Identification of novel genes determining early adipogenesis in 3T3-L1 cells using conditioned differentiation medium

Abstract

Confluent 3T3-L1 preadipocytes initiate differentiation into adipocytes when treated with 3-isobutyl-1-methylxanthine, dexamethasone, and insulin (MDI). After MDI induction, differentiating cells secrete various molecules into medium, and this medium is designated as conditioned medium (hereafter called CM). When CM was treated to 3T3-L1 preadipocytes, adipocyte differentiation was accelerated compared to when MDI alone was treated. In this regard, it was assumed that in the CM, there are some specific molecules secreted by adipocytes, which facilitate differentiation. On the basis of this assumption, this study was started to search for novel genes which are expected to promote 3T3-L1 differentiation. To investigate candidate genes, RNA sequencing was performed using cells treated with MDI or CM. The RNA sequencing data were filtered out and 51 candidate genes were selected which are highly expressed in CM compared with MDI. In sequence, through confirming actual gene expression in 3T3-L1 cells and distribution in mouse fat tissue, three genes, Serpina3c, Serpina3n, and Adamts15 were selected. The result from RNA interference study aimed at these three genes, Serpina3c was finally selected as a new molecule expected to be related with adipocyte differentiation. Serpina3c, serine protease inhibitor A3C, belongs to Serpin superfamily. In 3T3-L1 cells, knockdown of Serpina3c resulted in the inhibition of mitotic clonal expansion. In addition, Serpina3c-knockdown cells showed that integrin alpha 5 and ERK activation were decreased whereas AKT activation was increased. These results suggest that Serpina3c may play as a contributing factor in adipogenesis via IGF-1 signaling mediated by integrin alpha 5.