Armoni Michal, Harel Chava, Karni Shiri, Chen Hui, Bar-Yoseph Fabiana, Ver Marel R, Quon Michael J, Karnieli Eddy
Institute of Endocrinology, Diabetes and Metabolism, Rambam Medical Center, Haifa, Israel.
J Biol Chem. 2006 Jul 21;281(29):19881-91. doi: 10.1074/jbc.M600320200. Epub 2006 May 2.
FOXO1 and peroxisome proliferator-activated receptor-gamma (PPARgamma) are crucial transcription factors that regulate glucose metabolism and insulin responsiveness in insulin target tissues. We have shown that, in primary rat adipocytes, both factors regulate transcription of the insulin-responsive GLUT4 gene and that PPARgamma2 detachment from the GLUT4 promoter upon thiazolidinedione binding up-regulates GLUT4 gene expression, thus increasing insulin sensitivity (Armoni, M., Kritz, N., Harel, C., Bar-Yoseph, F., Chen, H., Quon, M. J., and Karnieli, E. (2003) J. Biol. Chem. 278, 30614-30623). However, the mechanisms regulating PPARgamma gene transcription are largely unknown. We studied the effects of FOXO1 on human PPARgamma gene expression in primary rat adipocytes and found that both genes are endogenously expressed. FOXO1 coexpression dose-dependently repressed transcription from either the PPARgamma 1 or PPARgamma2 promoter reporter by 65%, whereas insulin (100 nm, 20-24 h) either partially or completely reversed this effect. Phosphorylation-defective FOXO1 mutants T24A, S256A, S319A, and T24A/S256A/S319A still repressed the PPARgamma1 promoter and partially lost their effects on the PPARgamma2 promoter in either basal or insulin-stimulated cells. Use of DNA binding-defective FOXO1 (H215R) indicated that this domain is crucial for FOXO1 repression of the PPARgamma2 (but not PPARgamma1) promoter. Progressive 5'-deletion and gel retardation analyses revealed that this repression involves direct and specific binding of FOXO1 to the PPARgamma2 promoter; chromatin immunoprecipitation analysis confirmed that this binding occurs in cellulo. We suggest a novel paradigm to increase insulin sensitivity in adipocytes in which FOXO1 repression of PPARgamma, the latter being a repressor of the GLUT4 promoter, consequently leads to GLUT4 derepression/up-regulation, thus enhancing cellular insulin sensitivity. The newly identified FOXO1-binding site on the PPARgamma2 promoter may serve as a therapeutic target for type 2 diabetes.
FOXO1和过氧化物酶体增殖物激活受体γ（PPARγ）是关键的转录因子，它们调节胰岛素靶组织中的葡萄糖代谢和胰岛素反应性。我们已经表明，在原代大鼠脂肪细胞中，这两种因子都调节胰岛素反应性葡萄糖转运蛋白4（GLUT4）基因的转录，并且噻唑烷二酮结合后PPARγ2从GLUT4启动子上脱离会上调GLUT4基因表达，从而增加胰岛素敏感性（Armoni，M.，Kritz，N.，Harel，C.，Bar-Yoseph，F.，Chen，H.，Quon，M. J.，和Karnieli，E.（2003）J. Biol. Chem. 278，30614 - 30623）。然而，调节PPARγ基因转录的机制在很大程度上尚不清楚。我们研究了FOXO1对原代大鼠脂肪细胞中人类PPARγ基因表达的影响，发现这两个基因都是内源性表达的。FOXO1共表达以剂量依赖的方式使PPARγ1或PPARγ2启动子报告基因的转录抑制65%，而胰岛素（100 nM，20 - 24小时）部分或完全逆转了这种作用。磷酸化缺陷型FOXO1突变体T24A、S256A、S319A和T24A/S256A/S319A在基础或胰岛素刺激的细胞中仍能抑制PPARγ1启动子，并部分丧失其对PPARγ2启动子的作用。使用DNA结合缺陷型FOXO1（H215R）表明该结构域对于FOXO1对PPARγ2（而非PPARγ1）启动子的抑制至关重要。逐步5' - 缺失和凝胶阻滞分析表明这种抑制涉及FOXO1与PPARγ2启动子的直接和特异性结合；染色质免疫沉淀分析证实这种结合在细胞内发生。我们提出了一种在脂肪细胞中增加胰岛素敏感性的新范式，其中FOXO1对PPARγ的抑制，而PPARγ是GLUT4启动子的抑制因子，从而导致GLUT4去抑制/上调，进而增强细胞胰岛素敏感性。在PPARγ2启动子上新鉴定的FOXO1结合位点可能作为2型糖尿病的治疗靶点。
