Pulsatile LH release is diminished, whereas FSH secretion is normal, in hypocretin-deficient narcoleptic men

S. W. Kok,1 F. Roelfsema,2 S. Overeem,3 G. J. Lammers,3 M. Frölich,1 A. E. Meinders,1 and H. Pijl1

Departments of 1General Internal Medicine, 2Endocrinology, and 3Neurology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands

Submitted 9 February 2004 ; accepted in final form 27 May 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypocretin (orexin) peptides are involved in the regulation of energy balance and pituitary hormone release. Narcolepsy is a sleep disorder characterized by disruption of hypocretin neurotransmission. Pituitary LH secretion is diminished in hypocretin-deficient animal models, and intracerebroventricular administration of hypocretin-1 activates the hypothalamo-pituitary-gonadal axis in rats. We evaluated whether hypocretin deficiency affects gonadotropin release in humans. To this end, we deconvolved 24-h serum concentrations of LH and FSH in seven hypocretin-deficient narcoleptic males (N) and seven controls (C) matched for age, body mass index, and sex. Basal plasma concentrations of testosterone, estradiol, and sex hormone-binding globulin were similar in both groups. Mean 24-h LH concentration was significantly lower in narcolepsy patients [3.0 ± 0.4 (N) vs. 4.2 ± 0.3 (C) U/l, P = 0.01], which was primarily due to a reduction of pulsatile LH secretion [23.5 ± 1.6 (N) vs. 34.3 ± 4.9 (C) U·l–1·24 h–1, P = 0.02]. The orderliness of LH and FSH secretion, quantitated by the approximate entropy statistic, was greater in patients than in controls. In contrast, all other features of FSH release were similar in narcoleptic and control groups. Also, LH and FSH secretions in response to intravenous administration of 100 µg of GnRH were similar in patients and controls. These data indicate that endogenous hypocretins are involved in the regulation of the hypothalamo-pituitary-gonadal axis activity in humans. In particular, reduced LH release in the face of normal pituitary responsivity to GnRH stimulation in narcoleptic men suggests that hypocretins promote endogenous GnRH secretion.

orexin; luteinizing hormone; follicle-stimulating hormone; testosterone; estradiol; circadian rhythm; deconvolution analysis


NARCOLEPSY IS CHARACTERIZED by excessive daytime sleepiness, cataplexy, hypnagogic hallucinations, and sleep paralysis (30). It is caused by the progressive loss of hypocretin neurons in the brain (28, 31). Hypocretin neuronal cell bodies are located exclusively in the lateral hypothalamus/perifornical area (41), but axonal projections throughout the central nervous system predict diverse biological actions (7). Indeed, hypocretin peptides 1 and 2 (also called orexin A and B) appear to affect feeding behavior and energy expenditure, arousal, autonomic outflow, and a variety of neuroendocrine ensembles (9, 10, 43, 49). Therefore, apart from its well-recognized neurological manifestations, the clinical syndrome of hypocretin deficiency in humans may encompass a broad spectrum of behavioral, endocrine, and metabolic anomalies. In keeping with this postulate, we recently identified aberrations of the pituitary-adrenal and somatotropic ensembles and circulating leptin concentrations in hypocretin-deficient narcoleptic men (15, 17, 29).

The septal preoptic and arcuate nuclei are among the many brain sites receiving hypocretin inputs (7, 26, 32, 46). These nuclei are involved in the control of the hypothalamo-pituitary-gonadal (HPG) axis (50). Furthermore, intracerebroventricular administration of hypocretins acutely induces LH release in ovarian-steroid-primed ovariectomized rats (37). Also, hypocretin-1 promotes gonadotropin-releasing hormone (GnRH) release in hypothalamic explants from male rats (40), and intracerebroventricular administration of anti-hypocretin antibody completely abolishes the preovulatory LH surge in intact female rats (14). Thus the available data suggest that endogenous hypocretin peptides stimulate LH release in rodents. It is currently unknown whether these peptides exert analogous effects in humans. As far as we are aware, the impact of hypocretins on FSH secretion has not been investigated to date.

The present study was conducted to evaluate the role of endogenous hypocretins in the regulation of gonadotropin release in the human. We specifically hypothesized that hypocretin deficiency in narcoleptic men would blunt spontaneous pituitary LH release.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Subjects

We included seven male patients from the outpatient clinic of the Department of Neurology, Leiden University. The diagnosis of narcolepsy with cataplexy was made on clinical grounds by a physician experienced with narcolepsy (G. J. Lammers). Also, the result of a multiple sleep latency test was typical for narcolepsy in all patients (23). In addition, all patients were HLA-DR2/DQB1*0602 positive (22) and lacked hypocretin-1 in their cerebrospinal fluid [measurements as previously described (28)]. All subjects were free of medication or (3 patients) had discontinued medication for ≥2 wk before the study. Two of these three narcoleptic subjects had used psychostimulants (methylfenidate and modafinil) and one a tricyclic antidepressant (clomipramine).

