睾酮对3T3-L1脂肪细胞促酰化蛋白受体及下游信号蛋白的影响

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

目的:观察睾酮对3T3-L1(前)脂肪细胞促酰化蛋白(ASP)受体C5L2-mRNA和细胞表面C5L2蛋白表达的影响,以及睾酮对3T3-L1(前)脂肪细胞Gαq/11,Gβ,p-PKCα和p-PKCζ蛋白以及ASP刺激的Gαq/11,Gβ,p-PKCα和p-PKCζ蛋白表达的影响。方法:体外培养3T3-L1细胞,诱导细胞分化,用不同浓度睾酮作用于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ASP受体mRNA和蛋白表达情况;采用Western Blot法检测基础状态和ASP刺激的Gαq/11,Gβ,p-PKCα和p-PKCζ蛋白表达。结果:睾酮呈浓度依赖性均抑制3T3-L1成熟脂肪细胞C5L2-mRNA和蛋白的表达,10-6mol/L时mRNA和蛋白分别减少了60%(P<0.01)和27%(P<0.01)。但前脂肪细胞C5L2-mRNA和蛋白表达水平均无显著性差异。高浓度(10-6mol/L)睾酮在一定程度上抑制ASP刺激的成熟脂肪细胞Gαq/11,Gβ,p-PKCα和p-PKCζ的表达,分别减少了52%(P<0.05),27%(P>0.05),50%(P<0.01)和57%(P<0.05);在前脂肪细胞,ASP-C5L2下游信号分子Gαq/11,Gβ,p-PKCα和p-PKCζ表达无明显抑制作用。结论:睾酮抑制成熟脂肪细胞ASP信号分子表达,睾酮诱导ASP抵抗的发生机制与下调脂肪细胞表面ASP受体功能有关,与干扰ASP-C5L2信号转导途径有关。ASP抵抗参与了高睾酮引起的胰岛素抵抗状态的病理生理过程,ASP抵抗可能参与多囊卵巢综合征(PCOS)、肥胖症等多种疾病的病理生理过程。

Objective： To evaluate potential acylation stimulating protein （ASP） resistance in both adipocytes and preadipocytes under the conditions of insulin resistance induced by testosterone on both receptor level and post-receptor level. Methods： 3T3-L1 preadipocytes were cultured and differentiated, and the adipocytes and preadipocytes were treated with 0 （testosterone-free DMEM/ F12）, 10^-8 10^-7 and 10^-6 mol/L testosterone overnight. RT-PCR and flow cytometry were used to detected mRNA and cell surface expression of ASP receptor. Both non-testosterone treated and testosterone 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 Gβ, Gαq/11, p-PKCα, and p-PKCζ were measured by Western blot. Results： High dose testosterone suppressed the C5L2 mRNA and protein expression in 3T3-L1 adipocytes but not in preadipocytes. At 10^-6mol/L, testosterone inhibited C5L2 mRNA and cell surface C5L2 expression by 60G （P〈0.01） and 27G （P〈0.01）, respectively. After overnight incubation with testosterone （in adipocytes and preadipocytes）, Gαq/ 11, Gβ, p-PKCα, and p-PKCζ were downregulated in the presence of ASP treatment to a certain degree. In adipoeytes, testosterone effectively blocked the ASP-stimulated Gαq/11, p PKCα, and p-PKCα expression by 52%,50% and 57% （P〈0.05 to P〈0.01, respectively） at 10^-6mol/Lin adipocytes. In preadipocytes, testosterone did not influence the four proteins＇ expression. Conclusion： Testosterone inhibited ASP-stimulated signal proteins. The ASP resistance mechanism of action involves both changes in expression of C5L2 as well as signaling parameters to some extent. Testosterone-induced ASP resistance may contribute to the physiological abnormalities associated with insulin resistance and some abnormalities related with testosterone such as disorders in polycystic ovarian syndrome and so on.