Construction Of Screening Model For GLUT4 Gene Expression Modulators And Screening Of GLUT4 Activators From Natural Compounds

Diabetes mellitus is a kind of endocrine metabolic disease characterized by high blood glucose levels and metabolic disorders and caused by both genetic and environmental factors,which affect human life and health seriously and the number of diabetic patients continues to rise.Thus,it is really significant to research and develop more and more effective and low-toxic antidiabetic medicines.Diabetes mellitus occurs generally through two kinds of pathological processes,insulin secretion injury and insulin resistance,regardless of personal or environmental causative factors,eventually leading to imbalance of the body glucose metabolism and hyperglycemia.Insulin resistance occurs mainly in insulin responsive target organs,such as skeletal muscle and adipose tissue.GLUT4 is the major glucose transporter in skeletal muscle and adipose tissue.In skeletal muscles and adipose tissues of patients with type 2 diabetes or impaired glucose tolerance,the levels of GLUT4 proteins and mRNA reduce significantly compared with health persons,indicating that abnormal GLUT4 protein synthesis and gene transcription is one of the important molecular basises of insulin resistance.Therefore,we regard GLUT4 as a potential molecular target to screen effective compounds for the treatment of diabetes.The main methods and results of this thesis are shown as follows:First,we used GLUT4 as a target to construct a firefly luciferase reporter vector driven by GLUT4 promoter.We amplified a 1929 bp long fragment of GLUT4 promoter by PCR.Then we inserted it into the pGL3-basic vector to establish the luciferase reporter vector pGL3-GLUT4.pGL3-GLUT4 vector was shown to have high luciferase activity indicating the GLUT4 promoter fragment we identified had high transcriptional activity and it could be applied to subsequent compound screening experiments.Then,we screened the GLUT4 expression activators from natural compounds derived from Chinese medicine.More than 300 kinds of natural compounds were screened by adopting luciferase reporter assay method.At last,we found that compound TI432 could facilitate the GLUT4 promoter activity evidently through primary and secondary screening.TI432 was further confirmed to be able to promote the expression of GLUT4 protein in subsequent western blotting analysis.Moreover,we investigated the molecular mechanisms by which the candidate compound TI432 regulates GLUT4 expression.AMP-activated protein kinases(AMPK)signaling pathway and MEF2 transcription factor play important roles in regulating GLUT4 expression transcriptionally.Hence,we explored the effect of TI432 on AMPK signaling pathway and MEF2 transcriptional activity.As a result,TI432 was shown to increase the level of phosphorylated AMPK and MEF2 transcription factor activity.These results suggest that TI432 may promote GLUT4 expression via enhancing AMPK signaling pathway and MEF2 transcriptional activity.Finally,we further explored whether TI432 as an activator of GLUT4 expression affects the ability of insulin stimulated glucose uptake utilizing 2-NBDG fluorescent probe and the results showed that TI432 indeed enhanced insulin-induced glucose uptake in L6 and HepG2 cells.In summary,our studies have provided an effective model for antidiabetic drug screening with GLUT4 as a target,and successfully screened a natural compound TI432 which increases GLUT4 expression and the ability of insulin stimulated glucose uptake in L6 and HepG2 cells.These studies provide a theoretical and experimental foundation for developing TI432 as an antidiabetic drug in future.