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The relation of leptin and soluble leptin receptor levels with metabolic and clinical parameters in obese and healthy children
Peptides 56 (2014) 72–76
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The relation of leptin and soluble leptin receptor levels with metabolic and clinical parameters in obese and healthy children Gonul Catli a , Ahmet Anik a , Hale Ünver Tuhan a , Tuncay Kume b , Ece Bober a , Ayhan Abaci a,∗ a b
Department of Pediatric Endocrinology, Dokuz Eylul University Faculty of Medicine, Izmir, Turkey Department of Biochemistry, Dokuz Eylul University Faculty of Medicine, Izmir, Turkey
a r t i c l e
i n f o
Article history: Received 23 February 2014 Received in revised form 18 March 2014 Accepted 21 March 2014 Available online 2 April 2014 Keywords: Childhood Obesity Leptin Soluble leptin receptor Insulin resistance
a b s t r a c t We investigated the relation of serum leptin, soluble leptin receptor (sLR) and free leptin index (FLI) with metabolic and anthropometric parameters in obese and healthy children. Height, weight, waist circumference (WC), fasting serum glucose, insulin, lipid profile, leptin and sLR levels of 35 obese children and 36 healthy children were measured and FLI was calculated as the ratio of leptin to sLR. In obese children, serum leptin and FLI were found significantly higher, while sLR level was significantly lower than the healthy children. Comparison of obese children regarding the insulin resistance showed significantly higher serum leptin and FLI in the insulin resistant group; however sLR level was not different between the insulin resistant and non-resistant obese children. In obese children, sLR was not correlated with any of the metabolic parameters except total cholesterol, while FLI was significantly and positively correlated with BMI, WC, TC, fasting insulin, and HOMA-IR. However, regression analysis confirmed that the HOMAIR was the only independent variable significantly correlated with FLI in obese children. Findings of this study suggest that in obese children and adolescents (i) serum leptin and FLI were found significantly higher, while sLR level was significantly lower than the healthy children, (ii) increased FLI might be a compensatory mechanism for increasing leptin effect in peripheral tissue, (iii) FLI is a more accurate marker to evaluate leptin resistance than leptin or sLR alone, and (iv) increased FLI may contribute toward the development of hyperinsulinemia and insulin resistance. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Leptin, a cytokine which is secreted predominantly by the adipose tissue, is considered to be involved in satiety regulation in humans and animals [18]. In adults, circulating leptin levels are closely related to the amount of adipose tissue [16]. Leptin transports across the blood–brain barrier by a saturated system and acts within the brain regions, which regulate feeding behavior and thermogenic responses [2,5]. In addition to the above-mentioned effects, leptin also plays a role in the regulation of other body functions, including reproduction, liver and enteric metabolism, hematopoiesis, and immunity via leptin receptors, which are shown to be present in many peripheral tissues [5,26]. In humans, four different mRNA splice variants of the leptin
∗ Corresponding author at: Department of Pediatric Endocrinology, Dokuz Eylul University Faculty of Medicine, Balcova, Izmir 35340, Turkey. Tel.: +90 232 4126076; fax: +90 232 4126001. E-mail address: [email protected] (A. Abaci). http://dx.doi.org/10.1016/j.peptides.2014.03.015 0196-9781/© 2014 Elsevier Inc. All rights reserved.
receptor, including a membrane-bound receptor with a long cytoplasmic domain and three membrane-bound receptors with a short cytoplasmic domain have been identified. A circulating soluble form of the leptin receptor [soluble leptin receptor (sLR)], which is the main leptin-binding protein and determinant of free leptin index (FLI), also exists [3]. Although the source of sLR in the plasma is not known definitely, it (sLR) is generated by the proteolytic cleavage of membrane-anchored receptors, indicating that the leptin receptor might have other functions besides signal transduction [6,17,24]. In blood, leptin is suggested to circulate both in the free form as well as bound to sLR and possibly also to other as yet unidentified binding proteins [24]. Whilst binding of leptin with sLR has been suggested to increase the bioavailability of leptin in the plasma and to elongate its circulatory half-life [10,12,14,24,30], it was also proposed to decrease the free fraction of leptin, resulting in decreased binding of free leptin to the membrane-bound leptin receptors [15]. However, the role of the sLR in the regulation of the physiological function of leptin is until now unclear. Currently, limited data exists regarding the relation of sLR and FLI with metabolic and anthropometric parameters in childhood
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obesity. In this study, we aimed to investigate the relation of serum leptin, sLR and FLI with clinical and metabolic parameters in obese and healthy children.
