Manuel Serrano-Ríos and José Luis González-Sánchez, Internal Medicine II Department, Hospital Clinico San Carlos, 28040 Madrid, Spain, Tel: +34-913303387/8, Fax: +34-913303385, E-mail: mserrano.hcsc[at]salud.madrid.org

The metabolic syndrome (MS), is a cluster of cardiovascular risk factors that often accompany obesity and is associated with increased risk for both atherosclerotic cardiovascular disease and type 2 diabetes [1-2]. Most of risk factors for MS are of metabolic origin and mainly consist of atherogenic dyslipidemia, elevated blood pressure, elevated plasma glucose, prothrombotic and proinflammatory states [3]. Most individuals with this constellation will be insulin resistant and thus will be at higher risk for type 2 diabetes at the prediabetic state [4]. We and others feel that MS represents a powerful valid working hypothesis that unifies the metabolic factors underlying the development of both atherosclerotic cardiovascular disease and diabetes [1,4-5]. Still there is a hot debate about the reality of the concept of MS and its clinical utility has been very much questioned [3,6-9]. The current profusion of definition criteria for MS are based either on obeso-centricity or focused on insulin resistance [3,8-9].

MS has a multi-factorial causation as result of complex interactions between genetic and environmental factors, and several studies have suggested a heritable basis to the etiology of metabolic syndrome [10-13].

MS and Cardiovascular Risk: Reality or Fiction

Though largely debated in the past few years, current evidence from a recent extensive meta-analysis drawn from a large number (n = 37) of longitudinal studies including 172,573 people of diverse populations (including many rural and urban regions of the U.S., Spain, Italy, Finland, Sweden, Norway, The Netherlands, Scotland, England, Poland, Turkey, and Japan) clearly indicates a significantly increased risk of cardiovascular events and death in people with MS [14].

Endothelial Dysfunction and Atherosclerosis

Endothelial dysfunction is believed to be a link between complex phenomena at the molecular level and vascular pathologies like atherosclerosis [15]. Atherosclerosis is a chronic inflammatory disease that represents the primary cause of heart disease and stroke. Endothelial dysfunction is one of the key earliest manifestations of the many changes induced in the natural history of the chronic inflammation in arterial wall by the impact of metabolic abnormalities that contributes to the development and progression of atherosclerosis, and accompany diabetes and insulin resistance [16-18].

Nitric Oxide (NO)

The endothelial-derived vasorelaxation factor NO plays a critical role in mediating the vascular actions of insulin [19-20]. Arginine is converted to NO by the enzyme nitric oxide synthase (NOS) in the intact vascular endothelium. The activity of NOS is increased several fold by cytokines such as IL-1β, IL-6, TNF-α, interferon-γ, and adenosine [21].

Three isoforms of NOS have been characterized, purified, and cloned and, in chronological order, were neuronal NOS or NOS-1, inducible NOS or NOS-2, and endothelial NOS or NOS-3. These isoforms are encoded by three different genes on different chromosomes and have about 50-60% homology with each other and the cytochrome P-450 enzymes [22].

Endothelial nitric oxide synthase (eNOS) is the main enzyme that synthesizes NO in endothelial cells [2]. Decreased bioavailability of NO therefore results in impaired endothelium-dependent vasodilation and promotes atherosclerosis. Also NO facilitates the uptake and metabolism of glucose in skeletal muscle [23-24].

Moreover, NO acts as a key messenger to activate the mitochondrial biogenesis program in diverse cell types, which has been involved in the pathogenesis of MS [25].

Several studies have found decreased endothelium-independent vasodilation in patients with diabetes compared with nondiabetic control subjects after administration of NO donors like nitroprusside [17,26], suggesting decreased NO action in VSM cells. Moreover, in vivo evidence supports the major contribution of hyperglycemia in producing oxidative stress and, ultimately, acute endothelial dysfunction in blood vessels of patients with diabetes [27].

NOS3 Gene and MS

Although the more common forms of atherosclerosis manifest later in life, twin and adoption studies have shown that this more common form is also partly heritable as a result of environmental factors and many common gene variants with small-to-moderate effect [28]. Therefore, individual differences in endothelial function, and hence susceptibility to later development of atherosclerosis, might relate not only to different levels of exposure to risk factors but also to inter-individual differences in the carriage of risk alleles of genes expressed in the vascular endothelium. Thus, endothelial mediators such as NO suggest that genes involved in synthesis and inactivation of these mediators might be important.