Weight and height of the subjects were measured, as well as waist and hip circumference. The waist-to-hip ratio (WHR) was used as a relative measure of abdominal fat mass. Total body fat mass was determined by dual-energy X-ray absorptiometry (Hologic QDR4500, Waltham, MA). Seven male control subjects were recruited through advertisements in local newspapers. Controls were matched for age, body mass index, total fat mass, and WHR, since narcoleptics are (moderately) obese (16).

Subjects were eligible for the study after exclusion of hypertension (defined as a repeated blood pressure measurement of systolic >160 mmHg or diastolic >90 mmHg), any known (history of) pituitary disease, recent body weight change (>3 kg weight gain or loss within the previous 3 mo), and fasting blood glucose >7.0 mmol/l. Written informed consent was obtained from all subjects. The study was approved by the ethics committee of the Leiden University Medical Center.

Clinical Protocol

Subjects were admitted to the Clinical Research Center in the morning. Drawing of blood was performed under standardized dark-light and alimentary conditions. During the 24-h study occasion, three standardized meals were served, at 0900, 1300, and 1800 (Nutridrink 1.5 kcal/ml; 1,500–1,800 kcal/day; macronutrient composition per 100 ml: 5 g protein, 6.5 g fat, 17.9 g carbohydrate; Nutricia, Zoetermeer, Netherlands). Subjects remained sedentary throughout the study except for bathroom visits. Lights were switched off at 2300.

Upon arrival, an intravenous cannula was inserted in an antecubital vein 1 h before the start of blood sampling, which was performed at 10-min intervals for 24 h through a long line to prevent sleep disturbance. Samples were allowed to clot and were centrifuged at 4°C for 20 min, and serum was frozen at –20°C until assay. Sleep registration, using a portable electroencephalogram system (Porti; Twente Medical Systems, Enschede, Netherlands), confirmed abnormally distributed 24-h total sleep as well as REM sleep in narcoleptic subjects and normal distribution of sleep stages in control subjects.

On a separate occasion, blood was drawn at 0800 after an overnight fast for measurement of LH, FSH, testosterone, estradiol, and sex hormone-binding globulin (SHBG) concentrations. Subsequently, a GnRH test was performed (42), whereby 100 µg of gonadoreline (Aventis Pharma, Hoevelaken, Netherlands) were given as an intravenous bolus, and serum LH concentrations were measured every 10 min for 90 min thereafter.

Assays

Serum LH and FSH concentrations were measured with time-resolved immunofluorometric assays (Wallac, Turku, Finland), with coefficients of variation (CV) of 3.9–6.8% in the concentration range of 5.5–147 U/l for LH and 3.7–5.3% in the concentration range of 5.1–41.3 U/l for FSH. Testosterone was measured with a coated-tube radioimmunoassay (RIA; Diagnostic Products, Los Angeles, CA) with a CV of 10.6–18.6% in the concentration range of 2.7–44.3 nmol/l. SHBG was measured with an immunoradiometric assay (Spectria, Espoo, Finland) and serum estradiol with an RIA (Diagnostic Systems Laboratory, Webster, TX). All samples of each gonadotropin 24-h profile were processed in the same assay procedure. In addition, the leptin concentration was measured in every 20-min sample by RIA (Linco Research, St. Charles, MO). The detection limit of the assay was 0.5 µg/l, and the CV was 3.4–8.3% in the concentration range of 4.9–25.6 µg/l. Details on leptin secretion in narcolepsy were previously published, but here we report only the 24-h mean leptin concentration in relation to LH parameters and testosterone (15).

Calculations and Statistics

Deconvolution analysis. Deconvolution analysis (47) was used to estimate the four secretory and clearance measures of interest: 1) the number and locations of secretory events; 2) the amplitudes of secretory bursts; 3) the durations of randomly dispersed LH and FSH secretory bursts; and 4) the endogenous single-component, subject-specific plasma half-lives of LH and FSH. It was assumed that the gonadotropin distribution volumes and LH and FSH half-lives were time and concentration invariant. The following parameters were calculated: half-duration of secretory bursts (duration of the secretory burst at half-maximal amplitude), hormone half-life, burst frequency, amplitude of the secretory burst (maximal secretory rate attained within a burst), mass secreted per burst, basal secretion rate (only for LH), pulsatile secretion rate (product of burst frequency and mean burst mass) and total secretion (sum of basal and pulsatile). Based on recent validation studies in men, deconvolution analysis was carried out at 95% joint statistical confidence intervals for all calculated LH and FSH amplitudes (13, 25).

