Effects Of Metformin On Proliferation, Differentiation And Apoptosis Of SW872 Cells

Part I Study of the proliferation of SW872 cells and the differentiation ofSW872 cells induced by oleic acidObjective To investigate the proliferation of SW872 cells and the effect of oleic acid on SW872 cells differentiation.Methods SW872 cells were cultured in vitro. Cell counting was employed to find out the population doubling-time and growth curve of SW872 cells. The morphologic changes of SW872 cells were observed during the progress of differentiation. Fat droplets were observed in the cytoplasm by Oil red O staining. Triglyceride (TG) mass was detected by chemical colorimetry methods.Results The growth curve of SW872 cells were the shape of "s", and the population doubling-time of SW872 cells were 65.48±2.99 hours. SW872 cells were fibroblastic, and no fat droplet was noted in cytoplasm before differentiation. 24h after stimulated by oleic acid, The cells became bigger, rounder and contained numerous small lipid-filled inclusions. The differentiation was found in most all cells and the bulk of the cytoplasm was filled lipid storage droplets 48h after oleic acid stimulation. More and more fat drops were accumulated in these cells with time pass. Seventy-two hours after differentiation, SW872 cells differentiated into mature adipocytes with lots of fat droplets in the cells, which became more bigger and rounder .In the meantime, comparing with that of before differentiation, TG mass was significantly increased 72h after 0.6mM oleic acid stimulation (P<0.01). Conclusion These observations suggested that SW872 cells could be differentiated into typical mature adipocytes induced by 0.6mM oleic acid. Part II Effects of metformin on the proliferation and differentiationOf SW872 cellsObjective To observe the effects of metformin on the proliferation anddifferentiation of SW872 cells, so that to explore the possible mechanism of metforminreducing weight.Methods SW872 cells were cultured in vitro. Growth curve, MTT assay andPopulation doubling time were employed to discover the effects of metformin on theproliferation of SW872 cells. Fat droplets were observed in the cytoplasm by Oil red Ostaining. Triglyceride (TG) mass was detected by chemical colorimetry methods. Thelevels of (PPAR-γ and C/EBP-α) mRNA expressions were measured bysemi-quantitative RT-PCR assay.Results There was no influence of metformin on the growth curve and populationdoubling time of SW872 cells incubated with 0.01mM, 0.1mM, 0.5mM, 1.0mMmetformin for 72 hours, meanwhile Oil red O staining and triglyceride (TG) massgradually reduced in SW872 cells.The cells treated for 72h with metformin(0.01mM)showed no change on the expression of PPAR-γ mRNA(P>0.05), however, when thedose of metformin were increased upon 0.1mM and more, the level of PPAR-γ mRNAexpression significantly reduced(P<0.01). The same change was found on theexpression of C/EBP-α mRNA in SW872 cells.Conclusion Metformin had no effects on the proliferation of SW872 cells, butcould inhibit the differentiation of SW872 cells in a dose-dependent manner. PartIIIEffects and mechnism of metformin on the apoptosis of SW872 cellsObjective To observe the effects of metformin on the apoptosis of SW872 cells, sothat to explore the possible mechanism of metformin reducing weight.Methods The apoptosis rate of SW872 cells was detected by flow cytometry, andthe levels of (Bcl-2 and Bax) mRNA expression were measured by semi-quantitativeRT-PCR assay.Results 1.The basic apoptosis rate of SW872 cells was (3.54±0.28)%. Comparingwith the control group, the apoptotic rate of the group treated respectively by 0.1 mM,0.5mM, 1.0mM metformin was seriously distinct (P<0.01), but that of the grouptreated respectively by 0.5mM, 1.0mM was not distinct (P>0.05). Therefore, theoptimum concentration of metformin inducing apoptosis was 0.5mM.Treating SW872cells with 0.5mM metformin for 0,12,24,36,48 hours, the apoptotic rate of SW872 cellswas distinct comparing with that of the control group (P<0.01). However, thedifference of apoptosis rate between 36 and 48 hours was not distinct (P>0.05).Therefore, 36 hours may be the optimum time to induce adipocyte apoptosis bymetformin.2. Metformin inhibited the expression of Bcl-2 mRNA of SW872 cells in atime- and dose-dependent manner.3. The expression of Bax mRNA of SW872 cellswas no change after treated with metformin (P>0.05).Conclusion Metformin could promote the apoptosis of SW872 cells throughinhibiting the expression of Bcl-2 mRNA.Part IV Effects of metformin on the plasma levels of adipocyte derivedhormone in obese children with hyperinsulinism Objective To observe the changes of adipocyte derived hormone in obese children with hyperinsulinism after treated with metformin for 4-6 months.Methods 76 children, 5 ～ 17 years old, including 44 obese children withhyperinsulinism and 32 normal children. The standard to detect obesity is that weight ismore than the normal weight with the same gender and same stature by 20%. Thediagnostic criteria of hyperinsulinism is that the concentration of fasting plasmaglucose is more than 25mIU/L.ELISA technique was employed to determine theplasma IL-6, adiponectin and leptin concentrations.Results 1. The levels of plasma IL-6 and leptin increased in obese children withhyperinsulinism, while plasma adiponectin decreased significantly. 2.After treated withmetformin for 4-6 months, the levels of plasma IL-6(22.42±4.20 pg/mL vs 14.34±3.10pg/mL )and leptin(28.55±4.06 ng/mL vs 17.82±3.48 ng/mL) decreasedsignificantly(P<0.05), but the plasma adiponectin(7.54±1.87ug/mL vs 7.65±2.03ug/mL)was unchanged comparing with that of pretreatment(P>0.05).Conclusion The levels of plasma IL-6, leptin and adiponectin were abnormal inobese children with hyperinsulinism, while metformin can improve them partly.