Low physical activity is a rapidly growing health problem in children. Understanding the biochemical regulation of energy homeostasis in children at different stages of puberty will assist in the understanding and implementation of countermeasures and strategies for the prevention of obesity in children and adolescents. A particular focus of this investigation was the impact of peripheral signals, such as ghrelin (12) and leptin (36), which have been associated with appetite-regulating responses at the hypothalamic and pituitary levels (20,35). Ghrelin, a peptide secreted by the endocrine cells in the gastrointestinal tract (12), transfers information from the stomach to the hypothalamus and influences growth hormone release in response to changes in energy homeostasis (26,35). Leptin is a product of the LEP gene (previously denoted OB) and acts directly on the hypothalamus, where it regulates a large number of molecules that are involved in energy homeostasis (3,26). The interaction between circulating ghrelin and leptin concentrations in the blood has been the subject of interest for some years with mixed results. Some studies suggest that leptin apparently does not affect ghrelin secretion in a mixed group of obese boys and girls (7,9), while others have found an inverse association between ghrelin and leptin in healthy boys and girls (23,33).
Physical changes during puberty include accelerated growth and changes in anthropometric and body-composition variables that are mediated by many hormones, such as sex steroids, insulin-like growth factor-I (IGF-I) and leptin (6,24). Specifically, it has been suggested that leptin is involved in pubertal activation (3). However, it has also been proposed that the hormone ghrelin could influence growth and physical development (33). It has been reported that the initiation of puberty is associated with increased leptin (3) and decreased ghrelin (7,33) concentrations in blood. Moreover, there are some data to suggest that changes in plasma ghrelin concentrations are more pronounced in boys than in girls (33). However, data on ghrelin concentrations throughout childhood have not yet been fully elucidated, especially the effect of regular high-energy expenditure on ghrelin concentrations during puberty in girls.
Given the importance of leptin (3,22) and ghrelin (14,23) in the human organism during puberty, it was our aim to assess the influence of regular physical activity on plasma ghrelin concentrations after onset of puberty in girls. In addition, the influence of leptin, sex steroids and IGF-I on ghrelin concentration has not been studied well in pubertal girls. Accordingly, this study also examines the association of fasting plasma ghrelin concentrations with various plasma biochemical, body composition, and aerobic capacity variables in healthy adolescent girls. The detailed information about these relationships may provide further information about the possible role of circulating ghrelin during pubertal maturation in adolescent girls.
Fifty healthy pubertal schoolgirls ages 11-16 yr participated in this study. They were divided into physically active (N = 25) and physically inactive (N = 25) groups. The physically active group consisted of swimmers that were recruited from local training groups and had a training history of 3.7 ± 1.8 yr and had trained an average of 6.2 ± 2.0 h·wk−1 for the last 2 yr (4). During the testing period, the mean weekly training volume was 17.6 ± 5.9 km, performed mainly at an aerobic pace. Physically inactive subjects were recruited from the physical education classes, which they had twice a week. Participation in physical education classes only was the inclusion criteria for these subjects. The subjects were divided into two maturation groups on the basis of their self-assessment using an illustrated questionnaire of pubertal stage according to the Tanner classification method (29). Pubertal development assessment according to the method of Tanner, which uses self-assessment of breasts and pubic hair stage in girls, has been validated previously (5,18,27). The subjects were given photographs, figures and descriptions, and asked to choose the one that most accurately reflected their appearance. In case of discrepancies between the two variables (breast development and pubic hair stage), greater emphasis for the determination of Tanner stage was placed on the degree of breast development. Maturation I group (14 matched pairs) included pubertal stages 2 and 3, and the maturation II group (11 matched pairs) included pubertal stages 4 and 5. In the beginning, 29 physically inactive subjects were recruited to generate 25 matched pairs with physically active girls. Both groups were matched for age (±1 yr) and body mass index (BMI; ± 2 kg·m−2). None of the subjects were receiving any medications or had a history of bone or renal diseases. Throughout the study, norestrictions were placed on dietary intake and subjects consumed their ordinary everyday diet. The study was approved by the medical ethics committee of the University of Tartu in Estonia. The purpose, risks, and benefits were explained to the children and their parents who signed the consent form.
All testing was completed during the two visits of the subjects to the laboratory. During the first visit, participants had a venous blood sample taken in the morning following an overnight fast. In addition, main anthropometric parameters, biological age, and peak oxygen consumption (V˙O2peak) on the bicycle ergometer were measured after a light breakfast. The first measurement session was conducted during the early follicular phase of the menstrual cycle in menstruating girls (30). The second measurement session consisted of body composition and bone mineral assessments by dual-energy x-ray absorptiometry (DXA). Measurement sessions were separated by approximately 1 wk, dependent on the participant's schedule and DXA availability.