Approximate entropy. The univariate approximate entropy (ApEn) statistic was developed to quantify the degree of irregularity, or disorderliness, of a time series (33). Technically, ApEn quantifies the summed logarithmic likelihood that templates (of length m) of patterns in the data that are similar (within r) remain similar (within the same tolerance r) on next incremental comparison and has been formally defined elsewhere (34). The ApEn calculation provides a single nonnegative number, which is an ensemble estimate of relative process randomness, wherein larger ApEn values denote greater irregularity. Cross-ApEn (X-ApEn) quantifies joint pattern synchrony between two separate but parallel time series after standardization (z-score transformation) (36, 38). In the present analysis, we calculated X-ApEn assuming r = 20% of the SD of the individual time-series and m = 1; hence, use of the designation X-ApEn (1, 20%). This parameter set affords sensitive, valid, and statistically well-replicated ApEn and X-ApEn metrics for assessing hormone time series of this length (35, 36). Both ApEn and X-ApEn results are reported as the ratio of the absolute value to that of the mean of 1,000 randomly shuffled data series, values approaching 1.0 denoting complete randomness.

Statistical analysis. Results are expressed as means ± SE unless stated otherwise. Student's one-tailed t-test was used to evaluate the a priori hypothesis that LH secretion is reduced in narcoleptic men. All other comparisons were statistically evaluated by two-tailed Student's t-test or the Kolmogorov-Smirnov test for two samples, where appropriate. Statistical analysis was performed using Systat (release 10.0; Systat, Richmond, CA).


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Subjects

Narcoleptic patients (N) and controls (C) had a comparable age [46.1 ± 15.9 (N) vs. 46.9 ± 16.1 (C) yr] and body mass index [28.3 ± 2.6 (N) vs. 28.4 ± 2.2 (C) kg/m2].

Serum Hormone Concentrations

Gonadal steroid hormone concentrations were similar in narcoleptics and controls [testosterone, 23.3 ± 1.8 (N) vs. 19.9 ± 0.8 (C) nmol/l, P = 0.11; estradiol, 73.9 ± 5.8 (N) vs. 78.3 ± 8.2 (C) pmol/l, P = 0.18; SHBG, 28.5 ± 2.4 (N) vs. 27.6 ± 2.3 (C) nmol/l, P = 0.79]. The mean plasma LH concentration was significantly lower in narcoleptic patients [3.0 ± 0.4 (N) vs. 4.2 ± 0.3 (C) U/l, P = 0.01], whereas the FSH concentration was similar in both groups [3.7 ± 0.9 (N) vs. 4.0 ± 0.4 (C), P = 0.74]. Fig. 1 shows the 24-h serum LH and FSH concentration vs. time in narcoleptic subjects and controls.



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Fig. 1. A: serum LH concentrations in 7 narcoleptic subjects and 7 controls. Blood samples were drawn every 10 min for 24 h. B: FSH concentrations. Data are shown as means and SE. Lights were off from 2300 until 0800. Meals were served at 0900, 1300, and 1800.

 
LH and FSH Secretion

The deconvolution-derived parameter estimates of LH and FSH secretion and elimination are given in Tables 1 and 2, respectively. Pulsatile secretion of LH was diminished in narcoleptic subjects [23.5 ± 1.6 (N) vs. 34.3 ± 4.9 (C) U·l–1·24 h–1, P = 0.02] as a result of the concerted effects of reduced burst frequency and burst mass. Total LH secretion was also less in narcoleptic patients (P = 0.04). In contrast, all features of FSH release were similar in narcoleptics and controls. Also, plasma LH and FSH half-lives were not different in both groups. Representative 24-h LH- and FSH-secretory profiles calculated by deconvolution analysis with the corresponding curves of serum concentration data are shown in Figs. 2 and 3, respectively.


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Table 1. Deconvolution-derived features of LH secretion

 

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Table 2. Deconvolution-derived features of FSH secretion

 


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Fig. 2. Representative 24-h LH-secretory profiles (C and D) with their corresponding plasma concentration series and intrasample dose-dependent standard deviations and fitted curves calculated by deconvolution (A and B) in a narcoleptic patient (A and C) and his matched control (B and D). Note the decreased pulsatile secretion in the narcoleptic patient. Time is depicted in minutes elapsed after the 1st sample was taken.

