Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
College of Animal Science and Technology, Anhui Province Key Laboratory of Local Livestock and Poultry Genetic Resource Conservation and Bio-breeding, Anhui Agricultural University, Hefei 230036, China.
J Anim Sci. 2023 Jan 3;101. doi: 10.1093/jas/skad286.
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.
羊奶富含对人体健康有益的脂肪酸。先前的研究表明，98%的乳脂由三酰基甘油组成。然而，调节乳脂组成的机制尚不清楚。叉头框蛋白 O1 (FoxO1) 是一种关键的调节因子，涉及各种细胞类型的脂质代谢。染色质免疫沉淀测序 (ChIP-seq) 和 RNA 测序 (RNA-seq) 数据表明，FoxO1 与乳脂代谢密切相关，提示其与乳脂代谢密切相关。在乳用山羊泌乳期间。本研究旨在探讨 FoxO1 调节山羊乳腺上皮细胞 (GMEC) 脂质代谢的机制。FoxO1 敲低显著下调脂肪甘油三酯脂肪酶 (ATGL) 的表达，并抑制 ATGL 启动子的活性。一致地，FoxO1 过表达细胞中的脂滴数量显著减少，ATGL 敲低细胞中的脂滴数量增加。为了进一步验证 FoxO1 对 ATGL 启动子活性的影响，将细胞转染到不同长度的四个启动子片段中。我们发现 ATGL 启动子的核心区域位于-882 bp 和-524 bp 之间，包含两个 FoxO1 结合位点 (FKH1 和 FKH2)。FoxO1 结合位点的突变显著下调了 GMEC 中 ATGL 启动子活性。荧光素酶报告基因分析表明，FoxO1 过表达显著增强 ATGL 启动子活性。此外，定点突变证实 FKH1 和 FKH2 位点同时突变显著减弱了 FoxO1 对 ATGL 启动子活性的刺激作用。FKH1 和 FKH2 位点同时突变显著减弱了 FoxO1 对 ATGL 启动子活性的刺激作用。ChIP 分析表明，FoxO1 在内体中直接结合到位于 ATGL 启动子上的 FKH2 元件。最后，免疫荧光染色显示，胰岛素促进 FoxO1 从细胞核转位到细胞质，从而减弱 FoxO1 诱导的 ATGL 启动子激活。总之，这些发现揭示了一种新的途径，FoxO1 可能通过调节 ATGL 的转录活性来调节 GMEC 中的脂质代谢。