Anthropometry, body composition, and bone mineral measurements.
Body height was measured using a Martin metal anthropometer to the nearest 0.1 cm with a standardized technique. Body mass was measured with minimal clothing to the nearest 0.05 kg using a medical electronic scale (A&D Instruments, UK), and BMI was calculated as body mass (kg) divided by height (m2).
Whole-body fat and fat-free mass were measured by DXA using the DPX-IQ densitometer (Lunar Corporation, Madison, WI) equipped with proprietary software, version 3.6. Participants were scanned in light clothing while lying flat on their backs with arms at their sides. The fast scan mode and standard subject positioning were used for total body measurements and analyzed with the use of the extended analysis option.
Peak oxygen consumption.
Peak oxygen consumption (V˙O2peak), peak oxygen consumption per kilogram of body mass (V˙O2peak·kg−1), and maximal aerobic power (Pamax) were measured on an electronically braked cycle ergometer (Tunturi T8, Finland). The initial workload was set at 80 W followed by an increase of 20 W every 2 min. The participants were required to pedal at 70 ± 5 rpm and were strongly encouraged to continue until volitional fatigue. If participants were not able to pedal at 70 rpm, they were asked to pedal as fast as possible for 1 min (10,25). Heart rate was recorded every 5 s during the test using a Sporttester Polar Vantage NV (Kempele, Finland). Respiratory gas-exchange variables were measured throughout the test in a breath-by-breath mode, and data were stored in 10-s intervals for the measurement of oxygen consumption using a portable open-circuit system (Med Graphics VO200, St. Paul, MN) (10). To determine that V˙O2peak was reached, the attainment of a plateau in V˙O2 with increasing work rate and/or after 1 min of cycling as fast as possible was used as a criterion (10,25). When this plateau was not observed, a respiratory-exchange ratio exceeding 1.05 and a theoretical maximal cardiac frequency were used (10,25). All participants satisfied these criteria.
A 10-mL blood sample was obtained from an antecubital vein with the participant sitting in the upright position. The plasma was separated and frozen at −20°C for later analysis. Blood plasma analysis was completed within 6 months after collection. Ghrelin concentration was determined in duplicate using a commercially available radioimmunoassay (RIA) kit (Linco Research). The sensitivity of this kit was 93 pg·mL−1, and the intra- and interassay coefficients of variation (CV) were < 10% and < 14.7%, respectively. Leptin was determined in duplicate via RIA (Mediagnost GmbH, Germany). This assay has the intra- and interassay CV of less than 5%. Estradiol, IGF-I, and IGFBP-3 were analyzed in duplicate on an Immulite 2000 (DPC, Los Angeles, CA) with inter- and intraassay CV of less than 5%. Insulin was also determined in duplicate on an Immulite 2000 (DPC, Los Angeles, CA), and the intra- and interassay CV for insulin were 4.5 and 12.2%, respectively, at an insulin concentration of 6.6 μIU·mL−1.
Statistical analysis was performed with SPSS 11.0 for Windows (Chicago, IL). Means and standard deviations (± SD) were determined. Evaluation of normality was performed with the Shapiro-Wilk statistical method. The differences between groups were tested using the Wilcoxon matched-pairs signed rank test. When analyzing the matched pairs, swimmer-control comparisons were performed with paired t-tests. Spearman correlation analysis was performed to assess bivariate relationships. Partial correlation coefficients were used to estimate the relationships between ghrelin and other measured variables after adjustment for age and pubertal status. Multivariate linear regression analysis was performed to determine the predictors of ghrelin concentration and the variables that had a significant association with ghrelin concentration on the bivariate correlation analyses were selected as independent variables (20). Significance was set at P < 0.05.
Physical characteristics of the physically active and physically inactive subjects are presented in Table 1. Maturation I and II groups had significantly different ages, whereas physical activity groups did not differ by age. In addition, maturation I and II groups differed in height, body mass, BMI, fat mass, fat-free mass, V˙O2peak, and Pamax values. Ghrelin concentration was not different between the two maturation levels, but physically active girls in maturation I and II groups had significantly higher plasma ghrelin concentrations compared with the physically inactive subjects at the same maturation levels (Table 2). Leptin, estradiol, IGF-I, and IGFBP-3 levels were not different between maturation I and II groups in physically active and physically inactive groups. However, physically active subjects had significantly lower values for leptin and estradiol concentrations compared with physically inactive subjects in both maturation I and II groups.
