± 16.1 (C) yr], body mass index [28.3 ± 2.6 (N) vs. 28.4 ± 2.2 (C) kg/m2], or waist to hip circumference ratio ([0.96 ± 0.09 (N) vs. 0.97 ± 0.10 (C)].
Sleep analysis
The sleep period did not differ between patients (405 ± 30 min) and controls (383 ± 41 min; P = 0.67). Both groups had a relatively low sleep efficiency (N, 64 ± 6.4%; C, 72 ± 4.9%; P = 0.41), resulting in 263 ± 32 min of nocturnal sleep in the patients and 274 ± 36 min in the controls (P = 0.82).
Patients, however, had a 2-fold increased relative amount of nocturnal stage 1 sleep [21.8 ± 2.5% (N) vs. 14.5 ± 1.7% (C); P = 0.04], as an indication of more fragmented nocturnal sleep. The amount of slow wave sleep during the night did not differ between patients and controls [25.1 ± 9.3 min (N) vs. 22.4 ± 6.9 min (C); P = 0.82]. None of the control subjects slept during the daytime. In contrast, all narcoleptics took at least two daytime naps (average, 3.3 naps), and an average of 14.8 ± 6.1 min of these daytime sleep episodes were spent in slow wave sleep.
Plasma hormone concentrations
Figure 1
shows the average 24-h plasma ACTH (upper panel) and cortisol (lower panel) concentration vs. time series of all seven narcoleptic subjects and their matched controls. Plasma hormone concentrations were not significantly lower in narcolepsy patients, although the 24-h mean plasma concentrations of ACTH tended to be reduced in narcoleptic patients compared with controls [-24%; ACTH, 18.6 ± 2.6 (N) vs. 24.6 ± 2.7 (C) ng/liter], which was less apparent for cortisol [-12%; 170 ± 13 (N) vs. 194 ± 9 (C) nmol/liter]. The diurnal rhythms of plasma ACTH and cortisol concentrations were apparently normal in narcoleptics, with an early morning rise of hormone levels.
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Examples of plots of 24-h plasma hormone profiles with curves of raw data fitted by deconvolution and accompanying estimated secretory profiles are shown in Fig. 2. The deconvolution-derived parameter estimates for ACTH and cortisol are shown in Table 1. Basal and total secretion of ACTH were decreased in narcoleptic subjects, whereas all features of pulsatile ACTH secretion did not differ between narcoleptics and controls (i.e. the number, duration and height of bursts, and mass of ACTH secreted per burst). In contrast, basal, pulsatile, and total cortisol secretion values were similar in narcoleptic and control subjects. Also, estimated plasma half-lives of both ACTH and cortisol were not different between groups.
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shows deconvolution-derived data of pulsatile PA hormone secretion in distinct circadian timeframes. No significant differences between patients and controls were observed in these separate timeframes.
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The results of the cosinor analysis of plasma hormone time series are listed in Table 3. The diurnal rhythm of plasma ACTH concentrations could mathematically be characterized by a cosine function in narcoleptics and controls. Moreover, the acrophase of the cosine fit occurred at similar clock times in the early morning (Table 3). Also, the plasma cortisol times series could be fitted by a cosine function in both groups, where the acrophases occurred approximately 1 h later than those of ACTH.
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The results of the ApEn analysis are given in Table 4. The ApEn estimate of plasma ACTH time series was higher in narcoleptics than in controls, reflecting a less orderly corticotropin release in patients. The cross-ApEn value of the ACTH/cortisol ensemble was also elevated in narcoleptic subjects [1.26 ± 0.07 (N) vs. 1.07 ± 0.04 (C); P = 0.037], which suggests that the joint signal synchrony of the plasma PA hormone time series is reduced in these patients. Cortisol ApEn scores were similar in patients and controls.
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This paper presents the first detailed description of the secretory dynamics of the PA ensemble in hypocretin-deficient narcoleptic humans. We primarily hypothesized that hypocretin deficiency would blunt pituitary ACTH release and thereby dampen the activity of the PA axis. To test this hypothesis, we deconvolved 24-h ACTH and cortisol plasma concentration time series in patients with narcolepsy and in healthy controls. In addition, to unveil the circadian time-keeping function of the master pacemaker in narcolepsy, we mapped the timing of peaks and troughs of ACTH and cortisol in plasma in these subjects. Total pituitary ACTH release was diminished in narcoleptics, which was fully attributable to a considerable (60%) reduction in basal secretion, whereas pulsatile ACTH release did not differ from that in controls. Moreover, the ACTH release process appeared less regular in narcoleptics, as indicated by elevated ApEn estimates of the plasma ACTH concentration time series. In contrast, cortisol secretion (basal and pulsatile) and plasma cortisol levels were not different between groups. The apparent discrepancy between ACTH and cortisol secretion may indicate a disruption of the secretory dynamics of the ACTH/cortisol ensemble in narcolepsy. Indeed, the joint plasma ACTH and cortisol concentration time series was less synchronous in narcoleptic patients, which is in line with this postulate. The circadian timing of ACTH and cortisol concentration peaks and troughs was similar in both groups, which suggests that the time-keeping function of the biological clock is intact in narcolepsy.
