Vascular Endothelial and Smooth Muscle Function Relates to Body ...

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The Journal of Clinical Endocrinology & Metabolism 91(11):4467– 4471 Copyright © 2006 by The Endocrine Society doi: 10.1210/jc.2006-0863

Vascular Endothelial and Smooth Muscle Function Relates to Body Mass Index and Glucose in Obese and Nonobese Children Alexia Sophie Penã, Esko Wiltshire, Karen MacKenzie, Roger Gent, Lino Piotto, Craig Hirte, and Jennifer Couper Departments of Endocrinology and Diabetes (A.S.P., K.M., J.C.) and Pediatrics (J.C.), Medical Imaging (R.G., L.P.), and Public Health Research Unit (C.H.), University of Adelaide, Women’s and Children’s Hospital, North Adelaide, South Australia 5006, Australia; and Department of Pediatrics and Child Health (E.W.), Wellington School of Medicine and Health Sciences, Wellington South, Wellington, New Zealand Context: Endothelial and smooth muscle dysfunction are critical precursors of atherosclerosis. These can be detected in children at risk of cardiovascular disease. Objective: The objective of this study is to evaluate endothelial and smooth muscle function and their determinants using flow-mediated dilatation (FMD) and glyceryl trinitrate-mediated dilatation (GTN) in obese, nonobese, and type 1 diabetes mellitus (T1DM) children. Design: This is a cross-sectional study. Subjects: The study subjects were 270 children [140 males, mean age 13.7 (2.8) yr] including 58 obese, 53 nonobese, and 159 T1DM children. Measurements: Vascular function (FMD and GTN), body mass index (BMI) z-score, blood pressure, glucose, glycosylated hemoglobin, lipids, folate, homocysteine, and high sensitive C-reactive protein were measured.

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HE ENDOTHELIUM IS a key regulator of vascular function (1). Endothelial dysfunction is an early and fundamental event in the development of atherosclerosis (2, 3). Abnormal endothelial function, measured by brachial artery flow-mediated dilatation (FMD), correlates with abnormal coronary angiography in adults (4, 5). Children with cardiovascular risk factors such as diabetes, (6 – 8), hypercholesterolemia (9, 10), and obesity (11–13) have abnormal endothelial function measured by FMD. Vascular smooth muscle dysfunction is an independent risk factor for atherosclerosis in adults (14, 15). Smooth muscle function can be measured by glyceryl trinitrate-mediated dilatation (GTN). The majority of vascular function studies in childhood do not include smooth muscle function (GTN), but the limited data regarding vascular smooth muscle function in children with cardiovascular risk factors such as diFirst Published Online August 8, 2006 Abbreviations: BMI, Body mass index; FMD, flow-mediated dilatation; GTN, glyceryl trinitrate-mediated dilatation; HbA1c, glycosylated hemoglobin; HDL, high-density lipoprotein; hsCRP, high sensitive Creactive protein; LDL, low-density lipoprotein; T1DM, type 1 diabetes mellitus. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

Results: FMD and GTN were significantly lower in obese and T1DM compared with nonobese subjects (P ⬍ 0.001, P ⬍ 0.001). FMD and GTN were similarly reduced in obese and T1DM subjects (P ⫽ 0.22, P ⫽ 0.28). In all nondiabetic subjects (n ⫽ 111), both FMD and GTN were significantly and independently related to BMI z-score (r ⫽ ⫺0.28, P ⫽ 0.003, ␤ ⫽ ⫺0.36, P ⬍ 0.001) and weight z-score (␤ ⫽ ⫺0.31, P ⫽ 0.002; r ⫽ ⫺0.52, P ⬍ 0.001). FMD related independently to total cholesterol (␤ ⫽ ⫺0.22, P ⫽ 0.02). GTN related independently to vessel diameter (␤ ⫽ ⫺0.49, P ⬍ 0.001). GTN related to glucose within the normal range (r ⫽ ⫺0.34, P ⫽ 0.001). Conclusions: Children with obesity and T1DM have a similar degree of vascular dysfunction. BMI and weight adjusted for age and sex relate to endothelial and smooth muscle function in nonobese and obese children. Glucose relates to smooth muscle function in nonobese nondiabetic children. This suggests a continuum effect of BMI and glucose within the normal range on vascular function in childhood. (J Clin Endocrinol Metab 91: 4467– 4471, 2006)

