Alves-Wagner Ana Barbara, Yonamine Caio Yogi, de Fatima Luciana Alves, Festuccia William, Machado Ubiratan Fabres
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil,
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil.
Cell Physiol Biochem. 2019;52(3):580-594. doi: 10.33594/000000041.
BACKGROUND/AIMS: Studies have indicated that sympathetic activity enhances GLUT4 expression (Slc2a4 gene) by activating beta-adrenergic receptors. This could be mediated by a direct enhancer effect of cyclic AMP-responsive element binding protein (CREB) and family members upon Slc2a4 gene. However, a cAMP responsive element (CRE) in Slc2a4 promoter has never been demonstrated.
Slc2a4 CRE-site was searched by in silico analysis. In skeletal muscles from rats displaying high sympathetic activity (SHR), Slc2a4 CRE-site was investigated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay; and Slc2a4 expression was analyzed by RT-qPCR. Functional activity of the CRE-site was investigated by luciferase assay, 2 hours after 8-br-cAMP stimulation, in 3T3L1 adipocytes transientely transfected with native and mutated CRE-sites.
In silico analysis indicated the -480/-473 segment as a putative CRE-site, with 62.5% of identity to CRE consensus sequence, and highly preserved in mouse, rat and human. CREB/CREM binding in this CRE-site was confirmed to occur in vitro (EMSA) and in vivo (ChIP assay). Enhancer activity of this segment in Slc2a4 transcription was confirmed in 3T3-L1 cells. Finally, in extensor digitorum longus muscle from SHR, 80% increase in Slc2a4 mRNA expression was observed to be accompanied by increased CREB/CREM binding into the CRE-site both in vitro and in vivo.
This study demonstrates the presence of a functional CRE-site at -480/-473 sequence of the Slc2a4 gene. This CRE-site has an enhancing activity on Slc2a4 expression and participates in the Slc2a4 increased expression observed in glycolytic muscles of rats displaying high sympathetic activity.
背景/目的：研究表明，交感神经活动通过激活β-肾上腺素能受体增强葡萄糖转运蛋白4（GLUT4，Slc2a4基因）的表达。这可能是由环磷酸腺苷反应元件结合蛋白（CREB）及其家族成员对Slc2a4基因的直接增强子效应介导的。然而，Slc2a4启动子中的环磷酸腺苷反应元件（CRE）从未得到证实。
通过计算机分析搜索Slc2a4 CRE位点。在具有高交感神经活动的大鼠（SHR）的骨骼肌中，通过电泳迁移率变动分析（EMSA）和染色质免疫沉淀（ChIP）分析研究Slc2a4 CRE位点；并通过RT-qPCR分析Slc2a4表达。在用天然和突变的CRE位点瞬时转染的3T3L1脂肪细胞中，在8-溴环磷酸腺苷刺激2小时后，通过荧光素酶测定研究CRE位点的功能活性。
计算机分析表明，-480/-473片段为假定的CRE位点，与CRE共有序列的一致性为62.5%，在小鼠、大鼠和人类中高度保守。该CRE位点中CREB/CREM的结合在体外（EMSA）和体内（ChIP分析）均得到证实。该片段在Slc2a4转录中的增强子活性在3T3-L1细胞中得到证实。最后，在SHR的趾长伸肌中，观察到Slc2a4 mRNA表达增加80%，同时体外和体内CREB/CREM与CRE位点的结合均增加。
本研究证明在Slc2a4基因的-480/-473序列存在一个功能性CRE位点。该CRE位点对Slc2a4表达具有增强活性，并参与在具有高交感神经活动的大鼠的糖酵解肌肉中观察到的Slc2a4表达增加。
