Effects Of Cellular Activity And Autophagy Lysosome Induced By Lipid Overloading In C2C12 Cells

Background and objective:Neutral lipid storage disease with myopathy(NLSDM)is a genetic disorder in which excessive and abnormal neutral lipid in the form of lipid droplets accumulate in muscle fibers due to a disorder in the process of lipids breakdown.The pathogenesis is caused by the mutation of patatin like phospholipase domain 2(PNPLA2)gene,which encodes triglyceride hydrolase(ATGL).ATGL is responsible for the first step of triglyceride hydrolysis and the rate-limiting enzyme of triglyceride hydrolysis.The catabolism of lipids includes lipolysis and lipophagy.In the physiological state,lipolysis is the most important lipid mobilization pathway.When the cells are in a state of stress or the normal adipose lipolysis pathway is blocked,the cells degrade fat through the lipid autophagy pathway.Lipid autophagy is degraded by macroautophagy.Autophagy substrates are eventually degraded by lysosomal acid hydrolytic enzymes.Lysosomal hydrolytic enzymes are most active at pH between 4 and 5,while the acid environment in the lysosomal is maintained by vacuolar-type H+-atpase(v-atpase).Research shows ATGL defects within the muscle cells of triglycerides cannot lipolysis.And with the development of the course,the storage of lipid deposition also cannot effectively degrade by lipid autophagy pathway,it may caused by the decrease in the ability of lysosome hydrolysis.Then the cells turn into apoptosis pathway.In order to further explore the influence of neutral lipids metabolism disorder in skeletal muscle cells,we use Mouse myoblasts C2C12 to lipid overload experiment.And we use the ATGL siRNA interfere C2C12 cells at the same time.We simulate part of the NLSDM pathological process to observe the survival rate of C2C12 cells as well as the function of lysosomes in the cell to investigate the possible pathogenesis.Materials and methods:mouse myoblasts C2C12 used for the lipid overload experiment.Cells were treated with different concentrations of medium containing oleic acid and palmitic acid.Flow cytometry was used to detect the fluorescence intensity changes after lipid overload.Protein western blot was used to detect the change of ATGL protein expression.The number and size of intracellular lipid droplets were detected by bodipy 493/503 staining.CCK8 was used to detect cell activity.Immunofluorescence staining was used to detect the expression of autophagy and apoptosis-related proteins in cells.The activity of V-ATPase was detected by fluorescence quantitative PCR.Lysosensor yellow/blue was used to detects lysosomal acidity changes.Results:1.Treatment with medium containing oleic acid or palmitic acid resulted in an increase in the number and volume of intracellular lipid droplets,and the increase of lipid droplets in the oleic acid treatment group was more than that in the palmitic acid treatment group at the same concentration.2.down-regulation of intracellular ATGL enzyme activity in C2C12 can increase intracellular lipid droplets,which is similar to the results of loading of oleic acid and palmitic acid in the culture medium.3.The survival rate of the cells treated with 3-MA showed no significant change at 18 hours,while the survival rate decreased significantly at 30 hours.The survival rate of cells treated with oleic acid decreased at 18 hours and 30 hours,and decreased more significant at 30 hours.Palmitate-treated cells showed no significant change in survival rate at 18 h and increased survival rate at 30 h.Cells treated with both oleic acid and palmitic acid showed no significant change in survival rate at 18 h and increased survival rate at 30 h.4.The survival rate of cells treated with siRNA was lower than that of normal cells when treated with oleic acid,palmitic acid and 3-ma for 24 hours.When the treatment time was 48 hours,the survival rate of siRNA interfering cells in the oleic acid-treated group was significantly lower than that of normal cells.There was no significant change in cell survival rate after treatment with palmitic acid for 48 hours.5.The binding of ATP6V1 A on lysosomes treated with oleic acid and palmitic acid was reduced.6.After treatment with oleic acid after 48 hours,the PH value of lysosome in C2C12 cells increased,and the acid environment of lysosome was damaged.7.Expression of autophagy and apoptosis-related proteins increased after treatment with oleic acid.Conclusion:1.the damage of oleic acid to the activity of C2C12 cells increased with time.Palmitic acid can promote cell survival rate and resist the decrease of cell survival rate caused by oleic acid.2.Oleic acid overload increased the autophagy level of C2C12 cells,but the autophagy degradation ability was impaired.3.Oleic acid overload reduces the assembly of V-ATPase V1 subunit on the lysosome,and decreases the acidity in the lysosome,resulting in decreased lysosomal degradation ability.4.oleic acid loading increased the apoptosis level of C2C12 cells.