Christin Krause

Background: The development of obesity-associated comorbidities such as type 2 diabetes (T2D) and hepatic steatosis has been linked to selected microRNAs in individual studies; however, an unbiased genome-wide approach to map T2D induced changes in the miRNAs landscape in human liver samples, and a subsequent robust identification and validation of target genes are still missing.Methods: Liver biopsies from age- and gender-matched obese individuals with (n=20) or without (n=20) T2D were used for microRNA microarray analysis. The candidate microRNA and target genes were validated in 85 human liver samples, and subsequently mechanistically characterized in hepatic cells as well as by dietary interventions and hepatic overexpression in mice.Results: Here, we present the human hepatic microRNA transcriptome of type 2 diabetes in liver biopsies and use a novel seed prediction tool to robustly identify microRNA target genes, which were then validated in a unique cohort of 85 human livers. Subsequent mouse studies identified a distinct signature of T2D-associated miRNAs, partly conserved in both species. Of those, human-murine miR-182–5 p was the most associated with whole-body glucose homeostasis and hepatic lipid metabolism. Its target gene LRP6 was consistently lower expressed in livers of obese T2D humans and mice as well as under conditions of miR-182–5 p overexpression. Weight loss in obese mice decreased hepatic miR-182–5 p and restored Lrp6 expression and other miR-182–5 p target genes. Hepatic overexpression of miR-182–5 p in mice rapidly decreased LRP6 protein levels and increased liver triglycerides and fasting insulin under obesogenic conditions after only seven days.Conclusions: By mapping the hepatic miRNA-transcriptome of type 2 diabetic obese subjects, validating conserved miRNAs in diet-induced mice, and establishing a novel miRNA prediction tool, we provide a robust and unique resource that will pave the way for future studies in the field. As proof of concept, we revealed that the repression of LRP6 by miR-182–5 p, which promotes lipogenesis and impairs glucose homeostasis, provides a novel mechanistic link between T2D and non-alcoholic fatty liver disease, and demonstrate in vivo that miR-182–5 p can serve as a future drug target for the treatment of obesity-driven hepatic steatosis.Funding: This work was supported by research funding from the Deutsche Forschungsgemeinschaft (KI 1887/2-1, KI 1887/2-2, KI 1887/3-1 and CRC-TR296), the European Research Council (ERC, CoG Yoyo LepReSens no. 101002247; PTP), the Helmholtz Association (Initiative and Networking Fund International Helmholtz Research School for Diabetes; MB) and the German Center for Diabetes Research (DZD Next Grant 82DZD09D1G).

De novo lipogenesis (DNL) in visceral adipose tissue (VAT) is associated with systemic insulin sensitivity. DNL in VAT is regulated through ChREBP activity and glucose uptake through Glut4 (encoded by Slc2a4). Slc2a4 expression, ChREBP activity, and DNL are decreased in obesity, the underlying cause however remains unidentified. We hypothesize that increased DNA methylation in an enhancer region of Slc2a4 decreases Slc2a4 expression in obesity and insulin resistance. We found that SLC2A4 expression in VAT of morbidly obese subjects with high HbA1c (>6.5%, n = 35) is decreased, whereas DNA methylation is concomitantly increased compared to morbidly obese subjects with low HbA1c (≤6.5%, n = 65). In diet-induced obese (DIO) mice, DNA methylation of Slc2a4 persistently increases with the onset of obesity and insulin resistance, while gene expression progressively decreases. The regulatory impact of DNA methylation in the investigated enhancer region on SLC2A4 gene expression was validated with a reporter gene assay. Additionally, treatment of 3T3 pre-adipocytes with palmitate/oleate during differentiation decreased DNA methylation and increased Slc2a4 expression. These findings highlight a potential regulation of Slc2a4 by DNA methylation in VAT, which is induced by fatty acids and may play a role in the progression of obesity and insulin resistance in humans.