Introduction

Leptin is mainly secreted by adipose tissue and appears as an important regulator of energy balance. Circulating leptin concentrations correlate with body mass index (BMI) and even more highly with body fat content both in adults^{1, 2} and in children.^{3, 4} However, for a given level of adiposity, there are large interindividual variations in plasma leptin levels and many factors appear to affect it in addition to body fatness. Females have higher leptin concentrations compared to males.⁴ Leptin is also sensitive to changes in energy intake: fasting reduces leptin concentrations within 2 days, even in the absence of any significant change in body fat.⁵ Conversely, food intake stimulates leptin secretion in man.⁶ The relationship between leptin levels and energy expenditure remain unclear.⁷ Energy expenditure related to physical activity is the most variable part of total energy expenditure. There is some evidence that the habitual level of physical activity may be a significant determinant of leptin concentrations, independent of body fat content. In adults, higher levels of physical activity were associated with lower BMI-adjusted plasma leptin levels.^{8, 9} In children, there are discrepancies among studies. Nonsignificant,^{3, 10} positive,¹¹ and negative¹² relationships between plasma leptin and energy expenditure were reported. These discrepancies could be related to the fact that puberty stages, which affect leptin levels in children,^{13, 14, 15} were not taken into account. In addition, physical activity can decrease insulin levels^{7, 16} and improve insulin sensitivity¹⁷ and these parameters are associated with plasma leptin concentrations, both in adults¹⁸ and in children.¹⁹

The primary aim of the present study was to assess in a population-based sample of children at different pubertal stages the relationships between leptin levels and habitual physical activity, taking account others factors, which may affect leptin, such as adiposity, pubertal stage and fasting plasma insulin levels.

Subjects and methods

Subjects

A total of 579 families who had at least one child attending primary schools in 1992–1993 at Fleurbaix and Laventie (two towns in Northern France) were enrolled in 1992 in the Fleurbaix–Laventie Ville Santé (FLVS) II Study.²⁰ All the families still living in this area in 1999 (393) were invited to participate in the FLVS II Study, a longitudinal study of the determinants of weight gain among children and their parents, and 294 responded positively (75%). Study subjects were the 513 children (256 girls and 257 boys), aged 7–18 y, of the families participating in the FLVS II study. This study received the approval from the Ethical Committee at Lille University.

The data presented here were gathered at baseline examination of FLVS II in 1999. Anthropometric measurements and venous blood sampling were performed at home, between 0630 and 0730 hours, in the postabsorptive state.

Anthropometric measurements

Body weight was measured to the nearest 0.100 kg and height was measured to the nearest 0.5 cm. Skinfold thicknesses (bicipital, tricipital, subscapular and suprailiac skinfolds) were measured by trained investigators on the left side of the body by using a Harpenden skinfold caliper. Measurements were performed twice and averaged. The sum of the four skinfolds (SSK) was used as an indicator of subcutaneous adiposity. Pubertal stage was assigned by physical examination according to the criteria of Tanner.²¹

Biochemical assays

Glucose was determined by an enzymatic reaction (hexokinase method, Randox Laboratories Ltd, UK). Hormone levels were determined using commercially available assays. Insulin was measured by radioimmunoassay (Bi-Insulin RIA, Pasteur, France). Leptin levels were measured by radioimmunoassay (Human leptin RIA, kit DSL-23100, Webster USA GMBH, Germany). The lower limit of sensitivity of the assay is 0.10 ng/ml. For mean values of 2.75 ng/ml, the intra- and interassay variability were 3.7 and 6.6%, respectively.

