Yamaguchi University, Graduate School of Medicine, Division of Endocrinology, Metabolism, Hematological Sciences and Therapeutics, Department of Bio-Signal Analysis, Ube, Yamaguchi 755-8505, Japan.
Biochem Biophys Res Commun. 2013 May 3;434(2):370-5. doi: 10.1016/j.bbrc.2013.03.084. Epub 2013 Apr 6.
Aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia inducible factor-1β (HIF-1β) has emerged as a potential determinant of pancreatic β-cell dysfunction and type 2 diabetes in humans. An 82% reduction in Arnt expression was observed in islets from type 2 diabetic donors as compared to non-diabetic donors. However, few regulators of Arnt expression have been identified. Meanwhile, disruption of the clock components CLOCK and BMAL1 is known to result in hypoinsulinemia and diabetes, but the molecular details remain unclear. In this study, we identified a novel molecular connection between Arnt and two clock-controlled output genes, albumin D-element binding protein (Dbp) and E4 binding protein 4 (E4bp4). By conducting gene expression studies using the islets of Wfs1(-/-) A(y)/a mice that develop severe diabetes due to β-cell apoptosis, we demonstrated clock-related gene expressions to be altered in the diabetic mice. Dbp mRNA decreased by 50%, E4bp4 mRNA increased by 50%, and Arnt mRNA decreased by 30% at Zeitgever Time (ZT) 12. Mouse pancreatic islets exhibited oscillations of clock gene expressions. E4BP4, a D-box negative regulator, oscillated anti-phase to DBP, a D-box positive regulator. We also found low-amplitude circadian expression of Arnt mRNA, which peaked at ZT4. Over-expression of DBP raised both mRNA and protein levels of ARNT in HEK293 and MIN6 cell lines. Arnt promoter-driven luciferase reporter assay in MIN6 cells revealed that DBP increased Arnt promoter activity by 2.5-fold and that E4BP4 competitively inhibited its activation. In addition, on ChIP assay, DBP and E4BP4 directly bound to D-box elements within the Arnt promoter in MIN6 cells. These results suggest that in mouse pancreatic islets mRNA expression of Arnt fluctuates significantly in a circadian manner and that the down-regulation of Dbp and up-regulation E4bp4 contribute to direct suppression of Arnt expression in diabetes.
芳香烃受体核转位蛋白（ARNT）/缺氧诱导因子-1β（HIF-1β）已成为人类胰腺β细胞功能障碍和 2 型糖尿病的潜在决定因素。与非糖尿病供体相比，2 型糖尿病供体的胰岛中 ARNT 的表达减少了 82%。然而，已经确定了很少的 ARNT 表达调节剂。同时，时钟成分 CLOCK 和 BMAL1 的破坏已知会导致胰岛素分泌不足和糖尿病，但分子细节尚不清楚。在这项研究中，我们发现了 ARNT 与两个时钟控制输出基因白蛋白 D 元素结合蛋白（Dbp）和 E4 结合蛋白 4（E4bp4）之间的新的分子联系。通过使用由于β细胞凋亡而发生严重糖尿病的 Wfs1(-/-) A(y)/a 小鼠的胰岛进行基因表达研究，我们证明了糖尿病小鼠的时钟相关基因表达发生了改变。Dbp mRNA 减少了 50%，E4bp4 mRNA 增加了 50%，而 Arnt mRNA 在 Zeitgever Time (ZT) 12 时减少了 30%。小鼠胰腺胰岛表现出时钟基因表达的振荡。E4BP4，一种 D 框负调节剂，与 D 框正调节剂 DBP 反相振荡。我们还发现 Arnt mRNA 的低幅度昼夜节律表达，其峰值在 ZT4。在 HEK293 和 MIN6 细胞系中，Dbp 的过表达提高了 ARNT 的 mRNA 和蛋白水平。MIN6 细胞中 Arnt 启动子驱动的荧光素酶报告基因分析显示，Dbp 使 Arnt 启动子活性增加了 2.5 倍，而 E4BP4 竞争性抑制其激活。此外，在 ChIP 测定中，Dbp 和 E4BP4 在 MIN6 细胞中直接结合到 Arnt 启动子的 D 框元件上。这些结果表明，在小鼠胰腺胰岛中，Arnt 的 mRNA 表达以昼夜节律的方式显著波动，并且 Dbp 的下调和 E4bp4 的上调有助于直接抑制糖尿病中的 Arnt 表达。
