Metabolic syndrome (MS) in children has gained attention recently as the health consequences of the emerging obesity pandemic become known (1). MS is a cluster of cardiovascular risk factors related to obesity, insulin resistance, glucose intolerance, hypertension, and dyslipidemia. MS increases cardiovascular mortality and morbidity, and is considered as emerging and promoting atherosclerosis (2). Moreover, recent studies indicate that the process of atherosclerosis starts at an early age and is linked to obesity (3). As the largest endocrine organ of the body, adipose tissue not only stores excess body energy, but also secretes a variety of bioactive adipocytokines. Resistin is a novel adipokine and has been suggested to play a putative role in insulin resistance, inflammation, obesity, and cardiovascular disease (CVD) (4).
Resistin is a secreted peptide hormone and is highly expressed in human monocytes and macrophages, and at much lower levels in adipocytes. In humans, resistin is primarily expressed in inflammatory cells and has been shown to be involved in obesity-related subclinical inflammation, atherosclerosis and cardiovascular disease (5). Choi et al. showed that resistin may be useful in circulating biomarkers, reflecting vascular inflammation in obese subjects (6). A recent study also claimed that resistin is an inflammatory mediator and a biomarker of cardiovascular diseases, especially in atherosclerosis (7). Some human clinical studies and experiments suggested resistin was a hormone linking obesity with insulin resistance and atherosclerosis (4). Rajala et al. found a correlation between insulin resistance and endothelial dysfunction, suggesting insulin resistance play a key role in the pathogenesis of atherosclerosis (8). Additionally, resistin in humans appears to play a much larger role in inflammation than obesity or insulin resistance (9). However, inflammation plays a major role in the development of obesity, insulin resistance, MS, and atherosclerosis, therefore, we inferred that resistin may play a direct or indirect role in the progress of these diseases, but the exact mechanism is poorly understood so far.
This study was designed to investigate the relationship between resistin and MS risk factors, chronic inflammation markers (Hs-CRP) and vascular parameters characterized by carotid intima-media thickness (IMT), and then try to evaluate the value level of serum resistin in obese children with MS and early atherosclerosis.
Materials and methods
A total of 176 obese children [94 male, 82 female; body mass index (BMI)>95th percentile for age and gender] and 88 healthy children [normal weight, waist circumference (WC) less than the 90th percentile for age and sex] in which gender and age (9–12 years old) were matched were recruited through an ongoing epidemiological study. These included 3354 children (1698 male, 1656 female) for MS prevalence among children in five primary schools of five different districts in Jinan, China. The investigation was approved by the local education institution, informed parental consent was given, and subject assent were obtained from all participants before the study. All children with endocrine, genetic, or metabolic disorders, who smoked, had kidney diseases, or medical therapy were excluded from the study. The information on the children was provided by the students, parents, and head teachers. MS was defined according to International Diabetes Federation (IDF) consensus in 2007. The definition of obesity (95th percentile of BMI) was consulted from the Working Group on Obesity (WGOC) proposed BMI classification reference for screening overweight and obesity in Chinese school age (7–18 years old) children and adolescents.
All participants underwent physical examination, including staging of the puberty according to the criteria of Tanner and routine hematology and biochemical testing. Height and weight were measured, BMI was calculated, and blood pressure was measured according to the guidelines of the National High Blood Pressure Education Program (NHBPEP) (10). Systolic and diastolic blood pressure was measured twice on the right arm after a 10 min rest in the supine position using a standard mercury sphygmomanometer and averaged afterwards.
Venous blood sample were drawn after an overnight fast. Serum fasting triglyceride, high density lipoprotein cholesterol (HDL-c), low density lipoprotein cholesterol (LDL-c), total cholesterol were measured in all children using commercially available test kits. Plasma glucose levels was measured by the glucose oxidase method with a glucose analyzer, and insulin concentration was determined by radioimmunoassay method. High-sensitivity C-reactive protein (Hs-CRP) concentration was analyzed by latex turbidimetry immunoassay method. Resistin levels were measured by enzyme-linked immuno sorbent assay (ELISA) using commercially available kits (R&D systems Inc., Minneapolis, MN, USA). The intra-assay and inter-assay coefficients of variation were 4.7% and 8.1%.
Insulin resistance was expressed as homoeostasis model assessment for insulin resistance (HOMA-IR), HOMA-IR=[fasting insulin (m U/L)×fasting glucose (mmol /L)]/22.5.
The MS group was divided into five subgroups according to the number of components of MS. The metabolic syndrome definition was based on the International Diabetes Federation (IDF) cut-off values for children and adolescents in 2007, which are: 0, normal weight and waist circumference less than the 90th percentile for age and gender; 1, only central obesity (waist circumference ≥95th percentile of Chinese children for age and gender; Table 1); 2, number 1 plus one of following four criteria; (i) serum triglycerides ≥150 mg/L, (ii) plasma HDL cholesterol <40 mg/L, (iii) blood pressure: systolic blood pressure ≥130 mm Hg or diastolic blood pressure ≥85 mm Hg, (iv) plasma glucose >5.6 mmol/L; 3, number 1 plus two of above four criteria; 4, number 1 plus three or more of above four criteria.