Assessment of physical activity

Past-year leisure-time physical activity (LTPA) was assessed by using the Modifiable Activity Questionnaire (MAQ) for adolescents that was administered by trained interviewers. The younger children answered with the help of their parents. The MAQ for adolescents has been described in details.²² Validation and reliability testing of this questionnaire was previously published.²³ A French version, similar to the one used in this study, was published by French investigators.²⁴ Briefly, the questionnaire assesses the number of weekly hours spent in physical education classes and in being physically active outside school. Based on a list of leisure or sport activities common in the population under study, subjects identify activities performed at least 10 times in the past 12 months. Detailed information about the duration and frequency of each leisure activity is then collected. Total past-year leisure activity is expressed as hours per week. Ambulatory activity was concurrently measured by an electronic pedometer (Yamax, Digiwalker, Yamax, Japan). The pedometer was worn at the waist over the hip from morning to evening during seven consecutive days. The total number of steps recorded over the week was divided by seven to obtain an average daily step count. The Yamax Digiwalker was selected because previous studies²⁵ have shown that it was more reliable than other devices to record walking distances.

Statistical analysis

To normalize distributions, logarithmic transformations were performed for leptin and insulin concentrations and SSK. Analyses were carried out separately by gender. Anthropometric, biochemical and physical activity variables were compared across Tanner stages by analyses of variance. Leptin was compared across categorical variables (Tanner stages, quarters of daily step count and of weekly LTPA), by analysis of variance and covariance after adjustment for covariates when appropriate. Univariate relationships between leptin concentrations, anthropometric variables and insulin concentration were assessed by Pearson correlation coefficients. Multiple linear regression was used to determine whether physical activity variables were associated with leptin concentration independently of fasting insulin concentration and other covariates. Familial correlations between sibs were taken into account by using hierarchical models with a familial random effect at the level of intercept (Proc Mixed of SAS version 8.2 Cary, NC, USA).

Results

The description of the children according to pubertal stages is given in Tables 1 and 2.

Table 1 - Subjects characteristics according to Tanner stage (boys).

Full table

Table 2 - Subjects characteristics according to Tanner stage (girls).

Full table

In boys, plasma leptin concentrations differed according to pubertal stages: they were higher for stage 1 and 2, and decreased thereafter. The influence of pubertal stage remained significant after adjustment for age and SSK (P<0.03). In girls, leptin concentrations steadily increased with pubertal stages, but the relation was no longer significant after adjustment for SSK. Unadjusted relationships (Table 3) show that leptin was significantly correlated with age, BMI, SSK and insulin in both genders.

Table 3 - Unadjusted Pearson correlation coefficients between leptin and several variables.

Full table

Influence of physical activity (Figure 1 and 2)

In boys, after adjustment for age, SSK and Tanner stage, plama leptin concentrations were related neither to hours per week of physical activity as assessed by questionnaire, nor to steps per day as assessed by the pedometer (Figure 1 and 2).

Figure 1.

Plasma leptin concentrations (ng/ml), in boys (white bars) and girls (hatched bars), adjusted for age, tanner stage and SSK, according to levels of physical activity assessed by pedometer (steps/day): For trend: P=0.15 in boys; P=0.009 in girls. Cutoff points for quartiles were respectively, 7700, 9850, 11900, for boys and 6450, 8100, 9751, for girls.

Full figure and legend (15K)

Figure 2.

Plasma leptin concentrations (ng/ml) in boys (white bars) and girls (hatched bars), adjusted for age, tanner stage and SSK, according to LTPA (h/week). For trend: P=0.92 in boys; P=0.003in girls. Cutoff points for quartiles were, respectively, 3 h 12 min, 5 h 12 min, 8 h 44 min, for boys and 1 h 45 min, 3 h 13 min and 5 h 30 min for girls.

Full figure and legend (18K)

In girls, plasma leptin concentrations decreased with physical activity, these differences remained significant after adjustment for SSK, Tanner stage for both LTPA (P<0.005) and steps/day (P<0.002).

Multivariate regression analysis was performed by gender with leptin as a dependent variable and Tanner stage, SSK, insulin and number of steps/day as independent variables (Table 4). In both genders, the best predictor of leptin was SSK. Insulin was associated with leptin levels in both genders independently of other covariates. Higher ambulatory activity levels were associated with lower leptin levels in girls only: the girls in the three lowest quartiles of physical activity have higher leptin levels (as indicated by positive coefficients) than girls in the highest quartiles, which is the reference quartile. Similar results were obtained with LTPA as an indicator of physical activity in the multivariate analysis (not shown).