2. Subjects and methods The study included 35 obese children with a body mass index (BMI) above 95th percentile, who applied to our department with the complaint of weight gain and 36 healthy children with a BMI below 85th percentile, who had similar age and gender distribution. Obesity was defined according to the data from the Centers for Disease Control and Prevention (CDC), 2000, growth charts. For calculation of BMI-standard deviation score (SDS), data from the CDC were used [13]. Before the outset of the study, all of the obese and healthy children underwent a thorough physical examination and laboratory evaluation including thyroid function tests and serum cortisol measurement for probable endocrine pathology. Those with chronic diseases (cardiovascular, gastrointestinal, and respiratory), a history of drug use (steroids and antipsychotics), endocrine pathology (Cushing syndrome and hypothyroidism), or syndromes associated with obesity (Prader–Willi and Laurence–Moon–Biedle syndromes) were excluded from the study. Height was measured using a Harpenden stadiometer with a sensitivity of 0.1 cm and weight was measured using a SECA scale with a sensitivity of 0.1 kg. The weight of each subject was measured with all clothing removed except undergarments. BMI was calculated by dividing weight (kg) by height squared (m2 ). Waist circumference (WC) was measured between the lowest rib and the iliac crest, horizontally through the narrowest part of the torso. The percentage of body fat and fat mass were measured using bioelectric impedance analysis (Tanita BC-418, Tokyo, Japan). Findings for pubertal development were evaluated according to Tanner staging [25]. A testicular volume of ≥4 mL in males and breast development of stage 2 and over in females were considered to be findings of puberty. Blood samples for glucose, insulin, lipids, thyroid function tests, cortisol, leptin and sLR levels were drawn after 10–12 h of overnight fasting. The plane tubes were centrifuged at 1200 × g 10 min and serum samples were removed from clots into the Eppendorf tubes using plastic Pasteur pipettes. They were stored at −80 ◦ C until analysis. Serum glucose, triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) concentrations were measured enzymatically using DP Modular Systems (Roche Diagnostic Corp., Indianapolis, IN). Low-density lipoprotein cholesterol (LDL-C) levels were calculated using the Friedewald formula when plasma TG’s were <400 mg/dL. Serum insulin was measured according to the electro chemiluminescence immunoassay method, using an automated immunoassay analyzer (Immulite 2500 Insulin, Diagnostic Products Corporation, Los Angeles, CA). Cut-off points above the 95th percentile of healthy children were used to define dyslipidemia and impaired fasting glucose, according to the international recommendations [1,9]. Insulin resistance was evaluated according to the homeostasis model assessment-insulin resistance (HOMAIR) index. Different cut-off values for prepubertal and pubertal stages were used to determine the status of insulin resistance (prepubertal > 2.5, pubertal > 4) [27]. Serum leptin and sLR concentrations were measured by Enzyme Immunoassay (EIA) kit based on the principle of standard sandwich enzyme immunoassay (Catalog No. EK-0437, Boster Biological Tech., China and Catalog No. CSB-E04647h, CUSABIO Biotech Coop., USA, respectively). Standards and samples were pipetted into the wells, which were precoated with analyte specific antibody. The analytes were bound by the immobilized antibody. After
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removing unbound conjugates, biotinylated detection antibodies were added to the wells subsequently and then followed by washing with wash buffer. Avidin conjugated Horseradish Peroxidase (HRP) were added to the wells and then unbound conjugates were washed away. A substrate solution was added to the wells and color develops in proportion to the amount of bounded analyte and the intensity of the color was measured. The ELISA assays had a sensitivity of 10 pg/mL and 0.78 pg/mL; a detection range of 62.5–4000 pg/mL and 3.12–200 pg/mL, respectively; intraaasay CV of <10% and <8%, interassay CV of <15% and <10%, respectively. Free leptin index (FLI) was calculated as the ratio of leptin to sLR. Blood pressure was measured by one of the investigators using a validated protocol. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured twice at the right arm after a 10-min rest in the supine position using a calibrated sphygmomanometer. Hypertension was defined as blood pressure values above the 95th percentile for height, age, and gender [23]. The study was initiated upon approval of the local ethics committee of Dokuz Eylul University, Faculty of Medicine in light of the Helsinki Declaration. A written informed consent of the parent(s) of each subject was also obtained before the study. 3. Statistical analyses Statistical analysis of the data was conducted with SPSS 16.0.1 (SPSS Inc., Chicago, IL, USA). Homogeneity of the data was evaluated with the Kolmogorov–Smirnov test. Between study groups, the obtained data were compared by using Student’s t-test (for normally distributed data) and Mann–Whitney U test (for nonnormally distributed data) Categorical variables were compared using Chi-square test. The correlations between the independent variables were investigated with Pearson’s correlation analysis. Variables with a p-value < 0.05 in univariate correlation analysis were included in a multivariate linear regression analysis model to assess the independent determinants of FLI. p < 0.05 was considered statistically significant. 4. Results A total of 35 obese children (22 male; 16 prepubertal; mean age, 11.0 ± 3.2 years) and 36 healthy children (17 male; 19 prepubertal, mean age, 13.0 ± 2.5 years) were included in this study. The groups were similar for age and gender. There were significant differences between obese and healthy children in terms of BMI, BMI-SDS, WC, insulin, TG, HDL-C, HOMA-IR, leptin, sLR levels, FLI, and leptin/BMI ratio (p < 0.05), while TC and LDL-C levels were not different (p > 0.05). Obese children had significantly higher SBP and DBP values than those of the healthy children (p < 0.05) (Table 1). Comparison of the obese children regarding the insulin resistance showed statistically significant differences for age, pubertal status, BMI, WC, fat mass (kg), percentage of body fat (%), insulin, HOMA-IR, leptin, FLI, and leptin/BMI ratio (p < 0.05); however serum sLR, glucose, LDL-C, HDL-C, TG and TC levels were not different between the insulin resistant and non-resistant obese children (p > 0.05) (Table 2). In healthy children, FLI was positively correlated with BMI, BMISDS, and WC while in obese children FLI was positively correlated with BMI, WC, insulin, HOMA-IR, and TC levels (p < 0.05) (Table 3). In multivariate regression analysis, HOMA-IR (ˇ = 0.289; p = 0.042) was the only independent variable significantly correlated with FLI in obese children, which explained 28.8% of the variance (Table 4). In obese children, sLR significantly correlated only with TC (r = −0.416, p = 0.013), while sLR had a weak negative correlation with leptin, insulin and TG levels which failed to reach statistical significance (r = −0.224, p = 0.195; r = −0.334, p = 0.06; r = −0.260,
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Table 1 The clinical characteristics of obese and healthy children.