Plasma ghrelin concentration was inversely correlated with percent body fat, fat mass, V˙O2peak·kg−1, leptin, estradiol, insulin, and IGF-I values in the overall group (Table 3, Fig. 1). Partial correlation analysis revealed that plasma ghrelin concentration was significantly related to percent body fat, fat mass, V˙O2peak, V˙O2peak·kg−1, Pamax, leptin, and IGF-I after controlling for age and pubertal status (Table 3).
Using multivariate linear regression analysis, only IGF-I was significantly related to the levels of plasma ghrelin in the overall group (Table 4).
In this study, plasma ghrelin concentration did not change with advancing pubertal stage in adolescent girls. However, significantly higher values for circulating ghrelin were observed in physically active compared to physically inactive girls at different stages of puberty. This demonstrates that regular physical activity may have an influence on plasma ghrelin concentrations during puberty in girls. In addition, we found that plasma IGF-I concentration was the main predictor of circulating ghrelin concentration in adolescent girls.
A decrease in ghrelin concentration with increasing age during puberty has been previously reported (7,24,33). Moreover, the changes in circulating ghrelin levels are more pronounced in boys than girls with advancing pubertal stage (33). In contrast, a study by Bellone et al. (1) did not show ghrelin's dependence on sex or pubertal status. In our study, there was no change in ghrelin concentration after onset of puberty (pubertal stages 2 and 3 vs pubertal stages 4 and 5) in both physically active and physically inactive adolescent girls (see Table 2). Furthermore, no relationship between plasma ghrelin levels and age or pubertal status was observed in our adolescent girls. Similarly to the results of our study, Park et al. (23) found no correlation between plasma ghrelin concentration and age in girls, while other studies have observed a significant relationship between circulating ghrelin and age in boys (23,24) and in mixed groups of boys and girls (7,32,33). According to the results of our study, it could be suggested that in contrast to boys, ghrelin concentration remains relatively constant after the initiation of puberty in girls. However, further investigation is needed to fully elucidate the possible sex differences in plasma ghrelin concentration during puberty.
Physically active girls had significantly higher values of circulating ghrelin in both pubertal groups compared with the corresponding physically inactive girls (Table 2). This suggests that the elevated energy expenditure (i.e., training for a mean of 6.2 ± 2.0 h·wk−1) may have caused a significantly higher ghrelin concentration during puberty in our physically active girls. It is well recognized that appetite and food intake increase during puberty (33), and ghrelin is known to stimulate appetite (20). This anticipated increase in appetite and food intake caused by puberty may be associated with an elevation in plasma ghrelin concentration in our physically active adolescent girls (Table 2). Whatmore et al. (33) propose that there could be an increased sensitivity for appetite stimulation by ghrelin during puberty. The results of our investigation demonstrated that elevated energy expenditure, and therefore also an increased energy intake, in physically active girls was linked to higher circulating ghrelin levels during puberty. Accordingly, it could be suggested that regular physical activity increases plasma ghrelin concentrations to stimulate appetite and food intake to cover the higher energy expenditure. Our results support the idea that ghrelin may act as a hormone, signaling a need for energy consumption and that ghrelin secretion is triggered to counter a further deficit of energy storage by helping to maintain body mass (8,28). However, it should be noted that the effect of a given ghrelin concentration on appetite and food intake is not well defined (33), and further studies are needed to fully elucidate the effects of regular physical activity on ghrelin concentrations during puberty in girls.
The mechanism of ghrelin regulation has not yet been fully elucidated. It has been suggested that as ghrelin secretion is inhibited by ingestion of nutrients (31) and that various gut and pancreatic hormones may be candidate regulators of ghrelin (7). However, in our study, we observed a negative association between plasma ghrelin and leptin concentrations in pubertal girls with different physical activity levels (Fig. 1). This is in accordance with other studies in children (23,33) as both peptides are directly linked to energy expenditure (8,34). In contrast, other investigations have found no relationship between ghrelin and leptin in boys (25) and in a mixed groups of boys and girls which also included obese children and adolescents (7,9). However, in accordance with the Park et al. (23) study, the results of our study support the possibility that leptin regulates ghrelin secretion, at least in normal-weight healthy adolescent girls during puberty. Thus, the relationship between plasma ghrelin and leptin concentrations may depend on sex, pubertal stage, and obesity. However, further collection of data is needed to clarify the interrelationships of ghrelin and leptin at different stages of maturation during puberty.
