*This work was supported by grant from Polish Ministry of Science and Higher Education nr NN 407173534.

INTRODUCTION

Adverse health effects of simple obesity in children and adolescents require efficient dietary treatment connected with sufficient physical activity. Excessive weight causes metabolic disorders, orthopaedic problems and in the future can also influence the health of the adult (1). Anthropometric parameters may not identify all positive changes associated with lifestyle modifications. The concentrations of leptin and leptin receptor may have important physiological and therapeutical implications for human obesity. Leptin is a 16-kDa protein hormone composed of four α-helices and two short β-strands that contain an intra-chain disulphide bond responsible for its biological activity (2). Leptin is primarily secreted by adipose tissue and circulates in serum as free and bound forms. This protein plays a role in the control of body fat stores through coordinated regulation of feeding behavior, the autonomic nervous system and body energy balance (3). Higher leptin concentrations have been reported in obese than in lean children and a positive correlation has been found with BMI, fat mass and a negative correlation with fat-free mass (4-6).

Leptin plays an important role in appetite regulation and food intake by interaction with the specific receptor in the brain (OB-R). The leptin receptor, a member of the cytokine receptor family, exists in several isoforms (7). A soluble form of leptin receptor (sOB-R) represents the main leptin-binding activity in human blood. Circulating sOb-R is derived from ectodomain shedding of membrane-bound receptors. It modulates steady-state leptin levels by completing free leptin in the circulation and may prevent the hormone from degradation and clearance (8).

It is widely recognized that lifestyle intervention, including modification of nutritional habits and physical activity, is the most important therapy to reduce weight excess in childhood and adolescence. The relationship between leptin, sOB-R and this form of lifestyle intervention in obese children is not completely understood. Several studies have shown a relationship between leptin levels and energy balance in obese children. however the results are inconclusive with leptin levels that either decrease (9-12), increase (13) or remain unchanged (14) after exercise and/or dietary intervention.

The aim of this study was to investigate if leptin and soluble leptin receptor can be clinically useful markers for the monitoring of therapy efficacy in prepubertal obese children.

MATERIAL AND METHODS

The changes in clinical, anthropometric and metabolic parameters including leptin and leptin receptor in 26 prepubertal patients (mean age±SD 7.6±1.6 years) before and after the 3-months intervention programme were determined. Additionally, control visits of patients took place after 6 weeks of therapy. Healthy normal-weight children (n=30; mean age±SD 7.8±2.0 years) were the reference group. Physical examination was performed and body mass index (BMI) calculated (Table I). Children were considered as obese (z-score BMI >2) and non-obese (z-score BMI <-1+1>). Fat mass was measured by dual-energy X-ray absorptiometry (DXA). Patients with endocrine disorders or genetic syndromes, including syndromic obesity were excluded. Obese and non-obese children who were taking medications that could affect growth, pubertal development, nutritional status or dietary intake were not included. The lifestyle intervention programme consisted of dietary and physical activity modifications and behaviour therapy including individual psychological care of the child and its family. The dietary guidelines, recommending the low-energy diet based on a balanced distribution of carbohydrate, proteins and lipids for children and their parents were described in a previous study (15). The recommended daily intake from low-energy diet was 1200-1400 kcal/day (Tab. I). Patients had 3-5 meals every day. The dietary intake and physical activity data were collected using randomly selected 3-day records before and after treatment. Average daily food rations and their nutritional value were calculated using nutritional computer programme (Dieta2®, National Food and Nutrition Institute, Warsaw). Physical activity of children before and during therapy were estimated by their parents according to a questionnaire (15). Patients obtained the instruction concerning physical exercises which were described by Oblacińska and Weker (16). The children were advised to reduce sedentary behaviour including television and computer games to less than two hours a day.

Venous blood samples were collected between 8-10 am after overnight fasting and centrifuged (1000 g for 10 min at 4oC). Serum levels of leptin and leptin receptor were determined using ELISA kits (DRG, Germany). Glucose, total cholesterol, LDL and HDL-cholesterol, triglycerides were measured by kits from Roche (Switzerland). All parameters were determined twice in obese children before and after 3-months therapy and once in the control subjects. The Statistica (version 8.0) computer software was used for statistical analysis and the differences were regarded as significant at p<0.05.

This study has been approved by the Ethics Committee of the Institute of Mother and Child.

RESULTS

The average daily food rations of studied obese children are presented in Table I. The proportions of protein, fat and carbohydrates in energy intake of these diet were different than recommended daily intake. The diet of obese children was higher in protein and fat intake by about 15% and 10%, respectively. After 3-months of therapy energy intake in the diets of these patients were lower by about 30% (p<0.001). In average daily food ration, energy from protein was higher by about 15%, but that from fat was lower by about 10%.

In Table II clinical characteristics of prepubertal obese children and normal-weight controls are shown. Obese children had higher average BMI by about 50% and 2-fold higher fat mass than age-matched controls. We observed normal mean values of glucose, total cholesterol, HDL- and LDL-cholesterol and triglycerides in obese patients before and after the lifestyle intervention programme (Tab. III). However, the concentration of triglycerides was about 15% lower after therapy than before and it was similar to the value obtained in normal-weight children.

