The aim of the present study was to investigate the impact of acute hyperglycaemia on endothelial function in both normal-weight and overweight children. A total of 16 overweight [BMI (body mass index) ≥85th percentile] and 15 normal-weight (BMI <85th percentile) children were evaluated for FMD (flow-mediated dilation) at baseline and 30, 60 and 120 min after glucose ingestion. At 15 min following the measurement of the final FMD, 0.3 mg of sublingual nitroglycerine was administered and the brachial artery was imaged in order to assess endothelium-independent dilation. By design, the overweight children were significantly heavier (63.2±4.6 compared with 41.3±2.5 kg; P=0.0003) and had a greater percentage body fat (43.9±1.8 compared with 23.8±2.05%; P<0.0001) than the normal-weight children. The area under the curve in response to glucose administration was significantly (P<0.0001) greater in the overweight group for both glucose and insulin. The FMD area under the curve was not significantly different at baseline or between time points after glucose ingestion, nor was there a difference in response between the two groups. Endothelium-independent dilation in the normal-weight group was significantly greater compared with the overweight group (26.7±1.6 compared with 20.2±2.0% respectively; P=0.019). In conclusion, these results suggest that acute elevation of glucose and insulin in overweight and normal-weight children are not associated with impairment in endothelial function.
- endothelium-derived dilation
- glucose tolerance
- overweight children
A primary function of the vascular endothelium is the secretion of NO (nitric oxide), which is responsible for vasodilation [1,2] and inhibition of platelet and monocyte adhesions to the vessel wall . It has been hypothesized that dysfunction of the endothelium may be the initial step in the development of cardiovascular disease [4,5].
Previous investigations have assessed the role of acute hyperglycaemia in inducing transient endothelial dysfunction in adults [6–11]. The results of these studies have been mixed. Williams et al.  produced hyperglycaemia utilizing the hyperglycaemic clamp technique and reported a significant impairment in endothelium-dependent vasodilation in young non-diabetic adults. A decrease in endothelium-dependent vasodilation during acute oral ingestion of glucose has also been shown in middle- and older-aged non-diabetic adults [6,7]. In contrast, Siafarikas et al.  reported no adverse effect of acute hyperglycaemia following an OGTT (oral glucose tolerance test) on endothelial function in healthy nonobese young adults. Similarly, Reed et al.  reported no effect of hyperglycaemia, produced during a hyperglycaemic clamp, on endothelium-dependent vasodilation.
To date, studies examining the effect of hyperglycaemia on endothelial function have been done exclusively in adults. Whether the endothelium of children, who have not undergone puberty, has a similar response to glucose as adults has yet to be determined. It may well be that, prior to puberty, the endothelium of children is more resilient to the effects of a transient elevation in glucose. Therefore the aim of this study was to investigate the impact of acute hyperglycaemia on endothelial function in healthy normal-weight and overweight non-diabetic children. Because of the strong relationship of obesity to insulin resistance and development of the metabolic syndrome in children  and the fact that over 60% of overweight children have already developed at least one heart disease risk factor , we hypothesized that overweight children would have a significant impairment in endothelial function in response to acute hyperglycaemia when compared with normal-weight children.
MATERIALS AND METHODS
A total of 31 healthy children, 16 overweight and 15 normal-weight children, from the greater Minneapolis/St Paul, Minnesota metropolitan area agreed to participate in this study. Overweight was defined as a BMI (body mass index) ≥85th percentile for age and gender based on established criteria . The study protocol was reviewed and approved by the University of Minnesota Institutional Review Board, and all participants and parents/guardians gave written informed assent and consent. The procedures followed in the present study were in accordance with Institutional Review Board and HIPAA (Health Insurance Portability and Accountability Act) guidelines.
Glucose and vascular protocol
Participants fasted for 12 h and completed testing in the morning at the University of Minnesota General Clinical Research Center. Prior to testing, all subjects were questioned regarding any illness or injuries in the previous 2 weeks. Subjects were asked to lie in the supine position and, following a 15 min rest, BP (blood pressure) measurements were obtained at three separate times using a Vasotrac APM 205A automated wrist tonometer (Medwave). Mean BP was the average of the three consecutive measurements. Following BP measurement, a catheter was inserted into the antecubital fossa for future blood draws. At 15 min after the catheter placement, participants were assessed for FMD (flow-mediated dilation) of the brachial artery, as described previously in our laboratory and by others [15,16]. Briefly, a standard ultrasound instrument (Image Point Hx; Philips Medical) with a 7.5 MHz linear-array probe was used to obtain B-mode images of the left brachial artery (approx. 2–10 cm proximal to the elbow). After measuring resting artery diameter, a BP cuff was inflated below the elbow (distal to the imaged artery segment) to 200 mmHg for 5 min. Brachial artery diameter was measured for 3 min after cuff release to determine peak percentage dilation (FMD peak) and FMD AUC (area under the curve).