abetes (8, 9), hypercholesterolemia (9, 10), and obesity (11, 16) is inconsistent. Both FMD and GTN allow the first changes in vascular function, critical to the development of atherosclerosis, to be detected early in life. Vascular function can be measured by ultrasound that assesses brachial artery responses to an increase in flow (FMD) and to GTN. FMD induces an increase in vessel diameter dependent on nitric oxide release by endothelium (endothelium-dependent response). Glyceryl trinitrate is a nitric oxide donor that induces an increase in vessel diameter independent of the endothelium and, therefore, assesses vascular smooth muscle response (17). Body mass index (BMI) above the 95th percentile for age and sex defines obesity in children and adolescents (18) and relates to abnormal vascular function (11). BMI z-scores relate to FMD and GTN in severely obese children and to FMD but not GTN in less obese children (11, 12, 16, 19). BMI relates to other determinants of vascular function such as high sensitive C-reactive protein (hsCRP) (20) and insulin resistance (11). In this study, we aimed to measure vascular endothelial and smooth muscle function (FMD and GTN) and their determinants in nonobese healthy children, children with mild to moderate obesity, and children with type 1 diabetes.

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J Clin Endocrinol Metab, November 2006, 91(11):4467– 4471

Subjects and Methods Subjects A total of 270 children [140 males, mean age 13.7 (2.8) yr, 266 Caucasians] were studied. Fifty-eight subjects were mildly to moderately obese (BMI z-score 1.7–3.0) nondiabetic subjects and were recruited consecutively from pediatric outpatient clinics at the Women’s and Children’s Hospital (Adelaide, Australia). Subjects with syndromal obesity and/or endocrinological causes of obesity were excluded. Fiftythree age- and sex-matched subjects were nonobese (BMI ⬍ 95th percentile, BMI z-score ⬍ 1.7) and recruited from two sources: siblings or friends of the participating subjects and relatives of staff members. A total of 159 subjects with type 1 diabetes mellitus (T1DM) [86 males, mean age 13.8 (2.8) yr], 18 of whom were also obese, were recruited consecutively from the diabetes outpatient clinic at the Women’s and Children’s Hospital. No subjects with diabetes had background retinopathy on direct fundoscopy or microalbuminuria measured by overnight albumin excretion rate. Exclusion criteria were smoking, hypertension (defined as blood pressure at rest above 95th percentile for age, sex, and height), and lipid-lowering treatment. The Human Research Ethics Committee approved the study. Written informed consent was obtained from the parents/guardians of the subject and the subject if he/she was more than 16 yr old. Height was measured with a wall-mounted stadiometer to the nearest 0.1 cm. Weight with minimal clothing was taken on an electronic digital scale to the nearest 0.1 kg. BMI [weight (kg)/height (m)2], BMI z-score, weight z-score, and height z-score were calculated using EpiInfo database version 3.2.2 and Centers for Disease Control 2000 standardized reference charts (21). Waist circumference was measured at the midpoint between the lower edge of the ribs in the midaxillary line and the top of the iliac crest, at minimal respiration. Blood pressure was taken with appropriate size cuff on the left arm after 10 min of rest in the supine position.

