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

Abstract

In order to characterize the regulation of resistin gene expression, we explore the effect of tumornecrosis factor-alpha (TNF-alpha) on resistin mRNA expression and its underlying mechanism in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were treated for 24 h with 0-10 ng/ml of TNF-alpha or with 2.5 ng/ml of TNF-alpha 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-alpha-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, N(G)-nitro-L-arginine methyl ester (L-NAME), and S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea.2HBr (PBITU), on the TNF-alpha effect in adipocytes were examined. The effects of TNF-alpha on inducible NOS (iNOS) protein expression in adipocytes were also measured by western blotting. Our results showed that TNF-alpha caused a dose-dependent reduction in resistin mRNA levels. This effect seemed to be associated with the TNF-alpha-induced expression of iNOS. The results showed that TNF-alpha 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-alpha-induced downregulation of resistin expression, while treatment with SNP mimicked the inhibitory effect of TNF-alpha on resistin expression. In addition, pretreatment with protein tyrosine kinase (PTK) inhibitors, genistein and AG-1288, prevented TNF-alpha-induced iNOS expression and subsequent resistin downregulation. Our data suggest that TNF-alpha suppresses resistin expression by inducing iNOS expression, thus causing overproduction of NO, which downregulates resistin gene expression.