Following the baseline measurement of FMD, participants ingested a bolus of glucose consistent with a standard OGTT (1.75 g/kg of bodyweight; maximum of 75 g). Measurement of FMD was repeated at baseline and at 30, 60 and 120 min after glucose ingestion. Resting brachial artery diameters were measured immediately prior to each FMD to ensure that no changes occurred due to variations in glucose and insulin levels, as baseline diameter affects percentage dilation. At 15 min following the FMD measurement at 120 min, 0.3 mg of sublingual nitroglycerine was administered and the brachial artery was imaged 3 min later in order to assess endothelium-independent dilation.
Vascular image analyses
All brachial artery images were captured and triggered off of the R wave of the ECG (end-diastolic diameter), digitized and stored on a personal computer for later off-line analysis using electronic wall-tracking software (CVI; Information Integrity). The same trained reader analysed all of the digital files. Reproducibility of the FMD technique in our laboratory has been shown to have a mean difference of 0.53±0.28% for analyses separated by 1 week in ten young healthy individuals .
Glucose and insulin were collected and analysed at the following intervals: −10, −5, 0, 30, 60, 90 and 120 min during the OGTT. Baseline glucose and insulin levels were determined by averaging the samples obtained at the −10, −5 and 0 min time points. Glucose and insulin responses during the OGTT were determined by calculating the AUCs for glucose and insulin.
Fasting blood was collected for the measurement of total cholesterol, HDL (high-density lipoprotein)-cholesterol, LDL (low-density lipoprotein)-cholesterol, triacylglycerols (triglycerides), glucose, insulin, CRP (C-reactive protein), adiponectin, leptin, resistin, and 8-isoprostane. Assays for lipids, glucose, insulin and CRP were conducted at Fairview Diagnostic Laboratories, Fairview University Medical Center, Minneapolis, MN, U.S.A. Cholesterol, triacylglycerols and glucose were analysed by colorimetric reflectance spectrophotometry. Ultrasensitive CRP was analysed by rate nephelometry. Insulin was measured by chemiluminescent immunoassay. Adiponectin, leptin, resistin and 8-isoprostane were analysed in the University of Minnesota Cytokine Reference Laboratory by standard techniques using ELISA. HOMA (homoeostasis model assessment) for insulin resistance was calculated as (fasting glucose×fasting insulin)/22.5, as described by Matthews et al. . Body composition was measured by dual-energy X-ray absorptiometry system (Prodigy; GE). A trained pediatrician determined the Tanner stage for pubertal development.
On the basis of the reproducibility data from our laboratory , the sample size in this study provided 80% power to detect a difference in FMD of 1% between groups. Results are expressed as means±S.E.M. CRP and triacylglycerols were log-transformed for all analyses. FMD and FMD AUC were corrected for baseline diameter.
Unpaired Student's t tests were used to compare baseline variables between the overweight and normal-weight groups. Comparison of variables between groups following oral glucose ingestion was analysed by two-way repeated measures ANOVA with Bonferroni post-hoc tests, where appropriate. An α value of 0.05 was used to signify statistical significance. Statistical analyses were performed with GraphPad Prism version 4.0 (GraphPad Software) and Statview (Abacus Concepts).
Subject characteristics are shown in Table 1. A total of 15 normal-weight (seven males and eight females) and 16 overweight (seven males and nine females) (10.7±0.3 years) children were studied. There were no statistically significant differences in gender, Tanner stage or age. Although lean body mass was not significantly different between the two groups, the overweight children had a significantly greater BMI, fat body mass, waist circumference and percentage body fat. SBP (systolic BP), DBP (diastolic BP) and MABP (mean arterial BP) were significantly greater in the overweight children compared with the normal-weight children (Table 1). There were no significant differences between the two groups in fasting levels of total cholesterol, HDL-cholesterol, LDL-cholesterol, triacylglycerols, resistin or 8-isoprostane (Table 2). Adiponectin levels were significantly elevated in the normal-weight children, whereas both CRP and leptin levels were significantly lower, compared with the overweight group (Table 2).
Oral glucose tolerance
There was no significant difference in fasting plasma glucose between the normal-weight and overweight groups; however, plasma insulin levels were significantly lower (P=0.0388) in the normal-weight compared with the overweight group (Table 2). The AUC for both glucose and insulin in response to oral glucose administration was significantly (P<0.0001) greater in the overweight group (Figure 1). There were significant differences (P=0.0336) in insulin sensitivity as determined by HOMA between the normal-weight and overweight children (Table 2).
