Associations Of The NF-E2Related Factor2Gene Polymorphism With Oxidative Stress, Antioxidative Status,and Susceptibility To Type2Diabetes

Type2diabetes mellitus (T2DM) has been one of the biggest chronic diseases to threaten the public health. Increases in the prevalence of diabetes have occurred internationally, and it has been estimated that between1980and2008, the number of adults with diabetes rose from153million to347million. Moreover, according to the estimation of the World Health Organization (WHO), there will be366million diabetic people worldwide by2030.Research suggests that oxidative stress is a major risk factor in the onset and progression of type2diabetes mellitus (T2DM). Some of the consequences of an oxidative environment may be the development of impaired glucose tolerance,β-cell dysfunction, insulin resistance, and mitochondrial dysfunction, which can contribute ultimately to the diabetic disease state. Recent evidence also suggests that NF-E2related factor2(Nrf2) is a pivotal transcription factor to the antioxidant response in oxidative stress related illnesses.Nrf2is a member of the cap’n’collar (CNC) subfamily of basic leucine zipper transcription factors. Nrf2has highly conserved domains named Nrf2-erythroid-derived CNC homology protein homology (Neh) domains. Among them, Nehl domain is CNC and basic leucine zipper domain, which interacts with partner proteins for heterodimerization. Neh3domain, located at the extreme end of the carboxyl terminus of Nrf2, is related to Nrf2transactivation. Neh4and Neh5cooperatively bind with the cyclic adenosine monophosphate response element binding protein-binding protein. In the absence of Nrf2structural data, it is not clear how Neh4and Neh5, together with the Neh3domains, exert transactivation activity. The Neh2domain, which is located in the N-terminus of Nrf2, is a regulatory domain that responds to oxidative stress. Neh2mediates binding with Kelch-like erythroid-derived protein with CNC homology-associated protein1(Keapl), and negatively regulates Nrf2function.Keap1was originally thought to be an actin-binding protein that represses the function of Nrf2by simply sequestering Nrf2in the cytoplasm. Recently, Keapl has also been identified as an adaptor protein between Nrf2and Cullin3, a component of the E3ligase complex. Under normal conditions, Nrf2molecules may be subjected to continuous degradation by the proteasome. When induced by oxidative stress derived from accumulation of reactive oxygen species (ROS) or reactive nitrogen species, single or multiple reactive cysteine(s) in Keapl can be modified. This conformation change causes Nrf2to dissociate from Keapl. Nrf2quickly cumulates in the nucleus and elicits the antioxidant response by transactivating the antioxidant response element (ARE) in the promoter region of many antioxidant genes. The activation of Nrf2is an important clue for the inducible expression of cytoprotective genes. The antioxidant enzymes encoded by these genes may play important roles in scavenging oxygen free radicals.In the promoter region of the Nrf2gene, the promoter polymorphisms were found in a Japanese population by Yamamoto et al.. Several studies demonstrated the associations of the Nrf2polymorphisms with oxidative stress related illnesses, such as acute lung injury after major trauma and vitiligo. However, there is little research evaluating the association between Nrf2polymorphisms and oxidative stress and antioxidative status. The relationship between this polymorphism with β-cell function, insulin sensisity, and the risk of T2DM is largely unknown.Therefore, the objective of the present study was to evaluate the association between Nrf2gene polymorphisms and the susceptibility to T2DM in a relatively large Chinese population. We also evaluated the functional relevance of this polymorphism by measuring β-cell function, insulin sensitivity, oxidative stress and antioxidative status among the study populations.Part I Oxidative stress and antioxidant status and type2diabetes mellitusObjective:To investigate oxidative stress and antioxidative status in participants with and without diabetesMethods:The measure of plasma TAC was based on the ability of antioxidants in the samples to change Fe3+-tripyridyltriazine to Fe2+-tripyridyltriazine, a stable blue product proportional to the TAC, which was tested at593nm. Plasma CAT activity was assayed by a method of Goth. MDA as an index of lipid peroxidation was estimated by using the method described by Beuege and Aust. The activity of SOD