 


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Fig. 3. Representative 24-h FSH-secretory profiles (C and D) with their corresponding plasma concentration series and intrasample dose-dependent standard deviations and fitted curves calculated by deconvolution (A and B) in a narcoleptic patient (A and C) and his matched control (B and D). Time is depicted in minutes elapsed after the first sample was taken.

 
GnRH Test

Both basal serum gonadotropin levels [LH, 3.2 ± 0.4 (N) vs. 4.4 ± 0.4 (C) U/l, P = 0.18; FSH, 4.8 ± 1.1 (N) vs. 3.8 ± 5.9 (C) U/l, P = 0.43] and their maximal concentrations in response to GnRH stimulation [LH, 16.6 ± 2.8 (N) vs. 22.8 ± 4.9 (C) U/l, P = 0.27; FSH, 9.3 ± 3.0 (N) vs. 6.8 ± 0.9 (C) U/l, P = 0.43] were similar in both groups. The individual LH and FSH responses are displayed in Fig. 4.



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Fig. 4. Individual LH and TSH increases after iv injection of 100 µg of LHRH in 7 narcoleptic patients ({bullet}) and their matched controls ({blacktriangleup}). Mean increases of LH and FSH, respectively, were similar in the studied groups.

 
Leptin and LH Relationship

The mean 24-h leptin concentration in patients was 5.9 ± 1.1 µg/l, and in controls 11.2 ± 1.3 µg/l (P = 0.01). No significant correlations were present between 24-h LH secretion and mean 24-h leptin concentration either in the combined groups (R = 0.07) or separately (patients: R = 0.20; controls: R = 0.39).

Leptin and Testosterone

Between the 24-h leptin concentration and serum testosterone a significant inverse linear correlation was found, as displayed in Fig. 5.



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Fig. 5. Linear relationship between mean 24-h leptin concentration (20-min sampling) and serum testosterone concentration. {circ}, Patients; {triangleup}, controls. Note the inverse relationship between these measures.

 
ApEn

ApEn ratio values for LH were lower in patients compared with controls, denoting a more regular secretion (N 0.653 ± 0.035; C 0.718 ± 0.014, P = 0.046). The individual data are displayed in Fig. 6. Similarly, the ApEn ratio of FSH was also smaller in patients (0.837 ± 0.032 vs. 0.927 ± 0.020, P = 0.03). The X-ApEn ratio between the LH and FSH concentration series was lower in patients than in controls, indicating increased synchrony between both hormones in patients compared with controls (N, 0.725 ± 0.017; C, 0.786 ± 0.015, P = 0.02). X-ApEn ratio between the leptin concentration series (20-min data) and the LH series (with only the 20-min data) did not differ between the groups (N, 0.889 ± 0.021; C, 0.911 ± 0.026, P = 0.53).



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Fig. 6. Aproximate entropy (ApEn) ratios of LH and FSH secretion and cross-ApEn ratio between LH and FSH series. Lower values denote more orderliness of secretion.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study shows that the average 24-h plasma LH concentration is reduced in hypocretin- deficient narcoleptic men, whereas gonadal steroid hormone levels are normal. The reduction of the plasma LH concentration in narcoleptics is accounted for by a diminution of pulsatile LH release (by 30% compared with healthy controls), which cannot be attributed to insensitivity of the pituitary gonadotrophs to GnRH stimulation. LH burst frequency and burst mass were reduced in narcoleptic men. In contrast, plasma FSH concentrations and various features of FSH secretion were similar in narcoleptics and controls.

These data suggest that endogenous hypocretin peptides stimulate pulsatile LH release in men. In particular, the fact that spontaneous LH secretion is blunted, whereas pituitary responsiveness to GnRH stimulation is intact in narcoleptic subjects, suggests that hypocretin deficiency primarily inhibits hypothalamic GnRH release. This inference agrees with studies in male rats showing that hypocretin-1 promotes GnRH release from hypothalamic explants (40). Moreover, hypocretin inputs are abundant in the preoptic area that contains many GnRH neurons (50), which coexpress hypocretin-1 receptors and are in direct contact with hypocretin fibers (3). These physiological and anatomical observations obviously support the notion that hypocretin peptides are involved in the regulation of GnRH release. The fact that FSH secretion is not diminished (whereas LH release is) in narcoleptic subjects may imply that hypocretins specifically modulate GnRH burst frequency, as LH secretion is particularly enhanced by high-frequency bursts, whereas low-frequency bursts predominantly promote FSH release (20).

