LEPTIN, TESTOSTERONE, GH, BODY COMPOSITION AND NEUROMUSCULAR PERFORMANCE IN PREPUBESCENT FENCERS AND UNTRAINED BOYS

TSOLAKIS CHARIS PhD

Department of Physical Education and Sports Science, National & Kapodistrian University of Athens

Key words: training, hormones, children

Introduction

Leptin, the product of the obese gene, which acts in the hypothalamus regulating body mass (26) may play a physiological role in initiating androgen (28) and GH secretion (17) as puberty progresses and is closely related to fat and BMI in children (6,23) and adults (7,34). GnRH secretion leads to increases in gonadal and sex steroid hormone secretion (40), (testosterone, GH, and SHBG), which beside their anabolic and growth effects, regulate the relation between fat and lean body mass (11,18,24,38) and seem to be closely related to serum leptin concentrations in humans.

Exercise probably stimulates hormonal secretion in children (31,43) and adults (12). However there is no evidence that the growth and puberty processes are markedly influenced by training (2,27). There is limited information to describe neuromuscular performance in association with hormonal growth profile of prepubescent experienced athletes (29,30,31), while review of the related literature did not identify any studies on physical training and leptin concentrations in prepubertal - pubertal athletes.

The interval character of fencing, the short-span but of great intensity movements, the kinesthetic forms and the presence of an opponent, activate differently in each case the total of energy sources, a fact which denotes that a considerable number of biological and physiological factors may be responsible for the maximization of performance in fencing. (33).

Teaching the basic kinetics patterns of the lower limbs and in consequence the enhancement of great forms of strength and power, is an essential training pursuit, especially in the early stages of training taking into consideration that the level of physical is closely connected with trainees actual age. (5)

The aim of this study was to a) compare T, GH, leptin levels, body composition and neuromuscular performance in pre- and pubertal fencers versus a proper control group of untrained boys, b) determine whether moderate fencing training has the potential to alter the normal growth process, through modifications of the hormonal factors, c) examine the possible association between hormonal regulators of growth and leptin, in an attempt to clarify leptin contribution at the onset of growth and maturation process, as leptin and fat relation is adequate documented in children and adults.

Subjects – methods

The experimental group of the study (EG) consisted of nine, prepubescent and pubescent male fencers 11-14 years old. They were selected by trainers from regional sports academies of the national development system, they had been participating in systematic training for more than one year prior to the present study and they were still active at a competitive level, following appropriate designing for this age groups programs. 10 untrained pupils of the same age, participating only in sports school activities consisted the control group of the study (CG). The subjects' physical characteristics are given in Table 1. They participated voluntarily after being informed of the purpose of the study and after obtaining their parents written consent. The study was approved by the University of Athens, School of Biology, Department of Biochemistry and Molecular Biology. and deals with the first measurements of a longitudinal -1 year- study. 

 Maturation stage was evaluated according to Tanner's scale (1962), based on external genitals and pubic hair development. They all underwent medical evaluation prior to their selection so that children with abnormalities of endocrine function, metabolic diseases, or other known intervening factors are excluded.

Anthropometry: Subjects' height (Ht), and body mass (BM) were measured to the nearest 0.1 cm and 0.1 kg, respectively. Skin folds were taken with a Harpenden skin fold caliper while % body fat percentage was estimated using Slaughter's equation (38). Lean body mass (LBM) was calculated by subtracting fat mass from BM. Body Mass Index (BMI) was calculated as BM/hgt². Forearm muscle mass (arm muscle plus bone, CSA) and midthigh (midthigh muscle plus bone, CSA) was calculated with an anthropometrical formula incorporating limp circumference and skin folds, using an established approach (20,22). All anthropometrical and body composition raw data were measured by an investigator with a previously controlled for his test-retest reliability of r 0.92.

Jumping performance was estimated with a two leg maximal jump test using an ergojump contact platform as described by Bosco (4). Subjects were asked to perform two styles of vertical jumps: (a) squat jumps (SJ) from a semisquat angle 90^ο position, and (b) a countermovement jump (CMJ) from a standing position with a preliminary countermovement. Each subject performed three jumping trials with 30 -sec interval in between and the highest score was retained for analysis. (ICC: 0.93-0.94, p<0.0001)

Hand grip strength. Dominant hand grip strength was measured using a hand grip dynamometer. While holding the dynamometer each subject parallel to the side of the dial facing away from the body performed three maximal hand grip trials lasting 4-5 seconds with a 60-sec interval in between. Again the highest value was retained for analysis. (ICC: 0.91, p<0.001)

 Blood Tests. After 2 days of rest and 12 hours of fasting, approximately 10mL of blood was drawn from the antecubital forearm vein using a gauge needle 21G x 1^1/2΄΄/ vacutainer set up between 08.30-09.00 hours, in order to avoid the influence of the diurnal variations in serum hormones. The blood was allowed to clot at room temperature (22^oC) and the serum was separated by centrifugation at 3000 rpm for 15 min and then stored at –30^oC until analyzed (within 30 days). All samples were determined in duplicate.

