ZnT8 In Pathogenesis And Differential Diagnosis Of Diabetes And Regulation By TNFAIP3

BackgroudZinc is especially important in pancreatic β cells. Zinc transporter 8(Zn T8), encoded by the SLC30A8 gene, is responsible for Zn transport into and accumulation in the insulin-secreting granules. Zn T8 has been identified as a risk factor for type 2 diabetes, and autoantibodies to Zn T8(Zn T8A) have been recently identified as a reliable biomarker of autoimmune diabetes, which can distinguish it from type 2 diabetes and healthy status. Furthermore, the most common polymorphism at the 325 site(rs13266634, C/T) is associated with type 2 diabetes and epitope specificity of Zn T8 autoantibodies in type 1 diabetes.The autoantibodies against islet cells and self-antigens such as Zn T8, glutamic acid decarboxylase, insulinoma-associated protein 2, and insulin, commonly emerge at or even precede the onset of diabetes and persist for different time periods following the onset. Therefore, autoantibodies against islet cell antigens are useful clinical tools for the ri sk prediction and diagnosis of autoimmune diabetes. Although the enzyme-linked immunosorbent assay(ELISA) and radioimmunoassay are the most frequently used testing methods, they do not allow simultaneous detection of multiple autoantibodies. Another obstacle is the high cost of Zn T8 production for antibody assays.Studies about the role for rs13266634 variant of SLC30A8 in type 2 diabetes have been accumulating but inconsistency remains partly due to small sample sizes and non-identical ethnicity. Additionally, Zn T8 can be downregulated by activating nuclear factor-κB(NF-κB)-regulated cytokines. The zinc finger protein tumor necrosis factor α-induced protein-3(TNFAIP3) is a negative regulator of NF-κB activation and the central gatekeeper in inflammation and immunity. Although the role of TNFAIP3 in diabetes is widely studied, its expression in immune cells and its influence on Zn T8 are not fully elucidated.AimsThis study firstly aimed to cost-effectively produce two Zn T8 C-terminal fragments containing main Zn T8 antigen epitopes and establish an reliable and rapid assay for the detection of anti-Zn T8 antibodies with potential to simultaneously measure multiple autoantibodies. Another aim of this study was to meta-analyze the accumulating but inconsistent data between SLC30A8 rs13266634 variant and type 2 diabetes risk. Lastly in this study we sought to determine the expression of TNFAIP3 in immune cells in patients with diabetes and evaluate the effect of pro-inflammatory cytokines as well as TNFAIP3 overexpression on Zn T8 activities.Methods1. The mutation of Zn T8 was achieved using site-directed mutagenesis and two Zn T8 C-terminal antigens were expressed via a p Cold II expression system in Escherichia coli.The dot immunogold filtration assay(DIGFA) was established to detect autoantibodies and evaluated by comparing with ELISA as the “gold standard”.2. We searched Pub Med and Cochrane Library to identify eligible studies and extract data of baseline characteristics, genotype count, odds ratio(OR) and 95% confidence interval(CI). Both adjusted OR with 95% CI and genotype counts were employed to assess the association. Genotype data were further pooled estimate under different genetic models and the most appropriate model. Sensitivity and cumulative analysis were conducted to guarantee the strength of results.3. Using a population study, quantitative real-time PCR and western blot analysis were employed to determine the expression of TNFAIP3 m RNA and protein, respectively, in peripheral blood lymphocyte from patients with latent autoimmune diabetes in adults, type 2 diabetes, and matched healthy controls. Cell-based studies using NIT-1 cells overexpressing TNFAIP3 were used to assess the effect of cytokines on Zn T8 and NF-κB activation and the effect of TNFAIP3 on Zn T8 expression under inflammatory condition.Results1. Two Zn T8 antigens(arginin and tryptophan Zn T8 at position325) were successfully produced. We established a rapid DIGFA method for the simultaneous detec tion of anti-Zn T8 antibodies, with the sensitivity, specificity, accuracy, Youden index, and positive and negative likelihood ratio being 64.3%, 96.4%, 85.7%, 0.607, 18.0, and 0.370, respectively and the results did not significantly differed from those fo r ELISA(p= 0.22). Aims2. Fifty-five datasets of 39 studies(including 38 of 24 with genotype count) were included. Significant associations were found in allelic contrasts using adjusted ORs and raw genotype count, respectively, in overall, Asian and European(Overall: OR=1.147/1.157, 95% CI 1.114–1.181/1.135–1.180; Asian: OR= 1.186/1.165, 95% CI 1.150–1.222/1.132–1.198; European: OR=1.100/1.151, 95% CI 1.049–1.153/1.120–1.183; All p= 0.00) but not African populations(African: OR=1.255/1.111, 95% CI 0.964–1.634/0.908–1.360, p= 0.091/0.305). Further analysis with genotype count under different genetic models all showed that individuals with CC genotype had 33.0% and 16.5 % higher risk of type 2 diabetes than those carrying TT and CT genotypes under the most likely codominant model, respectively. Cumulative analysis indicated gradually improved precision of estimation after studies accumulated.3. The m RNA and protein expression of TNFAIP3 in patients with type 2 diabetes and latent autoimmune diabetes in adults was significantly decreased compared with healthy controls. Furthermore, the TNFAIP3 m RNA and protein expression in newly diagnosed T2 D patients( 1 year) was significantly lower than in patients with longstanding T2D( 1 year). Cytokine stimulation led to TNFAIP3 upregulation, Zn T8 downregulation, and NF-κB activation. TNFAIP3 overexpression also protected Zn T8 from cytokine-induced downregulation.Conclusions1. The p Cold II expression system is suitable for the production of bioactive Zn T8 antigens and that DIGFA can be a rapid, reliable, and highly specific method for the detection of Zn T8 antibodies, which can be potentially applied to identify a panel of diabetes-specific autoantibodies simutaneously.2. SNP rs13266634 may be an important genetic factor of type 2 diabetes risk among Asian and European but not African populations.3. Decreased expression of TNFAIP3 may be involved in the pathogenesis of diabetes, and that TNFAIP3 may protect against diabetes partly by inhibiting pro-inflammatory cytokine-induced downregulation of Zn T8.