Serum leptin and leptin receptor concentrations in patients and controls are shown on Figure 1. The mean level of leptin in patients before treatment was about 6-fold higher (p<0.0001) but the mean value of sOB-R was about 2-fold lower (p<0.0001) than in controls. In obese patients we observed positive correlation between percent of fat mass and concentrations of leptin (r= 0.501, p<0.01) and negative with concentration of leptin receptor (r=-0.560, p<0.005).

After 3-months therapy lower concentrations of leptin in obese children by about 50% (p<0.001) and higher of sOB-R by about 20% (p<0.05) in comparison to baseline were observed (Fig. 1). Significant negative correlations were obtained between both markers before (r= -0.506, p<0.001) and after therapy (r= -0.572, p<0.001).

Mean value of BMI was lower by 10% (p<0.05) in obese children after modification of their diet and physical activity (Fig. 2). The correlations between BMI and leptin, leptin receptor before (r=0.379, r=-0.401; p<0.001) and after therapy (r=0.531, r= - 0.500; p<0.001) were found.

DISCUSSION

In agreement with other authors our results have demonstrated significantly higher leptin levels and lower sOB-R levels in prepubertal obese children than in prepubertal normal-weight controls (4-6). Moreover, in obese children we observed correlations between both biochemical and anthropometric parameters such as BMI and percentage of fat mass.

Dielan et al. (17) found higher sOB-R concentrations in lean compared to obese individuals. It is known that, sOB-R in humans is considered as a potential reservoir for bioactive leptin, which only in free form can act on target sites to elicit biological responses (18). When sOB-R concentration decrease in obese individuals, there may be higher concentrations of liberated leptin and in overall higher than normal circulating leptin concentration. According to van Dielan et al. (17) and Sinha et al. (19) in lean subjects leptin circulates mainly in the bound form, whereas in obese subjects the majority of leptin circulates in the free form. Higher concentration of leptin and elevated leptin to sOB-R ratio were found in obese adults (17).

In our study leptin to sOB-R ratio in prepubertal obese children (1.48) was also higher than in the reference group (0.14). The changes of sOB-R and leptin levels were significantly correlated to the changes of body weight and percentage of body fat, pointing to a link between fat mass and the level of these biochemical markers. After 3-months weight reduction programme the ratio of leptin to sOB-R in obese children was about 3-fold lower (0.53) than before intervention (1.48), but still higher as compared to the normal-weight controls (0.14). However, our results suggest that short-time intervention may have been sufficient to detect greater changes in biochemical and anthropometric parameters. In obese children with a significant reduction in weight a marked increase of leptin receptor and the decrease of leptin concentrations were found. The lower levels of sOB-R before therapy may indicate the expression decrease of functional leptin receptors and be a sign of leptin resistance (20). Our results suggest that this type of leptin resistance is reversible and therefore does not seem to cause obesity since sOB-R levels increased in obese children after weight loss. Similar changes concerning these both parameters in obese children after exercise intervention programmes with and without dietary modification were observed by other authors (9, 21, 22).

Recent studies of Cambuli et al. (13) and Reinehr et al. (14) show that leptin cannot be used as a reliable biomarker of positive metabolic outcomes of lifestyle intervention in overweight and obese children. According to Cambuli et al. (13) changes of leptin levels are in disagreement with the positive outcomes after intervention. Leptin remained higher at 1 year of follow-up in overweight and obese children and increased significantly in those children without weight loss, but also in those children who did loose weight. No significant changes from baseline were observed, suggesting that leptin does not follow directly the changes in body weight. Reinehr et al. (14) showed that after 1-year therapy leptin levels did not significantly change in obese children that lost weight, and increased in those that did not change their weight status. This increase and the lacking of significance in the decrease of leptin levels in children with substantial weight loss can be connected with duration of studies. It is known, that leptin concentrations increase with age and pubertal development due to sex hormones (23). Moreover, in the above studies obese children were at the beginning of intervention at the age 10.7±3.2 years and 10.0±2.3 years and changed pubertal status during the follow-up. The increment of leptin value is expected in puberty and it is not modified by lifestyle intervention.

Contrary to Cambuli et al. (13) and Reinehr et al. (14), in our study leptin concentrations correlated to sOB-R levels in prepubertal obese children (at the age 7.6±1.6 years) at baseline, and most importantly, the changes of leptin levels correlated to the changes of sOB-R values in these patients after therapy. Since the changes of leptin and sOB-R concentrations in obese children tend to normalize after substantial weight loss in concordance with studies in adults, these changes are the consequence rather than the cause of overweight.

CONCLUSION

Our study confirms the positive effects of simple dietary and physical activity recommendations on metabolic and clinical parameters in obese children. We suggest that leptin and soluble leptin receptor concentration may identify the changes in metabolism associated with weight loss in obese children after short-term lifestyle intervention programme. Longitudinal observation of the studied patients is continued in order to confirm the value of these biochemical markers in the management of prepubertal obese children.

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Corresponding author:

Joanna Gajewska
Department of Newborn Screening,
Institute of Mother and Child
ul. Kasprzaka 17a, 01-211 Warszawa
tel. (+48 22) 32-77-260
biochem.imid@imid.med.pl