Hormone assays: GH concentrations were determined using an ELSA-HGH solid phase two-site immunoradiometric assay (Cis bio international, ORIS Group, France). Serum samples were incubated with an antiHGH tracer in antiHGH monoclonal antibody coated ELSA tubes for 2 hours at room temperature (18-25^ο C) under agitation (400 rpm). After aspiration and washing tree times the tubes the radioactivity bound to the ELSA tubes was measured. The sensitivity of the assay was 0.04 ng/mL. The intra- and inter-assay CVs were 2.4, 2.8, 2.8, 2.3 and 4.2, 3.2, 4.4, 4.0, for concentrations of 3.5, 7.3, 17, 47.4, and 3.3, 7.1, 16.4, 45.4 ng/mL respectively. There was not any cross reaction with LH, FSH, HCG, TSH, Prolactin.

Testosterone concentrations were determined using a Gammacoat (¹²⁵I) Testosterone radioimmunoassay kit (Dia Sorin, USA). Serum samples were incubated with ¹²⁵I Testosterone tracer in rabbit anti-testosterone coated tubes for 90 min. at 37^ο C. The separation of free from bound antigen was achieved by decanting the antibody coated tubes. The sensitivity of the assay was 0.059ng/mL The intra- and inter-assay CVs (%) were 6.2, 6.7, 8.6 and 6.9, 7.71, 13.6 respectively. The cross reactivity with dihydrotestosterone, androstendione, epitestosterone, cortisol was 3.8, 0.8, 0.4, 0.02 respectively.

SHBG concentrations were determined using an SHBG two-site immunoradiometric assay (DSL Inc, USA). Serum samples were incubated with anti SHBG (¹²⁵I) reagent in anti SHBG coated tubes at room temperature (25^ο C) for 2 hours under agitation (180 rpm). Unbound material were removed by decanting and washing three times the tubes. The sensitivity of the assay was 3 nmol/L. The intra- and inter-assay CVs were 3.7, 1.1, 3.4 and 11.5, 10.3, 8.7 for doses of 27, 92, 119 and 26, 87, 115 nmol/L, respectively. No human serum protein is known to cross react with the antibodies employed in the DSL SHBG IRMA system.

The Free Androgen Index (FAI) was computed using the formula:

• FAI  concentration of total T (nmol/L) x 100 (14)

Leptin concentrations were determined using a two-site immunoradiometric assay (DSL Inc., USA). Serum samples were incubated with an ¹²⁵I antileptin reagent in anti leptin coated tubes for 4 hours at room temperature (25^ο C) under agitation (200rpm). Unbound reagent was removed by decanting and washing three times the tubes. The sensitivity of the assay was 0.10 ng/mL. The intra- and inter-assay CVs were 3.7, 4.9, 2.6 and 6.6, 5.6, 3.7 for concentrations of 2.7, 13.5, 73.6 and 2.8, 14.3, 73.9 ng/mL respectively. No cross reactivity was observed at concentrations of 10 mg/mL.

All samples were counted in a gamma counter Packard Cobra Quantum for 1 min.

Statistical analysis: Plasma leptin, FAI, BMI and fat were not normally distributed and were log transformed. Differences between groups (EG, CG) were determined using the independent samples t-test. For all unvaried correlation analysis Pearson's coefficient was calculated. Stepwise multiple linear regression was used to determine explanatory variables. The data in the tables, are presented as meanSD. For each analysis statistical significance was tested at the a0.05 probability level.

Results: There were no statistical differences between EG and the CG in physical characteristics, BMI, LBM,. Fencers' grip strength, squat and counter jump were significantly different (p<0.05) than that of the controls (Tables 1 and 3).

Table 1

Physical characteristics and body composition of the subjects

Variables	EG (n:9)	CG (n: 10)
Age (Years)	12.581.34	12.281.28
Tanner stages	2.440.9	2.450.49
Height (m)	1.550.12	1.590.11
Weight (kg)	52.913.15	53.417.84
Fat (%)	17.3512.54	14.5510.79
BMI (Kg/m2)	20.533.45	20.884.33
LBM (Kg)	42.76.94	44.411.22

EG: experimental group, CG: control group, * p<0.05 : Significance of difference (EG vs CG).

