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Unité d’Endocrinologie Pédiatrique (V.B., H.T.T.M., M.M.), Department of Internal Medicine (F.Z.), Service de Radiologie Pédiatrique (P.C.), and Unité d’Endocrinologie et Métabolisme (S.M.B.), Université Catholique de Louvain, B-1200 Brussels, Belgium
Address all correspondence and requests for reprints to: Véronique Beauloye, M.D., Ph.D., Unité d’Endocrinologie Pédiatrique, Department of Pediatrics, Cliniques Universitaires Saint-Luc, UCL 1300, Avenue Hippocrate, 10, B-1200 Brussels, Belgium. E-mail: veronique.beauloye{at}pedi.ucl.ac.be.
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Abstract |
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Objective: The aim of the study was to investigate determinants of increased carotid intima-media thickness (IMT), an early marker of atherosclerosis, in obese children.
Design: A total of 104 obese children [age, 12.7 ± 0.2 yr; body mass index (BMI)-z-score, 2.8 ± 0.7] underwent an oral glucose tolerance test. Fasting levels of glucose, insulin, C-reactive protein and adhesion molecules (sICAM, sVCAM, sE-selectin), lipid profile, adiponectin, and resistin were determined. IMT was measured by ultrasound. Insulin resistance was estimated by the homeostatic model assessment index. Baseline measurements of blood parameters were obtained from 93 nonobese children (age, 13.0 ± 0.2 yr; BMI-z-score, –0.2 ± 0.9), and IMT was measured in 23 other control children with similar characteristics.
Results: Univariate analysis showed a significant positive correlation between IMT and relative BMI, the degree of systolic hypertension, fasting insulin levels, homeostatic model assessment-R index, and resistin concentrations, whereas an inverse correlation with adiponectin levels was found. No correlation was obtained between IMT and classical CV risk factors such as positive familial history of type 2 diabetes or precocious CV disease, visceral obesity, or the lipid profile. C-reactive protein and adhesion molecule levels were not associated with IMT in our obese population. When controlled for sex, Tanner stage, and relative BMI, only adiponectin levels remained an independent determinant of IMT.
Conclusion: Adiponectin more than conventional CV risk factors and inflammation status may be related to early atherosclerosis in obese children.
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Introduction |
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Persistent elevation of insulin levels is associated with increased CV risk in young adults (3), and a correlation between insulin resistance and endothelial dysfunction has been described in adults (4), suggesting a key role for insulin resistance in the pathogenesis of atherosclerosis.
Besides serving as energy storage, adipose tissue secretes various bioactive substances (adipokines), which might affect insulin sensitivity and/or vascular function. Adiponectin is one of such molecules exhibiting important antidiabetic and antiatherogenic properties. Its circulating levels are paradoxically inversely proportional to adipose tissue mass—mainly visceral—and proportional to insulin sensitivity. Moreover, low adiponectin levels promote the production of adhesion molecules in endothelial cells, proliferation of smooth muscle cells, and formation of foam cells (5). In line with these observations, hypoadiponectinemia has been shown to predict the occurrence of coronary artery disease and myocardial infarction in adults (6). Resistin is another adipokine that may play a role in the development of obesity-associated insulin resistance. In obese children, the effects of low adiponectin levels and high resistin concentrations on the early atherosclerotic process, independent of other confounding CV risk factors common in older populations, remain to be determined.
Up-regulation of endothelial adhesion molecules, including endothelial-leukocyte adhesion molecule (E-selectin), intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), plays a pivotal role in the earliest phases of atherosclerosis by mediating the binding and subsequent recruitment of monocytes into arterial intima (7). Another mechanism, which probably plays a fundamental role in atherogenesis, is low-grade systemic inflammation. Concentrations of high-sensitive C-reactive protein (hs-CRP) and soluble adhesion molecules have been found to be higher in obese than in lean children (8, 9). In adults, prospective studies have provided evidence for a predictive role of elevated circulating levels of soluble (s) ICAM-1 in initially healthy people, of sVCAM-1 in patients at high risk or with overt CV diseases, and of hs-CRP in both healthy subjects and patients with preexisting ischemic heart disease (10, 11, 12). The predictive role of hs-CRP and adhesion molecule levels in the earliest stage of atherosclerosis remains to be studied in obese children.
