The nuclear factor-erythroid 2- (NF-E2-) related factor 2 (Nrf2) is abated and its ability to reduce oxidative stress is impaired in type 2 diabetes and obesity. Thus, the aim of this study was to explore if polymorphisms in Nrf2 and target genes are associated with diabetes and obesity in Mexican mestizo subjects. The rs1800566 of NAD(P)H:quinone oxidoreductase 1 (NQO1) gene, rs7211 of thioredoxin interacting protein (TXNIP) gene, rs2071749 of heme oxygenase-1 (HMOX1) gene, and the rs6721961 and the rs2364723 from Nrf2 gene were genotyped in 627 diabetic subjects and 1020 controls. The results showed that the rs7211 polymorphism is a protective factor against obesity in nondiabetic subjects (CC + CT versus TT, OR = 0.40,
The International Diabetes Federation has reported that there are 382 million people living with diabetes worldwide [
The master antioxidant regulator, the nuclear factor-erythroid 2- (NF-E2-) related factor 2 (Nrf2) is a member of the cap’n’collar family of basic leucine zipper transcription factors that regulates the expression of many antioxidant genes including NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HMOX1) to avoid oxidative damage [
However, despite the recent efforts to understand the association of antioxidant gene polymorphism with T2DM, the number of studies is still very limited in Mexican population. Thus, the aim of this study was to investigate potential associations between Nrf2 (rs6721961 and rs2364723), NQO1 (rs1800566), HMOX1 (rs2071749), and TXNIP (rs7211) polymorphisms in a T2DM population.
A case control study was performed which included 1647 Mexican mestizo subjects. This study was approved by the Ethics Committee on Human Studies from the Committee on Research, Ethics and Safety of the Hospital Regional “Lic. Adolfo López Mateos” with registration number 254.2013 and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all subjects who were recruited in Family Medical Clinics of the Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado (ISSSTE) in Mexico City.
A standard questionnaire was applied to obtain demographic information, family and personal health history (diabetes and/or further diseases), and information about physical activity, alcohol consumption, smoking, and drugs consumption. Their medical history data, weight (kg), and height (m) were obtained. The body mass index (BMI) was estimated by dividing weight by the square of height. Criteria for inclusion/exclusion of participants were as follows: the study included men and women aged 35 and older, nonpregnant and nonlactating women, and subjects without excessive alcohol consumption. The criteria for classification and diagnosis of diabetes were according to the standards in medical care on diabetes from American Diabetes Association (ADA). Subjects of the control group (
Whole blood samples (20 mL) were collected from patients and controls (with fasting of 8–12 h) in order to determine the levels of glucose, triglycerides, total cholesterol, HDL-C, low-density lipoprotein cholesterol (LDL-C), and creatinine in serum using an automatized analyzer (Miura 200, ISE, Rome, Italy). Moreover, total blood with ethylenediaminetetraacetic (EDTA) acid as anticoagulant was used to obtain genomic DNA.
Genomic DNA from whole blood containing EDTA was isolated by standard techniques [
Fifteen autosomal STR markers (CSF1PO, FGA, THO1, TPOX, VWA, D3S11358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, D19S433, and D2S1338) along with amelogenin were genotyped in 200 controls and 200 cases, using the AmpFlSTR Identifiler Kit (Applied Biosystems, Foster City, CA, USA) as previously described [
We performed admixture estimations using the STR’s allele distribution by a model-based clustering method with the
Continuous variables were analyzed using a
The Hardy Weinberg equilibrium was calculated in controls using StatCalc software (Epi Info 2005 v3.3.2; Centers of Disease Control and Prevention, Atlanta, GA, USA). Multivariable linear regression models were carried out for adjustment of glucose for potential confounders like age, gender, BMI, and tobacco. In order to assess the genetic risk factor for diabetes and obesity, logistic regression analyses were applied to estimate the OR for each polymorphism. Because the associations between the SNPs and the outcomes have been previously reported, it is unlikely to detect effects due to statistical fluctuations only. Therefore, correction by multiple comparisons was not applied. Associations were considered statistically significant at a nominal
General characteristics of the study population are shown in Table
Clinical and anthropometric characteristics of the study groups.
