Introduction

Obesity is a health risk frequently associated with complications such as type 2 diabetes, dyslipidemia, high blood pressure, abnormal fibrinolysis and cardiovascular disease.^1,2,3,4 The syndrome known as metabolic syndrome or insulin resistance is associated with high plasma leptin concentrations⁵ and/or leptin resistance.

Insulin resistance is one of the main disorders present in obese individuals.⁶ The presence of hyperinsulinism and insulin resistance has been described in obese children of both sexes (mean age 10 y), using a hyperinsulinemic euglucemic clamp.⁷ Some of the disorders related to this syndrome have been described in children, suggesting that it may have a pre-pubertal origin.^{8,9,10,11,12,13,14}

A positive correlation has been described in children between insulin and plasma leptin concentrations.¹⁵ Independent of obesity, insulin resistance is associated with a high concentration of leptin,¹⁶ which suggests that persistently high plasma levels of this hormone stimulate leptin production.

In vitro leptin, meanwhile, lessens the action of insulin on isolated liver cells,¹⁷ increases the oxidation of fatty acids and the depletion of triglycerides in adipocytes,¹⁸ reduces the union of insulin and its receptors in adipocytes,¹⁹ and possibly inhibits the secretion of insulin from the pancreas.²⁰

Leptin may therefore play a role in modulating the action of insulin. The independent correlation between leptin and insulin sensitivity has been described. Leptin has been related to different variables of metabolic syndrome, however the results concerning the possible relationship between these variables and leptin have not been sufficiently addressed,^21,22,23,24 particularly in pre-pubertal obese children.

A recent study shows that leptin is an independent risk factor for coronary heart disease²⁵ and may participate in metabolic syndrome.

Hyperleptinemia and/or leptin resistance may be an important component in the etiopathogenesis of metabolic syndrome by participating in its origin either directly or by regulating insulin sensitivity.

The present study aims to analyze the variables involved in the increase of plasma leptin concentration in child obesity, and to assess the influence of leptin on the various components of metabolic syndrome.

Subjects and methods

Subjects

A case-control study was carried out in obese children of both sexes. One group comprised 41 obese children (body mass index (BMI) over 90th percentile in growth curves for the study population,²⁶ and the control group comprised the same number of non-obese children paired by age and sex (aged six–nine y). All subjects were at Tanner stage 1.

Several schools in the area were informed of the study. All parents submitted written consent and the study was authorized by the ethical investigation committee of our hospital.

Children with primary hyperlipidemia, hypertension, diabetes or glucose intolerance, and secondary obesity were excluded from the study. Any child receiving pharmacological treatment was also excluded. All children had similar lifestyles, with no significant physical training program.

Blood sampling and analysis

Blood samples were collected after 12 h of fasting from a vein in the antecubital fossa, without venous occlusion. All collections were made between 8.00 and 9.00 am. Whole blood specimens were collected in different tubes to obtain serum and plasma. The samples was separated into aliquots and frozen immediately at -45 °C until analyses could be performed.

The basal leptin concentration was determined for all the children, as well as different variables related to metabolic syndrome (insulin, lipids, blood pressure, hydrocarbonate metabolism, hemostasia, sex hormone binding globulin (SHBG), uric acid and biochemical markers of the intra-abdominal fat deposit).

Glucose, uric acid, alanine aminotransferase (ALT), cholinesterase (ChE), cholesterol and triglycerides (TG) were determined in a random access analyser (Axon, Bayer Diagnostics, Tarrytown, NY, USA) with Bayer Diagnostics reagents.

High-density lipoprotein cholesterol (HDL-C) was determined after precipitation of chylomicrons, very low-density lipoproteins and low-density lipoproteins, with phosphotungstic acid and magnesium ions. The concentration of low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald formula.²⁷ The non-esterified fatty acids (NEFA) were quantified by a colorimetric enzyme assay (NEFA C ACS-ACOD Method, Woko Chemicals GmbH, Nissanstr, Germany).

Insulin was quantified using a microparticle immunoassay (IMx system Insulin, Abbott Laboratories, Chicago, IL, USA) in an IMx automatic analyser (Abbott Laboratories, Chicago, IL, USA). Apolipoprotein A-I (Apo A-I) and apolipoprotein B (Apo B) were measured by nephelometry (N Antisera to Human Apolipoprotein A-I and Apolipoprotein B reagent, Behringwerke AG, Marburg, Germany) in a Dade Behring Analyzer II Nephelometer.

The sex hormone binding globulin was quantified using an enzyme immunoassay (Radim S.A., Liège, Belgium) in a microtiter plate analyser (Labotech, Chemila, Rome, Italy).

