Abstract
Aims/hypothesisIRS2 is an important molecular switch that mediates insulin signalling in the liver. IRS2 dysregulation 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 of IRS2 in the liver of obese and type 2 diabetic individuals.MethodsDNA 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 and IRS2 downregulation.ResultsWe found a significant downregulation of IRS2 expression in the liver of obese individuals with type 2 diabetes (0.84 ± 0.08-fold change; p = 0.0833; adjusted p value [pa] = 0.0417; n = 31) in comparison with non-diabetic obese participants (n = 50). This downregulation correlated with hepatic IRS2 DNA methylation at CpG5. Additionally, CpG6, which is located in intron 1 of IRS2, 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 the IRS2 promoter-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; pa = 0.0450), is likely to act synergistically with altered IRS2 DNA methylation to decrease IRS2 expression. Mechanistic in vitro experiments demonstrated an acute upregulation of let-7e-5p expression and simultaneous IRS2 downregulation in a liver (HepG2) cell line upon hyperinsulinaemic and hyperglycaemic conditions.Conclusions/interpretationOur study highlights a new multi-layered epigenetic network that could be involved in subtle dysregulation of IRS2 in the liver of individuals with type 2 diabetes. This might lead to fine-tuning of IRS2 expression and is likely to be supplementary to the already known factors regulating IRS2 expression. Thereby, our findings could support the discovery of new diagnostic and therapeutic strategies for type 2 diabetes.Graphical abstract
Highlights
IRS2 is one of the most important molecular switches mediating hepatic insulin receptor signalling and is involved in the phenomenon of selective insulin resistance, in which the lipogenic actions of insulin remain intact while glucose uptake and glycogen synthesis are disrupted [1, 2]
It was shown that IRS2 expression is altered in obese individuals and in people suffering from non-alcoholic fatty liver disease (NAFLD) [5, 6]; it currently remains unclear as to whether IRS2 contributes to the pathogenesis of type 2 diabetes and fatty liver disease in humans, and the mechanisms responsible for IRS2 dysregulation are unknown
Hepatic IRS2 expression is associated with liver health We measured hepatic IRS2 expression in liver tissue of obese individuals who were stratified by type 2 diabetes, as well as liver fibrosis and steatosis status (NAS)
Summary
IRS2 is one of the most important molecular switches mediating hepatic insulin receptor signalling and is involved in the phenomenon of selective insulin resistance, in which the lipogenic actions of insulin remain intact while glucose uptake and glycogen synthesis are disrupted [1, 2]. It was shown that IRS2 expression is altered in obese individuals and in people suffering from non-alcoholic fatty liver disease (NAFLD) [5, 6]; it currently remains unclear as to whether IRS2 contributes to the pathogenesis of type 2 diabetes and fatty liver disease in humans, and the mechanisms responsible for IRS2 dysregulation are unknown. A 3% increase in IRS2 DNA methylation within a CpG island of exon 1 (cg05514401) was identified in subcutaneous adipose tissue of 15 insulin-resistant obese women concomitant with decreased IRS2 gene expression, as compared with 14 non-obese normoglycaemic women [11], indicating that IRS2 expression may be epigenetically regulated in metabolic diseases. Functional consequences of these alterations in IRS2 DNA methylation remain unclear and whether they could lead to future glucose-lowering therapies is not yet known
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