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Obesity during pregnancy is associated with increased risk of nonalcoholic fatty liver disease (NAFLD) in the offspring; however, the underlying molecular mechanisms remain largely unknown. The aim of the current study was to explore the contribution of microRNAs (miRs) as mediators of programming by maternal obesity. To achieve this, we used a mouse model of maternal-diet induced obesity where dams develop gestational diabetes and offspring demonstrate increased hepatic triglyceride content. We carried out small RNA next generation sequencing analysis of liver from twelve-week-old male offspring and identified miR-582-5p as the most dysregulated, showing a threefold increase. Binding site prediction (TargetScan 7.2) and pathway enrichment analysis (Reactome v67) suggested a number of miR-582-5p target genes related to lipid metabolism including Insulin Induced Gene 2 (INSIG2) and Translocase of Inner Mitochondrial Membrane 23 (TIMM23). Overexpression of miR-582-5p in AML12 (mouse) and HEPG2 (human) cell lines caused increased triglyceride accumulation, downregulation of INSIG2 and TIMM23, increased nuclear localization of Sterol Regulatory Element-Binding Protein 1 (SREBP-1), and overexpression of lipogenic genes. These findings suggest that overexpression of miR-582-5p induces hepatic intracellular lipid accumulation through mechanisms that involve mitochondrial malfunction and activation of de novo lipogenesis. We therefore conclude that an epigenetically programmed increase in hepatic miR-582 is a likely contributing mechanism underlying the programming of fatty liver disease in the offspring of obese gestational diabetic mothers. Disclosure L. Pantaleao: None. D. Fernandez-Twinn: None. A. Carpenter: None. E. Loche: None. L. Dearden: None. D. Duque Guimaraes: None. N.C. Penfold: None. T.J. Ashmore: None. M. Bushell: None. S. Ozanne: Advisory Panel; Self; Nestlé. Funding Brazilian National Council for the Improvement of Higher Education (BEX 10594/13-2); Brazilian National Council for Scientific and Technological Development (PDE/204416/2014-0)

Strong evidence suggests that an adverse in utero and/or early postnatal environment impacts on long-term risk of developing type 2 diabetes. We showed previously that miR-126 is increased in adipose tissue of mouse offspring born to obese mothers which leads to impaired insulin signaling pathway by silencing IRS-1 at the translational level. However, the full spectrum of targets of miR-126 and consequently the consequences of its overexpression for adipose tissue function are unknown. The aim of the current study was therefore to identify novel targets of miR-126 using the proteomic technique, known as Pulsed Stable Isotope Labeling by Amino Acids (pSILAC). 3T3-L1-cells were transfected with miR-126 and their proteome compared to those transfected with a scrambled sequence. We detected 4567 proteins that were translated in adipocytes and of these 401 demonstrated a >1.3 fold decrease following over-expression of miR-126. Bioinformatic analysis revealed that 43 of these contained a miR-126 seed sequence in their 3’un-translated region. This included known miR-126 targets such as IRS-1 and VCAM-1 as well as novel targets. One of the largest changes in expression was observed for Lunapark and through the use of luciferase assays and western blotting we independently confirmed this was a direct target of miR-126. Lunapark is a key component for stabilization of nascent three-way junctions in the endoplasmic reticulum (ER). Consistent with this role we observed altered levels of mTOR and XBP1 in cells treated with miR-126, reflecting the presence of ER stress. Together, the results suggest that overexpression of miR-126 can lead to both ER stress and Insulin Resistance and therefore represent a novel link between two pathways that contribute to development and progression to T2DM. Disclosure J.A. Faria: None. D. Duque Guimaraes: None. L. Pantaleao: None. T.P. Ong: None. A. Ozanne: None.