Impact of in utero diabetes exposure on miRNA: effects on cellular metabolism

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Abstract Future risk for the development of cardiometabolic disease in youth, including type 2 diabetes, is increased by exposure to maternal diabetes (DM) in utero. These early exposures “program” the offspring toward cardiometabolic disease. Studies from my K23 indicate that a specific miRNA, miR-126, is highly abundant in human umbilical vein endothelial cells, placenta, and circulation, targeting elements in the insulin signaling pathway, potentially leading to insulin resistance. However, the effects of miR-126 on cell function and differences in mRNA targets based on cell type and diabetes exposure were not examined. My overall hypothesis is that maternal diabetes increases miR-126 expression in the infant, and this is an important epigenetic driver of insulin resistance and cellular metabolism. Therefore, the overarching goal of the proposed research is to determine how miR-126 disrupts cellular metabolism in fetal cells and to discover novel targets for these pathways. Perinatal studies of miRNA are limited in scope, with most focusing on candidate miRNA species and their putative targets without direct testing of the mechanistic impact. The current proposal will overcome these constraints and extend our current work by a) investigating the impact of maternal DM on cellular function, b) examining biological effects of miR-126 at the cellular level and c) identifying additional mRNA targets of miR-126 in a cell/context-specific fashion. Aim 1. To test the hypothesis that maternal diabetes exposure increases miR-126 abundance altering cellular metabolism. Aim 2. To test the hypothesis that the targets of miR-126 will be cell-type specific and altered by DM exposure. We hypothesize that DM exposure will result in decreased glucose uptake and proliferation, but an increase in senescence associated with increased abundance of miR-126; and in vitro, miR-126 will decrease glucose uptake in the adipocytes and decrease proliferation but increase senescence in the mesenchymal stem cell (MSC). Glucose uptake will be measured in adipocytes using glucose isotope. Proliferation will be assessed by MTT assay, and senescence will be assessed via flow cytometry in the MSCs. miR-126 will be transfected into the DM exposed and unexposed cells with repetition of the above studies to determine the direct effect of miR- 126. In the second aim, high-throughput sequencing of RNA by crosslinking immunoprecipitation (HITS-CLIP) in MSCs and differentiated adipocytes exposed and unexposed to DM will identify novel targets within each cell type as well as the impact of the DM exposure on target selection. These targets will then be examined by Western blot analysis. Understanding the biological and metabolic pathways altered by miRNAs will further elucidate their impact on cellular metabolism and increased risk of cardiometabolic complications, including obesity and diabetes, in youth exposed to DM. Understanding these alterations will be key to prevention.