It has been estimated that the worldwide prevalence of diabetes mellitus and metabolic syndrome (MetS) are 10.5% and 12.5 – 31.4%, respectively, and the values are predicted to increase during the following years (1,2).
Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder mainly characterized by insulin resistance and β-cell dysfunction (3). Besides insulin resistance, MetS is described by a cluster of conditions, namely high blood pressure, abdominal obesity, high triglyceride levels, low HDL cholesterol level, and impaired fasting glucose. Several risk factors have been identified for T2DM and MetS, such as high adiposity, abnormal blood biomarkers levels, medical history, regional and psychosocial factors. In addition, lifestyle factors such as daily caloric intake, smoking and alcohol consumption are considered to be related to the prevalence of T2DM and MetS (4,5).
Dysfunction of many biological pathways may be involved in the pathophysiology of the diseases. Mutations in protein-coding genes involved in oxidative stress (OS) reduction (SOD1 and CAT) (6,7), endothelial functions (eNOS) (8) and hemodynamics (ACE, ATR1) (9,10,11,12) or in the carbohydrate metabolism (OXTR) (13,14) may to be involved in predisposition for T2DM or MetS. There are gender-related differences regarding the relative contribution of risk factors for these diseases (15,16). Therefore, we evaluated the susceptibility in men of the association of common polymorphisms in six genes with T2DM and MetS.
The case-control study included Caucasian men considered healthy (n=120) or diagnosed with T2DM (n=120) or MetS (n=75). The American Diabetes Association 2016 and NCEP ATP III criteria were used for the diagnosis of these diseases (17). Healthy individuals were selected based on a standard clinical evaluation and on paraclinical data. Patients with a diagnosis of chronic kidney diseases, retinopathy, diabetic peripheral neuropathy or with an addiction to drugs were not included in the study. We collected clinical, paraclinical, and lifestyle data from all patients. Subjects were considered smokers if they smoked between 2 and 25 cigarettes per day for at least a year. Alcohol consumers were considered those who drunk at most 50 g alcohol per day for at least a year, but were not heavy drinkers (56g alcohol/day). The research was approved by the ethics committee of the National Research and Development Institute for Food Bioresources (966/27.08.2019).
Genomic DNA was extracted from peripheral venous blood using the Promega Wizard Genomic DNA purification kit (Promega Corporation, Madison, WI), followed by a Polymerase Chain Reaction (PCR) to genotype the rs4646994 (ACE I/D) (18), rs1799983 (eNOS VNTR 4a/b) (18), and rs53576 (OXTR A>G) (19) polymorphisms. A PCR-restriction fragment length polymorphism was used to genotype the ATR1 rs5186 (A1166C) (18), CAT rs7943316 (−21A/T) (20), and SOD1 rs2234694 (+35A/C) polymorphismes (20).
Statistical analysis was performed with the MedCalc software (version 20.111, Ostend, Belgium). After Bonferroni correction for multiple hypotheses (n=33) a p value at p<0.0015 was considered statistically significant.
The clinical data of subjects enrolled in the study are shown in Table 1. MetS patients have 3 (64%), 4 (32%) or 5 (4%) NCEP ATP III criteria for diagnosis.
Characteristics of the study participants
| Parameters | T2DM | MetS | HC |
|---|---|---|---|
| Number of subjects | 120 | 75 | 120 |
| Aged | 54.00 (51–56) a | 55.00 (54–58) | NA |
| Agei | 58 (54.5–59) a, b | 59 (57–61) b | 55 (53–59) |
| BMId | 32.99 (29.41–35.22) | 33.66 (29.74–35.29) | NA |
| BMIi | 30.95 (27.97–33.07)a,b | 32.83 (28.8–33.74) b | 24.55 (23.71–27.74) |
| Glycemiai (mg/dl) | 113.5 (102.5–123) a,b | 109 (98–116) | 98 (93–104.5) |
| Obesityd | 68 | 54 | 0 |
| Hypertension | 37 | 50 | 0 |
| Hyperglycemiad (≥110 mg/dl) | 120 | 33 | 0 |
| HDL cholesterold (<40 mg/dl) | 39 | 55 | 12 |
| Triglyceridesd (≥150 mg/dl) | 48 | 63 | 8 |
| Strokei | 10 | 9 | 0 |
| Coronary heart diseasesi | 9 | 6 | 0 |
| Sexual dysfunctionsi (yes/ no/ refused to respond) | 17/80/23 | 10/47/18 | 12/91/17 |
| Subjects with offspring | 45 | 25 | 100 |
| Smokersi | 19 | 42 | 57 |
| Alcohol consumersi | 43 | 28 | 27 |
Values are presented as number of subjects (n), median (range), or ratio as specified;
at study inclusion;
at disease diagnosis;
p<0.0015 compared to MetS group,
p<0.0015 compared to HC group.
