Effect Of O-GlcNAcylation On Glucose Metabolism In Skeletal Muscle Of Intermittent Cold Treated Mice And Its Regulatory Mechanism

Cold environment is one of the unavoidable stressors for animals in northern regions,which can easily trigger cold stress in animals and thus damage animal health and growth performance,greatly hindering the development of livestock breeding in northern regions.Long-term low feeding temperature or chronic cold exposure has a negative impact on animal muscle characteristics and muscle quality.Skeletal muscle accounts for 40% to 60% of body weight and is an important tissue for nutrient storage and energy utilization.Under cold exposure,skeletal muscle maintains body temperature homeostasis by altering its metabolism and enhancing contractile thermogenesis.Therefore,the energy metabolism and morphological function of skeletal muscle play a crucial role in the health of the body.It has been shown that O-GlcNAcylation can be involved in the regulation of insulin sensitivity and maintenance of glucose metabolic homeostasis in skeletal muscle as well as mediating TCA cycle and mitochondrial biogenesis.It has also been shown that O-GlcNAcylation can regulate skeletal muscle contractile and structural properties and influence skeletal muscle growth and differentiation.Meanwhile,O-GlcNAcylation can act as "stress receptors" and "trophic sensors" in a variety of stress and metabolic regulation by dynamically modifying target proteins to broadly regulate a variety of biological processes,such as signal transduction,immunity,autophagy and proteasome activity.In addition,our group has found that the target proteins can be dynamically modified to regulate a wide range of biological processes,such as signal transduction,immunity,autophagy and proteasome activity,as well as apoptosis,to maintain normal cellular physiological functions and thus promote cell survival.In addition,our group found that skeletal muscle atrophy was observed in skeletal muscle conditional OGlcNAc transferase(OGT)knockout mice under chronic cold exposure.What is the relationship between energy metabolism,structural morphology and O-GlcNAcylation in skeletal muscle under chronic cold exposure,and how O-GlcNAcylation act as "stress receptors" and "nutrient sensors" in chronic cold exposure? "How do O-GlcNAcylation act as cytoprotective agents against skeletal muscle atrophy under chronic cold exposure? These questions are still a gap in the field of animal cold stress research.Therefore,this study aims to investigate the effects of O-GlcNAcylation on skeletal muscle energy metabolism and structural morphology under chronic cold exposure and.This study can not only reveal the molecular regulatory mechanism of O-GlcNAcylation on skeletal muscle energy metabolism and tissue morphology under cold exposure,but also provide a theoretical basis for the prevention and control of cold damage in animal husbandry while safeguarding animal health,improving meat quality and increasing economic benefits.At the same time,this study has strong regional characteristics and is of great theoretical and practical significance to human health and animal husbandry development in cold regions.To investigate the effects of O-GlcNAcylation on skeletal muscle energy metabolism and structural morphology under chronic cold exposure,we constructed skeletal muscle conditional Ogt knockout mice and gave Wild type mice and Ogt mKO mice 3 h of cold stimulation at 4°C for 1 w per day,collected blood and skeletal muscle samples and recorded body weight and wet mass of gastrocnemius and flounder muscles.Physiological,biochemical and immunological parameters in plasma and global O-GlcNAcylation and OGT and OGA expression in skeletal muscle tissues were measured in each group of mice;high and low speed running exercise tests and HE,Masson and Tunnel staining and scanning and transmission electron microscopy were performed in skeletal muscle tissues of each group of mice,and MDA content and total antioxidant capacity were determined.Glucose and insulin tolerance tests were performed and fasting blood glucose and insulin levels were measured in each group of mice;glycogen,ATP and UDP-GlcNAc contents and AKT and its downstream glucose metabolism related proteins such as AS160 and GLUT4(key protein for glucose transport),GSK3β and GS(key protein for glycogen synthesis)and PFKFB2(key protein for glycolysis)were measured in each group of mice.mTOR,AMPK and its downstream autophagy-related proteins such as ULK1(autophagy initiation key protein),Beclin1(autophagy effect key protein),ATG5(autophagosome formation key protein),SQSTM1(autophagy degradation key protein),LC3(autophagy marker key protein)expression and global ubiquitin modifications in skeletal muscle tissues of all groups of mice.The results showed that under chronic cold exposure conditions,Ogt mKO mice showed different degrees of suppression of liver and kidney function and immune function,and a significant decrease in the wet mass ratio of gastrocnemius and flounder muscles as well as motility compared to Wild type mice(P<0.05).The muscle fiber disorganization,wrinkling and vacuolation degeneration,as well as the positive decrease in muscle fiber cross-section(P<0.001),the highly significant increase in fibrosis and apoptosis(P<0.001),the