Abdominal fat thickness measurements (11)
The maximum thickness of preperitoneal fat (P max) and the minimum thickness of subcutaneous fat (S min) in the upper median abdomen, respectively, were measured by ultrasonography using ultrasound equipment (Aloka SSD-α10; Hitachi Aloka Medical Ltd, Tokyo, Japan) and a linear-array frequency conversion probe (7–13 MHz). Subjects were kept in the supine position, the linear-array probe was kept perpendicular to the skin on the upper median abdomen, and a longitudinal scan was undertaken from the xiphoid process to the navel along the linea alba. Scanning was performed at the optimal position, and the surface of the liver was kept almost parallel to the skin by having the subjects hold their breath. The probe was touched to the skin as lightly as possible so that the fat layers were not compressed. P max and S min were determined as the visceral fat thickness of the hepatic falciform membrane, and the subcutaneous fat thickness of the upper abdomen, respectively. P max and S min were measured directly from the screen with electronic calipers. P max and S min were both related with visceral fat accumulation and subcutaneous fat accumulation in the upper abdomen.
Carotid intima-media thickness
The specific investigator measured the carotid IMT by B-mode ultrasound using 7.5 MHz linear transducer following a standardized protocol. The image was focused on the common carotid near the bifurcation at the posterior (far) wall of each side of the carotid artery after a 10 min rest, and gain settings were used to optimize image quality. A magnified image was recorded from the angle showing the greatest distance between the lumen-intima interface and the media-adventitia interface. The sonographer measured three values on each side and took the maximum value for statistical purposes as the strongest association between the different measurement of IMT and coronary risk factors is achieved by using the maximum and not the mean value of IMT (12). The participants were examined in the supine position with the head turned slightly to the side. The coefficient of variation of IMT measurements were 6% for IMT of 0.4 mm and 4% for IMT of 0.7 mm (13, 14).
Statistical procedures were performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Descriptive data were expressed as means±standard deviation. Non-parametric data not normally distributed were analyzed after being logarithmically transformed. Independent sample t-test was used to determine the difference of the descriptive metabolic characteristics, vascular measurement and serum resistin levels in obese and healthy children. Spearman univariate analysis was performed to evaluate relationships of the serum resistin levels with the atherosclerosis risk factors, inflammation marker and vascular measurements. All the subjects were divided into five subgroups (0, 1, 2, 3, 4) according to the number of MS components. Multi-group comparison was performed using one-way-ANOVA. Univariate correlation analysis was performed to evaluate the relationship of early atherosclerosis (IMT) with biomarkers of atherosclerosis risks. Path analysis was used to evaluate the value of resistin levels to early atherosclerosis. Statistical significance was taken as p-value<0.05.
Comparison of physical and biochemical parameters between obese and healthy groups
As indicated in Table 2, no difference in age and Tanner stage were observed between obese and no-obese children. However, BMI, BMI z-score, P max, S min, systolic blood pressure, diastolic blood pressure, triglyceride, total cholesterol and LDL-c, Hs-CRP, HOMA-IR, carotid IMT and resistin increased significantly, whereas HDL-c decreased in obese children. Resistin levels were higher among obese boys, while there was no difference between obese and healthy girls. There was no difference in gender.
|Tanner stage||1, 2||1, 2|
|P max, cm||1.12±0.37||0.50±0.23||0.000|
|SBP, mm Hg||121.75±11.37||103.76±10.38||0.000|
|DBP, mm Hg||71.21±7.06||64.20±6.10||0.000|
|Resistin, ng/mLa total||1.36±0.57||1.21±0.47||0.016|
|Carotid IMT, mm||0.49±0.008||0.40±0.005||0.003|
Adjusted for age and gender, the serum resistin levels are significantly and positively correlated with makers of obesity (BMI, WC, P max), systolic blood pressure, fasting insulin, HOMA-IR (r=0.345, p=0.001), Hs-CRP (r=0.293, p=0.006), and IMT(r=0.398, p=0.000). There was no correlation between serum resistin levels and fasting blood glucose, diastolic blood pressure and blood lipids (Table 3).
|SBP, mm Hg||0.290||0.006|
|DBP, mm Hg||0.301||0.054|
|P max, cm||0.415||0.006|
|S min, cm||0.325||0.053|
Figure 1 shows a positive liner correlation between resistin levels and MS components. The resistin levels in children with 1, 2, 3, 4 MS components (22.23±7.88 ng/mL, 22.78±7.88 ng/mL, 23.59±7.64 ng/mL, 24.28±6.89 ng/mL, p<0.01, respectively) was higher than those without MS component (13.88±5.3 ng/mL), except the first group, where no differences were found among the other four groups (p>0.05).
