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Background: Adipose tissue is a veritable "endocrine organ" due to its adipocytokines secretion implied in insulin sensitivity modulation and cardiovascular complications.
Objective: To identify the adipocytokines' plasmatic profile (adiponectin, leptin, resistin, IL-6, TNFα) in obese children and adolescents and to assess their relationship with "classic" clinical/paraclinical markers of metabolic syndrome and insulin resistance.
Material and Methods: A case-control study comparing a study group of 38 obese children and adolescents (age 13.5±2.3 years) to a normal weight age matched control group of 24 children.
We measured body mass index (BMI) and waist circumference (WC), systolic and diastolic blood pressure (BP). The classical metabolic parameters (fasting glycemia, total cholesterol and its fractions, serum triglycerides) were measured in both groups. Insulin sensitivity was evaluated using fasting insulinemia, HOMA-index and insulin-resistance summary score (IRS). Adiponectin, leptin, resistin, IL-6 and TNFα were measured using ELISA method.
Outcomes: Serum levels of leptin, resistin and IL-6 were signficantly higher (42.42±22.58 ng/ml versus 14.4±14.49 ng/ml, p <0.001; 9.69±3.47 ng/ml versus 7.92±2.13ng/ml, p = 0.029 and 2.66 ±2.87 pg/ml versus 0.89 ± 1.16 pg/ml, p = 0.006 respectively), while adiponectin levels were significantly lower (9.05±4.61 µg/ml versus 15.93±9.24 μg/ml, p <0.001) in the obese group compared to control group. TNFα was not statistical different between groups.
In multivariate regression analysis adiponectin was negatively and significantly correlated with WC (r = - 0.463, p = 0.003); leptin was positively and significantly related to WC, diastolic BP, fasting insulinemia and resistin (r = 0.775, p <0.001); resistin was positively related to leptin and IL-6 (r = 0.499, p <0.001), IL-6 was positively and significantly related to diastolic blood pressure (r = 0.333, p = 0.008).
Conclusions: Serum levels of adiponectin, leptin, resistin and IL-6 are significantly different in obese children compared to normal weight controls; leptin was the only adipokine correlated with insulin resistance in children. There are significant correlations between plasmatic levels of leptin, resistin and IL-6.
Simple plasmatic determination of TNFα is not a marker of the degree of obesity or its metabolic complications in pediatric population.
Pediatric obesity has become an epidemic health issue in industrialized regions. Data from the NHANES (National Health and Nutrition Examination Survey) survey (2007–2008) indicate that approximately 9.5% of infants and toddlers were at or above the 95th percentile of the weight-for-recumbent-length growth charts, 11.9% of children and adolescents aged 2 to 19 years were severely obese (at or above the 97th percentile of the BMI-for-age growth charts), 16.9% were obese and 31.7% were overweight (1); the European Association for the Study of Obesity (EASO) has recently shown that overweight prevalence in European pediatric population (age 7-17 years) was 16-22%, while 4-6% of children and adolescents were already obese (2). The proportion of overweight and obese children and adolescents near doubled in 2010 compared to that reported in 1990-2003. It is estimated that in European Union almost 1.3 million children become overweight annually and that obesity incidence in the pediatric population is 300 000 new cases/year (3).
Obesity is associated to a metabolic risk which evolves to a cardiovascular risk. Obesity complications, as type 2 diabetes, dyslipidemia, arterial hypertension - formerly recognized in adult obese population - have recently been described in obese children and adolescents, with continuous growing prevalence, paralleling those of overweight prevalence (4-7).
Insulin resistance has a defined role in metabolic complications of obesity. There are several molecular mechanisms concurring to its appearance – recent years papers have demonstrated that various adipokines and cytokine derived from adipocytes per se or from others cellular elements of stromal or vascular origins in adipose tissue are responsible for insulin resistance and metabolic alterations in obese persons.
