Multi-layered epigenetic regulation of IRS2 expression in the liver of obese individuals with type 2 diabetes

Aims/hypothesis IRS2 is an important molecular switch that mediates insulin signalling in the liver.IRS2dysregulation is responsible for the phenomenon of selective insulin resistance that is observed in type 2 diabetes. We hypothesise that epigenetic mechanisms are involved in the regulation ofIRS2in the liver of obese and type 2 diabetic individuals. Methods DNA methylation of seven CpG sites was studied by bisulphite pyrosequencing and mRNA and microRNA (miRNA) expression was assessed by quantitative real-time PCR in liver biopsies of 50 obese non-diabetic and 31 obese type 2 diabetic participants, in a cross-sectional setting. Methylation-sensitive luciferase assays and electrophoretic mobility shift assays were performed. Furthermore, HepG2 cells were treated with insulin and high glucose concentrations to induce miRNA expression andIRS2downregulation. Results We found a significant downregulation ofIRS2expression in the liver of obese individuals with type 2 diabetes (0.84 +/- 0.08-fold change;p = 0.0833; adjustedpvalue [p(a)] = 0.0417;n = 31) in comparison with non-diabetic obese participants (n = 50). This downregulation correlated with hepaticIRS2DNA methylation at CpG5. Additionally, CpG6, which is located in intron 1 ofIRS2, was hypomethylated in type 2 diabetes; this site spans the sterol regulatory element binding transcription factor 1 (SREBF1) recognition motif, which likely acts as transcriptional repressor. The adjacent polymorphism rs4547213 (G>A) was significantly associated with DNA methylation at a specificity-protein-1 (SP1) binding site (CpG3). Moreover, DNA methylation of cg25924746, a CpG site located in the shore region of theIRS2promoter-associated CpG island, was increased in the liver of individuals with type 2 diabetes, as compared with those without diabetes. A second epigenetic mechanism, upregulation of hepatic miRNA hsa-let-7e-5p (let-7e-5p) in obese individuals with type 2 diabetes (n = 29) vs non-diabetic obese individuals (n = 49) (1.2 +/- 0.08-fold change;p = 0.0332;p(a) = 0.0450), is likely to act synergistically with alteredIRS2DNA methylation to decreaseIRS2expression. Mechanistic in vitro experiments demonstrated an acute upregulation of let-7e-5p expression and simultaneousIRS2downregulation in a liver (HepG2) cell line upon hyperinsulinaemic and hyperglycaemic conditions. Conclusions/interpretation Our study highlights a new multi-layered epigenetic network that could be involved in subtle dysregulation ofIRS2in the liver of individuals with type 2 diabetes. This might lead to fine-tuning ofIRS2expression and is likely to be supplementary to the already known factors regulatingIRS2expression. Thereby, our findings could support the discovery of new diagnostic and therapeutic strategies for type 2 diabetes.