We evaluated total adiponectin, high-molecular weight (HMW), medium-molecular weight (MMW), low-molecular weight (LMW) adiponectin subfractions, clinical parameters, routine lab parameters, lipids, metabolic, inflammatory biomarkers, and intima-media thickness (IMT) of common carotid arteries in 70 obese juveniles and adolescents with preatherosclerosis and 55 normal weight controls of similar age and gender distribution. Compared with the controls, the obese probands had a significantly increased IMT (P < 0.001) and elevated ultra-sensitive C-reactive protein (P < 0.001) indicating early vascular burden. Total and HMW adiponectin were significantly decreased in the obese cohort. The ratio between HMW and total adiponectin was significantly decreased in obese probands whereas the LMW/total adiponectin ratio was increased. Overall, total-, HMW, and MMW adiponectin were significantly negatively correlated with carotid IMT. The HMW/total adiponectin ratio correlated significantly negatively, and the LMW/total adiponectin ratio significantly positively with the IMT. Furthermore, HMW adiponectin was significantly positively correlated with high-density lipoprotein (HDL)-cholesterol and serum apolipoprotein A1, and negatively with BMI, triglycerides, homeostatic model assessment (HOMA)-index, leptin, liver transaminases, and uric acid. This remained stable after controlling for gender. Multiple regression analysis of body measures and all other lab parameters showed the strongest correlation between HMW adiponectin and carotid IMT (β = −0.35, P < 0.001). Taken together, our study provides the first evidence that preatherosclerosis in obese juveniles and adolescents is associated with altered subfractions of adiponectin, whereas after multiple testing the HMW subfraction showed a better correlation to IMT compared with total adiponectin.
Ample evidence exists now that the adipose tissue represents a highly active endocrine organ (1). Adiponectin, a cytokine that is exclusively expressed in adipose tissue (2), was found to be decreased in obesity (3). Hypoadiponectinemia is associated with insulin resistance (4), type 2 diabetes (5), dyslipidemia (6), and hypertension (7). Adiponectin exerts protective effects also against atherosclerosis, because of antiinflammatory and antiatherogenic features (8). Thus, adiponectin levels are decreased in adults with advanced stages of atherosclerosis, such as coronary artery disease (9), and in subjects with endothelial dysfunction (10,11). Recently it was shown that adiponectin circulates in peripheral blood in low-molecular weight (LMW), medium-molecular weight (MMW), and high-molecular weight (HMW) complexes (12,13,14,15,16,17,18). These subfractions may exert markedly different biological functions (13). As compared with total adiponectin, HMW adiponectin was shown to better reflect early metabolic abnormalities found in childhood obesity (19). Thus, associations of adiponectin subfractions with metabolic and vascular diseases remain to be elucidated. In foregoing studies we detected an increased carotid intima-media thickness (IMT) paralleled by a subclinical inflammation in obese juveniles aged around 13 years (20). Additionally, we showed an influence of subcutaneous adipose tissue topography on total adiponectin serum levels in obese juveniles, and provided the first evidence that incipient atherosclerosis is associated with low serum levels of adiponectin (21). These observations are confirmed by a recent study of Beauloye et al., who showed that total adiponectin plasma levels are related to preatherosclerosis in juvenile obesity more than conventional cardiovascular risk factors (22). We hypothesize that the subfractions of adiponectin are associated with preatherosclerotic symptoms in early phases of obesity in a particular manner. To address this, we analyzed correlations of total adiponectin and LMW, MMW, HMW subfractions with carotid IMT, and cardiovascular risk factors in a cohort of obese juveniles and adolescents compared with normal weight, healthy controls.
Methods and Procedures
Study participants were from the STYrian Juvenile Obesity Study (STYJOBS), which is designed to investigate early stages of atherosclerosis and metabolic disorders in obese juveniles and adolescents. The STYrian Juvenile Obesity Study is registered at ClinicalTrials.gov (Identifier NCT00482924), where detailed information of the study is available. The inclusion criterium for the obese probands was BMI > 97th percentile if <18 years of age, BMI > 30 kg/m2 if >18 years of age. Exclusion criteria were endocrine diseases (e.g., hypothyreosis), infectious or any other chronic diseases. Controls were healthy juvenile volunteers recruited from the Department of Pediatric Surgery, where they underwent minor elective surgery. Fasted blood samples were collected before surgery and all controls had to be normal weight and free of infectious or endocrine diseases. Seventy obese juveniles and adolescents recruited from January to December 2006 (mean age 12.9 ± 3.3 (s.d.) years) and 55 normal weight healthy controls of similar age and gender distribution were investigated. The study was approved by the ethical committee of the Medical University of Graz. Blood collection and ultrasonography were performed after written informed consent was given by the probands if aged >18 years, or by their parents, if they were <18 years old. At the time of blood collection the probands were fasting. Venous puncture was performed in a standard procedure (cubital vein approach with butterfly), blood samples were immediately centrifugated at 3,500 rpm at ambient temperature and stored at −80 °C until analysis.