Studies of partial eNOS knockout mice demonstrate that these animals are hypertensive, hyperinsulinemic, dyslipidemic, and have decreased insulin-stimulated glucose uptake compared with wild-type mice [29]. Moreover, mice with deletion of the eNOS gene showed accelerated atherosclerosis [30], and also hypertension, hypertriglyceridemia, and insulin resistance [29]. Interestingly, it has recently been reported that genomic variation within the NO3-proximal region of chromosome 7q influences both insulin resistance and blood pressure [31], suggesting that this locus may influence traits associated with MS. In humans, eNOS is encoded by the NOS3 gene, which is located at 7q35-36. More than 15 polymorphisms exist in the NOS3 promoter that might influence mRNA transcription and reduce gene expression.

The effect of eNOS variants on the enzyme activity and on the production of NO is still a controversial issue. Some polymorphisms in the promoter region (-786T/C) have been shown to influence transcriptional activity in vitro and to be related with coronary arterial spasm [32]. Recent findings suggest, however, that this polymorphism does not have a significant effect on NO bioavailability [33]. Controversial evidence of polymorphisms in exon 7 (894G→T) on impaired enzyme function have also been reported [34-35]. Furthermore, it has been suggested that eNOS haplotypes, instead of eNOS genotypes alone, could have a major contribution on NO bioavailability [36].

However, variants at the NOS3 gene have been related to type 2 diabetes mellitus [37], insulin resistance [38-39], and MS [40]. In a comprehensive epidemiological study of the relationship between variation at the NOS3 gene, type 2 diabetes, and glucose intolerance in three populations (two from U.K and one from Spain) Franks et al. [37] suggested that variations at this gene influenced risk of diabetes and level of glucose intolerance and that total energy expenditure and variation within the NOS3 gene interacted to modify these phenotypes.

Recently we performed a NOS3 gene haplotypes study [40] showing that genetic variations at the NOS3 gene locus were associated with feature of the MS in the Spanish population. For the NOS3 IVS11-30 SNP, the AA genotype was associated with increased insulin resistance, and higher total cholesterol concentrations. For the rs3800787 SNP, the CC genotype was significantly more frequent in individuals with MS than in those without MS and in those with decreased HDL-cholesterol concentrations (16.1% versus 12.7%, P = 0.044).

In haplotype analysis, we observed that a haplotype (212) was associated with an increased OR for MS (OR 1.81, 95% CI 1.15-2.84) even after adjustment for variables that may affect NO metabolism such as smoking, alcohol intake, type 2 diabetes, and antihypertensive medication (OR 1.87, 95%CI 1.14-3.08). Moreover, this haplotype was also associated with a greater OR for decreased HDL-cholesterol concentrations (OR 1.52, 95% CI 1.01-2.29, p = 0.04) and with increased mean values of HOMA, and triglycerides compared with the most frequent haplotype “121.” However, the molecular mechanisms underlying the association between specific eNOS haplotypes and abnormalities in eNOS activity deserve further investigation. To our knowledge, this is the first study investigating the relationship between eNOS haplotypes and MS in a large population-based study.

Discrepancies between other NOS3 gene variants studies may be attributable to sample size, study design, and population stratification secondary to ethnic diversity. In this regard, marked interethnic differences in the distribution of eNOS genetic variants and haplotype frequencies have been reported [41-42]. In addition, discrepancies in association studies may also result from consideration limited to only one polymorphism rather than combinations of polymorphisms (haplotypes) [43,44]. Therefore, it is now generally accepted that analyzing combinations of genetic markers in a region of interest (haplotypes) can be much more informative than testing the effects of genetic markers one by one [44]. At variance with our study, the majority of published studies reporting association between NOS3 gene variants and features of the MS have used several single markers (SNPs) to characterize the genetic architecture. Thus, because haplotype approaches are less likely to lead to false discovery, the genetic associations identified in this way may be of greater value.

In addition to NOS3 gene, other genes controlling NO synthesis at the endothelium might affect the decreased NO bioavailability which could also result from increased generation of asymmetric dimethylarginine (ADMA; endogenous circulating inhibitor of NOS); decreased availability of 6R-tetrahydrobiopterin (BH4; and essential NOS co-factor); or increased inactivation of NO by reactive oxygen species such as superoxide. Additionally, variations in other genes whose products have been implicated in endothelial dysfunction such as angiotensin converting enzyme, methylene tetrahydrofolate reductase, neuropeptide Y, the bradykinin receptor, interleukine-6, and others, have given inconsistent results [45].

Conclusions

Endothelial dysfunction and insulin resistance commonly occur together and can be detected early in the pathogenesis of atherosclerosis. Insulin resistance can be inferred by the presence of a cluster of metabolic and cardiovascular abnormalities known as the MS. Lack of endothelial-derived NO may provide the link between insulin resistance and endothelial dysfunction.

Some data including ours suggest that genetic variations at the NOS3 locus may increase an individual’s susceptibility to certain components of the MS. Further studies focused on functional genomic and gene-environmental interactions are needed to identify individuals who are at high risk of MS.

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