Yokomori N, Tawata M, Onaya T
Third Department of Internal Medicine, Yamanashi Medical University, Tamaho, Japan.
Diabetes. 1999 Apr;48(4):685-90. doi: 10.2337/diabetes.48.4.685.
GLUT4 is the major glucose transporter in adipose tissue and skeletal and cardiac muscles. We examined the mechanisms underlying GLUT4 gene expression in 3T3-L1 cells, which express the gene during their differentiation from preadipocytes to adipocytes. In transient transfections, the activity of a mouse GLUT4 promoter extending to -100 bp in the 5'-flanking region did not differ significantly between 3T3-L1 preadipocytes and adipocytes. Promoter activity up to -590 bp in preadipocytes and adipocytes showed a 70% lower and 228% higher activity, respectively, than promoter activity extending to -100 bp. We also examined methylation status of the GLUT4 promoter. Up to -100 bp, there were five CpG sites at -11, -30, -58, -63, and -75 bp. Two CpG sites at -11 and -30 bp were highly methylated in preadipocytes (60 and 92%, respectively) and highly demethylated in adipocytes (28.6 and 25%, respectively). Conversely, three CpG sites at -58, -63, and -75 bp were highly demethylated in both preadipocytes and adipocytes (<12%). In gel mobility-shift assays, a fragment extending from -40 to -1 bp generated a methylation-sensitive band with nuclear extracts from both preadipocytes and adipocytes when the CpG sites were methylated. Southwestern analysis identified a protein of approximately 55 kDa that bound strongly to the methylated probe. Furthermore, methylation of the CpG sites inhibited promoters extending to -50 or -70 bp. These results suggest that in addition to cell type-specific transcription factor, methylation of specific CpG sites and the methylation-sensitive transcription factor contribute to GLUT4 gene regulation during 3T3-L1 differentiation.
葡萄糖转运蛋白4（GLUT4）是脂肪组织、骨骼肌和心肌中的主要葡萄糖转运体。我们研究了3T3-L1细胞中GLUT4基因表达的潜在机制，该细胞在从前脂肪细胞分化为脂肪细胞的过程中表达该基因。在瞬时转染实验中，5'侧翼区域延伸至-100 bp的小鼠GLUT4启动子活性在3T3-L1前脂肪细胞和脂肪细胞之间没有显著差异。前脂肪细胞和脂肪细胞中延伸至-590 bp的启动子活性分别比延伸至-100 bp的启动子活性低70%和高228%。我们还检测了GLUT4启动子的甲基化状态。在-100 bp范围内，在-11、-30、-58、-63和-75 bp处有五个CpG位点。-11和-30 bp处的两个CpG位点在前脂肪细胞中高度甲基化（分别为60%和92%），在脂肪细胞中高度去甲基化（分别为28.6%和25%）。相反，-58、-63和-75 bp处的三个CpG位点在前脂肪细胞和脂肪细胞中均高度去甲基化（<12%）。在凝胶迁移率变动分析中，当CpG位点被甲基化时，从-40至-1 bp延伸的片段与前脂肪细胞和脂肪细胞的核提取物产生了一条甲基化敏感带。蛋白质印迹分析鉴定出一种约55 kDa的蛋白质，它与甲基化探针强烈结合。此外，CpG位点的甲基化抑制了延伸至-50或-70 bp的启动子。这些结果表明，除细胞类型特异性转录因子外，特定CpG位点的甲基化和甲基化敏感转录因子在3T3-L1细胞分化过程中对GLUT4基因调控起作用。
