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Original Article |
Departments of Endocrinology and Metabolism (M.O., E.B., I.C.O., M.Ku.), Internal Medicine (G.K., C.O.), Urology (M.Ki.), and Biostatistics (S.Y.S.), Gulhane School of Medicine, Etlik-Ankara and Bayindir Medical Center (N.B.), Sogutozu-Ankara, Turkey 06018
Address all correspondence and requests for reprints to: Metin Ozata, M.D., Associate Professor of Medicine, Department of Endocrinology and Metabolism, Gulhane School of Medicine, TR-06018 Etlik-Ankara, Turkey. E-mail: mozata{at}gata.edu.tr or metinozata{at}hotmail.com.
Abstract
Circulating leptin shows a pulsatile secretory pattern along with a nocturnal rise. We have previously shown that circulating leptin concentrations are high in males with untreated idiopathic hypogonadotropic hypogonadism (IHH). However, circadian leptin secretion in IHH before and after gonadotropin treatment is not known. Thus, we studied 14 adult males with IHH who had no history of previous hormonal therapy, and 12 age- and body mass index-matched healthy men. Plasma leptin concentrations were measured with 1-h intervals for 24 h before and 6 months after gonadotropin treatment. The 24-h mean leptin concentration showed a significant decrease, from 11.78 ± 1.908 µg/liter at baseline to 10.85 ± 1.939 µg/liter after 6 months of therapy (z = 3.107; P = 0.002). Before and after treatment, 24-h mean leptin concentrations were also significantly higher in the patient group when compared with controls (4.275 ± 0.711 µg/liter) (z = 5.938; P = 0.0001). Hourly leptin levels demonstrated a diurnal pattern in hypogonadal patients, a surge in the midday, and a peak just after midnight, and this pattern did not differ before and after treatment. We observed a similar diurnal pattern in the control subjects too. Leptin levels were negatively and significantly correlated with free testosterone and total testosterone levels both before (r = -0.656, P = 0.011; and r = -0.639, P = 0.014, respectively) and after (r = -0.537, P = 0.048; and r = -0.563, P = 0.036, respectively) gonadotropin administration.
Our observations suggest that the diurnal rhythm of leptin is intact in males with IHH, and short-term gonadotropin treatment does not effect its diurnal rhythm. Moreover, testosterone produced under the influence of the gonadotropin treatment led to decreases in the leptin levels.
LEPTIN IS A HORMONE, secreted by adipose tissue, that circulates in the blood in proportion to the amount of body fat (1). In addition to its role as a hormonal regulator of body weight and energy expenditure, leptin is now implicated as a regulatory molecule in lipid metabolism, hematopoiesis, insulin action, ovarian function, reproduction, immune function, and angiogenesis (2, 3, 4, 5, 6, 7, 8).
Circulating leptin shows a pulsatile secretory pattern along with a nocturnal rise (9, 10, 11). Previous studies have demonstrated that leptin is secreted in a circadian fashion, with a single nocturnal peak, in lean and obese patients as well as in patients with type 2 diabetes mellitus (12). Changes in diurnal leptin levels may represent an increase and/or decrease in its production rate and/or metabolic clearance rate, respectively. Over the course of 24 h, leptin transcript levels exhibit cyclic variation, with an increase during the night, shortly after initiation of feeding, and a decrease during the day (13). It has been speculated that the nocturnal rise in serum leptin concentrations may be related to nighttime bed rest (i.e. inactivity) and the suppression of appetite during sleep. Consistently, lower concentrations of leptin during the day are related to increased activity and energy expenditure (14). However, the physiological or pathophysiological significance of circadian and pulsatile leptin secretion remains to be established (2). Both corticosteroids and insulin have been reported to up-regulate leptin mRNA expression, which could indicate either a circadian regulation related to cortisol, or a diurnal regulation reflecting changes in food consumption (15, 16, 17).
We have previously demonstrated that male hypogonadism is associated with elevated circulating leptin levels (18). However, the pattern of circadian leptin secretion in male hypogonadism is not known. Here, we provide data on diurnal changes in plasma leptin before and after gonadotropin treatment in adult men with idiopathic hypogonadotropic hypogonadism (IHH).