Characteristic | Control | Diabetes |
|
---|---|---|---|
|
1020 | 627 | |
Age (years) | 42.1 ± 7.3 | 53.2 ± 9.4 | <0.001 |
Males, |
474 (46) | 302 (48) | 0.759 |
BMI (kg⋅m−2) | 28.7 ± 4.9 | 29.7 ± 6.9 | 0.0016 |
Obesity, |
350 (34) | 289 (46) | <0.001 |
Glucose (mg⋅dL−1) | 95.5 ± 9.6 | 154 ± 73 | <0.001 |
HbA1c | 4.45 ± 0.8 | 7.27 ± 2.1 | <0.001 |
Triglycerides (mg⋅dL−1) | 183 ± 122 | 214 ± 140 | <0.001 |
Total cholesterol (mg⋅dL−1) | 201 ± 43 | 198 ± 52 | 0.2257 |
HDL-C (mg⋅dL−1) | 49.5 ± 12.5 | 45.4 ± 13 | <0.001 |
LDL-C (mg⋅dL−1) | 129.2 ± 36 | 152.5 ± 56 | <0.001 |
Creatinine (mg⋅dL−1) | 1.17 ± 0.22 | 1.2 ± 0.92 | 0.4225 |
SBP | 116 ± 14 | 121 ± 16 | <0.001 |
DBP | 77 ± 10 | 77.4 ± 10.6 | 0.102 |
Smoking, |
256 (25) | 159 (25.3) | 0.918 |
Continuous variables are presented as means ± SD and categorical variables as numbers (percentage). BMI, body mass index; HbA1c, glycated hemoglobin; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; SBP, systolic blood pressure; DBP: diastolic blood pressure.
The distribution of the Native American (NAM), the European (EUR), and the African (AFR) individual admixture proportions was comparable between the diabetic subjects and controls (NAM:
Table
Genotype frequencies of the polymorphisms studied in diabetic patients and controls.
Gene/polymorphism | Genotypes alleles | Diabetes | Controls | OR (95% CI) |
|
|
---|---|---|---|---|---|---|
TXNIP |
CC, |
345 (55.4) | 528 (54.5) | Reference | 0.880 | |
CT, |
239 (38.3) | 376 (38.8) | 0.88 (0.6–1.3) | 0.577 | ||
TT, |
39 (6.3) | 65 (6.7) | 0.9 (0.8–1.2) | 0.776 | ||
|
||||||
NQO1 |
CC, |
216 (34.7) | 327 (33) | Reference | 0.406 | |
CT, |
288 (46.2) | 483 (48.6) | 0.90 (0.7–1.1) | 0.373 | ||
TT, |
119 (19.1) | 183 (18.4) | 0.98 (0.7–1.3) | 0.915 | ||
|
||||||
HMOX1 |
AA, |
269 (43.8) | 413 (43.2) | Reference | 0.625 | |
AG, |
267 (43.5) | 417 (43.6) | 1.05 (0.76–1.4) | 0.757 | ||
GG, |
78 (12.7) | 126 (13.2) | 1.03 (0.74–1.4) | 0.837 | ||
|
||||||
NRF2 |
CC, |
210 (33.6) | 301 (30.3) | Reference | 0.092 | |
CG, |
286 (56.8) | 471 (47.5) | 0.87 (0.7–1.1) | 0.236 | ||
GG, |
129 (20.6) | 220 (22.2) | 0.84 (0.63–1.1) | 0.223 | ||
|
||||||
NRF2 |
CC, |
407 (65.3) | 618 (62.5) | Reference | 0.281 | |
CA, |
189 (30.4) | 317 (32) | 0.9 (0.7–1.1) | 0.374 | ||
AA, |
27 (4.3) | 54 (5.5) | 0.76 (0.5–1.2) | 0.259 |
CI, confidence interval; HWE, Hardy-Weinberg equilibrium; HMOX1, heme oxygenase-1; NQO1, NAD(P)H quinone oxidoreductase 1; NRF2, Nuclear factor-erythroid 2- (NF-E2-) related factor 2; OR, odds ratio; and TXNIP, thioredoxin interacting protein.
Genotype and allele frequencies of the polymorphisms studied in obese and nonobese subjects.
