The Correlated Research On Thyroid-Stimulating Hormone With Insulin Resistance In Adipocyte

Objective:Epidemiological studies have shown that serum thyroid-stimulating hormone (TSH) levels are positively related to insulin resistance. Since the thyroid-stimulating hormone receptor (TSHR) is not only expressed in thyrocytes but also in preadipocytes and adipocytes. It has been reported that TSH can stimulate 3T3-L1 adipocytes to release interleukin-6 through a cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway. All of the studies did not verify whether or not TSH can directly stimulate adipocytes to produce tumor necrosis factor-alpha (TNF-a). The objective of our study is to test whether TSH can suppress expression and translocation of GLUT4 by stimulating production of TNF-a in OLETF rats 3T3-L1 adipocytes through a cAMP-PKA pathway, and then result in the development of insulin resistance.Methods:1.Research of zoology:Experimental rats were divided into two groups: diabetes mellitus(DM) and normal control(N).There were 8 OLETF rats and 8 LETO rats were used as DM group and N group respectively. At the age of 42 weeks, the serum TSH levels were measured by using Enzyme-Linked Immunosorbent Assay (ELISA). The protein expression of TSHR, Phospho-NF-KBp65 Ser276, TNF-a and GLUT4 in epididymal adipose tissues were quantified by Western blotting. 2.Research of cytology:Mouse 3T3-L1 preadipocytes were induced to differentiate into adipocytes.?These adipocytes were treated with 0.1mIU/ml bovine TSH for up to 24 hours in order to choose the best timepoints. The concentration of TNF-a was measured by ELISA.?We treated cells with 0 to 1.0mIU/mL bovine TSH in order to monitor the production of TNF-a in the conditioned medium. The levels of GLUT4 mRNA were assessed by real time polymerase chain reaction(PCR). Protein levels of total and cell membrane GLUT4 and Phospho-NF-?Bp65 Ser276 were quantified by Western blotting.?fter stimulation with 1?M Forskolin and 1mIU/ml bovine TSH respectively, we monitored the production of TNF-a in the conditioned medium.?efore stimulation with 1mIU/ml bovine TSH, adipocytes were pretreated with 10?M H89,we monitored the production of TNF-a in the conditioned medium and Protein levels of Phospho-NF-KBp65 Ser276. Co-Immunoprecipitation was used to assess the I?B?/PKAc complex formation.?Before stimulation with 1mIU/ml bovine TSH, adipocytes were pretreated with 20 nM Rapamycin. Then we monitored the mRNA,total and cell membrane levels of GLUT4.Results:1.Research of zoology:The serum TSH concentration was higher in DM group than those in N group (P<0.01). The protein expression of TSHR, Phospho-NF-?Bp65 Ser276 and TNF-a in epididymal adipose tissues were increased (P<0.05, or P<0.01),but the protein expression of GLUT4 was decreased (P<0.01) compared with those of control. Correlate analysis showed that The serum TSH concentration was positively correlated with the protein expression of Phospho-NF-?Bp65 Ser276 and TNF-a (P<0.05),and negatively correlated with the protein expression of GLUT4 and IAI (P<0.05, or P<0.0l).2.Research of cytology: When 3T3-L1 adipocytes were treated with 0.1 mIU/ml TSH for 4 hours, the concentration of TNF-a in medium increased (p<0.05). When the concentration of TSH was increased from 0,1 to 1.0 mIU/ml, the level of TNF-a in medium and Phospho-NF-KBp65 Ser276 also increased in a dose dependent manner (p<0.05); meanwhile, the levels of GLUT4 mRNA and protein levels of total and cell membrane GLUT4 decreased in a dose dependent manner (P<0.05). H89 and Rapamycin could block the effects respectively. However, there was no statistical difference for the TNF-a levels between stimulation with 1?M Forskolin and 1mIU/ml bovine TSH (P>0.05). The I?B?/PKAc complex was present in the cells stimulated 1mIU/ml bovine TSH. The I?B?/PKAc complex formation was also inhibited by H89.Conclusion:?TSH may result in the development of insulin resistance by stimulating TSHR on extrathyroidal adipose cells.?SH can stimulate 3T3-L1 adipocytes to produce TNF-a in a dose dependent manner.?TSH can down-regulate GLUT4 expression and translocation in 3T3-L1 adipocytes via activting NF-?B, and then stimulate production of TNF-a through a cAMP-PKA pathway. TSH maybe attribute to the development of insulin resistance.