Age (year) Sex (male/female) Puberty (prepubertal/pubertal) BMI (kg/m2 ) BMI SDS WC (cm) Glucose (mg/dL) Insulin (uIU/mL) HOMA-IR TG (mg/dL) TC (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) Leptin (pg/mL) sLR (pg/mL) FLI Leptin/BMI SBP (mmHg) DBP (mmHg)
Obese children (n = 35)
Healthy children (n = 36)
p
10.9 ± 3.1 22/13 16/19 30.1 ± 5.3 2.4 ± 0.2 98.6 ± 18.2 88.7 ± 9.0 18.0 ± 12.4 3.8 ± 2.9 135.3 ± 98.7 180.34 ± 28.2 108.3 ± 25.9 46.5 ± 21.5 10.2 ± 4.7 17.1 ± 7.3 0.80 ± 0.71 0.33 ± 0.14 118.0 ± 14.1 71.9 ± 7.6
11.4 ± 3.2 17/19 19/17 18.0 ± 3.0 −0.04 ± 0.6 68.4 ± 8.9 84.0 ± 10.4 6.9 ± 4.0 1.5 ± 0.9 80.1 ± 32.8 177.3 ± 36.0 104.1 ± 28.1 56.0 ± 10.6 2.0 ± 1.6 22.2 ± 11.3 0.11 ± 0.12 0.10 ± 0.07 102.7 ± 14.2 65.4 ± 8.5
0.494a 0.236b 0.638b <0.001 a <0.001a <0.001a 0.052a <0.001c <0.001c 0.001c 0.602a 0.438a 0.020a <0.001a 0.028a <0.001c <0.001a 0.004a 0.030a
Obese children (n = 35)
Data are given as mean ± SD. HOMA-IR, homeostasis model assessment-insulin resistance; TG, triglyceride; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; sLR, soluble leptin receptor; FLI, free leptin index; BMI, body mass index; BMI-SDS, standard deviation score of body mass index; WC, waist circumference; SBP, systolic blood pressure; DBP, diastolic blood pressure. a Student’s t test. b Chi-square test. c Mann–Whitney U test. Table 2 The clinical and laboratory characteristics of obese children with and without insulin resistance.
Age (year) Sex (M/F) Puberty (prepubertal/pubertal) BMI (kg/m2 ) BMI SDS WC (cm) Fat mass (kg) Percentage of body fat (%) Glucose (mg/dL) Insulin (uIU/mL) HOMA-IR TG (mg/dL) TC (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) Leptin (pg/mL) sLR (pg/mL) FLI Leptin/BMI
Table 3 The correlations between free leptin index and anthropometric and laboratory parameters of obese and healthy children.
IR (−) obese children (n = 19)
IR (+) obese children (n = 16)
pa
8.5 10/9 13/6 25.5 2.36 80 16.7 35 81.5 10.7 1.8 103 159 98 46 6.5 18.4 0.39 0.25
13.0 12/4 3/13 34.5 2.46 114 38.2 40.8 86.0 24.7 5.0 111 180 108 39 14.1 14.8 0.81 0.42
0.010 0.293b 0.006b 0.001 0.230 <0.001 0.003 0.002 0.756 <0.001 <0.001 0.217 0.230 0.367 0.102 <0.001 0.230 0.002 0.005
2
BMI (kg/m ) BMI SDS WC (cm) Glucose (mg/dL) Insulin (uIU/mL) TG (mg/dL) TC (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) HOMA-IR Fat mass (kg) Percentage of body fat (%)
Healthy children (n = 36)
r
pa
r
pa
0.358 0.143 0.342 0.155 0.435 0.208 0.365 0.319 −0.136 0.509 0.279 0.200
0.035 0.412 0.044 0.373 0.009 0.230 0.031 0.062 0.435 0.002 0.104 0.249
0.568 0.432 0.524 −0.072 0.329 0.142 −0.099 −0.056 −0.212 0.247 NA NA
<0.001 0.011 0.003 0.678 0.054 0.422 0.576 0.754 0.218 0.146 NA NA
FLI, free leptin index; BMI, body mass index; BMI-SDS, standard deviation score of body mass index; WC, Waist circumference; HOMA-IR, homeostasis model assessment-insulin resistance; TG, triglyceride; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; HOMA-IR, homeostasis model assessment-insulin resistance. a Pearson correlation analysis. Table 4 Multivariate backward linear regression analysis (dependent variable: free leptin index). Variable 2
BMI (kg/m ) WC (cm) Insulin (uIU/mL) HOMA-IR TC (mg/dL)
B
SRC (ˇ)
T
P value
r2
p
0.007 0.001 −0.046 0.289 0.004
0.055 0.023 −0.823 1.198 0.192
0.138 0.059 −1.432 2.078 1.161
0.891 0.953 0.162 0.042 0.255
0.352
0.024
Abbreviations: B, coefficient of regression; SRC, standardized regression coefficient; BMI, body mass index; WC, waist circumference; HOMA-IR, homeostasis model assessment-insulin resistance.
