Endothelial nitric oxide synthase (eNOS) catalyzes NO production in vascular endothelial cells, and NO regulates local blood flow by inducing vasodilation (1). Enhancement of skeletal muscle glucose uptake occurs during elevation of muscle blood flow, which is induced by increased eNOS expression and activated by insulin stimulation (24). It was previously reported that low production of NO in eNOS knockout mice causes reduction of insulin-induced blood flow and glucose uptake in whole body (5). These findings suggest that eNOS plays an important role in the regulation of insulin-induced glucose uptake in whole body.

Polymorphism in eNOS exon 7 with G→T conversion at nucleotide position 894 results in amino acid substitution of glutamic acid for aspartic acid in amino acid residue 298 (Glu298Asp). Structural alteration in this variant affects the susceptibility to cleavage and reduces activity of this enzyme (6,7). Dysfunction of eNOS by this polymorphism may cause reduction of insulin-induced blood flow and glucose uptake. It was recently demonstrated that insulin secretion and sensitivity could be assessed by 75-g oral glucose tolerance test (8,9). Therefore, we examined the association of this polymorphism with fasting and postchallenge glucose and insulin levels in nondiabetic Japanese subjects by the 75-g oral glucose tolerance test.

This study comprised 247 Japanese nondiabetic volunteers (69 men and 178 women). Written informed consent was obtained from all subjects enrolled in this study. A 75-g glucose tolerance test was performed early in the morning after fasting overnight. Venous sampling was obtained before loading (0 min), at 30 min, and 120 min after glucose loading, and blood glucose and insulin levels were measured. The serum insulin levels were measured with an EIA kit (Eiken insulin kit; EIA, Tokyo, Japan). All subjects were nondiabetic according to American Diabetes Association criteria (10). For assessing the substitution of G→T at position 894 (Glu298Asp), genomic DNA isolated from peripheral blood leukocytes was amplified by PCR and digested with BanII restriction enzyme as previously described (11,12). Data are expressed as means ± SEM. Differences between each group were tested by two-tailed unpaired Student’s t test. A P < 0.05 was considered as statistically significant.

The allele frequency was 0.927 for G and 0.073 for T in all subjects. Genotype distribution was 86.6% (214 of 247) for Glu/Glu, 12.2% (30 of 247) for Glu/Asp, and 1.2% (3 of 247) for Asp/Asp. The frequency and distribution are compatible with previous data (11,12). Because the number of homozygous mutants was too small, the combined data from homozygous and heterozygous individuals were used in the following analysis. There was no significant difference in age (53.8 ± 0.5 vs. 56.0 ± 1.6 years), BMI (23.4 ± 0.2 vs. 24.4 ± 0.7 kg/m2), waist-to-hip ratio (1.01 ± 0.01 vs. 0.98 ± 0.04), systolic blood pressure (130.0 ± 1.3 vs. 130.1 ± 3.4 mmHg), diastolic blood pressure (77.6 ± 0.8 vs. 77.2 ± 2.0 mmHg), total cholesterol (5.46 ± 0.10 vs. 5.58 ± 0.21 mmol/l), triglyceride (1.28 ± 0.05 vs. 1.16 ± 0.10 mmol/l), HDL cholesterol (1.62 ± 0.03 vs. 1.63 ± 0.09 mmol/l), or HbA1c (5.0 ± 0.1 vs. 5.0 ± 0.2%) between Glu/Glu and Glu/Asp + Asp/Asp. The results of the glucose tolerance test were as follows: plasma glucose in Glu/Glu was not significantly different from Glu/Asp + Asp/Asp at 0 min (5.07 ± 0.05 vs. 5.12 ± 0.12 mmol/l), 30 min (8.36 ± 0.16 vs. 8.94 ± 0.30 mmol/l), and 120 min (6.36 ± 0.18 vs. 6.93 ± 0.35 mmol/l). However, serum insulin levels were significantly increased in Glu/Asp + Asp/Asp compared with Glu/Glu at 30 min (309.6 ± 40.8 vs. 236.4 ± 9.6 pmol/l, P < 0.02) and 120 min (342.0 ± 36.0 vs. 220.2 ± 10.8 pmol/l, P < 0.0005). There was no significant difference in serum insulin levels at 0 min (35.2 ± 1.2 vs. 41.8 ± 3.0 pmol/l), homeostasis model assessment for insulin resistance (HOMA-IR) (1.36 ± 0.06 vs. 1.59 ± 0.12), and insulinogenic index (ΔI30/ΔG30, 1.09 ± 0.22 vs. 0.74 ± 0.12) between Glu/Glu and Glu/Asp + Asp/Asp.

In the present study, elevation of insulin levels at 30 and 120 min after glucose loading test was observed in subjects with Glu/Asp + Asp/Asp polymorphism compared with wild-type. However, the blood levels of glucose were not significantly different between these two groups. These data showed that there is a remarkable difference in postchallenge insulin levels between Glu/Glu and Glu/Asp + Asp/Asp groups. Subjects with Glu/Asp + Asp/Asp require more insulin to maintain the same glucose levels than subjects with Glu/Glu during glucose loading test. It was reported (8,9) that insulin level during postchallenge (120 min) is correlated with insulin sensitivity as measured by the glucose clamp method in nondiabetic subjects. Thus, one explanation for the elevated postchallenge (120 min) insulin levels may be reduced insulin sensitivity due to impaired insulin-mediated local blood flow in subjects with Glu/Asp + Asp/Asp polymorphism.

However, no significant difference was observed in HOMA-IR, a marker of insulin sensitivity, between subjects with Glu/Asp + Asp/Asp and those with Glu/Glu, suggesting that another mechanism in addition to insulin sensitivity may affect postchallenge insulin levels. It was previously observed (13) that decreased insulin-mediated blood flow in muscles is associated with reduction of insulin clearance in obese subjects with insulin resistance. Also, remarkable difference in blood flow and insulin clearance has been observed between lean and obese subjects in hyperinsulinemic conditions (13). These observations suggest that decreased insulin-mediated blood flow reduces insulin clearance, which leads to increased circulating insulin levels. Therefore, impairment of insulin-mediated vasodilation with subsequent reduction of insulin clearance may be another explanation for the changes of postchallenge insulin levels in subjects with the Glu/Asp + Asp/Asp polymorphism.

In conclusion, the present study showed for the first time that eNOS Glu289Asp polymorphism affects postchallenge insulin levels in nondiabetic Japanese subjects.

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