Sample genotyping was performed for all patients included in the study. The distribution of the studied genotypes was in accordance with Hardy-Weinberg Equilibrium (Table 2).
Distribution of the genotypes in the studied groups
| Polymorphisms | Genotypes | T2DM | MetS | HC |
|---|---|---|---|---|
| ACE I/D (rs4646994) | DD | 44 | 25 | 31 |
| ID | 49 | 35 | 61 | |
| II | 27 | 15 | 28 | |
| ATR1 A1166C (rs5186) | AA | 70 | 43 | 65 |
| AC | 38 | 31 | 44 | |
| CC | 12 | 1 | 11 | |
| eNOS VNTR 4a/b (rs1799983) | bb | 90 | 52 | 82 |
| ba | 26 | 23 | 36 | |
| aa | 4 | 0 | 2 | |
| OXTR A>G (rs53576) | GG | 59 | 31 | 42 |
| GA | 47 | 34 | 49 | |
| AA | 14 | 10 | 29 | |
| SOD1 +35A/C (rs2234694) | AA | 27 | 13 | 33 |
| AC | 62 | 44 | 51 | |
| CC | 31 | 18 | 36 | |
| CAT-21A/T (rs7943316) | AA | 104 | 71 | 118 |
| AT | 15 | 4 | 2 | |
| TT | 1 | 0 | 0 |
We found that T2DM was diagnosed at an earlier age compared to MetS (p=0.0003). The statistical significance of difference was greater when comparing patients who are carriers of OXTR G (54.04 vs 56.05 years old, p=0.0002) or both OXTR G and eNOS b alleles (54.00 vs 56.05, p=0.00016).
Our results showed that the SOD1 AA genotype (p=0.0006) and the presence of both ACE I and OXTR A alleles (p=0.0005) are protective factors for T2DM. T2DM patients with lower triglyceride levels (<150 mg/dl) were more frequent carriers of SOD1 AA and AC genotypes when compared to HC subjects (p=0.0002 and p=0.0005, respectively). Similarly, SOD1 AA and AC genotypes were more frequent in T2DM patients with HDL levels over 40 mg/dl when comparing to HC (p=0.0002 and p=0.0004, respectively). Hypertensive T2DM patients were more frequent carriers of ACE DD genotype than HC (56.76% vs 25.83%, p=0.0005) (Table 3). In addition, this genotype, in association with eNOS bb or OXTR G, was found more frequently in alcohol consumers compared to those without this habit (p<0.0001) (Table 3). No other statistically significant associations were found between the investigated groups or subgroups of subjects.