Ultrasound assessment of vascular function Endothelial and smooth muscle function (FMD and GTN) were assessed after overnight fasting in a quiet and stable temperature environment, as previously reported (6, 22). The diameter of the brachial artery (2–15 cm above the elbow) was measured in longitudinal section from two-dimensional ultrasound images, with a 10.0-MHz linear array transducer (Advanced Technology Laboratories, Bothel, WA), using an Advanced Technology Laboratories HDI 3000 ultrasound system. Experienced pediatric vascular ultrasonographers performed all studies. A suitable site for imaging the vessel was first selected, with reproducible ultrasonic markers such as venous valves or vessel bifurcations, to ensure that the measurement occurred at the same place for each scan. An electrocardiogram was recorded with the ultrasound images. Each study included four scans. The first one was taken at rest. Reactive hyperemia was then induced by occluding arterial blood flow for 4 min using a sphygmomanometer inflated to 250 mm Hg. Arterial flow velocity was measured by a pulsed Doppler signal at 60 degrees to the vessel, during the resting scan and for the first 15 sec after deflation of the cuff. The second scan (reactive hyperemia or FMD) was recorded 30 –90 sec after cuff deflation, with measurements between 45 and 75 sec after deflation. Ten to 15 min were allowed for vessel recovery, and then the third (recontrol) scan was taken. The last scan was taken 4 min after the sublingual administration of the glyceryl trinitrate spray (400 ␮g, Nitro lingual Spray; G. Pohl-Boskamp, Hohenlockstedt, Germany). All images were recorded onto high-quality super VHS videotape and analyzed subsequently by an observer blinded to the stage of experiment. For each scan, measurements were made with ultrasonic calipers over four cardiac cycles, and the measurements were averaged. All measurements were made incident with the electrocardiogram R wave (i.e. at end diastole). The measurements were averaged and expressed as percentages of the first control (resting) scan. There were four final average measurements in total: resting, FMD, recontrol, and GTN. Reactive hyperemia was calculated as flow in the first 15 sec after cuff deflation divided by the flow during the resting scan. Our coefficient of variation between 20 subjects is 3.9% for FMD and 4.0% for GTN (6).

Penã et al. • Vascular Function and BMI and Glucose

Laboratory tests Overnight fasting venous blood samples were collected. hsCRP was measured using a near infrared particle immunoassay method using IMMAGE Immunochemistry Systems Reagent (Beckman Coulter Inc., Fullerton, CA). Triglycerides, total cholesterol, and high-density lipoprotein (HDL) were measured using enzyme-based assays on the Beckman Coulter Synchron CX5 analyzer. Low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald equation. Glycosylated hemoglobin (HbA1c) was measured using a latex immunoagglutination inhibition methodology (DCA 2000 Hemoglobin A1c Reagent Kit; Bayer, Toronto, Ontario). Glucose was measured by hexokinase spectrophotometry method (Synchron cx5ce system; Beckman Coulter). Serum folate and red cell folate were measured using Ion Capture technology (Abbott IMx analyzer; Abbott Laboratories, Oslo, Norway). Total homocysteine was measured by a fluorescence polarization immunoassay using the commercial IMx Homocysteine assay (Abbott Diagnostic Division). Obese subjects had an oral glucose tolerance test after an overnight fast using 1.75 g/kg of anhydrous dextrose to a maximum of 75 g in a volume of 300 ml.

Statistical analysis The data were analyzed using SPSS software version 13.0. Differences in measurements between groups were assessed with one-way ANOVA and least significant difference post hoc tests for normally distributed data and a Kruskal-Wallis test for data that were not normally distributed. Associations between FMD or GTN and their determinants were evaluated with Spearman’s correlations and Mann-Whitney U tests. Multiple linear regression analysis was undertaken to determine independent predictors of the vascular function variables, which were FMD and GTN. Predictor variables with a P value of less than 0.05 in their univariable test on FMD or GTN were included in the multiple linear regression, and vessel diameter was included to control for any effect it may have. A forward selection method was used to estimate the final linear regression. Statistical significance was inferred with a P value less than 0.05.

Results Vascular function in obese, nonobese, and T1DM children

Vascular endothelial (FMD) and smooth muscle function (GTN) were significantly lower in mildly to moderately obese subjects and those with T1DM compared with nonobese subjects adjusting for vessel diameter (P ⬍ 0.001, P ⬍ 0.001) (Table 1 and Fig. 1). These results were also confirmed by Kruskal-Wallis (FMD P ⬍ 0.001 and GTN P ⬍ 0.001). FMD (P ⫽ 0.25) and GTN (P ⫽ 0.28) were similarly reduced in obese and T1DM subjects. Characteristics of the study children by group are shown in Table 1. Vascular endothelial and smooth muscle function and BMI

