Mechanism Of Cold-induced O-GlcNAcylation Regulation Of Mouse Liver Lipid Metabolism

The cold stress caused by low temperature has seriously restricted the development of animal husbandry in China.Under cold stress,the body accelerates energy metabolism to resist the energy loss caused by cold stress.As a hub of lipid metabolism,the liver participates in the body’s energy supply under cold stress.O-GlcNAcylation is a post-translational modification(PTMs).And OGlcNAc mediated by OGT and OGA.As the "stress receptor" and "nutrition receptor" of the body,O-GlcNAcylation can realize the highly dynamic regulation of cell signals.Whether OGlcNAcylation is involved in cold-induced lipid metabolism in the liver.What is the mechanism of its regulation on liver lipid metabolism?This study aims to explore the role of O-GlcNAcylation in liver lipid metabolism under cold stress and its regulatory mechanism.To provide a reference for liver lipid metabolism under cold stress.Firstly,metabolic cage experiment was carried out on liver Ogt specific knockout mice(OgtLKO,KO)and wild type mice(Wild Type,WT).It was found that the daytime respiratory entropy of KO mice was significantly lower than that of WT mice(P<0.0001).Lipid consumption can reduce the level of respiratory entropy,indicating that OGT may affect the level of energy metabolism through liver lipid consumption.In order to further determine the effect of OGlcNAcylation on liver lipid metabolism under cold stress.Mice were exposed to chronic cold at 4℃,three hours a day for 21 days.The feed intake and weight gain of mice were detected during chronic cold exposure,and the blood and liver tissues of mice were collected after chronic cold exposure.Liver morphology,hepatic glycogen and lipid content were detected by HE,MASSON,PAS,oil red O staining;O-GlcNAcylation,lipid synthesis and lipid decomposition related proteins were detected by Western blot;lipid synthesis and lipid decomposition gene expression were detected by qRT-PCR.The results showed that the food intake and weight gain of KO mice were significantly higher than WT mice.Liver function index ALT(P<0.0001)and AST(P<0.001)were significantly increased and the liver shows slight fibrosis.The content of liver glycogen increased significantly(P<0.0001)and the lipid deposition in liver tissue increased significantly(P<0.01).The levels of O-GlcNAcylation-related proteins OGT,OGA,O-GlcNAc were significantly reduced(P<0.05),the expression levels of lipid synthesis-related proteins FASN,ACC1,SREBP1 were significantly increased(P<0.001),the expression levels of lipid breakdownrelated proteins CPT1,ACOX1,and PPARa were significantly reduced(P<0.001).It is suggested that liver Ogt gene specific knockout promotes lipid synthesis in liver and inhibits lipid decomposition.At the same time,liver injury and fibrosis occur.The food intake of WT mice increased under chronic cold exposure(P<0.001).The liver has vacuolar degeneration and slight fibrosis.The content of hepatic glycogen decreased significantly(P<0.01),but the content of lipid in liver had no significant change.The expression levels of O-GlcNAcylation-related proteins,lipid synthesis-related proteins,lipid decomposition-related proteins and genes increased significantly(P<0.05).Food intake and body weight gain of KO mice increased significantly under chronic cold exposure(P<0.0001),and AST increased more significantly(P<0.01).Liver tissue injury and fibrosis were further aggravated,liver glycogen content and liver tissue lipid deposition were significantly decreased(P<0.0001).The expression of O-GlcNAcylation-related proteins were significantly increased.The expression levels of proteins and genes related to lipid synthesis decreased(P<0.05).The expression levels of lipid decomposition-related proteins and genes increased(P<0.05).This shows that chronic cold exposure accelerates the body’s energy demand,cold induction inhibits fat transport in KO mice,further aggravates liver damage and fibrosis,and at the same time,the lipid metabolism of the liver increases with the increase of O-GlcNAcylation under cold induction;mass spectrometry and sWGA results show that the O-GlcNAcylation of ACOX1 is elevated under chronic cold exposure,indicating that the O-GlcNAcylation of ACOX1 under cold induction regulates the lipid decomposition of the liver.To further explore the regulatory mechanism of O-GlcNAcylation of ACOX1 in hepatocyte lipid decomposition,the hepatocyte hyperlipidemia model was established by using AML-12 mouse hepatocytes in vitro,and the best action time and concentration of OGT inhibitor alloxan(Alloxan)were selected to inhibit the expression of OGT and O-GlcNAcylation.The cells were divided into three groups:control group(NC),hyperlipidemia group(PA/OA),and hyperlipidemia combined with the Alloxan group(PA/OA+A).Oil red O staining,Western blot,and qRT-PCR were used to determine the changes of glycosylation modification and lipid synthesis and decomposition of O-GlcNAc in hepatocytes.The results showed that high lipid caused further aggravation of lipid deposition in hepatocytes under the action of Alloxan.Compared with the PA/OA group,the expression of lipolysis-related proteins and genes in the PA/OA+A group was lower than that in PA/OA group.It is further confirmed that the level of O-GlcNAcylation promotes lipid decomposition in the liver.sWGA results showed that inhibition of OGT expression could down-regulate the O-GlcNAcylation of ACOX1.Immunofluorescence and IP results showed that there was an interaction between ACOX1 and OGT in hepatocytes;Western blot results showed that the ubiquitin level of ACOX1 increased under high-fat conditions,especially at the K48 site,indicating that the interaction between OGT and ACOX1 regulated the glycosylation of O-GlcNAc,promoted the proteasome degradation of ACOX1,and regulated liver lipid decomposition.Conclusion:Cold-induced lipid decomposition in mouse liver is accelerated,and the increase of O-GlcNAcylation of lipid decomposing protein ACOX1 inhibits proteasome degradation at site K48 of ACOX1 and promotes lipid decomposition in mouse liver.