Insulin-Induced Serine 22 Phosphorylation of Retinoid X Receptor Alpha Is Dispensable For Adipogenesis in Brown Adipocytes

Insulin action initiates a series of phosphorylation events regulating cellular differentiation, growth and metabolism. We have previously discovered, in a mass spectrometry-based phosphoproteomic study, that insulin/IGF-1 signaling induces phosphorylation of retinoid x receptor alpha (RXRa) at S22 in mouse brown pre-adipocytes. Here, we show that insulin induces the phosphorylation of RXRa at S22 in both brown precursor and mature adipocytes through a pathway involving ERK, downstream of IRS-1 and -2. We also found that RXRa S22 phosphorylation is promoted by insulin and upon re-feeding in brown adipose tissue in vivo, and that insulin-stimulated S22 phosphorylation of RXRa is dampened by diet-induced obesity. We used Rxra knockout cells re-expressing wild type (WT) or S22A non-phosphorylatable forms of RXRa to further characterize the role of S22 in brown adipocytes. Knockout of Rxra in brown pre-adipocytes resulted in decreased lipid accumulation and adipogenic gene expression during differentiation, and re-expression of RxraWT alleviated these effects. However, we observed no significant difference in cells re-expressing the RxraS22A mutant as compared with the cells re-expressing RxraWT. Furthermore, comparison of gene expression during adipogenesis in the WT and S22A re-expressing cells by RNA sequencing revealed similar transcriptomic profiles. Thus, our data propose a dispensable role for RXRa S22 phosphorylation in adipogenesis and transcription in differentiating brown pre-adipocytes. Overall design: 72 samples. 2 genotypes (WT and S22A), 2 cell cultures (biological replicate). For each biological replicate 3 timepoints (D0, D2, D6), and 3 technical replicate.