In the present study, we examined in obese children and adolescents the association between increased IMT and the presence of conventional CV risk factors such as visceral obesity, hypertension, dyslipidemia, and insulin resistance. We also investigated in our population whether increased IMT correlated with subclinical inflammation or enhanced production of adhesion molecules. Finally, we explored the possible role of the dysregulated production of adipokines in the vascular changes observed in obese children and adolescents.
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Subjects and Methods |
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We studied 104 children or adolescents who had been referred to our pediatric obesity clinic between 2002 and 2004. Subjects were eligible if they were healthy, were between 8 and 18 yr of age, and had a body mass index (BMI) that exceeded the age- and gender-IOTF (International Obesity Task Force) BMI cutoff points for children (corresponding to an adult BMI > 25 kg/m2) (13). Exclusion criteria included: hepatic, infectious, or endocrine diseases [other than diabetes or impaired glucose tolerance (IGT)], syndromic obesity, craniopharyngioma, and the use of medication that alters blood pressure or glucose or lipid metabolism. All subjects were nonsmokers; 82% were Caucasian, 6% Hispanic, 4% Black, and 9% Arabian. A total of 93 volunteers, who were partially recruited from the 1-d pediatric surgery clinic and underwent minor elective surgery (e.g. varicocoele, dental extraction), were enrolled as controls. Fasting blood samples were collected before surgery, and subjects had to be normal weighted (BMI values corresponding for age and sex to an adult BMI < 25 kg/m2, according to the IOTF criteria). Apart from obesity, inclusion/exclusion criteria were identical to those used for the obese group. Twenty-three other nonobese control children (mean age, 11.7 ± 0.4 yr) were recruited for the measurement of their intima-media thickness (IMT) only. This second control group had similar characteristics for sex ratio and Tanner stage to the first control cohort. The study was conducted according to the Declaration of Helsinki, and the study protocol was approved by the Ethical Committee of the Cliniques Universitaires Saint-Luc. Written informed consent was obtained from the parents, and informed assent from the children and adolescents.
Procedures
Subjects were evaluated at 0800 h, after an overnight fast. The carotid intima-media thickness was measured first. Thereafter, a detailed medical and familial history was obtained for all subjects, and a physical examination was performed, including staging of the puberty according to the criteria of Tanner. Waist circumference (WC) was measured midway between the lowest rib and the iliac crest at the end of gentle expiration. In the absence of national references, the Dutch references of WC for children were used (14). Blood pressure was measured three times with an appropriate size cuff while the subjects were seated and the lowest measurement was used for analysis. Baseline blood samples were obtained by venipuncture (cubital vein approach with butterfly) for measurements of levels of glucose, insulin, C-reactive protein (CRP), adhesion molecules (sICAM, sVCAM, sE-selectin), lipid profile, adiponectin, leptin, and resistin. Serum (after allowing blood to clot at room temperature) or plasma (potassium EDTA) was rapidly obtained by refrigerated (4 C) centrifugation at 2000 x g for 15 min. Glucose, insulin, and lipids were immediately measured; aliquots for other assays were stored at –20 C until analysis. An oral glucose tolerance test was then performed with the administration of 1.75 g of glucose per kilogram of body weight (maximal dose, 75 g; pyrogen-free anhydrous D-glucose; Roquette, Lestrem, France). Blood samples for determination of glucose and insulin were collected just before and 30, 60, 90, and 120 min after the glucose load.
Definitions
The relative BMI of each patient was calculated as the BMI divided by the BMI at 50th percentile for age and gender (15) x 100. When the values were adjusted for sex and Tanner, our population was divided in three groups: boys and girls of Tanner stages 1 and 2, boys of Tanner stages 3 to 5, and girls of Tanner stages 3 to 5.
Subjects were considered as having a positive familial history of obesity, diabetes, or CV diseases if one parent of the first or second degree was obese (BMI > 30 kg/m2) or had type 2 diabetes or precocious CV disease.
Subjects were classified as hypertensive if their systolic and/or diastolic blood pressure exceeded the 90th percentile for age and sex after adjustment for height (16). The degree of hypertension was evaluated by the difference between the systolic or diastolic blood pressure measured in one subject and the 90th percentile for age and sex after adjustment for height.