HMW, MMW, and LMW subfractions as well as total full-length adiponectin concentrations were analyzed from human sera using the adiponectin (multimeric) enzyme-linked immunosorbent assay (ELISA; 47-ADPH-9755) from Alpco Diagnostics. Leptin and resistin were determined from human plasma by ELISAs from Biovendor Laboratory Medicine (Brno, Czech Republic) according to manufacturer's instructions. Intra- and interassay coefficients of variation for all ELISAs in our study were <10%. Cholesterol and triglycerides were measured by means of ECLIA (ElectroChemiLuminiscenceAssay) on an Elecsys 2010 analyser (Roche Diagnostics, Mannheim, Germany), and lipoproteins were separated by a combined ultracentrifugation-precipitation method (β-quantification). Blood lipids inclusive of fatty acids were analyzed as outlined elsewhere (23). Oxidized low-density lipoprotein (oxLDL) was measured by a commercially available ELISA (Mercodia oxLDL Competitive ELISA, SE-754 50; Uppsala, Sweden). High-sensitive C-reactive protein was analyzed using a particle-enhanced immunoturbidimetric assay (Tina-quant C-reactive protein latex ultra-sensitive assay (Roche diagnostics)). Homocysteine was determined by triple quadrupole mass spectrometry (Applied Biosystems; API 2000 LC/MS/MS-system) using a 3.3 × 0.46 cm2 HPLC column (SUPELCO LC-CN). Plasma insulin was measured by ELISA (Mercodia, Uppsala, Sweden), and plasma glucose was measured by the glucose hexokinase method on a Hitachi 917 chemical analyser. HOMA-IR (homeostatic model assessment-insulin resistance) was calculated as the product of the fasting plasma insulin value (in µU per ml) and the fasting plasma glucose value (in mmol/l), divided by 22.5 (ref. 24). Liver transaminases AST/GOT, ALT/GPT, γ-GT, creatinine, and uric acid were measured by routine laboratory methods on a Hitachi 917 chemical analyser.
Carotid artery ultrasound
The ultrasound protocol involved scanning of the bulbous near CCA (25) on both sides with a 12-to-5-MHz broad-band linear transducer on a HDI 5000 (ATL; Bothell, Washington, DC). All scans were performed by the same investigator to identify the greatest wall thickness. Longitudinal images directed through the center of the artery were taken at each vessel site. Measurements were made from stored digital images by an experienced reader. The IMT was assessed at the far wall as the distance between the interface of the lumen and intima, and the interface between the media and adventitia (25,26,27). The maximal IMT was recorded at each of the vessel segments and averaged for the left and right sides. The lumen diameter was calculated as the interadventitial diameter minus twice the maximum far wall IMT. All diameters were measured during diastole to avoid image blurring due to systolic arterial wall motion, and to minimize the influence of blood pressure (25,26,27).
Statistical analysis was performed using SPSS version 14. Kolmogorov-Smirnov test was used to examine for normal distribution. Means were compared by a two-tailed unpaired sample t-test or by Mann-Whitney test, depending on the distribution of the data. A value of P < 0.05 was considered statistically significant. Least squares regression analysis was performed to test the correlation between variables according to Pearson. Significance of correlation was determined by univariate ANOVA and subsequent multiple comparison analysis. Stepwise multiple regression analysis was applied to investigate associations between total adiponectin, adiponectin subfractions as dependent variables, and IMT as well as clinical and metabolic measures as independent variables.
