Leptin, one of the adipocytesecreted peptides, is involved in the control of appetite and body weight. Several studies have demonstrated that plasma leptin levels are elevated in obese subjects and are positively correlated with body weight. The arterial endothelin (ET) system plays an important role in the regulation of vascular tone, and ET-1 overexpression may be involved in the pathogenesis of the hypertension associated with insulin resistance. This study was performed to explore the regulatory effects of leptin on ET receptor expression and ET binding in A10 vascular smooth muscle cells (VSMCs) by use of Northern blotting, immunoblotting, and a 125 I-labeled ET-1 binding assay. The effect of leptin on ET receptor-mediated cell proliferation was also tested. The results showed that leptin caused a significant increase in [125 I]-ET-1 binding, which was time-and dose-dependent. Immunoblotting showed that expression of the ET type A receptor (ET AR) in leptin (10 Ϫ7 M)-treated cells was increased by up to 2.3-fold compared with controls. Levels of ET AR mRNA measured by Northern blotting were also increased by up to 2.2-fold in leptin (10 Ϫ7 M)-treated cells. Pretreatment with an ERK inhibitor, PD-98059 (2.5 ϫ 10 Ϫ5 M), blocked the leptin-induced increase in 125 I-ET-1 binding. Finally, ET-1 (10 Ϫ7 M)-stimulated cell proliferation was enhanced by leptin (10 Ϫ7 M) pretreatment, with a maximal increase of twofold compared with controls. In conclusion, leptin increases ET AR expression in VSMCs in a time-and dose-dependent manner. This effect is ERK dependent and is associated with increased ET-1-stimulated cell proliferation. These findings provide support for roles for leptin and the ET system in the pathogenesis of obesity-associated hypertension.endothelin-1; proliferation; leptin receptor; extracellular signal-regulated kinase

objective: In order to characterize the regulation of resistin gene expression, we explore the effect of tumor necrosis 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, N G -nitro-l-arginine methyl ester (l-NAME), and S,Sʹ-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.