Statistically significant results in the studied groups
| Groups compared | Genetic variants | Distribution | O.R., 95% CI, p value |
|---|---|---|---|
| T2DM vs HC | SOD1 AA | 104/16 vs 118/2 | 0.11, 0.03 – 0.49, 0.0006 |
| T2DM vs HC | ACE I and OXTR A | 32/88 vs 58/62 | 0.39, 0.23 – 0.67, 0.0005 |
| T2DM with triglycerides level <150 mg/dl vs HC | SOD1 AA | 60/12 vs 110/2 | 0.09, 0.02 – 0.42, 0.0002 |
| SOD1 AC | 11/61 vs 2/110 | 9.92, 2.13 – 46.21, 0.0005 | |
| T2DM with HDL level > 40 mg/dl vs HC | SOD1 AA | 67/14 vs 106/2 | 0.09, 0.02 – 0.41, 0.0002 |
| SOD1 AC | 13/68 vs 2/106 | 10.13, 2.22 – 46.31, 0.0004 | |
| T2DM with hypertension vs HC | ACE DD | 21/16 vs 31/89 | 3.77, 1.75 – 8.12, 0.0005 |
| T2DM alcohol drinkers vs T2DM non-drinkers | ACE DD | 30/13 vs 14/63 | 10.38, 4.35 – 24.82, <0.0001 |
| ACE DD and eNOS bb | 25/18 vs 10/67 | 9.31, 3.79 – 22.87, <0.0001 | |
| ACE DD and OXTR1 G | 26/17 vs 12/65 | 8.28, 3.48 – 19.73, <0.0001 |
In this study, we evaluated the association between six common polymorphisms in the ACE, eNOS, OXTR, ATR1, CAT, and SOD1 genes with characteristics of T2DM and MetS in Romanian Caucasian men. Thus, we tried to avoid the impact of gender on these associations. Patients with acute or chronic hyperglycemia have increased OS levels can be predisposed to long term complications of diabetes (21). SOD1 gene codes for an antioxidant enzyme and therefore, it is a functional candidate for obesity (22), T2DM, and its long-term complications (23). In our study, SOD1 AA was a protective factor for T2DM (p<0.0006). Concordant results were reported in the study by Flekac et al., where both Czech males and females were included. The authors reported that SOD1 +35A/C had potential effect on enzyme activity and genotype AA was protective for T2DM (p<0.05) (24). Additionally, in our study SOD1 AA and AC genotypes were associated with triglycerides (p=0.0002 and p=0.0005, respectively) and HDL cholesterol (p=0.0002 and p=0.0004, respectively) levels in T2DM patients. Our results are supported by previous publications in which SOD1 concentration was negatively correlated with HDL cholesterol concentration. However, unlike previously published data, we found no statistically significant association between SOD1 polymorphism and obesity (p<0.05) (25).
Ethnicity and gender may explain, at last partially, previous conflicting results regarding the impact of ACE I/D in predisposition for T2DM (26). The ACE DD genotype was found to increased risk of hypertension and/or diabetes in Egyptian (27), Malaysian (28), Chinese (29) populations, but not in Turkish (30) or Emirati (31). Meta-analyses have also described a positive association with subjects from Middle East, North Africa (26) or Asia (32), whereas, among Europeans, the results are more heterogenous (33,34). The number of ACE D variants was correlated with an increase in ACE activity (35) and angiotensin II signal transduction influences secretion of oxytocin (36,37). Long-term ACE hyperactivity may pre-dispose to insulin hypersecretion and impairment of vessel walls compliance which increase the risk for T2DM and hypertension development (38). Although the distribution of polymorphisms in ACE or OXTR did not differ between our groups, the presence of both ACE I and OXTR A alleles could be a protective factor for T2DM (OR=0.39, p<0.0005).
OXTR mediates the impact of stressful experience and influences social support seeking during distress (39). OXTR polymorphisms may influence the response to stress, via hypothalamic–pituitary–adrenal axis, and the risk for stress-related disorders, including T2DM (13,39,40). Carriers of the rs53576 G allele were more sensitive to both favorable or negative surroundings and individuals with GG genotype had altered cortisol levels and blood pressure after rejection (39).
Predisposition to T2DM involves the interaction between different genetic and non-genetic factors. It was considered that moderate alcohol consumption (24 g/day) is protective for T2DM development, while higher quantities (60 g/day alcohol) represents a risk factor (41). In our T2DM group, patients carrying the ACE DD genotype were more often alcohol consumers. The association of ACE DD with eNOS bb or with OXTR G were found more frequently in drinking compared to non-drinking T2DM patients. We found no association regarding the OXTR gene polymorphisms and smoking habits in T2DM patients.
No association was identified concerning gene polymorphisms in the MetS patients who reported being smokers or drinking alcohol.
Our study indicated no significant association between MetS and tested polymorphisms or between T2DM and ATR1 A1166C or CAT-21A/T.