Effect Of ApoM Gene Deletion On Glucose-related Functions In Liver And Kidney

Objective: To investigate the effects of ApoM-S1 P on hepatic glucose metabolism and insulin signaling pathway and the effects of down-regulation of ApoM gene expression on the apoptosis of mesangial cells,and to explore the potential association between ApoM and type 2 diabetes.Methods: ApoM gene-deficient mice and hepatocyte models,ApoM gene overexpressing mouse hepatocyte model,and ApoM gene expression-interfering mesangial cell model were used.The effect of ApoM gene deletion on glucose metabolism was detected by sugar stimulation test.qPCR and Western-blot were used to detect the expression changes of hepatic glucose metabolism-related enzymes and insulin signaling pathways after ApoM gene deletion.Western-blot was used to detect the changes of key proteins in hepatocyte insulin signaling pathway after overexpression of ApoM gene.The effect of S1 P bioactive preparation on the expression of key protein in insulin signaling pathway of ApoM-deficient hepatocytes was detected by Western-blot.The morphological changes of kidney in mice were observed by electron microscopy.The effects of ApoM expression down-regulation on the level of renal apoptosis and its molecular mechanism were observed by Western-blot,DAPI staining and cell flow imaging.Results:1.The continuous increase in glucose content down-regulated the expression level of ApoM in mouse hepatocyte AML12(P<0.05).2.After 24 hours of stimulation with different concentrations of glucose,the sugar concentration of the ApoM gene-deficient AML12 cell culture medium was higher than that of the normal control group(P<0.05).3.After the ApoM gene was deleted,the mRNA level of the gluconeogenesis key enzyme G6 PC in the liver tissue and liver cells of mice was increased(P<0.05).4.The expression levels of Akt,GSK3?,AS160 and AMPK? in mouse liver cell AML12 after ApoM gene deletion were not significantly different from those in normal control group(P>0.05).The expression levels of p-AKT ser473,p-GSK3? ser9,p-As160 ser588 and p-AMPK? Thr172 were significantly lower than those in the normal control group(P<0.05).5.Successfully constructed a hepatocyte model with overexpression of ApoM gene by using AdEasy recombinant adenovirus system.6.Overexpression of ApoM in ApoM knockout mouse hepatocyte AML12,p-GSK3? ser9,p-As160 ser588 regained to a level close to normal cells.7.Addition of S1 P to hepatocytes deficient in ApoM gene resulted in a significant up-regulation of the expression levels of p-AKT ser473,p-GSK3? ser9,p-As160 ser588 and p-AMPK? Thr172,which were down-regulated in the original expression level(P<0.05),basically consistent with normal cells.8.The deletion of ApoM gene in mice resulted in a significant decrease in the mRNA levels of S1PR1,S1PR3,S1PR4 and S1PR5(P<0.05),and the mRNA level of S1PR2 was not statistically significant.Cell levels were essentially consistent with animal level results.9.Normal mouse hepatocyte AML12 was incubated with S1PR1 and S1PR3 inhibitor VPC23019.The expression levels of p-AKT ser473,p-As160 ser588,and p-AMPK? Thr172 were significantly decreased(P<0.05).After using the S1PR2 inhibitor JTE013,the expression levels of p-AKT ser473,p-As160 ser588,and p-AMPK? Thr172 were not significantly changed.10.The mitochondria of the kidney of ApoM knockout mice showed swelling,vacuolization and myeloid changes,and a large number of rough endoplasmic reticulum expanded abnormally.The proliferative activity of mesangial cells inhibited by ApoM gene expression is decreased,and chromatin condensation,nuclear pyknosis,and nuclear lysis occur in the nucleus.11.Down-regulation of ApoM expression levels resulted in significant up-regulation of AIF,Bax,chop,p-JNK,clever-caspase9,clever-caspase12,clever-caspase7,and clever-caspase3 expression in mouse kidney and mesangial cells(P<0.05),the expression level of Bcl2 was significantly decreased(P<0.05).It is suggested that the decrease of ApoM expression level up-regulates the apoptosis level of mouse kidney and mesangial cells through mitochondria and endoplasmic reticulum pathway.Conclusion:1.The deletion of the ApoM gene leads to abnormal glucose metabolism in the liver of mice and an obstacle to the transduction of related signaling pathways.2.ApoM may regulate the transduction of mouse hepatic glucose metabolism-related signaling pathway through S1P/S1PR1/S1PR3.3.Down-regulation of ApoM expression level up-regulates the apoptosis of mouse kidney and mesangial cells through mitochondria and endoplasmic reticulum pathway.