highly significant increase in MDA content(P<0.001)and the significant decrease in total antioxidant capacity(P<0.05),no significant change in fasting glucose and a significant increase in insulin(P<0.05)and The insulin resistance index increased significantly(P<0.001)and insulin sensitivity index decreased significantly(P<0.01),UDP-GlcNAc content increased significantly(P<0.001)and ATP and glycogen content decreased significantly(P<0.05),AKT activation and downstream GLUT4,AS160,GSK3β expression were enhanced while GS and PFKFB2 expression were mTOR inhibition and AMPK activation and its downstream autophagy-related proteins ULK1,Beclin1,ATG5 and LC3II/LC3Ⅰ levels were significantly increased while SQSTM1 was significantly decreased,and Atroginl,MuRF1 and Myogenin expression was increased(P<0.05)while global ubiquitination levels were significantly decreased(P<0.01),while For the first time,SQSTM1 was found to interact with OGT and thus undergo O-GlcNAcylation in skeletal muscle.This part of the study showed that skeletal muscle Ogt deletion caused systemic insulin resistance and glucose metabolic homeostasis imbalance in intermittent cold treatment mice,activated autophagy flux and ubiquitin degradation,thereby disrupting skeletal muscle morphological structure and motor function in mice and inducing atrophy.In order to further investigate whether O-GlcNAcylation can act as a "trophic sensor" in response to changes in energy status and regulate autophagic fluxes under acute cold exposure,this study was conducted in C2C12 mouse adult myoblast cell line,and the cells were cultured in high and low glucose medium and subjected to mild hypothermia treatment at 32°C for 3 h,6h,9 h and 12 h.The global O-GlcNAcylation levels and OGT and OGA expressions as well as autophagy-related proteins such as ULK1,Beclin1,ATG5,SQSTM1,LC3II/LC3Ⅰ expressions were detected in each group of cells.The results showed that low glucose significantly inhibited the global O-GlcNAcylation level and OGT and OGA expression compared with high glucose cultured cells under mild hypothermia(P<0.01),and significantly enhanced ULK1,Beclin1,ATG5 expression and LC3II/LC3Ⅰ level while decreasing SQSTM1 expression(P<0.05).Subsequently,ATP,ADP,AMP and the residual glucose content in their medium were measured in cells treated with mild hypothermia for 3 h,6 h and 12 h under normal culture in this study.The results showed that the ATP/(ADP+AMP)levels and the residual glucose content in the medium showed a temporal decrease while ATP,ADP and AMP showed a temporal fluctuation under mild hypothermia.Finally,this study measured the changes of glucose metabolism histology in cells treated with mild hypothermia for 3 h under normal culture.The results showed that glycolysis and tricarboxylic acid cycle-related metabolites were increased to different degrees and c AMP was significantly lower in the subcooled cells compared to the control group at 37°C(P<0.01).This part of the study showed that mild hypothermia at 32℃ resulted in decreased energy levels,increased glucose consumption and enhanced glycolysis in C2C12 cells,while O-GlcNAcylation could sense and respond to glucose availability and regulate autophagy flux in C2C12 cells.To further investigate whether the O-GlcNAcylation could act as a "stress receptor" in response to acute cold exposure to maintain cellular energy homeostasis and inhibit autophagic flux and ubiquitin degradation,this study was conducted in a C2C12 mouse myogenic cell line,which was mild hypothermia at 32℃ for 3 h and treated with Thiamet G(OGA inhibitor)and Alloxan(OGT inhibitor)were used to enhance and attenuate the global O-GlcNAcylation in C2C12 cells,to detect the global O-GlcNAcylation and OGT and OGA expression in each group of cells,to detect GLUT4 expression and membrane translocation and glycolytic flux in each group of cells,and to detect autophagy-related proteins such as ubiquitin degradation in each group of cells.The results showed that mild hypothermia induced upregulation of global O-GlcNAcylation,and further enhancement of this upregulation significantly promoted GLUT4 expression and membrane translocation in cells and enhanced cellular glycolytic flux.In contrast,inhibition of global O-GlcNAcylation at mild hypothermia significantly increased the expression of autophagic flux and ubiquitin degradation-related proteins.Meanwhile,the present study again validated the O-GlcNAcylation of SQSTM1.This part of the study showed that mild hypothermia induced enhanced O-GlcNAcylation promoted glucose uptake and glycolysis in C2C12 cells and inhibited autophagy flux and ubiquitin degradationIn conclusion,skeletal muscle Ogt deletion caused systemic insulin resistance and glucose metabolic homeostasis imbalance in intermittent cold treatment mice,activated autophagy flux and ubiquitin degradation,which damaged skeletal muscle morphological structure and motor function.Mild hypothermia at 32℃ induced decreased energy levels and increased glucose consumption and glycolysis,while O-GlcNAcylation induced glucose availability and regulated autophagy flux.Mild hypothermia at 32℃ induced the enhancement of O-GlcNAcylation to promote glucose uptake and glycolysis and inhibit autophagy flux and ubiquitin degradation,thus achieving cell protection.