in erythrocyte lysates was evaluated on the basis of its ability to inhibit the oxidation of hydroxylamine, as described previously. Erythrocyte GSH-Px activity was measured by the method described by Paglia and Valentine. Erythrocyte GSH content was measured using the method described by Beutler et al. Descriptive statistics in the clinical and laboratory characteristics of healthy controls and patients with T2DM were calculated for the study subjects. Differences between diabetes cases and controls were tested by one-way analysis of variance, followed by Chi-square (categorical variables) or t test (continuous variables).Results:When compared with subjects with NGT, patients with T2DM had a significant increase in plasma MDA (P=0.001) and a significant decrease in plasma TAC levels (P=0.024) and CAT activity (P<0.001). Erythrocyte SOD activity (P=0.002), GSH-Px activity (P=0.002), and GSH content (P=0.040) were also significantly lower in patients with T2DM.Conclusion:The results also demonstrated that patients with T2DM had an increased free radical production and a reduced antioxidant capacity.Part II Association of the Nrf2rs6721961polymorphism with oxidative stress and antioxidative statusObjective:To investigate oxidative stress, and antioxidative status in relation to genotypes of rs6721961polymorphism in the study populations.Methods:The genotyping of single nucleotide polymorphisms (SNPs) of the Nrf2gene was done by using an allelic discrimination assay-by-design TaqMan method on ABI7900HT. The measure of plasma TAC was based on the ability of antioxidants in the samples to change Fe3+-tripyridyltriazine to Fe2+-tripyridyltriazine, a stable blue product proportional to the TAC, which was tested at593nm. Plasma CAT activity was assayed by a method of Goth. MDA as an index of lipid peroxidation was estimated by using the method described by Beuege and Aust. The activity of SOD in erythrocyte lysates was evaluated on the basis of its ability to inhibit the oxidation of hydroxylamine, as described previously. Erythrocyte GSH-Px activity was measured by the method described by Paglia and Valentine. Erythrocyte GSH content was measured using the method described by Beutler et al.Results:Compared to individuals with the CC genotype, those with the AA genotype had a significant decrease in plasma TAC levels (P=0.025), CAT activity (P=0.001), erythrocyte SOD activity (P=0.042), GSH-Px activity (P=0.020), and GSH content (P=0.042), whereas a significant increase in plasma MDA concentration (P=0.005).Conclusion:Polymorphisms in the Nrf2genes were significantly associated with decreased antioxidant activity and increased oxidative stress.Part Ⅲ Association of Nrf2rs6721961polymorphism with risk of type2diabetes mellitusObjective:To investigate the genotype and allele frequencies of the rs6721961polymorphism in the Nrf2gene in the study populations.Methods:The genotyping of SNPs of the Nrf2gene was done by using an allelic discrimination assay-by-design TaqMan method on ABI7900HT. Differences in allelic and genotypic frequencies of the gene polymorphisms in healthy controls and patients with T2DM were compared by Chi-square test, which was also used to evaluate Hardy-Weinberg equilibrium for each individual locus. We used logistic regression analysis to assess the association of diabetes events with the specific polymorphism. Odds ratios and95%confidence intervals were adjusted for known risk factors for T2DM, including age, body mass index, sex, family history of diabetes, and hypertension.Results:There were significant differences in the allelic frequency of the rs6721961polymorphism between T2DM cases and controls. The frequency of allele A was significantly higher in T2DM subjects (29.4%) compared to NGT subjects (26.1%)(P=0.019). The rs6721961was associated with increased risk of diabetes. The individuals carrying the AA genotype had a significantly higher risk for developing T2DM (OR1.77;95%CI1.26,2.49; P=0.011) relative to those with the CC genotype. After adjustment for age, sex, and body mass index, this association remained statistically significant (OR1.56;95%CI1.11,2.20; P=0.014). Individuals carrying the AA genotype had a lower HOMA-β (AA71.37±4.05vs. CC84.37±1.59, P=0.005) and a higher HOMA-IR values than those with the CC genotype (AA2.90±0.19vs. CC2.36±0.06, P=0.003).Conclusion:The rs6721961polymorphism of the Nrf2gene was associated with an increased risk of T2DM. The polymorphisms were also associated with impaired β-cell function and increasing insulin resistance.