These data suggest that hypocretins are involved in the regulation of basal, but not pulsatile, pituitary ACTH release in humans. ACTH secretion is primarily controlled via feedforward drive by CRH and arginine vasopressin (AVP) and feedback restraint by cortisol. CRH and AVP are themselves regulated by a host of neurotransmitter pathways (30). Hypocretin neurons project to CRH- and AVP-producing cells in the parvocellular part of the PVN (9), and intracerebroventricular injection of hypocretin-1 acutely stimulates the PA axis in rats (31) through activation of both CRH and AVP neurons (32). Thus, hypocretin deficiency might impact pituitary ACTH release through diminution of hypothalamic CRH tonus. The compromised orderliness of the plasma ACTH time series that we observed in narcoleptics is in keeping with this postulate. Diminution of CRH feedforward inputs into the PA ensemble could elevate ApEn estimates of ACTH secretory process regularity in humans (33). CRH administration stimulates arousal in humans as well as in experimental animals (34, 35, 36), whereas CRH deficiency in rats increases total sleeping time (35). Thus, a putative reduction of central CRH tonus induced by hypocretin deficiency could simultaneously contribute to the sleep phenotype and the reduction of pituitary ACTH release in narcoleptic patients. In this context, it is interesting to note that it has been suggested that idiopathic hypersomnia, a disorder characterized by a similar sleep phenotype, may also be associated with central CRH deficiency (37).
Despite the fact that the circadian distribution of sleep and wakefulness was severely disturbed in narcoleptics, the timing and amplitude of the acrophase of ACTH and cortisol in plasma were similar in patients and controls. These observations apparently contradict the idea that at least part of the clinical syndrome of narcolepsy is explained by anomalous circadian time-keeping by the biological clock (14). The master clock is considered to be the main force driving circadian fluctuations of PA axis activity (21). Thus, our data suggest that inputs from the master pacemaker into the PVN are normally timed in narcolepsy and that hypocretin neural circuits do not significantly contribute to the neuroendocrine link between the suprachiasmatic nuclei, the seat of the master pacemaker in mammals (including humans), and the PA axis. This inference is in line with previous observations indicating normal circadian distribution of body temperature fluctuations in narcoleptic humans (16), which is considered to be another reliable measure of the endogenous time-keeping function of the biological clock (38). To reconcile these observations, it has been proposed that inputs from the suprachiasmatic nuclei into hypocretin neurons drive clock-dependent alertness in healthy humans, and that destruction of these neurons therefore abrogates the impact of an intact master pacemaker on the circadian sleep-wakefulness cycle in narcoleptics (10).
It is uncertain whether the alterations in PA axis activity in narcoleptic patients we describe here are of clinical relevance. Main target tissue (adrenal) function seems largely unaffected by the changes in pituitary ACTH release. However, it remains to be established to what extent the apparent alterations impact PA activity in case the system is acutely appealed to (i.e. physical or mental stress or fasting). Subtle hypoactivity of the hypothalamic-PA axis has been proposed to increase the susceptibility to autoimmune disease (39, 40). Although the pathogenesis of narcolepsy itself is thought to be of an autoimmune nature, associations between narcolepsy and other immune disorders have not been found (41). In view of these considerations, the present study primarily provides novel insight into the putative role of hypocretins in neuroregulation of the PA ensemble in humans. These data are the first to suggest that endogenous hypocretins modulate PA axis activity in humans, whereas currently available information on the role of hypocretins in the regulation of ACTH secretion concerns the impact of exogenous hypocretin administration in rodents.
In conclusion, the present study suggests that hypocretin deficiency blunts basal ACTH release by the pituitary and slightly resets the dynamics of the ACTH/cortisol ensemble. Furthermore, the data indicate that the master pacemaker adequately times circadian fluctuations of PA activity in narcoleptic humans, which suggests that the endogenous clock ticks on time in these patients.
Acknowledgments
We gratefully acknowledge the efforts of E. J. M. Ladan-Eygenraam and E. C. Sierat-van der Steen for technical support during the study, and M. van Dijk-Besling and H. G. Haasnoot-van der Bent for performing the ACTH and cortisol concentration assays.
Footnotes
Abbreviations: ApEn, Approximate entropy; AVP, arginine vasopressin; C, controls; CRH, corticotropin-releasing hormone; N, narcoleptic males; PA, pituitary-adrenal; PVN, paraventricular nucleus.
Received April 24, 2002.
Accepted August 14, 2002.
References
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