| Adipose tissue is also an important endocrine organ in the animal body.The adipokines secreted by adipose tissue can affect the metabolic function of some organs(such as skeletal muscle,liver and brain)through paracrine,autocrine and blood circulation.Importantly,the content and distribution of adipose tissue is closely related to the quality of meat in the producing animals.Therefore,it is of great significance to analyze the mechanism of lipogenesis for the genetic improvement of meat quality in animal husbandry.As a multifunctional transcription factor,forkhead box transcription factor O1(FoxO1)is involved in biological processes such as cell cycle control,antioxidant response,apoptosis,glucose metabolism,lipid metabolism,and tumor inhibition.In recent years,FoxO1,as a target factor for weight loss,has attracted much attention due to its important role in cell cycle control and adipocyte differentiation.However,the specific mechanism of FoxO1 on the proliferation differentiation and lipid metabolism of adipose cells is still not clear.In view of this,this dissertation took Guyuan cattle as the research object,and explored the regulatory effects and mechanisms of FoxO1 on the proliferation,differentiation and lipid metabolism of bovine adipocytes.The research aimed to clarify the mechanism of FoxO1 on bovine lipogenesis,and also provide genetic material for the genetic improvement of bovine meat quality.The main research results are as follows:(1)In this study,bovine adipocytes with high purity and differentiation potential were successfully isolated by tissue mass method.Subcellular localization showed that FoxO1 played an important biological function in the nucleus and cytoplasm of bovine adipocytes.Real-time quantitative PCR(qPCR)found that FoxO1 was highly expressed in adult cattle subcutaneous fat and perirenal fat,and its expression in adult cattle lower fat was significantly higher than that in calf(P<0.O1).In addition,the expression of FoxO1 was gradually increased during the proliferation of bovine adipocytes.Moreover,the expression of FoxO1 on the 2 d,4 d and 8 d of bovine adipocyte differentiation was significantly higher than that on the Oth day of differentiation(P<0.01).(2)FoxO1 inhibits the G1/S phase transformation of bovine adipocytes and further inhibits the proliferation of bovine adipocytes by negatively regulating the expression of proliferation marker genes(PCNA,CDK1,CDK2,CCNA2,CCNB1 and CCNE2)and proteins(PCNA,CDK2 and CCNE2).Moreover,while negatively regulating the proliferation of bovine adipocytes,FoxO1 promotes the expression of pro-apoptotic marker protein BAD,thereby promoting the early and late apoptosis of bovineadipocytes.In addition,FoxO1 increases the triglyceride(TG)content in bovine adipocytes and promotes the generation of lipid droplets by positively regulating the expression of adipogenic differentiation marker genes(PPARy and C/EBPα)and lipid anabolism marker genes(ACC,FASN,SCD1,SREBP1,FABP4,ACSL1,LPL and DGAT1).(3)The bovine adipose cells were transfected with targeted FoxO1 small interfering RNA(siRNA),and induced to differentiate into the 4 d for RNA-sequencing(RNA-seq)and non-target metabolism sequencing.In total,222 DEGs and 52 differential metabolites(28 in POS mode and 24 in NEG mode)were screened.Joint analysis showed that FoxO1 could regulate bovine lipogenesis by regulating the activities of AMPK and PI3K/AKT pathways.In addition,downstream targets of FoxO1 for the regulation of bovine lipogenesis,CD36 and STEAP4,were screened based on sequencing.(4)Chromatin immunoprecipitation(ChIP)and double luciferase reporting experiments showed that FoxO1 could regulate bovine lipogenesis by binding to CD36 and STEAP4 promoter regions and affecting the transcriptional activities of CD36 and STEAP4.In summary,we investigated the regulatory effects of FoxO1 on the proliferation,differentiation and lipid metabolism of bovine adipocytes,and identified downstream targets of FoxO1 for the regulation of bovine lipogenesis,CD36 and STEAP4.This study not only enriched the molecular network of lipogenesis,but also provided a theoretical basis for the genetic improvement of bovine meat quality. |