Significant univariate correlations for FMD and GTN are shown in Tables 2 and 3, respectively. In all nondiabetic subjects (obese and nonobese, n ⫽ 111), both FMD and GTN were significantly related to BMI z-score and weight z-score (Fig. 2). GTN was related to waist and hip circumference (Table 3). In a multiple regression analysis, FMD was independently and significantly related to weight z-score (␤ ⫽ ⫺0.31, P ⫽ 0.002) and total cholesterol (␤ ⫽ ⫺0.22, P ⫽ 0.02). When weight z-score was replaced by BMI z-score in the multiregression model, there was also an independent correlation with FMD (␤ ⫽ ⫺0.11, P ⫽ 0.007). In multivariate analysis, GTN was independently and significantly related to BMI z-score (␤ ⫽ ⫺0.36, r2 ⫽ 0.48, P ⬍ 0.001) and vessel diameter (␤ ⫽ ⫺0.5, P ⬍ 0.001). BMI



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TABLE 1. Results of vascular function studies and other variables by group

FMD (%) GTN (%) Resting vessel diameter (cm) Age (yr) Sex (male/female) BMI z-score Height z-score Weight z-score Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Serum folate (ng/ml) Red cell folate (ng/ml) tHcy (␮mol/liter) Triglycerides (mg/dl) Total cholesterol (mg/dl) HDL cholesterol (mg/dl) LDL cholesterol (mg/dl) Glucose (mg/dl) HbA1c (%) hsCRP (mg/liter)

Nonobese (n ⫽ 53)

Obese (n ⫽ 58)

T1DM (n ⫽ 159)

P ANOVA

7.8 (4.6) 27.7 (7.5) 0.27 (0.04) 14.1 (3.2) 24/29 0.31 (0.87) 0.66 (1.03) 0.59 (0.79) 111.0 (11.4) 60.4 (7.0) 9.4 (3.8) 282 (134) 6.8 (2.6) 49 (30) 166 (39) 50 (12) 104 (35) 88 (7) 4.8 (0.4) 0.5 (0.2–5.0)a

4.9 (4.0) 21.7 (6.1) 0.29 (0.04) 13.3 (2.3) 30/28 2.28 (0.27) 0.74 (0.93) 2.50 (0.51) 116.1 (8.8) 60.4 (6.1) 11.1 (4.8) 307 (111) 6.9 (1.8) 83 (50) 170 (35) 42 (8) 112 (27) 88 (7) 5.0 (0.3) 2.3 (0.5–10)

3.8 (4.3) 20.5 (7.0) 0.29 (0.05) 13.8 (2.8) 86/73 0.64 (0.78) 0.34 (1.03) 0.68 (0.85) 117.7 (15.0) 63.1 (8.0) 12.7 (3.4) 457 (148) 6.0 (1.6) 45 (34) 170 (35) 62 (15) 97 (27) 245 (85) 8.7 (1.3) 0.6 (0.3– 0.7)

⬍0.001 ⬍0.001 0.01 0.24 0.54 ⬍0.001 0.02 ⬍0.001 0.01 0.01 ⬍0.001 ⬍0.001 0.001 ⬍0.001 0.80 ⬍0.001 0.01 ⬍0.001 ⬍0.001 ⬍0.001b

Conversion factor for SI units: folate and red cell folate, ⫻2.266 (nmol/liter); triglycerides, ⫻0.01129 (mmol/liter); total cholesterol, LDL, and HDL, ⫻0.02586 (mmol/liter); and glucose, ⫻0.05551 (mmol/liter). tHcy, Total homocysteine. Data are means (SD) unless otherwise stated. a Median (range). b Kruskal-Wallis test.

z-score related to vessel diameter (r ⫽ 0.31, P ⫽ 0.001). In the multivariate model there was no significant vessel diameter by BMI z-score interaction (P ⫽ 0.37, r2 ⫽ 0.48). In nonobese subjects (n ⫽ 53), GTN related to BMI z-score (r ⫽ ⫺0.30, P ⫽ 0.03), and GTN was independently and inversely related to vessel diameter (␤ ⫽ ⫺0.47, r2 ⫽ 0.42, P ⫽ 0.001). BMI z-score relationship was not significant after controlling for vessel diameter in the multiregression model (P ⫽ 0.89, r2 ⫽ 0.44). In obese subjects (n ⫽ 58), GTN related to weight z-score (r ⫽ ⫺0.33, P ⫽ 0.01) and inversely and independently related to vessel diameter (␤ ⫽ ⫺0.52, P ⬍ 0.001).

