The Function And Mechanism Of FoxO1 Regulated By O-GlcNAcase

Background and Objectives:Metabolic syndrome(MS),a complex syndrome caused by energy utilization and storage disorders,increases the risk of various diseases such as nonalcoholic fatty liver,diabetes and cardiovascular and cerebrovascular diseases.Among them,the main clinical characteristics of Type 2 Diabetes(T2DM)is hyperglycemia caused by insulin deficiency or decreased sensitivity,whose prominent feature is the excessive activation of hepatic gluconeogenesis.Numerous studies have shown that O-GlcNAcylation can be used as a nutrient receptor to participate in a variety of metabolic processes,especially associateds with abnormal glucose metabolism.Previous studies have shown that FoxO1 can be regulated by O-GlcNAc glycosyltransferase(OGT),which is a key transcriptional factor of hepatic gluconeoge-nesis.However,the role and molecular mechanism of O-GlcNAc deglycosylation mediated by OGA on FoxO1 remains to be confirmed.Methods:For phenotypic studies: the cell lines model of OGA overexpression and knockdown in human normal liver cells was established by lentiviral infectio,and the effects of overexpression of OGA on hepatic gluconeogenesis were verified by qRT-PCR and Western blot studies.For the terms of mechanism studies:(1)the effect of OGA on FoxO1 transcriptional activity was studied through siRNA transfection;(2)the interaction between OGA and FoxO1 was studied through co-immunoprecipitation;(3)the regulation of nuclear translocation of FoxO1 was identified by OGA via immunofluorescence;(4)the effect of OGA mediated on FoxO1 stability was detected through the half-life experiment of proteins;(5)the effect of insulin pathway on OGA regulation of FoxO1 was studied by PI3K/Akt inhibitor treatment;(6)the effect of AMPK pathway on OGA regulation of FoxO1 was studied by AMPK inhibitor and siRNA interference.Results:1.The OGA overexpression and knockout cell lines of human normal hepatocytes were successfully constructed.2.Overexpression of OGA inhibits excessive activation of hepatic gluconeogenesis by down-regulating FoxO1 protein expression.3.OGA directly interacts with FoxO1 and up-regulate O-GlcNAcylation level of FoxO1.4.There is no relationship between negative regulation of FoxO1 mediated by OGA and its nuclear translocation.5.Overexpression of OGA promotes FoxO1 degradation and reduces its stability.6.O-GlcNAcase negatively regulates FoxO1 activity individually in an independent of insulin and AMPK pathways.Conclusion:O-GlcNAc hydrolase interacts with FoxO1 to decrease its protein stability and inhibits FoxO1 transcriptional activation of key gluconeogenesis genes.This study may suggest a new research sight for the prevention of diabetes.