PI3K抑制剂LY294002对鼠前脂肪细胞分化和C/EBPα及PPARγ表达的影响

Effects of PI3K inhibitor LY294002 on the differentiation of mouse preadipocytes and the expression of C/EBPα and PPARγ

摘要:

目的：研究磷脂酰肌醇激酶（PI3K）抑制剂LY294002对小鼠前脂肪细胞分化和CCAAT增强子结合蛋白α（C/EBPα）及过氧化物酶体增殖物激活受体γ（PPARγ）表达的影响，探讨胰岛素受体底物（IRSs）/PI3K信号通路在前脂肪细胞分化中的作用。方法：小鼠3T3-L1细胞分为实验组和对照组，实验组加入LY294002（25 μmol/L），对照组加入同体积DMSO。应用0.5 mmol/L 3-异丁基-1-甲基黄嘌呤(IBMX)、10-6 mol/L地塞米松和5 μg/mL胰岛素诱导两组前脂肪细胞分化，在培养的0 d、2 d、4 d、8 d分别收集细胞，实时PCR法及Western blot法检测细胞中C/EBPα和PPARγ表达水平。培养8 d油红O染色，观察细胞分化情况。结果：小鼠3T3-L1细胞基础状态下两组C/EBPα及PPARγ表达差异无统计学意义（P＞0.05）；诱导分化时实验组C/EBPα及PPARγ表达低于对照组（分别P＜0.05，P＜0.01）。油红O染色示实验组细胞无明显脂滴。结论：PI3K抑制剂LY294002能抑制小鼠前脂肪细胞分化及C/EBPα和PPARγ的表达，提示IRSs/PI3K信号通路可通过调节PPARγ和C/EBPα的表达在3T3-L1前脂肪细胞的分化中发挥重要作用。

Abstract:

OBJECTIVE: This study examined the effects of PI3K inhibitor LY294002 on the differentiation of mouse preadipocytes and the expression of CCAAT enhancer binding protein α (C/EBPα) and peroxisome proliferation activated receptor γ (PPARγ), in order to study the possible roles of insulin receptor substrate (IRSs)/PI3K signal pathway in the differentiation of preadipocytes. METHODS: The mouse 3T3-L1 cells were cultured normally and divided into experimental and control groups. 3T3-L1 cells in the experimental group were treated with PI3K inhibitor LY294002 (25 μmol/L) and those in the control group were treated with DMSO culture medium. 3-isobutyl-1-methylxanthine (IBMX) (0.5 mmol/L), dexamethasone (10-6 mol/L) and insulin (5 μg/mL) were used to induce the differentiation of 3T3-L1 preadipocytes in both groups. Before culture, and 2, 4 and 8 days after culture, the cells were collected to detect the expression of C/EBPα and PPARγ by real-time PCR and Western blot assays. The lipid droplets of 3T3-L1 preadipocytes were observed by oil-red O staining. RESULTS: PI3K inhibitor LY294002 did not affect the expression of C/EBPα and PPARγ in un-induced 3T3-L1 preadipocytes (P>0.05), but decreased the expression of C/EBPα and PPARγ during the in vitro induced differentiation of 3T3-L1 preadipocytes compared with the control group (P<0.05 or 0.01). The lipid droplets count was greatly reduced by LY294002. CONCLUSIONS: PI3K inhibitor LY294002 can inhibit the differentiation of mouse 3T3-LI preadipocytes and the expression of C/EBPα and PPARγ in the differentiation of 3T3-LI preadipoeytes, suggesting that IRSs/PI3K signal pathway may play an important role in the differentiation of 3T3-L1 preadipocytes by regulating the expression of C/EBPα and PPARγ.