Adiponectin is the main adipokine secreted by adipocytes and the only adipokine down regulated in obesity. It has insulin-sensitizing effects through multiple mechanisms (inhibits hepatic gluconeogenesis (8), increases glucose uptake in adipocytes (9) and muscle cells (10) and protective cardiovascular effects (diminishes adhesion molecules expression, smooth muscle cells proliferation, suppress macrophage transformation in foam cells, has direct antithrombotic effects (11-14), stimulates nitric oxide production in small vessels (15,16).
Plasmatic levels of adiponectin were negatively correlated with BMI, adipose tissue proportion and fasting insulinemia in different adult populations (17-24) and also in pediatric populations: Pima Indians children (25), Japanese children (26), Taiwanese children (27).
The molecular mechanism of its lower secretion in obese people is not fully understood: its down-regulation may be induced by insulin resistance or its secretion may be suppressed by high plasmatic levels of TNFα and IL-6 (18).
In adults, a strong correlation of adiponectin to insulin-resistance biomarkers has been demonstrated and hypoadiponectinemia was associated to progression to type 2 diabetes (17, 28-31). A negative correlation between plasmatic levels of adiponectin and insulin-resistance was also demonstrated in pediatric populations: Taiwanese children (27), Hispanic children (32), Latino children and adolescents (33).
Leptin, primarily adipose tissue-derived protein product of the obesity (OB) gene, originally identified as an important regulator of energy metabolism, is a multifunctional polypeptide which may be associated with the occurrence of insulin resistance and diabetes in humans.
Plasma levels of leptin showed positive correlations with BMI, WC and adipose tissue proportion in various adult populations (34-36). In children, these correlations are demonstrated in a limited number of studies and for smaller groups (25,37). Plasmatic levels of leptin seem to be a better predictor of future weight gain in prospective studies (38-40).
Although in vitro and animal models studies have demonstrated the insulin-sensitizing effects of leptin (41-43) hyperleptinemia associated to obesity is associated in clinical studies with insulin-resistance in both obese adults and children (34,36,44,45); this apparent discordance can be explained by a state of leptin resistance described in obese persons (46).
Resistin is a protein secreted in human adipose tissue by preadipocytes (47) and mainly by monocytes from the stromal component (48). Resistin acquired initial attention as a potential link between obesity and glucose regulation. In rodents, resistin can induce insulin resistance, while its implication in the control of insulin sensitivity is still a matter of debate in humans. Several clinical studies have demonstrated that plasmatic levels of resistin are positively correlated to BMI (49-54) while others failed to identify its correlation to adiposity (47,55,56).
There are also controversies regarding resistin's correlation to insulin sensitivity in humans: several clinical studies on variable groups (obese or diabetic persons) have demonstrated that resistin is positively correlated to insulin-resistance (53,57), while other population studies disproved these remarks – resistin was not correlated to insulin-resistance in Pima Indiens (52) or healthy normal-weight individuals (55).
TNFα, a protein synthesized especially by macrophages, is a cytokine implied in systemic inflammation. It is also expressed in adipocytes and is associated with obesity (58), adipocytes cell volume (59), and inhibition of glucose uptake in adipocytes from lean individuals (58). TNFα is overproduced in adipose tissue of several rodent models of obesity and has a key role in the pathogenesis of insulin resistance in these species. However, its actual involvement in glucose metabolism disorders in humans remains controversial.
IL-6 production by human adipose tissue increases during obesity. It may induce hepatic C reactive protein synthesis and may promote the onset of cardiovascular complications.
Both TNF-a and IL-6 can alter insulin sensitivity by triggering different key steps in the insulin signaling pathway.
In vitro studies have shown that TNFα and IL-6 have pivotal roles in insulin resistance, acting directly at the insulin receptor (60,61). Both TNFα and IL-6 are positively related to adiposity, particularly visceral fat, and correlate with insulin resistance and other CVD risk factors in adults (60-64).
In children, data regarding IL-6 and TNFα plasmatic levels are sparse. Levels of these inflammatory markers seem to decrease with increasing age (65) and this can become a confounding factor in interpreting results in obese pediatric population.
