The Role Of SIRT3 In Metformin Improving Palmitic Acid-induced Autophagy And Oxidative Stress In Muscle Cells

Metformin is the first-line therapeutics for type 2 diabetes treatment,and its hypoglycemic mechanism may be related with inhibiting hepatic glucose output,improving insulin sensitivty and increasing glucose uptake and utilization.It is reported that metformin can alleviate oxidative stress and enhance autophagy.However,its exact mechanism remains unclear.This study is to investigate the mechanism of metformin improving lipid-induced oxidative stress and autophagy in L6 myoblasts,and provide the theoretical basis for clinical medication.Objectives: To explore the role of SIRT3 in metformin improving palmitic acid-induced autophagy and oxidative stress in muscle cells.Methods: To establish the insulin resistance model with 0.4mmol/L PA interference for 24 h.L6 rat myoblasts were recovered,cultured and differentiated into skeletal muscle cells,and randomly divided into two groups:The control group(Con)and PA group(PA).After 24 hours,the concentration of glucose in the culture medium was measured to determine whether the model was constructed successfully.After the modeling,the divided experimental groups was as follows: control group(Con),PA group(PA),metformin group(PA+Met),PA group transfected with negative control si RNA(PA+si RNA),PA group transfected with SIRT3 si RNA(PA+si SIRT3),metformin group transfected with negative control si RNA(PA+Met+si RNA),metformin group transfected with SIRT3 si RNA(PA+Met+si SIRT3).The expression level of SIRT3 m RNA and protein were detected by RT-PCR and Western Blot methods to determine whether the transfection is successful or not.The m RNA and protein expression of LC3Ⅱ/LC3Ⅰ,Beclin1,p62,SIRT3 in each group were detected by Real-Time PCR and Western Blot,respectively.Indicators of oxidative stress,such as superoxide dismutase(SOD),reducedglutathione(GSH),catalase(CAT),and liver peroxidation(LPO)were measured by matched test kit.Reactive oxygen radicals(ROS)was detected by dihydroethidium(DHE)staining.Significance of differences among many samples was determined using One-way ANOVA,and differences between two groups were determined using t-test.Results:1 The identification of L6 myoblasts differentiation Compared with undifferentiated L6 myoblasts,the m RNA expression of Desmin and Myogenin in differentiation group was significantly increased(P<0.05);2 In vitro model of insulin resistance in L6 muscle cells L6 muscle cells were cultured for 24 hours in Con and PA group respectively.There were an increased level of glucose concentration in PA group,indicating that the model of insulin resistance in vitro was established successfully;3 The effect of drug intervention on proliferation in muscle cells observed by CCK 8 assay Compared with control group,the inhibitory rate of cell proliferation was obviously decreased in muscle cells exposed to palmatic acid at different concentrations for 24 hours(P<0.05).Compared with PA group,cell inhibitory rate was lower in muscle cells cultured in Met(2mmol/L)(P<0.05),but failed to recover to normal level.According to CCK 8 result,ultimate concerntration of PA and metformin were 0.4mmol/L and 2mmol/L,respectively;4 The expression of SIRT3 after si RNA intervention Compared with the PA+si RNA group,the m RNA and protein expression of SIRT3 were significantly decreased in PA+si SIRT3 group;compared with the PA+Met+si RNA group,the m RNA and protein expression of SIRT3 were significantly decreased in PA+Met+si SIRT3 group.The difference was statistically significant(P<0.05),suggesting that SIRT3 si RNA was successfully transfected into muscle cells;5 Autophagy in muscle cells5.1 gene expression of key markers in autophagy Compared with control group,the m RNA levels of LC3Ⅱ,Beclin1 were significantly decreased,the difference was statistically significant(P<0.05),and the m RNA expression of p62 was significantly increased(P<0.05)in PA group.Compared with PA group,the m RNA expression of LC3Ⅱ,Beclin1 were significantly increased,and the m RNA level of p62 was significantly decreased(P<0.05)in PA+Met group.Compared with the PA+si RNA group,the m RNA expression of LC3Ⅱ(P<0.05),Beclin1(P>0.05)were decreased,and the m RNA expression of p62 was increased(P > 0.05)in PA+si SIRT3 group.Compared with the PA+Met+si RNA group,the m RNA expression of LC3 Ⅱ,Beclin1 were significantly decreased(P<0.05),and the m RNA expression of p62 was significantly increased(P<0.05)in PA+Met+si SIRT3group;5.2 the protein expression of key molecules in autophagy Compared with control group,the ratio of LC3 Ⅱ /LC3 Ⅰ,Beclin1/GAPDH were significantly decreased(P<0.05),and the ratio of p62/GAPDH was significantly increased(P<0.05)in PA group.Compared with PA group,the ratio of LC3Ⅱ/LC3Ⅰ,Beclin1/GAPDH were significantly increased,and the ratio of p62/GAPDH was significantly decreased(P<0.05)in PA+Met group.Compared with the PA+si RNA group,the rate of LC3Ⅱ/LC3Ⅰ,Beclin1/GAPDH were significantly decreased(P<0.05),and the rate of p62/GAPDH was significantly increased(P<0.05)in PA+si SIRT3 group.Compared with the PA+Met+si RNA group,the rate of LC3 Ⅱ /LC3 Ⅰ,Beclin1/GAPDH were significantly decreased(P<0.05),and the rate of p62/GAPDH was significantly increased(P<0.05)in PA+Met+si SIRT3 group;6 oxidative stress in L6 muscle cells Compared with Con group,the ROS generation of muscle cells and LPO were significantly increased in PA group,while the activities of anti-oxidative enzymes such as SOD,CAT and the content of GSH were significantly decreased(P < 0.05).Treatment with metformin significantly reduced ROSand LPO generation,while increased SOD(P<0.05),CAT activity(P>0.05)and GSH level(P < 0.05)when compared with PA group.Compared with PA+si RNA group,the ROS generation of muscle cells and LPO were significantly increased in PA+si SIRT3 group,while the activities of anti-oxidative enzymes such as SOD(P < 0.05),CAT(P > 0.05)and the content of GSH(P>0.05)were decreased.Compared with PA+Met+si RNA group,the ROS generation of muscle cells(P<0.05)and LPO(P>0.05)were increased in PA+Met+si SIRT3 group,while the activities of anti-oxidative enzymes such as SOD(P<0.05),CAT(P<0.05)and the content of GSH(P>0.05)were decreased.Conclusions:1 High fat incubation led to decreased glucose uptake,insulin resistance,and SIRT3 attenuation in skeletal muscle.While metformin could ameliorate insulin resistance in muscle cells exposed to high-fat intervention and increase the expression of SIRT3.2 High fat incubation could inhibit the levels of autophagy in L6 muscle cells.Metformin treatment could not only increase autophagy activity,but also improve the oxidative stress,reduce ROS production and LPO content,and recover the activity of SOD and CAT in muscle cells induced by high-fat treatment,thus improving insulin resistance.3 After SIRT3 knockdown the stimulatory effect of metformin on autophagy and the improvement of oxidative stress were decreased in L6 muscle cells exposed to palmitic acid.