Thyroid-Stimulating Hormone Inhibits Insulin Receptor Substrate-1 Expression and Tyrosyl Phosphorylation in 3T3-L1 Adipocytes by Increasing NF- κ B DNA-Binding Activity

Abstract

Background: Abundant evidence indicates that thyroid-stimulating hormone (TSH) levels are associated with insulin resistance in adipocytes. However, the potential mechanism of the association remains uncertain. The objective of this study was to determine the potential role of TSH in the suppression of insulin receptor substrate-1 (IRS-1) expression and IRS-1 tyrosyl phosphorylation, which might contribute to insulin resistance.

Methods: Mouse 3T3-L1 preadipocytes were differentiated into adipocytes. After treatment with 0.01, 0.1, and 1.0 mIU/ml bovine TSH, the TNF-α concentration in the medium was determined by enzyme-linked immunosorbent assay (ELISA). Nuclear factor-kappa B (NF-κB) DNA-binding activity was quantified by electrophoretic mobility shift assay (EMSA). IRS-1 levels in adipocytes were quantified by Western blotting, and tyrosine phosphorylation was measured by immunoprecipitation.

Results: TSH induced TNF-α secretion in a dose-dependent manner. There was a significant positive correlation between NF-κB DNA-binding activity and TNF-α secretion. This effect and correlation were weakened by BAY 11-7082 (a nuclear NF-κB inhibitor) and H89 (an inhibitor of cyclic adenosine monophosphate- (cAMP-) dependent protein kinase A (PKA)). Treatment of cultured adipocytes with TSH inhibited insulin-stimulated IRS-1 tyrosyl phosphorylation but promoted TSH-dependent secretion of TNF-α and activation of NF-κB DNA-binding activity. The effects of TSH were significantly inhibited by BAY 11-7082 and H89 and were completely blocked by the TNF-α antagonist WP9QY.

Conclusion: TSH inhibited IRS-1 protein expression and tyrosyl phosphorylation in 3T3-L1 adipocytes by stimulating TNF-α production via promotion of NF-κB DNA-binding activity. TSH might play a pivotal role in the development of insulin resistance.

Figure 1

Effect of TSH on the DNA-binding activity of NF-κB in 3T3-L1 adipocytes. (a) Representative autoradiogram showing the NF-κB DNA-binding activity in each group. 1: the control, 2: the TSH concentration of 0.01 mIU/ml, 3: the TSH concentration of 0.1 mIU/ml, 4: the TSH concentration of 1 mIU/ml, 5: after H89; and 6: after BAY 11-7082. (b) NF-κB DNA-binding activity was significantly and dose-dependently upregulated in cells treated with different concentrations of TSH (0.01, 0.1, and 1 mIU/ml) compared with control cells (P < 0.05), especially in cells stimulated with 0.1 mIU/ml and 1 mIU/ml TSH (P < 0.01). (c) Effects of H89 on NF-κB DNA binding. (d) Effects of BAY 11-7082 on NF-κB DNA-binding. Cells cultured under the same conditions were pretreated with H89 (an inhibitor of cAMP-dependent PKA) or BAY 11-7082 (a nuclear NF-κB inhibitor). NF-κB DNA-binding activity was suppressed after treatment with H89 and BAY 11-7082. The DNA-binding activity of NF-κB was quantified by densitometric analysis. The band intensities were normalized relative to the internal control and background. The number 1 represents the control group; 2, 3, and 4 represent the 0.01, 0.1, and 1 mIU/ml TSH groups, respectively; and 5 and 6 represent the H89- and BAY 11-7082-pretreated groups, respectively. The data are presented as the mean ± SD from three independent experiments (^∗∗P < 0.01 vs. the 1 mIU/ml TSH group).

Figure 2

TSH-stimulated 3T3-L1 adipocytes produce TNF-α. Differentiated adipocytes were treated with different concentrations of TSH (0.01, 0.1, and 1 mIU/ml) for 4 h, and the TNF-α concentration in the medium was measured by ELISA. Increasing doses of TSH stimulated 3T3-L1 adipocytes to secrete TNF-α. The data are presented as the mean ± SD (n = 3) (P < 0.05 for all comparisons).

Figure 3

Factors that affect TNF-α secretion from 3T3-L1 adipocytes. (a) Pretreatment of cells with the PKA inhibitor H89 (10 μM) for 15 minutes before treatment with 1 mIU/ml bovine TSH decreased TNF-α levels. (b) Pretreatment with the nuclear NF-κB inhibitor BAY 11-7082 (5 μM) reduced TSH-stimulated TNF-α production. These results suggested that H89 and BAY 11-7082 inhibited the effect of TSH on TNF-α secretion. The data demonstrated that TSH stimulated 3T3-L1 adipocytes to secrete TNF-α via the cAMP-PKA pathway. The data are presented as the mean ± SD (n = 3) (^∗∗P < 0.01).

Figure 4

TSH decreased tyrosine phosphorylation of IRS-1 in 3T3-L1 adipocytes. Cells were pretreated with 100 nM insulin for 10 min and then treated with different concentrations of TSH (0.01, 0.1, and 1 mIU/ml) for 24 h. IRS-1 levels in adipocytes were quantified by Western blotting, and tyrosine phosphorylation was measured by immunoprecipitation. The relative expression levels of IRS-1 and IRS-1-tyr in each treatment group were calculated using β-tubulin as the standard. Treatment with various concentrations of bovine TSH (0.01, 0.1, and 1 mIU/ml) increased the ratio of phosphorylated IRS-1 to total IRS-1 in a dose-dependent manner in 3T3-L1 adipocytes. These data indicated that TSH suppressed IRS-1 tyrosyl phosphorylation in 3T3-L1 adipocytes (P < 0.05 for all comparisons).

Figure 5

BAY 11-7082 and H89 inhibited the TSH-mediated downregulation of tyrosine phosphorylation of IRS-1 in 3T3-L1 adipocytes. (a) After pretreatment with 10 μM H89 for 15 minutes and treatment with 1 mIU/ml bovine TSH, phosphorylated IRS-1 levels were unchanged in 3T3-L1 adipocytes. (b) Pretreatment of cells with BAY 11-7082 inhibited the TSH-dependent increases in phosphorylated IRS-1 levels. The data are presented as the mean ± SD (n = 3) (^∗P < 0.05,  ^∗∗P < 0.01).

Figure 6

Effect of TSH on TNF-α-induced inhibition of insulin signals in 3T3-L1 adipocytes. Adipocytes were treated with 100 nM insulin and then coincubated with 1 mIU/ml TSH in the absence or presence of WP9QY. (a) Cells treated with insulin (the positive control group) exhibited higher IRS-1 tyrosyl phosphorylation levels than untreated control cells (the negative control group). TSH decreased IRS-1 tyrosyl phosphorylation. (b) Pretreatment with WP9QY significantly reversed the TSH-induced decrease in IRS-1 tyrosyl phosphorylation. The data are presented as the mean ± SD (n = 3) (^∗∗P < 0.01).