The Study Of The Association Between TLR4, RAGE Gene Polymorphisms And Diabetes, And The Association Between TLR4 Expression In White Adipose Tissue And Obesity In Wuhan Chineses Population

PartⅠThe Study of the Association between TLR4, RAGE Gene Polymorphisms and Diabetes in Wuhan Chinese populationObjective: To examine the associations between the single nucleotide polymorphisms (SNPs) of Asp299Gly in TLR4 gene, 1704 G/T and G82S in the RAGE gene and diabetes and its complications.Methods: The SNPs of Asp299Gly in TLR4 gene, 1704G/T and G82S in the RAGE gene were detected in 506 patients with type 2 diabetes mellitus (T2DM) and 182 subjects with normal glucose tolerance (NGT) by the methods of ligase detection reaction coupled polymerase chain reaction (LDR-PCR).Results: No mutation was found on Asp299Gly in TLR4 gene in 688 subjects which are all AA genotypes. There was no difference in the 1704G/T and G82S genotypic and allelic frequences between NGT and T2DM subjects. The homeostasis model assessment of B cell function index (HOMA-? ) values of carriers of 1704T allele were lower than those of carriers of G1704 allele in T2DM patients(1.68±0.43 VS 1.80±0.37,P =0.03). In addition, the carriers of 1704T allele had lower prevalence of hypertension than those of carriers of G1704 allele in T2DM (P =0.001). The HOMA-? values of carriers of 82S allele were also lower than those G82 allele carriers (1.70±0.4 VS 1.81±0.38,P =0.05). These differences were not found within NGT group. Furthermore, the percentage of diabetic retinopathy (DR) of 82S allele carriers was higher than that of G82 allele carriers in T2DM. No difference was found in other complications. The frequences of G/A + AA genotypes(60.6%) and A allele(36.4%) of G82S were significantly higher in T2DM with DR than those(38.8%; 20.9%, respectively) in T2DM without DR (P<0.05). Furthermore, the multiple logistic regression analysis indicated that G82S polymorphism and diabetes duration were independent risk factors for DR (OR=2.964, 95%CI:1.013-5.46,P =0.029; OR=1.013, 95% CI:1.007-1.02, P <0.001; respectively). Haplotype analysis revealed that G-A haplotype had significantly higher risk of DR than other haplotypes, with OR for 2.120, 95% CI:1.185-3.792, P =0.01.Conclusions: 82S allele of G82S polymorphism in the RAGE gene deteriorates the B cell function of pancreas and increases the risk of DR. G-A haplotype constructed across the 1704G/T and G82S polymorphisms would be a gene marker of DR in Wuhan Chinese T2DM patients. Moreover, 1704T variant predict deteriorative B cell function of pancreas. PartⅡThe Study of Association between TLR4 Expression in White Adipose Tissue and ObesityObjective: Recent studies suggest that adipose tissue plays a potential role in innate immunity. Furthermore, obesity is characterized by enriched macrophages and hypertrophic adipocytes in white adipose tissue (WAT). Our aim were to address the TLR4 protein expression and clarify the association among the level of TLR4 protein expression and obesity phenotype,inflammatory marker as well as the morphology of human WAT.Methods: Paired subcutaneous and omental WAT (oWAT) were obtained from 30 male subjects undergoing selective cholecystectomy [age, 34-60yr; body mass index(BMI), 23.8±3.5 kg/m2]. 30 participants were classified into the lean group, overweight without diabetes(DM) subgroup as well as overweight with DM subgroup according to the BMI and fasting plasma glucose. The level of TLR4 protein expression in WAT were examined by Western blot. High-sensitive C-reactive protein(hs-CRP) were detected by the method of immunoturbidimetry. The number of CD68+ cell and adipocyte size in WAT were quantified microscopically using the immunohistology of CD68 antibody stained. The association among the level of TLR4 expression in oWAT, clinic and morphology parameters were performed using multiple linear regression analysis.Result: Western blot confirmed the TLR4 protein expression in both oWAT and subcutaneous WAT(scWAT), but the level of TLR4 protein expression in scWAT was much lower than that in oWAT within same subject (0.20±0.03 VS 0.59±0.19, P<0.001). Higher level of TLR4 expression in oWAT of overweight without DM and overweight with DM subgroup were demonstrated than that of lean group(P<0.01). TLR4 expression of oWAT in overweight with DM subgroup was lower than that in overweight without DM subgroup(P <0.01). There were 4-fold as many CD68+ cells in oWAT as that in scWAT (24.4±3.2 VS 6.1±2.9,P <0.001). The number of CD68+ cells of either oWAT or scWAT in overweight without DM and overweight with DM subgroup was higher than that of lean group, but no difference was found between them. Average adipocytes diameter and adipocyte cross-sectonal area of oWAT in overweight without DM subgroup were greater compared with those in the lean group (69.2±19.37μm VS 44.2±10.4μm, P=0.003; 4014.1±2443.1μm2 VS 1613.4±755.6μm2, P=0.04; respectively.), and not different from overweight with DM subgroup. There was a strong positive correlation among the level of TLR4 protein expression in oWAT with the degree of macrophage infiltration in oWAT(b'=0.635, P<0.001), BMI (b'=0.201, P=0.02) and hs-CRP(b'=0.211, P=0.044).Conclusion: Our results clarified the presence of TLR4 in both oWAT and scWAT. Obesity may be the major contributor to TLR4 expression in WAT,especially in oWAT, and hyperglycemia attenuates this effect. TLR4 in WAT would be involved into the progression of chronic low degree inflammation of the obesity-related diseases.