Cornelius P, Marlowe M, Call K, Pekala P H
Department of Biochemistry, School of Medicine, East Carolina University, Greenville, North Carolina 27858.
J Cell Physiol. 1991 Feb;146(2):298-308. doi: 10.1002/jcp.1041460215.
In the present study we have examined the ability of 8-bromoadenosine cyclic 3',5'-phosphate (8-bromo-cAMP; the membrane permeant analog of cAMP which can activate protein kinase A) to mimic hormone action and stimulate glucose transport and glucose transporter (GLUT-1) gene expression as well as the expression of several growth-related protooncogenes in quiescent 3T3-L1 fibroblasts. 8-Bromo-cAMP induced a rapid and prolonged increase in the rate of hexose transport. Early activation of hexose transport (within 30 min) was associated with increased plasma membrane immunoreactive glucose transporters, which corresponded to a doubling in the number of D-glucose-displaceable, plasma membrane cytochalasin B binding sites. The time course for 8-bromo-cAMP-induced hexose transport preceded the accumulation of GLUT-1 mRNA, which peaked between 4 and 8 h after exposure to the agent, and subsequently declined to approach basal (control) levels. Expression of the immediate-early genes c-fos and jun-B was induced by 8-bromo-cAMP on a rapid, but sustained time course, whereas induction of c-jun expression was delayed. Alterations in specific mRNAs following exposure to 8-bromo-cAMP were due to increased gene transcription (as judged by nuclear transcription run-on assays), although with respect to GLUT-1, an increase in mRNA stability was also observed. Treatment of the cells with forskolin resulted in the induction of GLUT-1 expression as well as expression of the immediate early genes. Exposure of quiescent 3T3-L1 fibroblasts to 8-bromo-cAMP resulted in a substantial increase in rates of total protein and RNA synthesis, but had little effect on DNA synthesis. The results demonstrate that 8-bromo-cAMP initiated a G0/G1 transition, but did not permit progression into S-phase. The results further suggest that increased cytosolic cAMP results in the stimulation of glucose transport by three distinct mechanisms to include translocation of pre-existing transporters, increased transcription of the GLUT-1 gene and increased stability of GLUT-1 mRNA.
在本研究中，我们检测了8-溴腺苷环3',5'-磷酸（8-溴-cAMP；可激活蛋白激酶A的cAMP膜渗透类似物）模拟激素作用、刺激静止的3T3-L1成纤维细胞中葡萄糖转运及葡萄糖转运蛋白（GLUT-1）基因表达以及几种生长相关原癌基因表达的能力。8-溴-cAMP诱导己糖转运速率迅速且持续增加。己糖转运的早期激活（30分钟内）与质膜免疫反应性葡萄糖转运蛋白增加有关，这相当于D-葡萄糖可置换的质膜细胞松弛素B结合位点数量翻倍。8-溴-cAMP诱导己糖转运的时间进程先于GLUT-1 mRNA的积累，GLUT-1 mRNA在接触该试剂后4至8小时达到峰值，随后下降至接近基础（对照）水平。即刻早期基因c-fos和jun-B的表达由8-溴-cAMP以快速但持续的时间进程诱导，而c-jun表达的诱导则延迟。接触8-溴-cAMP后特定mRNA的改变是由于基因转录增加（通过核转录延伸分析判断），尽管对于GLUT-1，还观察到mRNA稳定性增加。用毛喉素处理细胞导致GLUT-1表达以及即刻早期基因的表达。静止的3T3-L1成纤维细胞暴露于8-溴-cAMP导致总蛋白和RNA合成速率大幅增加，但对DNA合成影响很小。结果表明8-溴-cAMP引发了G0/G1期转变，但不允许进入S期。结果进一步表明，胞质cAMP增加通过三种不同机制刺激葡萄糖转运，包括现有转运蛋白的易位、GLUT-1基因转录增加以及GLUT-1 mRNA稳定性增加。