Subjects and Methods
Patients
The study included 14 male patients with IHH (mean age, 21.1 ± 1.2 yr) who had no history of previous hormonal therapy, and 12 age- and body mass index (BMI)-matched healthy men (mean age, 20.6 ± 1.2 yr). The diagnosis of IHH was based on failure to undergo spontaneous puberty before 18 yr of age and was confirmed by a decreased serum testosterone concentration below normal range for adults, FSH and LH levels within or below the normal range, absence of a pituitary or hypothalamic mass lesion on computerized tomography (CT) or magnetic resonance imaging, presence of a gonadotropin response to repetitive doses of GnRH, normal smell test, and normal karyotypes (46, XY). None of the patients had hyposmia, anosmia, or a family history of IHH. All patients had scrotal testis. All controls had a history of spontaneous puberty, and their physical and biochemical findings were within the normal range.
All patients and the control subjects were informed about the aim and procedure of the study and gave their written consent. The study was approved by the ethics committee of the Gülhane School of Medicine.
Hormone measurements
After an overnight fast, an indwelling iv catheter was placed. A fasting blood sample at 0800 h (time 0) was withdrawn; and thereafter, 60-min samples were withdrawn, until 0800 h the next day, in all patients, before and 6 months after treatment, as well as control subjects. Blood samples were collected in ethylendiamine tetraacetate-coated venipuncture tubes, promptly centrifuged, and separated and stored at –70 C until leptin assay was performed. All plasma samples were run in the same assay. Plasma leptin levels were measured in duplicate by immunoradiometric assay (IRMA) (human leptin IRMA, DSL-23100; Diagnostic Systems Laboratories, Inc., Webster, TX). The assay sensitivity was 0.5 µg/liter. The intraassay coefficient of variation (CV) of the assay at 3.0 µg/liter was 3.9% (n = 7), and that at 12.0 µg/liter was 1.7% (n = 4).
Serum FSH, LH, prolactin (PRL), dehydroepiandrosterone sulfate (DHEAS), SHBG, free testosterone (FT), and total testosterone (TT) were measured on the day before scheduled injections of human menopausal gonadotropin (hMG)/human chorionic gonadotropin (hCG). Serum FSH, LH, and PRL were measured by IRMA with reagents from Radim Techland SA (Angleur, Belgium; FSH IRMA CT and PRL IRMA CT kits, respectively). The intra- and interassay CV were 4.4% and 6.0% for FSH, 4.8% and 5.4% for LH, and 4.6% and 6.0% for PRL, respectively. Serum FT was determined by a solid-phase 125I RIA with reagents from Diagnostic Products (Los Angeles, CA; Coat-A-Count FT Kit). The intra- and interassay CV for FT were 3.8% and 4.2%. Serum TT was determined by RIA with reagents from Diagnostic Systems Laboratories, Inc. (active testosterone kit). The intra- and interassay CV for TT were 9.3% and 11.0%. Serum DHEAS was determined by RIA with reagents from Diagnostic Systems Laboratories, Inc. (active DHEAS kit). The intra- and interassay CV were 7.9% and 5.8% for DHEAS. Serum SHBG was measured by RIA with reagent from Radim Techland SA (SHBG RIA 125 I kits). The intra- and interassay CV for SHBG were 2.4% and 2.9%. The normal range in our laboratory is less than 15 IU/liter for FSH, less than 20 IU/liter for LH, 52–1565 pM for FT, 9.37–37.13 nM for TT, 9–38 nM for SHBG, 0.54–9.09 µM for DHEAS, and 4–8 ng/ml for PRL.
Therapy and analysis
Patients were treated with hCG (Profasi; Serono Laboratories, Inc., Aubonne, Switzerland), 5000 IU, three times a week im injection, along with one vial hMG (Pergonal, 75 IU FSH plus 75 IU LH/vial; Serono Laboratories, Inc.). Clinical and biochemical data were assessed 6 months after therapy in all patients. Bone age was estimated using the radiological method of Greulich and Pyle (19).
Statistical analysis
All the statistical analyses were performed by using SPSS 10.0 software (SPSS, Inc., Chicago, IL). The leptin circadian rhythms were analyzed according to nonlinear regression analysis. The rhythms were characterized by the following parameters: 1) mesor (acronym for midline estimating statistics of rhythm), rhythm-adjusted mean; 2) amplitude, the difference between the maximum value measured at acrophase and the mesor in the cosine curve; and 3) acrophase, lag between local midnight and time of highest value of the cosine function used to approximate the rhythm. Iterative nonlinear least-squares analysis of biological rhythm data, using Marquardt’s modification of the Gauss-Newton algorithm, was used to analyze the circadian rhythms. Data were initially analyzed for the circadian rhythm parameters (20).
