The circadian clock repressor Cry2 promotes adipogenesis by suppressing Wnt signaling via interaction with Per2

Abstract

The circadian clock is driven by a transcriptional-translational feedback loop that elicits ~24 hour rhythms in behavior and physiology. The cell-autonomous clock exerts temporal regulation in adipocyte development and clock disruption leads to the development of obesity. There is current intense effort to identify clock-targeting interventions for metabolic disease therapy. Cryptochrome 2 (Cry2) is a circadian clock repressor, and it binds with Period proteins as a heterodimer complex to inhibit CLOCK/Bmal1-activated clock transcription that constitutes the negative feedback arm of the clock circuit. Our previous studies demonstrated that clock activator Bmal1 inhibits adipogenesis via the Wnt signaling pathway. To date, the role of Cry2 in adipogenic regulation remains unknown. Here we show that Cry2 inhibits adipogenesis and identify a critical residue that mediates interaction with Per2 required for this regulation. Cry2 transcript and protein are markedly induced during adipogenic differentiation. Via site-directed mutagenesis, we identified that Cysteine 432 within the lid domain of Cry2 protein is required for interaction with Per2, and a C432A mutant abolished this interaction. As a result, Cry2 repression activity of CLOCK/Bmal1-mediated transcription was abolished by the C432A mutant, as shown by Per2-luc reporter luciferase assay. Cry2 C432 mutation also led to elevated ubiquitination that accelerated protein degradation, an additional mechanism contributing to its loss of function. In 3T3L1 preadipocyte, we found that ectopic expression of Cry2 enhanced adipogenesis, whereas its loss-of-function suppressed adipocyte differentiation. In line with these findings, a Cry-stabilizing compound KL001 markedly augmented adipogenic differentiation. As compared to wild-type Cry2 effect on promoting adipogenesis, Cry2 C432A mutant largely abolished differentiation consistent with loss of repressive function in clock modulation. Mechanistically, we show that Cry2 induces adipogenesis via negative regulation of Wnt signaling pathway leading to inhibition of CCAAT/enhancer binding protein α (C/EBPα) expression. Together, our findings reveal a novel function of Cry2 in promoting adipocyte development via a critical residue involved in interaction with Per2. Our study thus provides a mechanistic basis for clock-targeting interventions to inhibit Cry2 activity for the treatment of obesity and its associated complications This work is supported by NIDDK grants 1R01DK112794, 1R01DK130499 and AR-DMRI 2023 T2D Innovation Grant. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.