Involvement of iNOS and NO in TNF-α–downregulated Resistin Gene Expression in 3T3-L1 Adipocytes

Objective: In order to characterize the regulation of resistin gene expression, we explore the effect of tumornecrosis factor-α (TNF-α) on resistin mRNA expression and its underlying mechanism in 3T3-L1 adipocytes. Methods and Procedures: Differentiated 3T3-L1 adipocytes were treated for 24 h with 0–10 ng/ml of TNF-α or with 2.5 ng/ml of TNF-α for 0–24 h, and then resistin mRNA levels were measured by northern blotting. To further explore the involvement of nitric oxide (NO) in TNF-α–regulated resistin expression, the effect of the NO donor, sodium nitroprusside (SNP), on resistin mRNA levels in adipocytes and the effect of the nitric oxide synthase (NOS) inhibitors, NG-nitro-l-arginine methyl ester (l-NAME), and S, S′−1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea·2HBr (PBITU), on the TNF-α effect in adipocytes were examined. The effects of TNF-α on inducible NOS (iNOS) protein expression in adipocytes were also measured by western blotting. Results: Our results showed that TNF-α caused a dose-dependent reduction in resistin mRNA levels. This effect seemed to be associated with the TNF-α–induced expression of iNOS. The results showed that TNF-α induced iNOS expression and release of NO after 24-h treatment of differentiated 3T3-L1 adipocytes. Pretreatment with l-NAME and PBITU significantly reversed the TNF-α–induced downregulation of resistin expression, while treatment with SNP mimicked the inhibitory effect of TNF-α on resistin expression. In addition, pretreatment with protein tyrosine kinase (PTK) inhibitors, genistein and AG-1288, prevented TNF-α–induced iNOS expression and subsequent resistin downregulation. Discussion: Our data suggest that TNF-α suppresses resistin expression by inducing iNOS expression, thus causing overproduction of NO, which downregulates resistin gene expression.