Paired sample t test was performed to determine the differences in 24-h mean leptin concentrations, data before and after treatment. Pearson’s test was performed to seek correlations. P < 0.05 was considered statistically significant. All results are given as the mean ± SD (21).
Results
All variables in the untreated patients and control subjects are given in Table 1
. No significant differences in age, BMI, and PRL levels were found between patient and control groups. As expected, serum FSH, LH, TT, and FT levels in all patients were significantly lower than those in the normal subjects. SHBG levels were significantly higher in the patient group than in the normal men.
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. Individual cosinor analyses demonstrated significant circadian rhythms for all patients before and after therapy. Individual leptin profiles before and after therapy are presented as the percentage change in leptin concentrations at each time point, in relation to the baseline 24-h mean in Fig. 1. The leptin mesor was 11.782 ± 0.127 µg/liter, with an amplitude of 2.512 ± 0.179 µg/liter before therapy. After therapy, the mesor decreased to 10.850 ± 0.131 µg/liter, and the amplitude to 2.546 ± 0.186 µg/liter. The acrophase of the circadian rhythm shifted from 0057 to 0142 h after therapy.
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Hourly leptin levels revealed a diurnal pattern in hypogonadal patients, a surge in the midday, and a peak just after midnight and did not differ before (Fig. 2) and after treatment (Fig. 3). Of note, a similar pattern was also observed in the control subjects, suggesting that diurnal leptin rhythm is intact in IHH (Fig. 4).
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Discussion
The main finding of this study is that patients with IHH have preserved circadian rhythmicity and that short-term gonadotropin replacement does not effect circadian rhythmicity. Our results also demonstrated that plasma leptin level is influenced by the testosterone produced under the influence of the gonadotropin treatment.
Little is known about the regulatory system responsible for the circadian variation of leptin levels in humans. Leptin levels in these subjects with IHH show a diurnal pattern of appearance similar to that demonstrated in healthy subjects, with the lowest levels during the early-to-late morning hours and highest around midnight (10, 12). Such a pattern is similar to the appearance of TSH (22) and is inverse to the appearance of ACTH and cortisol (11). This finding is consistent with the report that suppression of gonadotropin secretion by GnRH agonists did not alter the diurnal pattern of leptin secretion in children with central precocious puberty (23).
Several studies have reported a progressive increase in leptin levels during childhood and the prepubertal period in both sexes (24, 25, 26, 27, 28). Mantzoros et al. (4) documented a rise in leptin levels of about 50% in prepubertal boys that just preceded the testosterone increase, and they therefore considered that leptin could be a signal inducing the onset of puberty, although the stimulus for the leptin peak remains unidentified. In boys, leptin level rises until Tanner stage 2 and then decreases (4, 24, 25, 27). We have previously reported that leptin gene mutation is associated with morbid obesity and hypogonadotropic hypogonadism (29). Thus, human data indicate that leptin plays an important permissive role in the initiation of puberty and in the maintenance of reproduction function thereafter (30).
In boys with constitutional delay in growth and puberty, hypoleptinemia was found at the onset of puberty (31). Conversely, several reports observed increased leptin levels during precocious puberty in boys and girls (32, 33). Anorexia nervosa is characterized by extremely low levels of leptin, suppression of the diurnal rhythm and of the pulsatile leptin secretion, as well as amenorrhea (34, 35, 36, 37). Altered leptin secretion patterns with decreased serum leptin were described for other eating disorders such as binge eating and bulimia nervosa, which may have a possible implication in the frequently reported dysfunction of the reproductive axis in these conditions (38, 39).
