邻苯二甲酸二(2-乙基己基)酯对3T3-L1前脂肪细胞成脂分化和脂肪分解的影响

Effect of diethylhexyl phthalate on adipogenic differentiation and lipolysis of 3T3-L1 preadipocytes

目的观察邻苯二甲酸二(2-乙基己基)酯(DEHP)对3T3-L1前脂肪细胞脂质代谢的影响及其可能的作用机制。方法采用鸡尾酒诱导法对3T3-L1前脂肪细胞诱导脂肪分化,诱导的同时给予DEHP 0(细胞对照组),3.125,6.25,12.5,25和50μmol·L^(-1),诱导过程中每天显微镜下观察3T3-L1细胞脂滴大小和数量的变化。诱导完成(7 d)后细胞继续培养3 d(第10天),显微镜下观察油红O染色后细胞内脂滴大小和数量,甘油三酯(TG)测定试剂盒测定细胞培养液中(细胞外)TG水平,分光光度法测定油红O染色细胞洗脱液中(细胞内)脂肪含量,Western印迹法检测细胞中脂肪TG脂肪酶(ATGL)、激素敏感脂肪酶(HSL)、Ser660和Ser563位点磷酸化HSL及磷酸化蛋白激酶A底物蛋白表达水平,并采用Western印迹法和免疫荧光法检测细胞中解偶联蛋白1(UCP1)蛋白表达水平。结果显微镜下观察发现,诱导分化第5天,细胞对照组细胞内尚未发现脂滴,DEHP 3.125~50μmol·L^(-1)组细胞内均出现小脂滴;诱导分化第6天,细胞对照组开始出现脂滴,DEHP 3.125~50μmol·L^(-1)组脂滴数量增多;第10天,DEHP 12.5,25和50μmol·L^(-1)组油红O着色细胞与细胞对照组相比增多,尤其DEHP 50μmol·L^(-1)组可见较大脂滴和“指环”样脂肪细胞。与细胞对照组相比,DEHP 3.125~50μmol·L^(-1)组细胞外TG水平和细胞内脂肪含量均明显升高(P<0.01);DEHP12.5~50μmol·L^(-1)组细胞ATGL蛋白水平明显下降(P<0.01);DEHP 3.125~50μmol·L^(-1)组细胞中蛋白激酶A活性、HSL磷酸化水平、UCP1表达水平和UCP1表达阳性细胞数目明显降低(P<0.05,P<0.01)。结论DEHP促进3T3-L1前脂肪细胞成脂分化,并抑制脂肪分化细胞脂肪分解。

OBJECTIVE To observe the effect of diethylhexyl phthalate(DEHP)on lipid metabolism of3T3-L1 preadipocytes,and the possible mechanism.METHODS Adipogenic differentiation of 3T3-L1preadipocytes was induced using the cocktail method and DEHP 0(cell control group),3.125,6.25,12.5,25 and 50μmol·L^(-1)were added simultaneously.The changes in the lipid droplet size and number of 3T3-L1 cells were observed every day under a microscope during induction.After 7 d of induction,the 3T3-L1 cells continued to be cultured for 3 d(the10thday).Oil red O staining was used to reveal the intracellular lipid droplets,the intracellular fat content in the eluent of oil red O stained cells and triglyceride(TG)levels in the cell culture medium were detected by spectrophotometry respectively.Western blotting was used to detect the protein expressions of adipose triglyceride lipase(ATGL),hormone sensitive lipase(HSL)and phosphorylated HSL(p-HSL)at Ser660 and Ser563 sites,and phosphorylated protein kinase A(p-PKA)substrate in 3T3-L1 differentiated adipocytes.The protein level of uncoupling protein 1(UCP1)in 3T3-L1 differentiated adipocytes was also analyzed by immunofluorescence and Western blotting.RESULTS On the 5th day of induction of differentiation,some tiny lipid droplets were observed in the DEHP 3.125-50μmol·L^(-1)group,but not in the cell control group.On the 6thday,the lipid droplets appeared in the cel control group,and the numbers of lipid droplets in the DEHP 3.125-50μmol·L^(-1)groups were increased compared with the cell control group.On the10thday,oil red O staining showed that the number of oil red O stained cells in the DEHP treated cells was much larger than that of the cell control group,especially in DEHP 12.5,25 and 50μmol·L^(-1)groups.Larger lipid droplets and ringlike adipocytes could be observed in DEHP 50μmol·L^(-1)treated 3T3-L1 cel s.Compared with the cel control group,the extracellular fat content and intracellular TG level in DEHP 3.125-50μmol·L^(-1)groups significantly increased(P<0.01),the protein expression of ATGL in DEHP 12.5,25 and 50μmol·L^(-1)groups was obviously down-regulated(P<0.01),and the PKA active level,HSL phosphorylation,UCP1 protein expreesion and the number of UCP1-positive cells in DEHP 3.125-50μmol·L^(-1)groups were also significantly decreased(P<0.05,P<0.01).CONCLUSION DEHP may promote 3T3-L1 preadipocytes into adipocytes and inhibit the lipolysis of adipogenic cells.