Effect Of Metformin On Proliferation And Differentiation Of Preadipocytes

Objective:The effect of different concentration of Met on the proliferation and differentiation of preadipocytes was studied by using 3T3-L1 preadipocytes as the research object.To observe the effect of different concentrations of metformin on the proliferation and differentiation of preadipocytes,and to detect the changes of various transcription factors.Method:We cultured the 3T3-L1 and subcultured for twice,and then seed it on the six or ninety-six well plates.After cultured for 24 hours,added1mmol/l,2mmol/l,5mmol/l,10mmol/l metformin in the different plates and co-cultured for 48 h.(1)detected the proliferation of 3T3-L1 preadipocytes with CCK-8.(2)Havest the adipose and detected the mRNA expression of GATA3,PPAR-?,C/EBP?,?-catenin.(3)After confluent cultured for another48 h,removed the growth medium and added differentiation medium.Medium A for three days,medium B for one day,continued for 12 days.To judge the lipid droplets formation we used oil red O staining.Experimental data applications SPSS13.0software for statistical analysis,differences between groups using single factor analysis of variance,multiple comparisons between groups using LSD test,test level taking ?=0.05.Result(1)CCK-8 proliferation experiment:1mmol/l,2mmol/l,5mmol/l metformin promote the preadipocytes proliferation,the promotion for proliferaiton shows no difference.While the 10mmol/l metfomin show less promotion action compared with 1mmol/l metformin.(2)Oil red O staining:Compared with cotrol group,different concetrations both show inhibiton.Amomg them,the 10mmol/l shows the strongest inhibitory effect.(3)m RNA expression:10mmol/l metformin promotes the GATA3 m RNA expression,and inhibits the PPAR-?mRNA expression compared with 5mmol/l metformin.As for the C/EBP?expression,different concentration both inhibit the C/EBP?expression.(4)The ?-catenin expression shows no differecein all groups.Conclusion10mmol/l metfomin inhibit the transformation between preadipocytes and mature adipose.It influence the preadipocytes maturity mainly through the pomotion of GATA3 and the inhabiton of PPAR-?,C/EBP?.Recent studies have shown that AICAR activates the Wnt classical pathway by activating the AMPK signaling pathway and promotes the expression of GATA3 to inhibit adipocyte adipogenesis.PPAR-? and C / EBP? are key transcript molecules in adipocytes that regulate adipocyte differentiation.This study suggests that high concentrations of Met may activate the AMPK pathway,promote GATA3 expression,inhibit PPAR-? and C / EBP?,but no change in ?-catenin,may be related to?-catenin-independent nonclassical pathways,specific molecular mechanisms To further protein levels and gene knockout and other experimental steps in-depth study.