ChREBP rather than USF2 regulates glucose stimulation of endogenous L-pyruvate kinase expression in insulin-secreting cells
ChREBP rather than USF2 regulates glucose stimulation of endogenous L-pyruvate kinase expression in insulin-secreting cells
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DOI：
10.1074/jbc.m201635200
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发表时间：
2002-09-06
影响因子：
4.8
通讯作者：
Wollheim, CB
中科院分区：
文献类型：
--
作者：
Wang, HY;Wollheim, CB
上游刺激因子（USF）是否调节肝细胞中L-丙酮酸激酶（L-PK）启动子活性的葡萄糖响应性存在争议。已经表明USF-2是葡萄糖刺激单个胰岛β细胞和INS-1细胞中L-PK启动子活性所必需的（Kennedy，H. J.，Viollet，B.，拉菲克岛，Kahn，A.，和Rutter，G. A.（1997）J. Biol. Chem 272，20636-20640）。在本研究中，tet-on系统已被用于实现在INS-1细胞中USF-1和USF-2及其显性负突变体DN-USF-1（DeltabTDU 1）和DN-USF-2（TDU 2）的严格控制和诱导表达。定量北方印迹分析显示，USF-1和USF-2的过表达既不影响内源性L-PK mRNA的基础水平，也不影响内源性L-PK mRNA的葡萄糖响应性。同样，L-PK表达不受USF功能的显性负抑制的影响。Western blotting证实USF-1和USF-2以及DN-USF-1和DN-USF-2蛋白在INS-1细胞的核组分中稳定表达。免疫荧光染色表明这些转基因编码的蛋白在细胞核中的均匀诱导。使用L-PK启动子片段的电泳迁移率变动分析揭示USF-1和-2的诱导显著增强USF结合活性，而DN-USF-1和-2消除结合。DN-USF-1和DN-USF-2通过与内源性USF蛋白形成非功能性异源二聚体发挥其显性-负效应。碳水化合物反应元件结合蛋白（ChREBP）最近被证明可以调节肝细胞中L-PK启动子活性的葡萄糖反应性。我们现在报告这种转录因子在大鼠胰岛和INS-1细胞中的存在。葡萄糖刺激INS-1细胞中的ChREBP转录，如核运行实验所示。在INS-1细胞中使用tet-on系统过表达ChREBP导致L-PK表达的葡萄糖响应性左移和增强的L-PK启动子活性。内源性和多西环素诱导的ChREBP蛋白都以葡萄糖依赖性方式与L-PK启动子结合。这些前所未有的结果表明，ChREBP而不是USF介导葡萄糖促进的胰岛素分泌细胞中的L-PK表达。
There is controversy whether or not upstream stimulatory factors (USF) regulate the glucose responsiveness of L-pyruvate kinase (L-PK) promoter activity in hepatocytes. It has been suggested that USF-2 is required for glucose stimulation of L-PK promoter activity in single islet beta-cells and INS-1 cells (Kennedy, H. J., Viollet, B., Rafiq, I., Kahn, A., and Rutter, G. A. (1997) J. Biol. Chem 272, 20636-20640). In the present study, the tet-on system has been employed to achieve tightly controlled and inducible expression of USF-1 and -2 and their dominant-negative mutants DN-USF-1 (DeltabTDU1) and.2 (TDU2) in INS-1 cells. Quantitative Northern blot analysis shows that neither basal level nor glucose responsiveness of endogenous L-PK m-RNA is affected by overexpression of USF-1 and -2. Likewise, the L-PK expression is unaltered by dominant-negative suppression of USF function. Western blotting demonstrates that USF-1 and -2 and DN-USF-1 and -2 proteins are stably expressed in nuclear fractions of INS-1 cells. Immunofluorescence staining indicates the uniform induction of these transgene-encoded proteins in the cell nuclei. Electrophoretic mobility shift assays using the L-PK promoter segment reveal that induction of USF-1 and -2 dramatically enhances the USF binding activity, whereas DN-USF-1 and -2 abolish binding. DN-USF-1 and -2 exert their dominant-negative effect by forming non-functional heterodimers with endogenous USF proteins. Carbohydrate response element-binding protein (ChREBP) was recently shown to regulate the glucose responsiveness of the L-PK promoter activity in hepatocytes. We now report the presence of this transcription factor in rat islets and INS-1 cells. Glucose stimulates ChREBP transcription in INS-1 cells, as shown by nuclear run-on experiments. Overexpression of ChREBP in INS-1 cells using the tet-on system results in a left shift of glucose responsiveness of L-PK expression and an enhanced L-PK promoter activity. Both endogenous and doxycycline-induced ChREBP proteins bind to the L-PK promoter in a glucose-dependent manner. These unprecedented results suggest that ChREBP rather than USF mediates glucose-promoted L-PK expression in insulin-secreting cells.