BMI, body mass index; BMI-SDS, standard deviation score of body mass index; WC, Waist circumference; BF%, percentage of body fat; HOMA-IR, homeostasis model assessment-insulin resistance; TG, triglyceride; TC, total cholesterol; LDL-C, lowdensity lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; sLR, soluble leptin receptor; FLI, free leptin index. a Mann–Whitney U test (Data are given as median). b Chi-square test.
p = 0.131) (Fig. 1). On the other hand, in healthy children, sLR was not significantly correlated with any of the laboratory or anthropometric parameters (p > 0.05). 5. Discussion Leptin circulates mainly bounded to the sLR and shows its effect by binding to the membrane-bound leptin receptor, which is a
Fig. 1. The relation of leptin and soluble leptin receptor in obese subjects.
member of class I cytokine receptor family. sLR, which is a determinant of FLI, was first measured in normal and diabetic pregnant women in 1999 by using the radioimmunoassay method [15]. In 2002, Ogier et al. [19] measured sLR concentration in obese and
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healthy individuals (age 18–50) by using a direct double monoclonal sandwich enzyme-linked immunosorbent method. In the present study, we quantitatively measured sLR and leptin levels in obese children and investigated its relation with anthropometric and metabolic parameters. To the best of our knowledge, there is limited number of studies regarding childhood obesity, which investigated the relation of sLR and FLI with metabolic and anthropometric parameters [4,8,21,22]. Various studies have reported higher leptin and lower sLR levels in obese adults in relation to BMI and fat mass [19,22,24,28]. In a study conducted in healthy children and adolescents, it was shown that sLR level was negatively correlated with age, pubertal stage, height, weight, BMI, WC, subscapular skin fold thickness, systolic and diastolic blood pressures, leptin, IGF-1 levels, and leptin/BMI ratio [12]. Previously, Cinaz et al. [4] reported higher leptin and lower sLR levels in obese children than the healthy controls, and they also showed a negative correlation between leptin and sLR levels in obese subjects. Popruk et al. [21] investigated obese children and adolescents (116 males and 65 females; age range of 5–19 years), and reported that serum leptin level was high, whereas sLR level was significantly low in obese children. In the same study, a significant negative correlation was detected between leptin and sLR levels in the obese group. In our study, similar to the findings of previous studies, serum leptin level was significantly high, whereas sLR was significantly low in the obese group. However, distinct from previous studies, the negative correlation between sLR and leptin levels could not reach statistical significance which might be due to the small number of patients. Levels of sLR can provide the information for free leptin concentration. However, FLI may be a more accurate determinant of leptin activity and resistance than the only evaluation of sLR and leptin levels [7,20,29]. van Dielen et al. [28] calculated molar ratios of these proteins and found that leptin/sLR ratio was 1/1 (max: 2/1) in the healthy group, whereas it was 25/1 in the morbid obese group. In the present study, FLI was found approximately eight folds (0.58/0.07 = 8.28) higher in obese children when compared to healthy children. In a previous study, it was shown that in obese adults sLR level was negatively correlated with serum leptin level and percentage of body fat, whereas FLI was positively correlated with percentage of body fat [19]. The authors have suggested that in morbid obese cases, sLR level was decreased as a result of the negative feedback effect of increased leptin levels [28]. Owecki et al. [20] have demonstrated that serum leptin, sLR, and FLI were not correlated with insulin resistance in obese and non-obese humans. Reiner et al. [22] evaluated the serum sLR, leptin, and insulin resistance before and after weight loss in obese children and showed that sLR is not correlated with insulin resistance, while leptin levels are associated with insulin resistance. In the present study, sLR was not significantly correlated with any of the laboratory or anthropometric parameters, except TC, in either healthy or obese children. Besides, FLI, one of the markers for leptin activity and resistance, showed significant positive correlation with some of the metabolic parameters (TC, insulin, HOMA-IR) and anthropometric parameters (BMI, WC) in obese children. In agreement with the literature, these findings suggest that as compared to serum leptin and sLR levels, FLI may be a more accurate parameter to evaluate leptin resistance in obese children. Furthermore, we suggest that the increased FLI in obese children may be a compensatory mechanism that the organism develops in order to overcome leptin resistance. In the literature, the relationship between serum leptin and insulin levels is not clear. Previously it has been suggested that leptin release was regulated in part by nutritional status and its expression in adipose tissue was up-regulated by insulin. In the same study binding of leptin to its receptor in beta-cells was suggested to modulate insulin expression in a negative feedback loop,
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and thereby might have an anti-obesity effect [11]. In the present study, it was found that obese children with insulin resistance had higher FLI than the insulin non-resistant obese children. Besides, multivariate regression analysis revealed that HOMA-IR was the only independent variable significantly correlated with FLI. These knowledge and the results of the present study suggest that in obese children increased FLI contribute toward the development of hyperinsulinemia and insulin resistance. There are some limitations that need to be acknowledged regarding the present study. First is the small number of study participants; second is the lack of fat mass and percentage of body fat measurements in the healthy children; third is the unequal gender distribution when groups were compared according to the pubertal status; and finally, the absence of re-evaluations of leptin and sLR levels after weight losing in obese subjects. In conclusion, the results of the present study showed that sLR was lower; leptin and FLI were significantly higher in obese children when compared to healthy children. Besides, it was reported for the first time in obese children that while sLR was not correlated with any of the anthropometric and metabolic parameters except TC levels, FLI had significant positive correlations with BMI, WC, HOMA-IR, fasting insulin and TC levels. However, multivariate regression analysis confirmed that the insulin resistance index was the only independent variable significantly correlated with FLI in obese children. All of these findings suggest that in obese children: (i) FLI is a more accurate marker to evaluate leptin resistance and function when compared to serum leptin and sLR levels, and (ii) increased FLI may contribute toward the development of hyperinsulinemia and insulin resistance.
