| Abstract | Introduction: Type 2 diabetes mellitus (T2DM) is a global health problem and its complications are a significant cause of morbidity and mortality worldwide. T2DM is the common type of diabetes and it affects mainly adults. Insulin resistance is considered as a main factor contributing to T2DM. Different substances are implicated in development of insulin resistance, among them vitamin D, resistin and homocysteine which can interrupt the insulin signalling pathway leading to insulin resistance. Elevated levels of homocysteine have been observed in patients with T2DM and metabolic syndrome, while resistin (a hormone) was suggested to play a role in the pathogenesis of T2DM. Increased levels of resistin have been linked with adiposity/inflammation and insulin resistance in T2DM patients. Vitamin D deficiency is another global problem and elevated levels of blood glucose and vitamin D deficiency have been linked to complications associated with T2DM. Recently studies conducted in ethnic groups in Asia and Europe suggest that vitamin D deficiency, resistin and homocysteine may also be implicated in the pathogenesis of T2DM. However, limited numbers of studies were from African subjects and none investigated vitamin D, resistin and homocysteine together in association with T2DM. T2DM is a common disease in Sudan and it causes considerable morbidity and mortality. Few studies have been conducted in Sudan to explain the increase in prevalence of diabetes but no studies looked at the role of homocysteine, resistin and vitamin D. Methyl tetrahydrofolate reductase (MTHFR) is an enzyme that contribute to the formation of methionine from homocysteine and it has taken all these attention because the present of the genetic mutation which led to hyperhomocysteinemia.
Objective: The aim of this study was to investigate possible associations between homocysteine, resistin and vitamin D with T2DM in a representative study population from Sudan. In addition, associations between the Methyl tetrahydrofolate reductase (MTHFR) gene polymorphisms (C677T and A1298C) with homocysteine levels and with T2DM, as well as, the prevalence of VDR gene polymorphism (T56058C) in patients with T2DM and their healthy controls were investigated.
Material and Methods: This is a case-control hospital-based study conducted at the Diabetes clinic with samples obtained from the Military hospital, Omdurman, Sudan. Two hundred patients with T2DM were consecutively enrolled during 2013-2014. The included patients were known to have T2DM for a minimum of one year and were under-regular follow-up at the diabetes clinic. Patients with chronic illness (especially heart and kidney diseases) and other types of diabetes (including type 1 diabetes) were excluded. The control group (n=195) consisted of healthy individuals with no family history of diabetes. A questionnaire was used to acquire demographic data (age, gender), anthropometric measurements (weight/height and waist circumference), basic biochemical tests (HbA1c, fasting blood glucose (BG), lipid profile) and blood pressure (systolic/diastolic). Plasma levels of resistin and homocysteine were measured using ELISA-based methods, while total Vitamin D (D2/D3) levels were measured using an UHPLC method. The gene variants MTHFR C677T, MTHFR A1298C and VDR T56058C were typed using real time PCR methods. Associations between plasma levels of resistin, homocysteine and total vitamin D with T2DM, and their associations with different variables (age, gender, BMI, HbA1c, fasting blood glucose, lipid profile) were statistically analysed. This was in addition to the associations between the MTHFR gene polymorphisms and homocysteine levels, and the associations between VDR gene polymorphism and T2DM.