ApEn quantitates the relative orderliness or reproducibility of subordinate (nonpulsatile) secretory patterns in neurohormone time series and reflects feedforward and feedback adjustments driven by (patho)physiological changes in interglandular communication. In view of the unchanged testosterone feedback signal in the patients, the increased LH and FSH regularity and increased synchrony of both hormones might reflect a diminished GnRH input signal with unchanged pituitary responsiveness toward this neurohormone (48).

Alternatively, hypoleptinemia may blunt LH release in narcoleptic patients. We have previously shown (15) that the plasma leptin concentration is considerably reduced in narcoleptic men. Leptin has emerged as an important modulator of the HPG axis (5). Indeed, leptin-deficient ob/ob mice are hypogonadotropic and sterile, and recombinant leptin administration rescues these defects in ob/ob males (24) and females (6). Moreover, the circadian rhythm of plasma leptin levels is synchronous with that of LH concentrations (18), and the reduction of LH secretion induced by fasting in the rat and in humans can be reversed by leptin administration (1, 4, 8). These data indicate that leptin stimulates LH release and that genetic and/or physiological alterations of circulating leptin levels can impact pituitary gonadotropin release. In this context, it is conceivable that the reduction of the plasma leptin concentration in narcoleptics (15) is involved in the diminution of pituitary LH release in these patients, but it should be mentioned that we could not demonstrate a (linear) correlation between mean circulating leptin concentration and LH production.

Notwithstanding the diminished LH production, testosterone concentration was unchanged in patients. Recently, it has become clear that leptin is involved in the (local) regulation of testosterone synthesis and secretion in the male rat gonad (44). Direct evidence for such action in humans is not available, but an inverse relation between serum testosterone and leptin has been demonstrated (19). Therefore, the inhibitory effect of leptin on testosterone (and adrenal hormone) secretion might be relevant in narcoleptic patients, because narcoleptics exhibit a 50% reduction in circulating leptin concentration, which may unleash gonadal testosterone secretion to compensate for diminished LH drive (15). The finding of an inverse relationship between testosterone and leptin in our subjects reinforces this view.

Prepro-orexin mRNA and the orexin receptors are expressed in various endocrine and nonendocrine tissues (11), and, for instance, orexins can modulate directly human and porcine adrenal stereoidogenic function (21, 27). Relevant for the present investigation was the recent finding of the mRNA expression of the OX1 but not of the OX2 receptor in the male rat gonad (2). Activation of the OX1 receptor stimulates testosterone secretion, both in vivo and in vitro, suggesting a physiological (modulating) role of the orexin system in concert with leptin and ghrelin at the testicular level (45). The significance of these findings for human physiology needs to be explored. However, OX1 and OX2 receptor expression was demonstrated in the male human reproductive system, including the testis, suggesting that orexins can directly impact gonadal hormone release in humans as well as in rodents (12).

The impact of the alterations in the HPG ensemble described here on the fertility of narcoleptic men remains to be definitely determined. Some early reports have suggested that narcolepsy is associated with impaired fertility (39), but since then these suggestions have not been confirmed, as far as we are aware. Moreover, it is not our clinical impression that narcoleptic (male) patients are less fertile, and gonadal hormone concentrations in plasma were normal in narcoleptics despite the reduction of LH levels. Thus it seems unlikely that the partial hypogonadotropism observed here has clinical sequelae. Nevertheless, in view of our findings in males, it is warranted to investigate narcoleptic females across the menstrual cycle for putative defects in ovarian function.

In conclusion, hypocretin-deficient narcoleptic men have reduced circulating LH levels, which are brought about by a diminution of pulsatile LH release. In contrast, FSH secretion is normal in these patients. The pituitary sensitivity to GnRH stimulation is normal in narcoleptics, which suggests that a reduction of hypothalamic GnRH release underlies their blunted LH secretion, and in accordance with this view is the increased orderliness of secretion of gonadotropins. Both hypocretin deficiency per se and hypoleptinemia may be involved in changes in the HPG axis in narcoleptic males.


    ACKNOWLEDGMENTS
 
We gratefully acknowledge the skillful assistance of E. J. M. Ladan-Eygenraam and E. C. Sierat-van der Steen during the study occasions.