Conflict of interest The authors declare there was no any conflict of interest
Funding None declared.
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Peptides journal homepage: www.elsevier.com/locate/peptides
The relation of leptin and soluble leptin receptor levels with metabolic and clinical parameters in obese and healthy children Gonul Catli a , Ahmet Anik a , Hale Ünver Tuhan a , Tuncay Kume b , Ece Bober a , Ayhan Abaci a,∗ a b
Department of Pediatric Endocrinology, Dokuz Eylul University Faculty of Medicine, Izmir, Turkey Department of Biochemistry, Dokuz Eylul University Faculty of Medicine, Izmir, Turkey
a r t i c l e
i n f o
Article history: Received 23 February 2014 Received in revised form 18 March 2014 Accepted 21 March 2014 Available online 2 April 2014 Keywords: Childhood Obesity Leptin Soluble leptin receptor Insulin resistance
a b s t r a c t We investigated the relation of serum leptin, soluble leptin receptor (sLR) and free leptin index (FLI) with metabolic and anthropometric parameters in obese and healthy children. Height, weight, waist circumference (WC), fasting serum glucose, insulin, lipid profile, leptin and sLR levels of 35 obese children and 36 healthy children were measured and FLI was calculated as the ratio of leptin to sLR. In obese children, serum leptin and FLI were found significantly higher, while sLR level was significantly lower than the healthy children. Comparison of obese children regarding the insulin resistance showed significantly higher serum leptin and FLI in the insulin resistant group; however sLR level was not different between the insulin resistant and non-resistant obese children. In obese children, sLR was not correlated with any of the metabolic parameters except total cholesterol, while FLI was significantly and positively correlated with BMI, WC, TC, fasting insulin, and HOMA-IR. However, regression analysis confirmed that the HOMAIR was the only independent variable significantly correlated with FLI in obese children. Findings of this study suggest that in obese children and adolescents (i) serum leptin and FLI were found significantly higher, while sLR level was significantly lower than the healthy children, (ii) increased FLI might be a compensatory mechanism for increasing leptin effect in peripheral tissue, (iii) FLI is a more accurate marker to evaluate leptin resistance than leptin or sLR alone, and (iv) increased FLI may contribute toward the development of hyperinsulinemia and insulin resistance. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Leptin, a cytokine which is secreted predominantly by the adipose tissue, is considered to be involved in satiety regulation in humans and animals [18]. In adults, circulating leptin levels are closely related to the amount of adipose tissue [16]. Leptin transports across the blood–brain barrier by a saturated system and acts within the brain regions, which regulate feeding behavior and thermogenic responses [2,5]. In addition to the above-mentioned effects, leptin also plays a role in the regulation of other body functions, including reproduction, liver and enteric metabolism, hematopoiesis, and immunity via leptin receptors, which are shown to be present in many peripheral tissues [5,26]. In humans, four different mRNA splice variants of the leptin
∗ Corresponding author at: Department of Pediatric Endocrinology, Dokuz Eylul University Faculty of Medicine, Balcova, Izmir 35340, Turkey. Tel.: +90 232 4126076; fax: +90 232 4126001. E-mail address: [email protected] (A. Abaci). http://dx.doi.org/10.1016/j.peptides.2014.03.015 0196-9781/© 2014 Elsevier Inc. All rights reserved.
receptor, including a membrane-bound receptor with a long cytoplasmic domain and three membrane-bound receptors with a short cytoplasmic domain have been identified. A circulating soluble form of the leptin receptor [soluble leptin receptor (sLR)], which is the main leptin-binding protein and determinant of free leptin index (FLI), also exists [3]. Although the source of sLR in the plasma is not known definitely, it (sLR) is generated by the proteolytic cleavage of membrane-anchored receptors, indicating that the leptin receptor might have other functions besides signal transduction [6,17,24]. In blood, leptin is suggested to circulate both in the free form as well as bound to sLR and possibly also to other as yet unidentified binding proteins [24]. Whilst binding of leptin with sLR has been suggested to increase the bioavailability of leptin in the plasma and to elongate its circulatory half-life [10,12,14,24,30], it was also proposed to decrease the free fraction of leptin, resulting in decreased binding of free leptin to the membrane-bound leptin receptors [15]. However, the role of the sLR in the regulation of the physiological function of leptin is until now unclear. Currently, limited data exists regarding the relation of sLR and FLI with metabolic and anthropometric parameters in childhood
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obesity. In this study, we aimed to investigate the relation of serum leptin, sLR and FLI with clinical and metabolic parameters in obese and healthy children.
