O-GlcNAcylation Regulates Mitochondrial Quality Control In Cold Stimulated Skeletal Muscle Of Mice Through SIRT1

O-GlcNAcylation is considered to be a“nutritional and stress sensor”that is widely involved in biological processes of signal transduction,transcription,translation,cell division,metabolism and stress sensitivity in cells.Our team’s previous results showed that O-GlcNAcylation modification,as a“nutrient and stress receptor”during cold stress,has regulatory effects on skeletal muscle glucose metabolism,insulin sensitivity and apoptosis.Moreover,the loss of O-GlcNAcylation signal will directly interfere with glucose metabolism and mitochondrial quality control.However,the regulatory mechanism of O-GlcNAcylation modification on mitochondrial quality control remains unclear.This study targeted SIRT1 to explore the mechanism of O-GlcNAcylation modification in the quality control process of skeletal muscle mitochondria.To elucidate the role of O-GlcNAcylation modification in the regulation of skeletal muscle mitochondrial quality.To clarify the effect of O-GlcNAcylation modification on the regulation of skeletal muscle mitochondrial quality and identify its target.To study and reveal the mechanism of O-GlcNAcylation modification regulating skeletal muscle mitochondrial quality control through target proteins.In this study,the skeletal muscle tissues of wild type（WT）mice and skeletal muscle-specific knockout Ogt（Ogt m KO）mice were observed by electron microscopy and histological staining.The results showed that compared with WT mice,the skeletal muscle of Ogt m KO mice showed abnormal skeletal muscle morphological structure and mitochondrial structure.At the same time,compared with WT mice,the expression level of SIRT1 in skeletal muscle of Ogt m KO mice was significantly down-regulated.In order to further explore the mechanism of SIRT1 in the regulation of skeletal muscle mitochondria in Ogt m KO mice,this study gave mice cold stimulation treatment to make the mice in a state of energy mobilization.The experimental results showed that the absence of O-GlcNAcylation signal in mouse skeletal muscle resulted in abnormal skeletal muscle morphology and structure,mitochondrial ridge breakage,significantly upregulated expression levels of autophagy related proteins,mitophagy related proteins,and mitochondrial division/fusion proteins,and abnormal mitochondrial function and other mitochondrial quality regulation phenomena.Moreover,cold stimulation accelerated the abnormal regulation of mitochondrial quality induced by loss of O-GlcNAcylation.At the same time,the expression of SIRT1 is inhibited.However,whether O-GlcNAcylation directly acts on SIRT1 to participate in the above process is unclear.Next,this study took C2C12 cells as experimental objects in vitro and treated them with mild low temperature at 32℃to make them in a state of energy mobilization,so as to explore the above mechanism.The experimental results showed that mild hypothermia caused significantly up-regulated expression levels of autophagy and mitophagy related proteins in C2C12 cells,as well as abnormalities in mitochondrial structure and function.However,inhibition of O-GlcNAcylation signal aggravated the abnormal regulation of mitochondrial quality in C2C12 cells.The above abnormal regulation of mitochondrial quality has been significantly alleviated and improved.However,the downstream target protein of O-GlcNAcylation modification at mild hypothermia remains unclear.In this study,SIRT1 expression level and O-GlcNAcylation modification of SIRT1 were detected in C2C12 cells under mild hypothermia.The results showed that compared with the control group,NAD⁺content,SIRT1 enzyme activity,Sirt 1 m RNA and Sirt1 protein expression levels of C2C12 cells in mild hypothermia group were significantly down-regulated.After the O-GlcNAcylation was inhibited,the above indexes were further decreased,while the enhanced O-GlcNAcylation signal was significantly increased.Immunocoprecipitate assay,metabolic labeling assay and s WGA assay showed that endogenous SIRT1 in C2C12 cells interacts with OGT.The results of co-incubation of SIRT1-HIS recombinant protein and OGT-GST recombinant protein showed that exogenous SIRT1 also interacted with OGT.Yin OYang 1.2 system prediction results showed that SIRT1 could undergo O-GlcNAcylation at sites 160（Thr）and 161（Ser）.In this study,SIRT1 wild-type plasmid and SIRT1-modified site mutant plasmid were constructed and transfected into C2C12 cells,respectively,to evaluate the regulatory mechanism of SIRT1 on skeletal muscle cells at low temperature.The results showed that mitochondrial dysfunction and oxidative stress were significantly alleviated in C2C12 cells after SIRT1 overexpression,and the expression levels of autophagy related proteins,mitophagy related proteins,and mitochondrial division/fusion related proteins were significantly down-regulated.Transfection of SIRT1-E2^mut-AAplasmid did not alleviate the above abnormal phenomenon in C2C12 cells caused by mild hypothermia.This study combined in vivo and in vitro experiments to explore the regulatory mechanism of O-GlcNAcylation modification in the quality control process of mouse skeletal muscle mitochondria with SIRT1 as the target.In vivo experiments showed that Ogt gene knockout mediated loss of O-GlcNAcylation signal in mouse skeletal muscle resulted in increased fibrosis and senescence,decreased glycogen accumulation,significantly decreased mitochondrial function,increased autophagy and mitophagy,and increased oxidative stress level through inhibition of SIRT1 expression.The above phenomenon of skeletal muscle of Ogt m KO mice was more serious under the condition of cold stimulation.The results of in vitro experiments showed that the mitochondrial structure and function of C2C12 cells were abnormal under mild hypothermia conditions,and the levels of autophagy,mitophagy and oxidative stress were significantly up-regulated.However,inhibition of O-GlcNAcylation signal accelerated the mitochondrial structure and function abnormalities induced by mild hypothermia in C2C12 cells,and enhancement of O-GlcNAcylation signal improved the mitochondrial structure and function abnormalities in C2C12 cells.At mild hypothermia,the m RNA expression level of Sirt 1,the protein expression level of SIRT1 and the deacetylation activity of SIRT1 were significantly down-regulated.Inhibition of O-GlcNAcylation signal further down-regulated the expression level and deacetylation activity of SIRT1,while enhancement of O-GlcNAylation signal up-regulated the expression level and deacetylation activity of SIRT1.Overexpression of SIRT1 improved the mitochondrial dysfunction of C2C12 cells caused by mild hypothermia,and the deletion of O-GlcNAcylation in SIRT1 at Thr¹⁶⁰and Ser¹⁶¹could not improve the above phenomenon.In summary,this study elucidation the role and mechanism of O-GlcNAcylation modification in regulating skeletal muscle mitochondrial quality control through Thr¹⁶⁰and Ser¹⁶¹sites of SIRT1,providing a theoretical basis for revealing biological environmental adaptability.To determine the target of O-GlcNAcylation modification in the regulation of skeletal muscle mitochondrial quality,and provide a new intervention target and strategy for the prevention and treatment of diseases.