Gene/polymorphism | Genotype | Obesity | No obesity | OR (95% CI) |
|
---|---|---|---|---|---|
TRXNIP |
CC, |
350 (56.6) | 523 (53.7) | Reference | |
CT, |
239 (38.7) | 376 (38.6) | 0.95 (0.7–1.2) | 0.627 | |
TT, |
29 (4.7) | 75 (7.7) | 0.56 (0.35–0.87) | 0.012 | |
|
|||||
NQO1 |
CC, |
212 (34.2) | 331 (33.3) | Reference | |
CT, |
302 (48.8) | 469 (47) | 1 (0.8–1.25) | 0.963 | |
TT, |
106 (17) | 196 (19.7) | 0.84 (0.6–1.1) | 0.257 | |
|
|||||
HMOX1 |
AA, |
261 (40) | 419 (45.7) | Reference | |
AG, |
306 (46.9) | 378 (41.3) | 1.3 (1–1.6) | 0.019 | |
GG, |
85 (13.1) | 119 (13) | 1.1 (0.8–1.5) | 1.14 | |
|
|||||
NRF2 |
CC, |
194 (31) | 317 (32) | Reference | |
CG, |
300 (48) | 457 (46) | 1.1 (0.85–1.35) | 0.551 | |
GG, |
131 (21) | 210 (22) | 0.98 (0.7–1.3) | 0.899 | |
|
|||||
NRF2 |
CC, |
390 (63.1) | 635 (63.9) | Reference | |
CA, |
195 (31.6) | 311 (31.3) | 1 (0.8–1.3) | 0.853 | |
AA, |
33 (5.3) | 48 (4.8) | 1.1 (0.7–1.7) | 0.631 |
CI, confidence interval; HMOX1, heme oxygenase-1; NQO1, NAD(P)H quinone oxidoreductase 1; NRF2, Nuclear factor-erythroid 2- (NF-E2-) related factor 2; OR, odds ratio; and TXNIP, thioredoxin interacting protein.
When CC + CT genotype of the rs7211 polymorphism was compared with TT, lower frequency of TT genotype associated with lower BMI (TT: 27.6 ± 4 versus CC + CT: 29 ± 5,
Genotype frequency of the rs7211 polymorphism in subjects without diabetes and women.
Obese | Nonobese | Crude OR (95% CI) |
|
Adjusted |
| |
---|---|---|---|---|---|---|
Nondiabetic | ||||||
CC | 189 (56.6) | 339 (53.4) | Reference | Reference | ||
CT | 133 (40) | 241 (38) | 0.98 (0.74–1.3) | 0.903 | 1 (0.76–1.4) | 0.863 |
TT | 55 (8.6) | 12 (3.6) | 0.3 (0.2–0.7) | 0.004 | 0.3 (0.15–0.7) | 0.003 |
CC + CT | 322 (96.4) | 580 (91.5) | 0.4 (0.2–0.76) | 0.005 | 0.3 (0.15–0.7) | 0.003 |
|
||||||
Women | ||||||
CC | 197 (59) | 259 (51) | Reference | Reference | ||
CT | 118 (36) | 203 (40) | 0.7 (0.6–1) | 0.072 | 0.9 (0.6–1.2) | 0.418 |
TT | 17 (5) | 47 (9) | 0.5 (0.26–0.85) | 0.013 | 0.5 (0.25–0.96) | 0.04 |
CT + TT | 135 (41) | 250 (49) | 0.70 (0.5–0.9) | 0.016 | 0.7 (0.5–0.96) | 0.028 |
CI, confidence interval; OR, odds ratio.
Multiple linear regression of BMI as dependent variable in nondiabetic subjects and women.
|
|
| |
---|---|---|---|
Nondiabetic subjects | |||
HDL-C |
|
<0.001 | 0.11 |
Glucose |
|
<0.001 | |
rs7211 (CT + TT: 0, TT: 1) |
|
0.033 | |
|
|||
Women | |||
HDL |
|
0.003 | 0.048 |
Glucose |
|
0.001 | |
rs7211 (CC: 0, CT + TT: 1) |
|
0.037 |
Models also include age, gender (in nondiabetic subjects model), LDL-C, and triglycerides. Significant variables were presented. BMI, body mass index; HDL-C, high-density lipoprotein cholesterol.