2. Subjects and methods The study included 35 obese children with a body mass index (BMI) above 95th percentile, who applied to our department with the complaint of weight gain and 36 healthy children with a BMI below 85th percentile, who had similar age and gender distribution. Obesity was defined according to the data from the Centers for Disease Control and Prevention (CDC), 2000, growth charts. For calculation of BMI-standard deviation score (SDS), data from the CDC were used [13]. Before the outset of the study, all of the obese and healthy children underwent a thorough physical examination and laboratory evaluation including thyroid function tests and serum cortisol measurement for probable endocrine pathology. Those with chronic diseases (cardiovascular, gastrointestinal, and respiratory), a history of drug use (steroids and antipsychotics), endocrine pathology (Cushing syndrome and hypothyroidism), or syndromes associated with obesity (Prader–Willi and Laurence–Moon–Biedle syndromes) were excluded from the study. Height was measured using a Harpenden stadiometer with a sensitivity of 0.1 cm and weight was measured using a SECA scale with a sensitivity of 0.1 kg. The weight of each subject was measured with all clothing removed except undergarments. BMI was calculated by dividing weight (kg) by height squared (m2 ). Waist circumference (WC) was measured between the lowest rib and the iliac crest, horizontally through the narrowest part of the torso. The percentage of body fat and fat mass were measured using bioelectric impedance analysis (Tanita BC-418, Tokyo, Japan). Findings for pubertal development were evaluated according to Tanner staging [25]. A testicular volume of ≥4 mL in males and breast development of stage 2 and over in females were considered to be findings of puberty. Blood samples for glucose, insulin, lipids, thyroid function tests, cortisol, leptin and sLR levels were drawn after 10–12 h of overnight fasting. The plane tubes were centrifuged at 1200 × g 10 min and serum samples were removed from clots into the Eppendorf tubes using plastic Pasteur pipettes. They were stored at −80 ◦ C until analysis. Serum glucose, triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) concentrations were measured enzymatically using DP Modular Systems (Roche Diagnostic Corp., Indianapolis, IN). Low-density lipoprotein cholesterol (LDL-C) levels were calculated using the Friedewald formula when plasma TG’s were <400 mg/dL. Serum insulin was measured according to the electro chemiluminescence immunoassay method, using an automated immunoassay analyzer (Immulite 2500 Insulin, Diagnostic Products Corporation, Los Angeles, CA). Cut-off points above the 95th percentile of healthy children were used to define dyslipidemia and impaired fasting glucose, according to the international recommendations [1,9]. Insulin resistance was evaluated according to the homeostasis model assessment-insulin resistance (HOMAIR) index. Different cut-off values for prepubertal and pubertal stages were used to determine the status of insulin resistance (prepubertal > 2.5, pubertal > 4) [27]. Serum leptin and sLR concentrations were measured by Enzyme Immunoassay (EIA) kit based on the principle of standard sandwich enzyme immunoassay (Catalog No. EK-0437, Boster Biological Tech., China and Catalog No. CSB-E04647h, CUSABIO Biotech Coop., USA, respectively). Standards and samples were pipetted into the wells, which were precoated with analyte specific antibody. The analytes were bound by the immobilized antibody. After
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removing unbound conjugates, biotinylated detection antibodies were added to the wells subsequently and then followed by washing with wash buffer. Avidin conjugated Horseradish Peroxidase (HRP) were added to the wells and then unbound conjugates were washed away. A substrate solution was added to the wells and color develops in proportion to the amount of bounded analyte and the intensity of the color was measured. The ELISA assays had a sensitivity of 10 pg/mL and 0.78 pg/mL; a detection range of 62.5–4000 pg/mL and 3.12–200 pg/mL, respectively; intraaasay CV of <10% and <8%, interassay CV of <15% and <10%, respectively. Free leptin index (FLI) was calculated as the ratio of leptin to sLR. Blood pressure was measured by one of the investigators using a validated protocol. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured twice at the right arm after a 10-min rest in the supine position using a calibrated sphygmomanometer. Hypertension was defined as blood pressure values above the 95th percentile for height, age, and gender [23]. The study was initiated upon approval of the local ethics committee of Dokuz Eylul University, Faculty of Medicine in light of the Helsinki Declaration. A written informed consent of the parent(s) of each subject was also obtained before the study. 3. Statistical analyses Statistical analysis of the data was conducted with SPSS 16.0.1 (SPSS Inc., Chicago, IL, USA). Homogeneity of the data was evaluated with the Kolmogorov–Smirnov test. Between study groups, the obtained data were compared by using Student’s t-test (for normally distributed data) and Mann–Whitney U test (for nonnormally distributed data) Categorical variables were compared using Chi-square test. The correlations between the independent variables were investigated with Pearson’s correlation analysis. Variables with a p-value < 0.05 in univariate correlation analysis were included in a multivariate linear regression analysis model to assess the independent determinants of FLI. p < 0.05 was considered statistically significant. 4. Results A total of 35 obese children (22 male; 16 prepubertal; mean age, 11.0 ± 3.2 years) and 36 healthy children (17 male; 19 prepubertal, mean age, 13.0 ± 2.5 years) were included in this study. The groups were similar for age and gender. There were significant differences between obese and healthy children in terms of BMI, BMI-SDS, WC, insulin, TG, HDL-C, HOMA-IR, leptin, sLR levels, FLI, and leptin/BMI ratio (p < 0.05), while TC and LDL-C levels were not different (p > 0.05). Obese children had significantly higher SBP and DBP values than those of the healthy children (p < 0.05) (Table 1). Comparison of the obese children regarding the insulin resistance showed statistically significant differences for age, pubertal status, BMI, WC, fat mass (kg), percentage of body fat (%), insulin, HOMA-IR, leptin, FLI, and leptin/BMI ratio (p < 0.05); however serum sLR, glucose, LDL-C, HDL-C, TG and TC levels were not different between the insulin resistant and non-resistant obese children (p > 0.05) (Table 2). In healthy children, FLI was positively correlated with BMI, BMISDS, and WC while in obese children FLI was positively correlated with BMI, WC, insulin, HOMA-IR, and TC levels (p < 0.05) (Table 3). In multivariate regression analysis, HOMA-IR (ˇ = 0.289; p = 0.042) was the only independent variable significantly correlated with FLI in obese children, which explained 28.8% of the variance (Table 4). In obese children, sLR significantly correlated only with TC (r = −0.416, p = 0.013), while sLR had a weak negative correlation with leptin, insulin and TG levels which failed to reach statistical significance (r = −0.224, p = 0.195; r = −0.334, p = 0.06; r = −0.260,
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Table 1 The clinical characteristics of obese and healthy children.