Brachial arterial endothelial function
Resting brachial artery diameters in overweight and normal-weight children at baseline and at 30, 60 and 120 min were not significantly (P=0.0861) different, nor was there any affect of time on these measurements (P=0.6166; Figure 2). In addition, there was no significant (P=0.270) interaction between the brachial diameter in the two groups and time. FMD AUC was not significantly (P=0.1029) different between the two groups after oral glucose ingestion, and there was no significant (P=0.1129) difference in response over time between the two groups (Figure 3). In addition, there was no significant (P=0.8784) interaction between the FMD AUC in the two groups and time. Although there was no significant (P=0.1625) difference in peak FMD between the two groups, there was a significant (P=0.0116) increase in peak FMD over time in both groups (Figure 4). However, there was no significant (P=0.5696) interaction between peak FMD in the two groups and time. The normal-weight group had a significantly greater arterial dilation in response to nitroglycerine when compared with the overweight group (26.7±1.6 compared with 20.2±2.0% respectively; P=0.019).
The present study demonstrates that acute hyperglycaemia in both normal-weight and overweight children does not adversely affect endothelium-dependent vasodilation, despite a significantly greater insulin and glucose response in overweight children. To the best of our knowledge, this is the first study to report the effects of acute hyperglycaemia on arterial endothelial function in normal-weight as well as overweight children. The results of the present study in normal-weight children agree with previous studies [8,9] reporting no adverse effects of hyperglycaemia on endothelial function in healthy non-obese young adults. Although other studies have reported a decline in endothelial function with acute hyperglycaemia, a number of these studies were done in middle-aged and older adults [6,7], or involved a very small number of subjects [10,11].
In the present study, normal-weight and overweight children had a similar endothelial function response to acute hyperglycaemia, despite a greater increase in both glucose and insulin concentrations in the overweight children. In addition, the overweight children had a higher degree of insulin resistance, as evidenced by the higher HOMA values. Although insulin resistance and diabetes [19,20] have been linked to endothelial dysfunction, it may be that the effects of insulin resistance need to take place over a period of time before the endothelium is affected.
In addition to differences in glucose and insulin response to oral glucose ingestion, the overweight children had significantly higher resting DBP, MABP, CRP, adiponectin and leptin levels than the normal-weight children. Previous studies in adults have demonstrated that both hypertension [21,22] and elevated CRP levels [23,24] are associated with impaired endothelial function. In addition, leptin has been shown to promote vascular injury and induce atherosclerosis and thrombosis in mice , and adiponectin  has been associated with coronary artery calcification in humans. It is possible, in children who are in the early development phases of glucose intolerance and other cardiovascular risk factors, that the endothelium is still able to maintain a high degree of normal function. As these children mature and are exposed to a number of metabolic and cardiovascular risk factors for longer periods of time, the endothelium may experience a decline in function .
It should be noted that in the present study we did not observe any significant differences in endothelium-dependent vasodilation between the normal-weight and overweight children. Previous studies in normal-weight and overweight children have reported impaired FMD in overweight children [28,29]. Differences between the present and these previous studies may be due to subject populations. The relative difference in body weight between our overweight and normal-weight control groups were much smaller compared with these previous studies.
Although there was no significant difference in endothelium-dependent vasodilation between the normal-weight and overweight children, there was a trend to-ward a greater endothelium-independent response to nitroglycerine in the normal-weight children compared with the overweight children. Similar findings have been reported in a comparison of normal-weight and severely obese children . In a large population study in adults , the smooth muscle response to nitroglycerine administration was impaired in individuals with risk factors for atherosclerosis and this was independent of endothelial function. It may be that changes in smooth muscle function precede functional changes in the endothelium in children. In the present study, we did not examine the effect of acute hyperglycaemia on endothelium-independent vasodilation. It is possible that acute hyperglycaemia in overweight children has an effect on the arterial smooth muscle, resulting in dysfunctional endothelium-independent vasodilation. Finally, the methods employed in the present study do not measure microcirculation, and it is possible that the microcirculation may be affected by acute hypoglycaemia.
In conclusion, despite increased glucose and insulin responses to acute hyperglycaemia in overweight compared with normal-weight children, we did not observe a significant change in arterial endothelial function. Children may have the capacity to maintain adequate endothelial function despite the presence of early cardiovascular disease risk factors (e.g. obesity, hyperinsulinaemia etc.) Endothelial dysfunction from acute glucose loading may not occur until later in life.
This work was supported in part by American Heart Association Pre-Doctoral Grant 0315213Z (A.S.K.), University of Minnesota Grant-in-Aid (D.R.D.), and M01-RR00400 from the General Clinical Research Center Program (GCRC), NCRR/NIH (National Center for Research Resources/National Institutes of Health).
Abbreviations: AUC, area under the curve; BMI, body mass index; BP, blood pressure; CRP, C-reactive protein; DBP, diastolic BP; FMD, flow-mediated dilation; HDL, high-density lipoprotein; HOMA, homoeostasis model assessment; LDL, low-density lipoprotein; MABP, mean arterial BP; OGTT, oral glucose tolerance test; SBP, systolic BP
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