To identify the underlying mechanisms mediating the anti-adipogenic role played by ARS in TED-OFs, we used bulk RNA sequencing (RNAseq). At the beginning, we focused on identifying key signaling pathways that are differentially expressed during adipogenic differentiation of TED-OFs in vitro. There were 1398 upregulated genes and 1709 downregulated genes in the TED-OFs induced by DM compared to those in PM (
Supplementary Fig. S3A). As depicted in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, multiple regulatory signaling of differentially expressed genes (DEGs), such as proliferation (PI3K/AKT and cell cycle), and energy metabolism (AMPK and FOXO) were enriched in the DM group, apart from the specific adipogenic signaling, such as PPAR signaling and unsaturated fatty acid biosynthesis (see
Supplementary Fig. S3B). Using Protein Analysis Through Evolutionary Relationships (PANTHER) enrichment analysis, insulin/IGF1-protein kinase B (PKB or AKT) were observed in the DM group (see
Supplementary Fig. S3C). Compared with those in the DM treated group without ARS, a total of 86 upregulated DEGs and 74 downregulated DEGs were detected in TED-OFs in the DM treated group with ARS (
Fig. 6A). As analyzed by KEGG of DEGs, PPAR signaling and fatty acid metabolism were affected in the DM+ARS group (
Fig. 6B). Using PANTHER analysis, insulin/IGF1 cascade signaling were highlighted (
Fig. 6C). Furthermore, upregulated DEGs in the DM versus the PM data set and downregulated ones in the ARS versus the DM data set were compared via online Venn diagram tools (Venny version 2.1). As illustrated in
Figure 6D and
Supplementary Table S3, we identified 50 oppositely regulated DEGs. Similarly, we compared downregulated DEGs in the DM versus the PM data set and those upregulated by ARS to identify 19 oppositely regulated DEGs (see
Fig. 6D,
Supplementary Table S4). Given that the remarkable impacts of ARS on insulin/IGF1 signaling and fatty acid metabolism which are involved in adipogenesis and lipogenesis, we focused on IGF1R and stearoyl-CoA desaturase (SCD). As shown in
Figure 6E, the qPCR was performed to confirm that mRNA levels of IGF1R were suppressed by ARS, DHA, and ART in a concentration-dependent manner (ARS versus DMSO: 10 µM [
P = 0.0808], 50 µM [
P = 0.0269], 200 µM [
P = 0.0086]; ART versus DMSO: 0.5 µM [
P = 0.1578], 2 µM [
P = 0.0379], 10 µM [
P = 0.0235]; DHA versus DMSO: 1 µM [
P = 0.0527], 5 µM [
P = 0.0277], and 20 µM [
P = 0.0152]). Consistently, mRNA levels of SCD were suppressed by ARS, DHA, and ART concentration dependently. We perform Western blot analysis to analyze whether ARSs interfere with the signaling of PI3K-AKT-FOXO1 pathway, which is downstream of IGF1R. As expected, phosphorylation of AKT and FOXO1 was inhibited by ARS, DHA, and ART in TED-OFs instead of non-TED-OFs, confirmed by Western blot analysis (
Fig. 6F). It was suggested that the inhibitory effect of ARSs on TED-OFs was potentially mediated by the IGF1R-PI3K-AKT-FOXO1 pathway by decreasing IGF1R expression.