Results: Resistin levels were significantly higher in the healthy controls compared to patients with T2DM (p<0.0001). Levels were significantly higher in healthy males compared to healthy females (p=0.007) as well as in males in the study population in general compared to females in the study population (p=0.002). However, no significant difference in resistin levels was found between male and female in individuals with T2DM (p=0.09). My results showed also that healthy controls had significantly higher resistin levels compared to patients with T2DM in groups with BMI>25 and BMI<25 (p=0.03, for both comparisons). In addition, the results also showed significantly higher levels of resistin in male healthy controls compared with male patients with T2DM in groups with BMI>25 and BMI<25 (p<0.001 and p=0.007, respectively), and there was no significant difference in resistin levels between female healthy controls and female patients with T2DM in groups with BMI>25 and BMI<25 ((p=0.45 and p=0.16, respectively). Resistin levels were significantly higher in patients with T2DM not using vitamin D supplementation compared with those using it (p=0.0039). There is a positive correlation between resistin levels and 2hBG and HbA1c% in the study population (all participants) (r=0.121; p=0.035 and r= 0.237p<0.0001, respectively) and also with FBG, BMI (log), homocysteine levels in individuals with T2DM (r=0.188; p=0.008; r=0.140; p=0.048 and r=0.335; p<0.0001, respectively), while there is a negative correlation between resistin levels and vitamin D in patients with T2DM (r= -0.261; p<0.0001). Homocysteine levels were significantly higher in patients with T2DM compared to healthy controls (p<0.0001). In addition, its levels were significantly higher in females compared with males (p<0.0001) and there was a positive correlation between plasma homocysteine levels and HbA1c% (r=0.298; p<0.0001) and 2hBG (r=0.361; p<0.001) in all participants. The percentage of patients carrying the MTHFR C677T genotypes CC, TT and CT is 85.5%, 0.5% and 14.0% respectively, while among healthy controls it is 88.7%, 0.0% and 11.3%. There is no significant difference in the genotype (CC, CT and TT) and allele (C and T) frequencies between patients and healthy controls (χ2= 0.909, p=0.340; χ2= 0.660, p=417; χ2= N/A, p=N/A for CC, CT and TT genotypes, respectively; and χ2= 1.110, p=0.292 for the C and T alleles, the % of patients carrying the MTHFR A1298C genotypes AA, CC and AC is 65.0%, 6.0% and 29.0% respectively, while among healthy controls it is 65.1%, 5.1% and 29.8%. There is no statistically significant difference in the genotype (AA, CC and AC) and allele (A and C) frequencies between patients and healthy controls (χ2=0.001 p=0.098; χ2= 0.26, p=0.87; χ2= 0.143, p=0.71 for the AA, CC and AC genotypes, respectively; and χ2= 1.031, p=0.86 for the A and T alleles). Our results showed that the levels of homocysteine are significance higher in patients with T2DM carrying the three genotypes, 677CT, 1298AC and 1298CC, and carrying the heterozygous haplotype C677T/A1298C compared to the healthy controls carrying the same genotypes/haplotype.
Vitamin D levels was significantly higher in patients with T2DM compared to healthy controls (p<0.0001), and its levels have negative correlation with FBG in patients with T2DM (r= -0.177; p=0.013). The % of patients carrying the VDR T56058C genotypes CC, TC and TT is 36.5%, 46% and 17.5% respectively, while among healthy controls it is 28.7%, 52.8% and 18.5%. There are no significant differences in the VDR T56058C genotype (CC, TC and TT) and allele (C and T) frequencies between the patients and their healthy controls (χ2= 2.719, p=0.099; χ2= 1.838, p=0.175; χ2= 0.062, p=0.803 for CC, TC and TT genotypes; and χ2= 2.084, p=0.149 for the C and T allele, respectively).
Conclusion: Previous studies have suggested a role for Vitamin D deficiency and elevated levels of plasma resistin and homocysteine levels in the pathogenesis of insulin resistance and T2DM. Our results confirmed previously reported high levels of homocysteine in patients with T2DM. A positive correlation was observed between homocysteine and FBG, HbA1c and resistin, as well as a positive correlation between resistin and FBG and HbA1c, and a negative correlation between vitamin D and resistin, homocysteine and FBG levels in patients with T2DM. The negative association between vitamin D and resistin/blood glucose levels suggest that vitamin D might have an impact on resistin levels in patients with T2DM and improved insulin action/blood glucose levels and T2DM. Taken together, our results suggest a possible role for vitamin D, resistin and homocysteine in the development of insulin resistance and T2DM. |
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