IGT was defined, according to the American Diabetes Association guidelines, as a 2-h glucose level between 140 and 200 mg/dl, and type 2 diabetes was defined as a 2-h glucose level of more than 200 mg/dl. Insulin resistance was determined by the homeostatic model assessment (HOMA) and calculated as the product of the fasting plasma insulin level (in microunits per milliliter) and the fasting plasma glucose level (in millimoles per liter), divided by 22.5. The HOMA index was validated as a reliable measure of insulin sensitivity in obese and nonobese children and adolescents (17, 18).
Obese children and adolescents were classified as having the metabolic syndrome if they met three or more of the following criteria for age and sex: a BMI above a BMI corresponding for age and sex to an adult BMI above 30 kg/m2 (IOTF criteria); systolic or diastolic blood pressure above the 90th percentile; a triglyceride level above the 95th percentile; a high-density lipoprotein (HDL)-cholesterol level below the 5th percentile (19); and IGT.
Biochemical analysis
Serum adiponectin and leptin levels were determined by RIA (Linco Research Inc., St. Charles, MO), according to the manufacturer’s instructions. Plasma resistin and serum adhesion molecules (sICAM, sVCAM, sE-selectin) concentrations were measured by ELISA (Linco Research Inc., and R&D Systems, Minneapolis, MN, respectively). The hs-CRP was measured by immunonephelometry (Dade-Behring, Marburg, Germany). Serum levels of apolipoprotein (Apo) A and B were assessed by immunoturbidimetry (Roche Diagnostics, Mannheim, Germany). Fasting plasma glucose was measured using a glucose oxidase method. Serum insulin concentrations were measured by an immunoassay (Elecsys, Roche Diagnostics). Plasma total cholesterol, HDL cholesterol, and triglycerides were determined by automated enzymatic methods (Unicel DxC800 Synchron System, Beckman Coulter, Fullerton, CA). Low-density lipoprotein (LDL)-cholesterol concentrations were calculated using Friedewald’s equation.
Carotid artery ultrasound
Subjects were examined by two trained radiologists, lying with the head turned 45° away from the side being scanned. The ultrasound system used was a high-resolution appliance (HDI 5000, ATL, Bothell, WA) equipped with a 12- to 5-MHz broadband linear transducer. Both the left and right carotid arteries were scanned at four sites: at 1 and 2 cm from the beginning of the dilatation of the carotid bulb using the longitudinal axis of the common carotid artery and at the level of the carotid artery bulb and the internal carotid artery, on a transversal scan. The IMT was assessed at the far wall as the distance between the interface of the lumen and intima (first echogenic line) and the interface between the media and adventitia (second echogenic line) (illustrated in Fig. 3
). The maximal IMT of two measurements done at each of the four segments vessels was recorded on both sides and averaged for the left and right sides. We thus obtained four IMT values (one value for each site) in a given subject. Because the four IMT values were highly correlated with each other and were not statistically different, the mean of the four IMT values was used for analysis. Our IMT values for both common and internal carotid arteries lay within the range of previously reported data for both obese and control subjects (20, 21, 22).
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The data were expressed as percentage, median (Tanner stage), or mean ± SEM. All of the continuous variables that were not normally distributed were transformed to a logarithm or an x-root according to Box and Cox before any statistical analysis. Comparisons of continuous values between groups were done by Student’s t test or ANOVA. Comparisons of dichotomic values between groups were done by 
2 test. To adjust for sex and Tanner stage or other parameters, we used multiple regression analysis for continuous variables and logistic regression for dichotomic parameters. Two-sided P < 0.05 was considered as statistically significant.
