The new findings of this study are: (1) Ach-induced and SNP-induced maximal FBFs were impaired in subjects with metabolic syndrome. In particular, we first found that the NOS-dependent component of Ach-induced maxFBF was selectively decreased, while the NOS-independent component remained unchanged. (2) Ach-induced maximal FBF and ∆Ach-induced maximal FBF by L-NMMA, and SNP-induced maximal FBF were correlated strongly with M value than with individual metabolic components. (3) Multivariate regression models clearly indicated that defining metabolic syndrome, as compared to individual metabolic components, predicts impairment of endothelial dysfunction.
Vascular reactivity
As observed in previous studies of obese subjects [
15,
16], Ach-induced maxFBF, a marker of endothelial function, was reduced in MS group. We further investigated vascular NO bioavailability by comparing the dose–response curves of Ach with and without pharmacological NOS inhibition. In the NO clamp technique, the NOS-dependent and NOS-independent components of FBF can be accurately determined [
17]. Co-infusion of L-NMMA and Ach caused a decrease in the level of inhibition of Ach-induced maxFBF in subjects with metabolic syndrome, and equalized the Ach-induced maximum FBF during NOS inhibition between the two groups. This implies that the NOS-dependent component of Ach-induced maxFBF was selectively decreased in subjects with metabolic syndrome, while the NOS-independent component remained relatively unchanged.
Classically, it has been believed that endothelial function is impaired, even in the first step of atherosclerosis, but smooth muscle cell function is preserved even in the advanced stages of atherosclerosis [
5]. Conversely, current study showed that SNP-induced maximal FBF, a marker of smooth muscle responsiveness, was reduced in the MS group. The data obtained during NO inhibition by L-NMMA, suggest that the whole difference in Ach reactivity between two groups can be mainly due to a defect in the NOS-dependent NO synthesis. However, the impaired smooth muscle vasorelaxation suggests an impaired bioavailability and/or responsiveness to endogenous NO [
18]. Given that the response to Ach was superimposable at the levels in NO synthesis and/or its biological activities, the contradictory findings may be partially explained by a converse compensation for the loss in NO bioavailability [
18]. Endothelium-derived hyperpolarizing factor (EDHF) pathway, in which alterations was reported under pathological conditions, might be one such candidate [
19]. Fernandes et al. reported that time-to-peak after hyperemia rather than flow mediated dilation (FMD) distinguished metabolic syndrome from healthy controls [
20]. In the time-course analysis of FMD [
21], time to peak after hyperemia is not influenced by L-NMMA inhibition, suggesting that other factors, such as differences in vascular compliance and transduction independently of the NOS pathway [
20,
22].
Vascular reactivity, abdominal fat distribution, adiponectin and insulin sensitivity
In the current study, Ach-induced maximal FBF, and ∆Ach-induced maximal FBF by L-NMMA were correlated with waist circumference, glucose and triglycerides, and more strongly with
M value. This result agrees with a previous study [
15] where Steinberg et al. report that obesity/insulin resistance is associated with both blunted endothelium-dependent vasodilation and failure of euglycemic hyperinsulinemia to augment endothelium-dependent vasodilation. This suggests that obese/insulin-resistant subjects are characterized by endothelial dysfunction and endothelial resistance to insulin’s effect on enhancement of endothelium-dependent vasodilation. Our results further indicated that each of abdominal fat distribution, adiponectin, and insulin sensitivity was correlated with Ach-induced maximal FBF and ∆Ach-induced maximal FBF by L-NMMA more greatly than each of the MS components. Therefore, endothelial function in metabolic syndrome can be explained mostly by mutually dependent insulin resistance, visceral obesity and hypoadiponectinemia through increased production of reactive oxidative species (ROS) and proinflammatory cytokines [
11,
23‐
29].
Impairment in the SNP-induced maximal FBF was also correlated with
M value as well as in Ach-induced maximal FBF. The observation may be supported by Schinzari et al. showing that the vascular responsiveness to both Ach and SNP was not enhanced during hyperinsulinemia in patients with metabolic syndrome [
30]. They suggests that insulin’s facilitator action on the vasodilator machinery was caused by endothelium unresponsiveness (a decrease in NO synthesis and/or a loss of its biological activities) and by defective sensitization of smooth muscle vasorelaxation (a loss in NO bioavailability) [
19]. Aoqui et al. found distinct patterns of microvascular dysfunction in metabolic syndrome, with augmented vasoconstriction present in the initial phase of metabolic syndrome independent of endothelial dysfunction [
31]. Reportedly, locally produced ROS [
25] and/or fat-derived ROS [
23] can react with NO, generate peroxynitrite, and impair cyclic GMP-dependent vasodilatation; this mechanism may partially explain these smooth muscle cell dysfunction [
18,
31].
Since metabolic syndrome is a cluster of relatively heterogeneous atherosclerotic risk factors, which may solely impair endothelial function, we evaluated the relationship between vascular reactivity and individual components or its clustering. In a simple regression model (Fig.