Table 4 - Multivariate association between plasma leptin^a and plasma insulin, SSK, age, Tanner stage and quartiles of increasing number of steps per day.

Full table

When multivariate regression analysis were performed with insulin as dependant variable, physical activity was negatively correlated to insulin in boys, this association persisted after adjustment for pubertal stage but disappeared after adjustment for SSK, this relationship was not found in girls (data non shown).

Discussion

Our results confirm the differential pattern of changes in plasma leptin across stages of puberty in boys and girls. In girls, there is a steady increase in leptin levels that parallels the increase in adiposity, whereas in boys, leptin concentrations reach a peak at Tanner stage 2 and then decrease independently of changes in adiposity.^{4, 12, 13, 14, 26} This study also confirms in a population of healthy children that plasma insulin is associated with leptin levels independently of body fat. In adults, plasma leptin concentration were found to be related to fasting insulin, independently of fat mass in several reports.^{18, 27, 28} In children there are fewer studies and this relationships was found in some¹⁹ but not all studies.²⁹ These observations are in accordance with experimental studies showing that physiological insulin levels acutely increase plasma leptin.³⁰ As described in other studies, fasting insulinemia was associated with physical activity,¹⁷ this relationship was significant only in boys and disappeared after adjustment for fat mass. It has been suggested¹⁷ that using fasting insulin underestimates the magnitude of the potential for physical activity to improve insulin sensitivity, which could explain that we failed to detect such a relationship.

The major finding of this study is the association between leptin levels and physical activity in girls, independently of pubertal stage and adiposity. In boys, no such relationship was found. However, their lower leptin concentrations, and, in some slim boys, the proximity of the sensitivity threshold may have hampered the detection of the effects of physical activity. It has also been suggested that the MAQ might be more valid and reliable in girls than boys.²³ However in boys, ambulatory physical activity, as assessed by pedometer recordings, did not appear related to leptin levels, either. Few studies have considered the relationships between leptin levels and physical activity in children. In 127 5-y old Pima Indian children, a positive relationship between leptin concentrations and physical activity levels calculated as the ratio of total energy expenditure on resting metabolic rate (TEE:RMR) was described.¹¹ Other studies failed to find any association between total or resting energy expenditure and leptin concentrations in prepubertal children. However, in these cases, either the number of enrolled subjects was insufficient,³ or puberty stages were not taken into account.¹⁰ In adults, experimental as well as epidemiological studies have been performed to study the relationships between leptin and physical activity and also revealed a gender difference. In adult males, several studies have described negative relationships between leptin concentrations and physical activity, but in these studies there was no adjustment for fat mass.^{8, 9} When fat mass is taken into account, daily physical activity^{23, 31, 32} as well as moderate intensity exercise training^{31, 33} are associated with changes in leptin levels in females but not in males. Exercise produces changes in leptin levels in men only in case of strenuous exercise³⁴ or when a concomitant modification of the dietary intake results in an energy deficit.³⁵ These data suggest that plasma leptin levels are more sensitive to change in energy balance in females that in males. Low levels of plasma leptin independent of fat mass have been described in young athletes, and are associated with a delayed puberty in girls but not in boys.³⁶ Decreasing leptin levels by physical activity may help females maintain the long-term energy balance by allowing adjustment of energy intake to energy expenditure.

This study is the first to describe gender differences in the plasma leptin in relation to daily physical activity in children, independently of pubertal stage. This difference was established in a sample of healthy children by using two different methods to assess physical activity, both giving concordant results.

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Acknowledgements

Financial support for the study was obtained from: Knoll, CEDUS, Groupe Fournier, Lesieur, Nestlé France, Produits Roche and TEPRAL. We express our gratitude to Agnes Lommez for technical assistance and Nadine Thibult for statistical help.