Chatzopoulou Antonia, Roy Upasana, Meijer Annemarie H, Alia A, Spaink Herman P, Schaaf Marcel J M
Department of Animal Sciences and Health (A.C., A.H.M., H.P.S., M.J.M.S.), Institute of Biology, Leiden University, 2333CC Leiden, The Netherlands; Department of Biophysical Organic Chemistry/Solid State NMR (U.R., A.A.), Leiden Institute of Chemistry, Leiden University, 2333CC Leiden, The Netherlands; and Institute of Medical Physics and Biophysics (A.A.), University of Leipzig, D-04107 Leipzig, Germany.
Endocrinology. 2015 May;156(5):1757-69. doi: 10.1210/en.2014-1941. Epub 2015 Mar 10.
In humans and zebrafish, 2 glucocorticoid (GC) receptor (GR) splice variants exist: the canonical GR α-isoform (GRα), and the GRβ. In the present study, we have used the zebrafish model system in order to reveal genes affected by each of these 2 receptor isoforms. By injecting zebrafish embryos with different splice-blocking morpholinos, we could knock down both GR isoforms or could target the alternative splicing of the GR pre-mRNA in favor of the GRβ. In addition, specific GRβ overexpression was achieved by injecting mRNA. Embryos were treated with the synthetic GC dexamethasone, and transcriptome analysis was performed. Two distinct gene clusters were found that were regulated by GRα: one that was regulated by GRα under basal conditions (presence of endogenous cortisol only), and one that was regulated upon increased activation of GRα (using a pharmacological dose of dexamathasone). GRβ may act as a dominant-negative inhibitor of GRα when GRβ is overexpressed and the GRα expression level is knocked down simultaneously. However, without GRα knockdown, no evidence for this activity was found. In addition, the data indicate regulation of gene transcription through other mechanisms of action by GRβ. We also investigated the concentrations of several metabolites using nuclear magnetic resonance spectroscopy. We found that dexamethasone treatment and knockdown of GRα together with overexpression of GRβ had opposite effects on glucose, amino acid, and fatty acid levels. Thus, we have shed new light on the molecular mechanisms of GC-induced effects on metabolism, which are known to increase the risk of obesity, hyperglycemia, and diabetes.
在人类和斑马鱼中，存在两种糖皮质激素（GC）受体（GR）剪接变体：经典的GRα异构体（GRα）和GRβ。在本研究中，我们使用斑马鱼模型系统来揭示受这两种受体异构体各自影响的基因。通过向斑马鱼胚胎注射不同的剪接阻断吗啉代寡核苷酸，我们可以敲低两种GR异构体，或者靶向GR前体mRNA的可变剪接以有利于GRβ。此外，通过注射mRNA实现了特异性GRβ过表达。用合成糖皮质激素地塞米松处理胚胎，并进行转录组分析。发现了两个由GRα调节的不同基因簇：一个在基础条件下（仅存在内源性皮质醇）由GRα调节，另一个在GRα激活增加时（使用药理剂量的地塞米松）受到调节。当GRβ过表达且GRα表达水平同时被敲低时，GRβ可能作为GRα的显性负抑制剂发挥作用。然而，在没有敲低GRα的情况下，未发现这种活性的证据。此外，数据表明GRβ通过其他作用机制调节基因转录。我们还使用核磁共振光谱研究了几种代谢物的浓度。我们发现地塞米松处理以及GRα敲低与GRβ过表达一起对葡萄糖、氨基酸和脂肪酸水平产生相反的影响。因此，我们为糖皮质激素诱导的代谢效应的分子机制提供了新的见解，已知这些效应会增加肥胖、高血糖和糖尿病的风险。
