Identification of transcription factor which regulates obesity-linked downregulation of adiponectin gene and development of treatment modality for metabolic syndrome

为了阐明肥胖导致脂联素（Adiponectin，Ad）基因下调的机制，我们首先在体外肥大脂肪细胞模型中，利用功能启动子研究和电泳迁移率变动分析（Electrophoretic Mobility Shift Assay，EMSAs）技术，鉴定了Adiponectin基因的启动子区及其相关转录因子。我们鉴定了一个这样的启动子区域，我们将其命名为糖尿病/肥胖相关元件（DRE）及其结合蛋白（Dre结合蛋白; Deb）。酵母单杂交筛选结果表明，Deb 1基因是KLF家族的转录因子，EMSA和染色质免疫沉淀实验表明，Deb 1确实与DRE结合。Deb 1剂量依赖性和特异性地增强Ad启动子活性。通过Deb 1 siRNA和Deb 1过表达在体外对内源性Ad mRNA的相应变化证实了Deb 1对Ad转录的调节。有针对性地破坏Deb 1导致了部分但显著的逆转。 ...更多信息 WAT中Ad表达和血浆Ad水平的排出。有趣的是，Deb 1的表达及其与DRE的结合在肥胖小鼠如ob/ob小鼠中减少。此外，我们发现，在肥大脂肪细胞内观察到的氧化应激和炎症增加以及激素受体信号传导的变化减少了Deb 1和Ad，并且这些细胞内改变的正常化增加了Deb 1和Ad。总之，这些结果表明：1）肥胖通过多种途径减少Deb 1，例如氧化应激和炎症，这导致Ad减少，2）WAT中的Deb 1激活剂可能为代谢综合征提供一种新的治疗模式（论文正在撰写中）。此外，我们发现肥胖患者不仅血浆脂联素水平降低，而且AdipoR 1/R2表达水平也降低，这似乎在胰岛素抵抗的发生中起着因果作用。此外，我们表明，不仅AdipoR 1/R2的激动作用，而且在脂联素存在下增加AdipoR 1/R2的策略确实导致胰岛素抵抗和2型糖尿病的改善（Nat.Med.13：332，2007）。少

To clarify the mechanism by which obesity results in downregulation of Adiponectin (Ad) gene, we first tried to identify the promoter region of the Ad gene that confers the response to obesity and its responsible transcription factor(s) by using a combination of functional promoter studies and electrophoretic mobility shift assays (EMSAs) techniques in hypertrophic adipocytes model in vitro. We identified one such promoter region, which we named Diabetes/obesity-Related Element (DRE), and its binding protein(s) (Dre binding protein; Deb). Deb1 gene turned out to be a KLF family transcription factor by yeast one-hybrid screen.EMSAs and chromatin immunoprecipitation assays revealed that Deb1 indeed bound to DRE. Deb1 dose-dependently and specifically enhanced Ad promoter activity. Regulation of Ad transcription by Deb1 was confirmed by corresponding changes in endogenous Ad mRNA by Deb1 siRNA and Deb1 overexpression in vitro. Targeted disruption of Deb1 caused a partial but significant r … More eduction of Ad expression in WAT and plasma Ad levels. Interestingly, expression of Deb1 and its binding to DRE were reduced in obese mice such as ob/ob mice. Moreover, we found that increased oxidative stress and inflammation as well as changes in hormonal receptor signalling observed within hypertrophic adipocytes reduced Deb1 and Ad, and that normalization of these intracellular alterations increased Deb1 and Ad.Taken together, these results suggested that 1) obesity decreases Deb1 via multiple pathways such as oxidative stress and inflammation, which leads to Ad reduction, 2) Deb1 activators in WAT may provide a novel treatment modality for metabolic syndrome (manuscript in preparation).Moreover, we showed that not only plasma adiponectin levels but also expression levels of AdipoR1/R2 were decreased in obesity, which appeared to play causal roles in the development of insulin resistance. Furthermore, we showed that not only agonism of AdipoR1/R2 but also strategies to increase AdipoR1/R2 in the presence of adiponectin indeed resulted in amelioration of insulin resistance and type 2 diabetes (Nat. Med. 13:332,2007). Less