College of Animal Science & Lingnan Guangdong Laboratory of Modern Agriculture, South China Agricultural University, Guangzhou 510642, China; Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, and Key Laboratory of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture, Guangzhou 510642, China.
College of Animal Science & Lingnan Guangdong Laboratory of Modern Agriculture, South China Agricultural University, Guangzhou 510642, China; Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, and Key Laboratory of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture, Guangzhou 510642, China.
Life Sci. 2021 Jan 15;265:118858. doi: 10.1016/j.lfs.2020.118858. Epub 2020 Dec 5.
Previous studies have shown that the forkhead transcription factor FoxO6 involved in memory consolidation and hepatic glucose homeostasis. Here we asked whether chicken FoxO6 may regulate preadipocyte proliferation, apoptosis and early adipogenesis.
Overexpression and knockdown of FoxO6 were performed and evaluated through cell proliferation methods, Oil-Red-O staining, and specific marker expression. Chromatin immunoprecipitation (ChIP) assay was performed to confirm cyclin G2 (CCNG2) as a direct target gene of FoxO6.
FoxO6 is ubiquitously expressed in different chicken tissues and highly expressed in liver, abdominal fat, and preadipocytes in cultured cell. FoxO6 overexpression decreased preadipocyte proliferation by causing G1-phase cell-cycle arrest, whereas inhibition of FoxO6 showed the opposite effects. Overexpression or knockdown of FoxO6 significantly altered the mRNA and protein levels of cell-cycle related markers, such as CCNG2, cyclin dependent kinase inhibitor 1B (CDKN1B), cyclin dependent kinase inhibitor 1A (CDKN1A) and cyclin D2 (CCND2). During preadipocyte proliferation, FoxO6 targets and induces expression of CCNG2, as confirmed by ChIP assay and qPCR. In addition, FoxO6 induces preadipocyte apoptosis through increasing the protein expression levels of cleaved caspase-3 and cleaved caspase-8. Moreover, FoxO6 at the early stage of adipogenesis suppressed mRNA and protein levels of the key early regulators of adipogenesis, such as PPARγ and C/EBPα.
The results demonstrate that FoxO6 controls preadipocyte proliferation, apoptosis and early adipogenesis, and point to new approaches for further studies related to obesity.
先前的研究表明，叉头转录因子 FoxO6 参与记忆巩固和肝葡萄糖稳态。在这里，我们询问鸡 FoxO6 是否可以调节前体脂肪细胞的增殖、凋亡和早期脂肪生成。
通过细胞增殖方法、油红-O 染色和特定标志物表达来进行 FoxO6 的过表达和敲低，并进行评估。进行染色质免疫沉淀（ChIP）实验以确认细胞周期蛋白 G2（CCNG2）是 FoxO6 的直接靶基因。
FoxO6 在不同的鸡组织中广泛表达，在培养细胞中的肝脏、腹部脂肪和前体脂肪细胞中高度表达。FoxO6 的过表达通过引起 G1 期细胞周期停滞来减少前体脂肪细胞的增殖，而 FoxO6 的抑制则表现出相反的效果。FoxO6 的过表达或敲低显著改变了细胞周期相关标志物（如 CCNG2、细胞周期蛋白依赖性激酶抑制剂 1B（CDKN1B）、细胞周期蛋白依赖性激酶抑制剂 1A（CDKN1A）和细胞周期蛋白 D2（CCND2）的 mRNA 和蛋白水平。在前体脂肪细胞增殖过程中，FoxO6 通过靶向并诱导 CCNG2 的表达，ChIP 实验和 qPCR 得到了证实。此外，FoxO6 通过增加 cleaved caspase-3 和 cleaved caspase-8 的蛋白表达水平诱导前体脂肪细胞凋亡。此外，FoxO6 在脂肪生成的早期阶段抑制脂肪生成的关键早期调节因子 PPARγ 和 C/EBPα 的 mRNA 和蛋白水平。
这些结果表明 FoxO6 控制前体脂肪细胞的增殖、凋亡和早期脂肪生成，并为进一步研究肥胖相关问题提供了新的方法。
