The FoxO1-ATGL axis alters milk lipolysis homeostasis through PI3K/AKT signaling pathway in dairy goat mammary epithelial cells

Abstract

Abstract Goat milk is enriched in fatty acids which are beneficial to human health. Previous research has revealed that 98% of milk fat is composed of triglycerides. However, the mechanisms regulating milk fat composition remain unclear. Forkhead box protein O1 (FoxO1) is a crucial regulatory factor involved in lipid metabolism across various cell types. Chromatin immunoprecipitation sequencing (ChIP)—seq data) and RNA sequencing (RNA-seq) data revealed that have indicated a close association between FoxO1 was closely related to lipid metabolism during lactation in dairy goats. The objective of this study was to investigate the mechanisms by which FoxO1 regulates lipid metabolism in goat mammary epithelial cells (GMECs). FoxO1 knockdown significantly downregulated the expression of adipose triglyceride lipase (ATGL) and suppressed the activity of the ATGL promoter. Consistently, the number of lipid droplets decreased significantly in FoxO1-overexpressing cells and increased in ATGL-knockdown cells. To further verify the effect of FoxO1 on ATGL promoter activity, cells were transfected with four promoter fragments of different lengths. We found that the core region of the ATGL promoter was located between −882 bp and −524 bp, encompassing two FoxO1 binding sites (FKH1 and FKH2). Mutations in the FoxO1 binding sites significantly downregulated ATGL promoter activity in GMECs. Luciferase reporter assays demonstrated that FoxO1 overexpression markedly enhanced ATGL promoter activity. Furthermore, site-directed mutation confirmed that FKH1 and FKH2 sites were simultaneously mutated significantly attenuated the stimulatory effect of FoxO1 on ATGL promoter activities simultaneous mutation of FKH1 and FKH2 sites significantly attenuated the stimulatory effect of FoxO1 on ATGL promoter activity. ChIP assays showed that FoxO1 directly binds to the FKH2 element located in the ATGL promoter in vivo. Finally, immunofluorescence staining revealed that insulin promotes the translocation of FoxO1 from the nucleus to the cytoplasm, thereby attenuating the FoxO1-induced activation of the ATGL promoter. Collectively, these findings uncover a novel pathway where by FoxO1 may regulate lipid metabolism in GMECs specifically by modulating the transcriptional activity of ATGL.