促甲状腺激素受体对3T3-L1脂肪细胞葡萄糖转运体4表达的抑制作用

摘要

目的 探讨促甲状腺激素(TSH)是否通过激活环腺苷酸-蛋白激酶A(eAMP-PKA)通路,促进3T3-L1脂肪细胞分泌肿瘤坏死因子-α(TNF-α),进一步影响脂肪细胞葡萄糖转运蛋白4(GLUT4)的基因及蛋白的表达和转位,参与脂肪细胞胰岛素抵抗发生的慢性非特异性炎症过程.方法 体外诱导3T3-L1前脂肪细胞成为成熟脂肪细胞.采用牛促甲状腺素(TSH)刺激脂肪细胞,并分别予以腺苷酸环化酶激活剂(Forskolin)、蛋白激酶A抑制剂(H89)、雷帕霉素(Rapamycin)干预,酶联免疫吸附试验(ELISA)法检测培养基TNF-α水平,实时荧光定量聚合酶链反应(Real time PCR)法检测细胞GLUT4 mRNA表达水平,Western blotting方法检测细胞GLUT4总蛋白及膜蛋白表达变化. 结果 随着TSH刺激时间的延长和刺激浓度的增加,3T3-L1脂肪细胞培养基内TNF-α浓度呈上升趋势(P＜0.05)；Forskolin组与牛TSH组(0.04 μmmol/L)比较,TNF-α水平变化差异无统计学意义[(481.9±28.4)ng/L比(522.7±36.2)ng/L,P＞0.05]；与空白对照组比较,H89干预组(10 μmmol/L) TNF-α水平无明显升高[(326.7±43.2)ng/L比(341.9±12.0) ng/L,P＞0.05]；随着TSH刺激浓度的增加(0.0004、0.0040和0.0400 μmol/L),与对照组比较GLUT4 mRNA表达(0.25±0.11、0.10±0.03、0.04±0.02比0.40±0.13)、总蛋白(1.37±0.19、1.01±0.05、0.46±0.11比1.79±0.22)表达及膜蛋白的相对表达水平呈下降趋势,差异有统计学意义(P＜0.05或P＜0.01),Rapamycin可以逆转TSH对GLUT4的下调作用. 结论 TSH与3T3-L1脂肪细胞表面TSH受体结合后,可能通过激活eAMP-PKA途径,促进TNF-α分泌,进一步下调GLUT4的表达并抑制其转位,并有可能参与胰岛素抵抗的发生.%Objective To test thyroid-stimulating hormone (TSH) suppress GLUT4 expression and translocation by stimulating TNF-α secretion in 3T3-L1 adipocytes via a cAMP-PKA pathway.Methods 3T3-L1 preadipocytes were induced to differentiate into adipocytes.The adipocytes were treated with bovine TSH,Forskolin,H89 and Rapamycin,respectively.The concentration of TNF-α in the cell culture medium was measured by ELISA.The level of GLUT4 mRNA in adipocytes was assessed by real time polymerase chain reaction.Protein levels of GLUT4 in total cell lysates and plasma membrane lysates were quantified by Western blotting.Results Incubating 3T3-L1 adipocytes with TSH markedly increased the concentration of TNF-α in medium in a time-and dosedependent manner (P ＜ 0.05); meanwhile,the levels of GLUT4 mRNA and total and plasma membrane GLUT4 protein were decreased in a dose-dependent manner (P＜0.05 or ＜0.01).H89 and rapamycin could block the above effects respectively (326.7±43.2 vs.341.9±12.0,P＞0.05).However,there was no statistical difference in the TNF-α levels between stimulation with 1 μmmol/L forskolin versus 0.04 μmmol/L bovine TSH (481.9± 28.4 vs.522.7± 36.2,P＞0.05).Conclusions TSH can down-regulate GLUT4 expression and translocation in 3T3-L1 adipocytes by stimulating TNF-α secretion through a cAMP-PKA pathway.