ChREBP Rather than USF2 Regulates Glucose Stimulation of Endogenous L-pyruvate Kinase Expression in Insulin-secreting Cells

• Published in: The Journal of biological chemistry 2002-09, Vol.277 (36), p.32746-32752
• Main Authors: Wang, Haiyan, Wollheim, Claes B.
• Format: Article
• Language: English
• Subjects: Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Blotting, Northern; Blotting, Western; Cell Nucleus - metabolism; Cloning, Molecular; DNA, Complementary - metabolism; DNA-Binding Proteins - metabolism; Dose-Response Relationship, Drug; Down-Regulation; Genes, Dominant; Glucose - metabolism; Glucose - pharmacology; Hepatocytes - metabolism; Immunoblotting; Insulin - metabolism; Luciferases - metabolism; Mice; Microscopy, Fluorescence; Plasmids - metabolism; Promoter Regions, Genetic; Protein Binding; Pyruvate Kinase - metabolism; RNA, Messenger - metabolism; Subcellular Fractions; Time Factors; Transcription Factors - metabolism; Transcription, Genetic; Transcriptional Activation; Transfection; Up-Regulation; Upstream Stimulatory Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M201635200

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 β-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 (ΔbTDU1) and -2 (TDU2) in INS-1 cells. Quantitative Northern blot analysis shows that neither basal level nor glucose responsiveness of endogenous L-PK mRNA 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.