Age (year) Sex (male/female) Puberty (prepubertal/pubertal) BMI (kg/m2 ) BMI SDS WC (cm) Glucose (mg/dL) Insulin (uIU/mL) HOMA-IR TG (mg/dL) TC (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) Leptin (pg/mL) sLR (pg/mL) FLI Leptin/BMI SBP (mmHg) DBP (mmHg)
Obese children (n = 35)
Healthy children (n = 36)
p
10.9 ± 3.1 22/13 16/19 30.1 ± 5.3 2.4 ± 0.2 98.6 ± 18.2 88.7 ± 9.0 18.0 ± 12.4 3.8 ± 2.9 135.3 ± 98.7 180.34 ± 28.2 108.3 ± 25.9 46.5 ± 21.5 10.2 ± 4.7 17.1 ± 7.3 0.80 ± 0.71 0.33 ± 0.14 118.0 ± 14.1 71.9 ± 7.6
11.4 ± 3.2 17/19 19/17 18.0 ± 3.0 −0.04 ± 0.6 68.4 ± 8.9 84.0 ± 10.4 6.9 ± 4.0 1.5 ± 0.9 80.1 ± 32.8 177.3 ± 36.0 104.1 ± 28.1 56.0 ± 10.6 2.0 ± 1.6 22.2 ± 11.3 0.11 ± 0.12 0.10 ± 0.07 102.7 ± 14.2 65.4 ± 8.5
0.494a 0.236b 0.638b <0.001 a <0.001a <0.001a 0.052a <0.001c <0.001c 0.001c 0.602a 0.438a 0.020a <0.001a 0.028a <0.001c <0.001a 0.004a 0.030a
Obese children (n = 35)
Data are given as mean ± SD. HOMA-IR, homeostasis model assessment-insulin resistance; TG, triglyceride; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; sLR, soluble leptin receptor; FLI, free leptin index; BMI, body mass index; BMI-SDS, standard deviation score of body mass index; WC, waist circumference; SBP, systolic blood pressure; DBP, diastolic blood pressure. a Student’s t test. b Chi-square test. c Mann–Whitney U test. Table 2 The clinical and laboratory characteristics of obese children with and without insulin resistance.
Age (year) Sex (M/F) Puberty (prepubertal/pubertal) BMI (kg/m2 ) BMI SDS WC (cm) Fat mass (kg) Percentage of body fat (%) Glucose (mg/dL) Insulin (uIU/mL) HOMA-IR TG (mg/dL) TC (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) Leptin (pg/mL) sLR (pg/mL) FLI Leptin/BMI
Table 3 The correlations between free leptin index and anthropometric and laboratory parameters of obese and healthy children.
IR (−) obese children (n = 19)
IR (+) obese children (n = 16)
pa
8.5 10/9 13/6 25.5 2.36 80 16.7 35 81.5 10.7 1.8 103 159 98 46 6.5 18.4 0.39 0.25
13.0 12/4 3/13 34.5 2.46 114 38.2 40.8 86.0 24.7 5.0 111 180 108 39 14.1 14.8 0.81 0.42
0.010 0.293b 0.006b 0.001 0.230 <0.001 0.003 0.002 0.756 <0.001 <0.001 0.217 0.230 0.367 0.102 <0.001 0.230 0.002 0.005
2
BMI (kg/m ) BMI SDS WC (cm) Glucose (mg/dL) Insulin (uIU/mL) TG (mg/dL) TC (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) HOMA-IR Fat mass (kg) Percentage of body fat (%)
Healthy children (n = 36)
r
pa
r
pa
0.358 0.143 0.342 0.155 0.435 0.208 0.365 0.319 −0.136 0.509 0.279 0.200
0.035 0.412 0.044 0.373 0.009 0.230 0.031 0.062 0.435 0.002 0.104 0.249
0.568 0.432 0.524 −0.072 0.329 0.142 −0.099 −0.056 −0.212 0.247 NA NA
<0.001 0.011 0.003 0.678 0.054 0.422 0.576 0.754 0.218 0.146 NA NA
FLI, free leptin index; BMI, body mass index; BMI-SDS, standard deviation score of body mass index; WC, Waist circumference; HOMA-IR, homeostasis model assessment-insulin resistance; TG, triglyceride; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; HOMA-IR, homeostasis model assessment-insulin resistance. a Pearson correlation analysis. Table 4 Multivariate backward linear regression analysis (dependent variable: free leptin index). Variable 2
BMI (kg/m ) WC (cm) Insulin (uIU/mL) HOMA-IR TC (mg/dL)
B
SRC (ˇ)
T
P value
r2
p
0.007 0.001 −0.046 0.289 0.004
0.055 0.023 −0.823 1.198 0.192
0.138 0.059 −1.432 2.078 1.161
0.891 0.953 0.162 0.042 0.255
0.352
0.024
Abbreviations: B, coefficient of regression; SRC, standardized regression coefficient; BMI, body mass index; WC, waist circumference; HOMA-IR, homeostasis model assessment-insulin resistance.
