Studies On Mechanisms Of The Role And Celastrol’s Regulation Of Skeletal Muscle OGT In Mouse Model Of High-fat Obesity

The present study,using in vivo experimental evaluation as well as cellular and molecular biology techniques and methods including animal behavior experiment,metabolic cage analysis,insulin and glucose tolerance test,Western blotting,enzyme-linked immunosorbent assay（ELISA）,cell culture,RNA interference,immunofluorescence labeling,GFP-LC3 staining,intracellular ROS imaging,and assay for glucose uptake capacity,ATP content and citric acid synthase activity,etc.,and employing the OGT skeletal muscle specific knockout（OGT m KO）C57BL/6mice,C2C12 cells as experimental subjects,systematically studied the regulatory effect of celastrol and its relation to OGT function activity in the models of high-fat diet-induced obesity（DIO）mice and sodium palmitate-induced C2C12 myotube cells.The detailed results were summarized as follows:1 Celastrol plays an action in controlling obesity via OGTF2 generation HSA^+/+/OGT^{Lox P/Y} wild-type（OGT WT）and HSA^Cre/+/OGT^{Lox P/Y}muscle-specific OGT knockout（OGT m KO）male mice were obtained by breeding HSA^Cre/+/OGT^+/Y male mice and HSA^+/+/OGT^{Lox P/Lox P} female mice.After grow and developed to 4 weeks of age,the mice were fed with high-fat diet for 3 months to establish the mouse models of high-fat diet-induced obesity（DIO）/insulin resistance.Then,the mice were intraperitoneally injected with celastrol daily for 21 days,followed by behavioral experiment,metabolic cage analysis,insulin and glucose tolerance test,tissue immunohistochemistry and Western blotting.The results showed that celastrol failed to relieve obesity,insulin resistance,muscular endurance,grip strength,metabolic function and muscle mass in DIO OGT m KO mice,suggesting that celastrol plays an action in controlling obesity via OGT.2 Celastrol’s amelioration of insulin resistance and restoration of mitochondrial function is closely related to OGT function activity in high-fat induction of myotube cellsC2C12 myoblast cells or C2C12 myoblast cells infected with shRNA OGT,shRNA GFP（as control）,FLAG-OGT or EGFP control（as control）,respectively,were cultured in medium with 2%horse serum for 5 days to form multinucleated C2C12 myotube cells.Subsequently,the cells were starved in serum-free medium for24 h and then treated with sodium palmitate for 48 h to establish a cellular model of high-fat.For the experiment,the indicated cells were treated with celastrol for 6 h and then with insulin for 0.5 h.After that,the glucose uptake capacity,ATP content and citrate synthase activity in the cells were analyzed,and the expressions of insulin/glucose transporter signaling pathway-and inflammation-related proteins were detected by Western blotting.The results showed that celastrol significantly reversed insulin resistance and restored mitochondrial function in sodium palmitate-induced C2C12 myotube cells.Celastrol had almost no effects on the events in OGT-downregulated C2C12 myotube cells induced by sodium palmitate.However,celastrol exerts a stronger effect on that in OGT-overexpressed C2C12 myotube cells induced by sodium palmitate.The data imply that celastrol’s amelioration of insulin resistance and restoration of mitochondrial function is closely related to OGT function activity in high-fat induction of C2C12 myotube cells.3 Celastrol regulates cell atrophy of insulin resistance in high-fat induction of myotube cells by an OGT-dependent mechanismC2C12 myoblast cells or C2C12 myoblast cells infected with shRNA OGT,shRNA GFP（as control）,FLAG-OGT or EGFP control（as control）,respectively,were cultured in medium with 2%horse serum for 5 days to form multinucleated C2C12 myotube cells.Subsequently,the cells were starved in serum-free medium for24 h and then treated with sodium palmitate for 48 h to establish a cellular model of high-fat.After the cells were treated with celastrol for 6 h and then insulin for 0.5 h,cellular morphology was photographed,cellular diameter and reactive oxygen species（ROS）level were detected,autophagosomes were labeled using GFP-LC3 staining,MHC expression was analyzed using immunofluorescence,and the expressions of related proteins were determined by Western blotting.The results showed that cellullar atrophy was blocked by celastrol in sodium palmitate-induced C2C12myotube cells.The cell atrophy was not restored by celastrol in OGT-downregulated C2C12 myoduct cells induced by sodium palmitate.The more serious atrophy was powerfully reversed by celastrol in OGT-overexpressed C2C12 myoduct cells induced by sodium palmitate.The findings suggest that celastrol regulates cell atrophy of insulin resistance in high-fat induction of myotube cells by an OGT-dependent mechanism.