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Obesity in children continues to be a significant public health burden, with a >100% increase in the number of children diagnosed as being overweight in the past 30 y (1). The increasing prevalence of obesity has been accompanied by an increase in complications of obesity such as type 2 diabetes mellitus (T2DM). At our own institution, for example, we have observed a 10-fold increase in the number of pediatric patients with T2DM over the 10-y period from 1990 to 2000 (2). Blood pressure (BP) levels and the prevalence of hypertension in the young are also increasing, with the obesity epidemic responsible for much of the increase (3).

Obesity is accompanied by generalized inflammation, characterized by increased plasma C-reactive protein levels as well as by dysregulation of cytokine production by monocytes, lymphocytes, and other immune cells (4). Simultaneously, the presence of obesity has long been associated with the presence of endothelial and vascular dysfunction (5–7). Adipose tissue itself secretes several hormones, particularly leptin and adiponectin, and a variety of other proteins that are collectively known as adipokines. Unlike most adipokines, adiponectin expression and serum concentration are reduced in obesity, insulin resistance, and T2DM. Adiponectin levels are also modulated by the renin-angiotensin system (RAS). Its concentration is inversely correlated with other traditional cardiovascular (CV) risk factors, such as BP, low-density lipoprotein (LDL), and triglyceride (TG) levels (8,9). Furthermore, there is evidence that adiponectin may protect the endothelium via its insulin sensitizing, anti-inflammatory, antiatherogenic, antioxidant signals, and by impeding arterial wall thickening (10).

Adiponectin, a 30 kDa complement C1Q-related protein, also known as Acrp30, is the most abundant gene product secreted by fat cells of white adipose tissue. This 247 amino acid protein monomer forms trimers, which further polymerize into larger polymeric complexes varying in size between 180 kDa (hexameres; LMW) or 400-600 kDa (16-meres; HMW) (11). Adiponectin modulates several physiologic processes, such as metabolism of glucose and fatty acids, and immune responses. Decreased plasma adiponectin levels are associated with insulin resistance, T2DM, obesity, and atherosclerosis. Adiponectin has been associated with several other factors known to contribute to hypertension in obese individuals, including increased sympathetic nervous system and RAS activity, insulin resistance, elevated FFA and atherosclerosis and impaired vasoreactivity (12).

Our objective in this study was to determine whether plasma adiponectin and other inflammatory markers associated with obesity and insulin resistance are also associated with abnormal 24-h ambulatory blood pressure (ABP) patterns in obese adolescents.

MATERIALS AND METHODS

Setting and study design.

Patients who fulfilled the following criteria were recruited from the Pediatric Diabetes and Pediatric Nephrology Clinics at the Children's Hospital at Montefiore in the Bronx, NY: age 10-21 y, obesity defined as BMI >95% for age and gender (13), a diagnosis within the past 3 y of T2DM according to the standard American Diabetes Association criteria (14), and serum test results negative for antibodies to glutamic acid decarboxylase-65 or insulin autoantibodies. Obese nondiabetic subjects, defined as BMI >95% for age and gender, and normal glucose tolerance, were recruited from primary care, nephrology, and pediatric endocrinology clinics.

Patients with T2DM were ineligible when they were metabolically unstable, as defined by the history of an episode of diabetic ketoacidosis or of hospitalization for uncontrolled diabetes within the previous 2 mo. Patients with comorbid inflammatory disease, secondary hypertension, other endocrine disorders, or any genetic syndrome that would independently predispose them to either diabetes mellitus or to kidney disease were also excluded. Additionally, patients on treatment with insulin-sensitizing agents known to affect adiponectin levels (15), statins, and treatment with any antihypertensive medication including angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARB) were excluded.

Written informed consent was obtained from the parent or guardian of each subject. Subjects who were 13-21 y old also provided written informed consent. Written informed assent was obtained from children aged 10-13 y. The protocol was approved by the Montefiore Medical Center Institutional Review Board.