In the T1DM subjects (n ⫽ 159) FMD was not related to BMI z-score or weight, but GTN was related to BMI z-score (r ⫽ ⫺0.16, P ⫽ 0.03) and weight (r ⫽ ⫺0.29, P ⬍ 0.001). A comparison of FMD and GTN between obese and nonobese within the T1DM subjects was made; there was no significant difference between FMD in obese and nonobese T1DM subjects (P ⫽ 0.76). GTN was significantly lower in obese subjects compared with nonobese T1DM subjects (P ⫽ 0.03). Vascular endothelial and smooth muscle function and glucose homeostasis

In all nondiabetic subjects (obese and nonobese, n ⫽ 111) GTN related inversely to fasting glucose within the normal range, fasting insulin, homeostasis model assessment, and HbA1c (Table 3). FMD did not relate to fasting glucose, fasting insulin, or HbA1c. In nonobese subjects (n ⫽ 53), GTN related inversely to fasting glucose (r ⫽ ⫺0.44, P ⫽ 0.003). In obese subjects (n ⫽ 58), vascular function did not relate to fasting glucose, fasting insulin, HbA1c, or the presence of acanthosis nigricans (27 of 58). Five obese children had impaired glucose tolerance without impaired fasting glucose [mean fasting glucose 94 (4) mg/dl [5.2 (0.23) mmol/liter] and mean 2-h postglucose load 142 (23) mg/dl [7.9 (1.3) mmol/liter]] unrelated to vascular function. TABLE 2. Significant correlations between vascular function (FMD) and cardiovascular risk factors in a univariate analysis in all subjects without diabetes (n ⫽ 111) FMD

FIG. 1. FMD (%) and GTN (%) in nonobese and obese children and children with type 1 diabetes. The horizontal solid line is the median, the edges of the box plots represent the 25th and 75th percentiles, the bars represent values within 1.5 times the interquantile range, and open circles are the outliers.

BMI z-score Weight z-score Total cholesterol LDL cholesterol

r

P value

⫺0.28 ⫺0.27 ⫺0.21 ⫺0.27

0.003 0.005 0.03 0.022

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TABLE 3. Significant correlations between vascular function (GTN) and cardiovascular risk factors in a univariate analysis in all subjects without diabetes (n ⫽ 111) GTN

Vessel diameter BMI z-score Weight z-score Waist circumference Hip circumference Systolic blood pressure hsCRP HDL cholesterol Triglycerides Total homocysteine Fasting glucose Fasting insulin HOMA HbA1c

r

P value

⫺0.61 ⫺0.50 ⫺0.52 ⫺0.57 ⫺0.50 ⫺0.37 ⫺0.23 0.29 ⫺0.35 ⫺0.21 ⫺0.31 ⫺0.36 ⫺0.39 ⫺0.20

⬍0.001 0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 0.029 0.003 ⬍0.001 0.036 0.001 ⬍0.001 ⬍0.001 0.050

HOMA, Homeostasis model assessment.

In the T1DM subjects (age range 7.9 –19.3 yr), the median age of diabetes onset was 9.2 (0.6 –16.9) yr, and the median diabetes duration was 5 (0.3–14.4) yr. Their mean insulin dose was 1.16 (0.35) U/kg䡠d. Their vascular function did not relate to fasting glucose, HbA1c, diabetes duration, or insulin dose. Discussion

We have shown that children with mild to moderate obesity had a similar degree of endothelial and vascular smooth muscle dysfunction to children with type 1 diabetes, in whom metabolic control was relatively poor, although comparable to adolescent populations (23). Vascular endothelial and smooth muscle function was primarily determined by BMI and weight adjusted for age and sex in obese and nonobese children. There was no threshold BMI for this effect. Leanness in children appears to confer an advantage in vascular health over heavier but normal-weight children. Interestingly, children with mild to moderate obesity had a similar degree of endothelial and vascular smooth muscle

FIG. 2. The relationship between GNT (%) and BMI z-score in nonobese and obese children (n ⫽ 111) (r ⫽ ⫺0.5, P ⫽ 0.001).