There were no significant differences between AG and CG in Testosterone, GH, SHBG, FAI and leptin (Table 2). SHBG concentration of the EG were significantly related to leptin levels (r: -0.66, p<0.001). Grip strength, squat and counter jump of EG were significantly correlated with testosterone levels (r: 0.77-0.90, p<0.05-0.001).

Table 2

Hormonal levels of the subjects

Variables	EG (n:9)	CG (n: 10)
Testosterone (nmol/L)	10.2110.7	10.5811.82
SHBG (nmol/L)	136.3382.31	107.445.31
FAI	25.3959.64	11.9914.82
GH (ng/ml)	3.535.96	23.58
Leptin (ng/ml)	107.04	15.2713.28

EG: experimental group, CG: control group, * p<0.05: Significance of difference (EG vs CG).

Table 3

Arm, midthigh muscle CSA, hand grip strength and jumping performance of the subjects

Variables	EG (n:9)	CG (n: 10)
Arm CSA (cm2)	29.988.51	29.985.67
Thigh CSA (cm2)	128.1028.74	127.8633.86
Hand grip strength (kg)	309.48	28.158.66 *
Squat jump (cm)	21.23.65	20.765.52 *
Counter jump (cm)	24.365.90	21.765.81 *

EG: experimental group, CG: control group* p<0.05: Significance of difference (EG vs CG).

Leptin levels of EG and CG were significantly related to BMI and fat (r0.67-0.94, p<0.05-0.01) respectively, but were not related to age.

Stepwise multiple regression analysis was used to assess the prediction of leptin concentrations from body fat %, BMI and age as independent variables. In EG 87% of the variation of leptin was attributed to BMI % while in CG 73% was attributed to fat (p<0.001-0.001) (Table 4).

To assess the predictors of leptin, -Testosterone, GH, SHBG and FAI- were entered as independent variables. SHBG accounted for 26% of the variability of leptin in EG +CG (n:19) subjects (Table 5).

Table 4

Stepwise regression analysis of plasma leptin levels on % body fat, age,

BMI for EG and CG

Groups	Predicting variables	Significance	R2	Excluded variables
EG	BMI	<0.001	0.87	Age, Fat %
CG	Fat %	<0.001	0.73	BMI, age

* EG: athletes (n:9), CG: control group (n:10)

Table 5

Stepwise regression analysis of plasma leptin on Testosterone, SHBG, FAI

and GH for EG and CG

Groups	Predicting variables	Significance	R2	Excluded variables
CG / EG n:19	SHBG	<0.05	0.26	Testosterone, GH, FAI

* EG: athletes (n:9), CG: control group (n:10)

Discussion: The main findings of this study have shown that: (a) Fencers' grip strength, squat and counter jump were significantly different (p<0.05) than that of the controls and were significantly correlated with testosterone levels (r: 0.77-0.90, p<0.05-0.001). (b) Leptin levels of the fencers and the controls were related to body fat and BMI. In addition it was found that there is little evidence that leptin has a positive effect on growth and anabolic factors (SHBG explain the variation of leptin in EG+CG subjects).

 In this study the fencers and the controls were of the same chronological and biological age (according to Tanner), with similar anthropometric variables, BMI, and LBM. In this respect there is a contrast between our work and relative previous studies (2,10,15,27) in which athletes were significantly different in anthropometric measurement and body composition compared to controls. The boys of the present study, had all similar mean values of T, SHBG, GH, FAI and leptin which were within the reference value range (42).

The existing data on hormonal responses to exercise in male children and adolescent are conflicting. For instance serum testosterone levels have been shown to increase (31,43), or remain unchanged (13,32), following different physical training protocols. In addition exercise seems to stimulate GH release in adolescent boys (46). The data on the growth hormonal profile of young athletes are limited (9,29,30,31) and therefore there is a lack of information especially on leptin concentration of pre- and pubescent athletes. The growth hormonal parameters of this study were unaffected from the training patterns followed by the subjects, are consistent to those of (9,30), and seem to indicate that experimental and control groups were at a similar phase of biological maturation. Hand grip strength and jumping performance of the fencers were significantly higher (p<0.05) than that of the controls, probably because the athletes were more experienced in training (>1 year) and they underwent 3-5 trainings per week. This is in accordance to the findings of (29,30,31) who suggested that the training background of athletic group affects their strength capacity. Significant correlation were identified between hand grip strength, jumping performance and testosterone, which are in accordance with the findings of (29), and seem to imply that strength and power are affected by anabolic/androgenic activity although the intensity of the training programs for these age groups was moderate, as it is well established that the primary aim of the relative training programs is mostly associated to the practice of the basic motor fencing skills.