After the univariate correlation analysis, carotid IMT was positive with BMI, WC, HOMA-IR, Hs-CRP and resistin (Table 4). For contribution of multiple independent variables (HOMA-IR, Hs-CRP, and resistin levels) on one dependent variable (IMT), path analysis was used. This model explained 38.7% (R2) of variance, it means that the influence in three factors on IMT account for 38.7% (p<0.05). As shown in Table 5, path analysis indicated that serum resistin can directly (β=0.304, p=0.001) and indirectly via HOMA-IR (β=0.085, p=0.008) and Hs-CRP (β=0.047, p=0.029) contribute to early atherosclerosis in obese children (Figure Figure 2).
|SBP, mm Hg||0.158||0.092|
|DBP, mm Hg||–0.112||0.237|
|P max, cm||0.058||0.537|
|S min, cm||–0.009||0.923|
|Variables||B||β (direct)||β (indirect)||t||R2||F|
Our study demonstrates that resistin levels were higher in obese children. The serum resistin levels were positively associated with several obesity markers (BMI, WC, P max), blood pressure, fasting insulin, HOMA-IR, Hs-CRP and IMT, independent for age and gender, suggesting a potential role in the development of MS and atherosclerosis.
Resistin is an adipocyte-derived collagen like protein, highly specific to adipose tissue and is increased in obesity. A study performed on adolescents in Switzerland showed that serum resistin levels were higher in obese children (15). Similar data are reported by Li et al. in obese Chinese children (16). Additionally, resistin was correlated with carotid intima media thickness and systolic blood pressure, whereas no correlation was observed with fasting blood glucose and lipid levels (17). This study was in agreement with those studies, furthermore, observed resistin levels were positive with HOMA-IR and the number of MS components. However, in Polish obese children, no significant correlations were found between resistin concentration and IR (18). This difference may be because the children were affected by severe obesity. In adults, serum resistin levels were positive with insulin resistance (19–21). This suggested that resistin, as an adipocytokine, might play a certain role in the presence of MS in the obese children.
Atherosclerosis is a slow and progressive disease that can start in childhood (22). Measuring the IMT of the common carotid artery, as a non-invasive marker for early atherosclerotic changes, has been reported to be reliable and predictive for later CVD (23). Enhanced carotid IMT is an established marker for early preclinical atherosclerosis (24). Hs-CRP, biochemical markers for the chronic inflammation, HOMA-IR, a marker of insulin resistance and IMT, a maker of early atherosclerosis are also significantly increased in the obese children. Inflammation has a central role in the pathogenesis of atherosclerosis and in mediating its impact on the development of cardiovascular disease. Hs-CRP was identified as a useful screening marker for evaluating and estimating the degree of atherosclerosis in obese children (25). Inflammatory cells, rather than adipocytes, seem to be the major source of resistin in humans. It was suggested that resistin could contribute to the development of atherosclerosis. Serum resistin levels were significantly and positively correlated with IMT in obese Belgium children (3), and the obese Chinese children in this study are no exception. Moreover, a recent study claimed insulin resistance may be the main risk predictors of increased IMT (26). It appears that increased IMT was associated with serum resistin levels, inflammation and insulin resistance. However, the exact pathophysiology mechanism between resistin and IMT is poorly understood.
In path analysis, the results showed that serum resistin levels were directly (β=0.304, p=0.001), and indirectly via HOMA-IR (β=0.085, p=0.008) and Hs-CRP (β=0.047, p=0.029), contributing to early atherosclerosis. These three factors can account for 38.7% of all reasons in IMT, other related risk factors need to further studied. Basic research (vitro research) also proved resistin can impact on atherosclerosis directly and indirectly. The direct mechanism was as follows: resistin is highly expressed in atherosclerotic lesions and mainly from monocytes and macrophages. It can accelerate lipid accumulation and affect vascular cells, promote the formation of foam cells (27), and increase the proliferation and migration of vascular endothelial and smooth muscle cells (28). Additionally, resistin exerts direct effects on human coronary artery endothelial cells by promoting tissue factor expression to contribute to atherosclerosis (29). Resistin also indirectly impacts on elevated IMT by insulin resistance and inflammation. Resistin induces insulin resistance in pancreatic islet beta-cells at least partly via suppress cytokine signaling (SOCS-3) expression, reduces Akt phosphorylation, and also impairs glucose-induced insulin secretion (30). IR can induce an increased thrombocytes activation and aggregation, promote the smooth muscle cells proliferation, increase the monocytes adhesion molecules expression, and reduce the NO bioavailability of the endothelium (31). Inflammation also plays an important role in atherosclerotic plaque maturation until final rupture. In humans, resistin has general pro-inflammatory properties. It stimulates the secretion of tumor necrosis factor-α and interleukin-6, playing a direct role in the development of CVD (15). Resistin by inducing insulin resistance, and pro-inflammatory indirectly, contribute to IMT in obese children.
In conclusion, resistin was considered as an adipokine that is linked to MS and early atherosclerosis. Resistin not only plays a role in the presence of MS, but also indirectly via insulin resistance and Hs-CRP related to early atherosclerosis in obese Chinese children.
These studies were supported by Science and Technology Project of Shandong Province (2006GG3202024).
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