dysfunction to those with type 1 diabetes. In our experience, families of children with mild to moderate obesity often have difficulty appreciating that their children are at high risk of vascular disease, unlike families of children with diabetes. Whereas children with obesity and type 1 diabetes had a similar degree of vascular dysfunction, our results suggest the mechanisms underlying this may be different in these two groups. For example, obese children had significantly lower HDL cholesterol, higher triglycerides, and higher hsCRP than both children with diabetes and controls, and FMD was independently associated with total cholesterol in the nondiabetic group, suggesting that these lipid abnormalities and inflammation may have more of a role in vascular dysfunction in obesity. Obesity in children with T1DM confers an additional negative effect on vascular function; therefore, weight management needs to be reinforced in the care of children with T1DM. Endothelial dysfunction has been related to obesity in children (11–13), but not previously to BMI less than 95th percentile. A previous study does not suggest an association between FMD and BMI (without correction for age) in healthy children ages 8 –17 or those with type 1 diabetes or hypercholesterolemia, but does show that smooth muscle function relates to BMI (9). In 13- to 15-yr-old children, adiposity relates more to decreased arterial distensibility than to serum cholesterol levels (24). In adults from the community, including smokers and hypertensive subjects, BMI and smoking relates to endothelial and smooth muscle function (25, 26). Visceral obesity relates to vascular dysfunction in obese adults, (27, 28) but this has not been studied in normal-weight children. In this study, BMI z-score in obese and nonobese children related directly to blood vessel size, which in turn, related inversely to smooth muscle function. Juonala et al. (29) have shown a similar relationship between BMI and both endothelial function and blood vessel size in adults with a mean BMI of 25 kg/m2, but smooth muscle function was not measured in this study. Blood vessel size may be a further marker of vascular dysfunction. Higher levels of hsCRP that relate to obesity were related to vascular function as has been shown before (19, 29), but this association was not independent of BMI in our subjects. Our finding that smooth muscle function relates to plasma glucose within the normal range in nonobese, nondiabetic children is original to our knowledge but adds further to the known relationship between glucose and HbA1c and atherosclerosis detected postmortem in nondiabetic youth without cardiovascular risk factors (30, 31). A prospective study in adults has also identified an association between HbA1c within the normal range and cardiovascular disease and mortality (32). On the other hand, plasma glucose did not relate to FMD (endothelial function) in our nondiabetic subjects, possibly due to the number of subjects studied. These results, along with the BMI z-score correlations described above, do suggest that GTN is another sensitive marker of early vessel disease in children and may be more sensitive than FMD. In nondiabetic adults, glucose relates to endothelial function, including a multiethnic cohort of 579 nondiabetic adults in whom endothelial function related to glucose, including impaired fasting glucose, after adjustment for age, gender, BMI, and hypertensive status (33). In 228 Chi-


nese adults, including a majority with impaired fasting glucose, endothelial dysfunction also relates to glucose. Our obese subjects were only mildly insulin resistant, which may explain why we did not see a relationship between vascular function (endothelial and smooth muscle function) and markers of insulin resistance, in comparison with severely obese children and overweight children with metabolic syndrome (11, 24). In addition, the sample size of obese children may have been inadequate to detect a relationship. In summary, children with obesity and T1DM have a similar degree of vascular dysfunction. Vascular endothelial and smooth muscle function were primarily determined by BMI z-score and weight z-score in both obese and nonobese subjects. Smooth muscle function also related to glucose within the normal range; endothelial function related to glucose in the total (diabetic and nondiabetic) cohort. Lifestyle modifications early in childhood to lower BMI and glucose throughout the whole childhood population, including those without recognizable disease or risk factors, may impact on the development of atherosclerosis. Acknowledgments Received April 21, 2006. Accepted August 2, 2006. Address all correspondence and requests for reprints to: Alexia Sophie Penã, Department of Endocrinology, Women’s and Children’s Hospital, 72 King William Road, North Adelaide, SA 5006, Australia. E-mail: [email protected]. This work was supported by research grants from Channel 7 Research Foundation and Women’s and Children’s Research Foundation. Disclosure statement: The authors have nothing to declare.

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