It has been previously demonstrated that ghrelin gene expression is age dependent and is influenced by the level of circulating IGF-I concentration (16). Furthermore, Whatmore et al. (33) have argued that a negative relationship of ghrelin concentration with IGF-I would suggest that a decrease in ghrelin facilitates growth acceleration in puberty. In the present study, we found that IGF-I was the most important determinant of ghrelin concentration in our pubertal adolescent girls (Table 4). It could be suggested that the increase in estradiol levels at the beginning of puberty stimulates IGF-I secretion (11), and, thus, via negative feedback, IGF-I may suppress ghrelin secretion (24). Accordingly, our results support a negative feedback mechanism between ghrelin and IGF-I levels in healthy normal-weight adolescent girls in puberty.
Plasma ghrelin concentration in our study was positively related to the V˙O2peak values in our healthy adolescent pubertal girls (Table 3). In contrast, St-Pierre et al. (28) did not find a significant relationship between V˙O2peak and ghrelin in young healthy females, whereas our previous study has demonstrated a negative association between V˙O2peak and ghrelin in boys at different maturation stages (25). The results of these investigations indicate that different sex hormones may have different impacts on the possible interaction between circulating ghrelin and V˙O2peak values in boys and girls. However, because relatively little is known about the relationship between plasma estradiol and ghrelin concentrations in girls (15), additional longitudinal studies are needed to elucidate the physiological interaction of sex hormones and ghrelin with aerobic capacity in girls throughout puberty.
The present study has some limitations. The cross-sectional nature of our study limits determination of temporality or causality. An additional limitation of the present study is that single fasting blood biochemical measurements, including plasma ghrelin concentrations, might not represent the full nature of the reciprocal relationships, because of the multifactorial dynamic nature of the measurements (23). Finally, we assessed total plasma ghrelin and not acylated and desacylated ghrelin separately. Of the two circulating ghrelin forms, the acylated one is thought to be essential for ghrelin biological activity (13,19), although it has been demonstrated that the desacylated form is not biologically inactive (2,19). Desacylated ghrelin shares with the acylated form some neuroendocrine actions such as cardiovascular effects, modulation of cell proliferation and also some influence on adipogenesis (2). However, because total and active ghrelin are positively correlated (17), the results of the present study are a step further in understanding the influence of regular physical activity on ghrelin during puberty in girls.
In conclusion, this study suggests that regular physical activity influences plasma ghrelin concentrations in girls at different pubertal maturation levels. In addition, plasma IGF-I concentration seems to be the main determinant of circulating ghrelin in healthy, normal-weight adolescent girls.
This study was supported by Estonian Science Foundation Grant No 6220.
1. Bellone, S., A. Rapa, D. Vivenza, et al. Circulating ghrelin levels as function of gender, pubertal status and adiposity in childhood. J. Endocrinol. Invest.
2. Broglio, F., C. Gottero, F. Prodam, et al. Non-acylated ghrelin counteracts the metabolic but not the neuroendocrine response to acylated ghrelin in humans. J. Clin. Endocrinol. Metab.
3. Clayton, P. E., M. S. Gill, C. M. Hall, V. Tillmann, A.J.Whatmore, and D. A. Price. Serum leptin through childhood and adolescence. Clin. Endocrinol.
4. Dowthwaite, J. D., J. G. Distefano, R. J. Ploutz-Snyder, J.A.Kanaley, and T. A. Scerpella. Maturity and activity-related differences in bone mineral density: Tanner I vs. II and gymnasts vs. non-gymnasts. Bone
5. Duke, P. M., I. F. Litt, and R. T. Gross. Adolescents' self-assessment of sexual maturation
6. Garnett, S. P., W. Högler, B. Blades, et al. Relation between hormones and body composition, including bone, in prepubertal children. Am. J. Clin. Nutr.
7. Haqq, A. M., I. S. Farooqi, S. O'Rahilly, et al. Serum ghrelin levels are inversely correlated with body mass index, age, and insulin
concentrations in normal children and are markedly increased in Prader-Willi syndrome. J. Clin. Endocrinol. Metab.
8. Horvath, T. L., S. Diano, P. Sotonyi, M. Heiman, and M. Tschöp. Ghrelin and the regulation of energy balance-a hypothalamic perspective. Endocrinology
9. Ikezaki, A., H. Hosoda, K. Ito, et al. Fasting plasma ghrelin levels are negatively correlated with insulin
resistance and PAI-1, but not with leptin, in obese children and adolescents. Diabetes
10. Jürimäe, J., K. Haljaste, A. Cicchella, et al. Analysis of swimming performance from physical, physiological and biomechanical parameters in young swimmers. Pediatr. Exerc. Sci.
11. Kanbur-Oksuz, N., O. Derman, and E. Kinik. Correlation of sex steriods with IGF-1 and IGFBP-3 during different pubertal stages. Turk. J. Pediatr.