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. Pijl, Leiden Univ. Medical Center, Dept. of General Internal Medicine (C1-R39), PO Box 9600, 2300 RC Leiden, The Netherlands (E-mail: h.pijl{at}lumc.nl)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ahima RS, Prabakaran D, Mantzoros C, Qu D, Lowell B, Maratos-Flier E, and Flier JS. Role of leptin in the neuroendocrine response to fasting. Nature 382: 250–252, 1996.[CrossRef][ISI][Medline]
  2. Barreirro ML, Pineda R, Navarro VM, Lopez M, Suominen JS, Pinella L, Searis R, Toppara J, Aguilar E, Diéguez C, and Tena-Sempera M. Orexin 1 receptor messenger ribonucleic acid expression and stimulation of testosterone secretion by orexin A in rat testis. Endocrinology 145: 2297–2306, 2004.[Abstract/Free Full Text]
  3. Campbell RE, Grove KL, and Smith MS. Gonadotropin-releasing hormone neurons coexpress orexin 1 receptor immunoreactivity and receive direct contacts by orexin fibers. Endocrinology 144: 1542–1548, 2003.[Abstract/Free Full Text]
  4. han JL, Heist K, DePaoli AM, Veldhuis JD, and Montzoros CS. The role of falling leptin levels in the neuroendocrine and metabolic adaptation to short-term starvation in healthy men. J Clin Invest 111: 1409–1421, 2003.[Abstract/Free Full Text]
  5. Chehab FF. Leptin as a regulator of adipose mass and reproduction. Trends Pharmacol Sci 21: 309–314, 2000.[CrossRef][ISI][Medline]
  6. Chehab FF, Lim ME, and Lu R. Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet 12: 318–320, 1996.[ISI][Medline]
  7. Date Y, Ueta Y, Yamashita H, Yamaguchi H, Matsukura S, Kangawa K, Sakurai T, Yanagisawa M, and Nakazato M. Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems. Proc Natl Acad Sci USA 96: 748–753, 1999.[Abstract/Free Full Text]
  8. Gonzalez LC, Pinilla L, Tena-Sempere M, and Aguilar E. Leptin (116–130) stimulates prolactin and luteinizing hormone secretion in fasted adult male rats. Neuroendocrinology 70: 213–220, 1999.[CrossRef][ISI][Medline]
  9. Hagan JJ, Leslie RA, Patel S, Evans ML, Wattam TA, Holmes S, Benham CD, Taylor SG, Routledge C, Hemmati P, Munton RP, Ashmeade TE, Shah AS, Hatcher JP, Hatcher PD, Jones DN, Smith MI, Piper DC, Hunter AJ, Porter RA, and Upton N. Orexin A activates locus coeruleus cell firing and increases arousal in the rat. Proc Natl Acad Sci USA 96: 10911–10916. 1999.[Abstract/Free Full Text]
  10. Hara J, Beuckmann CT, Nambu T, Willie JT, Chemelli RM, Sinton CM, Sugiyama F, Yagami K, Goto K, Yanagisawa M, and Sakurai T. Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity. Neuron 30: 345–354, 2001.[CrossRef][ISI][Medline]
  11. Jöhren O, Neidert SJ, Kummer M, Dendorfer A, and Dominiak P. Preproorexin and orexin receptor mRNAs are differentially expressed in peripheral tissues of male and female rats. Endocrinology 142: 3324–3331, 2001.[Abstract/Free Full Text]
  12. Karteris E, Chen J, and Randeva HS. Expression of human prepro-orexin and signalling characteristics of orexin receptors in the male reproductive system. J Clin Endocrinol Metab 89: 1957–1962, 2004.[Abstract/Free Full Text]
  13. Keenan DM and Veldhuis JD. Disruption of the hypothalamic luteinizing hormone pulsing mechanism in aging men. Am J Physiol Regul Integr Comp Physiol 281: R1917–R1924, 2001.[Abstract/Free Full Text]
  14. Kohsaka A, Watanabe H, Kakizaki Y, Suda T, and Schioth HB. A significant participation of orexin-A, a potent orexigenic peptide, in the preovulatory luteinizing hormone and prolactin surges in the rat. Brain Res 898:166–170, 2001.[CrossRef][ISI][Medline]
  15. Kok SW, Meinders AE, Overeem S, Lammers GJ, Roelfsema F, Frölich M, and Pijl H. Reduction of plasma leptin levels and loss of its circadian rhythmicity in hypocretin (orexin) deficient narcoleptic humans. J Clin Endocrinol Metab 87:805–809, 2002.[Abstract/Free Full Text]