Study procedures and definitions.

Subjects had a study visit that included a history and a physical examination. Family health history, smoking status, and medication and vitamin usage were assessed. Families self-identified the race/ethnicity of the subject.

Anthropometric and body composition measurements were performed in the morning, before breakfast, with the subject wearing light clothing, without footwear. Body weight and standing height were measured by the same observer using a digital scale and a wall-mounted stadiometer to the nearest 0.1 kg and 0.5 cm, respectively. Waist and hip circumference was measured in a standard fashion (16) and the waist to hip ratio (WHR), was calculated as the ratio of waist and hip circumferences. BMI was calculated by dividing the weight in kilograms by the square of the height in meters, and was further used to calculate the BMI z score.

Three resting BP measurements were obtained from the right upper arm using an aneroid sphygmomanometer and appropriate size cuff (17). The first measurement was discarded and the average of the other two measurements was recorded as the study visit BP. Subjects were classified as having casual hypertension if this mean value was ≥95th percentile for age, gender, and height (17).

The subjects underwent a 24-h ABP recording using the Spacelabs 90217 ambulatory monitor (Spacelabs Medical, Issaquah, WA), fitted and programmed as previously described (18). Awake, or daytime, and sleep, or nocturnal, periods were determined by means of a diary provided by the subjects. Ambulatory hypertension was diagnosed when the average ambulatory BP for the period was ≥95th percentile BP on the basis of the subject's sex and height according to normative values for ABP (19). BP load was defined as the percentage of readings for a given period that exceeded the 95th percentile for that individual. A BP load >40% was considered to be elevated (20). Percent dipping was calculated for both average systolic (SBP) and diastolic BP (DBP) with the following formula: [(daytime BP−nocturnal BP)/daytime BP] × 100. Each subject was categorized as a “dipper” (decrease in average SBP and DBP ≥10% during sleep) or a “nondipper” (decrease <10%) (21). BP index (mean ambulatory BP divided by the 95th percentile for pediatric ambulatory BP) was calculated.

Fasting blood was drawn for adiponectin, glucose, insulin, liver enzymes, high sensitivity C-reactive protein (hs-CRP), creatinine, renin, aldosterone, HA1c, LDL, high-density lipoprotein, TG, and cholesterol levels. Urinary creatinine and microalbumin measurements were obtained from two random mid-stream urinary specimens obtained from all subjects. All tests except adiponectin measurement were performed in the Montefiore Medical Center's clinical laboratory.

The lipid panel and Hb A1c were interpreted according to the American Diabetes Association clinical practice recommendations (22). The remaining serum tests were compared with standardized normal values. Microalbuminuria was defined as an albumin-to-creatinine ratio ranging between 30 and 300 mg/g creatinine (23). Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the formula [fasting serum insulin (uIU/mL) × fasting serum glucose (mg/dL)] × 0.002246 (24). The estimated glomerular filtration rate (eGFR) was calculated with the Schwartz formula (25).

Serum total adiponectin concentration (μg/mL) was measured at the Diabetes Research Center at the Albert Einstein College of Medicine using a commercially available radio immunoassay (Linco Research, St. Louis, MO). Blood samples obtained at the study visit were allowed to clot at room temperature for 30 min, then centrifuged at 4°C for 15 min at 2500 rpm. Serum was stored at −80°C until the day of measurement. The assay was carried out in duplicate at the same time in one batch to minimize interassay variability. The assay (26) has a sensitivity of 1 ng/mL. The interassay and the intra-assay coefficients are (1.78-6.21%) and 3.9%, respectively. To ensure reproducibility, samples were analyzed at two different occasions using the same technique. The correlation between results was 0.72, p < 0.01.

Data analysis.

Categorical variables are presented as relative frequencies, and their associations with obese subgroups were tested for significance using Fisher's exact tests. Continuous variables, not normally distributed, are presented as medians and interquartile ranges, and associations with subgroups were tested for significance using the Mann-Whitney tests. Normally distributed continuous variables are presented as means ± standard deviations, with differences between subgroups were assessed by two-tailed t tests. Bivariate correlations between various measures of BP indexes and adiponectin levels were examined using Spearman correlation.

