Abstract
ABSTRACT 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 signalling induces phosphorylation of retinoid x receptor alpha (RXRα) at S22 in mouse brown pre-adipocytes. Here, we show that insulin induces the phosphorylation of RXRα at S22 in both brown precursor and mature adipocytes through a pathway involving ERK, downstream of IRS-1 and −2. We also found that RXRα S22 phosphorylation is promoted by insulin and upon re-feeding in brown adipose tissue in vivo, and that insulin-stimulated S22 phosphorylation of RXRα is dampened by diet-induced obesity. We used Rxra knockout cells re-expressing wild type (WT) or S22A non-phosphorylatable forms of RXRα 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 RXRα S22 phosphorylation in adipogenesis and transcription in differentiating brown pre-adipocytes.
Highlights
Insulin action in adipose tissue is one of the most potent signals regulating cell metabolism and differentiation [1]
To test whether RXRα phosphorylation on serine 22 (S22), detected in our phosphoproteomic study, occurs in response to insulin, we applied an antibody directed against phosphorylated-S22 RXRα for protein analysis using protein lysates from brown preadipocytes stimulated with insulin or vehicle
Using a newly generated antibody raised against the phosphorylated form of RXRα, we show that RXRα phosphorylation on S22 occurs in brown precursor and mature adipocytes in response to insulin or insulin-like growth factor 1 (IGF-1)
Summary
Insulin action in adipose tissue is one of the most potent signals regulating cell metabolism and differentiation [1]. The RXRα S22 phosphorylation site is located in an autonomous, ligandindependent transcriptional activation domain in the N-terminal A/B region of RXRα [5] This domain is called ‘activation function 1’ (AF-1) and is required for the transduction of retinoic acid signals during development [6]. It has been reported that RXRα constitutive (without ligand) phosphorylation on S22 is required for the anti-proliferative effect of retinoic acid and induction of several retinoic acid-responsive genes [7]. Phosphorylation at this site is the most commonly detected post-translational modification of RXRα in high-throughput studies in mouse, human and rat [8]. The physiological relevance and impact of this phosphorylation event in adipocytes have not been investigated
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
