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 tothreaten the public health. Increases in the prevalence of diabetes have occurredinternationally, and it has been estimated that between1980and2008, the number of adultswith diabetes rose from153million to347million. Moreover, according to the estimationof the World Health Organization (WHO), there will be366million diabetic peopleworldwide by2030.Research suggests that oxidative stress is a major risk factor in the onset andprogression of type2diabetes mellitus (T2DM). Some of the consequences of an oxidativeenvironment may be the development of impaired glucose tolerance, β-cell dysfunction,insulin resistance, and mitochondrial dysfunction, which can contribute ultimately to thediabetic disease state. Recent evidence also suggests that NF-E2related factor2(Nrf2) is apivotal transcription factor to the antioxidant response in oxidative stress related illnesses.Nrf2is a member of the cap ‘n’ collar (CNC) subfamily of basic leucine zippertranscription factors. Nrf2has highly conserved domains named Nrf2-erythroid-derivedCNC homology protein homology (Neh) domains. Among them, Neh1domain is CNC andbasic leucine zipper domain, which interacts with partner proteins for heterodimerization.Neh3domain, located at the extreme end of the carboxyl terminus of Nrf2, is related toNrf2transactivation. Neh4and Neh5cooperatively bind with the cyclic adenosinemonophosphate response element binding protein-binding protein. In the absence of Nrf2structural data, it is not clear how Neh4and Neh5, together with the Neh3domains, exerttransactivation 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-likeerythroid-derived protein with CNC homology-associated protein1(Keap1), andnegatively regulates Nrf2function.Keap1was originally thought to be an actin-binding protein that represses the functionof Nrf2by simply sequestering Nrf2in the cytoplasm. Recently, Keap1has also beenidentified as an adaptor protein between Nrf2and Cullin3, a component of the E3ligasecomplex. Under normal conditions, Nrf2molecules may be subjected to continuousdegradation by the proteasome. When induced by oxidative stress derived fromaccumulation of reactive oxygen species (ROS) or reactive nitrogen species, single ormultiple reactive cysteine(s) in Keap1can be modified. This conformation change causesNrf2to dissociate from Keap1. Nrf2quickly cumulates in the nucleus and elicits theantioxidant response by transactivating the antioxidant response element (ARE) in thepromoter region of many antioxidant genes. The activation of Nrf2is an important clue forthe inducible expression of cytoprotective genes. The antioxidant enzymes encoded bythese genes may play important roles in scavenging oxygen free radicals.In the promoter region of the Nrf2gene, the promoter polymorphisms were found in aJapanese population by Yamamoto et al.. Several studies demonstrated the associations ofthe Nrf2polymorphisms with oxidative stress related illnesses, such as acute lung injuryafter major trauma and vitiligo. However, there is little research evaluating the associationbetween Nrf2polymorphisms and oxidative stress and antioxidative status. The relationshipbetween this polymorphism with β-cell function, insulin sensisity, and the risk of T2DM islargely unknown.Therefore, the objective of the present study was to evaluate the association betweenNrf2gene polymorphisms and the susceptibility to T2DM in a relatively large Chinesepopulation. We also evaluated the functional relevance of this polymorphism by measuringβ-cell function, insulin sensitivity, oxidative stress and antioxidative status among the studypopulations.PartⅠ Oxidative stress and antioxidant status and type2diabetes mellitusObjective: To investigate oxidative stress and antioxidative status in participants withand 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 productproportional to the TAC, which was tested at593nm. Plasma CAT activity was assayed bya method of Goth. MDA as an index of lipid peroxidation was estimated by using themethod described by Beuege and Aust. The activity of SOD in erythrocyte lysates wasevaluated on the basis of its ability to inhibit the oxidation of hydroxylamine, as describedpreviously. Erythrocyte GSH-Px activity was measured by the method described by Pagliaand Valentine. Erythrocyte GSH content was measured using the method described byBeutler et al. Descriptive statistics in the clinical and laboratory characteristics of healthycontrols and patients with T2DM were calculated for the study subjects. Differencesbetween diabetes cases and controls were tested by one-way analysis of variance, followedby Chi-square (categorical variables) or t test (continuous variables).Results: When compared with subjects with NGT, patients with T2DM had asignificant increase in plasma MDA (P=0.001) and a significant decrease in plasma TAClevels (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 inpatients with T2DM.Conclusion: The results also demonstrated that patients with T2DM had an increasedfree radical production and a reduced antioxidant capacity.PartⅡ Association of the Nrf2rs6721961polymorphism with oxidative stressand antioxidative statusObjective: To investigate oxidative stress, and antioxidative status in relation togenotypes 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 onABI7900HT. The measure of plasma TAC was based on the ability of antioxidants in thesamples to change Fe3+-tripyridyltriazine to Fe2+-tripyridyltriazine, a stable blue productproportional to the TAC, which was tested at593nm. Plasma CAT activity was assayed bya method of Goth. MDA as an index of lipid peroxidation was estimated by using themethod described by Beuege and Aust. The activity of SOD in erythrocyte lysates wasevaluated on the basis of its ability to inhibit the oxidation of hydroxylamine, as describedpreviously. Erythrocyte GSH-Px activity was measured by the method described by Paglia and Valentine. Erythrocyte GSH content was measured using the method described byBeutler et al.Results: Compared to individuals with the CC genotype, those with the AA genotypehad 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 withdecreased 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 allelicdiscrimination assay-by-design TaqMan method on ABI7900HT. Differences in allelic andgenotypic frequencies of the gene polymorphisms in healthy controls and patients withT2DM were compared by Chi-square test, which was also used to evaluateHardy-Weinberg equilibrium for each individual locus. We used logistic regression analysisto 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 wassignificantly higher in T2DM subjects (29.4%) compared to NGT subjects (26.1%)(P=0.019). The rs6721961was associated with increased risk of diabetes. The individualscarrying 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 forage, 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 thanthose 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-cellfunction and increasing insulin resistance.