Dual Effects Of Metformin On Adipogenic Differentiation Of 3T3-L1 Preadipocyte In AMPK-Dependent And Independent Manners

Adipocyte accumulation is associated with the development of obesity and obesity-related diseases,including type 2 diabetes.Metformin has been widely used to treat type 2 diabetes,especially is favored for its body weight lowering effects.However,very few in vitro studies examined the effects of metformin on adipocyte differentiation and its potential signaling targets.Moreover,contradictory results about the dose and effects of metformin warrants more studies.In this study we examined the effects of a wide range of doses of metformin on adipogenesis and found metformin exhibits a biphasic effect on adipogenesis.Objective To study the effects of metformin at different concentrations on the differentiation of 3T3-L1 preadipocytes and the expression of intracellular adipogenic and lipogenic factors.And to determine the involvement of signaling pathways of AMPK,MAPK family and Akt in metformin-regulated adipogenic differentiation to explore the mechanism of metformin on adipogenesis in 3T3-L1 cells.Methods(1)MTT assay was used to detect the effect of metformin on the proliferation of3T3-L1 preadipocytes.(2)Oil red O staining and isopropanol extraction were used to observe the formation of lipid droplets in cells.(3)q RT-PCR was used to detect the adipogenic and lipogenic factors in cells under treatment with metformin during differentiation.(4)The changes of three key proteins C/EBP α,FASN and PPAR-γ in cell differentiation were detected by Western-blot,as well as the expression of signal pathways of AMPK / ERK /JNK/ p38 / Akt et al.Results We demonstrated that metformin at lower concentrations(1.25-2.5 m M)significantly induced while at higher concentrations(5-10 m M)significantly inhibited adipogenesis in3T3-L1 cells respectively,as evidenced by Oil red O staining and microscopic examinations.Similar effects were observed for the intracellular and extracellular triglycerides contents.In addition,the biphasic effect of different doses of metformin on adipogenesis is accompanied by a concurrent increase or decrease of the expression of aidpogenic and lipogenic genes including PPARγ,C/EBPα,and FASN at both m RNA and protein levels in 3T3-L1 cells.Furthermore,treatment with higher concentrations of metformin induced the phosphorylation of AMPK,p38 and JNK,while reduced the phosphorylation of ERK and Akt,but lower concentrations in general had not much effects on these signalings.Pretreatment with compound C,a specific AMPK inhibitor,significantly rescued metformin-induced inhibition of adipogenesis.Conclusion Taken together,our research estimates the biphasic effects of metformin on adipocyte differentiation,that is,lower concentration of metformin will induce adipogenesis.whereas higher concentration of metformin reduced adipogenesis.In addition,our data suggest that the effect of metformin at lower concentration on 3T3-L1 cells was independent of AMPK,while the higher concentration of metformin-induced inhibition of adipogenesis was mediated at least in part by AMPK activation.