The Study On Mechanism Of Glutathione S-Transferase M2 Regulating Lipid Droplets Content In Cells

Fat is one of the most economically important traits in pigs,and studies have shown that lipids in cells are stored in organelles called "lipid droplets".Lipid droplets are approximately spherical in shape and consist of a neutral lipid core and a single phospholipid membrane on the surface,the number and size of which determine the lipid content of the cell.The production and size of lipid droplets are influenced by various factors,such as nutritional status,hormone levels and oxidative stress.Previously,our group isolated the porcine glutathione S-transferase mu2(GSTM2)gene and found that there was a C27 T transition(CGA→TGA)in exon 5,leading to nonsense-mediated m RNA degradation of the transcript.Further analysis of the effect of altered GSTM2 expression levels on the phenotype revealed that the number of lipid droplets was significantly increased in cells with knockdown GSTM2.By constructing a knockout mouse model,it was found that GSTM2 deletion resulted in increased lipid content in liver and skeletal muscle of mice.High-fat feeding,histomorphological assay,immunoprecipitation combined with immunoblotting assay,signaling pathway analysis,laser confocal assay,and live cell workstation observation revealed that GSTM2 regulates cellular lipid droplet content.The main findings are as follows.1.GSTM2 regulates cellular lipid droplet content through the ASK1-p38-JNK signaling pathway.(1)Inhibition of GSTM2 expression leads to increased lipid droplet content in cells.Using RNAi to knock down GSTM2 in cells,BODIPY493/503 labeled lipid droplets,fluorescence microscopy showed a significant increase in lipid droplets in the interfering group of cells.Skeletal muscle and liver tissues from GSTM2 knockdown mice were taken for oil red O staining analysis,and the results showed a significant increase in lipid content in GSTM2 knockdown tissues.(2)GSTM2 knockout mice were more sensitive to high-fat feeding.Mice were fed with high-fat diet and choline-methionine-deficient diet,respectively,and liver tissues were taken for HE and Oil Red O staining,and triglyceride content was measured.The results showed that GSTM2 knockout mice had higher lipid content and faster accumulation in hepatocytes;in addition,GSTM2 knockout mice had higher levels of liver fibrosis.(3)GSTM2 binds to ASK1 and thus regulates the p38-JNK signaling pathway.The level of ASK1 phosphorylation was increased and p38-JNK signaling was activated after GSTM2 interference in cells;overexpression of GSTM2 inhibited ASK1 phosphorylation and suppressed p38-JNK signaling.Immunoprecipitation experiments showed that GSTM2 and ASK1 bound to each other,and further protein truncation experiments revealed that GSTM2 bound to ASK1 through the C-terminus.(4)GSTM2 promotes lipid droplet production and growth by activating the ASK1-p38-JNK signaling pathway.Using palmitic acid and Selonsertib(GS-4997)to activate and inhibit ASK1 activity,respectively,the expression levels of lipid synthesis genes such as SREBP1 and PLIN2 were elevated,and the localization of GPAT4 and DGAT2 in lipid droplets surface increased,thus promoting lipid droplet generation and growth.In addition,GSTM2 regulates PCYT1 A expression,which affects the rate of phosphatidylcholine synthesis on the surface of lipid droplets,thus affecting the surface tension of lipid droplets to regulate lipid droplet growth.(5)Overexpression of GSTM2 inhibits high-fat feeding-induced liver fat accumulation.The GSTM2 overexpression vector was mixed with in vivo transfection test and injected into the peritoneal cavity of mice once a week for one month,during which high-fat feeding was performed,and liver tissues were taken for HE and oil red O assay,and the results showed that the liver lipid content of the overexpression group was significantly lower than that of the control group.In addition,the fibrosis level of liver tissues in the overexpression group was also significantly reduced.(6)Inhibition of ASK1-p38-JNK signaling pathway reduced the liver lipid content of GSTM2 knockout mice.Using Selonsertib for in vivo injection once a week for one month,liver tissues were taken for HE and Oil Red O assay,and the results showed that the fat content in liver tissues of GSTM2 knockout mice was significantly reduced.2.GSTM2 regulates cellular lipid droplet content by decreasing ROS levels in cells.(1)GSTM2 regulates ROS levels in cells.Using RNAi to interfere with GSTM2 expression,ROS levels were detected to be elevated;while overexpression of GSTM2 reduced ROS levels.(2)ROS regulates lipid droplet content in cells by promoting PLIN2 expression.The number of lipid droplets increased when cells were treated with hydrogen peroxide and did not change with increasing treatment concentration.Detection of lipid droplet-related gene expression revealed a significant increase in PLIN2 expression levels(P<0.05).Overexpression of PLIN2 increased the number of lipid droplets in cells;whereas,interference with PLIN2 expression led to a decrease in lipid droplet content in cells,and reducing PLIN2 expression inhibited the hydrogen peroxide-induced increase in cellular lipid droplet content.Analysis of PLIN2 expression from 0-7 hours after hydrogen peroxide treatment and the number of lipid droplets showed that PLIN2 expression increased from 2 hours(P<0.05)and lipid droplets increased from 3 hours 30 minutes(P<0.05).(3)Reducing ROS levels inhibits the increase in lipid droplets caused by GSTM2 knockdown.The exogenous addition of acetylcysteine after GSTM2 interference in cells showed a significant decrease in ROS levels(P<0.05)and a significant decrease in the number of lipid droplets.The above experimental results suggest that the N-terminal end of GSTM2 functions as a GST transferase to reduce ROS levels in cells and inhibit the ROS-induced increase in lipid droplet content;the C-terminal end of the protein can bind and inhibit ASK1 phosphorylation,inhibit the activation of downstream p38-JNK signaling,and regulate the expression and localization of genes related to lipid synthesis.In this study,the molecular mechanism of GSTM2 regulation of cellular lipid droplet content was resolved,and the potential role of phase II detoxification enzymes in cellular lipid metabolism was identified,which provides new ideas for subsequent studies related to cellular lipid production and deposition.