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Results |
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. No difference in age, sex ratio, and Tanner stage was observed between the control and the obese groups. Fasting glucose, insulin, HOMA for insulin resistance (HOMA-R) index, and LDL-cholesterol levels increased significantly, whereas HDL cholesterol and Apo A levels decreased in the obese cohort. All of these trends persisted after adjustment for sex and Tanner stage. hs-CRP, leptin, and sE-selectin levels were increased, and adiponectin levels were decreased in the obese patients (Table 1), and again these changes persisted after adjustment for gender and Tanner stage (data not shown). sICAM levels tended to increase in the obese group, but the difference was not statistically significant. In our obese population, several variables were correlated with the degree of obesity. Thus, fasting triglyceride (r = 0.261; P = 0.009) and insulin (r = 0.421; P < 0.001) levels, HOMA-R-index (r = 0.420; P < 0.001), WC-z-score (r = 0.741; P < 0.001), the degree of hypertension (r = 0.458; P < 0.001), hs-CRP (r = 0.276; P = 0.009), leptin (r = 0.442; P < 0.001), resistin (r = 0.295; P = 0.006), and sE-selectin (r = 0.243; P = 0.025) concentrations increased significantly, whereas adiponectin (r = –0.351; P = 0.001) levels decreased with increasing BMI values. All these trends persisted after adjustment for sex and Tanner stage (data not illustrated). The obese children also demonstrated significantly increased mean carotid IMT values as compared with nonobese children matched for age, sex, and pubertal stage. Thus, obese children and adolescents had a worse CV risk profile, enhanced concentrations of markers of future CV disease, and an increased carotid IMT, in agreement with their degree of obesity.
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). In our obese population, IMT values were not affected by sex and pubertal stage. In contrast, univariate analysis showed a statistically significant positive correlation with the relative BMI, the degree of systolic hypertension, resistin concentrations, fasting insulin levels, and HOMA-R index, and a strong inverse correlation with adiponectin levels (Table 2 and Figs. 1 and 2). These correlations remained significant even after adjustment for sex and Tanner stage. No correlation was observed between carotid IMT and other classical CV risk factors such as positive familial history of type 2 diabetes or precocious CV disease, visceral obesity (WC-z-score), triglycerides, Apo B, or HDL- and LDL-cholesterol levels. CRP and adhesion molecule levels also did not predict IMT in our obese population.
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To compare further the clinical and laboratory profile of the obese children according to their IMT, we stratified the obese cohort into quartiles of IMT values (Table 3 and Fig. 3
). Obese children in the top quartile (IV) displayed higher relative BMI and lower adiponectin levels than those in the bottom IMT quartile (I). Adiponectin was the only biomarker that gradually declined along the different quartiles. Lipid concentrations did not significantly differ between children with different IMT (data not shown).
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Discussion |
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Enhanced carotid IMT is an established marker for early, preclinical atherosclerosis (23). This technique has been widely used and validated in children and shows strong dose-response associations in children with known CV risk factors, such as familial hypercholesterolemic children (23) and children with type 1 diabetes (24) or hypertension (25). A study of Tounian et al. (26) first reported that severe obesity in children is associated with impaired arterial stiffness and endothelial dysfunction, other markers of early atherosclerosis. Our work demonstrates, in agreement with recent studies (20, 21, 22), that carotid IMT is increased in obese children and further extends those data by showing that carotid IMT increases with the degree of obesity and positively correlates with the relative BMI. The difference in weight rather than sex or Tanner stage explains the difference in carotid IMT. In a large group of normal children and young adults, aged 10–25 yr, Sass et al. (27) found that carotid IMT was not affected by age and sex until 18 yr of age. After 18 yr, carotid IMT increased sharply in men, leading to significantly greater IMT in men than in women. The results of our study demonstrate that carotid IMT also does not change with age in our pediatric population and for the first time that carotid IMT is not affected by pubertal status.
We confirm the observation of a recent study that the increased carotid IMT observed in obese children is related to classical risk factors of obesity, especially hypertension and impaired insulin sensitivity (28). In addition, we investigated the role of more recently described factors implicated in early atherosclerosis such as subclinical inflammation, endothelial activation, and adipokines. hs-CRP levels were significantly elevated in obese juveniles compared with the control group and correlated with BMI values. However, no correlation between hs-CRP and IMT was observed when our whole population of obese children was taken into consideration (lean control subjects could not be included in this analysis). In line with this lack of correlation, Whincup et al. (29) showed that CRP levels made only a modest contribution to the reduced arterial distensibility observed in obese adolescents and that this weak contribution was almost completely abolished after adjustment for adiposity. In addition, the association between the presence of metabolic changes and the increased vascular stiffness in obese children has recently been shown to be independent of the differences in CRP levels (30). Yet, when stratifying our obese population into quartiles of IMT values, we observed a trend toward higher levels of hs-CRP in the top quartile when compared with others. This suggests that chronic inflammation might be more powerfully associated with more severe atherosclerotic stages. The significant correlation between circulating E-selectin levels and BMI shown in our obese children [as also found in adults (Ref. 31)] suggests that obesity per se promotes early endothelial activation. However, in our obese population, no correlation between soluble adhesion molecule concentrations and IMT has been observed. Whether the observed endothelial activation in obese children plays an independent role in obesity-related atherogenesis needs further clarification by performing longitudinal follow-up studies.
