Mechanism Of Pyruvate Dehydrogenase Kinase 4 Mediated FXR Regulation Of Hepatic Lipid Metabolism

?Objective?Farnesoid X receptor?FXR?plays an important role in glucose and lipid metabolism.FXR knockout can cause hepatic steatosis in mice,but the mechanism is not very clear.This study was to investigate the molecular mechanism of FXR gene as a bile acid nuclear receptor transcription factor regulating hepatic lipid metabolism.?Method?1)FXR^-/-mice and C57BL/6J mice were fed with high-fat diet and normal diet to establish the model of non-alcoholic fatty liver disease.The insulin sensitivity and glucose tolerance of the mice were measured at after the 71 and 80 days.The animals were sacrificed after three months.Western blotting,Real-time PCR and immunohistochemistry were used to detect the expression of PDK4 in liver.H&E staining and oil red O staining were used to observe the morphological changes and lipid droplet of mouse liver.Blood lipids and liver enzymes of mice in each group were detected by automatic biochemical analyzer.2)FXR^-/-mice were fed intraperitoneally with DCA?inhibitor of PDKs?and a high-fat diet.The mice were tested for glucose tolerance at week 4,and the animals were sacrificed at week 5,and the same site of liver tissue was taken for H&E and oil red O staining.The automatic biochemical analyzer was used to detect the levels of blood lipids and liver enzymes in each group,and the expression of key enzymes in liver lipid metabolism was detected by western blot.3)CRISPR/Cas9 technology was used to construct FXR^-/-LO-2 cell line.The expression of PDK4 in FXR knockout LO-2 cell line was detected by western blot,sodium oleate/sodium palmitate was used to induce a high-fat environment,and BODIPY reagent was used to detect accumulation of lipid droplets in the cell.?Results?1)Compared with the weight of WT mice,there was no difference in body weight ratio between FXR^-/-mice in the normal diet.The high-fat diet,FXR^-/-mice weight?39.46±3.23g?was significantly less than WT mouse weight?50.72±2.16g?,and the weight gain was slow or even decreased.The liver of the FXR^-/-mice became larger?1.24 times that of the normal diet,2 times that of the high-fat diet compared with the liver of WT mice?,and the surface of the liver is tough,uneven pleats,the color is light,the cut surface is slightly greasy,and the high fat induction will aggravate its pathological state.H&E and oil red O staining revealed vacuolization and lipid droplet aggregation in the liver of FXR^-/-mice.High fat induction increased liver vacuoles and lipid droplets,and aggravated liver damage.FXR knockout also caused a significant increase in plasma levels of TG,NAFA,and LDL,decreased HDL,and increased ALT,AST,and ALP.It is suggested that the deletion of FXR gene leads to hyperlipidemia in mice,disorder of liver lipid metabolism,and finally liver damage.RNA-seq was used to detect the expression levels of various genes in the liver of FXR^-/-mice under normal diet.It was found that there was a significant difference in the expression of PDK4compared with the liver of WT mice.Western blot,immunohistochemistry and qRT-PCR further confirmed the results.In the high-fat diet,the expression of PDK4 in the liver of FXR knockout mice was elevated but not statistically significant.2)Intraperitoneal injection of DCA improved the diet and body weight of FXR^-/-mice,reduced the ratio liver and abdominal fat to body weight,improved glucose levels,reduced plasma LDL levels,and decreased liver lipid droplets.Western blot was used to detect the key proteases of lipid and triglyceride synthesis in mouse liver tissue.It was found that DCA can promote the expression of FASN and ACC,decrease the expression of ACLY and reduce the expression of lipid metabolism-related proteases such as HSL,PPAR?and PPAR?.Loss of FXR leads to an increase in mTOR levels,whereas DCA may regulate hepatic lipid metabolism by inhibiting mTOR expression.3)The expression of PDK4 was increased in FXR knockout LO-2 cells,and FXR knockout cells induced aggravation of lipid droplet aggregation induced by high fat medium,and lipid droplet aggregation decreased after interfering with PDK4.?Conclusion?Loss of bile acid receptor FXR can cause hepatocyte lipid metabolism disorder,liver lipid droplets,and elevated serum lipids.The specific mechanism is related to the up-regulation of PDK4 gene expression in hepatocytes.Inhibition of PDK4 expression can improve hepatocyte lipid droplet aggregation and alleviate hepatic lipid metabolism disorder caused by FXR deficiency.