Abstract
Aims/hypothesisLevels of the microRNA (miRNA) miR-126-3p are programmed cell-autonomously in visceral adipose tissue of adult offspring born to obese female C57BL/6J mice. The spectrum of miR-126-3p targets and thus the consequences of its dysregulation for adipocyte metabolism are unknown. Therefore, the aim of the current study was to identify novel targets of miR-126-3p in vitro and then establish the outcomes of their dysregulation on adipocyte metabolism in vivo using a well-established maternal obesity mouse model.MethodsmiR-126-3p overexpression in 3T3-L1 pre-adipocytes followed by pulsed stable isotope labelling by amino acids in culture (pSILAC) was performed to identify novel targets of the miRNA. Well-established bioinformatics algorithms and luciferase assays were then employed to confirm those that were direct targets of miR-126-3p. Selected knockdown experiments were performed in vitro to define the consequences of target dysregulation. Quantitative real-time PCR, immunoblotting, histology, euglycaemic–hyperinsulinaemic clamps and glucose tolerance tests were performed to determine the phenotypic and functional outcomes of maternal programmed miR-126-3p levels in offspring adipose tissue.ResultsThe proteomic approach confirmed the identity of known targets of miR-126-3p (including IRS-1) and identified Lunapark, an endoplasmic reticulum (ER) protein, as a novel one. We confirmed by luciferase assay that Lunapark was a direct target of miR-126-3p. Overexpression of miR-126-3p in vitro led to a reduction in Lunapark protein levels and increased Perk (also known as Eif2ak3) mRNA levels and small interference-RNA mediated knockdown of Lunapark led to increased Xbp1, spliced Xbp1, Chop (also known as Ddit3) and Perk mRNA levels and an ER stress transcriptional response in 3T3-L1 pre-adipocytes. Consistent with the results found in vitro, increased miR-126-3p expression in adipose tissue from adult mouse offspring born to obese dams was accompanied by decreased Lunapark and IRS-1 protein levels and increased markers of ER stress. At the whole-body level the animals displayed glucose intolerance.Conclusions/interpretationConcurrently targeting IRS-1 and Lunapark, a nutritionally programmed increase in miR-126-3p causes adipose tissue insulin resistance and an ER stress response, both of which may contribute to impaired glucose tolerance. These findings provide a novel mechanism by which obesity during pregnancy leads to increased risk of type 2 diabetes in the offspring and therefore identify miR-126-3p as a potential therapeutic target.Graphical abstract
Highlights
Overweight and obesity have reached epidemic proportions in both developed and developing countries [1]
Lunapark is a direct target of miR-126-3p In order to define the panel of proteins targeted by miR-126-3p, we transfected 3T3-L1 pre-adipocytes with either a fluorescent commercial scrambled miRNA or miR-126-3p mimic followed by 24 h of differential labelling with medium (2H4-L-lysine) or heavy (13C615N2-L-lysine) lysine, respectively (Fig. 1)
We have shown previously that maternal obesity programmes epididymal fat of young male mice offspring to overexpress miR-126-3p, resulting in impairment of the insulin signalling pathway via direct downregulation of IRS-1 protein levels [15]
Summary
Overweight and obesity have reached epidemic proportions in both developed and developing countries [1]. Obesity is associated with an increased risk of a number of metabolic diseases including type 2 diabetes [2] In part, this is thought to be the result of obesity-associated insulin resistance and activation of a variety of stress-related pathways in adipose tissue [3,4,5,6,7]. In addition to the well-established detrimental effects of obesity on the current health of an individual, there is accumulating evidence to suggest that obesity during pregnancy is detrimental This is because does obesity during pregnancy have an immediate detrimental impact on the mother (including increased risk of gestational diabetes and pregnancy-associated hypertension), it has long-term programmed impacts on offspring health [8, 9] including increased risk of cardiometabolic disease [10]. This programming effect is encompassed by the developmental origins of health and disease (DOHaD) hypothesis [11]
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