Antigenic immunoassay methods were used for the quantification of tissue-plasminogen activator (t-PA) (Coliza t-Pa, Chromogenix AB, Mölndal, Sweden), plasminogen activator inhibitor-1 (PAI-1) (Asserachrom PAI-1, Stago Diagnoses, Asnieres-south-Seine, France) and leptin (Quantikine human leptin, RD systems, Wiesbaden-Nordenstadt, Germany), carried out in a microtiter plate analyser (Labotech, Cormédica, Barcelona, Spain). Fibrinogen was measured by quantitative assay using thrombin in an automatic analyser (Electra 1600, Ortho Clinical Diagnostics, Madrid, Spain).

Anthropometric measurements

Weight was measured to the nearest 0.1 kg and height to the nearest 0.1 cm. The BMI was calculated as weight (kg)/height (m)². Waist circumferences were measured at the level of the umbilicus and hip circumferences at the level of the greater trochanters and pubic symphisis to the nearest 0.1 cm.

Blood pressure

The blood pressure of all the children was measured with a mercury sphygmomanometer (Pymah Corporation, Sommerville, NJ, USA) after 20 min of rest and in a supine position. One measurement was taken on each of three days, and the mean calculated.

Statistical analysis

Statistical assessment was conducted using Microstat (Ecosoft, Indianapolis, Inc.) or GraphPAD InStat (GraphPAD Software, San Diego, CA). Abnormal values (outliers) were excluded. Results were expressed as means.e.m. with a 95% confidence interval (95% CI). The distribution of each variable was tested for departure from Gaussian distribution and variance equality was controlled by Snedecor's F-test. The mean values of the groups were compared using Student's unpaired t-test. Statistical significance was set at P<0.05.

Correlation between variables was evaluated using Pearson's correlation coefficient and regression analysis. Multivariant regression analysis was performed using the Stepwise method. For each variable, potential confounding factors (0.05<P<0.2) were evaluated by an analysis of raw and adjusted regression coefficients.

Results

Table 1 shows the anthropometric data of both groups. The median age was 7.89 y (obese) and 7.85 y (control), with a range of 6–9 y.

Table 1 - Descriptive statistic and selected biochemical parameters of the study groups.

Full table (19K)

Mean plasma leptin concentration was significantly higher in the obese children, at 15.47 ng/ml (95% CI 12.9–18.05), in comparison to the control group, at 4.73 ng/ml (95% CI 3.39–6.08; Table 1). In the obese group, sex-related differences in plasma leptin concentrations were evaluated (boys 12.93 ng/ml2.08 vs 17.10 ng/ml1.64 girls; P=0.122). The comparisons between the biochemical parameters related to metabolic syndrome are summarized in Table 2.

Table 2 - Selected biochemical parameters related to metabolic cardiovascular syndrome.

Full table (27K)

Leptin vs insulin and BMI

In the single linear correlation, for the obese group, plasma leptin concentration was positively correlated with insulin (r=0.5779; P<0.001) and BMI (r=0.5382; P<0.001), but not with WHR. Using multivariant regression analysis, insulin (P partial=0.003) and BMI (P partial=0.018) were independent predictive factors for plasma leptin concentration (R=0.449; P<0.001). Leptin (P partial=0.002), corrected for BMI and WHR, is also an independent predictive factor for basal insulin concentration (R=0.3452; P<0.001).

In the control group, insulin (r=0.5838; P<0.001) and BMI (r=0.5193; P<0.001) also correlated with leptin, and both are independent predictive factors. As occurs in the obese group, leptin (P partial<0.001) corrected for BMI and WHR is an independent predictive factor for basal insulin (R=0.4335; P<0.001).

Figure 1 shows the single linear correlation of leptin with BMI and insulin for all of the children (obese and non-obese). In the combined group, corrected for BMI and WHR, leptin (P partial<0.001) is an independent predictive factor for basal insulin (R=0.4485; P<0.001).

Figure 1.

Serum leptin concentrations as a function of insulin (r=0.6636; P<0.001) and BMI (r=0.7525; P<0.001) in obese and non-obese (together) children.

Full figure and legend (30K)

Leptin and metabolic syndrome

With regard to the metabolic syndrome (MS) components, the univariant correlation analysis is summarized in Table 3 (obese) and Table 4 (obese and non-obese together). In the obese group, plasma leptin concentrations correlate positively with triglycerides, PAI-1 and t-PA, and negatively with Apo A-I, HDL-C and SHBG. Blood pressure, cholesterol, LDL-C, Apo B and NEFA levels did not correlate with leptin. The study with multivariant regression analysis showed that leptin corrected for BMI and insulin is not an independent predictive factor for the MS variables analysed.

Table 3 - Coefficients of simple correlation (r) between different variables of the obese group.

Full table (29K)

Table 4 - Coefficients of simple correlation (r) between different variables of the total group (obese and non-obese together).

Full table (31K)

In the obese group, using stepwise multivariant regression analysis, insulin was selected as a predictive variable for triglycerides, Apo A-I, ALT and SHBG, just as BMI was for HDL-C, ChE, PAI-1 and t-PA. Plasma leptin concentration was not selected as a predictive variable for the analysed metabolic syndrome parameters (with the exception of insulin).