The effect of leptin on LHRH involves direct and indirect pathways. Direct modulation of leptin on LHRH neurons is possible, because the presence of functional leptin receptors and induction of LHRH release was demonstrated in the GT1–7 LHRH cells (40). However, an indirect effect of leptin on LHRH neurons may be predominant, because coexpression of the leptin receptor long isoform and LHRH was only found in very few rat or ovine neurons (41, 42). One of the possible indirect pathways of leptin acting on LHRH neurons involves cocaine- and amphetamine-regulated transcript (CART), independently of neuropeptide Y (NPY) (43). Thus, leptin was shown to stimulate CART expression in vitro (44), and this peptide was able to shorten the LHRH interpulse interval of explants of the retrochiasmatic hypothalamus from prepubertal male and female rats (45). NPY could also act as a mediator of the effect of leptin on the reproductive axis. NPY was reported to exert inhibitory influence on the LHRH pulse generator (46, 47, 48, 49). Leptin inhibits the expression of NPY in the arcuate nucleus, with consecutive reduction of the prepubertal inhibitory tone of this neuropeptide on pulsatile LHRH release (46, 47, 48, 49). A recent work also supports the important role that NPY plays in leptin’s communication with the reproductive endocrine axis by demonstrating that Y4 receptor knockout rescues fertility in ob/ob mice (50). Thus, accumulating evidence suggests that leptin can regulate gonadotropin levels, and its secretion may be influenced by GnRH or gonadal steroids (51).
Our data demonstrate that leptin concentrations did not correlate with FSH, LH, or PRL. However, leptin levels were negatively and significantly correlated with testosterone levels. An inverse relationship between leptin levels and testosterone has been reported previously (52, 53). Moreover, Wabitsch et al. (54) demonstrated that both testosterone and dihydrotestosterone are able to suppress leptin production in human adipocytes. These findings suggest that testosterone is a direct negative modulator of serum leptin level. On the other hand, Haffner et al. (55) demonstrated that sex hormones were not important independent modifiers of blood leptin concentration in men. In our study, high levels of plasma leptin were associated with very low serum testosterone levels before gonadotropin treatment. However, 6 months after therapy, plasma leptin levels decrease, together with an increase in testosterone level. Previous studies have also demonstrated direct leptin lowering effect of testosterone (53, 56, 57). Thus, it is more likely that the testosterone produced under the influence of the gonadotropin led to very modest decreases in the leptin levels (58).
Although testosterone levels were similar in treated IHH patients and controls, the duration of treatment may be too short to detect a large change in leptin. Supporting this view, Sih et al. (57) demonstrated that testosterone supplementation in older hypogonadal men lowered serum leptin levels at the end of 12 months of therapy. Moreover, Elbers et al. (59) demonstrated that testosterone administration increases visceral fat in female-to-male transsexuals after 3 yr of therapy, but they did not observe an absolute increase in mean visceral fat after 1 yr of testosterone administration. Because direct measures of adipose mass were not performed in our study, it is possible that leptin levels may be related to regional deposition of adipose mass. Supporting this view, Katznelson et al. (60) demonstrated that acquired hypogonadism in adult men is associated with increased sc and skeletal muscle fat. Mauras et al. (61) also demonstrated that testosterone deficiency in young men is associated with alterations with whole-body protein kinetics, strength, and adiposity. Thus, it is also possible to consider that longer-term therapy leads to more profound alterations in body composition that are not detected by BMI. Moreover, Blum et al. (24) suggested that the BMI is the most precise measure for short-term longitudinal changes of body fat in a single patient. Therefore, as suggested by Jockenhovel et al. (53), the lack of information on the percentage body fat in the hypogonadal men does not degrade this observation. Thus, it is more likely that longer-term therapy may be needed to normalize leptin production.
In summary, our data show that in IHH, diurnal rhythm of leptin is preserved, and short-term gonadotropin treatment does not effect its diurnal rhythm. Moreover, testosterone produced under the influence of the gonadotropin treatment led to decreases in the leptin levels.
Acknowledgments
We are grateful to Dr. Johannes D. Veldhuis (General Clinical Research Center, University of Virginia, Charlottesville, VA) and to Dr. Gokhan Ozisik (Department of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Medical School, Chicago, IL) for critical review of the manuscript.
Footnotes
This work was supported in part by Research Center of Gulhane School of Medicine.
Abbreviations: BMI, Body mass index; CART, cocaine- and amphetamine-regulated transcript; CT, computerized tomography; CV, coefficient(s) of variation; DHEAS, dehydroepiandrosterone sulfate; FT, free testosterone; hCG, human chorionic gonadotropin; hMG, human menopausal gonadotropin; IHH, idiopathic hypogonadotropic hypogonadism; IRMA, immunoradiometric assay; NPY, neuropeptide Y; PRL, prolactin; TT, total testosterone.
Received April 1, 2002.
Accepted July 31, 2002.
References
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