APN/AdipoRon regulates luteal steroidogenesis through AMPK/EZH2/H3K27me3 in goats

AMPK plays a crucial role in cellular energy metabolism and is involved in the regulation of luteal steroidogenesis by APN and its analog AdipoRon. To further explore the regulatory mechanism of AMPK in goat luteal steroidogenesis mediated by APN, cyclic and pregnant CL were utilized to assess the localization and expression of AMPK, EZH2, H3K27me3 and H3K27ac by WB and mIHC, and the interaction between AMPK and EZH2 by Co-IP. Then, isolated luteal cells were treated with APN/AdipoRon to evaluate the expression levels of AMPK, EZH2, H3K27me3 and H3K27ac. Results showed that AMPK and EZH2 were co-localized to the cytoplasm of luteal cells, and interacted as detected by Co-IP. H3K27me3 and H3K27ac were localized to the nucleus of goat luteal cells. H3K27me3 expression in late CL was significantly higher than that in early and middle CL, while the expressions of AMPK, H3K27ac and EZH2 in middle CL were significantly higher than those in early and late CL. Notably, all these proteins were expressed at similar levels between pregnancy and middle cycle, with the exception of EZH2. Following incubation with AdipoRon (25 μM) and APN (1 μg/mL) for 24 h, the expressions of AMPK and H3K27ac decreased, while H3K27me3 increased in luteal cells. Compound C (AMPK activity inhibitor) reversed the AdipoRon - induced decrease in EZH2 expression and the increase in H3K27me3 expression. The increased H3K27me3 expression and decreased steroidogenic protein (CYP11A1 and HSD3B) expression after GSK126 (EZH2 inhibitor) treatment were consistent with the effects seen after AdipoRon treatment. In conclusion, APN/AdipoRon inhibits luteal steroidogenesis by inhibiting the interaction between AMPK and EZH2, thereby promoting H3K27me3 expression.