1G). When incubated in presence of radiolabeled glucose, we also saw a significant reduction in newly synthesized triacylglycerol but not in phospholipids (Fig. 1H). These results suggest that impairing biosynthesis of miRNAs and action of miRNAs negatively influences adipocyte lipid production. Microarray more info analysis of miRNAs in preadipocytes and adipocytes. Given our results showing an impairment in adipocyte lipid synthesis following alterations in global miRNA processing, we next sought to identify specific miRNAs that modulate adipocyte function. To screen for miRNAs that were differentially regulated during adipocyte differentiation, we analyzed two different experimental systems: the 3T3-L1 preadipocyte line and the ST2 mesenchymal precursor line.

Both cell lines can be efficiently induced to undergo adipocyte differentiation. The Ambion miRNA probe set was spotted on Nexterion glass slides and hybridized with small RNA fractions isolated from 3T3-L1 and ST2 preadipocytes and differentiated adipocytes. MiRNAs whose expression decreases during the transition between 3T3-L1 preadipocytes and adipocytes are ranked in Fig. 2A. By Northern blot, we confirmed that miRNA-199b (Fig. 2B), miRNA-197 (data not shown), and miRNA-34b (Fig. 2B) are downregulated during adipocyte differentiation. In contrast, miRNAs induced during adipogenesis were not as numerous. The most upregulated miRNAs were miRNA-378/378*, let-7c, miRNA-103, miRNA-107, and miRNA-210 (Fig. 2C). We confirmed by Northern blots using preadipocyte and adipocyte RNAs from two different cell lines (3T3-L1 and ST2) that miRNA-378/378*, miRNA-10a, miRNA-103, and miRNA-107 are induced during adipogenesis (Fig.

2D). Fig. 2. Regulated expression of micro-RNAs (miRNAs) during adipocyte differentiation. A: downregulated miRNAs during 3T3-L1 cell differentiation. The table shows the ratio of the level of expression in adipocytes vs. preadipocytes, as well as the coefficient … To establish whether some of these miRNAs might play a role in adipocyte differentiation, we generated retroviral vectors that contain an ~500-bp genomic region encompassing the indicated miRNAs. In one instance, inhibition of proliferation by miRNA34bc overexpression hampered its analysis during adipogenesis (1). Morphological changes such as increased lipid droplet size during adipocyte differentiation were only observed on overexpression of miRNA 378/378*.

We therefore decided to focus our further analysis on these miRNAs. Induction of miRNA378/378* during adipogenesis. Among the miRNAs identified as regulated during preadipocyte differentiation, miRNA378/378* was of particular interest due to its peculiar genomic localization, since these miRNAs Drug_discovery are localized in the first intron of PGC-1�� (Fig. 3A, indicated by the filled square) and are highly conserved between species.