  16. Kok SW, Overeem S, Visscher TL, Lammers GJ, Seidell JC, Pijl H, and Meinders AE. Hypocretin deficiency in narcoleptic humans is associated with abdominal obesity. Obes Res 11: 1147–1154, 2003.[Abstract/Free Full Text]
  17. Kok SW, Roelfsema F, Overeem S, Lammers GJ, Strijers RL, Frölich M, Meinders AE, and Pijl H. Dynamics of the pituitary-adrenal ensemble in hypocretin-deficient narcoleptic humans: blunted basal adrenocorticotropin release and evidence for normal time-keeping by the master pacemaker. J Clin Endocrinol Metab 87: 5085–5091, 2002.[Abstract/Free Full Text]
  18. Licinio J, Negrao AB, Mantzoros C, Kaklamani V, Wong ML, Bongiorno PB, Mulla A, Cearnal L, Veldhuis JD, Flier JS, McCann SM, and Gold PW. Synchronicity of frequently sampled, 24-h concentrations of circulating leptin, luteinizing hormone, and estradiol in healthy women. Proc Natl Acad Sci USA 95: 2541–2546, 1998.[Abstract/Free Full Text]
  19. Luukkaa V, Pesonen U, Huhtaniemi I, Lehtonen A, Tilvis R, Tuomiletho J, Koulu M, and Huupponen R. Inverse correlation between serum testosterone and leptin in men. J Clin Endocrinol Metab 83: 3243–3246, 1998.[Abstract/Free Full Text]
  20. Marshall JC, Eagleson CA, and McCartney CR. Hypothalamic dysfunction. Mol Cell Endocrinol 186: 227–230, 2002.[CrossRef][ISI]
  21. Mazzochi G, Malendowicz LK, Gottardo L, Aragona F, and Nussdorfer GG. Orexin A stimulates cortisol secretion from human adrenal cortical cells through activation of adenylate cyclase-dependent signalling cascade. J Clin Endocrinol Metab 86: 778–782, 2001.[Abstract/Free Full Text]
  22. Mignot E. Genetic and familial aspects of narcolepsy. Neurology 50, Suppl 1: S16–S22, 1998.
  23. Mitler MM, Van den Hoed J, Carskadon MA, Richardson G, Park R, Guilleminault C, and Dement WC. REM sleep episodes during the multiple sleep latency test in narcoleptic patients. Electroencephalogr Clin Neurophysiol 46: 479–481, 1979.[CrossRef][ISI][Medline]
  24. Mounzih K, Lu R, and Chehab FF. Leptin treatment rescues the sterility of genetically obese ob/ob males. Endocrinology 138: 1190–1193, 1997.[Abstract/Free Full Text]
  25. Mulligan T, Iranmanesh A, and Veldhuis JD. Pulsatile iv infusion of recombinant human LH in leuprolide-suppressed men unmasks impoverished Leydig-cell secretory responsiveness to midphysiological LH drive in the aging male. J Clin Endocrinol Metab 86: 5547–5553, 2001.[Abstract/Free Full Text]
  26. Nambu T, Sakurai T, Mizukami K, Hosoya Y, Yanagisawa M, and Goto K. Distribution of orexin neurons in the adult rat brain. Brain Res 827: 243–260, 1999.[CrossRef][ISI][Medline]
  27. Nanmoku T, Isobe K, Sakurai T, Yamanaka A, Takekoshi Kawakami Y, Goto K, and Nakai T. Effects of orexin on cultured porcine adrenal medullary and cortex cells. Regul Pept 104: 125–130, 2002.[CrossRef][ISI][Medline]
  28. Nishino S, Ripley B, Overeem S, Lammers GJ, and Mignot E. Hypocretin (orexin) deficiency in human narcolepsy. Lancet 355: 39–40, 2000.[CrossRef][ISI][Medline]
  29. Overeem S, Kok SW, Lammers GJ, Vein AA, Frölich M, Meinders AE, Roelfsema F, and Pijl H. The somatotropic axis in hypocretin-deficient narcoleptic humans: altered circadian distribution of GH secretory events. Am J Physiol Endocrinol Metab 284: E641–E647, 2003.[Abstract/Free Full Text]
  30. Overeem S, Mignot E, van Dijk JG, and Lammers GJ. Narcolepsy: clinical features, new pathophysiologic insights, and future perspectives. J Clin Neurophysiol 18: 78–105, 2001.[ISI][Medline]
  31. Peyron C, Faraco J, Rogers W, Ripley B, Overeem S, Charnay Y, Nevsimalova S, Aldrich M, Reynolds D, Albin R, Li R, Hungs M, Pedrazzoli M, Padigaru M, Kucherlapati M, Fan J, Maki R, Lammers GJ, Bouras C, Kucherlapati R, Nishino S, and Mignot E. A mutation in a case of early onset narcolepsy and a generalized absence of hypocretin peptides in human narcoleptic brains. Nat Med 6: 991–997, 2000.[CrossRef][ISI][Medline]