This is also the first study that investigated resistin as a determinant of early atherosclerosis in children. We did not find any significant difference in resistin levels between obese and lean children. This is in agreement with recent studies performed in children where no correlation was demonstrated between changes in resistin levels and changes in BMI (32). In fact, in humans, inflammatory cells more than adipocytes seem to be the major source of resistin. It has been suggested that resistin could contribute to the development of atherosclerosis because progressively increased resistin mRNA and protein expressions are detectable in the developing aortic atherosclerotic lesions of Apo E–/– mice (33). The fact that plasma resistin levels are associated with the presence of coronary heart disease in women (34) and with coronary artery calcification (35) also supports this hypothesis. One recent study has investigated whether resistin levels are associated with the development of early atherosclerosis assessed by carotid IMT measurements in 525 Finnish middle-aged subjects (36). The authors reported that resistin levels were correlated with carotid IMT but that this association did not remain significant after adjustments for BMI or other risk factors, an observation in line with our results in obese children. Thus, resistin does not appear to be a significant independent predictor of early atherosclerosis measured by IMT.
In contrast, our observation that adiponectin levels differed between extreme IMT quartile groups and were independently associated with increased IMT in obese children suggests that this adipokine may play an early role in the pathophysiology of atherosclerosis. Our results are consistent with background data showing that adiponectin suppresses various mechanisms contributing to atherogenesis (see introductory section). Moreover, in adults, hypoadiponectinemia has been shown to predict the occurrence of coronary artery disease and myocardial infarction (6). This association has also been recently shown to be independent of other well-known risk factors such as dyslipidemia, insulin sensitivity index, smoking, and blood pressure in elderly men (37). A recent study has also demonstrated in 1515 middle-aged healthy subjects an independent negative association between adiponectin levels and common carotid IMT, suggesting hypoadiponectinemia as a risk factor in the development of early atherosclerosis (38). In children, adiponectin has been associated to increased IMT in severely obese individuals (mean BMI-z-score, 6.0 compared with 2.8 in our study) (39) but did not predict vascular function (measured by brachial artery flow-mediated endothelial-dependent dilatation) in healthy ones (40). Our study shows a significant negative correlation between adiponectin (but not other variables) and carotid IMT in obese children after adjustment for sex, pubertal stage, and relative BMI. We thus confirm and extend the data of Pilz et al. (39) by showing that such an association occurs not only in severely obese children, but also in less obese ones (who best reflect the population of European obese children). We further demonstrate that this association is obtained after measurements performed at four sites of the carotid artery (not only at the bulb) and after taking into account additional novel CV risk factors (adhesion molecules).
In conclusion, the degree of obesity, hypertension, insulin resistance, and high resistin and low adiponectin levels form a risk profile for increased IMT in obese children. However, adiponectin more than conventional CV risk factors and other novel parameters (inflammation status, adhesion molecules, other adipokines) may emerge as a biomarker of early atherosclerosis.
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Acknowledgments |
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Footnotes |
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Disclosure Summary: The authors have nothing to disclose
First Published Online May 22, 2007
Abbreviations: Apo, Apolipoprotein; BMI, body mass index; CRP, C-reactive protein; CV, cardiovascular; E-selectin, endothelial-leukocyte adhesion molecule; HDL, high-density lipoprotein; HOMA, homeostatic model assessment; HOMA-R, HOMA for insulin resistance; hs-CRP, high-sensitive CRP; ICAM-1, intercellular cell adhesion molecule-1; IGT, impaired glucose tolerance; IMT, intima-media thickness; IOTF, International Obesity Task Force; LDL, low-density lipoprotein; s, soluble; VCAM-1, vascular cell adhesion molecule-1; WC, waist circumference.
Received March 19, 2007.
Accepted May 14, 2007.
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