Variables with significant correlations with the outcome variable (BP parameter) of magnitude 0.2 or greater were selected for the initial multivariate linear regression model, along with those variables considered clinically important or potential effect modifiers of adiponectin. Two multivariate regression models were derived to examine the relationships of adiponectin and other variables with outcomes: 1) 24-h mean SBP and 2) 24-h mean DBP. The confounding effects of gender and race (27–29) were examined, and were found not to be statistically significant. Female gender had a significantly negative correlation with SBP and remained in the model as an independent predictor.

Statistical and database software used included STATA 9.2 (Stata Corporation, College Station, TX) and Microsoft Office Excel 2003 (Microsoft Corporation, Redmond, WA), respectively. Statistical significance for all analyses was set at two-sided p < 0.05.

RESULTS

Study population.

Forty-one obese adolescents were studied, 15 of whom had T2DM (Table 1). Mean age was 14.2 ± 2.3 y (range, 10-20 y). Fifty-one person were male. Thirty-nine percent were African American and 61% were Hispanic. Mean BMI z score was 2.34 ± 0.32. Fifty-four percent, 19%, and 27% had casual BP in the hypertensive, prehypertensive, and normotensive range, respectively. Gender distribution between the two groups differed, with a significantly greater percentage of females in the T2DM group (p = 0.02). There were no significant differences between the groups in regard to age, eGFR, ethnicity, or prevalence of casual hypertension. Although BMI z score was not statistically different between the two groups, WHR was significantly greater in those with T2DM.

Table 1 Characteristics of adolescents in study by diagnosis
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Biochemical parameters.

Compared with published normative data (29), adiponectin levels were reduced in our obese cohort (6.6 μg/mL ± 2.6); levels were lower but not significantly different in black adolescents compared with Hispanics, and in males compared with females. Adiponectin levels were significantly lower in obese adolescents with blunted SBP dipping compared with those with normal SBP dipping (5.96 ± 1.7 and 7.71 ± 3.5), respectively, p = 0.059. Obese diabetics tended to have lower adiponectin levels and statistically different HOMA, HbA1c, urinary microalbumin compared with obese nondiabetics (mean adiponectin levels 5.6 ± 2.0, 7.2 ± 2.8, respectively, p = 0.07). hsCRP levels were elevated in this cohort.

BP measurements and parameters.

Although 22 subjects (53.7%) were diagnosed to be hypertensive using casual BP readings, only 8 (19.5%) had ABP measurements above 95% for height and gender. Most subjects (82%) classified to have casual hypertension had systolic hypertension, 9% had diastolic hypertension, and 9% had both systolic and diastolic hypertension.

Twenty-one study participants (51%) and 11 (27%) had impaired SBP and DBP dipping, respectively. The mean SBP dipping was impaired in this obese cohort with a mean dip of just 8.7 ± 6.9%. Comparing 24-h ABP variables of obese diabetic to obese nondiabetic subgroup, only 24-h heart rate was statistically different (p = 0.04). A trend toward higher DBP parameters in the obese group with T2DM was observed.

Table 2 presents bivariate correlations between adiponectin and various ABP parameters for this obese cohort. ABP parameters and indexes significantly correlated with adiponectin levels are presented. Similar statistically significant correlations were observed when these relationships were examined in each group separately (Figs. 1 and 2).

Table 2 Bivariate correlations between ambulatory BP indexes and parameters and adiponectin levels
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Figure 1
figure 1

Inverse correlation between SBP index and adiponectin levels. (A) All study participants, Rho = −0.4, p < 0.05. Obese (B) and Obese + T2DM (C) study participants.

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Figure 2
figure 2

Inverse correlation between DBP index and adiponectin levels. (A) All study participants, Rho = −0.42, p < 0.05. Obese (B) and Obese + T2DM (C) study participants.

