ORF29-02 - Quantifying the Protein Abundance and Circadian Rhythm of Nuclear Receptors in Metabolic Tissues by Mass Spectrometry

Objective: Nuclear receptors (NRs) play pivotal roles in regulating endocrine function. Circadian rhythm disruption has been shown to be closely associated with metabolic dysregulation and activation of inflammatory pathways. Comprehensive quantitative analyses of circadian expression of NRs at the protein level remain limited. We aimed to use an LC‑MS/MS assay to quantitatively analyze NRs in multiple metabolism-related tissues and to examine their circadian rhythms.

Methods: Nuclear proteins were extracted from eye, brain (whole brain or cerebral cortex, cerebellum, and hippocampus), inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), ileum, adrenal, kidney, testis, and ovary of C57BL/6 mice at ZT8. Samples from liver, cerebral cortex and iWAT of both sexes were collected at six time points (ZT0, ZT4, ZT8, ZT12, ZT16 and ZT20). After protein digestion and peptide desalting, endogenous NRs were detected using an Easy nLC‐1200 system coupled to a Thermo QExactive HF mass spectrometer. Circadian rhythmicity was assessed using cosinor regression, with significance determined by Benjamini-Hochberg adjustment.

Results: We successfully identified a total of 39 distinct nuclear receptors (NRs) (out of 49 possible) across all tissues. The male liver at ZT20 contained the highest number of detectable NRs (18). The estimated rank order abundance of NRs in male liver is listed in descending order: RXRα, HNF4α, EAR2, RXRβ, PPARα, LXRα, TR4, TRβ, RORα, RXRg, FXRα, GR, PXR, LRH1, LXRβ, CAR, SHP, and PPARg. In female liver, CAR, EAR2, LRH1, PPARα, PXR, RORα, RXRα, RXRγ, and SHP were rhythmic (P < 0.05). The peak expression of LRH1 was at ZT0, SHP at ZT8, CAR at ZT12, and PPARα at ZT16. In male iWAT, ER and GR showed significant circadian rhythmicity (both P< 0.001) with peaks at ZT20 and ZT16, respectively. Meanwhile, in female iWAT, COUP-TFI (P=0.020), COUP-TFII (P=0.045), TR2 (P=0.008), TR4 (P=0.020), and RXRα (P < 0.001) were rhythmic. The peak expression of COUP-TFI and COUP-TFII occurred at ZT16 and ZT20, respectively. TR2 and TR4 showed a significant increase after their nadirs between ZT8–ZT12, and RXRα peaked at ZT20. In the male cerebral cortex, GR (P=0.006), TRα (P < 0.001), and TRβ (P=0.017) exhibited significant rhythmicity, with all of them peaking at ZT8. In contrast, although EAR2, GR, and RARβ showed significant differences across time points in the female cerebral cortex, no NRs remained rhythmic after adjusting for multiple comparisons.

Conclusion: LC-MS/MS quantification of NR protein abundance in these nine tissues brings us one step closer to establishing a proteome atlas of NRs. This protocol enables systematic analysis of the circadian rhythms of NRs, providing evidence for time-targeted therapeutic strategies and rhythm-related mechanistic insights into metabolic diseases.