BMI, body mass index; BMI-SDS, standard deviation score of body mass index; WC, Waist circumference; BF%, percentage of body fat; HOMA-IR, homeostasis model assessment-insulin resistance; TG, triglyceride; TC, total cholesterol; LDL-C, lowdensity lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; sLR, soluble leptin receptor; FLI, free leptin index. a Mann–Whitney U test (Data are given as median). b Chi-square test.
p = 0.131) (Fig. 1). On the other hand, in healthy children, sLR was not significantly correlated with any of the laboratory or anthropometric parameters (p > 0.05). 5. Discussion Leptin circulates mainly bounded to the sLR and shows its effect by binding to the membrane-bound leptin receptor, which is a
Fig. 1. The relation of leptin and soluble leptin receptor in obese subjects.
member of class I cytokine receptor family. sLR, which is a determinant of FLI, was first measured in normal and diabetic pregnant women in 1999 by using the radioimmunoassay method [15]. In 2002, Ogier et al. [19] measured sLR concentration in obese and
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healthy individuals (age 18–50) by using a direct double monoclonal sandwich enzyme-linked immunosorbent method. In the present study, we quantitatively measured sLR and leptin levels in obese children and investigated its relation with anthropometric and metabolic parameters. To the best of our knowledge, there is limited number of studies regarding childhood obesity, which investigated the relation of sLR and FLI with metabolic and anthropometric parameters [4,8,21,22]. Various studies have reported higher leptin and lower sLR levels in obese adults in relation to BMI and fat mass [19,22,24,28]. In a study conducted in healthy children and adolescents, it was shown that sLR level was negatively correlated with age, pubertal stage, height, weight, BMI, WC, subscapular skin fold thickness, systolic and diastolic blood pressures, leptin, IGF-1 levels, and leptin/BMI ratio [12]. Previously, Cinaz et al. [4] reported higher leptin and lower sLR levels in obese children than the healthy controls, and they also showed a negative correlation between leptin and sLR levels in obese subjects. Popruk et al. [21] investigated obese children and adolescents (116 males and 65 females; age range of 5–19 years), and reported that serum leptin level was high, whereas sLR level was significantly low in obese children. In the same study, a significant negative correlation was detected between leptin and sLR levels in the obese group. In our study, similar to the findings of previous studies, serum leptin level was significantly high, whereas sLR was significantly low in the obese group. However, distinct from previous studies, the negative correlation between sLR and leptin levels could not reach statistical significance which might be due to the small number of patients. Levels of sLR can provide the information for free leptin concentration. However, FLI may be a more accurate determinant of leptin activity and resistance than the only evaluation of sLR and leptin levels [7,20,29]. van Dielen et al. [28] calculated molar ratios of these proteins and found that leptin/sLR ratio was 1/1 (max: 2/1) in the healthy group, whereas it was 25/1 in the morbid obese group. In the present study, FLI was found approximately eight folds (0.58/0.07 = 8.28) higher in obese children when compared to healthy children. In a previous study, it was shown that in obese adults sLR level was negatively correlated with serum leptin level and percentage of body fat, whereas FLI was positively correlated with percentage of body fat [19]. The authors have suggested that in morbid obese cases, sLR level was decreased as a result of the negative feedback effect of increased leptin levels [28]. Owecki et al. [20] have demonstrated that serum leptin, sLR, and FLI were not correlated with insulin resistance in obese and non-obese humans. Reiner et al. [22] evaluated the serum sLR, leptin, and insulin resistance before and after weight loss in obese children and showed that sLR is not correlated with insulin resistance, while leptin levels are associated with insulin resistance. In the present study, sLR was not significantly correlated with any of the laboratory or anthropometric parameters, except TC, in either healthy or obese children. Besides, FLI, one of the markers for leptin activity and resistance, showed significant positive correlation with some of the metabolic parameters (TC, insulin, HOMA-IR) and anthropometric parameters (BMI, WC) in obese children. In agreement with the literature, these findings suggest that as compared to serum leptin and sLR levels, FLI may be a more accurate parameter to evaluate leptin resistance in obese children. Furthermore, we suggest that the increased FLI in obese children may be a compensatory mechanism that the organism develops in order to overcome leptin resistance. In the literature, the relationship between serum leptin and insulin levels is not clear. Previously it has been suggested that leptin release was regulated in part by nutritional status and its expression in adipose tissue was up-regulated by insulin. In the same study binding of leptin to its receptor in beta-cells was suggested to modulate insulin expression in a negative feedback loop,
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and thereby might have an anti-obesity effect [11]. In the present study, it was found that obese children with insulin resistance had higher FLI than the insulin non-resistant obese children. Besides, multivariate regression analysis revealed that HOMA-IR was the only independent variable significantly correlated with FLI. These knowledge and the results of the present study suggest that in obese children increased FLI contribute toward the development of hyperinsulinemia and insulin resistance. There are some limitations that need to be acknowledged regarding the present study. First is the small number of study participants; second is the lack of fat mass and percentage of body fat measurements in the healthy children; third is the unequal gender distribution when groups were compared according to the pubertal status; and finally, the absence of re-evaluations of leptin and sLR levels after weight losing in obese subjects. In conclusion, the results of the present study showed that sLR was lower; leptin and FLI were significantly higher in obese children when compared to healthy children. Besides, it was reported for the first time in obese children that while sLR was not correlated with any of the anthropometric and metabolic parameters except TC levels, FLI had significant positive correlations with BMI, WC, HOMA-IR, fasting insulin and TC levels. However, multivariate regression analysis confirmed that the insulin resistance index was the only independent variable significantly correlated with FLI in obese children. All of these findings suggest that in obese children: (i) FLI is a more accurate marker to evaluate leptin resistance and function when compared to serum leptin and sLR levels, and (ii) increased FLI may contribute toward the development of hyperinsulinemia and insulin resistance.
Conflict of interest The authors declare there was no any conflict of interest
Funding None declared.
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