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Determinants of 24-h SBP and DBP.

In a multivariable linear regression model, lower adiponectin and albumin levels; female gender; and higher hs-CRP and cholesterol levels were associated with higher 24-h SBP measurements. On the other hand, lower HOMA and adiponectin levels; and higher hs-CRP and HbA1c levels were associated with higher 24-h DBP, Table 3.

Table 3 Multivariable linear regression for determinants of 24-hr SBP and 24-hr DBP
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The two obese subgroups of this cohort were comparable in many demographic, biochemical, and BP parameters, and were treated as one group when building the regression models. Besides the known CV risk factors and the rapid phase reactants such as hs-CRP, albumin, cholesterol, and male gender shown in Table 3; 24-hr SBP, a reduction of 1.3 μg/mL in adiponectin level was associated with a 1 mm Hg increase in SBP.

Determinants of DBP included higher hs-CRP and AST, and lower adiponectin levels; a reduction of 0.7 μg/mL in adiponectin level was associated with a 1 mm Hg increase in DBP (Table 3; 24-hr DBP).

DISCUSSION

We demonstrate in this young cohort of obese adolescents with and without T2DM that adiponectin levels were independently associated with 24-h SBP and DBP. We also show that several adverse markers of CV risk, including impaired BP dipping, elevated hs-CRP levels, and reduced adiponectin levels affected both diabetic and nondiabetic obese adolescents almost to the same extent. Adiponectin levels were reduced, with no statistically significant difference between obese adolescents with or without T2DM. This might be due to the comparable demographics of the two subgroups, especially age, ethnicity, and BMI z scores, which are all known to affect adiponectin levels (25–27). Adiponectin levels also did not differ by gender in our study; this may be attributed to the morbid obesity and its independent effect on reduced adiponectin levels. In addition, gender was neither a confounding factor nor had an interaction with other variables and remained statistically significant in the 24-h SBP model.

The important role played by adiponectin in the pathogenesis of CV disease is highlighted by the relationships we found between ABP parameters and adiponectin. Adiponectin levels were decreased in adolescents with blunted SBP dipping compared with those without blunted dipping. This is consistent with previous data from Della et al. (30) showing lower adiponectin levels in nondippers. Other investigators (31) have examined the relationship between adiponectin and left ventricular mass index (LVMI) in an adult hypertensive population and showed that plasma adiponectin concentration in the hypertensive group was significantly lower than that in the nonhypertensive group, and that LVMI was significantly higher in the hypertensive group, with a significant inverse correlation between plasma adiponectin and LVMI. The significant potential CV risk of decreased adiponectin in obesity is further underscored by our finding that adiponectin was an independent determinant of 24-h SBP in this relatively young population.

Although BMI z score and WHR were significantly correlated in this study, WHR and not BMI z score was significantly different between the two obese groups, as the second reflects the relationship between height and weight whereas the first measures the proportion by which fat is distributed around the torso. In our study, BMI and WHR were inversely correlated to adiponectin levels and positively to BP levels, but did not reach sufficient significance to remain in the model.

Our study was limited by its small sample size; this may have affected which variables could be included in the final model based on their significance level. The cross-sectional study design, while demonstrating an independent association between adiponectin and systolic and diastolic ABP, does not allow a cause-and-effect relationship to be established. Additionally, the racial composition of the study population was limited to minorities, which may limit the generalizability of our findings. However, the study also has unique strengths, especially the use of ambulatory BP monitoring, which provides superior assessment of BP compared with office measurements (19). Additionally, using ABPM allowed us to show blunted SBP dipping, which may represent one of the earliest abnormalities in BP patterns in this obese cohort.

Adiponectin levels inversely correlate with ABP parameters in obese adolescents. Larger studies are needed to examine the relationship between adiponectin and mechanisms of BP regulation. In addition, further studies to examine interventions with potential BP lowering effect via modulating adiponectin levels are warranted.