Thyroid-Stimulating Hormone Induces the Secretion of Tumor Necrosis Factor-α from 3T3-L1 Adipocytes via a Protein Kinase A-Dependent Pathway

Abstract

Objective:

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.

Methods:

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-immunopreci­pitations were performed to detect the formation of the IκBα/PKAc complex.

Results:

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.

Conclusion:

TSH stimulated the cAMP-PKA pathway in 3T3-L1 adipocytes to increase TNF-α secretion.

Key words

adipocyte - tumor necrosis factor-alpha - thyroid-stimulating ­hormone - nuclear factor-kappa B - protein kinase A

^* Both authors contributed equally to this study

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