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Effect of Sex Hormone Administration on Circulating Ghrelin Levels in Peripubertal Children

Institute for Endocrinology and Diabetes (Y.L., G.G.-Y., B.S., M.P., L.L.), National Center of Childhood Diabetes, Schneider Children’s Medical Center of Israel and Felsenstein Medical Research Center, Petah Tiqva 49202, Israel; Sackler Faculty of Medicine (Y.L., G.G.-Y., B.S., A.P., M.P., L.L.), Tel Aviv University, Tel Aviv 69978, Israel; and Department of Obstetrics and Gynecology (A.P.), Assaf Harofe Medical Center, Zerifin 70300, Israel

Address all correspondence and requests for reprints to: Moshe Phillip, M.D., Institute for Endocrinology and Diabetes, National Center for Childhood Diabetes, Schneider Children’s Medical Center of Israel, 14 Kaplan Street, Petah Tiqva 49202, Israel. E-mail: mosheph{at}post.tau.ac.il.

	Abstract

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Background: Ghrelin levels gradually decrease throughout childhood and with advancing pubertal stage. The change during puberty is more pronounced in boys than girls.

Objective: The objective of the study was to investigate whether the pubertal drop in ghrelin secretion is modified by the increase in sex hormones.

Patients and Methods: Ghrelin levels were measured in 34 short peripubertal children (17 boys and 17 girls) aged 8–12.5 yr before and after sex hormone priming for GH stimulation testing.

Results: In boys, priming with testosterone increased testosterone to pubertal levels (23.7 ± 7.1 nmol/liter), which in turn induced a marked decrease in ghrelin (from 1615.8 ± 418.6 to 1390.0 ± 352.0 pg/ml) and leptin (from 8.0 ± 4.5 to 5.8 ± 3.2 ng/ml) and an increase in IGF-I (from 162.7 ± 52.8 to 291.1 ± 101.6 ng/ml) (P < 0.001 for all parameters). In girls, priming with estrogen led to a supraphysiological increase in estradiol levels (1313.8 ± 438.0 pmol/liter), which had no effect on ghrelin, leptin, or IGF-I. There was no correlation between ghrelin levels and levels of sex hormones, leptin, or body mass index in either boys or girls.

Conclusions: A pharmacological increase in sex hormones is associated with a marked decline in circulating levels of ghrelin in boys but not girls. Additional longitudinal studies through puberty are needed to elucidate the physiological interaction between sex hormones and ghrelin.

	Introduction

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GHRELIN IS A peptide hormone predominantly synthesized by the stomach and involved mainly in food-intake regulation (1). Recently ghrelin was identified as the endogenous ligand of the GH-secretagogue receptor, possessing potent GH-releasing activity (2).

Cross-sectional studies conducted in healthy children and adolescents revealed that ghrelin levels peak in early postnatal life and then gradually decrease during childhood and adolescence (3, 4, 5). The precise mechanisms underlying these changes have not been fully elucidated. Researchers have noted that in patients with endocrine dysfunction, sex hormones modulate ghrelin levels: in hypogonadal men, testosterone replacement therapy induced a marked increase in plasma ghrelin levels (6); in women with polycystic ovary syndrome, the high androgen levels associated with the disorder, especially androstenedione, suppressed ghrelin levels (7, 8). Furthermore, the significant decrease observed in plasma ghrelin levels of pregnant rats (9) and normal pregnant women (10) suggested that increased estrogen levels directly induce a down-regulation of ghrelin expression. Hence, the increase in sex hormones during puberty may modify ghrelin secretion.

To test this assumption we measured ghrelin levels before and after sex hormone administration in peripubertal short children undergoing GH evaluation, as a model of hormonal changes in puberty.

	Patients and Methods

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Study group

The study sample comprised 34 prepubertal children (17 boys and 17 girls) aged 8–12.5 yr with short stature (height at or < 10th percentile) and decreased growth rate referred to our Pediatric Endocrine Unit for GH testing. It included children with genetic short stature and those with constitutional growth delay. The initial routine laboratory evaluation, including complete blood count, hepatic and renal function tests, urinalysis, thyroid function tests, and screening for celiac disease, was within normal limits in all cases.

Children with systemic disease, including gastrointestinal disturbances and malabsorption, eating disorders, genetic defects, and congenital syndromes and children born small for gestational age were excluded from the study.

The study protocol was approved by the hospital’s ethics committee, and informed consent was obtained from the children’s parents.