12. Kojima, M., H. Hosoda, Y. Date, M. Nakazato, H. Matsuo, and K. Kangawa. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature
13. Kojima, M., H. Hosoda, H. Matsuo, and K. Kangawa. Ghrelin: discovery of the natural endogeneous ligand for the growth hormone secretagogoue receptor. Trends Endocrinol. Metab.
14. Korbonits, M., A. P. Goldstone, M. Guerguiev, and A. B. Grossman. Ghrelin-a hormone with multiple functions. Front. Neuroendocrinol.
15. Lebenthal, Y., G. Gat-yablonski, B. Shtaif, A. Padoa, M. Phillip, and L. Lazar. Effect of sex hormone administration on circulating ghrelin levels in peripubertal children. J. Clin. Endocrinol. Metab.
16. Liu, Y. L., S. Yakar, V. Otero-corchon, M. J. Low, and J. L. Liu. Ghrelin gene expression is age-dependent and influenced by gender and the level of circulating IGF-I. Mol. Cell. Endocrinol.
17. Marzullo, P., B. Verti, G. Savia, et al. The relationship between active ghrelin levels and human obesity involves alterations in resting energy expenditure. J. Clin. Endocrinol. Metab.
18. Matsudo, S. M. M., and V. K. R. Matsudo. Self assessment and physician assessment of sexual maturation
in Brazilian boys and girls: concordance and reproducibility. Am. J. Hum. Biol.
19. Muccioli, G., M. Tschöp, M. Papotti, R. Deghenghi, M. Heiman, and E. Ghio. Neuroendocrine and peripheral activities of ghrelin: implications in metabolism and obesity. Eur. J. Pharmacol.
20. Nakazato, M., N. Murakami, Y. Date, et al. A role for ghrelin in the central regulation of feeding. Nature
21. Oh, K. W., W. Y. Lee, E. J. Rhee, et al. The relationship between serum resistin, leptin, adiponectin, ghrelin levels and bone mineral density in middle-aged men. Clin. Endocrinol.
22. Ong, K. K., M. L. Ahmed, and D. B. Dunger. The role of leptin in human growth and puberty. Acta Paediatr.
23. Park, H. S., K. U. Lee, Y. S. Kim, and C. Y. Park. Relationships between fasting plasma ghrelin levels and metabolic parameters in children and adolescents. Metabolism
24. Pomerants, T., V. Tillmann, J. Jürimäe, and T. Jürimäe. Relationship between ghrelin and anthropometrical, body composition parameters and testosterone levels in boys at different stages of puberty. J. Endocrinol. Invest.
25. Pomerants, T., V. Tillmann, K. Karelson, J. Jürimäe, and T.Jürimäe. Ghrelin response to acute aerobic exercise
in boys at different stages of puberty. Horm. Metab. Res.
26. Popovic, P., and L. H. Duntas. Leptin TRH and ghrelin: influence on energy homeostasis at rest and during exercise
. Horm. Metab. Res.
27. Saito, M. T. Sexual maturation
: self-evaluation of the adolescent. Pediatrica
28. St-Pierre, D. H., A. D. Karelis, K. Cianflone, et al. Relationship between ghrelin and energy expenditure in healthy young women. J. Clin. Endocrinol. Metab.
29. Tanner, J. Growth at Adolescence
. 2nd ed. Oxford, United Kingdom: Blackwell Scientific Publications, 1962.
30. Thong, F. S., C. Mclean, and T. E. Graham. Plasma leptin in female athletes: relationship with body fat, reproductive, nutritional, and endocrine factors. J. Appl. Physiol.
31. Tschöp, M., D. Smiley, and M. Heiman. Ghrelin induces adiposity in rodents. Nature
32. Vivenza, D., A. Rapa, N. Castellino, et al. Ghrelin gene polymorphisms and ghrelin, insulin
, IGF-I, leptin and anthropometric data in children and adolescents. Eur. J. Endocrinol.
33. Whatmore, A. J., C. M. Hall, J. Jones, M. Westwood, and P.E.Clayton. Ghrelin concentrations in healthy children and adolescents. Clin. Endocrinol.
34. Wilding, J. P. H. Neuropeptides and appetite control. Diabet. Med.
35. Wren, A. M., C. J. Small, H. J. Ward, et al. The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion. Endocrinology
36. Zhang, Y., R. Proenca, M. Maffei, M. Barone, L. Lori, and J.M. Friedman. Positional cloning of the mouse obese gene and its human homologue. Nature
Keywords:©2007The American College of Sports Medicine
MATURATION; EXERCISE; INSULIN; LEPTINE; ESTRADIOL