In the control group plasma leptin concentration correlated positively with triglycerides (r=0.4244; P<0.01), ALT (r=0.4227; P<0.01), ChE (r=0.3272; P<0.05), t-PA (r=0.3121; P<0.05) and fibrinogen (r=0.4981; P<0.01), and negatively with SHBG (r=-0.4306; P<0.01). As occurs with the obese children, in the control group plasma leptin concentration was not correlated with blood pressure, cholesterol, LDL-C or Apo B levels. The study with multivariant regression analysis showed that plasma leptin is not an independent predictive factor for the variables studied. In the combined group (obese and non-obese together), plasma leptin (Table 4) correlates positively with triglycerides, SBP, ALT, ChE, PAI-1, t-PA and fibrinogen, and negatively with HDL-C and SHBG. The study with multivariant regression analysis showed that plasma leptin corrected for BMI and insulin is not an independent predictive factor for the MS variables analysed.

Discussion

Obesity is a chronic pathology with a high morbidity–mortality rate which, due to eating habits and the lifestyle of western civilization, may become a veritable epidemic in coming years. It is associated with hypertension and various metabolic disorders such as type 2 diabetes mellitus, dyslipidemia, hyperuricemia, inappropriate fibrinolysis and hyperinsulinemia.^{4,13,14,28,29,30}

Hyperinsulinemia and/or insulin resistance have been described as the key factors underlying the association of this group of metabolic disorders, known as metabolic syndrome or insulin resistance syndrome (IRS).^4,30

Peripheral insulin resistance is one of the main disorders present in obese subjects.^6,7 In obese children, high insulin levels and insulin resistance were described when compared with non-obese children.^7,31 Some metabolic alterations described in IRS are present in most obese children,^8,14,32 and begin at an early age.

As well as the disorders traditionally related to IRS we have described, in obese children in the same age group as those studied here, and in comparison with non-obese children, significantly high levels of PAI-1, t-PA, fibrinogen and insulin.¹³ We have also observed a significant decrease of SHBG.¹² All these parameters are related to IRS.

Obesity is frequently associated with high plasma leptin concentrations and leptin resistance. A positive correlation has been described in children between levels of insulin and leptin.¹⁵ The concentration of leptin in serum is independently correlated with insulin sensitivity.²¹ Elevated plasma leptin levels strongly predict first-ever acute myocardial infarction in men³³ and recently it has been shown as an independent risk factor for coronary heart disease.²⁵

In our study we describe, in pre-pubertal obese children of both sexes, high concentrations of plasma leptin, which are dependent both on insulin concentration and on BMI. For the group of non-obese children, leptin is also correlated with insulin and BMI, suggesting that both parameters are determinants of leptin concentration in both physiological and pathological situations. In the combined group (obese and non-obese together), leptin is also correlated with insulin and BMI. For this age group, fat distribution (WHR) appears to have less influence on this syndrome. The leptin–insulin association therefore begins before puberty and may be connected to the onset of metabolic syndrome. In our results, leptin corrected for BMI and WHR is an independent predictive factor for the basal concentration of insulin.

Although PAI-1 has been correlated with leptin, indicating that leptin per se may increase PAI-1 concentration in adults aged 18–45 y,³⁴ other authors have not found it to be independently associated with the parameters of fibrinolysis.³⁵ Our results coincide with these findings, although our age group is lower than that studied by these other authors. In the single linear correlation analysis, for the obese group, leptin correlated positively with PAI-1, although in a multivariant regression analysis leptin is not an independent predictive factor for PAI-1.

Leptin has also been related to levels of glucose, uric acid and lipids.^21,22,23,24 For some authors leptin would intervene in the development of metabolic syndrome, even more than insulin.^21,22,23,24 Hypertension has also been related to high levels of leptin.^36,37,38

With the group of obese children that we studied, in a single linear correlation analysis, leptin correlated with different metabolic syndrome variables (insulin, triglycerides, HDLc, Apo-AI, SHBG and PAI-1); however leptin was not an independent predictive factor for any of them (except insulin).

Chronic insulin increase, described in obesity, favours an increase in leptin. Due to the action of leptin on insulin sensitivity (leptin favours the development of resistance to insulin and hyperinsulinism), it would perpetuate the increase of insulin and favour the onset of insulin resistance.

The risk of developing obesity-related diseases may be in part a function of resistance to both insulin and leptin among susceptible persons.

To summarize, for this age group high plasma leptin concentrations depend on both insulin and BMI levels. Hyperleptinemia and/or leptin resistance may be one more component of metabolic syndrome, and may be involved in its etiopathogenesis. The involvement of leptin in this syndrome may be indirect, modulating the insulin's action.

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Acknowledgements

This study was supported by grants from the Fondo de Investigación Sanitaria (FIS 183/97), Spanish Ministry of Health and the Andalusian Health Service (44/00). We thank Gabriela de Torres, Consuelo López, Carmen Segura and María Dolores Bonilla for their technical assistance.