Zhang Y-J, Zhao W, Zhu M-Y, Tang S-S, Zhang H
2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, The Key Laboratory of Hormones and Development (Ministry of Health), Metabolic Diseases Hospital & Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, China.
Exp Clin Endocrinol Diabetes. 2013 Aug;121(8):488-93. doi: 10.1055/s-0033-1347266. Epub 2013 Jul 17.
Numerous reports have suggested that thyroid-stimulating hormone (TSH) contributes to insulin resistance in adipocytes by directly stimulating the production of adipokines, such as tumor necrosis factor α (TNF-α). The objective of this study was to clarify how TSH regulates TNF-α secretion in 3T3-L1 adipocytes and to determine which cell signaling pathways were involved.
Mouse 3T3-L1 preadipocytes were differentiated into adipocytes and then exposed to 0.1 mIU/ml bovine TSH. The optimal treatment duration was determined by measuring the TNF-α concentration in the medium by ELISA. Thereafter, adipocytes were treated with 0.01, 0.1, and 1.0 mIU/ml bovine TSH, and the optimal TSH dose was determined. To decrease TSHR expression, TSHR shRNA was transfected into adipocytes, and the silencing was confirmed by Western blotting. The TSH signaling pathways responsible for regulating TNF-α secretion were studied. Phospho-NF-κBp65 Ser276 was quantified by Western blotting, and co-immunopreci-pitations were performed to detect the formation of the IκBα/PKAc complex.
TNF-α secretion from adipocytes peaked 4 h after TSH treatment. TSH induced TNF-α secretion in a dose-dependent manner, which was almost completely inhibited by TSHR shRNA. There was a significant positive correlation between phospho-NF-κBp65 Ser276 levels and TNF-α secretion. H89, a cAMP-dependent protein kinase A inhibitor, significantly inhibited the effects of TSH. Bovine TSH and forskolin, which increases intracellular cAMP, simultaneously stimulated TNF-α secretion. The IκBα/PKAc complex could be detected in TSH-treated cells, but complex formation was inhibited by H89.
TSH stimulated the cAMP-PKA pathway in 3T3-L1 adipocytes to increase TNF-α secretion.
众多报告表明，促甲状腺激素（TSH）通过直接刺激脂肪因子如肿瘤坏死因子α（TNF-α）的产生，导致脂肪细胞中的胰岛素抵抗。本研究的目的是阐明TSH如何调节3T3-L1脂肪细胞中TNF-α的分泌，并确定涉及哪些细胞信号通路。
将小鼠3T3-L1前脂肪细胞分化为脂肪细胞，然后用0.1 mIU/ml牛TSH处理。通过ELISA测量培养基中的TNF-α浓度来确定最佳处理持续时间。此后，用0.01、0.1和1.0 mIU/ml牛TSH处理脂肪细胞，并确定最佳TSH剂量。为降低TSHR表达，将TSHR shRNA转染到脂肪细胞中，并通过蛋白质印迹法确认沉默效果。研究了负责调节TNF-α分泌的TSH信号通路。通过蛋白质印迹法定量磷酸化的NF-κBp65 Ser276，并进行免疫共沉淀以检测IκBα/PKAc复合物的形成。
TSH处理后4小时，脂肪细胞中TNF-α的分泌达到峰值。TSH以剂量依赖性方式诱导TNF-α分泌，TSHR shRNA几乎完全抑制了这种分泌。磷酸化的NF-κBp65 Ser276水平与TNF-α分泌之间存在显著正相关。cAMP依赖性蛋白激酶A抑制剂H89显著抑制了TSH的作用。牛TSH和增加细胞内cAMP的福斯高林同时刺激TNF-α分泌。在TSH处理的细胞中可检测到IκBα/PKAc复合物，但H89抑制了复合物的形成。
TSH刺激3T3-L1脂肪细胞中的cAMP-PKA途径以增加TNF-α分泌。