  32. Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, and Kilduff TS. Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 18: 9996–10015, 1998.[Abstract/Free Full Text]
  33. Pincus SM. Approximate entropy as a measure of system complexity. Proc Natl Acad Sci USA 88: 2297–2301, 1991.[Abstract]
  34. Pincus SM and Goldberger AL. Physiological time-series analysis: what does regularity quantifies? Am J Physiol Heart Circ Physiol 266: H1643–H1656, 1994.[Abstract/Free Full Text]
  35. Pincus SM, Hartman ML, Roelfsema F, Thorner MO, and Veldhuis JD. Hormone pulsatility via coarse and short time sampling. Am J Physiol Endocrinol Metab 277: E948–E957, 1999.[Abstract/Free Full Text]
  36. Pincus SM, Mulligan T, Iranmanesh A, Gheorghiu S, Godschalk M, and Veldhuis JD. Older males secrete luteinizing hormone more irregularly, and jointly more asynchronously, than younger males. Proc Natl Acad Sci USA 93: 14100–14105, 1996.[Abstract/Free Full Text]
  37. Pu S, Jain MR, Kalra PS, and Kalra SP. Orexins, a novel family of hypothalamic neuropeptides, modulate pituitary luteinizing hormone secretion in an ovarian steroid-dependent manner. Regul Pept 78: 133–136, 1998.[CrossRef][ISI][Medline]
  38. Roelfsema F, Pincus SM, and Veldhuis JD. Patients with Cushing's disease secrete adrenocorticotropin and cortisol jointly more asynchronously than healthy subjects. J Clin Endocrinol Metab 83: 688–692, 1998.[Abstract/Free Full Text]
  39. Roth B. Other symptoms of narcolepsy. In: Narcolepsy and Hypersomnia, edited by Roth B and Broughton R. Basel: Karger, 1980, p. 82–93.
  40. Russell SH, Small CJ, Kennedy AR, Stanley SA, Seth A, Murphy KG, Taheri S, Ghatei MA, and Bloom SR. Orexin A interactions in the hypothalamo-pituitary gonadal axis. Endocrinology 142: 5294–5302, 2001.[Abstract/Free Full Text]
  41. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, Williams SC, Richardson JA, Kozlowski GP, Wilson S, Arch JR, Buckingham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terrett JA, Elshourbagy NA, Bergsma DJ, and Yanagisawa M. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92: 573–585, 1998.[ISI][Medline]
  42. Snyder PJ, Reitano JF, and Utiger RD. Serum LH and FSH responses to synthetic gonadotropin-releasing hormone in normal men. J Clin Endocrinol Metab 41: 938–945, 1975.[Abstract]
  43. Taylor MM and Samson WK. The other side of the orexins: endocrine and metabolic actions. Am J Physiol Endocrinol Metab 284: E13–E17, 2003.[Abstract/Free Full Text]
  44. Tena-Sempere M and Barreiro ML. Leptin in male reproduction: the testis paradigm. Mol Cell Endocrinol 188: 9–13, 2002.[CrossRef][ISI][Medline]
  45. Tena-Sempere M, Barreiro ML, Gonzales LC, Gaytan F, Zhang FP, Caminos JE, Pinilla L, Casanueva FF, Dieguez C, and Aguilar E. Novel expression and functional role of ghrelin in rat testis. Endocrinology 143: 717–725, 2002.[Abstract/Free Full Text]
  46. Trivedi P, Yu H, MacNeil DJ, Van der Ploeg LH, and Guan XM. Distribution of orexin receptor mRNA in the rat brain. Febs Lett 438: 71–75, 1998; Corrigenda Febs Lett 442: 122, 1999.[CrossRef][ISI][Medline]
  47. Veldhuis JD, Carlson ML, and Johnson ML. The pituitary gland secretes in bursts: appraising the nature of glandular secretory impulses by simultaneous multiple-parameter deconvolution of plasma hormone concentrations. Proc Natl Acad Sci USA 84: 7686–7690, 1987.[Abstract]
  48. Veldhuis JD, Johnson ML, Veldhuis OL, Straume M, and Pincus SM. Impact of pulsatility on the ensemble orderliness (approximate entropy) of neurohormone secretion. Am J Physiol Regul Integr Comp Physiol 281: R1975–R1985, 2001.[Abstract/Free Full Text]
  49. Willie JT, Chemelli RM, Sinton CM, and Yanagisawa M. To eat or to sleep? Orexin in the regulation of feeding and wakefulness. Annu Rev Neurosci 24: 429–458, 2001,.[CrossRef][ISI][Medline]
  50. Wray S. Development of gonadotropin-releasing hormone-1 neurons. Front Neuroendocrinol 23: 292–316, 2002.[ISI][Medline]