17β-雌二醇对脂肪细胞促酰化蛋白受体和下游信号蛋白表达的影响

Effect of 17β-Estradiol on Acylation Stimulating Protein-Receptor C5L2 Pathway in 3T3-L1 Adipocytes and Preadipocytes

目的观察17β-雌二醇对3T3-L1(前)脂肪细胞促酰化蛋白受体C5L2 mRNA、细胞表面C5L2蛋白和促酰化蛋白下游信号蛋白表达的影响。方法体外培养3T3-L1细胞,"鸡尾酒"方法诱导细胞分化,用不同浓度17β-雌二醇作用于3T3-L1(前)脂肪细胞,孵育过夜后收获细胞,采用RT-PCR和流式细胞仪检测促酰化蛋白受体mRNA和蛋白表达情况;采用Western Blot法检测基础状态和促酰化蛋白刺激时Gαq/11、Gβ、p-PKCα和p-PKCζ蛋白表达。结果10-8mol/L17β-雌二醇轻度增加3T3-L1成熟脂肪细胞和前脂肪细胞C5L2 mRNA和蛋白的表达,10-6mol/L时3T3-L1成熟脂肪细胞C5L2 mRNA和蛋白表达分别下降了40%(P<0.05)和21%(P<0.05)。但前脂肪细胞C5L2mRNA和蛋白表达水平差异均无显著性。高浓度(10-6mol/L)17β-雌二醇组在一定程度上抑制促酰化蛋白刺激的成熟脂肪细胞Gαq/11、Gβ、p-PKCα和p-PKCζ的表达,促酰化蛋白刺激的蛋白表达分别减少了17%、23%、15%和15%(P均>0.05);在前脂肪细胞,10-6mol/L17β-雌二醇仅抑制促酰化蛋白刺激的Gαq/11蛋白表达24%(P<0.05),对Gβ、p-PKCα和p-PKCζ的表达无明显差异。但17β-雌二醇作用后,在一定程度上"中和"促酰化蛋白对Gαq/11、Gβ、p-PKCα和p-PKCζ的促进作用。结论高浓度雌激素可诱导促酰化蛋白抵抗发生,促酰化蛋白抵抗可能参与了高浓度雌激素引起的脂肪细胞胰岛素抵抗状态的病理生理过程。

Ahn To evaluate potential aeylation stimulating protein （ASP} resistance in both adipocytes and preadipoeytes under the conditions which produce insulin resistance by 17β-estradiol on both receptor level and post-receptor level. Methods 3T3-L1 preadipocytes were induced differentiated and 0 mol/L （ 17β-estradiol-free DMEM/F12）, 10^-8mol/L, 10-7 mol/L and 10-6 mol/L 17β-estradiol was added to cultured 3T3-L1 adipocytes and preadipocytes overnight. RT-PCR and flow cytometry were used to detect mRNA and cell surface expression of ASP receptor. Both non-17β-estradiol treated and 17β-estradiol treated 3T3-L1 cells were cultured with 5.0 μmol/L ASP for 4 hours. Then the cell proteins were extracted and the expressions of C512, Gaq/11, p-PKCα and p-PKC ξ were measured by Western Blot. Results High dose 17β-estradiol suppressed C5L2 mRNA and protein expression in 3T3-L1 adipocytes but not preadipocytes. At 10-8 mol/L, 17/3-estradiol increased C5L2 mRNA and protein expression slightly in both adipocytes and preadipocytes （P 〉 0.05, respectively）. At 10-6 mol/L 17β-estradiol in- hibited CSL2 mRNA and cell surface C5L2 expression by 40% （P〈0.05） and 21% （P〈0.05}, respectively. Afterovernight incubation with 1713-estradiol （in adipocytes and preadipocytes）, Gaq/11, Gβ, p-PKCα and p-PKCξ were downregulated in the presence of ASP treatment to a certain degree. In adipocytesl at 10-6 mol/L, 171β-estradiol inhibited the ASP-induced Gaq/11, Gβ, p-PKCα and p-PKCξ expression by 17% ,23% ,15% and15% （P〉0.05, respectively}. Whereas high dose 1713-es- tradiol effectively blocked ASP-stimulated Gaq/ll significantly by 24% （P 〈 0.05 ） in preadipocytes. However, 17β-estradiol counteracted ASP-stimulated Gaq/11, Gβ, p-PKCα and p-PKCξ to some extent. Conclusion 17β-estradiol induces ASP re- sistance in adipocytes and preadipocytes. ASP resistance may contribute to the physiological abnormalities associated with insulin resistance induced by 17β-estradiol.