2), the Ach-induced maxFBF and ∆maxFBF by L-NMMA were lineally correlated with waist circumference, glucose and triglycerides. However, multivariate regression analysis indicated that individual components explained only partially Ach-induced maxFBF by 4–21 %, ∆maxFBF by L-NMMA by 6–31 %, and SNP-induced maxFBF by 8–17 % (data not shown). In model 3, including the 5 components of visceral obesity, high fasting glucose, elevated blood pressure, hypertriglyceridemia, and a low level of HDL cholesterol, increased the assumption to 35 % in Ach-induced maxFBF and 46 % in ∆maxFBF by L-NMMA, but only 16 % in SNP-induced maxFBF. These results support the notion that clustering of the components more greatly estimates endothelial dysfunction than individual components, but not smooth muscle cell function. Notably, the presence of metabolic syndrome, while still slightly less than the clustering of 5 components (model 2), explained 30 % of Ach-induced maxFBF and 41 % of ∆maxFBF by L-NMMA (model 4). These findings support the notion that defining metabolic syndrome is effective in predicting endothelial dysfunction, which can subsequently predict future cardiovascular events [
5,
7]. In the current study, type 2 diabetes mellitus was prevalent in the MS group (12/18, 67 %) than in the control group (4/19, 21 %) (p < 0.05). When all patients divided into diabetic and non-diabetic groups, Ach-induced maxFBF (21.5 ± 1.7 vs 16.0 ± 2.0 mL/min/100 mL, p = 0.040), ∆maxFBF by L-NMMA (8.3 ± 6.8 vs 2.8 ± 4.2 mL/min/100 mL, p = 0.008) and SNP-induced maxFBF (19.4 ± 1.4 vs 13.6 ± 1.7 mL/min/100 mL, p = 0.011) were all impaired in diabetic groups. Vascular function in type 2 diabetes mellitus is also confounded by cardiovascular risk factors, such as obesity, hypertension and dyslipidemia, suggesting significance of cardiovascular risk clustering [
32].
In our study, endothelial function is not strongly correlated with individual metabolic risk components, but strongly with insulin resistance. Thus, it is assumed that recovery of vascular function can be obtained less effectively by improvement of individual metabolic parameters [
33], but more effectively by improvement of metabolic syndrome or insulin resistance [
34]. This notion may be supported by the fact in patients with metabolically healthy obesity (MHO), a medical condition characterized by obesity which does not produce metabolic complications such as dyslipidemia, impaired glucose tolerance or hypertension [
35]. The MHO had abnormal vascular reactivity, although their endothelial dysfunction was less pronounced than in patients with metabolic syndrome, indicating that obesity is associated with vascular damage independent of those metabolic abnormalities underlying metabolic syndrome [
36].
We also evaluated the relationship between vascular reactivity and the number of metabolic syndrome components (Fig.
4). In subjects without visceral obesity, there were no differences in Ach-induced maxFBF and ∆maxFBF by L-NMMA among patients with 0, 1 and 2≥ of the 4 selected components. In contrast, in subjects with visceral obesity, Ach-induced maxFBF and ∆maxFBF by L-NMMA decreased in subjects with ≥2 components. As such, one or two components cannot be sufficient to cause impairment of endothelial function even in subjects with visceral obesity, but the clustering of ≥2 components can be sufficient. Interestingly, subjects without visceral obesity that have ≥2 components showed a subtler endothelial dysfunction than subjects with visceral obesity. This notion agrees with the previous study of Li et al., where they showed impaired endothelial function in subjects with metabolic syndrome as compared to individuals with a similar burden of traditional cardiovascular risk factors, but without metabolic syndrome [
37]. Their multivariate regression model found that after adjustment for covariates and 6 traditional cardiovascular risk factors, the presence of metabolic syndrome had a significant and independent influence on endothelial function (p < 0.01).
In our multivariate model, addition of M value increased the corrected R2 for Ach-induced maxFBF to 51 % in the model including all the components (model 3) and to 43 % in the model including the presence of metabolic syndrome (model 5). Insulin resistance should play a pivotal role in causing endothelial dysfunction by comorbidity of the metabolic syndrome components. We also found that endothelial function, indicated by Ach-induced maxFBF and ∆maxFBF by L-NMMA, was impaired in the group with ≥2 components, and M value decreased according to the number of components. Collectively, our data suggest that in subjects with visceral fat obesity, endothelial function is impaired by multiple cardiovascular risk factors exclusively when under the condition of insulin insensitivity.
Study limitations
First, we obtained data from a small number of subjects; therefore, there was the risk of type II errors. The strain gauge plethysmography and the euglycemic hyperinsulinemic clamp techniques, used in the study for measurement of vascular function and insulin sensitivity, are sensitive and solid, but the time-consuming and invasive characteristics limits number of participants. In contrast, alternative simplified methods such as flow-mediated dilation (FMD) [
38] and HOMA-IR [
39] are good for recruiting participants, but the reliability is limited. Thus, current results should be confirmed by combinations of multiple techniques and clinical studies with different size. Second, this cross-sectional study has shown only a correlation between vascular dysfunction and insulin resistance in subjects with metabolic syndrome, and has not indicated a cause-effect relationship. In future studies, we need to confirm that a therapeutic approach to improve insulin sensitivity by such as reductions in visceral fat obesity and ectopic fat deposition can recover vascular dysfunction in metabolic syndrome. Third, since metabolic syndrome is a cluster of relatively heterogeneous atherosclerotic risk factors, which solely affect endothelial function, careful consideration should be taken to evaluate underlying mechanisms. Forth, we could not determine the molecular mechanisms by which insulin resistance occurs and impairs endothelial function from the current study.