The clinical and laboratory characteristics of the study probands are summarized in Table 1. Obese juveniles had a significantly increased IMT (P < 0.001) and elevated high- sensitive C-reactive protein (P < 0.001) indicating early stages of atherosclerosis and chronic low grade inflammation (Table 1). Total cholesterol, triglycerides, oxLDL, fasted glucose, insulin, HOMA index, free fatty acids, homocysteine, systolic blood pressure, diastolic blood pressure, resistin, leptin, ALT/GPT, AST/GOT, and uric acid were significantly increased, and high-density lipoprotein (HDL) cholesterol, apolipoprotein A1 significantly decreased in the obese cohort (Table 1). Total adiponectin and HMW adiponectin were decreased (Table 2). The ratio between HMW and total adiponectin was significantly decreased in the obese group whereas the LMW/total adiponectin ratio was increased (Table 3).
Table 1. Baseline characteristics of the study subjects
Table 2. Adiponectin and subfractions
Table 3. Ratios between subfractions and total adiponectin
Total, HMW, and MMW adiponectin were significantly negatively correlated with carotid IMT (Table 4, Figure 1a-c). The ratio between HMW and total adiponectin correlated significantly negatively with the carotid IMT (Figure 2a) whereas the LMW/total adiponectin ratio showed a significant positive correlation (Figure 2b). Further, total and HMW adiponectin were significantly positively correlated with HDL-cholesterol, serum apolipoprotein A1, and negatively with BMI, triglycerides, glucose, insulin, HOMA index, AST, ALT, and uric acid (Table 4). A significant negative correlation was seen between leptin and HMW adiponectin (Table 4). The LMW adiponectin subfraction was positively associated with oxLDL, diastolic blood pressure, resistin, and leptin. These correlations remained stable after controlling for gender (Table 4). A multiple stepwise regression analysis encompassing all significant correlations shown in Table 4 revealed a significant negative correlation between HMW adiponectin and carotid IMT followed by uric acid (Table 5). Stable positive correlations were also seen between HMW adiponectin and apolipoprotein A1 as well as LMW adiponectin and oxLDL (Table 5).
Table 4. Correlation analysis of adiponectin serum levels of all study participants with several other parameters
Table 5. Multiple stepwise regression analysis to evaluate correlations of total adiponectin and subfractions
The adipose tissue secretes a broad range of hormone-like molecules called adipokines, which are involved in obesity-related metabolic abnormalities and chronic inflammation. Adiponectin is an exceptional adipokine because low plasma concentrations are associated with obesity, insulin resistance, and cardiovascular disease (28). Adiponectin seems to be protective against atherosclerosis, and the determination of plasma levels may help to assess the risk of coronary artery disease (8,9,10,11,20,29,30). A vasoprotective effect of adiponectin is supported by in vitro studies showing that adiponectin decreases the expression of adhesion molecules on endothelial cells (31), suppresses foam cell formation by macrophages (32), and inhibits vascular smooth muscle migration (33). Adiponectin knock out mice develop pronounced atherosclerotic lesions (34). However, the data concerning adiponectin plasma levels and increased IMT in obese adults as well as the association with mortality after cardiovascular events are contradictory (35,36). In particular, the role of adiponectin in very early stages of atherosclerosis remains unclear. It was recently shown that adiponectin circulates in peripheral blood in LMW, MMW, and HMW, which are formed by oligomerization from adiponectin monomers (12,13,14,15,16,17). Increasing evidence exists that these adiponectin subfractions exert different biological functions (13). In a foregoing study we showed an influence of subcutaneous adipose tissue topography on adiponectin serum levels in obese juveniles, and provided the first evidence that incipient atherosclerosis, identified by an increased IMT of common carotid arteries, was significantly correlated with low serum levels of adiponectin (21). To investigate these observations more in depth, we analyzed the associations of LMW, MMW, and HMW adiponectin isoforms with carotid IMT, cardiovascular risk factors, conventional laboratory markers, and other adipokines in a large cohort of obese juveniles and adolescents compared with normal-weight healthy controls.