The results of the stepwise multiple regression analysis, where FAI was the only determinant of strength and power variables among all relative hormones (explanatory variables), would be an indicator of the potential trainability status not only for adults but for the children also (43)

 The results of leptin concentration in our study are in contrast with the results of (44). In this study leptin levels of elite gymnasts were lower compared to normal controls and were correlated with the amount of fat mass. Additionally four months of physical training resulted in a significant decrease of leptin concentration in obese 7-11 year old children and the fat mass was highly correlated with baseline leptin (21). The lower leptin concentrations of the above mentioned studies may probably be attributed to changes in energy balance resulting from physical training (25,44). However, the precise mechanisms through which physical training may influence leptin concentration are yet uncertain.

In the present study leptin levels correlated positively with fat and BMI. Additionally in EG 87% of the variation of leptin was attributed to BMI % while in CG 73% was attributed to fat % (p<0.001-0.001), and this findings are in contrast with results of previews relative studies (1,23). The decline of leptin levels with age may reflects the process of pubertal events: decrease in fat, increase in LBM (35) which take place under the influence of androgens (3). Although the association between BMI and leptin is strong in adults (7,26), studies in children have yielded conflicting results (28,37). Therefore BMI may not provide an adequate assessment of adiposity during puberty (1), or between pubertal groups undergoing different degrees of physical exercise (45) perhaps because of the divergent pattern of pubertal and exercise related changes of fat and LBM

 The processes and the factors that initiate the onset of puberty remain are not yet fully known. Leptin levels increase just before the progressive rise of testosterone and the appropriate development of secondary sexual characteristics (16), but no association exists between leptin and adrenal androgen production (28). In the present study leptin levels correlated negatively with SHBG for all subjects. Specifically these correlations confirmed by the regression analyses, where SHBG was the main determinant of leptin accounted for 26 % of its variability. The age related decline of SHBG in prepuberty and puberty (8), being closely related to the progressive rise of sex steroids at the onset of puberty (19), and the increase of GH due to the gonadal steroid concentration (36), may consist evidence that leptin is associated with the hormonal regulators of the growth and maturation process.

It can then be concluded that a) that the training background of the fencers affects their strength capacity, b) leptin may be a permissive factor for the onset of puberty.

More research is required to clarify whether extension (until midpuberty at least) of the experimental design may influence maturity status, growth hormonal regulators, leptin concentration and the relative strength and power parameters of the exercised subjects. Additionally, numerous questions remain to be answered regarding the influence of different kind of exercise and the optimal combinations of the training factors to obtain maximal training effect, taking into consideration subjects' biological maturation process.

Summary

Physical exercise is a potent stimulus for hormonal changes in children and adults. However there is no evidence that the growth and puberty processes are markedly influenced by training. On the other hand there is limited information to describe neuromuscular performance in association with hormonal growth profile of prepubescent experienced athletes. In addition while experimental data indicate leptin concentrations decreases in men as a result of regular training, review of the related literature did not identify any studies on physical training and leptin concentrations in prepubertal athletes and in consequence in fencers too.

Hormonal regulators of growth and development, leptin levels, body composition, neuromuscular performance and their association were investigated in nine prepubertal fencers (experimental group-EG) and an untrained (n: 10) control group (CG). Informed consent was obtained from the children and their parents. Their maturation stage was evaluated according to Tanner's criteria. There were no differences between EG and CG in physical characteristics, body mass index, (BMI), lean body mass (LBM), testosterone (T), sex hormone binding globulin (SHBG), free androgen index (FAI), growth hormone (GH), and leptin. Fencers' grip strength, squat and counter jump were significantly different (p<0.05) than that of the controls', and were significantly correlated with testosterone levels (r: 0.77-0.90, p<0.05-0.001). Fencers' leptin levels were found to be significantly correlated to BMI (R²: 0.87, p<0.001). There is a little evidence that leptin has a positive effect on growth and anabolic factors. SHBG explain the variation of leptin in both EG and CG groups with (R²0.26, p<0.05) respectively. Leptin seems to be a permissive factor for the onset of puberty, while the training background needs an optimal biological maturation to produce significant differences in muscle and power performance.

Acknowledgements

We would like to thank all the participants and their parents for their enthusiastic contribution and patience shown during the project. We are also thankful to Dr. D.A Adamopoulos, MD, PhD, Chief Endocrinologist, Helena Venizelos Hospital, for reviewing and correcting our manuscript, and to Mr K. Tsolakis, ex director of Biochemistry Laboratory, in Sotiria Hospital as well the stuff of the endocrine department of Helena Venizelos Hospital for their expert technical assistance.

This study was supported by grants from the Greek State Scholarships Foundation.

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