Although in vitro and animal models studies have indicated a direct relationship between plasmatic TNFα and the degree of adiposity, in vivo studies have shown that mRNA expression for TNFα in adipose tissue is not directly dependent to BMI (66); leptin and other adipokines overexpressed in obese persons seem to stimulate TNFα production in the monocyte-macrophage system. This may be an explanation for the paucity of clinical data regarding its role in metabolic complications associated to obesity.
The adipocytokines' plasmatic levels and their correlations to classical metabolic and cardiovascular risk factors are far to be defined in adults and even less in children. ❑
The aim of this study is to define the adipocytokines' plasmatic profile in a study group of obese children and adolescents compared to normal-weight controls and to identify possible correlations to classical metabolic and insulin-resistance risk factors. ❑
This was a case–control observational study; we analyzed 38 obese children and adolescents (BMI above 95th percentile for age and sex - according to CDC - "Center of disease control" growth charts (67), evaluated for possible metabolic and cardiovascular complications over a six months period in the Pediatric Endocrinology Department of the "C.I.Parhon" National Institute of Endocrinology. We excluded from analysis children and adolescents with primary hypothyroidism (TSH values above 10 µUI/ml), children with Cushing syndrome, female adolescents with polycystic ovary syndrome. We did not examine patients with syndrome obesity (as Down syndrome or Prader Willi syndrome) or patients treated with insulin sensitizers like metformin.
The control group comprised 24 healthy age-matched normal weight (BMI between 5th and 85th percentiles for age and sex) children and adolescents, with similar pubertal status as the study group.
The study was conducted according to the Declaration of Helsinki, and the study protocol was approved by the Ethical Committee of "C.I.Parhon" National Institute of Endocrinology". Written informed consent was obtained from the parents and informed assent from the children and adolescents.
Children were evaluated in the morning, after 12 hours of fasting, dressed in light clothes and without shoes; we measured height, weight, waist circumference (WC) and blood pressure (BP); the pubertal status according to Tanner criteria was noted.
Height was measured using a Harpenden stadiometer: an average of 3 determinations at 5 minutes intervals was recorded; body weight was measured with a balance scale to the nearest 0.1 kg and waist circumference was measured with a tape just above the superior iliac crest at the end of normal expiration, according to U.S. Third National Health and Nutrition Examination Survey" recommendations.
Adiposity was evaluated by BMI (calculated as the weight in kilograms divided by the square of the height in meters). BMI variability with age and sex was adjusted using Z-score (standard deviation score) for BMI – we used for automatic calculation an online software application based on CDC growth charts (68).
Blood pressure was measured twice daily (in the morning and in the evening) with an appropriate sized cuff and after at least 10 minutes resting in the supine position; the highest systolic and diastolic value respectively were used for analysis.
We obtained a blood sample after a 12 h over-night fast. Lipid profile (total cholesterol, LDL-cholesterol and HDL-cholesterol, triglyceride), glucose, insulin and adipocytokines levels (adiponectin, leptin, resistin, TNFα and IL-6) were determined.
Plasma insulin was measured using RIA method (Radim, Italia, sensitivity = 1 µUI/ml) on a Gamma-Counter; plasma adiponectin, leptin, resistin, TNFα and IL-6 were measured by the ELISA Quantikine Human Adiponectin (R&D System, sensitivity = 0.891 ng/ml), Quantikine Human Leptin (R&D System, minimum detectable dose = 7.8 pg/ml), Quantikine Human Resistin (R&D System, sensitivity = 0.026 ng/mL), Quantikine Human TNFα (R&D System, sensitivity = 1.6 pg/mL), Quantikine Human IL-6 (R&D System, minimum detectable dose = 0.039 pg/mL).
For insulin sensitivity assessment we used:
Data were expressed as mean ± SD for quantitative variables.
Independent t-test student was used to compare differences between variables in the two groups. Bivariate correlations were evaluated with Pearson's coefficient.