Clinical data

Height and weight were measured and body mass index (BMI; weight in kilograms/height in square meters) was calculated. Pubertal breast and pubic hair stage was assessed according to Marshal and Tanner (11, 12), and testicular volume was determined with the Prader orchidometer. After determination of bone age (BA), BA values were converted to SD scores (SDS) according to the atlas of Greulich and Pyle (13). Height and BMI values were converted to SDS using the updated Centers for Disease Control and Prevention reference values (14).

GH assessment

Physiological exercise test. According to the practice in our clinic, each child was first subjected to a GH exercise stimulation test. The children were instructed to run up and down the stairs for 15 min without pausing. After a recovery period of 5–10 min, a blood sample was drawn for measurement of GH levels. An increase in GH above the cut-off point (10 ng/ml) was considered sufficient. Children with an insufficient response underwent further GH evaluation by pharmacological stimulation testing after sex hormone priming.

Pharmacological provocative test. The lag time between the exercise and the pharmacological test was 3–6 wk. Sex hormone priming was performed according to accepted practice in pediatric endocrinology (15). Sex steroids were administered 10 d preceding the GH stimulation test in boys [im testosterone enanthate (Testoviron Depot), 100 mg] and 2 d preceding the test in girls [conjugated estrogen (T. Premaril), 2.5 mg/m²·d for 2 d]. GH reserve was evaluated after a single dose of clonidine (Normopressan), 0.15 mg/m².

All tests were carried out in the morning (0800–0900 h) after an overnight fast. Before initiation of the physiological exercise test and at zero time of the pharmacological provocative test, a blood sample was drawn for measurement of basal levels of ghrelin, leptin, prolactin (PRL), cortisol, LH, FSH, testosterone (T), or estradiol (E2), and IGF-I.

Hormonal assays

Blood samples were immediately separated and stored at –70 C until assayed without freeze/thaw cycles. Testing for ghrelin and leptin was performed at the Molecular Research Laboratory of the Endocrinology Unit. Serum ghrelin levels were measured by a commercial radioimmunoassay kit (Linco Research, Inc., St. Charles, MO) according to the manufacturer’s instructions. The assay detects both octanoylated and des-octanoylated human ghrelin with a sensitivity of 100 pg/ml. All samples were assayed on the same day in duplicates, using a pool of two kits of the same lot number. The within-assay coefficient of variation was 3.3–10.0% and the between-assay coefficient of variation was 14.7–17.8%, depending on the concentration of ghrelin in the specimen. Serum leptin was determined using a human immunoradiometric assay kit (Diagnostic Systems Laboratories, Webster, TX). The sensitivity of the assay is 0.1 ng/ml.

All other hormonal examinations were performed with commercial kits in the endocrine laboratory of our hospital. E2 was measured by ultrasensitive assays (double-antibody kit; Diagnostic Products Corp., Los Angeles, CA) and T by RIA (Coat A Count kit, Diagnostic Products).

Serum GH and IGF-I concentrations were determined with a solid phase enzyme-labeled chemiluminescent immunoassay using an Immunolite automated analyzer (Immulite 2000 kits, Diagnostic Products). Chemiluminescent immunometric assays were also used to measure TSH, free T₄, and cortisol (Immulite 2000 kits, Diagnostic Products) as well as PRL, LH, and FSH (Euro/Diagnostic Products Llanberis, Gwynedd, UK).

Statistical analysis

All analyses were done with the BMDP program (16) and the results were expressed as mean ± SD. ANOVA with repeated measures was applied to assess the effect of sex hormones on serum levels of ghrelin, leptin, IGF-I, cortisol, and PRL. All data except those for LH, FSH, and ghrelin had normal (Gaussian) distributions. For these hormonal levels, we therefore applied log transformation before analysis. Pearson’s and Spearman’s correlations were applied to examine relationships among the variables at baseline, after priming, and the delta between them.

	Results

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Clinical characteristics

The chronological age range of the short boys and girls was 9.3–12.5 (11.4 ± 1.3) and 8.2–12.1(10.6 ± 1.4) yr, and the anthropometric characteristics were: BA-SDS, –2.5 ± 1.2 and –1.7 ± 0.7; height-SDS, –2.2 ± 0.4 and –2.1 ± 0.5; BMI-SDS, –0.4 ± 0.3 and –0.4 ± 0.5, respectively.

