Angiopoietin-like 4 (Angptl4) is a glucocorticoid receptor (GR) primary target gene in hepatocytes and adipocytes. It encodes a secreted protein that inhibits extracellular LPL and promotes adipocyte lipolysis. In Angptl4 null mice, glucocorticoid-induced adipocyte lipolysis and hepatic steatosis are compromised. Markedly, insulin suppressed glucocorticoid-induced Angptl4 transcription. To unravel the mechanism, we utilized small molecules to inhibit insulin signaling components and found that phosphatidylinositol 3-kinase and Akt were vital for the suppression in H4IIE cells. A forkhead box transcription factor response element (FRE) was found near the 15 bp Angptl4 glucocorticoid response element (GRE). Mutating the Angptl4 FRE significantly reduced glucocorticoid-induced reporter gene expression in cells. Moreover, chromatin immunoprecipitation revealed that GR and FoxO1 were recruited to Angptl4 GRE and FRE in a glucocorticoid-dependent manner, and cotreatment with insulin abolished both recruitments. Furthermore, in 24 h fasted mice, significant occupancy of GR and FoxO1 at the Angptl4 GRE and FRE was found in the liver. In contrast, both occupancies were diminished after 24 h refeeding. Finally, overexpression of dominant negative FoxO1 mutant abolished glucocorticoid-induced Angptl4 expression, mimicking the insulin suppression. Overall, we demonstrate that both GR and FoxO1 are required for Angptl4 transcription activation, and that FoxO1 negatively mediates the suppressive effect of insulin.
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机译:血管生成素样4(Angptl4)是肝细胞和脂肪细胞中糖皮质激素受体(GR)的主要靶基因。它编码一种抑制细胞外LPL并促进脂肪细胞脂解的分泌蛋白。在Angptl4 null小鼠中,糖皮质激素诱导的脂肪细胞脂解和肝脂肪变性受到损害。明显地,胰岛素抑制了糖皮质激素诱导的Angptl4转录。为了阐明这一机制,我们利用小分子抑制胰岛素信号传导成分,发现磷脂酰肌醇3-激酶和Akt对抑制H4IIE细胞至关重要。在15 bp Angptl4糖皮质激素反应元件(GRE)附近发现了一个叉头盒转录因子反应元件(FRE)。突变Angptl4 FRE显着降低了糖皮质激素诱导的报告基因在细胞中的表达。此外,染色质免疫沉淀显示GR和FoxO1以糖皮质激素依赖性方式被募集到Angptl4 GRE和FRE,并且与胰岛素的共同治疗消除了这两个募集。此外,在禁食的24小时小鼠的肝脏中,Angptl4 GRE和FRE上大量占有GR和FoxO1。相反,重新喂食24小时后,这两种占用率均降低。最后,显性负FoxO1突变体的过表达消除了糖皮质激素诱导的Angptl4表达,模仿了胰岛素的抑制作用。总的来说,我们证明了GR和FoxO1都是Angptl4转录激活所必需的,并且FoxO1负调节了胰岛素的抑制作用。
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