After gender stratification, CC genotype was higher in obese women compared with nonobese women (Table
The rs2071749 polymorphism in the HMOX1 gene was significantly associated with obesity when it was analyzed under a dominant model (Table
Association of the rs2071749 polymorphism of the HMOX1 gene with obesity.
Variable | Obese, |
Nonobese, |
|
BMI |
|
---|---|---|---|---|---|
AA | 261 (40) | 410 (46) | 0.026 | 28.9 ± 4.9 | 0.027 |
AG + GG | 390 (60) | 497 (54) | 29.5 ± 5.3 | ||
|
|||||
Crude OR (95% CI) | 1.25 (1.02–1.54) | 0.026 | |||
OR adjusted |
1.34 (1.06–1.7) | 0.013 | |||
Beta coefficient/ |
0.063/0.03 | 0.023 |
BMI values are presented as mean ± SD. BMI, body mass index, CI: confidence interval, and OR: odds ratio.
The rs6721961 polymorphism of the Nrf2 gene was associated with diabetes in men but not in women, after stratification by gender (Table
Association of the rs6721961 polymorphism of the Nrf2 gene with diabetes in men.
Variable | Diabetic, |
Nondiabetic, |
|
Glucose |
|
---|---|---|---|---|---|
CC | 203 (69) | 271 (58) | 0.011 | 126.6 ± 59 | 0.056 |
CA | 79 (27) | 171 (37) | 116 ± 56 | ||
AA | 12 (4) | 23 (5) | 105 ± 32 | ||
CA + AA | 91 (31) | 194 (42) | 0.003 | 114.4 ± 53 | 0.031 |
|
|||||
Crude OR (95% CI) | 0.62 (0.46–0.85) | 0.003 | |||
OR adjusted |
0.56 (0.38–0.82) | 0.003 | |||
Crude beta coefficient | −0.078 | 0.031 |
Glucose values (mg⋅dL−1) are presented as mean ± SD. CI: confidence interval; OR: odds ratio.
Oxidative stress is one of the major metabolic factors that lead to the onset of chronic disease like insulin resistance, hypertension, metabolic syndrome, prediabetes, diabetes, and its complications. It has been recognized that the antioxidant system is abated in diabetes and obesity, accompanied by an increased production of inflammatory cytokines. A defect in Nrf2 activation in many organs has been documented widely in experimental models of insulin resistance and diabetes leading to decreased expression of its target genes [
Wang et al. [
The rs7211 has been poorly studied in diabetic and obese subjects. First, van Greevenbroek et al. [
The SNP rs2071746 (T-413A) and
NRF2 gene regulates the enzymes studied here. Many SNPs have been identified in this gene. The rs35652124 and rs6721961 SNPs are predicted to affect Nrf2 myeloid zinc finger 1 (MZF1) and antioxidant response elements- (ARE-) like promoter binding sites, respectively. These SNPs affect the efficient binding of proteins such as Nrf2 to the MZF1 and ARE-like promoter binding sites [
In metabolic diseases, the rs6721961 polymorphism has been associated with blood pressure in Japanese subjects [
We know that this research has some limitations. The gene expressions and activities were not determined and just candidate genes were included. Dietary information was not available in all patients. However, this study opens the gate to new researches about the role of HMOX1, TXNIP, and Nrf2 in obesity, diabetes, and worse metabolic traits.
This study shows that the rs7211 polymorphism in the TXNIP gene and the rs2071749 of the HMOX1 gene are associated with obesity in Mexican mestizo people. In this sense, rs2071749 increases the risk of having obesity and the rs7211 polymorphism may be a protective factor to develop obesity and to have lower concentrations of HDL-C in Mexican mestizo women. In addition, rs6721961 gen polymorphism in Nrf2 was negatively associated with diabetes in men. All of these results open the doors for new research, taking into account the effect on metabolic traits, and may be useful tools to design new therapeutic strategies in obesity and diabetes type 2.
The authors declare that they have no competing interests.
The authors would like to acknowledge the Medical Family Clinics from ISSSTE: Ignacio Chávez, Del Valle, Xochimilco, and Fuentes Brotantes. This work was financially supported by CONACyT-Mexico (Grant no. SALUD-2013-01-201519).