Mechanism Of The Effect Of O-GlcNAc Glycosylation On The Expression Of IL-6 In Skeletal Muscle Of Mice Under Cold Exposure

Climate environment is one of the important factors that affect the production and development of animal husbandry.However,cold stress is easily induced by long winter and low temperature,which leads to abnormal energy metabolism,neuroendocrine,immunity and behavior in cold regions of northern China.As one of the important energy storage and utilization organizations,skeletal muscle can maintain the body’s movement and heat production in low temperature environment.In addition,as a secretory organ,skeletal muscle secretes a variety of muscle cytokines in response to environmental and metabolic changes.The IL-6secreted by skeletal muscle can participate in glucose and lipid metabolism of skeletal muscle.At the same time,O-GlcNAcylation mediated by OGT and OGA is a highly dynamic type of protein post-translational modification,which plays a role of "stress and nutrient receptor" in the process of stress and metabolism,thus maintaining the normal physiological function of cells.Therefore,the purpose of this study is to clarify the changes of IL-6 expression in the skeletal muscle of mice under acute cold exposure,to explore the potential relationship between O-GlcNAc ylation and skeletal muscle IL-6,and to clarify the sub mechanism of O-GlcNAcylation modification in the skeletal muscle of mice under acute cold exposure,and lay a theoretical foundation for further exploring the mechanism of cold stress molecular regulation.Firstly,the C2C12 cells were divided into control group(37℃)and cold exposure group(32℃ mild hypothermia treatment for 3 h,6 h,9 h,12 h)in this study.Western bolt was used to detect the expression of IL-6,OGT,OGA and O-GlcNAcylation were detected.Then primary skeletal muscle cells were isolated and treated with cold exposure model in vitro.The transcription levels of IL-6 and O-GlcNAcylation related enzymes were detected by qRT-PCR.Alloxan and TMG were used to regulate the O-GlcNAcylation,so as to verify the effect of O-GlcNAcylation modification on the expression of IL-6 under cold exposure.Western bolt was used to detect the expression of OGT and OGA protein,O-GlcNAcylation,IL-6 expression,p38-MAPK,JNK protein and its phosphorylation level,NF-κB signaling pathway protein and downstream signal protein expression.At the same time,the expression of GLUT-4 protein,AMPK,GS and phosphorylation in cytoplasm and cell membrane were detected.In addition,this study constructed skeletal muscle conditional OGT knockout mice,and used them as the research object for in vivo retrospective verification.WT and KO mice were divided into normal temperature control group and cold exposure group.The expression of IL-6,OGT,OGA,glycosylation of O-GlcNAcylation and the expression of NF-κB were detected by Western blot.The protein of skeletal muscle was treated with sWAG-Agarose,and the glycosylation level of p65 protein was detected by Western blot.The results showed that: compared with the normal temperature control group,the expression of OGT was significantly increased at 3 h of mild hypothermia(P < 0.05),and the expression of OGA was significantly increased at 6 h of mild hypothermia(P < 0.05).The glycosylation of O-GlcNAc and the expression of IL-6 were significantly increased under mild hypothermia for 3 h(P < 0.01),and the change of IL-6 expression under mild hypothermia was the same as that of O-GlcNAcylation at the same time.Therefore,an in vitro cold exposure model was established under mild hypothermia at 32℃ for 3 h.Inhibition of OGT significantly decreased the expression of IL-6(P < 0.05),and inhibition of OGA significantly increased the expression of IL-6(P < 0.05).At the same time,the expression of IL-1β and TNF-α increased,but the inhibition of OGT and OGA had no significant effect on the expression of IL-1β and TNF-α(P > 0.05).Cold exposure activated p38-MAPK/NF-κB pathway.meanwhile,after OGT was inhibited,p65 expression in cytoplasm and nucleus decreased(P < 0.05),and p65 expression increased(P < 0.05)after inhibition of OGA(P < 0.05),and significantly increased the nuclear displacement of p65(P < 0.05).In addition,the Western blot test showed that the O-GlcNAcylation of p65 protein in C2C12 was increased by the acute cold exposure,while the O-GlcNAcylation of p65 protein was also changed after OGT or OGA was inhibited.Wild type(WT)and skeletal muscle conditioned Ogt knockout(KO)mice were divided into normal temperature control group and cold exposure group.The results showed that the glycogen content of the skeletal muscle in mice was significantly lower than that in the normal temperature group under cold exposure,while the glycogen content of the skeletal muscle was significantly lower than that of wild type after the OGT gene was conditionally knocked out.The results of immunohistochemistry showed that the expression of IL-6 in skeletal muscle increased after cold exposure,and the expression of IL-6 in skeletal muscle of KO mice was less than WT.the expression of IL-6,OGT,OGA protein and O-GlcNAcylation in skeletal muscle of mice were significantly increased after cold exposure.NF-κB signaling pathway was activated,and KO mice were opposite.In addition,the Western blot results of s WAG-Agarose showed that the glycosylation level of p65 in skeletal muscle p65 of WT mice was significantly increased under cold exposure,while the O-GlcNAcylation of p65 in KO mice was significantly lower than that of WT mice.In conclusion,acute cold exposure can induce global O-GlcNAcylation and up-regulation of IL-6 expression in skeletal muscle of mice,and regulate glucose metabolism;OGT mediated p65 in acute cold exposure O-GlcNAcylation of subunit promoted p65 activity and nuclear translocation,resulting in up-regulation of downstream protein IL-6.