We also used stepwise multivariate regression models using either adiponectin, leptin, resistin, TNFα or IL-6 as a dependent variable and including Z-score for BMI, WC, systolic and diastolic BP, metabolic and insulin resistance biomarkers as independent variables. All analyzes were performed using SPSS 16.0 version (SPSS, Chicago, IL, USA). A p<0.05 was considered statistical significant and the confidence interval was 95%. ❑
There were 38 children and adolescents in the study group (17 girls and 21 boys), aged 13.51±2.31 years; the control group comprised 24 children and adolescents (12 girls, 12 boys), aged 14.27±2.36 years.
Body mass index (BMI), Z-score for BMI, waist circumference (WC), systolic blood pressure and diastolic blood pressure were significantly higher in the study group compared to the control group (Table (Table11).
As for metabolic and biochemical profile there were statistical significant differences between groups regarding plasmatic levels of triglycerides and the paraclinical markers of hyperinsulinism (fasting insulinemia, HOMA-index and IRS) (Table (Table22).
Plasmatic levels of leptin, resistin and IL-6 were significantly higher in the obese group compared to control group, while plasmatic levels of adiponectin were significantly lower in obese children compared to normal-weight ones (Table (Table3,3, Figure Figure11).
TNFα was also higher in obese children and adolescents but the difference did not reach statistical significance (Table (Table33).
Univariate regression analysis revealed several significantly correlations between studied adipocytokines and clinical, anthropometric, metabolic parameters and insulin resistance markers (Table (Table44).
We also found positive correlations between leptin and resistin (r = 0.444. p <0.001) and between IL-6 and resistin (r = 0.307. p = 0.015) and a weak negative but significant correlation between adiponectin and leptin (r = - 0.267. p = 0.036).
TNFα was the only cytokine with no correlation to any of the clinical, metabolic or insulin resistance markers; it had a week, but significant correlation to IL-6 (Table (Table44).
Stepwise multiple regression analysis finally indicated that:
The adipocytokines' plasmatic profile was not different according to pubertal stages, and the only significant difference between sexes was those of leptin plasmatic levels (40.29 ±28.23 pg/ml in females versus 23.91±16.56 pg/ml in males, p = 0.006). ❑
In this study, we showed that biomarkers of an increased risk of adverse CV outcomes are already altered in obese children and adolescent: plasmatic levels of HDL-cholesterol, triglycerides and insulin-resistance biomarkers. Not only classical biomarkers of metabolic and CV risk are altered, but also the adipokines' plasmatic profile showed a parallel variation with the degree of obesity.
Excepting TNFα all the other adipocytokines showed significant correlations (negative for adiponectin and positive for others) with clinical biomarkers of obesity and its complications: Z-score for BMI, systolic and diastolic blood pressure.
Adiponectin and leptin were also negatively and respectively positively related to central adiposity quantified by waist circumference.
The only significant correlation between adipokines' plasmatic levels and lipids' profile was found for adiponectin, which positively correlated to HDL-cholesterol and negatively correlated to plasmatic triglycerides; those correlations disappeared in multivariate analysis, being explained by central adiposity.
Although in univariate analysis adiponectin, leptin and Il-6 were correlated to different paraclinical markers of insulin resistance, in multivariate analysis the only adipokine positively and significantly correlated to insulin resistance was leptin; interestingly, plasmatic levels of resistin showed no correlations with any of the paraclinical markers of the insulin resistance.
Others interesting findings were correlations between plasmatic adipokines' profiles: leptin correlated positively with resistin independent to adiposity degree, resistin was also positively correlated to IL-6 (independent to Z-score for BMI or WC), adiponectin was negatively correlated to leptin, but the correlation was partially explained by the parallel variation with central adiposity.
Simple plasmatic determination of TNFα was not a marker of the degree of obesity or its metabolic complications in our study.
Adipocytokines' plasmatic profile is a good marker of central obesity in children and a possible marker of future metabolic and cardiovascular complications.