The Effects Of Leptin On Proliferation And Differentiation Of Preadipocytes

Objective To investigate the effects of leptin on the proliferation, differentiation and the lipid accumulation of 3T3-L1 preadipocytes as to elucidate the mechanisms underlying the role of leptin in enhancing the survival rate of autologous grainy fat transplantation.Methods Adopt preadipocyte cultured technique, 3T3-L1 preadipocytes were cultured in vitro. The expression of leptin receptor (OB-R) in cells was detected by fluoroimmunoassay. 3T3-L1 preadipocytes were treated by leptin with various concentrations (0ng/ml, 10ng/ml, 50ng/ml, 100ng/ml, 200ng/ml,500ng/ml) for 24h. The effect of leptin on proliferation of cells was examined by the colorimetric thiazolyblue assay (MTT assay) and the incorporation of 3H-thymidine. Meanwhile, inhibitory effect of ERK1/2 selective inhibitor PD98059, which blocks ERK1/2 phosphorylation, on the proliferation of 3T3-L1 preadipocytes induced by 50ng/ml leptin was also studied. Cells were treated with leptin (0ng/ml,50ng/ml,100ng/ml,200ng/ml) for 12h, then the level of phosphorylated ERK1/2 (p-ERK1/2) and ERK1/2 were examined by Western Blot. 3T3-L1 preadipocytes are stimulated by Leptin 50ng/ml+IBMX 0.5mmol/L+DEX 1.0μmol/L. On the 6th day and 9th day after inducing differentiation, the triglyeride (TG) in cells were detected by chemical colorimetry methods. And on the 9th day after inducing differentiation, the activity of glycerol-3-phosphate dehydrogenase (GPDH)-a terminal marker enzyme of adipose differentiation was measured.Results Fluoroimmunoassay showed the presence of OB-R in 3T3-L1 preadipocytes. Both MTT assay and the incorporation of 3H-thymidine showed that 10ng/ml, 50ng/ml and 100ng/ml leptin promoted proliferation of 3T3-L1 preadipocytes, but 200ng/ml and 500ng/ml leptin inhibited proliferation of the cells. Blocking ERK1/2 phosphorylation by PD98059 significantly reduced the proliferation of 3T3-L1 preadipocytes stimulated by leptin (50ng/ml). ERK1/2 phosphorylation was significantly increased stimulated by leptin, in a dose-dependent manner. During the course of differentiation, there were significantly increased in TG mass and the activity of GPDH in 3T3-L1 preadipocytes, compared with the control group and IBMX+DEX group.Conclusion Leptin has both positive and negative effects on the proliferation of 3T3-L1 preadipocytes, and the possible mechanisms of the role of leptin relate to the ERK1/2 signal pathway. 50ng/ml leptin can promote the differentiation and the lipid accumulation of preadipocytes.