Results of pharmacological stimulation testing after sex steroid priming (Table 1 and Fig. 1)

GH reserve was normal in all patients. Basal levels of LH and FSH were prepubertal. In boys, T measured 10 d after priming was increased from prepubertal levels to midpubertal or late pubertal levels, accompanied by significant modifications in all the hormones assayed. There was a marked decrease in ghrelin and leptin levels and an increase in IGF-I levels (P < 0.001 for all three parameters); PRL and cortisol levels also increased but to a lesser extent (P = 0.05 for both parameters). In girls, estrogen measured 2 d after priming was increased from prepubertal levels to extremely high values. No significant changes were detected in any of the other hormones tested.

View larger version (17K): [in this window] [in a new window]   FIG. 1. Individual ghrelin concentrations before (basal hormone levels drawn before sex steroid exposure before initiation of the GH exercise stimulation test) and after (basal hormone levels drawn after sex steroid exposure at 0 time of the clonidine test) sex hormone priming: comparison between 17 boys and 17 girls. NS, Nonsignificant.

No correlation was found between ghrelin levels and levels of T or E2, IGF-I, PRL, cortisol, or leptin and BMI in short boys and girls, either before or after sex hormone administration.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
The present study demonstrates that the increase in sex hormones after a short course of sex hormone priming for GH studies in children with short stature has a different effect on ghrelin levels in boys and girls. Boys showed a significant decline in ghrelin serum levels (P < 0.001), whereas in girls no clear trend was noted. This study extends previous findings by linking the changes in ghrelin levels to the rise in sex hormones.

The T priming in boys resulted in an increase in T up to levels comparable with those seen in midpuberty. Hence, the decrease in ghrelin could represent the changes witnessed during puberty. These findings agree with recent cross-sectional studies in which plasma ghrelin levels were shown to decrease with age, particularly through puberty (3, 4, 5). However, they contrast with those of Pagotto et al. (6), who reported an increase in ghrelin levels in hypogonadal men after administration of T replacement therapy. This discrepancy may be due to the differences in age and metabolic and hormonal status between the two populations studied.

In boys the T priming was also followed by a decrease in leptin levels and an increase in IGF-I levels concomitant with the decrease in serum ghrelin levels. These findings suggest that the drop in serum ghrelin levels may not be attributable solely to T but that the changes in IGF-I and leptin levels may have had an indirect effect on the ghrelin levels. Indeed, Liu et al. (17) reported that IGF-I modifies ghrelin gene expression; Haqq et al. (18), however, found that leptin apparently does not affect ghrelin secretion. The lack of significant statistical correlation among ghrelin, T, leptin, and IGF-I despite the significant changes in the levels of all the hormones assayed could stem from the small size of our sample.

Data from rodent models clearly indicate that estrogen directly induces ghrelin gene expression (9, 10, 19). In our study, however, the increase in estrogen levels in the prepubertal girls after priming did not alter serum ghrelin levels. A plausible explanation is that ghrelin is less sensitive to changes in estrogen levels. Reports of only a moderate or slight decrease in ghrelin with advancing pubertal stage in girls provide further support for this theory (4). In addition, the estrogen levels measured in our girls after estrogen administration were much above the normal pubertal range. Thus, the documented modifications in ghrelin secretion may not reflect the changes that take place in ghrelin levels through the course of physiological puberty.

The dissimilarity between boys and girls in the response of ghrelin to sex hormones could stem from a difference in underlying physiological mechanisms. It could, however, also be due to the difference in methods of priming: the sex hormone administered (T vs. estrogen) and the time elapsed from priming to ghrelin testing (10 vs. 2 d). Also to be taken into consideration is the fact that our study group was heterogenous in nature, most likely containing both children with idiopathic/familial short stature and children with constitutional growth delay. This is a point that can be verified only with time, and it is quite possible that children with different underlying causes for their short stature may respond differently to sex hormone administration.

In conclusion, our pharmacological model of puberty suggests that sex hormones modulate ghrelin secretion. Short-term administration of T to peripubertal boys caused a marked drop in serum ghrelin levels, whereas no similar effect of estrogen on serum ghrelin levels was found in girls. Longitudinal studies throughout physiological puberty are needed to confirm our findings and to elucidate the interaction between sex hormones and ghrelin.

	Acknowledgments

 
The authors thank Pnina Lilos for performing the statistical analysis and Gloria Ginzach (Editorial Board, Rabin Medical Center, Beilinson Campus) for her assistance.

	Footnotes

 
The authors have no conflict of interest.

First Published Online October 25, 2005

Abbreviations: BA, Bone age; BMI, body mass index; E2, estradiol; PRL, prolactin; SDS, SD score; T, testosterone.

Received January 31, 2005.

Accepted October 19, 2005.

	References

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