There has been limited study of metabolic correlates of adiponectin isoforms in humans, especially in very early stages of obesity (19). To the best of our knowledge, this is the first investigation of adiponectin subfractions in preatherosclerotic obese juveniles and adolescents diagnosed by an increased IMT. By a multiple stepwise regression analysis, we showed a significant negative association between HMW adiponectin and carotid IMT (Table 5). This fact underlines an important role of HMW adiponectin for vascular protection, as early as in childhood. The data by Torigoe et al., showing a predictive value of HMW adiponectin for endothelial dysfunction estimated by flow-mediated dilatation of the brachial artery in young men, support this notion (37). Adiponectin was found to be involved in the development of insulin resistance and metabolic syndrome in several studies (4,12,34). In accordance with these observations, we found negative correlations between HMW adiponectin, insulin resistance, and uric acid, which confirms the close relationship between HMW adiponectin and the metabolic syndrome (38), even as early as in childhood. After controlling by multiple regression analysis stable negative associations were observed between uric acid, total, and HMW adiponectin (Table 5). A negative correlation between uric acid and total adiponectin was also found in young adults investigated by the Bogalusa Heart Study (39). However, the functional metabolic role of adiponectin in this inverse relationship remains to be clarified (39). An increase in uric acid levels may indicate unfavorable changes of factors involved in preatherosclerosis (40). Decreased adiponectin plasma levels were also reported in fatty liver disease (41) and nonalcoholic hepatic steatosis (42). Putative hepatoprotective effects of adiponectin include the induction of hepatic fatty acid oxidation, inhibition of fatty acid synthesis, and suppression of tumor necrosis factor α production (43). We found a significant inverse correlation between HMW adiponectin, total adiponectin and ALT, which remained stable after controlling by gender. As the obese cohort showed markedly increased liver transaminases, indicative for incipient fatty liver disease, these significant negative correlations suggest an involvement of adiponectin in hepatic pathology associated with juvenile obesity. This is supported by recent data showing that HMW adiponectin, but not the MMW and LMW isoforms, correlated significantly negatively with markers of liver injury associated with the metabolic syndrome (44). However, after multiple regression analysis, SPSS did not choose ALT as significant independent variable with respect to either total, HMW, MMW, or LMW adiponectin as dependent variable.
It was reported that adiponectin is positively correlated with HDL-cholesterol, apolipoprotein A1, and negatively with triglycerides (6,45). The HMW subfraction was found closely correlated with HDL-cholesterol and apolipoprotein A1 (46). Concerning apolipoprotein A1, which showed a stable positive correlation with total and HMW adiponectin after multiple regression analysis, the present results confirm these results in cases of obese juveniles and adolescents.
Levels of adiponectin and subfractions were also shown to be affected by pubertal stage and gender (47). Thus, different Tanner stages may significantly influence the distribution of adiponectin multimers. At the moment, a possible limitation of our work is given by the fact that all Tanner stages were broadly present in our experimental groups (i.e., female/male controls, female/male obese juveniles) leading to the effect that the number of probands with certain Tanner stages was far too small for a reliable statistical analysis. It will be interesting to analyze differences between pre-, intra- and postpuberal phases in the future after increasing the number of investigated subjects in all subgroups.
The process of oligomerization of adiponectin monomers to the HMW adiponectin oligomeric complex may be critical for the physiological effects of this adipokine. The LMW trimeric complex forms through noncovalent interactions of the collagenous domains of three adiponectin monomers (48). Disulfide bonds are essential for further oligomerization of two LMW complexes to the MMW hexameric complex (49). Posttranslational glycosylation and hydroxylation of four conserved lysine residues within the collagenous domains are required to form HMW oligomeric complexes of adiponectin (50). While the HMW complex is assumed to act beneficial against insulin resistance and chronic inflammation (13,51,52,53), the function of the LMW form is controversial (13,51,54,55,56). In obese individuals the oligomerization may be disturbed (e.g., by oxidative stress induced by chronic low grade inflammation). This is supported by our observation that the LMW adiponectin subfraction was significantly positively correlated with oxLDL which remained stable after multiple stepwise regression analysis (Table 5). Furthermore, the decreased HMW/total adiponectin and the increased LMW/total adiponectin ratio found in obese cohort suggest a disturbed oligomerization process from LMW to HMW adiponectin.
In summary, we showed that HMW adiponectin exerts a significant negative correlation with carotid IMT after multiple testing. On the other hand, a positive correlation between LMW adiponectin, and oxLDL suggests negative effects of the LMW subfraction. Our study provides the first evidence that incipient atherosclerosis in obese juveniles and adolescents is associated with specifically altered subfractions of adiponectin.
We gratefully acknowledge the laboratory work of Ms. Kerstin Hingerl. This work was funded by the “Zukunftsfond Steiermark” Project “STYJOBS-Extension”. Furthermore, the Austrian Nano-Initiative co-financed this work as part of the Nano-Health project (no. 0200), the sub-project NANO-PLAQUE being financed by the Austrian FWF (Fonds zur Förderung der Wissenschaftlichen Forschung, Project no. N212-NAN). This study is registered at ClinicalTrials.gov (Identifier NCT00482924).